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The thesis provides insights in reconstruction and analysis pipelines for processing of
three-dimensional cell and vessel images of megakaryopoiesis in intact murine bone.
The images were captured in a Light Sheet Fluorescence Microscope. The work
presented here is part of Collaborative Research Centre (CRC) 688 (project B07) of
the University of Würzburg, performed at the Rudolf-Virchow Center. Despite ongoing
research within the field of megakaryopoiesis, its spatio-temporal pattern of
megakaryopoiesis is largely unknown. Deeper insight to this field is highly desirable to
promote development of new therapeutic strategies for conditions related to
thrombocytopathy as well as thrombocytopenia. The current concept of
megakaryopoiesis is largely based on data from cryosectioning or in vitro studies
indicating the existence of spatial niches within the bone marrow where specific stages
of megakaryopoiesis take place. Since classic imaging of bone sections is typically
limited to selective two-dimensional views and prone to cutting artefacts, imaging of
intact murine bone is highly desired. However, this has its own challenges to meet,
particularly in image reconstruction. Here, I worked on processing pipelines to account
for irregular specimen staining or attenuation as well as the extreme heterogeneity of
megakaryocyte morphology. Specific challenges for imaging and image reconstruction
are tackled and solution strategies as well as remaining limitations are presented and
discussed. Fortunately, modern image processing and segmentation strongly benefits
from continuous advances in hardware as well as software-development. This thesis
exemplifies how a combined effort in biomedicine, computer vision, data processing
and image technology leads to deeper understanding of megakaryopoiesis. Tailored
imaging pipelines significantly helped elucidating that the large megakaryocytes are
broadly distributed throughout the bone marrow facing a surprisingly dense vessel
network. No evidence was found for spatial niches in the bone marrow, eventually
resulting in a revised model of megakaryopoiesis.
Studies on the role of platelet serotonin in platelet function, hemostasis, thrombosis and stroke
(2019)
Platelet activation and aggregation are important processes in hemostasis resulting in reduction of blood loss upon vessel wall injury. However, platelet activation can lead to thrombotic events causing myocardial infarction and stroke. A more detailed understanding of the regulation of platelet activation and the subsequent formation of thrombi is essential to prevent thrombosis and ischemic stroke. Cations, platelet surface receptors, cytoskeletal rearrangements, activation of the coagulation cas-cade and intracellular signaling molecules are important in platelet activation and thrombus formation. One such important molecule is serotonin (5 hydroxytryptamin, 5 HT), an indolamine platelet agonist, biochemically derived from tryptophan. 5 HT is secreted from the enterochromaffin cells into the gastrointestinal tract (GI) and blood. Blood borne 5 HT has been proposed to regulate hemostasis by acting as a vaso-constrictor and by triggering platelet signaling through 5 HT2A receptor. Although platelets do not synthetize 5 HT, they take it up from the blood and store it in their dense granules which are secreted upon platelet activation. To identify the molecu-lar composite of the 5 HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke, 5 HT transporter knock out mice (5Htt / ) were analyzed in different in vitro and in vivo assays and in a model of is-chemic stroke. In 5Htt / platelets, 5 HT uptake from the blood was completely abol-ished and agonist-induced Ca2+ influx through store operated Ca2+ entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein (GP) VI and C type lectin-like receptor 2 (CLEC 2) were reduced. These observed in vitro defects in 5Htt / platelets could be normalized by the addition of exogenous 5 HT. Moreover, reduced 5 HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt / mice. Surprisingly, in the transient middle cerebral artery occlusion model (tMCAO) of ischemic stroke 5Htt / mice showed near-ly normal infarct volumes and a neurological outcome comparable to control mice. Although secreted platelet 5 HT does not appear to play a crucial role in the devel-opment of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and thus plays an important role in thrombus stabilization.
To further investigate the role of cations, granules and their contents and regulation of integrin activation in the process of thrombus formation, genetically modified mice were analyzed in the different in vivo thrombosis models. Whereas Tph1 / mice (lacking the enzyme responsible for the production of 5 HT in the periphery), Trpm7KI (point mu-tation in the kinase domain of Trpm7 channel, lacking kinase activity) and Unc13d / /Nbeal2 / mice (lacking α granules and the release machinery of dense granules) showed a delayed thrombus formation in vivo, MagT1y/ mice (lacking a specific Mg2+ transporter) displayed a pro thrombotic phenotype in vivo. Trpm7fl/fl Pf4Cre (lacking the non specific Mg2+ channel) and RIAM / mice (lacking a potential linker protein in integrin “inside out” signaling) showed no alterations in thrombus formation upon injury of the vessel wall.
Localized electron spins can couple magnetically via the Ruderman–Kittel–Kasuya–Yosida interaction even if their wave functions lack direct overlap. Theory predicts that spin–orbit scattering leads to a Dzyaloshinskii–Moriya type enhancement of this indirect exchange interaction, giving rise to chiral exchange terms. Here we present a combined spin-polarized scanning tunneling microscopy, angle-resolved photoemission, and density functional theory study of MnO2 chains on Ir(100). Whereas we find antiferromagnetic Mn–Mn coupling along the chain, the inter-chain coupling across the non-magnetic Ir substrate turns out to be chiral with a 120° rotation between adjacent MnO2 chains. Calculations reveal that the Dzyaloshinskii–Moriya interaction results in spin spirals with a periodicity in agreement with experiment. Our findings confirm the existence of indirect chiral magnetic exchange, potentially giving rise to exotic phenomena, such as chiral spin-liquid states in spin ice systems or the emergence of new quasiparticles.
Objective: In two independent study arms, we determine the effects of strength training (ST) and high-intensity interval training (HIIT) overload on cardiac autonomic modulation by measuring heart rate (HR) and vagal heart rate variability (HRV).
Methods: In the study, 37 well-trained athletes (ST: 7 female, 12 male; HIIT: 9 female, 9 male) were subjected to orthostatic tests (HR and HRV recordings) each day during a 4-day baseline period, a 6-day overload microcycle, and a 4-day recovery period. Discipline-specific performance was assessed before and 1 and 4 days after training.
Results: Following ST overload, supine HR, and vagal HRV (Ln RMSSD) were clearly increased and decreased (small effects), respectively, and the standing recordings remained unchanged. In contrast, HIIT overload resulted in decreased HR and increased Ln RMSSD in the standing position (small effects), whereas supine recordings remained unaltered. During the recovery period, these responses were reversed (ST: small effects, HIIT: trivial to small effects). The correlations between changes in HR, vagal HRV measures, and performance were weak or inconsistent. At the group and individual levels, moderate to strong negative correlations were found between HR and Ln RMSSD when analyzing changes between testing days (ST: supine and standing position, HIIT: standing position) and individual time series, respectively. Use of rolling 2–4-day averages enabled more precise estimation of mean changes with smaller confidence intervals compared to single-day values of HR or Ln RMSSD. However, the use of averaged values displayed unclear effects for evaluating associations between HR, vagal HRV measures, and performance changes, and have the potential to be detrimental for classification of individual short-term responses.
Conclusion: Measures of HR and Ln RMSSD during an orthostatic test could reveal different autonomic responses following ST or HIIT which may not be discovered by supine or standing measures alone. However, these autonomic changes were not consistently related to short-term changes in performance and the use of rolling averages may alter these relationships differently on group and individual level.
Background
While the coordination of oculomotor and manual behavior is essential for driving a car, surprisingly little is known about this interaction, especially in situations requiring a quick steering reaction. In the present study, we analyzed oculomotor gaze and manual steering behavior in approach and avoidance tasks. Three task blocks were implemented within a dynamic simulated driving environment requiring the driver either to steer away from/toward a visual stimulus or to switch between both tasks.
Results
Task blocks requiring task switches were associated with higher manual response times and increased error rates. Manual response times did not significantly differ depending on whether drivers had to steer away from vs toward a stimulus, whereas oculomotor response times and gaze pattern variability were increased when drivers had to steer away from a stimulus compared to steering toward a stimulus.
Conclusion
The increased manual response times and error rates in mixed tasks indicate performance costs associated with cognitive flexibility, while the increased oculomotor response times and gaze pattern variability indicate a parsimonious cross-modal action control strategy (avoiding stimulus fixation prior to steering away from it) for the avoidance scenario. Several discrepancies between these results and typical eye–hand interaction patterns in basic laboratory research suggest that the specific goals and complex perceptual affordances associated with driving a vehicle strongly shape cross-modal control of behavior.
In the past few years, two-dimensional quantum liquids with fractional excitations have been a topic of high interest due to their possible application in the emerging field of quantum computation and cryptography. This thesis is devoted to a deeper understanding of known and new fractional quantum Hall states and their stabilization in local models. We pursue two different paths, namely chiral spin liquids and fractionally quantized, topological phases.
The chiral spin liquid is one of the few examples of spin liquids with fractional statistics. Despite its numerous promising properties, the microscopic models for this state proposed so far are all based on non-local interactions, making the experimental realization challenging. In the first part of this thesis, we present the first local parent Hamiltonians, for which the Abelian and non-Abelian chiral spin liquids are the exact and, modulo a topological degeneracy, unique ground states. We have developed a systematic approach to find an annihilation operator of the chiral spin liquid and construct from it a many-body interaction which establishes locality. For various system sizes and lattice geometries, we numerically find largely gapped eigenspectra and confirm to an accuracy of machine precision the uniqueness of the chiral spin liquid as ground state of the respective system. Our results provide an exact spin model in which fractional quantization can be studied.
Topological insulators are one of the most actively studied topics in current condensed matter physics research. With the discovery of the topological insulator, one question emerged: Is there an interaction-driven set of fractionalized phases with time reversal symmetry? One intuitive approach to the theoretical construction of such a fractional topological insulator is to take the direct product of a fractional quantum Hall state and its time reversal conjugate. However, such states are well studied conceptually and do not lead to new physics, as the idea of taking a state and its mirror image together without any entanglement between the states has been well understood in the context of topological insulators. Therefore, the community has been looking for ways to implement some topological interlocking between different spin species. Yet, for all practical purposes so far, time reversal symmetry has appeared to limit the set of possible fractional states to those with no interlocking between the two spin species.
In the second part of this thesis, we propose a new universality class of fractionally quantized, topologically ordered insulators, which we name “fractional insulator”. Inspired by the fractional quantum Hall effect, spin liquids, and fractional Chern insulators, we develop a wave function approach to a new class of topological order in a two-dimensional crystal of spin-orbit coupled electrons. The idea is simply to allow the topological order to violate time reversal symmetry, while all locally observable quantities remain time reversal invariant. We refer to this situation as “topological time reversal symmetry breaking”. Our state is based on the Halperin double layer states and can be viewed as a two-layer system of an ↑-spin and a ↓-spin sphere. The construction starts off with Laughlin states for the ↑-spin and ↓-spin electrons and an interflavor term, which creates correlations between the two layers. With a careful parameter choice, we obtain a state preserving time reversal symmetry locally, and label it the “311-state”. For systems of up to six ↑-spin and six ↓-spin electrons, we manage to construct an approximate parent Hamiltonian with a physically realistic, local interaction.
RsaE is a conserved small regulatory RNA (sRNA) which was previously reported to represent a riboregulator of central carbon flow and other metabolic pathways in Staphylococcus aureus and Bacillus subtilis. Here we show that RsaE contributes to extracellular (e)DNA release and biofilm-matrix switching towards polysaccharide intercellular adhesin (PIA) production in a hypervariable Staphylococcus epidermidis isolate. Transcriptome analysis through differential RNA sequencing (dRNA-seq) in combination with confocal laser scanning microscopy (CLSM) and reporter gene fusions demonstrate that S. epidermidis protein- and PIA-biofilm matrix producers differ with respect to RsaE and metabolic gene expression. RsaE is spatiotemporally expressed within S. epidermidis PIA-mediated biofilms, and its overexpression triggers a PIA biofilm phenotype as well as eDNA release in an S. epidermidis protein biofilm matrix-producing strain background. dRNA-seq and Northern blot analyses revealed RsaE to exist as a major full-length 100-nt transcript and a minor processed species lacking approximately 20 nucleotides at the 5'-end. RsaE processing results in expansion of the mRNA target spectrum. Thus, full-length RsaE interacts with S. epidermidis antiholin-encoding lrgA mRNA, facilitating bacterial lysis and eDNA release. Processed RsaE, however, interacts with the 5'-UTR of icaR and sucCD mRNAs, encoding the icaADBC biofilm operon repressor IcaR and succinyl-CoA synthetase of the tricarboxylic acid (TCA) cycle, respectively. RsaE augments PIA-mediated biofilm matrix production, most likely through activation of icaADBC operon expression via repression of icaR as well as by TCA cycle inhibition and re-programming of staphylococcal central carbon metabolism towards PIA precursor synthesis. Additionally, RsaE supports biofilm formation by mediating the release of eDNA as stabilizing biofilm matrix component. As RsaE itself is heterogeneously expressed within biofilms, we consider this sRNA to function as a factor favoring phenotypic heterogeneity and supporting division of labor in S. epidermidis biofilm communities.
All living organisms need timekeeping mechanisms to track and anticipate cyclic changes in their environment. The ability to prepare for and respond to daily and seasonal changes is endowed by circadian clocks. The systemic features and molecular mechanisms that drive circadian rhythmicity are highly conserved across kingdoms. Therefore, Drosophila melanogaster with its relatively small brain (ca. 135.000 neurons) and the outstanding genetic tools that are available, is a perfect model to investigate the properties and relevance of the circadian system in a complex, but yet comprehensible organism.
The last 50 years of chronobiological research in the fruit fly resulted in a deep understanding of the molecular machinery that drives circadian rhythmicity, and various histological studies revealed the neural substrate of the circadian system. However, a detailed neuroanatomical and physiological description on the single-cell level has still to be acquired. Thus, I employed a multicolor labeling approach to characterize the clock network of Drosophila melanogaster with single-cell resolution and additionally investigated the putative in- and output sites of selected neurons.
To further study the functional hierarchy within the clock network and to monitor the “ticking clock“ over the course of several circadian cycles, I established a method, which allows us to follow the accumulation and degradation of the core clock genes in living brain explants by the means of bioluminescence imaging of single-cells.
This dissertation deals with composite-based methods for structural equation models with latent variables and their enhancement. It comprises five chapters. Besides a brief introduction in the first chapter, the remaining chapters consisting of four essays cover the results of my PhD studies.Two of the essays have already been published in an international journal.
The first essay considers an alternative way of construct modeling in structural equation modeling.While in social and behavioral sciences theoretical constructs are typically modeled as common factors, in other sciences the common factor model is an inadequate way construct modeling due to its assumptions. This essay introduces the confirmatory composite analysis (CCA) analogous to confirmatory factor analysis (CFA). In contrast to CFA, CCA models theoretical constructs as composites instead of common factors. Besides the theoretical presentation of CCA and its assumptions, a Monte Carlo simulation is conducted which demonstrates that misspecifications of the composite model can be detected by the introduced test for overall model fit.
The second essay rises the question of how parameter differences can be assessed in the framework of partial least squares path modeling. Since the standard errors of the estimated parameters have no analytical closed-form, the t- and F-test known from regression analysis cannot be directly used to test for parameter differences. However, bootstrapping provides a solution to this problem. It can be employed to construct confidence intervals for the estimated parameter differences, which can be used for making inferences about the parameter difference in the population. To guide practitioners, guidelines were developed and demonstrated by means of empirical examples.
The third essay answers the question of how ordinal categorical indicators can be dealt with in partial least squares path modeling. A new consistent estimator is developed which combines the polychoric correlation and partial least squares path modeling to appropriately deal with the qualitative character of ordinal categorical indicators. The new estimator named ordinal consistent partial least squares combines consistent partial least squares with ordinal partial least squares. Besides its derivation, a Monte Carlo simulation is conducted which shows that the new estimator performs well in finite samples. Moreover, for illustration, an empirical example is estimated by ordinal consistent partial least squares.
The last essay introduces a new consistent estimator for polynomial factor models.
Similarly to consistent partial least squares, weights are determined to build stand-ins for the latent variables, however a non-iterative approach is used.
A Monte Carlo simulation shows that the new estimator behaves well in finite samples.
Platelet collagen interactions at sites of vascular injuries predominantly involve glycoprotein VI (GPVI) and the integrin α2β1. Both proteins are primarily expressed on platelets and megakaryocytes whereas GPVI expression is also shown on endothelial and integrin α2β1 expression on epithelial cells. We recently showed that depletion of GPVI improves stroke outcome without increasing the risk of cerebral hemorrhage. Genetic variants associated with higher platelet surface integrin α2 (ITGA2) receptor levels have frequently been found to correlate with an increased risk of ischemic stroke in patients. However until now, no preclinical stroke study has addressed whether platelet integrin α2β1 contributes to the pathophysiology of ischemia/reperfusion (I/R) injury. Focal cerebral ischemia was induced in C57BL/6 and Itga2\(^{−/−}\) mice by a 60 min transient middle cerebral artery occlusion (tMCAO). Additionally, wild-type animals were pretreated with anti-GPVI antibody (JAQ1) or Fab fragments of a function blocking antibody against integrin α2β1 (LEN/B). In anti-GPVI treated animals, intravenous (IV) recombinant tissue plasminogen activator (rt-PA) treatment was applied immediately prior to reperfusion. Stroke outcome, including infarct size and neurological scoring was determined on day 1 after tMCAO. We demonstrate that targeting the integrin α2β1 (pharmacologic; genetic) did neither reduce stroke size nor improve functional outcome on day 1 after tMCAO. In contrast, depletion of platelet GPVI prior to stroke was safe and effective, even when combined with rt-PA treatment. Our results underscore that GPVI, but not ITGA2, is a promising and safe target in the setting of ischemic stroke.
Deep brain stimulation of the mesencephalic locomotor region (MLR) improves the motor symptoms in Parkinson’s disease and experimental stroke by intervening in the motor cerebral network. Whether high-frequency stimulation (HFS) of the MLR is involved in non-motor processes, such as neuroprotection and inflammation in the area surrounding the photothrombotic lesion, has not been elucidated. This study evaluates whether MLR-HFS exerts an anti-apoptotic and anti-inflammatory effect on the border zone of cerebral photothrombotic stroke. Rats underwent photothrombotic stroke of the right sensorimotor cortex and the implantation of a microelectrode into the ipsilesional MLR. After intervention, either HFS or sham stimulation of the MLR was applied for 24 h. The infarct volumes were calculated from consecutive brain sections. Neuronal apoptosis was analyzed by TUNEL staining. Flow cytometry and immunohistochemistry determined the perilesional inflammatory response. Neuronal apoptosis was significantly reduced in the ischemic penumbra after MLR-HFS, whereas the infarct volumes did not differ between the groups. MLR-HFS significantly reduced the release of cytokines and chemokines within the ischemic penumbra. MLR-HFS is neuroprotective and it reduces pro-inflammatory mediators in the area that surrounds the photothrombotic stroke without changing the number of immune cells, which indicates that MLR-HFS enables the function of inflammatory cells to be altered on a molecular level.
Blood–brain barrier (BBB) disruption is a critical event after ischemic stroke, which results in edema formation and hemorrhagic transformation of infarcted tissue. BBB dysfunction following stroke is partly mediated by proinflammatory agents. We recently have shown that high frequency stimulation of the mesencephalic locomotor region (MLR-HFS) exerts an antiapoptotic and anti-inflammatory effect in the border zone of cerebral photothrombotic stroke in rats. Whether MLR-HFS also has an impact on BBB dysfunction in the early stage of stroke is unknown. In this study, rats were subjected to photothrombotic stroke of the sensorimotor cortex and implantation of a stimulating microelectrode into the ipsilesional MLR. Thereafter, either HFS or sham stimulation of the MLR was applied for 24 h. After scarifying the rats, BBB disruption was assessed by determining albumin extravasation and tight junction integrity (claudin 3, claudin 5, and occludin) using Western blot analyses and immunohistochemistry. In addition, by applying zymography, expression of pro-metalloproteinase-9 (pro-MMP-9) was analyzed. No differences were found regarding infarct size and BBB dysfunction between stimulated and unstimulated animals 24 h after induction of stroke. Our results indicate that MLR-HFS neither improves nor worsens the damaged BBB after stroke. Attenuating cytokines/chemokines in the perilesional area, as mediated by MLR-HFS, tend to play a less significant role in preventing the BBB integrity.
Introduction
Multidisciplinary, complex rehabilitation interventions are an important part of the treatment of chronic diseases. However, little is known about the effectiveness of routine rehabilitation interventions within the German healthcare system. Due to the nature of the social insurance system in Germany, randomised controlled trials examining the effects of rehabilitation interventions are challenging to implement and scarcely accessible. Consequently, alternative pre-post designs can be employed to assess pre-post effects of medical rehabilitation programmes. We present a protocol of systematic review and meta-analysis methods to assess the pre-post effects of rehabilitation interventions in Germany.
Methods and analysis
The respective study will be conducted within the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. A systematic literature review will be conducted to identify studies reporting the pre-post effects (start of intervention vs end of intervention or later) in German healthcare. Studies investigating the following disease groups will be included: orthopaedics, rheumatology, oncology, pulmonology, cardiology, endocrinology, gastroenterology and psychosomatics. The primary outcomes of interest are physical/mental quality of life, physical functioning and social participation for all disease groups as well as pain (orthopaedic and rheumatologic patients only), blood pressure (cardiac patients only), asthma control (patients with asthma only), dyspnoea (patients with chronic obstructive pulmonary disease only) and depression/anxiety (psychosomatic patients only). We will invite the principal investigators of the identified studies to provide additional individual patient data. We aim to perform the meta-analyses using individual patient data as well as aggregate data. We will examine the effects of both study-level and patient-level moderators by using a meta-regression method.
Ethics and dissemination
Only studies that have received institutional approval from an ethics committee and present anonymised individual patient data will be included in the meta-analysis. The results will be presented in a peer-reviewed publication and at research conferences. A declaration of no objection by the ethics committee of the University of Würzburg is available (number 20180411 01).
Background: According to current guidelines, stroke patients treated with rt-PA should undergo brain imaging to exclude intracerebral bleeding 24 h after thrombolysis, before the start of medical secondary prevention. However, the usefulness of routine follow-up imaging with regard to changes in therapeutic management in patients without neurological deterioration is unclear. We hypothesized that follow up brain imaging solely to exclude bleeding in patients who clinically improved after rt-PA application may not be necessary.
Methods: Retrospective single-center analysis including stroke patients treated with rt-PA. Records were reviewed for hemorrhagic transformation one day after systemic thrombolysis and brain imaging-based changes in therapeutic management. Twenty-four hour after thrombolysis patients were divided into four groups: (1) increased NIHSS score; (2) unchanged NIHSS score; (3) improved NIHSS score and; (4) NIHSS score = 0.
Results: Out of 188 patients (mean age 73 years, 100 female) receiving rt-PA, 32 (17%) had imaging-proven hemorrhagic transformation including 11 (6%) patients with parenchymal hemorrhage. Patients in group (1, 2) more often had hypertension (p = 0.015) and more often had parenchymal hemorrhage (9 vs. 4%; p < 0.206) compared to group (3, 4) and imaging-based changes in therapeutic management were more frequent (19% vs. 6%; p = 0.007). Patients of group (3, 4) had no changes in therapeutic management in 94% of the cases. Patients in group (4) had no hemorrhagic transformation in routine follow-up brain imaging.
Conclusions: Frequency of hemorrhagic transformation in Routine follow-up brain imaging and consecutive changes in therapeutic management were different depending on clinical course measured by NHISS score.
The cytoskeletal crosslinking protein MACF1 is dispensable for thrombus formation and hemostasis
(2019)
Coordinated reorganization of cytoskeletal structures is critical for key aspects of platelet physiology. While several studies have addressed the role of microtubules and filamentous actin in platelet production and function, the significance of their crosstalk in these processes has been poorly investigated. The microtubule-actin cross-linking factor 1 (MACF1; synonym: Actin cross-linking factor 7, ACF7) is a member of the spectraplakin family, and one of the few proteins expressed in platelets, which possess actin and microtubule binding domains thereby facilitating actin-microtubule interaction and regulation. We used megakaryocyte- and platelet-specific Macf1 knockout (Macf1fl/fl, Pf4-Cre) mice to study the role of MACF1 in platelet production and function. MACF1 deficient mice displayed comparable platelet counts to control mice. Analysis of the platelet cytoskeletal ultrastructure revealed a normal marginal band and actin network. Platelet spreading on fibrinogen was slightly delayed but platelet activation and clot traction was unaffected. Ex vivo thrombus formation and mouse tail bleeding responses were similar between control and mutant mice. These results suggest that MACF1 is dispensable for thrombopoiesis, platelet activation, thrombus formation and the hemostatic function in mice.
The isolation and structural characterization of the cyanido-substituted metalated ylides [Ph3P−C−CN]M (1-M; M=Li, Na, K) are reported with lithium, sodium, and potassium as metal cations. In the solid-state, most different aggregates could be determined depending on the metal and additional Lewis bases. The crown-ether complexes of sodium (1-Na) and potassium (1-K) exhibited different structures, with sodium preferring coordination to the nitrogen end, whereas potassium binds in an unusual η2-coordination mode to the two central carbon atoms. The formation of the yldiide was accompanied by structural changes leading to shorter C−C and longer C−N bonds. This could be attributed to the delocalization of the free electron pairs at the carbon atom into the antibonding orbitals of the CN moiety, which was confirmed by IR spectroscopy and computational studies. Detailed density functional theory calculations show that the changes in the structure and the bonding situation were most pronounced in the lithium compounds due to the higher covalency.
The culture of human induced pluripotent stem cells (hiPSCs) at large-scale becomes feasible with the aid of scalable suspension setups in continuously stirred tank reactors (CSTRs). Suspension cul- tures of hiPSCs are characterized by the self-aggregation of single cells into macroscopic cell aggre- gates that increase in size over time. The development of these free-floating aggregates is dependent on the culture vessel and thus represents a novel process parameter that is of particular interest for hiPSC suspension culture scaling. Further, aggregates surpassing a critical size are prone to spon- taneous differentiation or cell viability loss. In this regard, and, for the first time, a hiPSC-specific suspension culture unit was developed that utilizes in situ microscope imaging to monitor and to characterize hiPSC aggregation in one specific CSTR setup to a statistically significant degree while omitting the need for error-prone and time-intensive sampling. For this purpose, a small-scale CSTR system was designed and fabricated by fused deposition modeling (FDM) using an in-house 3D- printer. To provide a suitable cell culture environment for the CSTR system and in situ microscope, a custom-built incubator was constructed to accommodate all culture vessels and process control devices. Prior to manufacture, the CSTR design was characterized in silico for standard engineering parameters such as the specific power input, mixing time, and shear stress using computational fluid dynamics (CFD) simulations. The established computational model was successfully validated by comparing CFD-derived mixing time data to manual measurements. Proof for system functionality was provided in the context of long-term expansion (4 passages) of hiPSCs. Thereby, hiPSC aggregate size development was successfully tracked by in situ imaging of CSTR suspensions and subsequent automated image processing. Further, the suitability of the developed hiPSC culture unit was proven by demonstrating the preservation of CSTR-cultured hiPSC pluripotency on RNA level by qRT-PCR and PluriTest, and on protein level by flow cytometry.
Automated real-time monitoring of human pluripotent stem cell aggregation in stirred tank reactors
(2019)
The culture of human induced pluripotent stem cells (hiPSCs) at large scale becomes feasible with the aid of scalable suspension setups in continuously stirred tank reactors (CSTRs). Innovative monitoring options and emerging automated process control strategies allow for the necessary highly defined culture conditions. Next to standard process characteristics such as oxygen consumption, pH, and metabolite turnover, a reproducible and steady formation of hiPSC aggregates is vital for process scalability. In this regard, we developed a hiPSC-specific suspension culture unit consisting of a fully monitored CSTR system integrated into a custom-designed and fully automated incubator. As a step towards cost-effective hiPSC suspension culture and to pave the way for flexibility at a large scale, we constructed and utilized tailored miniature CSTRs that are largely made from three-dimensional (3D) printed polylactic acid (PLA) filament, which is a low-cost material used in fused deposition modelling. Further, the monitoring tool for hiPSC suspension cultures utilizes in situ microscopic imaging to visualize hiPSC aggregation in real-time to a statistically significant degree while omitting the need for time-intensive sampling. Suitability of our culture unit, especially concerning the developed hiPSC-specific CSTR system, was proven by demonstrating pluripotency of CSTR-cultured hiPSCs at RNA (including PluriTest) and protein level.
The aim of the work was the development of thiol-ene cross-linked hydrogels based on functionalized poly(glycidol)s (PG) and hyaluronic acid (HA) for extrusion based 3D bioprinting. Additionally, the functionalization of the synthesized PG with peptides and the suitability of these polymers for physically cross-linked gels were investigated, in a proof of principle study in order to demonstrate the versatile use of PG polymers in hydrogel development.
First, the precursor polymers of the different hydrogel systems were synthesized. For thiol-ene cross-linked hydogels, linear allyl-functionalized PG (P(AGE-co-G)) and three different thiol-(SH-)functionalized polymers, ester-containing PG-SH (PG SHec), ester-free PG-SH (PG-SHef) and HA-SH were synthesized and analysed, The degree of functionalization of these polymers was adjustable.
For physically cross-linked hydrogels, peptide-functionalized PG (P(peptide-co-G)), was synthesized through polymer analogue thiol-ene modification of P(AGE-co-G).
Subsequently, thiol-ene cross-linked hydrogels were prepared with the synthesized thiol- and allyl-functionalized polymers. Depending on the origin of the used polymers, two different systems were obtained: on the one hand synthetic hydrogels consisting of PG-SHec/ef and P(AGE-co-G) and on the other hand hybrid gels, consisting of HA-SH and P(AGE-co-G). In synthetic gels, the degradability of the gels was determined by the applied PG-SH. The use of PG-SHec resulted in hydrolytically degradable hydrogels, whereas the cross-linking with PG-SHef resulted in non-degradable gels.
The physical properties of these different hydrogel systems were determined by swelling, mechanical and diffusion studies and subsequently compared among each other. In swelling studies the differences of degradable and non-degradable synthetic hydrogels as well as the differences of synthetic compared to hybrid hydrogels were demonstrated.
Next, the stiffness and the swelling ratios (SR) of the established hydrogel systems were examined in dependency of different parameters, such as incubation time, polymer concentration and UV irradiation. In general, these measurements revealed the same trends for synthetic and hybrid hydrogels: an increased polymer concentration as well as prolonged UV irradiation led to an increased network density. Moreover, it was demonstrated that the incorporation of additional non-bound HMW HA hampered the hydrogel cross-linking resulting in gels with decreased stiffness and increased SR. This effect was strongly dependent on the amount of additional HMW HA.
The diffusion of different molecular weight fluorescein isothiocyanate-dextran (FITC-dextran) through hybrid hydrogels (with/without HMW HA) gave information about the mesh size of these gels. The smallest FITC-dextran (4 kDa) completely diffused through both hydrogel systems within the first week, whereas only 55 % of 40 kDa and 5-10 % HMW FITC-dextrans (500 kDa and 2 MDa) could diffuse through the networks.
The applicability of synthetic and hybrid hydrogels for cartilage regeneration purpose was investigated through by biological examinations. It was proven that both gels support the survival of embedded human mesenchymal stromal cells (hMSCs) (21/28 d in vitro culture), however, the chondrogenic differentiation was significantly improved in hybrid hydrogels compared to synthetic gels. The addition of non-bound HMW HA resulted in a slightly less distinct chondrogenesis.
Lastly the printability of the established hydrogel systems was examined. Therefore, the viscoelastic properties of the hydrogel solutions were adjusted by incorporation of non-bound HMW HA. Both systems could be successfully printed with high resolution and high shape fidelity.
The introduction of the double printing approach with reinforcing PCL allowed printing of hydrogel solutions with lower viscosities. As a consequence, the amount of additional HMW HA necessary for printing could be reduced allowing successful printing of hybrid hydrogel solutions with embedded cells. It was demonstrated that the integrated cells survived the printing process with high viability measured after 21 d. Moreover, by this reinforcing technique, robust hydrogel-containing constructs were fabricated.
In addition to thiol-ene cross-linked hydrogels, hydrogel cross-linking via ionic interactions was investigated with a hybrid hydrogel based on HMW HA and peptide-functionalized PG. Rheological measurements revealed an increase in the viscosity of a 2 wt.% HMW HA solution by the addition of peptide-functionalized PG. The increase in viscosity could be attributed to the ionic interactions between the positively charge PG and the negatively charge HMW HA.
In conclusion, throughout this thesis thiol-ene chemistry and PG were introduced as promising cross-linking reaction and polymer precursor for the field of biofabrication. Furthermore, the differences of hybrid and synthetic hydrogels as well as chemically and physically cross-linked hydrogels were demonstrated.
Moreover, the double printing approach was demonstrated to be a promising tool for the fabrication of robust hydrogel-containing constructs. It opens the possibility of printing hydrogels that were not printable yet, due to too low viscosities.
The indepth metabolic profiling of the crude extracts of two African Ancistrocladus species viz. A. likoko from Central Africa and A. abbreviatus from West Africa, resulted in a total of 87 alkaloids among them 54 new ones. All of the compounds were intensely elucidated by 1D and 2D NMR, HRESIMS, as well as chemical and chiroptical techniques.
Among the newly discovered compounds are quinoid naphthylisoquinolines with an ortho-diketone in the naphthalene portion, nor-naphthylisoquinoline alkaloid lacking the always present methyl group at C-1, seco-(ring cleaved) naphthylisoquinolines, and a newly discovered class of natural products called the naphthylisoindolinones.
Some of the compounds displayed strong antitumoral activities against human pancreatic cancer cells and leukemia cells in-vitro.
Defeat of the antibiotic resistance of pathogenic bacteria is one great challenge today and for the future. In the last century many classes of effective antibacterials have been developed, so that upcoming resistances could be met with novel drugs of various compound classes. Meanwhile, there is a certain lack of research of the pharmaceutical companies, and thus there are missing developments of novel antibiotics. Gram-positive bacteria are the most important cause of clinical infections. The number of novel antibacterials in clinical trials is strongly restricted. There is an urgent need to find novel antibacterials. We used synthetic chemistry to build completely novel hybrid molecules of substituted indoles and benzothiophene. In a simple one-pot reaction, two novel types of thienocarbazoles were yielded. Both indole substituted compound classes have been evaluated as completely novel antibacterials against the Staphylococcus and Enterococcus species. The evaluated partly promising activities depend on the indole substituent type. First lead compounds have been evaluated within in vivo studies. They confirmed the in vitro results for the new classes of small-molecule antibacterials.
Mammalian haloacid dehalogenase (HAD)-type phosphatases are a large and ubiquitous family of at least 40 human members. Many of them have important physiological functions, such as the regulation of intermediary metabolism and the modulation of enzyme activities, yet they are also linked to diseases such as cardiovascular or metabolic disorders and cancer.
Still, most of the mammalian HAD phosphatases remain functionally uncharacterized.
This thesis reveals novel cell biological and physiological functions of the phosphoglycolate phosphatase PGP, also referred to as AUM. To this end, PGP was functionally characterized by performing analyses using purified recombinant proteins to investigate potential protein substrates of PGP, cell biological studies using the spermatogonial cell line GC1, primary mouse lung endothelial cells and lymphocytes, and a range of biochemical techniques to characterize Pgp-deficient mouse embryos.
To characterize the cell biological functions of PGP, its role downstream of RTK- and integrin signaling in the regulation of cell migration was investigated. It was shown that PGP inactivation elevates integrin- and RTK-induced circular dorsal ruffle (CDR) formation, cell spreading and cell migration. Furthermore, PGP was identified as a negative regulator of directed lymphocyte migration upon integrin- and GPCR activation.
The underlying mechanisms were analyzed further. It was demonstrated that PGP regulates CDR formation and cell migration in a PLC- and PKC-dependent manner, and that Src family kinase activities are required for the observed cellular effects. Upon integrin- and RTK activation, phosphorylation levels of tyrosine residues 1068 and 1173 of the EGF receptor were elevated and PLCγ1 was hyper-activated in PGP-deficient cells. Additionally, PGP-inactivated lymphocytes displayed elevated PKC activity, and PKC-mediated cytoskeletal remodeling was accelerated upon loss of PGP activity. Untargeted lipidomic analyses revealed that the membrane lipid phosphatidylserine (PS) was highly upregulated in PGP-depleted cells.
These data are consistent with the hypothesis that the accumulation of PS in the plasma membrane leads to a pre-assembly of signaling molecules such as PLCγ1 or PKCs that couple the activation of integrins, EGF receptors and GPCRs to accelerated cytoskeletal remodeling.
Thus, this thesis shows that PGP can affect cell spreading and cell migration by acting as a PG-directed phosphatase.
To understand the physiological functions of PGP, conditionally PGP-inactivated mice were analyzed. Whole-body PGP inactivation led to an intrauterine growth defect with developmental delay after E8.5, resulting in a gradual deterioration and death of PgpDN/DN embryos between E9.5 and E11.5. However, embryonic lethality upon whole-body PGP inactivation was not caused by a primary defect of the (cardio-) vascular system. Rather, PGP inactivated embryos died during the intrauterine transition from hypoxic to normoxic conditions.
Therefore, the potential impact of oxygen on PGP-dependent cell proliferation was investigated. Analyses of mouse embryonic fibroblasts (MEFs) generated from E8.5 embryos and GC1 cells cultured under normoxic and hypoxic conditions revealed that normoxia (~20% O2) causes a proliferation defect in PGP-inactivated cells, which can be rescued under
hypoxic (~1% O2) conditions. Mechanistically, it was found that the activity of triosephosphate isomerase (TPI), an enzyme previously described to be inhibited by phosphoglycolate (PG) in vitro, was attenuated in PGP-inactivated cells and embryos. TPI constitutes a critical branch point between carbohydrate- and lipid metabolism because it catalyzes the isomerization of the glycolytic intermediates dihydroxyacetone phosphate (DHAP, a precursor of the glycerol backbone required for triglyceride biosynthesis) and glyceraldehyde 3’-phosphate (GADP).
Attenuation of TPI activity, likely explains the observed elevation of glycerol 3-phosphate levels and the increased TG biosynthesis (lipogenesis). Analyses of ATP levels and oxygen consumption rates (OCR) showed that mitochondrial respiration rates and ATP production were elevated in PGP-deficient cells in a lipolysis-dependent manner. However under hypoxic conditions (which corrected the impaired proliferation of PGP-inactivated cells), OCR and ATP production was indistinguishable between PGP-deficient and PGP-proficient cells. We therefore propose that the inhibition of TPI activity by PG accumulation due to loss of PGP activity shifts cellular bioenergetics from a pro-proliferative, glycolytic metabolism to a lipogenetic/lipolytic metabolism.
Taken together, PGP acts as a metabolic phosphatase involved in the regulation of cell migration, cell proliferation and cellular bioenergetics. This thesis constitutes the basis for further studies of the interfaces between these processes, and also suggests functions of PGP for glucose and lipid metabolism in the adult organism.
Mitofusin 2 is essential for IP3-mediated SR/Mitochondria metabolic feedback in ventricular myocytes
(2019)
Aim: Endothelin-1 (ET-1) and angiotensin II (Ang II) are multifunctional peptide hormones that regulate the function of the cardiovascular and renal systems. Both hormones increase the intracellular production of inositol-1,4,5-trisphosphate (IP\(_3\)) by activating their membrane-bound receptors. We have previously demonstrated that IP\(_3\)-mediated sarcoplasmic reticulum (SR) Ca\(^{2+}\) release results in mitochondrial Ca\(^{2+}\) uptake and activation of ATP production. In this study, we tested the hypothesis that intact SR/mitochondria microdomains are required for metabolic IP\(_3\)-mediated SR/mitochondrial feedback in ventricular myocytes.
Methods: As a model for disrupted mitochondrial/SR microdomains, cardio-specific tamoxifen-inducible mitofusin 2 (Mfn2) knock out (KO) mice were used. Mitochondrial Ca\(^{2+}\) uptake, membrane potential, redox state, and ATP generation were monitored in freshly isolated ventricular myocytes from Mfn2 KO mice and their control wild-type (WT) littermates.
Results: Stimulation of ET-1 receptors in healthy control myocytes increases mitochondrial Ca\(^{2+}\) uptake, maintains mitochondrial membrane potential and redox balance leading to the enhanced ATP generation. Mitochondrial Ca\(^{2+}\) uptake upon ET-1 stimulation was significantly higher in interfibrillar (IFM) and perinuclear (PNM) mitochondria compared to subsarcolemmal mitochondria (SSM) in WT myocytes. Mfn2 KO completely abolished mitochondrial Ca\(^{2+}\) uptake in IFM and PNM mitochondria but not in SSM. However, mitochondrial Ca2+ uptake induced by beta-adrenergic receptors activation with isoproterenol (ISO) was highest in SSM, intermediate in IFM, and smallest in PNM regions. Furthermore, Mfn2 KO did not affect ISO-induced mitochondrial Ca\(^{2+}\) uptake in SSM and IFM mitochondria; however, enhanced mitochondrial Ca\(^{2+}\) uptake in PNM. In contrast to ET-1, ISO induced a decrease in ATP levels in WT myocytes. Mfn2 KO abolished ATP generation upon ET-1 stimulation but increased ATP levels upon ISO application with highest levels observed in PNM regions.
Conclusion: When the physical link between SR and mitochondria by Mfn2 was disrupted, the SR/mitochondrial metabolic feedback mechanism was impaired resulting in the inability of the IP\(_3\)-mediated SR Ca\(^{2+}\) release to induce ATP production in ventricular myocytes from Mfn2 KO mice. Furthermore, we revealed the difference in Mfn2-mediated SR-mitochondrial communication depending on mitochondrial location and type of communication (IP\(_3\)R-mRyR1 vs. ryanodine receptor type 2-mitochondrial calcium uniporter).
Infectious diseases are still a significant cause of morbidity and mortality worldwide. Despite the progress in drug development, the occurrence of microbial resistance is still a significant concern. Alternative therapeutic strategies are required for non-responding or relapsing patients. Chimeric antigen receptor (CAR) T cells has revolutionized cancer immunotherapy, providing a potential therapeutic option for patients who are unresponsive to standard treatments. Recently two CAR T cell therapies, Yescarta® (Kite Pharma/Gilead) and Kymriah® (Novartis) were approved by the FDA for the treatments of certain types of non-Hodgkin lymphoma and B-cell precursor acute lymphoblastic leukemia, respectively. The success of adoptive CAR T cell therapy for cancer has inspired researchers to develop CARs for the treatment of infectious diseases. Here, we review the main achievements in CAR T cell therapy targeting viral infections, including Human Immunodeficiency Virus, Hepatitis C Virus, Hepatitis B Virus, Human Cytomegalovirus, and opportunistic fungal infections such as invasive aspergillosis.
Sand mines represent anthropogenically impacted habitats found worldwide, which bear potential for bee conservation. Although floral resources can be limited at these habitats, vegetation free patches of open sandy soils and embankments may offer good nesting possibilities for sand restricted and other bees. We compared bee communities as found in three reclaimed sand mines and at adjacent roadside meadows in Maryland, USA, over two years. Both sand mines and roadsides hosted diverse bee communities with 111 and 88 bee species, respectively. Bee abundances as well as richness and Shannon diversity of bee species were higher in sand mines than at roadsides and negatively correlated with the percentage of vegetational ground cover. Species composition also differed significantly between habitats. Sand mines hosted a higher proportion of ground nesters, more uncommon and more ‘sand loving’ bees similar to natural sandy areas of Maryland. Despite the destruction of the original pre-mining habitat, sand mines thus appear to represent a unique habitat for wild bees, particularly when natural vegetation and open sand spots are encouraged. Considering habitat loss, the lack of natural disturbance regimes, and ongoing declines of wild bees, sand mines could add promising opportunities for bee conservation which has hitherto mainly focused on agricultural and urban habitats.
The two main collagen receptors on platelets, GPVI and integrin α2β1, play an important role for the recognition of exposed collagen at sites of vessel injury, which leads to platelet activation and subsequently stable thrombus formation. Both receptors are already expressed on megakaryocytes, the platelet forming cells within the bone marrow. Megakaryocytes are in permanent contact with collagen filaments in the marrow cavity and at the basal lamina of sinusoids without obvious preactivation. The role of both collagen receptors for megakaryocyte maturation and thrombopoiesis is still poorly understood. To investigate the function of both collagen receptors, we generated mice that are double deficient for Gp6 and Itga2. Flow cytometric analyses revealed that the deficiency of both receptors had no impact on platelet number and led to the expected lack in GPVI responsiveness. Integrin activation and degranulation ability was comparable to wildtype mice. By immunofluorescence microscopy, we could demonstrate that both wildtype and double-deficient megakaryocytes were overall normally distributed within the bone marrow. We found megakaryocyte count and size to be normal, the localization within the bone marrow, the degree of maturation, as well as their association to sinusoids were also unaltered. However, the contact of megakaryocytes to collagen type I filaments was decreased at sinusoids compared to wildtype mice, while the interaction to type IV collagen was unaffected. Our results imply that GPVI and α2β1 have no influence on the localization of megakaryocytes within the bone marrow, their association to the sinusoids or their maturation. The decreased contact of megakaryocytes to collagen type I might at least partially explain the unaltered platelet phenotype in these mice, since proplatelet formation is mediated by these receptors and their interaction to collagen. It is rather likely that other compensatory signaling pathways and receptors play a role that needs to be elucidated.
Background
Inherited pathogenic variants in BRCA1 and BRCA2 are the most common causes of hereditary breast and ovarian cancer (HBOC). The risk of developing breast cancer by age 80 in women carrying a BRCA1 pathogenic variant is 72%. The lifetime risk varies between families and even within affected individuals of the same family. The cause of this variability is largely unknown, but it is hypothesized that additional genetic factors contribute to differences in age at onset (AAO). Here we investigated whether truncating and rare missense variants in genes of different DNA-repair pathways contribute to this phenomenon.
Methods
We used extreme phenotype sampling to recruit 133 BRCA1-positive patients with either early breast cancer onset, below 35 (early AAO cohort) or cancer-free by age 60 (controls). Next Generation Sequencing (NGS) was used to screen for variants in 311 genes involved in different DNA-repair pathways.
Results
Patients with an early AAO (73 women) had developed breast cancer at a median age of 27 years (interquartile range (IQR); 25.00–27.00 years). A total of 3703 variants were detected in all patients and 43 of those (1.2%) were truncating variants. The truncating variants were found in 26 women of the early AAO group (35.6%; 95%-CI 24.7 - 47.7%) compared to 16 women of controls (26.7%; 95%-CI 16.1 to 39.7%). When adjusted for environmental factors and family history, the odds ratio indicated an increased breast cancer risk for those carrying an additional truncating DNA-repair variant to BRCA1 mutation (OR: 3.1; 95%-CI 0.92 to 11.5; p-value = 0.07), although it did not reach the conventionally acceptable significance level of 0.05.
Conclusions
To our knowledge this is the first time that the combined effect of truncating variants in DNA-repair genes on AAO in patients with hereditary breast cancer is investigated. Our results indicate that co-occurring truncating variants might be associated with an earlier onset of breast cancer in BRCA1-positive patients. Larger cohorts are needed to confirm these results.
The pharmacokinetics in patients with cystic fibrosis (CF) has long been thought to differ considerably from that in healthy volunteers. For highly protein bound β-lactams, profound pharmacokinetic differences were observed between comparatively morbid patients with CF and healthy volunteers. These differences could be explained by body weight and body composition for β-lactams with low protein binding. This study aimed to develop a novel population modeling approach to describe the pharmacokinetic differences between both subject groups by estimating protein binding. Eight patients with CF (lean body mass [LBM]: 39.8 ± 5.4kg) and six healthy volunteers (LBM: 53.1 ± 9.5kg) received 1027.5 mg cefotiam intravenously. Plasma concentrations and amounts in urine were simultaneously modelled. Unscaled total clearance and volume of distribution were 3% smaller in patients with CF compared to those in healthy volunteers. After allometric scaling by LBM to account for body size and composition, the remaining pharmacokinetic differences were explained by estimating the unbound fraction of cefotiam in plasma. The latter was fixed to 50% in male and estimated as 54.5% in female healthy volunteers as well as 56.3% in male and 74.4% in female patients with CF. This novel approach holds promise for characterizing the pharmacokinetics in special patient populations with altered protein binding.
Heat Shock Protein 90 as a Prognostic Marker and Therapeutic Target for Adrenocortical Carcinoma
(2019)
Background: Adrenocortical carcinoma (ACC) is a rare tumor entity with restricted therapeutic opportunities. HSP90 (Heat Shock Protein 90) chaperone activity is fundamental for cell survival and contributes to different oncogenic signaling pathways. Indeed, agents targeting HSP90 function have shown therapeutic efficacy in several cancer types. We have examined the expression of HSP90 in different adrenal tumors and evaluated the use of HSP90 inhibitors in vitro as possible therapy for ACC.
Methods: Immunohistochemical expression of HSP90 isoforms was investigated in different adrenocortical tumors and associated with clinical features. Additionally, a panel of N-terminal (17-allylamino-17-demethoxygeldanamycin (17-AAG), luminespib, and ganetespib) and C-terminal (novobiocin and silibinin) HSP90 inhibitors were tested on various ACC cell lines.
Results: Within adrenocortical tumors, ACC samples exhibited the highest expression of HSP90β. Within a cohort of ACC patients, HSP90β expression levels were inversely correlated with recurrence-free and overall survival. In functional assays, among five different compounds tested luminespib and ganetespib induced a significant decrease in cell viability in single as well as in combined treatments with compounds of the clinically used EDP-M scheme (etoposide, doxorubicin, cisplatin, mitotane). Inhibition of cell viability correlated furthermore with a decrease in proliferation, in cell migration and an increase in apoptosis. Moreover, analysis of cancer pathways indicated a modulation of the ERK1/2—and AKT—pathways by luminespib and ganetespib treatment.
Conclusions: Our findings emphasize HSP90 as a marker with prognostic impact and promising target with N-terminal HSP90 inhibitors as drugs with potential therapeutic efficacy toward ACC.
The helical distribution of the electronic density in chiral molecules, such as DNA and bacteriorhodopsin, has been suggested to induce a spin–orbit coupling interaction that may lead to the so-called chirality-induced spin selectivity (CISS) effect. Key ingredients for the theoretical modelling are, in this context, the helically shaped potential of the molecule and, concomitantly, a Rashba-like spin–orbit coupling due to the appearance of a magnetic field in the electron reference frame. Symmetries of these models clearly play a crucial role in explaining the observed effect, but a thorough analysis has been largely ignored in the literature. In this work, we present a study of these symmetries and how they can be exploited to enhance chiral-induced spin selectivity in helical molecular systems.
Although accepted agents in chorioamnionitis and preterm birth, the role of Ureaplasma species (spp.) in inflammation-driven morbidities of prematurity, including the development of bronchopulmonary dysplasia, remains controversial. To add to scarce in vitro data addressing the pro-inflammatory capacity of Ureaplasma spp., pulmonary epithelial-like A549 cells and human pulmonary microvascular endothelial cells (HPMEC) were incubated with Ureaplasma (U.) urealyticum, U. parvum, and Escherichia coli lipopolysaccharide (LPS). Ureaplasma isolates down-regulated caspase mRNA levels in A549 cells (caspase 8: p<0.001, 9: p<0.001, vs. broth), while increasing caspase protein expression, enzyme activity, and cell death in HPMEC (active caspase 3: p<0.05, caspase 8: p<0.05, active caspase 9: p<0.05, viability: p<0.05). LPS, contrarily, induced caspase mRNA expression in HPMEC (caspase 3: p<0.01, 4: p<0.001, 5: p<0.001, 8: p<0.001, vs. control), but not in A549 cells, and did not affect enzyme activity or protein levels in either cell line. LPS, but neither Ureaplasma isolate, enhanced mRNA expression of pro-inflammatory interleukin (IL)-6 in both A549 (p<0.05, vs. control) and HPMEC (p<0.001) as well as tumor necrosis factor-α (p<0.01), IL-1β (p<0.001), and IL-8 (p<0.05) in HPMEC. We are therefore the first to demonstrate a differential modulation of pulmonary caspases by Ureaplasma spp. in vitro. Ureaplasma-driven enhanced protein expression and activity of caspases in pulmonary endothelial cells result in cell death and may cause structural damage. Down-regulated caspase mRNA in pulmonary epithelial cells, contrarily, may indicate Ureaplasma-induced inhibition of apoptosis and prevent effective immune responses. Both may ultimately contribute to chronic Ureaplasma colonization and long-term pulmonary inflammation.
Background
Ureaplasma species (spp.) are commonly regarded as low-virulent commensals but may cause invasive diseases in immunocompromised adults and in neonates, including neonatal meningitis. The interactions of Ureaplasma spp. with host defense mechanisms are poorly understood. This study addressed Ureaplasma-driven cell death, concentrating on apoptosis as well as inflammatory cell death.
Methods
Human brain microvascular endothelial cells (HBMEC) were exposed to Ureaplasma (U.) urealyticum serovar 8 (Uu8) and U. parvum serovar 3 (Up3). Resulting numbers of dead cells as well as mRNA levels and enzyme activity of key agents in programmed cell death were assessed by flow cytometry, RNA sequencing, and qRT-PCR, respectively. xCELLigence data were used for real-time monitoring of changes in cell adhesion properties.
Results
Both Ureaplasma isolates induced cell death (p < 0.05, vs. broth). Furthermore, Ureaplasma spp. enhanced mRNA levels for genes in apoptosis, including caspase 3 (Up3 p < 0.05, vs. broth), caspase 7 (p < 0.01), and caspase 9 (Up3 p < 0.01). Caspase 3 activity was increased upon Uu8 exposure (p < 0.01). Vice versa, Ureaplasma isolates downregulated mRNA levels for proteins involved in inflammatory cell death, namely caspase 1 (Uu8 p < 0.01, Up3 p < 0.001), caspase 4 (Uu8 p < 0.05, Up3 p < 0.01), NOD-like receptor pyrin domain-containing 3 (Uu8 p < 0.05), and receptor-interacting protein kinase 3 (p < 0.05).
Conclusions
By inducing apoptosis in HBMEC as main constituents of the blood-brain barrier, Ureaplasma spp. may provoke barrier breakdown. Simultaneous suppression of inflammatory cell death may additionally attenuate host defense strategies. Ultimate consequence could be invasive and long-term CNS infections by Ureaplasma spp.
Ureaplasma species are common colonizers of the adult genitourinary tract and often considered as low-virulence commensals. Intraamniotic Ureaplasma infections, however, facilitate chorioamnionitis and preterm birth, and cases of Ureaplasma-induced neonatal sepsis, pneumonia, and meningitis raise a growing awareness of their clinical relevance. In vitro studies are scarce but demonstrate distinct Ureaplasma-driven impacts on immune mechanisms. The current study addressed cytokine and chemokine responses upon exposure of native or lipopolysaccharide (LPS) co-stimulated human brain microvascular endothelial cells (HBMEC) to Ureaplasma urealyticum or U. parvum, using qRT-PCR, RNA sequencing, multi-analyte immunoassay, and flow cytometry. Ureaplasma exposure in native HBMEC reduced monocyte chemoattractant protein (MCP)-3 mRNA expression (p < 0.01, vs. broth). In co-stimulated HBMEC, Ureaplasma spp. attenuated LPS-evoked mRNA responses for C-X-C chemokine ligand 5, MCP-1, and MCP-3 (p < 0.05, vs. LPS) and mitigated LPS-driven interleukin (IL)-1α protein secretion, as well as IL-8 mRNA and protein responses (p < 0.05). Furthermore, Ureaplasma isolates increased C-X-C chemokine receptor 4 mRNA levels in native and LPS co-stimulated HBMEC (p < 0.05). The presented results may imply immunomodulatory capacities of Ureaplasma spp. which may ultimately promote chronic colonization and long-term neuroinflammation.
The evolutionary conserved Myb-MuvB (MMB) multiprotein complex is a transcriptional master regulator of mitotic gene expression. The MMB subunits B-MYB, FOXM1 as well as target genes of MMB are often overexpressed in different cancer types. Elevated expression of these genes correlates with an advanced tumor state and a poor prognosis for patients. Furthermore, it has been reported that pathways, which are involved in regulating the mitotic machinery are attractive for a potential treatment of cancers harbouring Ras mutations (Luo et al., 2009).
This suggest that the MMB complex could be required for tumorigenesis by mediating overactivity of mitotic genes and that the MMB could be a useful target for lung cancer treatment. However, although MMB has been characterized biochemically, the contribution of MMB to tumorigenesis is largely unknown in particular in vivo.
In this thesis, it was demonstrated that the MMB complex is required for lung tumorigenesis in vivo in a mouse model of non small cell lung cancer. Elevated levels of B-MYB, NUSAP1 or CENPF in advanced tumors as opposed to low levels of these proteins levels in grade 1 or 2 tumors support the possible contribution of MMB to lung tumorigenesis and the oncogenic potential of B-MYB.The tumor growth promoting function of B-MYB was illustrated by a lower fraction of KI-67 positive cells in vivo and a significantly high impairment in proliferation after loss of B-Myb in vitro. Defects in cytokinesis and an abnormal cell cycle profile after loss of B-Myb underscore the impact of B-MYB on proliferation of lung cancer cell lines. The incomplete recombination of B-Myb in murine lung tumors and in the tumor derived primary cell lines illustrates the selection pressure against the complete loss of B-Myb and further demonstrats that B-Myb is a tumor-essential gene. In the last part of this thesis, the contribution of MMB to the proliferation of human lung cancer cells was demonstrated by the RNAi-mediated depletion of B-Myb. Detection of elevated B-MYB levels in human adenocarcinoma and a reduced proliferation, cytokinesis defects and abnormal cell cycle profile after loss of B-MYB in human lung cancer cell lines underlines the potential of B-MYB to serve as a clinical marker.
Adipocytes are specialized cells found in vertebrates to ensure survival in terms of adaption to food deficit and abundance. However, their dysfunction accounts for the pathophysiology of metabolic diseases such as T2DM. Preliminary data generated by Mona Löffler suggested that PKD1 is involved in adipocyte function. Here, I show that PKD1 expression and activity is linked to lipid metabolism of murine adipocytes. PKD1 gene expression and activity was reduced in murine white adipose tissue upon fasting, a physiological condition which induces lipolysis. Isoproterenol-stimulated lipolysis in adipose tissue and 3T3-L1 adipocytes reduced PKD1 gene expression. Silencing ATGL in adipocytes inhibited isoproterenol-stimulated lipolysis, however, the β-adrenergic
stimulation of ATGL-silenced adipocytes lowered PKD1 expression levels as well. Adipose tissue of obese mice exhibited high PKD1 RNA levels but paradoxically lower protein levels of phosphorylated PKD1-Ser916. However, HFD generated a second
PKD1 protein product of low molecular weight in mouse adipose tissue. Furthermore, constitutively active PKD1 predominantly displayed nuclear localization in 3T3-L1 adipocytes containing many fat vacuoles. However, adipocytes
overexpressing non-functional PKD1 contained fewer lipid droplets and PKD1-KD was distributed in cytoplasm. Most importantly, deficiency of PKD1 in mouse adipose tissue caused expression of genes involved in adaptive thermogenesis such as UCP-1 and thus generated brown-like phenotype adipocytes. Thus, PKD1 is implicated in adipose tissue function and presents an interesting target for therapeutic approaches in the prevention of obesity and associated diseases.
In 2006, 0.18 Mio pediatric nuclear medicine diagnostic exams were performed worldwide. However, for most of the radiopharmaceuticals used data on biokinetics and, as a consequence on dosimetry, are missing or have not been made publicly available. Therefore, most of the dosimetry assessments presented today for diagnostic agents in children and adolescents rely on the biokinetics data of adults. Even for one of the most common nuclear medicine exams for this patient group, renal scintigraphy with 99mTc-MAG3 for assessing renal function measured data on biokinetics is available only from a study performed on four children of different ages. In particular, renal scans are among the most frequent exams performed on infants and toddlers. Due to the young age, this patient group can be classified as a risk group with a higher probability of developing stochastic radiation effects compared to adults. As there are only limited data on biokinetics and dosimetry in this patient group, the aim of this study is to reassess the dosimetry and the associated radiation risk for a larger number of infants undergoing 99mTc-MAG3 renal scans based on a retrospective analysis of existing patient data.
Data were collected retrospectively from 34 patients younger than 20 months with normal (20 patients) and abnormal renal function (14 patients) undergoing 99mTc-MAG3 scans. The patient-specific organ activity was estimated based on a retrospective calibration which was performed based on a set of two 3D-printed infant kidneys (newborns: 8.6 ml; 1-year-old: 23.4 ml) filled with known activities. Both phantoms were scanned at different positions along the anteroposterior axis inside a water phantom, providing depth- and size-dependent attenuation correction factors for planar imaging. Time-activity curves were determined by drawing kidney, bladder, and whole body regions-of-interest for each patient, and subsequently applying the calibration factor for conversion of counts to activity. Patient-specific time-integrated activity coefficients were obtained by integrating the organ-specific time-activity curves. Absorbed and effective dose coefficients for each patient were assessed with OLINDA/EXM for the provided newborn and 1-year-old phantom. Based on absorbed dose values, the radiation risk estimation was performed individually for each of the 34 patients with the National Cancer Institute’s Radiation Risk Assessment Tool.
The patients’ organ-specific mean absorbed dose coefficients for the patients with normal renal function were 0.04±0.03 mGy/MBq for the kidneys and 0.27±0.24 mGy/MBq for the bladder. This resulted in a mean effective dose coefficient of 0.02±0.02 mSv/MBq. Based on the dosimetry results, the evaluation of the excess lifetime risk (ELR) for the development of radiation-induced cancer showed that the group of newborns has an ELR of 16.8 per 100,000 persons, which is higher in comparison with the 1-year-old group with an ELR of 14.7 per 100,000 persons. With regard to the 14 patients with abnormal renal function, the mean values for the organ absorbed dose coefficients for the patients were: 0.40±0.34 mGy/MBq for the kidneys and 0.46±0.37 mGy/MBq for the bladder. The corresponding effective dose coefficients (mSv/MBq) was: 0.05±0.02 mSv/MBq. The mean ELR (per 100,000 persons) for developing cancer from radiation exposure for patients with abnormal renal function was 29.2±18.7 per 100,000 persons.
As a result, the radiation-associated stochastic risk increases with the organ doses, taking age- and gender-specific influences into account. Overall, the lifetime radiation risk associated with the 99mTc-MAG3 scans is very low in comparison to the general population risk for developing cancer.
Furthermore, due to the increasing demand for PET-scans in children and adolescents with 68Ga-labelled peptides, in this work published data sets for those compounds were analyzed to derive recommendations for the administered activities in children and adolescents. The recommendation for the activities to be administered were based on the weight-independent effective dose model, proposed by the EANM Pediatric Dosage Card for application in pediatric nuclear medicine. The aim was to derive recommendations on administered activities for obtaining age-independent effective doses. Consequently, the corresponding weight-dependent effective dose coefficients were rescaled according to the formalism of the EANM dosage card, to determine the radiopharmaceutical class of 68Ga-labeled peptides (“multiples”), and to calculate the baseline activities based on the biokinetics of these compounds and an upper limit of the administered activity of 185 MBq for an adult. Analogous to 18F-fluoride, a minimum activity of 14 MBq is recommended. As a result, for those pediatric nuclear medicine applications involving 68Ga-labeled peptides, new values for the EANM dosage card were proposed and implemented based on the results derived in this work.
Overall, despite the low additional radiation-related cancer risk, all efforts should be undertaken to optimize administered activities in children and adolescents for obtaining sufficient diagnostic information with minimal associated radiation risk.
One of the challenges in the field of quantum sensing and information processing is to selectively address and coherently manipulate highly homogeneous qubits subject to external perturbations. Here, we present room-temperature coherent control of high-dimensional quantum bits, the so-called qudits, associated with vacancy-related spins in silicon carbide enriched with nuclear spin-free isotopes. In addition to the excitation of a spectrally narrow qudit mode at the pump frequency, several other modes are excited in the electron spin resonance spectra whose relative positions depend on the external magnetic field. We develop a theory of multipole spin dynamics and demonstrate selective quantum control of homogeneous spin packets with sub-MHz spectral resolution. Furthermore, we perform two-frequency Ramsey interferometry to demonstrate absolute dc magnetometry, which is immune to thermal noise and strain inhomogeneity.
Recent research revealed the superiority of artificial intelligence over dermatologists to diagnose melanoma from images. However, 30–50% of all melanomas and more than half of those in young patients evolve from initially benign lesions. Despite its high relevance for melanoma screening, neither clinicians nor computers are yet able to reliably predict a nevus’ oncologic transformation. The cause of this lies in the static nature of lesion presentation in the current standard of care, both for clinicians and algorithms. The status quo makes it difficult to train algorithms (and clinicians) to precisely assess the likelihood of a benign skin lesion to transform into melanoma. In addition, it inhibits the precision of current algorithms since ‘evolution’ image features may not be part of their decision. The current literature reveals certain types of melanocytic nevi (i.e. ‘spitzoid’ or ‘dysplastic’ nevi) and criteria (i.e. visible vasculature) that, in general, appear to have a higher chance to transform into melanoma. However, owing to the cumulative nature of oncogenic mutations in melanoma, a more fine-grained early morphologic footprint is likely to be detectable by an algorithm. In this perspective article, the concept of melanoma prediction is further explored by the discussion of the evolution of melanoma, the concept for training of such a nevi classifier and the implications of early melanoma prediction for clinical practice. In conclusion, the authors believe that artificial intelligence trained on prospective image data could be transformative for skin cancer diagnostics by (a) predicting melanoma before it occurs (i.e. pre-in situ) and (b) further enhancing the accuracy of current melanoma classifiers. Necessary prospective images for this research are obtained via free mole-monitoring mobile apps.
Background
Fabry disease is caused by a deficiency of the lysosomal enzyme α-galactosidase, resulting in progressive accumulation of globotriaosylceramide (GL-3). The disease can manifest early during childhood and adolescence. Enzyme replacement therapy (ERT) with recombinant human α-galactosidase is the first specific treatment for Fabry disease and has been available in Europe since 2001. This paper presents the findings of a systematic literature review of clinical outcomes with ERT in paediatric patients with Fabry disease.
Methods
A comprehensive systematic review of published literature on ERT in Fabry disease was conducted in January 2017. The literature analysis included all original articles reporting outcomes of ERT in paediatric patients.
Results
Treatment-related outcomes in the paediatric population were reported in six publications derived from open-label clinical trials and in 10 publications derived from observational or registry-based studies. ERT was shown to significantly reduce plasma and urine GL-3 levels in paediatric patients with Fabry disease. The effect of ERT on GL-3 clearance from renal podocytes appeared to be agalsidase dose-dependent. ERT relieved pain and improved gastrointestinal symptoms and quality of life.
Conclusions
Based on the published literature, the use of ERT in paediatric patients can significantly clear GL-3 accumulation, ameliorate the early symptoms of Fabry disease, and improve quality of life. Treatment with ERT in paediatric patients with Fabry disease may be important to prevent further disease progression and overt organ damage.
The mold Fusarium is a ubiquitous fungus causing plant, animal and human infections. In humans, Fusarium spp. are the major cause of eye infections in patients wearing contact lenses or after local trauma. Systemic infections by Fusarium spp. mainly occur in immunosuppressed patients and can disseminate throughout the human body. Due to high levels of resistance to antifungals a fast identification of the causative agent is an urgent need. By using a probe-based real-time PCR assay specific for the genus Fusarium we analysed several different clinical specimens detecting Fusarium spp. commonly found in clinical samples in Germany. Also, a large collection of lung fluid samples of haematological patients was analysed (n = 243). In these, two samples (0.8%) were reproducibly positive, but only one could be confirmed by sequencing. For this case of probable invasive fungal disease (IFD) culture was positive for Fusarium species. Here we describe a rapid, probe-based real-time PCR assay to specifically detect DNA from a broad range of Fusarium species and its application to clinically relevant specimens.
Dendritic cells (DCs) are antigen presenting cells which serve as a passage between the innate and the acquired immunity. Aspergillosis is a major lethal condition in immunocompromised patients caused by the adaptable saprophytic fungus Aspergillus fumigatus. The healthy human immune system is capable to ward off A. fumigatus infections however immune-deficient patients are highly vulnerable to invasive aspergillosis. A. fumigatus can persist during infection due to its ability to survive the immune response of human DCs. Therefore, the study of the metabolism specific to the context of infection may allow us to gain insight into the adaptation strategies of both the pathogen and the immune cells. We established a metabolic model of A. fumigatus central metabolism during infection of DCs and calculated the metabolic pathway (elementary modes; EMs). Transcriptome data were used to identify pathways activated when A. fumigatus is challenged with DCs. In particular, amino acid metabolic pathways, alternative carbon metabolic pathways and stress regulating enzymes were found to be active. Metabolic flux modeling identified further active enzymes such as alcohol dehydrogenase, inositol oxygenase and GTP cyclohydrolase participating in different stress responses in A. fumigatus. These were further validated by qRT-PCR from RNA extracted under these different conditions. For DCs, we outlined the activation of metabolic pathways in response to the confrontation with A. fumigatus. We found the fatty acid metabolism plays a crucial role, along with other metabolic changes. The gene expression data and their analysis illuminate additional regulatory pathways activated in the DCs apart from interleukin regulation. In particular, Toll-like receptor signaling, NOD-like receptor signaling and RIG-I-like receptor signaling were active pathways. Moreover, we identified subnetworks and several novel key regulators such as UBC, EGFR, and CUL3 of DCs to be activated in response to A. fumigatus. In conclusion, we analyze the metabolic and regulatory responses of A. fumigatus and DCs when confronted with each other.
BACKGROUND: The barrier to diffusion of organic solutes across the plant cuticle is composed of waxes consisting of very long-chain aliphatic (VLCA) and, to varying degrees, cyclic compounds like pentacyclic triterpenoids. The roles of both fractions in controlling cuticular penetration by organic solutes, e.g. the active ingredients (AI) of pesticides, are unknown to date. We studied thepermeabilityof isolated leaf cuticularmembranes from Garcinia xanthochymus andPrunus laurocerasus for lipophilic azoxystrobin and theobromine as model compounds for hydrophilic AIs.
RESULTS: The wax of P. laurocerasus consists of VLCA (12%) and cyclic compounds (88%), whereas VLCAs make up 97% of the wax of G. xanthochymus.We showthat treating isolated cuticles with methanol almost quantitatively releases the cyclic fraction while leaving the VLCA fraction essentially intact. All VLCAs were subsequently removed using chloroform. In both species, the permeance of the two model compounds did not change significantly after methanol treatment, whereas chloroform extraction had a large effect on organic solute permeability.
CONCLUSION: The VLCA wax fractionmakes up the permeability barrier for organic solutes, whereas cyclic compounds even in high amounts have a negligible role. This is of significance when optimizing the foliar uptake of pesticides.
Sensory processing and attention allocation are shaped by threat, but the role of trait-anxiety in sensory processing as a function of threat predictability remains incompletely understood. Therefore, we measured steady-state visual evoked potentials (ssVEPs) as an index of sensory processing of predictable and unpredictable threat cues in 29 low (LA) and 29 high (HA) trait-anxious participants during a modified NPU-paradigm followed by an extinction phase. Three different contextual cues indicated safety (N), predictable (P) or unpredictable threat (U), while foreground cues signalled shocks in the P-condition only. All participants allocated increased attentional resources to the central P-threat cue, replicating previous findings. Importantly, LA individuals exhibited larger ssVEP amplitudes to contextual threat (U and P) than to contextual safety cues, while HA individuals did not differentiate among contextual cues in general. Further, HA exhibited higher aversive ratings of all contexts compared to LA. These results suggest that high trait-anxious individuals might be worse at discriminating contextual threat stimuli and accordingly overestimate the probability and aversiveness of unpredictable threat. These findings support the notion of aberrant sensory processing of unpredictable threat in anxiety disorders, as this processing pattern is already evident in individuals at risk of these disorders.
Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, the rapid recanalization of occluded cranial vessels is the primary therapeutic aim. However, experimental data (obtained using mostly the transient middle cerebral artery occlusion model) indicates that progressive stroke can still develop despite successful recanalization, a process termed “reperfusion injury.” Mounting experimental evidence suggests that platelets and T cells contribute to cerebral ischemia/reperfusion injury, and ischemic stroke is increasingly considered a thrombo-inflammatory disease. The interaction of von Willebrand factor and its receptor on the platelet surface, glycoprotein Ib, as well as many activatory platelet receptors and platelet degranulation contribute to secondary infarct growth in this setting. In contrast, interference with GPIIb/IIIa-dependent platelet aggregation and thrombus formation does not improve the outcome of acute brain ischemia but dramatically increases the susceptibility to intracranial hemorrhage. Here, we summarize the current understanding of the mechanisms and the potential translational impact of platelet contributions to cerebral ischemia/reperfusion injury.
Flagellin hypervariable region determinessymbiotic properties of commensalEscherichia coli strains
(2019)
Escherichia coli represents a classical intestinal gram-negative commensal. Despite this commensalism, different E. coli strains can mediate disparate immunogenic properties in a given host. Symbiotic E. coli strains such as E. coli Nissle 1917 (EcN) are attributed beneficial properties, e.g., promotion of intestinal homeostasis. Therefore, we aimed to identify molecular features derived from symbiotic bacteria that might help to develop innovative therapeutic alternatives for the treatment of intestinal immune disorders. This study was performed using the dextran sodium sulphate (DSS)-induced colitis mouse model, which is routinely used to evaluate potential therapeutics for the treatment of Inflammatory Bowel Diseases (IBDs). We focused on the analysis of flagellin structures of different E. coli strains. EcN flagellin was found to harbor a substantially longer hypervariable region (HVR) compared to other commensal E. coli strains, and this longer HVR mediated symbiotic properties through stronger activation of Toll-like receptor (TLR)5, thereby resulting in interleukin (IL)-22–mediated protection of mice against DSS-induced colitis. Furthermore, using bone-marrow–chimeric mice (BMCM), CD11c+ cells of the colonic lamina propria (LP) were identified as the main mediators of these flagellin-induced symbiotic effects. We propose flagellin from symbiotic E. coli strains as a potential therapeutic to restore intestinal immune homeostasis, e.g., for the treatment of IBD patients.
Patients with relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL) following allogeneic hematopoietic stem cell transplantation (alloHSCT) have a poor prognosis, and alternative therapies are needed for this patient population. Blinatumomab, a bispecific T cell engager immunotherapy, was evaluated in an open-label, single-arm, phase II study of adults with R/R Philadelphia chromosome-negative B cell precursor ALL and resulted in a rate of complete remission (CR) or CR with partial hematologic recovery of peripheral blood counts (CRh) of 43% within 2 treatment cycles. We conducted an exploratory analysis to determine the efficacy and safety of blinatumomab in 64 patients who had relapsed following alloHSCT before enrollment in the phase II study. Forty-five percent of the patients (29 of 64) achieved a CR/CRh within the first 2 cycles of treatment, 22 of whom had a minimal residual disease (MRD) response (including 19 with a complete MRD response). After 1 year and 3 years of follow-up, the median relapse-free survival was 7.4 months for patients who achieved CR/CRh in the first 2 cycles, and the median overall survival was 8.5 months; overall survival rate (Kaplan-Meier estimate) was 36% at 1 year and 18% at 3 years. Grade 3 and 4 adverse events were reported in 20 patients (31%) and 28 patients (44%), respectively, with grade 3 and 4 neurologic events in 8 and 2 patients, respectively, and grade 3 cytokine release syndrome in 2 patients. Eight patients had fatal adverse events, including 5 due to infections. Seven patients had grade ≤ 3 graft-versus-host disease during the study, none of which resulted in the discontinuation of blinatumomab or hospitalization. Our data suggest that blinatumomab is an effective salvage therapy in this patient population.
Advanced Analytics in Operations Management and Information Systems: Methods and Applications
(2019)
The digital transformation of business and society presents enormous potentials for companies across all sectors. Fueled by massive advances in data generation, computing power, and connectivity, modern organizations have access to gigantic amounts of data. Companies seek to establish data-driven decision cultures to leverage competitive advantages in terms of efficiency and effectiveness. While most companies focus on descriptive tools such as reporting, dashboards, and advanced visualization, only a small fraction already leverages advanced analytics (i.e., predictive and prescriptive analytics) to foster data-driven decision-making today. Therefore, this thesis set out to investigate potential opportunities to leverage prescriptive analytics in four different independent parts.
As predictive models are an essential prerequisite for prescriptive analytics, the first two parts of this work focus on predictive analytics. Building on state-of-the-art machine learning techniques, we showcase the development of a predictive model in the context of capacity planning and staffing at an IT consulting company. Subsequently, we focus on predictive analytics applications in the manufacturing sector. More specifically, we present a data science toolbox providing guidelines and best practices for modeling, feature engineering, and model interpretation to manufacturing decision-makers. We showcase the application of this toolbox on a large data-set from a German manufacturing company.
Merely using the improved forecasts provided by powerful predictive models enables decision-makers to generate additional business value in some situations. However, many complex tasks require elaborate operational planning procedures. Here, transforming additional information into valuable actions requires new planning algorithms. Therefore, the latter two parts of this thesis focus on prescriptive analytics. To this end, we analyze how prescriptive analytics can be utilized to determine policies for an optimal searcher path problem based on predictive models. While rapid advances in artificial intelligence research boost the predictive power of machine learning models, a model uncertainty remains in most settings. The last part of this work proposes a prescriptive approach that accounts for the fact that predictions are imperfect and that the arising uncertainty needs to be considered. More specifically, it presents a data-driven approach to sales-force scheduling. Based on a large data set, a model to predictive the benefit of additional sales effort is trained. Subsequently, the predictions, as well as the prediction quality, are embedded into the underlying team orienteering problem to determine optimized schedules.
Structure-fluorescence activation relationships of a large Stokes shift fluorogenic RNA aptamer
(2019)
The Chili RNA aptamer is a 52 nt long fluorogen-activating RNA aptamer (FLAP) that confers fluorescence to structurally diverse derivatives of fluorescent protein chromophores. A key feature of Chili is the formation of highly stable complexes with different ligands, which exhibit bright, highly Stokes-shifted fluorescence emission. In this work, we have analyzed the interactions between the Chili RNA and a family of conditionally fluorescent ligands using a variety of spectroscopic, calorimetric and biochemical techniques to reveal key structure - fluorescence activation relationships (SFARs). The ligands under investigation form two categories with emission maxima of ~540 nm or ~590 nm, respectively, and bind with affinities in the nanomolar to low-micromolar range. Isothermal titration calorimetry was used to elucidate the enthalpic and entropic contributions to binding affinity for a cationic ligand that is unique to the Chili aptamer. In addition to fluorescence activation, ligand binding was also observed by NMR spectroscopy, revealing characteristic signals for the formation of a G-quadruplex only upon ligand binding. These data shed light on the molecular features required and responsible for the large Stokes shift and the strong fluorescence enhancement of red and green emitting RNA-chromophore complexes.
Objective
To identify and characterize patients with autoantibodies against different neurofascin (NF) isoforms.
Methods
Screening of a large cohort of patient sera for anti-NF autoantibodies by ELISA and further characterization by cell-based assays, epitope mapping, and complement binding assays.
Results
Two different clinical phenotypes became apparent in this study: The well-known clinical picture of subacute-onset severe sensorimotor neuropathy with tremor that is known to be associated with IgG4 autoantibodies against the paranodal isoform NF-155 was found in 2 patients. The second phenotype with a dramatic course of disease with tetraplegia and almost locked-in syndrome was associated with IgG3 autoantibodies against nodal and paranodal isoforms of NF in 3 patients. The epitope against which these autoantibodies were directed in this second phenotype was the common Ig domain found in all 3 NF isoforms. In contrast, anti–NF-155 IgG4 were directed against the NF-155–specific Fn3Fn4 domain. The description of a second phenotype of anti–NF-associated neuropathy is in line with some case reports of similar patients that were published in the last year.
Conclusions
Our results indicate that anti–pan-NF-associated neuropathy differs from anti–NF-155-associated neuropathy, and epitope and subclass play a major role in the pathogenesis and severity of anti–NF-associated neuropathy and should be determined to correctly classify patients, also in respect to possible differences in therapeutic response.