51 research outputs found
Establishment and improvement of a high-resolution method to analyse the phosphorylation state of the mitogen activated protein kinases ERK1 and ERK2
Fehlregulationen der ERK1/2-Signalkaskade könnten nicht nur in der Onkogenese, sondern auch im Kontext der Alzheimer-Krankheit und der Amyothrophen Lateralsklerose involviert sein (Kim und Choi, 2010; Deschenes-Simard et al., 2014). VollstĂ€ndig aktiviert werden die Mitogen-aktivierten Proteinkinasen ERK1 und ERK2 ĂŒber eine doppelte Phosphorylierung an einem konservierten Threonin-Glutamat-Tyrosin (TEY)-Motiv. Monophosphorylierte Isoformen von ERK1/2 sind ebenfalls in lebenden Zellen nachzuweisen und besitzen in vitro bei physiologischen ATP-Konzentrationen eine messbare KinaseaktivitĂ€t. Eine hochauflösende Trennung und Detektion der unphosphorylierten, einfach- und zweifach-phosphorylierten Isoformen von ERK1 und ERK2 sowie ihre semi-quantitative Analyse sind durch den Einsatz der isoelektrischen Fokussierung in Mikrokapillaren mit nachgeschaltetem Immunnachweis (CIEF-Immunoassay) möglich.
Im ersten Teil der vorliegenden Arbeit wurde ein optimiertes Arbeitsprotokoll entwickelt, um die Phosphorylierung von ERK1/2 in Zelllysaten mit dem CIEF-Immunoassay zu analysieren. Es wurden verschiedene Lysesysteme getestet, wobei sich herausstellte, dass Reduktionsmittel die ERK1/2-Detektion erheblich beeinflussen können. Das kommerziell erhĂ€ltliche M-PER-Reagenz eignete sich am besten, da es eine höhere Effizienz zur Lyse der Zellkerne zeigte als andere Lysepuffer, keine Reduktionsmittel enthielt und eine hohe Ausbeute an phosphorylierten ERK1/2-Isoformen ermöglichte. Die Reproduzierbarkeit der relativen Quantifizierungen im CIEF-Immunoassay erwies sich fĂŒr solche Signale als akzeptabel (Variationskoeffizient < 15 %), deren relativer FlĂ€cheninhalt mindestens 1 % der aufsummierten FlĂ€cheninhalte aller ERK1- bzw. ERK2-Signale betrug. Insgesamt lagen die Abweichungen in einem Bereich, der in der Regel sowohl einen Vergleich von Proben innerhalb eines Laufes als auch zwischen mehreren LĂ€ufen erlaubte.
Als wichtige Weiterentwicklung der bisher publizierten analytischen Möglichkeiten des verwendeten CIEF-Immunoassays gelang im Rahmen der hier vorgestellten Arbeit die zusÀtzliche Unterscheidung und eindeutige Zuordnung von Threonin-monophosphoryliertem und Tyrosin-monophosphoryliertem ERK1 und ERK2. Dieser Nachweis wurde durch die Kombination synthetischer phospho-ERK1/2-Peptide mit verschiedenen anti-phospho-ERK1/2-Antikörpern in kompetitiven Blockierungsexperimenten, welche in dieser Form erstmalig im CIEF-Immunoassay eingesetzt wurden, erreicht.
Um die praktische Anwendbarkeit des optimierten Arbeits- und Analyseprotokolls zu ĂŒberprĂŒfen, wurde im zweiten Teil der Arbeit zunĂ€chst exemplarisch die Kinetik der ERK1/2-Phosphorylierung in den Zelllinien SH-SY5Y und THP-1 sowie in humanen peripheren mononukleĂ€ren Blutzellen nach Stimulation der Zellen zeitabhĂ€ngig untersucht. Die ERK1/2-phospho-Form-Verteilung schien dabei abhĂ€ngig vom jeweiligen Zelltyp und eingesetzten Aktivator zu variieren.
Die erfolgreich etablierte Methode wurde schlieĂlich in Form einer orientierenden Pilot-Studie mit einer klinischen Kohorte an humanen peripheren mononukleĂ€ren Blutzellen von Patienten mit Alzheimer-Demenz, leichten kognititven EinschrĂ€nkungen, Amyotropher Lateralsklerose und nicht-dementen Kontrollprobanden angewandt. Trotz der sehr kleinen Stichprobe konnten statistisch signifikante Gruppenunterschiede, nach erfolgter Aktivierung durch Gabe eines Stimulus, hinsichtlich der Inaktivierung (Dephosphorylierung) der doppelt-phosphorylierten und Threonin-monophosphorylierten Formen von ERK1 beobachtet werden. Dies könnte auf eine mögliche krankheitsassoziierte VerĂ€nderung in der AktivitĂ€t von Phosphatasen hinweisen. Die Ergebnisse sind allerdings aufgrund der kleinen GruppengröĂen als vorlĂ€ufig zu betrachten. FĂŒr den Einsatz mit einer gröĂeren Kohorte ist die Optimierung weiterer Parameter erforderlich. Insgesamt ermöglicht der CIEF-Immunoassay eine detaillierte Studie zur relativen Verteilung der phospho-ERK1/2-Isoformen in Zelllysaten.The dysregulation of the ERK1/2 signalling cascade appears to be involved not only in oncogenesis but also in the context of Alzheimerâs disease and amyotrophic lateral sclerosis. The mitogen activated protein kinases ERK1 and ERK2 are fully activated by dual phosphorylation at a conserved threonine-glutamate-tyrosine (TEY) motif. Monophosphorylated isoforms of ERK1/2 are also present in living cells and have appreciable kinase activities in vitro at physiological ATP concentrations. The highly sensitive detection and differentiation of the unphosphorylated, monophosphorylated and diphosphorylated isoforms of ERK1 and ERK2 as well as their semi-quantitative analysis can be achieved by capillary isoelectric focussing followed by immunodetection (CIEF-immunoassay).
In the first part of the present work an optimised working procedure was elaborated for the detailed analysis of phosphorylation of ERK1/2 in cell lysates by CIEF-immunoassay. When different lysing systems were tested, it turned out, that reducing agents can substantially influence the ERK1/2 detection. The commercially available M-PER reagent was most suitable as it possessed higher efficiency in lysing nuclei than other buffers, it contained no reducing agents and offered a high recovery of ERK1/2-phospho-forms in the resulting cell lysates. The reproducibility of the relative quantifications in the CIEF-immunoassay was acceptable (coefficient of variation < 15 %) for signals with a relative abundancy greater than 1 % of the added abundancies of all ERK1 and ERK2 signals, respectively. Generally, the observed variation allowed for reasonable comparison of samples within the same assay as well as in different, independent assays.
An important improvement of previously published analytical possibilities of the CIEF-Immunoassay is the additional differentiation and classification of the threonine- and tyrosine-monophosphorylated isoforms of ERK1 and ERK2, which is presented within this work. The differentiation and unequivocal peak identification were achieved with a combination of synthetic phospho-ERK1/2 peptides and several anti-phospho-ERK1/2 antibodies in competitive blocking experiments, which were performed for the first time in the CIEF-Immunoassay in this form.
As a proof of principle the optimised standard operating procedures were applied to exemplarily analyse the kinetics of ERK1/2 phosphorylation and the time dependent occurrence of phospho-ERK1/2 after stimulation in SH-SY5Y and THP-1 cell lines as well as in peripheral blood mononuclear cells in the second part of the work. The ERK1/2-phospho-form distribution seemed to depend on the particular cell type and the applied stimulus.
The optimised method was furthermore applied to an orienting pilot study on human peripheral blood mononuclear cells from a small clinical cohort of patients with Alzheimerâs disease, mild cognitive impairment, amyotrophic lateral sclerosis and non-demented controls. Despite of the very small sample size, statistically significant differences between the diagnostic groups could be observed, after activation by applying a stimulus, for the inactivation (dephosphorylation) of the diphosphorylated and threonine-monophosphorylated forms of ERK1. These changes could possibly be linked to a disease related dysregulation of phosphatase activity. Due to the small size of the groups within this study, the results have to be considered as preliminary. For additional confirmative studies with a greater sample size an optimisation of several parameters will be necessary. Altogether, the CIEF-immunoassay enables detailed studies concerning the relative ERK1/2-phospho-form distribution in cell lysates
A Study of Language Learning Style and Teaching Style Preferences of Hong Kong Community College Students and Teachers in English for Academic Purposes (EAP) Contexts
In English language classrooms, students use different approaches to carry out
English learning tasks. Language learning styles, which generally refers to learnersâ
preferred modes of language learning, have been widely researched and discussed in
the fields of second language acquisition (SLA) and educational psychology.
Understanding the learning style preferences of students can help teachers cope with
studentsâ course-related learning difficulties and ultimately help alleviate their
frustration levels. Another important concept is teaching styles, which refers to
teachersâ classroom behaviour based on their teaching beliefs, is commonly
associated with learning styles in language education research. Teaching style is vital
for providing students with good learning experiences and improving studentsâ
academic outcomes.
This study explores the English language learning and teaching style preferences
in English for Academic Purposes (EAP) classrooms at community college level in
Hong Kong. The present study adopted a mixed method approach involving both
questionnaire surveys and semi-structured interviews, in attempt to investigate the
factors influencing learning styles and teaching styles, and the relationship between
them. It aims at providing valuable information for curriculum design and teacher
training in order to offer Hong Kong community college students adequate and
effective academic English language learning support.
A total of 637 students and 10 EAP teachers from two community colleges in Hong Kong participated in this research. The quantitative and qualitative findings of
this study show that the community college students in EAP classrooms have multiple
learning style preferences. A plethora of factors such as cultural and educational
backgrounds are related to their development of learning styles. This research also
explores the nature of teaching styles and the possible variables, including studentsâ
English language proficiency and their learning styles, influencing their teaching
styles in EAP classrooms.
This study attempts to explain the relationship between learning styles and
teaching styles in English language classrooms with reference to the interview
findings from both students and teachers. It is argued that both learning styles and
teaching styles are flexible and have a reciprocal influence on each other. Learners
may adjust their learning styles in order to meet academic requirements, while
teachers may adjust their teaching styles so as to provide students with an affective
learning environment. When learners and teachers have more interaction with each
other, their styles may become similar to each other. This study also identifies the
importance of improving learnersâ flexibility for developing learning styles and
accepting unfamiliar teaching styles.
Based on the evidence drawn from this research, educational implications on
teaching and learning in EAP classrooms, and recommendations for future research
on learning styles and teaching styles are proposed
The genomes of two key bumblebee species with primitive eusocial organization
Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation
The German National Pandemic Cohort Network (NAPKON): rationale, study design and baseline characteristics
Schons M, Pilgram L, Reese J-P, et al. The German National Pandemic Cohort Network (NAPKON): rationale, study design and baseline characteristics. European Journal of Epidemiology . 2022.The German government initiated the Network University Medicine (NUM) in early 2020 to improve national research activities on the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic. To this end, 36 German Academic Medical Centers started to collaborate on 13 projects, with the largest being the National Pandemic Cohort Network (NAPKON). The NAPKON's goal is creating the most comprehensive Coronavirus Disease 2019 (COVID-19) cohort in Germany. Within NAPKON, adult and pediatric patients are observed in three complementary cohort platforms (Cross-Sectoral, High-Resolution and Population-Based) from the initial infection until up to three years of follow-up. Study procedures comprise comprehensive clinical and imaging diagnostics, quality-of-life assessment, patient-reported outcomes and biosampling. The three cohort platforms build on four infrastructure core units (Interaction, Biosampling, Epidemiology, and Integration) and collaborations with NUM projects. Key components of the data capture, regulatory, and data privacy are based on the German Centre for Cardiovascular Research. By April 01, 2022, 34 university and 40 non-university hospitals have enrolled 5298 patients with local data quality reviews performed on 4727 (89%). 47% were female, the median age was 52 (IQR 36-62-) and 50 pediatric cases were included. 44% of patients were hospitalized, 15% admitted to an intensive care unit, and 12% of patients deceased while enrolled. 8845 visits with biosampling in 4349 patients were conducted by April 03, 2022. In this overview article, we summarize NAPKON's design, relevant milestones including first study population characteristics, and outline the potential of NAPKON for German and international research activities.Trial registration https://clinicaltrials.gov/ct2/show/NCT04768998 . https://clinicaltrials.gov/ct2/show/NCT04747366 . https://clinicaltrials.gov/ct2/show/NCT04679584. © 2022. The Author(s)
Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study
Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008â11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003â13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 Ă 10â10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 Ă 10â8 DHFR p=8·37 Ă 10â7 MTRNR2L2 p=2·15 Ă 10â9) and to a lesser extent in REGISTRY (MSH3 p=9·36 Ă 10â4 DHFR p=8·45 Ă 10â4 MTRNR2L2 p=1·20 Ă 10â3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 Ă 10â8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16â0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06â0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation
Etablierung und Weiterentwicklung einer Methode zur hochauflösenden Untersuchung des Phosphorylierungszustandes der Mitogen-aktivierten Proteinkinasen ERK1 und ERK2
Fehlregulationen der ERK1/2-Signalkaskade könnten nicht nur in der Onkogenese, sondern auch im Kontext der Alzheimer-Krankheit und der Amyothrophen Lateralsklerose involviert sein (Kim und Choi, 2010; Deschenes-Simard et al., 2014). VollstĂ€ndig aktiviert werden die Mitogen-aktivierten Proteinkinasen ERK1 und ERK2 ĂŒber eine doppelte Phosphorylierung an einem konservierten Threonin-Glutamat-Tyrosin (TEY)-Motiv. Monophosphorylierte Isoformen von ERK1/2 sind ebenfalls in lebenden Zellen nachzuweisen und besitzen in vitro bei physiologischen ATP-Konzentrationen eine messbare KinaseaktivitĂ€t. Eine hochauflösende Trennung und Detektion der unphosphorylierten, einfach- und zweifach-phosphorylierten Isoformen von ERK1 und ERK2 sowie ihre semi-quantitative Analyse sind durch den Einsatz der isoelektrischen Fokussierung in Mikrokapillaren mit nachgeschaltetem Immunnachweis (CIEF-Immunoassay) möglich. Im ersten Teil der vorliegenden Arbeit wurde ein optimiertes Arbeitsprotokoll entwickelt, um die Phosphorylierung von ERK1/2 in Zelllysaten mit dem CIEF-Immunoassay zu analysieren. Es wurden verschiedene Lysesysteme getestet, wobei sich herausstellte, dass Reduktionsmittel die ERK1/2-Detektion erheblich beeinflussen können. Das kommerziell erhĂ€ltliche M-PER-Reagenz eignete sich am besten, da es eine höhere Effizienz zur Lyse der Zellkerne zeigte als andere Lysepuffer, keine Reduktionsmittel enthielt und eine hohe Ausbeute an phosphorylierten ERK1/2-Isoformen ermöglichte. Die Reproduzierbarkeit der relativen Quantifizierungen im CIEF-Immunoassay erwies sich fĂŒr solche Signale als akzeptabel (Variationskoeffizient < 15 %), deren relativer FlĂ€cheninhalt mindestens 1 % der aufsummierten FlĂ€cheninhalte aller ERK1- bzw. ERK2-Signale betrug. Insgesamt lagen die Abweichungen in einem Bereich, der in der Regel sowohl einen Vergleich von Proben innerhalb eines Laufes als auch zwischen mehreren LĂ€ufen erlaubte. Als wichtige Weiterentwicklung der bisher publizierten analytischen Möglichkeiten des verwendeten CIEF-Immunoassays gelang im Rahmen der hier vorgestellten Arbeit die zusĂ€tzliche Unterscheidung und eindeutige Zuordnung von Threonin-monophosphoryliertem und Tyrosin-monophosphoryliertem ERK1 und ERK2. Dieser Nachweis wurde durch die Kombination synthetischer phospho-ERK1/2-Peptide mit verschiedenen anti-phospho-ERK1/2-Antikörpern in kompetitiven Blockierungsexperimenten, welche in dieser Form erstmalig im CIEF-Immunoassay eingesetzt wurden, erreicht. Um die praktische Anwendbarkeit des optimierten Arbeits- und Analyseprotokolls zu ĂŒberprĂŒfen, wurde im zweiten Teil der Arbeit zunĂ€chst exemplarisch die Kinetik der ERK1/2-Phosphorylierung in den Zelllinien SH-SY5Y und THP-1 sowie in humanen peripheren mononukleĂ€ren Blutzellen nach Stimulation der Zellen zeitabhĂ€ngig untersucht. Die ERK1/2-phospho-Form-Verteilung schien dabei abhĂ€ngig vom jeweiligen Zelltyp und eingesetzten Aktivator zu variieren. Die erfolgreich etablierte Methode wurde schlieĂlich in Form einer orientierenden Pilot-Studie mit einer klinischen Kohorte an humanen peripheren mononukleĂ€ren Blutzellen von Patienten mit Alzheimer-Demenz, leichten kognititven EinschrĂ€nkungen, Amyotropher Lateralsklerose und nicht-dementen Kontrollprobanden angewandt. Trotz der sehr kleinen Stichprobe konnten statistisch signifikante Gruppenunterschiede, nach erfolgter Aktivierung durch Gabe eines Stimulus, hinsichtlich der Inaktivierung (Dephosphorylierung) der doppelt-phosphorylierten und Threonin-monophosphorylierten Formen von ERK1 beobachtet werden. Dies könnte auf eine mögliche krankheitsassoziierte VerĂ€nderung in der AktivitĂ€t von Phosphatasen hinweisen. Die Ergebnisse sind allerdings aufgrund der kleinen GruppengröĂen als vorlĂ€ufig zu betrachten. FĂŒr den Einsatz mit einer gröĂeren Kohorte ist die Optimierung weiterer Parameter erforderlich. Insgesamt ermöglicht der CIEF-Immunoassay eine detaillierte Studie zur relativen Verteilung der phospho-ERK1/2-Isoformen in Zelllysaten
Membrane Region M2C2 in Subunit KtrB of the K+ Uptake System KtrAB from Vibrio alginolyticus Forms a Flexible Gate Controlling K+ Flux: AN ELECTRON PARAMAGNETIC RESONANCE STUDY*
Transmembrane stretch M2C from the bacterial K+-translocating protein KtrB is unusually long. In its middle part, termed M2C2, it contains several small and polar amino acids. This region is flanked by the two α-helices M2C1 and M2C3 and may form a flexible gate at the cytoplasmic side of the membrane controlling K+ translocation. In this study, we provide experimental evidence for this notion by using continuous wave and pulse EPR measurements of single and double spin-labeled cysteine variants of KtrB. Most of the spin-labeled residues in M2C2 were shown to be immobile, pointing to a compact structure. However, the high polarity revealed for the microenvironment of residue positions 317, 318, and 327 indicated the existence of a water-accessible cavity. Upon the addition of K+ ions, M2C2 residue Thr-318R1 (R1 indicates the bound spin label) moved with respect to M2B residue Asp-222R1 and M2C3 residue Val-331R1 but not with respect to M2C1 residue Met-311R1. Based on distances determined between spin-labeled residues of double-labeled variants of KtrB in the presence and absence of K+ ions, structural models of the open and closed conformations were developed
PDF Copy Of Online Survey
This is a PDF copy of an online-survey sent to members of UMG within the GRAcE-project.
Link to a copy of the online-survey: Online Survey</a
Identifikation von Kostenfaktoren und SchĂ€tzung des Ressourcenbedarfs fĂŒr das Forschungsdatenmanagement am Beispiel der UniversitĂ€tsmedizin Göttingen
Im Rahmen des vom BMBF geförderten Projektes Göttingen Research Data Exploratory (GRAcE) verfasster Projektbericht
- âŠ