Adaptive evolution of transcription factor binding sites

The regulation of a gene depends on the binding of transcription factors to specific sites located in the regulatory region of the gene. The generation of these binding sites and of cooperativity between them are essential building blocks in the evolution of complex regulatory networks. We study a theoretical model for the sequence evolution of binding sites by point mutations. The approach is based on biophysical models for the binding of transcription factors to DNA. Hence we derive empirically grounded fitness landscapes, which enter a population genetics model including mutations, genetic drift, and selection. We show that the selection for factor binding generically leads to specific correlations between nucleotide frequencies at different positions of a binding site. We demonstrate the possibility of rapid adaptive evolution generating a new binding site for a given transcription factor by point mutations. The evolutionary time required is estimated in terms of the neutral (background) mutation rate, the selection coefficient, and the effective population size. The efficiency of binding site formation is seen to depend on two joint conditions: the binding site motif must be short enough and the promoter region must be long enough. These constraints on promoter architecture are indeed seen in eukaryotic systems. Furthermore, we analyse the adaptive evolution of genetic switches and of signal integration through binding cooperativity between different sites. Experimental tests of this picture involving the statistics of polymorphisms and phylogenies of sites are discussed.Comment: published versio

Dipeptidylpeptidase IV (CD26) defines leukemic stem cells (LSC) in chronic myeloid leukemia

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although mechanisms of BCR/ABL1-induced transformation are well-defined, little is known about effector-molecules contributing to malignant expansion and the extramedullary spread of leukemic SC (LSC) in CML. We have identified the cytokine-targeting surface enzyme dipeptidylpeptidase-IV (DPPIV/CD26) as a novel, specific and pathogenetically relevant biomarker of CD34+/CD38─ CML LSC. In functional assays, CD26 was identified as target enzyme disrupting the SDF-1-CXCR4-axis by cleaving SDF-1, a chemotaxin recruiting CXCR4+ SC. CD26 was not detected on normal SC or LSC in other hematopoietic malignancies. Correspondingly, CD26+ LSC decreased to low or undetectable levels during successful treatment with imatinib. CD26+ CML LSC engrafted NOD-SCID-IL-2Rγ−/− (NSG) mice with BCR/ABL1+ cells, whereas CD26─ SC from the same patients produced multilineage BCR/ABL1– engraftment. Finally, targeting of CD26 by gliptins suppressed the expansion of BCR/ABL1+ cells. Together, CD26 is a new biomarker and target of CML LSC. CD26 expression may explain the abnormal extramedullary spread of CML LSC, and inhibition of CD26 may revert abnormal LSC function and support curative treatment approaches in this malignancy

Plant responses to photoperiod

Photoperiod controls many developmental responses in animals, plants and even fungi. The response to photoperiod has evolved because daylength is a reliable indicator of the time of year, enabling developmental events to be scheduled to coincide with particular environmental conditions. Much progress has been made towards understanding the molecular mechanisms involved in the response to photoperiod in plants. These mechanisms include the detection of the light signal in the leaves, the entrainment of circadian rhythms, and the production of a mobile signal which is transmitted throughout the plant. Flowering, tuberization and bud set are just a few of the many different responses in plants that are under photoperiodic control. Comparison of what is known of the molecular mechanisms controlling these responses shows that, whilst common components exist, significant differences in the regulatory mechanisms have evolved between these responses

Herstellung und Evaluation spezifischer PET-Liganden unter Anwendung moderner Radiofluorierungsmethoden

Die molekulare Bildgebung mit Hilfe der Positronen-Emissions-Tomographie (PET) ist wesentlich von der Verfügbarkeit geeigneter Radiotracer abhängig. Der erste Schritt im Entwicklungsprozess eines Radiotracers ist die Identifikation eines geeigneten molekularen Targets, welches durch den Radiotracer adressiert werden soll. Molekulare Targets sind beispielsweise Rezeptorproteine, die Erkrankungen aufzeigen können, da das regionale Rezeptorexpressionsprofil aufgrund pathologischer Prozesse verändert sein kann. Auch Enzyme eignen sich als molekulare Targets, da deren veränderte Aktivität mit Hilfe von Radiotracern detektiert werden kann. Nach Targetidentifikation, müssen Radioliganden entwickelt und deren Eignung für die Bildgebung evaluiert werden. Ein Ziel der vorliegenden Arbeit war die Radiosynthese und Evaluation geeigneter 18 F-markierter Radioliganden für die Bildgebung der Dopamin-D 4 sowie der Serotonin 5-HT 2A -Rezeptoren, deren Funktion und Beteiligung bei der Entstehung neurologischer und psychischer Erkrankungen, wie der Schizophrenie oder der Aufmerksamkeitsdefizit-/Hyperaktivitätsstörung (ADHS), heute noch nicht ausreichend geklärt ist. In-vivo-PET-Studien könnten einen entscheidenden Beitrag zur Aufklärung der Funktion dieser Rezeptoren liefern und dazu beitragen neue Therapieansätze zu entwickeln. Besonderer Klärungsbedarf besteht dabei für die Funktionen des Dopamin-D 4 -Rezeptors (D 4 R), der aufgrund seiner geringen Verteilungsdichte im Gehirn eine besondere Herausforderung für die Bildgebung darstellt. Die aus der aktuellen Literatur bekannten, D 4 R-spezifischen Liganden, wurden als nicht-radioaktive Referenzverbindungen hergestellt und ihr Bindungsverhalten gegenüber den Subtypen der Dopaminrezeptoren D 1-5 R, hinsichtlich Bindungsaffinität und Selektivität, in Bindungsstudien evaluiert. In dieser Arbeit wurden zwei Leitstrukturen ausgewählt und mit Fluor-18 markiert. In In-vitro-Autoradiographien wurde für eine der 18 F-markierten Verbindungen ein spezifisches Bindungsverhalten gezeigt. Für PET-Studien des 5-HT 2A -Rezeptors sind die Radioliganden [ 18 F]Altanserin und [ 11 C]MDL100,907 im klinischen Einsatz. Allerdings ist deren Anwendung mit einigen Nachteilen verbunden, wie z.B. der Bildung hirngängiger Radiometaboliten bei [ 18 F]Altanserin oder einer (zu) kurzen Halbwertszeit bei [ 11 C]MDL100,907, die den Einsatz in klinischen Studien erschweren. R91150 ist ein spezifischer 5-HT 2A R-Ligand, der als radioiodiertes Derivat [ 123 I]Iod-R91150 allerdings aufgrund seiner hohen Lipophilie ein unspezifisches Bindungsverhalten zeigt. Das entsprechende radiofluorierte Analogon sollte mit knapp zwei Stunden Halbwertszeit und geringerer Lipophilie günstigere Bindungseigenschaften aufweisen. Vor ca. 10 Jahren wurde bereits eine 6-stufige Radiosynthese von [ 18 F]R91150 vorgestellt. Trotz vielversprechender Resultate aus ersten Metabolismusstudien hat die aufwendige und schwierig zu automatisierende Radiosynthese bisher weitere Studien verhindert. Die Radiosynthese von [ 18 F]R91150 konnte in vorliegender Arbeit durch Anwendung moderner Radiofluorierungsmethoden deutlich vereinfacht werden, sodass erstmals der In-vitro-Einsatz von [ 18 F]R91150 möglich wurde. Der 18 F-markierte 5-HT 2A R-Ligand hat in den In-vitro-Autoradiographien eine klar abgegrenzte, spezifische Bindung in Hirnarealen mit hoher 5-HT 2A R-Expression gezeigt. Ein weiteres Ziel der vorliegenden Arbeit war die Entwicklung neuer, 18 F-markierter prosthetischer Gruppen für die indirekte Radiofluorierung empfindlicher Biomoleküle. Dazu wurden Amin-reaktive sowie für die „Click-Chemie“ funktionalisierte Azaisatosäureanhydride mit 18 F-markiert und ihre Reaktivität gegenüber verschiedenen Aminen, bzw. im Fall einer Propargyl-funktionalisierten Verbindung, die Konjugation mit Aziden in der Kupfer(I)-vermittelten Azid-Alkin-Cycloaddition (CuAAC) entwickelt. Durch Adaption neuer Methoden der Radiofluorierung konnten die neuen Markierungsbausteine innerhalb von nur 1-2 min direkt auf einer Anionenaustauscherkartusche mit Fluor-18 markiert werden. Die Reaktion mit verschiedenen Modellaminen lieferte überwiegend moderate bis quantitative radiochemische Ausbeuten (RCY = 15-98%). Durch Konjugation an die PSMSA-spezifische Glu-C(O)-Lys Struktur konnte ein neuer PSMA-selektiver Tracer [ 18 F]JK-PSMA-15 erhalten werden. Die Azid-funktionalisierten Azaisatosäureanhydride wurden mittels CuAAC mit verschiedenen Propargyl-funktionalisierten Modellverbindungen in radiochemischen Umsätzen von 38-56% dargestellt. Mit dieser Methode wurden zwei weitere, auf der Glu-C(O)-Lys Struktur basierende PSMA-Tracer, [ 18 F]JK-PSMA-16 und [ 18 F]JK-PSMA-17, synthetisiert. [ 18 F]JK-PSMA-15 wurde in In-vivo-PET-Studien an gesunden Ratten (PSMA-positives Ganglien-Modell) evaluiert, wobei der Tracer eine PSMA-spezifische Anreicherung mit hohem Signal-Hintergrund-Verhältnis, ohne Hinweise auf Defluorierung oder unspezifischem Bindungsverhalten aufwies. Der neue Markierungsbaustein lässt sich schneller markieren und isolieren als vergleichbare prosthetische Gruppen für die indirekte Radiofluorierung und kann sehr effektiv zur Konjugation mit Aminen und Aziden eingesetzt werden

Dynamically simulating the interaction of midazolam and the CYP3A4 inhibitor itraconazole using individual coupled whole-body physiologically-based pharmacokinetic (WB-PBPK) models

BACKGROUND: Drug-drug interactions resulting from the inhibition of an enzymatic process can have serious implications for clinical drug therapy. Quantification of the drugs internal exposure increase upon administration with an inhibitor requires understanding to avoid the drug reaching toxic thresholds. In this study, we aim to predict the effect of the CYP3A4 inhibitors, itraconazole (ITZ) and its primary metabolite, hydroxyitraconazole (OH-ITZ) on the pharmacokinetics of the anesthetic, midazolam (MDZ) and its metabolites, 1' hydroxymidazolam (1OH-MDZ) and 1' hydroxymidazolam glucuronide (1OH-MDZ-Glu) using mechanistic whole body physiologically-based pharmacokinetic simulation models. The model is build on MDZ, 1OH-MDZ and 1OH-MDZ-Glu plasma concentration time data experimentally determined in 19 CYP3A5 genotyped adult male individuals, who received MDZ intravenously in a basal state. The model is then used to predict MDZ, 1OH-MDZ and 1OH-MDZ-Glu concentrations in an CYP3A-inhibited state following ITZ administration. RESULTS: For the basal state model, three linked WB-PBPK models (MDZ, 1OH-MDZ, 1OH-MDZ-Glu) for each individual were elimination optimized that resulted in MDZ and metabolite plasma concentration time curves that matched individual observed clinical data. In vivo K(m )and V(max )optimized values for MDZ hydroxylation were similar to literature based in vitro measures. With the addition of the ITZ/OH-ITZ model to each individual coupled MDZ + metabolite model, the plasma concentration time curves were predicted to greatly increase the exposure of MDZ as well as to both increase exposure and significantly alter the plasma concentration time curves of the MDZ metabolites in comparison to the basal state curves. As compared to the observed clinical data, the inhibited state curves were generally well described although the simulated concentrations tended to exceed the experimental data between approximately 6 to 12 hours following MDZ administration. This deviations appeared to be greater in the CYP3A5 *1/*1 and CYP3A5 *1/*3 group than in the CYP3A5 *3/*3 group and was potentially the result of assuming that ITZ/OH-ITZ inhibits both CYP3A4 and CYP3A5, whereas in vitro inhibition is due to CYP3A4. CONCLUSION: This study represents the first attempt to dynamically simulate metabolic enzymatic drug-drug interactions via coupled WB-PBPK models. The workflow described herein, basal state optimization followed by inhibition prediction, is novel and will provide a basis for the development of other inhibitor models that can be used to guide, interpret, and potentially replace clinical drug-drug interaction trials

Longitudinal cell-free DNA characterization by low-coverage whole-genome sequencing in patients undergoing high-dose radiotherapy

BACKGROUND AND PURPOSE: Current radiotherapy guidelines rely heavily on imaging-based monitoring. Liquid biopsy monitoring promises to complement imaging by providing frequent systemic information about the tumor. In particular, cell-free DNA (cfDNA) sequencing offers a tumor-agnostic approach, which lends itself to monitoring heterogeneous cohorts of cancer patients. METHODS: We collected plasma cfDNA from oligometastatic patients (OMD) and head-and-neck cancer patients (SCCHN) at six time points before, during, and after radiotherapy, and compared them to the plasma samples of healthy and polymetastatic volunteers. We performed low-pass (on average 7x) whole-genome sequencing on 93 plasma cfDNA samples and correlated copy number alterations and fragment length distributions to clinical and imaging findings. RESULTS: We observed copy number alterations in 4/7 polymetastatic cancer patients, 1/7 OMD and 1/7 SCCHN patients, these patients' imaging showed progression following radiotherapy. Using unsupervised learning, we identified cancer-specific fragment length features that showed a strong correlation with copy number-based tumor fraction estimates. In 4/4 HPV-positive SCCHN patient samples, we detected viral DNA that enabled the monitoring of very low tumor fraction samples. CONCLUSIONS: Our results indicate that an elevated tumor fraction is associated with tumor aggressiveness and systemic tumor spread. This information may be used to adapt treatment strategies. Further, we show that by detecting specific sequences such as viral DNA, the sensitivity of detecting cancer from cell-free DNA sequencing data can be greatly increased

Cable Robotic 3D-printing: additive manufacturing on the construction site

This paper outlines an important step in characterizing a novel field of robotic construction research where a cable-driven parallel robot is used to extrude cementitious material in three-dimensional space, and thus offering a comprehensive new approach to computational design and construction, and to robotic fabrication at larger scales. Developed by the Faculty of Art and Design at Bauhaus-University Weimar (Germany), the faculty of Architecture at the University of Applied Sciences Dortmund (Germany) and the Chair of Mechatronics at the University of Duisburg-Essen (Germany), this approach offers unique advantages over existing additive manufacturing methods: the system is easily transportable and scalable, it does not require additional formwork or scaffolding, and it offers digital integration and informational oversight across the entire design and building process. This paper considers 1) key research components of cable robotic 3D-printing (such as computational design, material exploration, and robotic control), and 2) the integration of these parameters into a unified design and building process. The demonstration of the approach at full-scale is of particular concern

Stereotactic body radiotherapy to defer systemic therapy in patients with oligorecurrent disease

Background Patients who develop oligorecurrent disease may be treated with metastasis-directed stereotactic body radiotherapy (SBRT) to defer the start of systemic therapy and delay its potential side effects. We report oncological outcomes and patterns of failure in patients with oligorecurrent disease treated with SBRT and determine which factors impact the interval to initiation of systemic therapy. Material/Methods This retrospective study included patients with oligorecurrent disease (≤5 lesions) from any solid organ malignancy, treated with SBRT to all metastases and no systemic therapy for a minimum one month after SBRT between 01/2014 and 12/2019. The Kaplan-Meier method was used to analyze overall survival (OS) and progression-free survival (PFS), and the cumulative incidence of initiation of systemic therapy was analyzed assuming death without systemic therapy as a competing risk. Univariable and multivariable analyses are used to assess predictors of the systemic therapy-free interval. Results Among 545 patients treated with SBRT for oligometastatic disease, 142 patients were treated with SBRT only for oligorecurrent disease. The most common primary tumors were lung and gastrointestinal cancer in 47 (33.1 %) and 28 (19.7 %) patients, respectively. After a median follow-up of 25 months, the median PFS and OS was 6.1 months and 48.9 months, respectively. Distant metastases were the most common first failure, and oligometastatic distant failure occured in 86 patients (60.6 %). New metastases were treated with repeat SBRT in 48 patients (33.8 %). The 1- and 2-year cumulative incidence of initiation of systemic therapy was 24.6 % and 36.8 %, respectively. In multivariable analysis, the number of previous lines of systemic therapy and the cumulative volume of metastases were significantly associated with the interval to initiation of systemic therapy. Conclusion Selected patients with oligorecurrence achieved favorable OS and low cumulative incidence of initiation of systemic therapy. Prospective studies are warranted to determine how the deferral of systemic therapy impacts OS compared with immediate systemic therapy in combination with SBRT

Leukoencephalopathy after prophylactic whole-brain irradiation with or without hippocampal sparing: a longitudinal magnetic resonance imaging analysis

PURPOSE Neurocognitive changes are well described after prophylactic or therapeutic whole-brain radiotherapy (WBRT) and have been reported as early as 3 months after radiotherapy (RT). Therefore, WBRT with protection of the hippocampal region (hippocampal avoidance, HA) has been proposed to preserve neurocognition. Our aim was to compare the risk of leukoencephalopathy after prophylactic cranial irradiation (PCI) with or without HA. METHODS Patients with small-cell lung cancer who received either lateral-opposed field PCI (non-HA-PCI; n = 9) or hippocampus avoidance PCI (HA-PCI; n = 9) with available magnetic resonance imaging (MRI) follow-up were identified and age matched. Pre-therapeutic and follow-up MRI after RT was analysed for leukoencephalopathy based on the Fazekas score. Bilateral cortical and subcortical brain structures were segmented and analysed for alterations in dosimetric parameters and volumes. RESULTS There was no significant difference of Fazekas scores between groups at baseline. Fazekas score differed in post-treatment with a median of 1 in the HA-PCI group and 2 in the non-HA-PCI group (p = 0.007). Significant increase of Fazekas score over time after RT was observed for HA-PCI patients (p = 0.001) but not for non-HA-PCI patients. Dmax (highest radiation dose) and brain volume receiving doses >25Gy were higher in HA-PCI patients. There were no significant volumetric differences for segmented brain structures between groups. CONCLUSION Radiological changes are more prominent after HA-PCI than after non-HA-PCI. Although no standardised neurocognitive testing was performed, the significantly increased Fazekas scores after HA-PCI are expected to interfere with neurocognitive function. Prospective long-term neurocognitive studies are warranted before HA-PCI is implemented in routine clinical practice