Affinitätschromatographie mit orientiert und nicht-orientiert immobilisiertem Calmodulin am Beispiel der Aufreinigung der Ubiquityl-Calmodulin-Synthetase Faktor 2

Im Rahmen dieser Dissertation gelang es eine zielgerichtete Immobilisierung des Calmodulins durch Verwendung des Glutathion-S-Transferase-Calmodulin-Fusionspro-teins (GST-Calmodulin) zu erreichen. Das neue GST-Calmodulin-Gel wurde zur Auf-reinigung von Ubiquityl-Calmodulin-Synthetase Faktor 2 (uCaM Syn-F2) eingesetzt. Da-durch konnte die Ausbeute um Faktor zwei gegenüber der bisher verwendeten Methode mit ungerichtet immobilisiertem Divinylsulfon-Calmodulin gesteigert werden. Von den Ausgangsüberlegungen her hätte dieser Faktor aber etwa sechs betragen müssen, da den sechs verschiedenen Lysinen des Calmodulins im Falle der Divinylsulfon-Kopplung nur ein N-Terminus im Falle der Immobilisierung des GST-Calmodulins gegenüber stand. Die Diskrepanz zwischen dem erwarteten Ergebnis und dem tatsächlich beobachteten Befund wurde so gedeutet, dass im Falle der ungerichteten Immobilisierung möglicherweise nur zwei Lysine des Calmodulins an der Kopplung teilnehmen. Von diesen sich ergebenden Orientierungen würde nur eine die Interaktion mit calmodulinbindenden Proteinen ermöglichen. Diese Vorstellung wiederum erklärt das ähnliche Verhalten im Vergleich zur streng orientierten Immobilisierung des Fusionsproteins am N-Terminus. Auf der Basis bioinformatorischer Untersuchungen wurden verschiedene Modellvorstellungen, wie z.B. Force-Field und Zugänglichkeit, überprüft um so die Bevorzugung bestimmter Lysine zu erklären. Als Anwendungsbeispiel diente hierbei die Ribonuclease, von der bekannt war, dass sie nur über zwei Lysine an die Sepharose gebunden wird. Jedoch konnte keine Modellvorstellung die Bevorzugung einzelner Lysine erklären. Als abschließender Ansatz könnte die unterschiedliche Aktivierungsenergie der einzelnen Lysinseitengruppen eine Erklärung für die eingeschränkte Zahl reaktiver Lysine liefern. Die Aufreinigung der uCaM Syn-F2 an dem neuen Gel ergab, dass uCaM Syn-F2 durch proteolytische Einflüsse während des Lagerns in seinen biochemischen Eigenschaften (z.B. Molekulargewicht und Affinität zum Anionenaustauschergel MonoQ) verändert wurde. Die Proteolyse wurde durch die Anwesenheit von Calcium und der damit bedingten Aktivierung von Calpain verstärkt. Erst durch den Einsatz von Proteaseinhibitoren und kurzen Lagerungszeiten konnten die proteolytischen Einflüsse minimiert werden. Auf Grund hoher Verlusten bei der Anionenaustauschchromatographie wurde versucht, uCaM Syn-F2 durch die, in der Literatur beschriebenen, Blot-Overlay-Verfahren nachzuweisen. Diese Methoden ließen sich im Falle der uCaM Syn-F2 nicht reproduzieren, erst durch eine, in dieser Arbeit neue entwickelten, Overlay-Methode ließ sich eine 31 kDa große Bande als mögliche calmodulinbindende Komponente der uCaM Syn-F2 identifizieren

GFI1 (growth factor independent 1 transcription repressor)

Review on GFI1 (growth factor independent 1 transcription repressor), with data on DNA, on the protein encoded, and where the gene is implicated

The Role of Clonal Evolution on Progression, Blood Parameters, and Response to Therapy in Multiple Myeloma

IntroductionA variety of biomarkers are considered for diagnosis (e.g., β2-microgobulin, albumin, or LDH) and prognosis [e.g., cytogenetic aberrations detected by fluorescence in situ hybridization (FISH)] of multiple myeloma (MM). More recently, clonal evolution has been established as key. Little is known on the clinical implications of clonal evolution.MethodsWe performed in-depth analyses of 25 patients with newly diagnosed MM with respect to detailed clinical information analyzing blood samples collected at several time points during follow-up (median follow-up: 3.26 years since first diagnosis). We split our cohort into two subgroups: with and without new FISH clones developing in the course of disease.ResultsEach subgroup showed a characteristic chromosomal profile. Forty-three percent of patients had evidence of appearing new clones. The patients with new clones showed an increased number of translocations affecting chromosomes 14 (78% vs. 33%; p = 0.0805) and 11, and alterations in chromosome 4 (amplifications and translocations). New clones, on the contrary, were characterized by alterations affecting chromosome 17. Subsequent to the development of the new clone, 6 out of 9 patients experienced disease progression compared to 3 out of 12 for patients without new clones. Duration of the therapy applied for the longest time was significantly shorter within the group of patients developing new clones (median: 273 vs. 406.5 days; p = 0.0465).DiscussionWe demonstrated that the development of new clones, carrying large-scale alterations, was associated with inferior disease course and shorter response to therapy, possibly affecting progression-free survival and overall survival as well. Further studies evaluating larger cohorts are necessary for the validation of our results

Efficacy of COVID-19 Booster Vaccines in Patients with Hematologic Malignancies: Experiences in a Real-World Scenario.

BACKGROUND Two-dose COVID-19 vaccination often results in poor humoral response rates in patients with hematologic malignancies (HMs); yet responses to COVID-19 booster vaccines and the risk of COVID-19 infection post-booster are mostly uncertain. METHODS We included 200 outpatients with HMs and predominantly lymphoid neoplasms (96%, 191/200) in our academic center and reported on the humoral responses, which were assessed by measurement of anti-spike IgG antibodies in peripheral blood as early as 14 days after mRNA-based prime-boost vaccination, as well as factors hampering booster efficacy. Previous basic (double) immunization was applied according to the local recommendations with mRNA- and/or vector-based vaccines. We also report on post-booster COVID-19 breakthrough infections that emerged in the Omicron era and the prophylaxis strategies that were applied to poor and non-responders to booster vaccines. RESULTS A total of 55% (110/200) of the patients achieved seroconversion (i.e., anti-spike protein IgG antibody titer > 100 AU/mL assessed in median 48 days after prime-boost vaccination) after prime-boost vaccination. Multivariable analyses revealed age, lymphocytopenia, ongoing treatment and prior anti-CD20 B-cell depletion to be independent predictors for booster failure. With each month between anti-CD20-mediated B-cell depletion and booster vaccination, the probability of seroconversion increased by approximately 4% (p < 0.001) and serum-antibody titer (S-AbT) levels increased by 90 AU/mL (p = 0.011). Notably, obinutuzumab treatment was associated with an 85% lower probability for seroconversion after prime-boost vaccination compared to rituximab (p = 0.002). Of poor or non-responders to prime-boost vaccination, 41% (47/114) underwent a second booster and 73% (83/114) underwent passive immunization. COVID-19 breakthrough infections were observed in 15% (29/200) of patients after prime-boost vaccination with predominantly mild courses (93%). Next to seroconversion, passive immunization was associated with a significantly lower risk of COVID-19 breakthrough infections after booster, even in vaccine non-responders (all p < 0.05). In a small proportion of analyzed patients with myeloid neoplasms (9/200), the seroconversion rate was higher compared to those with lymphoid ones (78% vs. 54%, accordingly), while the incidence rate of COVID-19 breakthrough infections was similar (22% vs. 14%, respectively). Following the low frequency of myeloid neoplasms in this study, the results may not be automatically applied to a larger cohort. CONCLUSIONS Patients with HMs are at a high risk of COVID-19 booster vaccine failure; yet COVID-19 breakthrough infections after prime-boost vaccination are predominantly mild. Booster failure can likely be overcome by passive immunization, thereby providing immune protection against COVID-19 and attenuating the severity of COVID-19 courses. Further sophistication of clinical algorithms for preventing post-vaccination COVID-19 breakthrough infections is urgently needed

Maximum parsimony analysis of gene expression profiles permits the reconstruction of developmental cell lineage trees

AbstractSpatiotemporal control of gene expression lies at the heart of generating several hundred distinct cell types required for the development of higher order animals. Different cell types within complex organs are often characterised by means of genome-wide gene expression profiling, but analogous information for early developmental as well as adult stem and progenitor cells is largely missing because their identity is commonly unknown or they are present in prohibitively small numbers. Here we show that maximum parsimony approaches previously used to reconstruct evolutionary trees from gene content of extant species can be adapted to reconstruct cellular hierarchies both during development and steady state homeostasis of complex mammalian tissues. Using haematopoiesis as a model, we show that developmental trees reconstructed from expression profiles of mature cells are not only consistent with current experimentally validated trees but also have predictive value in determining progenitor cell specific transcriptional programmes and lineage determining transcription factors. Subsequent analysis across diverse developmental systems such as neuronal development and endoderm organogenesis demonstrated that maximum parsimony-based reconstruction of developmental trees represents a widely applicable approach to infer developmental pathways as well as the transcriptional control mechanisms underlying cell fate specification

Selective embolization of the internal iliac arteries for the treatment of intractable hemorrhage in children with malignancies

PurposeAcute internal hemorrhage is an occasionally life-threatening complication in pediatric cancer patients. Many therapeutic approaches have been used to control bleeding with various degrees of success. In this study, we evaluated the efficacy of selective internal iliac artery embolization for controlling acute intractable bleeding in children with malignancies.MethodsWe retrospectively evaluated the cases of 6 children with various malignancies (acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, T-cell prolymphocytic leukemia, Langerhans cell histiocytosis, and rhabdomyosarcoma), who had undergone selective arterial embolization (SAE) of the internal iliac artery at the Chonnam National University Hwasun Hospital between January 2004 and December 2009. SAE was performed by an interventional radiologist using Gelfoam® and/or Tornado® coils.ResultsThe patients were 5 boys and 1 girl with median age of 6.9 years (range, 0.7-14.8 years) at the time of SAE. SAE was performed once in 4 patients and twice in 2, and the procedure was unilateral in 2 and bilateral in 4. The causes of hemorrhage were as follows: hemorrhagic cystitis (HC) in 3 patients, procedure-related internal iliac artery injuries in 2 patients, and tumor rupture in 1 patient. Initial attempt at conservative management was unsuccessful. Of the 6 patients, 5 (83.3%) showed improvement after SAE without complications.ConclusionSAE may be a safe and effective procedure for controlling acute intractable hemorrhage in pediatric malignancy patients. This procedure may obviate the need for surgery, which carries an attendant risk of morbidity and mortality in cancer patients with critical conditions

Extracellular laminin regulates hematopoietic potential of pluripotent stem cells through integrin β1-ILK-β-catenin-JUN axis

血液細胞へ効率よく変化させる弱い接着を明らかに --血液細胞の産生効率を向上--. 京都大学プレスリリース. 2021-03-31.Stuck stem cells are no good at making blood. 京都大学プレスリリース. 2021-03-31.Recombinant matrices have enabled feeder cell-free maintenance cultures of human pluripotent stem cells (hPSCs), with laminin 511-E8 fragment (LM511-E8) being widely used. However, we herein report that hPSCs maintained on LM511-E8 resist differentiating to multipotent hematopoietic progenitor cells (HPCs), unlike hPSCs maintained on LM421-E8 or LM121-E8. The latter two LM-E8s bound weakly to hPSCs compared with LM511-E8 and activated the canonical Wnt/β-catenin signaling pathway. Moreover, the extracellular LM-E8-dependent preferential hematopoiesis was associated with a higher expression of integrin β1 (ITGB1) and downstream integrin-linked protein kinase (ILK), β-catenin and phosphorylated JUN. Accordingly, the lower coating concentration of LM511-E8 or addition of a Wnt/β-catenin signaling activator, CHIR99021, facilitated higher HPC yield. In contrast, the inhibition of ILK, Wnt or JNK by inhibitors or mRNA knockdown suppressed the HPC yield. These findings suggest that extracellular laminin scaffolds modulate the hematopoietic differentiation potential of hPSCs by activating the ITGB1-ILK-β-catenin-JUN axis at the undifferentiated stage. Finally, the combination of low-concentrated LM511-E8 and a revised hPSC-sac method, which adds bFGF, SB431542 and heparin to the conventional method, enabled a higher yield of HPCs and higher rate for definitive hematopoiesis, suggesting a useful protocol for obtaining differentiated hematopoietic cells from hPSCs in general

Joint Metabonomic and Instrumental Analysis for the Classification of Migraine Patients with 677-MTHFR Mutations

Migraine is a neurological disorder that correlates with an increased risk of cerebrovascular lesions. Genetic mutations of the MTHFR gene are correlated to migraine and to the increased risk of artery pathologies. Also, migraine patients show altered hematochemical parameters, linked to an impaired platelet aggregation mechanism. Hence, the vascular assessment of migraineurs is of primary importance