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- J-8841-2015 (1)
- M-1240-2017 (1)
- N-2030-2015 (1)
- N-3741-2015 (1)
Highlights
• Loss of DNAJC19's DnaJ domain disrupts cardiac mitochondrial structure, leading to abnormal cristae formation in iPSC-CMs.
• Impaired mitochondrial structures lead to an increased mitochondrial respiration, ROS and an elevated membrane potential.
• Mutant iPSC-CMs show sarcomere dysfunction and a trend to more arrhythmias, resembling DCMA-associated cardiomyopathy.
Background
Dilated cardiomyopathy with ataxia (DCMA) is an autosomal recessive disorder arising from truncating mutations in DNAJC19, which encodes an inner mitochondrial membrane protein. Clinical features include an early onset, often life-threatening, cardiomyopathy associated with other metabolic features. Here, we aim to understand the metabolic and pathophysiological mechanisms of mutant DNAJC19 for the development of cardiomyopathy.
Methods
We generated induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) of two affected siblings with DCMA and a gene-edited truncation variant (tv) of DNAJC19 which all lack the conserved DnaJ interaction domain. The mutant iPSC-CMs and their respective control cells were subjected to various analyses, including assessments of morphology, metabolic function, and physiological consequences such as Ca\(^{2+}\) kinetics, contractility, and arrhythmic potential. Validation of respiration analysis was done in a gene-edited HeLa cell line (DNAJC19tv\(_{HeLa}\)).
Results
Structural analyses revealed mitochondrial fragmentation and abnormal cristae formation associated with an overall reduced mitochondrial protein expression in mutant iPSC-CMs. Morphological alterations were associated with higher oxygen consumption rates (OCRs) in all three mutant iPSC-CMs, indicating higher electron transport chain activity to meet cellular ATP demands. Additionally, increased extracellular acidification rates suggested an increase in overall metabolic flux, while radioactive tracer uptake studies revealed decreased fatty acid uptake and utilization of glucose. Mutant iPSC-CMs also showed increased reactive oxygen species (ROS) and an elevated mitochondrial membrane potential. Increased mitochondrial respiration with pyruvate and malate as substrates was observed in mutant DNAJC19tv HeLa cells in addition to an upregulation of respiratory chain complexes, while cellular ATP-levels remain the same. Moreover, mitochondrial alterations were associated with increased beating frequencies, elevated diastolic Ca\(^{2+}\) concentrations, reduced sarcomere shortening and an increased beat-to-beat rate variability in mutant cell lines in response to β-adrenergic stimulation.
Conclusions
Loss of the DnaJ domain disturbs cardiac mitochondrial structure with abnormal cristae formation and leads to mitochondrial dysfunction, suggesting that DNAJC19 plays an essential role in mitochondrial morphogenesis and biogenesis. Moreover, increased mitochondrial respiration, altered substrate utilization, increased ROS production and abnormal Ca\(^{2+}\) kinetics provide insights into the pathogenesis of DCMA-related cardiomyopathy.
To fire action-potential-like electrical signals, the vacuole membrane requires the two-pore channel TPC1, formerly called SV channel. The TPC1/SV channel functions as a depolarization-stimulated, non-selective cation channel that is inhibited by luminal Ca\(^{2+}\). In our search for species-dependent functional TPC1 channel variants with different luminal Ca\(^{2+}\) sensitivity, we found in total three acidic residues present in Ca\(^{2+}\) sensor sites 2 and 3 of the Ca\(^{2+}\)-sensitive AtTPC1 channel from Arabidopsis thaliana that were neutral in its Vicia faba ortholog and also in those of many other Fabaceae. When expressed in the Arabidopsis AtTPC1-loss-of-function background, wild-type VfTPC1 was hypersensitive to vacuole depolarization and only weakly sensitive to blocking luminal Ca\(^{2+}\). When AtTPC1 was mutated for these VfTPC1-homologous polymorphic residues, two neutral substitutions in Ca\(^{2+}\) sensor site 3 alone were already sufficient for the Arabidopsis At-VfTPC1 channel mutant to gain VfTPC1-like voltage and luminal Ca\(^{2+}\) sensitivity that together rendered vacuoles hyperexcitable. Thus, natural TPC1 channel variants exist in plant families which may fine-tune vacuole excitability and adapt it to environmental settings of the particular ecological niche.
Interleukin-4 (IL-4) plays a key role in atopic diseases. It coordinates T-helper cell differentiation to subtype 2, thereby directing defense toward humoral immunity. Together with Interleukin-13, IL-4 further induces immunoglobulin class switch to IgE. Antibodies of this type activate mast cells and basophilic and eosinophilic granulocytes, which release pro-inflammatory mediators accounting for the typical symptoms of atopic diseases. IL-4 and IL-13 are thus major targets for pharmaceutical intervention strategies to treat atopic diseases. Besides neutralizing antibodies against IL-4, IL-13, or its receptors, IL-4 antagonists can present valuable alternatives. Pitrakinra, an Escherichia coli-derived IL-4 antagonist, has been evaluated in clinical trials for asthma treatment in the past; however, deficits such as short serum lifetime and potential immunogenicity among others stopped further development. To overcome such deficits, PEGylation of therapeutically important proteins has been used to increase the lifetime and proteolytic stability. As an alternative, glycoengineering is an emerging strategy used to improve pharmacokinetics of protein therapeutics. In this study, we have established different strategies to attach glycan moieties to defined positions in IL-4. Different chemical attachment strategies employing thiol chemistry were used to attach a glucose molecule at amino acid position 121, thereby converting IL-4 into a highly effective antagonist. To enhance the proteolytic stability of this IL-4 antagonist, additional glycan structures were introduced by glycoengineering utilizing eucaryotic expression. IL-4 antagonists with a combination of chemical and biosynthetic glycoengineering could be useful as therapeutic alternatives to IL-4 neutralizing antibodies already used to treat atopic diseases.
Graphs provide a key means to model relationships between entities.
They consist of vertices representing the entities,
and edges representing relationships between pairs of entities.
To make people conceive the structure of a graph,
it is almost inevitable to visualize the graph.
We call such a visualization a graph drawing.
Moreover, we have a straight-line graph drawing
if each vertex is represented as a point
(or a small geometric object, e.g., a rectangle)
and each edge is represented as a line segment between its two vertices.
A polyline is a very simple straight-line graph drawing,
where the vertices form a sequence according to which the vertices are connected by edges.
An example of a polyline in practice is a GPS trajectory.
The underlying road network, in turn, can be modeled as a graph.
This book addresses problems that arise
when working with straight-line graph drawings and polylines.
In particular, we study algorithms
for recognizing certain graphs representable with line segments,
for generating straight-line graph drawings,
and for abstracting polylines.
In the first part, we first examine,
how and in which time we can decide
whether a given graph is a stick graph,
that is, whether its vertices can be represented as
vertical and horizontal line segments on a diagonal line,
which intersect if and only if there is an edge between them.
We then consider the visual complexity of graphs.
Specifically, we investigate, for certain classes of graphs,
how many line segments are necessary for any straight-line graph drawing,
and whether three (or more) different slopes of the line segments
are sufficient to draw all edges.
Last, we study the question,
how to assign (ordered) colors to the vertices of a graph
with both directed and undirected edges
such that no neighboring vertices get the same color
and colors are ascending along directed edges.
Here, the special property of the considered graph is
that the vertices can be represented as intervals
that overlap if and only if there is an edge between them.
The latter problem is motivated by an application
in automated drawing of cable plans with vertical and horizontal line segments,
which we cover in the second part.
We describe an algorithm that
gets the abstract description of a cable plan as input,
and generates a drawing that takes into account
the special properties of these cable plans,
like plugs and groups of wires.
We then experimentally evaluate the quality of the resulting drawings.
In the third part, we study the problem of abstracting (or simplifying)
a single polyline and a bundle of polylines.
In this problem, the objective is to remove as many vertices as possible from the given polyline(s)
while keeping each resulting polyline sufficiently similar to its original course
(according to a given similarity measure).
Recent studies link increased ozone (O\(_3\)) and carbon dioxide (CO\(_2\)) levels to alteration of plant performance and plant-herbivore interactions, but their interactive effects on plant-pollinator interactions are little understood. Extra floral nectaries (EFNs) are essential organs used by some plants for stimulating defense against herbivory and for the attraction of insect pollinators, e.g., bees. The factors driving the interactions between bees and plants regarding the visitation of bees to EFNs are poorly understood, especially in the face of global change driven by greenhouse gases. Here, we experimentally tested whether elevated levels of O\(_3\) and CO\(_2\) individually and interactively alter the emission of Volatile Organic Compound (VOC) profiles in the field bean plant (Vicia faba, L., Fabaceae), EFN nectar production and EFN visitation by the European orchard bee (Osmia cornuta, Latreille, Megachilidae). Our results showed that O\(_3\) alone had significant negative effects on the blends of VOCs emitted while the treatment with elevated CO\(_2\) alone did not differ from the control. Furthermore, as with O\(_3\) alone, the mixture of O\(_3\) and CO\(_2\) also had a significant difference in the VOCs’ profile. O\(_3\) exposure was also linked to reduced nectar volume and had a negative impact on EFN visitation by bees. Increased CO\(_2\) level, on the other hand, had a positive impact on bee visits. Our results add to the knowledge of the interactive effects of O\(_3\) and CO\(_2\) on plant volatiles emitted by Vicia faba and bee responses. As greenhouse gas levels continue to rise globally, it is important to take these findings into consideration to better prepare for changes in plant-insect interactions.
Idiopathic Parkinson’s disease (PD) is characterized by a progredient degeneration of the brain, starting at deep subcortical areas such as the dorsal motor nucleus of the glossopharyngeal and vagal nerves (DM) (stage 1), followed by the coeruleus–subcoeruleus complex; (stage 2), the substantia nigra (SN) (stage 3), the anteromedial temporal mesocortex (MC) (stage 4), high-order sensory association areas and prefrontal fields (HC) (stage 5) and finally first-order sensory association areas, premotor areas, as well as primary sensory and motor field (FC) (stage 6). Autoimmunity might play a role in PD pathogenesis. Here we analyzed whether anti-brain autoantibodies differentially recognize different human brain areas and identified autoantigens that correlate with the above-described dissemination of PD pathology in the brain. Brain tissue was obtained from deceased individuals with no history of neurological or psychiatric disease and no neuropathological abnormalities. Tissue homogenates from different brain regions (DM, SN, MC, HC, FC) were subjected to SDS-PAGE and Western blot. Blots were incubated with plasma samples from 30 PD patients and 30 control subjects and stained with anti-IgG antibodies to detect anti-brain autoantibodies. Signals were quantified. Prominent autoantigens were identified by 2D-gel-coupled mass spectrometry sequencing. Anti-brain autoantibodies are frequent and occur both in healthy controls and individuals with PD. Glial fibrillary acidic protein (GFAP) was identified as a prominent autoantigen recognized in all plasma samples. GFAP immunoreactivity was highest in DM areas and lowest in FC areas with no significant differences in anti-GFAP autoantibody titers between healthy controls and individuals with PD. The anti-GFAP autoimmunoreactivity of different brain areas correlates with the dissemination of histopathological neurodegeneration in PD. We hypothesize that GFAP autoantibodies are physiological but might be involved as a cofactor in PD pathogenesis secondary to a leakage of the blood–brain barrier.
Background
Labelled with lutetium-177, the urea-based small molecules PSMA I&T and PSMA-617 are the two agents most frequently used for radioligand therapy (RLT) in patients with advanced metastatic castration-resistant and prostate-specific membrane antigen (PSMA) expressing prostate cancer (mCRPC). In this matched-pair analysis, we aimed to compare the toxicity and efficacy of both agents for PSMA-directed RLT.
Materials and methods
A total of 110 mCRPC patients from two centres were accrued, 55 individuals treated with [\(^{177}\)Lu]Lu-PSMA I&T, and a matched cohort of 55 patients treated with [\(^{177}\)Lu]Lu-PSMA-617. Matching criteria included age at the first cycle, Gleason score, prostate-specific antigen (PSA) values, and previous taxane-based chemotherapy. Using common terminology criteria for adverse events (CTCAE v. 5.0), toxicity profiles were investigated (including bone marrow and renal toxicity). Overall survival (OS) between both groups was compared.
Results
Toxicity assessment revealed grade III anaemia in a single patient (1.8%) for [\(^{177}\)Lu]Lu-PSMA I&T and five (9.1%) for [\(^{177}\)Lu]Lu-PSMA-617. In addition, one (1.9%) grade III thrombopenia for [\(^{177}\)Lu]Lu-PSMA-617 was recorded. Apart from that, no other grade III/IV toxicities were present. A median OS of 12 months for patients treated with [\(^{177}\)Lu]Lu-PSMA I&T did not differ significantly when compared to patients treated with [\(^{177}\)Lu]Lu-PSMA-617 (median OS, 13 months; P = 0.89).
Conclusion
In this matched-pair analysis of patients receiving one of the two agents most frequently applied for PSMA RLT, the rate of clinically relevant toxicities was low for both compounds. In addition, no relevant differences for OS were observed.
Natural DNA storage allows cellular differentiation, evolution, the growth of our children and controls all our ecosystems. Here, we discuss the fundamental aspects of DNA storage and recent advances in this field, with special emphasis on natural processes and solutions that can be exploited. We point out new ways of efficient DNA and nucleotide storage that are inspired by nature. Within a few years DNA-based information storage may become an attractive and natural complementation to current electronic data storage systems. We discuss rapid and directed access (e.g. DNA elements such as promotors, enhancers), regulatory signals and modulation (e.g. lncRNA) as well as integrated high-density storage and processing modules (e.g. chromosomal territories). There is pragmatic DNA storage for use in biotechnology and human genetics. We examine DNA storage as an approach for synthetic biology (e.g. light-controlled nucleotide processing enzymes). The natural polymers of DNA and RNA offer much for direct storage operations (read-in, read-out, access control). The inbuilt parallelism (many molecules at many places working at the same time) is important for fast processing of information. Using biology concepts from chromosomal storage, nucleic acid processing as well as polymer material sciences such as electronical effects in enzymes, graphene, nanocellulose up to DNA macramé , DNA wires and DNA-based aptamer field effect transistors will open up new applications gradually replacing classical information storage methods in ever more areas over time (decades).
Objectives
To evaluate whether a multimodal intervention in general practice reduces the proportion of second line antibiotic prescriptions and the overall proportion of antibiotic prescriptions for uncomplicated urinary tract infections in women.
Design
Parallel, cluster randomised, controlled trial.
Setting
General practices in five regions in Germany. Data were collected between 1 April 2021 and 31 March 2022.
Participants
General practitioners from 128 randomly assigned practices.
Interventions
Multimodal intervention consisting of guideline recommendations for general practitioners and patients, provision of regional data for antibiotic resistance, and quarterly feedback, which included individual first line and second line proportions of antibiotic prescribing, benchmarking with regional or supra-regional practices, and telephone counselling. Participants in the control group received no information on the intervention.
Main outcome measures
Primary outcome was the proportion of second line antibiotics prescribed by general practices, in relation to all antibiotics prescribed, for uncomplicated urinary tract infections after one year between the intervention and control group. General practices were randomly assigned in blocks (1:1), with a block size of four, into the intervention or control group using SAS version 9.4; randomisation was stratified by region. The secondary outcome was the prescription proportion of all antibiotics, relative within all cases (instances of UTI diagnosis), for the treatment of urinary tract infections after one year between the groups. Adverse events were assessed as exploratory outcomes.
Results
110 practices with full datasets identified 10 323 cases during five quarters (ie, 15 months). The mean proportion of second line antibiotics prescribed was 0.19 (standard deviation 0.20) in the intervention group and 0.35 (0.25) in the control group after 12 months. After adjustment for preintervention proportions, the mean difference was −0.13 (95% confidence interval −0.21 to −0.06, P<0.001). The overall proportion of all antibiotic prescriptions for urinary tract infections over 12 months was 0.74 (standard deviation 0.22) in the intervention and 0.80 (0.15) in the control group with a mean difference of −0.08 (95% confidence interval −0.15 to −0.02, P<0.029). No differences were noted in the number of complications (ie, pyelonephritis, admission to hospital, or fever) between the groups.
Conclusions
The multimodal intervention in general practice significantly reduced the proportion of second line antibiotics and all antibiotic prescriptions for uncomplicated urinary tract infections in women.
Trial registration
German Clinical Trials Register (DRKS), DRKS00020389
Summary
Blood oxygen saturation is an important clinical parameter, especially in postoperative hospitalized patients, monitored in clinical practice by arterial blood gas (ABG) and/or pulse oximetry that both are not suitable for a long-term continuous monitoring of patients during the entire hospital stay, or beyond. Technological advances developed recently for consumer-grade fitness trackers could—at least in theory—help to fill in this gap, but benchmarks on the applicability and accuracy of these technologies in hospitalized patients are currently lacking. We therefore conducted at the postanaesthesia care unit under controlled settings a prospective clinical trial with 201 patients, comparing in total >1,000 oxygen blood saturation measurements by fitness trackers of three brands with the ABG gold standard and with pulse oximetry. Our results suggest that, despite of an overall still tolerable measuring accuracy, comparatively high dropout rates severely limit the possibilities of employing fitness trackers, particularly during the immediate postoperative period of hospitalized patients.
Highlights
•The accuracy of O2 measurements by fitness trackers is tolerable (RMSE ≲4%)
•Correlation with arterial blood gas measurements is fair to moderate (PCC = [0.46; 0.64])
•Dropout rates of fitness trackers during O2 monitoring are high (∼1/3 values missing)
•Fitness trackers cannot be recommended for O2 measuring during critical monitoring
Summary
Here we describe a novel neuro-mesodermal assembloid model that recapitulates aspects of peripheral nervous system (PNS) development such as neural crest cell (NCC) induction, migration, and sensory as well as sympathetic ganglion formation. The ganglia send projections to the mesodermal as well as neural compartment. Axons in the mesodermal part are associated with Schwann cells. In addition, peripheral ganglia and nerve fibers interact with a co-developing vascular plexus, forming a neurovascular niche. Finally, developing sensory ganglia show response to capsaicin indicating their functionality.
The presented assembloid model could help to uncover mechanisms of human NCC induction, delamination, migration, and PNS development. Moreover, the model could be used for toxicity screenings or drug testing. The co-development of mesodermal and neuroectodermal tissues and a vascular plexus along with a PNS allows us to investigate the crosstalk between neuroectoderm and mesoderm and between peripheral neurons/neuroblasts and endothelial cells.
Highlights
•Novel neuro-mesodermal assembloid model of peripheral nervous system development
•Model covers neural crest cell induction, migration, and ganglion formation
•Ganglia send projections to the mesodermal as well as neural compartment
•Peripheral ganglia and nerve fibers interact with a co-developing vascular plexus
Background
Haemophilus influenzae (Hi) is a Gram-negative bacterium that may cause sepsis or meningitis, treatment of which mainly includes β-lactam antibiotics. Since 2019 EUCAST breakpoints for piperacillin/tazobactam have been available. Little is known about the prevalence and mechanisms of piperacillin/tazobactam resistance in Hi.
Objectives
To provide reliable prevalence data for piperacillin/tazobactam resistance in Hi in Germany, to evaluate different antibiotic susceptibility testing methods and to examine possible resistance mechanisms.
Methods
According to EUCAST breakpoints, the MIC for piperacillin/tazobactam resistance is >0.25 mg/L. All invasive Hi in Germany from 2019 were examined by gradient agar diffusion (GAD) for piperacillin/tazobactam susceptibility. Piperacillin/tazobactam broth microdilution (BMD), piperacillin GAD on tazobactam-containing agar [piperacillin GAD on Mueller–Hinton agar with horse blood (MH-F)/tazobactam) and piperacillin/tazobactam agar dilution (AD) were used for confirmation. Phenotypic testing was complemented by ftsI sequencing.
Results
Piperacillin/tazobactam GAD resulted in 2.9% (21/726) resistant Hi. BMD did not confirm piperacillin/tazobactam resistance. Two strains were found resistant by AD, of which one was also resistant using piperacillin GAD on MH-F/tazobactam. Overall, we found two strains with a piperacillin/tazobactam MIC >0.25 mg/L in at least two different tests (0.3%). Both were β-lactamase-producing amoxicillin/clavulanate-resistant with PBP3 mutations characterized as group III-like+. Relevant PBP3 mutations occurred in six strains without phenotypic piperacillin/tazobactam resistance. These mutations suggest a reduced efficacy of β-lactam antibiotics in these isolates.
Conclusions
Piperacillin/tazobactam resistance prevalence in invasive Hi is low in Germany. Reduced susceptibility was correlated with PBP3 mutations, in particular with group III mutations.
The genomes of both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV) were first sequenced over 20 years ago. Similar to HCMV, the MCMV genome had initially been proposed to harbor ≈170 open reading frames (ORFs). More recently, omics approaches revealed HCMV gene expression to be substantially more complex comprising several hundred viral ORFs. Here, we provide a state-of-the art reannotation of lytic MCMV gene expression based on integrative analysis of a large set of omics data. Our data reveal 365 viral transcription start sites (TiSS) that give rise to 380 and 454 viral transcripts and ORFs, respectively. The latter include 200 small ORFs, some of which represented the most highly expressed viral gene products. By combining TiSS profiling with metabolic RNA labelling and chemical nucleotide conversion sequencing (dSLAM-seq), we provide a detailed picture of the expression kinetics of viral transcription. This not only resulted in the identification of a novel MCMV immediate early transcript encoding the m166.5 ORF, which we termed ie4, but also revealed a group of well-expressed viral transcripts that are induced later than canonical true late genes and contain an initiator element (Inr) but no TATA- or TATT-box in their core promoters. We show that viral upstream ORFs (uORFs) tune gene expression of longer viral ORFs expressed in cis at translational level. Finally, we identify a truncated isoform of the viral NK-cell immune evasin m145 arising from a viral TiSS downstream of the canonical m145 mRNA. Despite being ≈5-fold more abundantly expressed than the canonical m145 protein it was not required for downregulating the NK cell ligand, MULT-I. In summary, our work will pave the way for future mechanistic studies on previously unknown cytomegalovirus gene products in an important virus animal model.
Background
To cover soft tissue defects, the perforator-based propeller flap offers the option to rotate healthy tissue into complex wounds. By rotating the flap, the perforator is torqued. As a result, perfusion changes are possible.
Methods
A retrospective data analysis of patients was done, who received a propeller flap to cover soft tissue defects of the lower extremity as well as a peri- and postoperative perfusion monitoring with a laser-Doppler-spectrophotometry system. Additionally, patient-specific data were collected.
Results
Seven patients were identified. Four patients experienced early complications, two epidermolysis of the distal flap areas, three wound healing disorders, and one partial flap necrosis. Intraoperative perfusion monitoring showed a decline of blood flow after incision of the flap, especially at distal flap site. In case of complications, there were prolonged blood flow declines up to the first postoperative day.
Conclusion
Torqueing the perforator by rotating the flap can cause an impairment in inflow and outflow. If the impairment is prolonged, perfusion-associated complications are possible. The identification of a viable perforator is particularly important. In addition, a conservative postoperative mobilization is necessary to compensate for the impaired and adapting outflow.
Background
Healthcare workers and medical students faced new challenges during the COVID-19 pandemic. Processes within many hospitals were completely disrupted. In addition, the face to face teaching of medical students was drastically reduced. Those at risk of developing mental health problems appear to be younger health care workers and women.
Objective
To investigate potential COVID-19 pandemic-related gender differences in psychological distress among medical students and physicians in their first years of practice.
Design and setting
An anonymous survey was carried out online between December 1, 2021, and March 31, 2022, at the Mannheim Medical Faculty and the Würzburg Medical Faculty, Germany, after obtaining informed consent. Primary outcome measures were changes in anxiety and depression symptoms using the Hospital Anxiety and Depression Scale (HADS), and changes in participants' current quality of life using the WHO Quality of Life BREF.
Results
The results show wave-like courses for perceived anxiety and burden overlapping with the course of the COVID-19 incidence. In comparison to men, women showed a significant higher increase in HADS (p = 0.005) and a reduced life quality (p = 0.007) after COVID-19. Both sexes showed different frequencies of the factors influencing quality of life, with the presence of a previous mental illness and mean anxiety having a significant higher negative impact in women.
Conclusion
Future and young female physicians reported a disproportionate higher burden during COVID-19 compared to their male colleges. These observations suggest an increased need for support and prevention efforts especially in this vulnerable population.
Background
Guideline-directed medical therapy (GDMT) is the cornerstone in the treatment of patients with heart failure and reduced ejection fraction (HFrEF) and novel substances such as sacubitril/valsartan (S/V) and sodium-glucose co-transporter-2 inhibitors (SGLT2i) have demonstrated marked clinical benefits. We investigated their implementation into real-world HF care in Germany before, during, and after the COVID-19 pandemic period.
Methods
The IQVIA LRx data set is based on ∼80% of 73 million people covered by the German statutory health insurance. Prescriptions of S/V were used as a proxy for HFrEF. Time trends were analysed between Q1/2016 and Q2/2023 for prescriptions for S/V alone and in combination therapy with SGLT2i.
Findings
The number of patients treated with S/V increased from 5260 in Q1/2016 to 351,262 in Q2/2023. The share of patients with combination therapy grew from 0.6% (29 of 5260) to 14.2% (31,128 of 219,762) in Q2/2021, and then showed a steep surge up to 54.8% (192,429 of 351,262) in Q2/2023, coinciding with the release of the European Society of Cardiology (ESC) guidelines for HF in Q3/2021. Women and patients aged >80 years were treated less often with combined therapy than men and younger patients. With the start of the COVID-19 pandemic, the number of patients with new S/V prescriptions dropped by 17.5% within one quarter, i.e., from 26,855 in Q1/2020 to 22,145 in Q2/2020, and returned to pre-pandemic levels only in Q1/2021.
Interpretation
The COVID-19 pandemic was associated with a 12-month deceleration of S/V uptake in Germany. Following the release of the ESC HF guidelines, the combined prescription of S/V and SGLT2i was readily adopted. Further efforts are needed to fully implement GDMT and strengthen the resilience of healthcare systems during public health crises.
Electrochemical impedance spectroscopy (EIS) is a valuable technique analyzing electrochemical behavior of biological systems such as electrical characterization of cells and biomolecules, drug screening, and biomaterials in biomedical field. In EIS, an alternating current (AC) power signal is applied to the biological system, and the impedance of the system is measured over a range of frequencies.
In vitro culture models of endothelial or epithelial barrier tissue can be achieved by culturing barrier tissue on scaffolds made with synthetic or biological materials that provide separate compartments (apical and basal sides), allowing for further studies on drug transport. EIS is a great candidate for non-invasive and real-time monitoring of the electrical properties that correlate with barrier integrity during the tissue modeling. Although commercially available transendothelial/transepithelial electrical resistance (TEER) measurement devices are widely used, their use is particularly common in static transwell culture. EIS is considered more suitable than TEER measurement devices in bioreactor cultures that involve dynamic fluid flow to obtain accurate and reliable measurements. Furthermore, while TEER measurement devices can only assess resistance at a single frequency, EIS measurements can capture both resistance and capacitance properties of cells, providing additional information about the cellular barrier's characteristics across various frequencies. Incorporating EIS into a bioreactor system requires the careful optimization of electrode integration within the bioreactor setup and measurement parameters to ensure accurate EIS measurements. Since bioreactors vary in size and design depending on the purpose of the study, most studies have reported using an electrode system specifically designed for a particular bioreactor. The aim of this work was to produce multi-applicable electrodes and established methods for automated non-invasive and real-time monitoring using the EIS technique in bioreactor cultures. Key to the electrode material, titanium nitride (TiN) coating was fabricated on different substrates (materials and shape) using physical vapor deposition (PVD) and housed in a polydimethylsiloxane (PDMS) structure to allow the electrodes to function as independent units. Various electrode designs were evaluated for double-layer capacitance and morphology using EIS and scanning electron microscopy (SEM), respectively. The TiN-coated tube electrode was identified as the optimal choice. Furthermore, EIS measurements were performed to examine the impact of influential parameters related to culture conditions on the TiN-coated electrode system. In order to demonstrate the versatility of the electrodes, these electrodes were then integrated into in different types of perfusion bioreactors for monitoring barrier cells. Blood-brain barrier (BBB) cells were cultured in the newly developed dynamic flow bioreactor, while human umblical vascular endothelial cells (HUVECs) and Caco-2 cells were cultured in the miniature hollow fiber bioreactor (HFBR). As a result, the TiN-coated tube electrode system enabled investigation of BBB barrier integrity in long-term bioreactor culture. While EIS measurement could not detect HUVECs electrical properties in miniature HFBR culture, there was the possibility of measuring the barrier integrity of Caco-2 cells, indicating potential usefulness for evaluating their barrier function. Following the bioreactor cultures, the application of the TiN-coated tube electrode was expanded to hemofiltration, based on the hypothesis that the EIS system may be used to monitor clotting or clogging phenomena in hemofiltration. The findings suggest that the EIS monitoring system can track changes in ion concentration of blood before and after hemofiltration in real-time, which may serve as an indicator of clogging of filter membranes. Overall, our research demonstrates the potential of TiN-coated tube electrodes for sensitive and versatile non-invasive monitoring in bioreactor cultures and medical devices.
Barth Syndrome (BTHS) is an inherited X-chromosomal linked disorder, characterized by early development of cardiomyopathy, immune system defects, skeletal muscle myopathy and growth retardation. The disease displays a wide variety of symptoms including heart failure, exercise intolerance and fatigue due to the muscle weakness. The cause of the disease are mutations in the gene encoding for the mitochondrial transacylase Tafazzin (TAZ), which is important for remodeling of the phospholipid cardiolipin (CL). All mutations result in a pronounced decrease of the functional enzyme leading to an increase of monolysocardiolipin (MLCL), the precursor of mature CL, and a decrease in mature CL itself. CL is a hallmark phospholipid of mitochondrial membranes, highly enriched in the inner mitochondrial membrane (IMM). It is not only important for the formation of the cristae structures, but also for the function of different protein complexes associated with the mitochondrial membrane. Reduced levels of mature CL cause remodeling of the respiratory chain supercomplexes, impaired respiration, defects in the Krebs cycle and a loss of mitochondrial calcium uniporter (MCU) protein. The defective Ca2+ handling causes impaired redox homeostasis and energy metabolism resulting in cellular arrhythmias and defective electrical conduction. In an uncompensated situation, blunting mitochondrial Ca2+ uptake provokes increased mitochondrial emission of H2O2 during workload transitions, related to oxidation of NADPH, which is required to regenerate anti-oxidative enzymes. However, in the hearts and cardiac myocytes of mice with a global knock-down of the Taz gene (Taz-KD), no increase in mitochondrial ROS was observed, suggesting that other metabolic pathways may have compensated for reduced Krebs cycle activation.
The healthy heart produces most of its energy by consuming fatty acids. In this study, the fatty acid uptake into mitochondria and their further degradation was investigated, which showed a switch of the metabolism in general in the Taz-KD mouse model. In vivo studies revealed an increase of glucose uptake into the heart and decreased fatty acid uptake and oxidation. Disturbed energy conversion resulted in activation of retrograde signaling pathways, implicating overall changes in the cell metabolism. Upregulated integrated stress response (ISR) was confirmed by increased levels of the downstream target, i.e., the activating transcription factor 4 (ATF4). A Tafazzin knockout mouse embryonal fibroblast cell model (TazKO) was used to inhibit the ISR using siRNA transfection or pharmaceutical inhibition. This verified the central role of
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the ISR in regulating the metabolism in BTHS. Moreover, an increased metabolic flux into glutathione biosynthesis was observed, which supports redox homeostasis. In vivo PET-CT scans depicted elevated activity of the xCT system in the BTHS mouse heart, which transports essential amino acids for the biosynthesis of glutathione precursors. Furthermore, the stress induced signaling pathway also affected the glutamate metabolism, which fuels into the Krebs cycle via -ketoglutarate and therefore supports energy converting pathways. In summary, this thesis provides novel insights into the energy metabolism and redox homeostasis in Barth syndrome cardiomyopathy and its regulation by the integrated stress response, which plays a central role in the metabolic alterations. The aim of the thesis was to improve the understanding of these metabolic changes and to identify novel targets, which can provide new possibilities for therapeutic intervention in Barth syndrome.
Platelets play an important role in haemostasis by mediating blood clotting at sites of blood vessel damage. Platelets, also participate in pathological conditions including thrombosis and inflammation. Upon vessel damage, two glycoprotein receptors, the GPIb-IX-V complex and GPVI, play important roles in platelet capture and activation.
GPIb-IX-V binds to von Willebrand factor and GPVI to collagen. This initiates a signalling cascade resulting in platelet shape change and spreading, which is dependent on the actin cytoskeleton. This thesis aimed to develop and implement different super-resolution microscopy techniques to gain a deeper understanding of the conformation and location of these receptors in the platelet plasma membrane, and to provide insights into their signalling pathways. We suggest direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM) as the best candidates for imaging single platelets, whereas expansion microscopy (ExM) is ideal for imaging platelets aggregates.
Furthermore, we highlighted the role of the actin cytoskeleton, through Rac in GPVI signalling pathway. Inhibition of Rac, with EHT1864 in human platelets induced GPVI and GPV, but not GPIbα shedding. Furthermore, EHT1864 treatment did not change GPVI dimerisation or clustering, however, it decreased phospholipase Cγ2 phosphorylation levels, in human, but not murine platelets, highlighting interspecies differences. In summary, this PhD thesis demonstrates that; 1) Rac alters GPVI signalling pathway in human but not mouse platelets; 2) our newly developed ExM protocol can be used to image platelet aggregates labelled with F(ab’) fragments
Compared to other countries, China was particularly early in developing a comprehensive set of policies to promote electric mobility (e-mobility). The aim of this study is to examine how China’s e-mobility development – through changes in formal institutions as well as market forces – has affected German passenger car manufacturers and their competitive environment and positions. The study is guided by two strands of research: new institutional economics and strategic management literature. A holistic multiple-case design is used to analyze five German case study firms. Qualitative interview data are collected through interviews and analyzed using a thematic analysis. The results show that the electric transformation in China has been shaped by changes in formal institutions at the macro, meso, and micro levels. Interestingly, the case study firms were affected not only by changes in China’s formal institutions but also by disparities between institutions in China and Europe. Furthermore, the data suggest that German car manufacturers are facing an increasingly competitive environment in China: at least four forces in Porter’s five-forces model seem to have intensified in recent years. The extent to which the case study firms have been affected by these developments may depend on the industry segments in which they are positioned. However, it can be argued that the electric transition has blurred the lines between traditional segments of the car industry to some extent. The interview data do not provide evidence that any of the German car brands have substantially changed their positioning, but they do suggest that some of the case study companies did not have an adequate offering for the Chinese market at the time of the interviews. In addition, the study finds that China’s transition to e-mobility has led to changes in various parts of the German automakers’ value chains, including production, sales, marketing, services, research and development, and procurement. Whether these changes will ultimately result in competitive advantage, parity, or disadvantage remains to be seen.
We have investigated optical properties of hybrid two-dimensional-zero-dimensional (2D-0D) tunnel structures containing strongly elongated InAs/InP(001) quantum dots (called quantum dashes), emitting at 1.55 μm. These quantum dashes (QDashes) are separated by a 2.3 nm-width barrier from an InGaAs quantum well (QW), lattice matched to InP. We have tailored quantum-mechanical coupling between the states confined in QDashes and a QW by changing the QW thickness. By combining modulation spectroscopy and photoluminescence excitation, we have determined the energies of all relevant optical transitions in the system and proven the carrier transfer from the QW to the QDashes, which is the fundamental requirement for the tunnel injection scheme. A transformation between 0D and mixed-type 2D-0D character of an electron and a hole confinement in the ground state of the hybrid system have been probed by time-resolved photoluminescence that revealed considerable changes in PL decay time with the QW width changes. The experimental discoveries have been explained by band structure calculations in the framework of the eight-band k·p model showing that they are driven by delocalization of the lowest energy hole state. The hole delocalization process from the 0D QDash confinement is unfavorable for optical devices based on such tunnel injection structures.
While there is abounding literature on virus-induced pathology in general and coronavirus in particular, recent evidence accumulates showing distinct and deleterious brain affection. As the respiratory tract connects to the brain without protection of the blood–brain barrier, SARS-CoV-2 might in the early invasive phase attack the cardiorespiratory centres located in the medulla/pons areas, giving rise to disturbances of respiration and cardiac problems. Furthermore, brainstem regions are at risk to lose their functional integrity. Therefore, long-term neurological as well as psychiatric symptomatology and eventual respective disorders cannot be excluded as evidenced from influenza-A triggered post-encephalitic Parkinsonism and HIV-1 triggered AIDS–dementia complex. From the available evidences for coronavirus-induced brain pathology, this review concludes a number of unmet needs for further research strategies like human postmortem brain analyses. SARS-CoV-2 mirroring experimental animal brain studies, characterization of time-dependent and region-dependent spreading behaviours of coronaviruses, enlightening of pathological mechanisms after coronavirus infection using long-term animal models and clinical observations of patients having had COVID-19 infection are calling to develop both protective strategies and drug discoveries to avoid early and late coronavirus-induced functional brain disturbances, symptoms and eventually disorders. To fight SARS-CoV-2, it is an urgent need to enforce clinical, molecular biological, neurochemical and genetic research including brain-related studies on a worldwide harmonized basis.
Purpose
The purpose of this study is to investigate changes over time in quality of life (QoL) in incurable lung cancer patients and the impact of determinants like molecular alterations (MA).
Methods
In a prospective, longitudinal, multicentric study, we assessed QoL, symptom burden, psychological distress, unmet needs, and prognostic understanding of patients diagnosed with incurable lung cancer at the time of the diagnosis (T0) and after 3 (T1), 6 (T2) and 12 months (T3) using validated questionnaires like FACT-L, National Comprehensive Cancer Network (NCCN) Distress Thermometer (DT), PHQ-4, SCNS-SF-34, and SEIQoL.
Results
Two hundred seventeen patients were enrolled, 22 (10%) with reported MA. QoL scores improved over time, with a significant trend for DT, PHQ-4, and SCNS-SF-34. Significant determinants for stable or improving scores over time were survival > 6 months, performance status at the time of diagnosis, and presence of MA. Patients with MA showed better QoL scores (FACT-L at T1 104.4 vs 86.3; at T2 107.5 vs 90.0; at T3 100.9 vs 92.8) and lower psychological distress (NCCN DT at T1 3.3 vs 5; at T2 2.7 vs 4.5; at T3 3.7 vs 4.5; PHQ-4 at T1 2.3 vs 4.1; at T2 1.7 vs 3.6; at T3 2.2 vs 3.6), but also a worsening of the scores at 1 year and a higher percentage of inaccurate prognostic understanding (27 vs 17%) compared to patients without MA.
Conclusion
Patients with tumors harboring MA are at risk of QoL deterioration during the course of the disease. Physicians should adapt their communication strategies in order to maintain or improve QoL.
Background
The psychometric properties of an instrument, the Activity Scale for Kids-performance (ASKp), were assessed which was proposed to capture physical functioning after allogeneic hematopoietic stem cell transplantation (HSCT). Additionally, this multicenter observational prospective study investigated the influence of clinical correlates focusing on chronic graft-versus-host disease (cGVHD).
Methods
Patient-reported ASKp, clinician-reported Karnofsky/Lansky status (KPS/PSS), patient characteristics and cGVHD details were assessed of 55 patients with a median age of 12 years at baseline after day +100 post-HSCT and every 3 months during the next 18 months. The psychometric properties were evaluated and ASKp and KPS/PSS status was compared using ANOVAS and multiple regression models.
Results
The German version of the ASKp showed good psychometric properties except for ceiling effects. Discrimination ability of the ASKp was good regarding the need for devices but failed to predict cGVHD patients. Both the ASKp and the KPS/PSS were associated with patients after adoptive cell therapy being in need for devices, suffering from overlap cGVHD and from steroid side effects but not with patients’ age and gender. In contrast to the KPS/PSS the ASKp only showed significant differences after merging moderate and severe cGHVD patients when comparing them to No-cGVHD (F = 4.050; p = 0.049), being outperformed by the KPS/PSS (F = 20.082; p < 0.001).
Conclusion
The ASKp showed no clear advantages compared to KPS/PSS even though economical and patients’ effort was higher. Further application range may be limited through ceiling effects. Both should be taken into consideration. Therefore, the results may not support the usage of ASKp after HSCT and rather suggest KPS/PSS, both patient and clinician reported.
Key message
Mobile laser scanning and geometrical analysis revealed relationships between tree geometry and seed dispersal mechanism, latitude of origin, as well as growth.
Abstract
The structure and dynamics of a forest are defined by the architecture and growth patterns of its individual trees. In turn, tree architecture and growth result from the interplay between the genetic building plans and environmental factors. We set out to investigate whether (1) latitudinal adaptations of the crown shape occur due to characteristic solar elevation angles at a species’ origin, (2) architectural differences in trees are related to seed dispersal strategies, and (3) tree architecture relates to tree growth performance. We used mobile laser scanning (MLS) to scan 473 trees and generated three-dimensional data of each tree. Tree architectural complexity was then characterized by fractal analysis using the box-dimension approach along with a topological measure of the top heaviness of a tree. The tree species studied originated from various latitudinal ranges, but were grown in the same environmental settings in the arboretum. We found that trees originating from higher latitudes had significantly less top-heavy geometries than those from lower latitudes. Therefore, to a certain degree, the crown shape of tree species seems to be determined by their original habitat. We also found that tree species with wind-dispersed seeds had a higher structural complexity than those with animal-dispersed seeds (p < 0.001). Furthermore, tree architectural complexity was positively related to the growth performance of the trees (p < 0.001). We conclude that the use of 3D data from MLS in combination with geometrical analysis, including fractal analysis, is a promising tool to investigate tree architecture.
Background
Research in the field of surgery is mainly driven by aiming for trauma reduction as well as for personalized treatment concepts. Beyond laparoscopy, other proposed approaches for further reduction of the therapeutic trauma have failed to achieve clinical translation, with few notable exceptions. We believe that this is mainly due to a lack of flexibility and high associated costs. We aimed at addressing these issues by developing a novel minimally invasive operating platform and a preoperative design workflow for patient-individual adaptation and cost-effective rapid manufacturing of surgical manipulators. In this article, we report on the first in-vitro cholecystectomy performed with our operating platform.
Methods
The single-port overtube (SPOT) is a snake-like surgical manipulator for minimally invasive interventions. The system layout is highly flexible and can be adapted in design and dimensions for different kinds of surgery, based on patient- and disease-specific parameters. For collecting and analyzing this data, we developed a graphical user interface, which assists clinicians during the preoperative planning phase. Other major components of our operating platform include an instrument management system and a non-sterile user interface. For the trial surgery, we used a validated phantom which was further equipped with a porcine liver including the gallbladder.
Results
Following our envisioned preoperative design workflow, a suitable geometry of the surgical manipulator was determined for our trial surgery and rapidly manufactured by means of 3D printing. With this setup, we successfully performed a first in-vitro cholecystectomy, which was completed in 78 min.
Conclusions
By conducting the trial surgery, we demonstrated the effectiveness of our PLAFOKON operating platform. While some aspects – especially regarding usability and ergonomics – can be further optimized, the overall performance of the system is highly promising, with sufficient flexibility and strength for conducting the necessary tissue manipulations.
Climate change is increasing the frequency and intensity of warming and drought periods around the globe, currently representing a threat to many plant species. Understanding the resistance and resilience of plants to climate change is, therefore, urgently needed. As date palm (Phoenix dactylifera) evolved adaptation mechanisms to a xeric environment and can tolerate large diurnal and seasonal temperature fluctuations, we studied the protein expression changes in leaves, volatile organic compound emissions, and photosynthesis in response to variable growth temperatures and soil water deprivation. Plants were grown under controlled environmental conditions of simulated Saudi Arabian summer and winter climates challenged with drought stress. We show that date palm is able to counteract the harsh conditions of the Arabian Peninsula by adjusting the abundances of proteins related to the photosynthetic machinery, abiotic stress and secondary metabolism. Under summer climate and water deprivation, these adjustments included efficient protein expression response mediated by heat shock proteins and the antioxidant system to counteract reactive oxygen species formation. Proteins related to secondary metabolism were downregulated, except for the P. dactylifera isoprene synthase (PdIspS), which was strongly upregulated in response to summer climate and drought. This study reports, for the first time, the identification and functional characterization of the gene encoding for PdIspS, allowing future analysis of isoprene functions in date palm under extreme environments. Overall, the current study shows that reprogramming of the leaf protein profiles confers the date palm heat- and drought tolerance. We conclude that the protein plasticity of date palm is an important mechanism of molecular adaptation to environmental fluctuations.
The carbohydrate D-glucose is the main source of energy in living organisms. In contrast to animals, as well as most fungi, bacteria, and archaea, plants are capable to synthesize a surplus of sugars characterizing them as autothrophic organisms. Thus, plants are de facto the source of all food on earth, either directly or indirectly via feed to livestock. Glucose is stored as polymeric glucan, in animals as glycogen and in plants as starch. Despite serving a general source for metabolic energy and energy storage, glucose is the main building block for cellulose synthesis and represents the metabolic starting point of carboxylate- and amino acid synthesis. Finally yet importantly, glucose functions as signalling molecule conveying the plant metabolic status for adjustment of growth, development, and survival. Therefore, cell-to-cell and long-distance transport of photoassimilates/sugars throughout the plant body require the fine-tuned activity of sugar transporters facilitating the transport across membranes. The functional plant counterparts of the animal sodium/glucose transporters (SGLTs) are represented by the proton-coupled sugar transport proteins (STPs) of the plant monosaccharide transporter(-like) family (MST). In the framework of this special issue on “Glucose Transporters in Health and Disease,” this review gives an overview of the function and structure of plant STPs in comparison to the respective knowledge obtained with the animal Na+-coupled glucose transporters (SGLTs).
Purpose
Diaschisis of cerebrocerebellar loops contributes to cognitive and motor deficits in pediatric cerebellar brain tumor survivors. We used a cerebellar white matter atlas and hypothesized that lesion symptom mapping may reveal the critical lesions of cerebellar tracts.
Methods
We examined 31 long-term survivors of pediatric posterior fossa tumors (13 pilocytic astrocytoma, 18 medulloblastoma). Patients underwent neuronal imaging, examination for ataxia, fine motor and cognitive function, planning abilities, and executive function. Individual consolidated cerebellar lesions were drawn manually onto patients’ individual MRI and normalized into Montreal Neurologic Institute (MNI) space for further analysis with voxel-based lesion symptom mapping.
Results
Lesion symptom mapping linked deficits of motor function to the superior cerebellar peduncle (SCP), deep cerebellar nuclei (interposed nucleus (IN), fastigial nucleus (FN), ventromedial dentate nucleus (DN)), and inferior vermis (VIIIa, VIIIb, IX, X). Statistical maps of deficits of intelligence and executive function mapped with minor variations to the same cerebellar structures.
Conclusion
We identified lesions to the SCP next to deep cerebellar nuclei as critical for limiting both motor and cognitive function in pediatric cerebellar tumor survivors. Future strategies safeguarding motor and cognitive function will have to identify patients preoperatively at risk for damage to these critical structures and adapt multimodal therapeutic options accordingly.
We steered the soil microbiome via applications of organic residues (mix of cover crop residues, sewage sludge + compost, and digestate + compost) to enhance multiple ecosystem services in line with climate-smart agriculture. Our result highlights the potential to reduce greenhouse gases (GHG) emissions from agricultural soils by the application of specific organic amendments (especially digestate + compost). Unexpectedly, also the addition of mineral fertilizer in our mesocosms led to similar combined GHG emissions than one of the specific organic amendments. However, the application of organic amendments has the potential to increase soil C, which is not the case when using mineral fertilizer. While GHG emissions from cover crop residues were significantly higher compared to mineral fertilizer and the other organic amendments, crop growth was promoted. Furthermore, all organic amendments induced a shift in the diversity and abundances of key microbial groups. We show that organic amendments have the potential to not only lower GHG emissions by modifying the microbial community abundance and composition, but also favour crop growth-promoting microorganisms. This modulation of the microbial community by organic amendments bears the potential to turn soils into more climate-smart soils in comparison to the more conventional use of mineral fertilizers.
Purpose
To investigate the association of patients’ sex with recurrence and disease progression in patients treated with intravesical bacillus Calmette–Guérin (BCG) for T1G3/HG urinary bladder cancer (UBC).
Materials and methods
We analyzed the data of 2635 patients treated with adjuvant intravesical BCG for T1 UBC between 1984 and 2019. We accounted for missing data using multiple imputations and adjusted for covariate imbalance between males and females using inverse probability weighting (IPW). Crude and IPW-adjusted Cox regression analyses were used to estimate the hazard ratios (HR) with their 95% confidence intervals (CI) for the association of patients’ sex with HG-recurrence and disease progression.
Results
A total of 2170 (82%) males and 465 (18%) females were available for analysis. Overall, 1090 (50%) males and 244 (52%) females experienced recurrence, and 391 (18%) males and 104 (22%) females experienced disease progression. On IPW-adjusted Cox regression analyses, female sex was associated with disease progression (HR 1.25, 95%CI 1.01–1.56, p = 0.04) but not with recurrence (HR 1.06, 95%CI 0.92–1.22, p = 0.41). A total of 1056 patients were treated with adequate BCG. In these patients, on IPW-adjusted Cox regression analyses, patients’ sex was not associated with recurrence (HR 0.99, 95%CI 0.80–1.24, p = 0.96), HG-recurrence (HR 1.00, 95%CI 0.78–1.29, p = 0.99) or disease progression (HR 1.12, 95%CI 0.78–1.60, p = 0.55).
Conclusion
Our analysis generates the hypothesis of a differential response to BCG between males and females if not adequately treated. Further studies should focus on sex-based differences in innate and adaptive immune system and their association with BCG response.
Purpose
While [\(^{18}\)F]-fluorodeoxyglucose ([\(^{18}\)F]FDG) is the standard for positron emission tomography/computed tomography (PET/CT) imaging of oral squamous cell carcinoma (OSCC), diagnostic specificity is hampered by uptake in inflammatory cells such as neutrophils or macrophages. Recently, molecular imaging probes targeting fibroblast activation protein α (FAP), which is overexpressed in a variety of cancer-associated fibroblasts, have become available and might constitute a feasible alternative to FDG PET/CT.
Methods
Ten consecutive, treatment-naïve patients (8 males, 2 females; mean age, 62 ± 9 years) with biopsy-proven OSCC underwent both whole-body [\(^{18}\)F]FDG and [\(^{68}\)Ga]FAPI-04 (FAP-directed) PET/CT for primary staging prior to tumor resection and cervical lymph node dissection. Detection of the primary tumor, as well as the presence and number of lymph node and distant metastases was analysed. Intensity of tracer accumulation was assessed by means of maximum (SUV\(_{max}\)) and peak (SUV\(_{peak}\) standardized uptake values. Histological work-up including immunohistochemical staining for FAP served as standard of reference.
Results
[\(^{18}\)F]FDG and FAP-directed PET/CT detected all primary tumors with a SUVmax of 25.5 ± 13.2 (FDG) and 20.5 ± 6.4 (FAP-directed) and a SUVpeak of 16.1 ± 10.3 ([\(^{18}\)F]FDG) and 13.8 ± 3.9 (FAP-directed), respectively. Regarding cervical lymph node metastases, FAP-directed PET/CT demonstrated comparable sensitivity (81.3% vs. 87.5%; P = 0.32) and specificity (93.3% vs. 81.3%; P = 0.16) to [\(^{18}\)F]FDG PET/CT. FAP expression on the cell surface of cancer-associated fibroblasts in both primary lesions as well as lymph nodes metastases was confirmed in all samples.
Conclusion
FAP-directed PET/CT in OSCC seems feasible. Future research to investigate its potential to improve patient staging is highly warranted.
Scalability is often mentioned in literature, but a stringent definition is missing. In particular, there is no general scalability assessment which clearly indicates whether a system scales or not or whether a system scales better than another. The key contribution of this article is the definition of a scalability index (SI) which quantifies if a system scales in comparison to another system, a hypothetical system, e.g., linear system, or the theoretically optimal system. The suggested SI generalizes different metrics from literature, which are specialized cases of our SI. The primary target of our scalability framework is, however, benchmarking of two systems, which does not require any reference system. The SI is demonstrated and evaluated for different use cases, that are (1) the performance of an IoT load balancer depending on the system load, (2) the availability of a communication system depending on the size and structure of the network, (3) scalability comparison of different location selection mechanisms in fog computing with respect to delays and energy consumption; (4) comparison of time-sensitive networking (TSN) mechanisms in terms of efficiency and utilization. Finally, we discuss how to use and how not to use the SI and give recommendations and guidelines in practice. To the best of our knowledge, this is the first work which provides a general SI for the comparison and benchmarking of systems, which is the primary target of our scalability analysis.
Purpose
FAPI ligands (fibroblast activation protein inhibitor), a novel class of radiotracers for PET/CT imaging, demonstrated in previous studies rapid and high tumor uptake. The purpose of this study is the head-to-head intra-individual comparison of \(^{68}\)Ga-FAPI versus standard-of-care \(^{18}\)F-FDG in PET/CT in organ biodistribution and tumor uptake in patients with various cancers.
Material and Methods
This international retrospective multicenter analysis included PET/CT data from 71 patients from 6 centers who underwent both \(^{68}\)Ga-FAPI and \(^{18}\)F-FDG PET/CT within a median time interval of 10 days (range 1–89 days). Volumes of interest (VOIs) were manually drawn in normal organs and tumor lesions to quantify tracer uptake by SUVmax and SUVmean. Furthermore, tumor-to-background ratios (TBR) were generated (SUVmax tumor/ SUVmax organ).
Results
A total of 71 patients were studied of, which 28 were female and 43 male (median age 60). In 41 of 71 patients, the primary tumor was present. Forty-three of 71 patients exhibited 162 metastatic lesions. \(^{68}\)Ga-FAPI uptake in primary tumors and metastases was comparable to 18F-FDG in most cases. The SUVmax was significantly lower for \(^{68}\)Ga-FAPI than \(^{18}\)F-FDG in background tissues such as the brain, oral mucosa, myocardium, blood pool, liver, pancreas, and colon. Thus, \(^{68}\)Ga-FAPI TBRs were significantly higher than 18F-FDG TBRs in some sites, including liver and bone metastases.
Conclusion
Quantitative tumor uptake is comparable between \(^{68}\)Ga-FAPI and \(^{18}\)F-FDG, but lower background uptake in most normal organs results in equal or higher TBRs for \(^{68}\)Ga-FAPI. Thus, \(^{68}\)Ga-FAPI PET/CT may yield improved diagnostic information in various cancers and especially in tumor locations with high physiological \(^{18}\)F-FDG uptake.
Purpose
To evaluate whether a deep learning model (DLM) could increase the detection sensitivity of radiologists for intracranial aneurysms on CT angiography (CTA) in aneurysmal subarachnoid hemorrhage (aSAH).
Methods
Three different DLMs were trained on CTA datasets of 68 aSAH patients with 79 aneurysms with their outputs being combined applying ensemble learning (DLM-Ens). The DLM-Ens was evaluated on an independent test set of 104 aSAH patients with 126 aneuryms (mean volume 129.2 ± 185.4 mm3, 13.0% at the posterior circulation), which were determined by two radiologists and one neurosurgeon in consensus using CTA and digital subtraction angiography scans. CTA scans of the test set were then presented to three blinded radiologists (reader 1: 13, reader 2: 4, and reader 3: 3 years of experience in diagnostic neuroradiology), who assessed them individually for aneurysms. Detection sensitivities for aneurysms of the readers with and without the assistance of the DLM were compared.
Results
In the test set, the detection sensitivity of the DLM-Ens (85.7%) was comparable to the radiologists (reader 1: 91.2%, reader 2: 86.5%, and reader 3: 86.5%; Fleiss κ of 0.502). DLM-assistance significantly increased the detection sensitivity (reader 1: 97.6%, reader 2: 97.6%,and reader 3: 96.0%; overall P=.024; Fleiss κ of 0.878), especially for secondary aneurysms (88.2% of the additional aneurysms provided by the DLM).
Conclusion
Deep learning significantly improved the detection sensitivity of radiologists for aneurysms in aSAH, especially for secondary aneurysms. It therefore represents a valuable adjunct for physicians to establish an accurate diagnosis in order to optimize patient treatment.
Purpose
Tiapride is commonly used in Europe for the treatment of tics. The aim of this study was to examine the relationship between dose and serum concentrations of tiapride and potential influential pharmacokinetic factors in children and adolescents. In addition, a preliminary therapeutic reference range for children and adolescents with tics treated with tiapride was calculated.
Methods
Children and adolescents treated with tiapride at three university hospitals and two departments of child and adolescents psychiatry in Germany and Austria were included in the study. Patient characteristics, doses, serum concentrations, and therapeutic outcome were assessed during clinical routine care using standardised measures.
Results
In the 49 paediatric patients (83.7% male, mean age = 12.5 years), a positive correlation was found between tiapride dose (median 6.9 mg/kg, range 0.97–19.35) and serum concentration with marked inter-individual variability. The variation in dose explained 57% of the inter-patient variability in tiapride serum concentrations; age, gender, and concomitant medication did not contribute to the variability. The symptoms improved in 83.3% of the patients. 27.1% of the patients had mild or moderate ADRs. No patient suffered from severe ADRs.
Conclusions
This study shows that tiapride treatment was effective and safe in most patients with tics. Compared with the therapeutic concentration range established for adults with Chorea Huntington, our data hinted at a lower lower limit (560 ng/ml) and similar upper limit (2000 ng/ml).
The EU chemicals strategy for sustainability (CSS) asserts that both human health and the environment are presently threatened and that further regulation is necessary. In a recent Guest Editorial, members of the German competent authority for risk assessment, the BfR, raised concerns about the scientific justification for this strategy. The complexity and interdependence of the networks of regulation of chemical substances have ensured that public health and wellbeing in the EU have continuously improved. A continuous process of improvement in consumer protection is clearly desirable but any initiative directed towards this objective must be based on scientific knowledge. It must not confound risk with other factors in determining policy. This conclusion is fully supported in the present Commentary including the request to improve both, data collection and the time-consuming and bureaucratic procedures that delay the publication of regulations.
Since the addition of fluoride to drinking water in the 1940s, there have been frequent and sometimes heated discussions regarding its benefits and risks. In a recently published review, we addressed the question if current exposure levels in Europe represent a risk to human health. This review was discussed in an editorial asking why we did not calculate benchmark doses (BMD) of fluoride neurotoxicity for humans. Here, we address the question, why it is problematic to calculate BMDs based on the currently available data. Briefly, the conclusions of the available studies are not homogeneous, reporting negative as well as positive results; moreover, the positive studies lack control of confounding factors such as the influence of well-known neurotoxicants. We also discuss the limitations of several further epidemiological studies that did not meet the inclusion criteria of our review. Finally, it is important to not only focus on epidemiological studies. Rather, risk analysis should consider all available data, including epidemiological, animal, as well as in vitro studies. Despite remaining uncertainties, the totality of evidence does not support the notion that fluoride should be considered a human developmental neurotoxicant at current exposure levels in European countries.
The work proposes possible designs of active regions for a mode-locked interband cascade laser emitting in the mid infrared. For that purpose we investigated the electronic structure properties of respectively modified GaSb-based type II W-shaped quantum wells, including the effect of external bias in order to simultaneously fulfil the requirements for both the absorber as well as the gain sections of a device. The results show that introducing multiple InAs layers in type II InAs/GaInSb quantum wells or introducing a tensely-strained GaAsSb layer into “W-shaped” type II QWs offers significant difference in optical transitions’ oscillator strengths (characteristic lifetimes) of the two oppositely polarized parts of such a laser, being promising for utilization in mode-locked devices.
Healthy functioning of the female reproductive tract (FRT) depends on balanced and dynamic regulation by hormones during the menstrual cycle, pregnancy and childbirth. The mucosal epithelial lining of different regions of the FRT—ovaries, fallopian tubes, uterus, cervix and vagina—facilitates the selective transport of gametes and successful transfer of the zygote to the uterus where it implants and pregnancy takes place. It also prevents pathogen entry. Recent developments in three-dimensional (3D) organoid systems from the FRT now provide crucial experimental models that recapitulate the cellular heterogeneity and physiological, anatomical and functional properties of the organ in vitro. In this review, we summarise the state of the art on organoids generated from different regions of the FRT. We discuss the potential applications of these powerful in vitro models to study normal physiology, fertility, infections, diseases, drug discovery and personalised medicine.
Purpose
In Germany, Austria, and Switzerland, pretreatment radiotherapy quality control (RT-QC) for tumor bed boost (TB) in non-metastatic medulloblastoma (MB) was not mandatory but was recommended for patients enrolled in the SIOP PNET5 MB trial between 2014 and 2018. This individual case review (ICR) analysis aimed to evaluate types of deviations in the initial plan proposals and develop uniform review criteria for TB boost.
Patients and methods
A total of 78 patients were registered in this trial, of whom a subgroup of 65 patients were available for evaluation of the TB treatment plans. Dose uniformity was evaluated according to the definitions of the protocol. Additional RT-QC criteria for standardized review of target contours were elaborated and data evaluated accordingly.
Results
Of 65 initial TB plan proposals, 27 (41.5%) revealed deviations of target volume delineation. Deviations according to the dose uniformity criteria were present in 14 (21.5%) TB plans. In 25 (38.5%) cases a modification of the RT plan was recommended. Rejection of the TB plans was rather related to unacceptable target volume delineation than to insufficient dose uniformity.
Conclusion
In this analysis of pretreatment RT-QC, protocol deviations were present in a high proportion of initial TB plan proposals. These findings emphasize the importance of pretreatment RT-QC in clinical trials for MB. Based on these data, a proposal for RT-QC criteria for tumor bed boost in non-metastatic MB was developed.
Objective
The current article encompasses a literature review and recommendations for radiotherapy in nodal oligorecurrent prostate cancer.
Materials and methods
A literature review focused on studies comparing metastasis-directed stereotactic ablative radiotherapy (SABR) vs. external elective nodal radiotherapy (ENRT) and studies analyzing recurrence patterns after local nodal treatment was performed. The DEGRO Prostate Cancer Expert Panel discussed the results and developed treatment recommendations.
Results
Metastasis-directed radiotherapy results in high local control (often > 90% within a follow-up of 1–2 years) and can be used to improve progression-free survival or defer androgen deprivation therapy (ADT) according to prospective randomized phase II data. Distant progression after involved-node SABR only occurs within a few months in the majority of patients. ENRT improves metastases-free survival rates with increased toxicity in comparison to SABR according to retrospective comparative studies. The majority of nodal recurrences after initial local treatment of pelvic nodal metastasis are detected within the true pelvis and common iliac vessels.
Conclusion
ENRT with or without a boost should be preferred to SABR in pelvic nodal recurrences. In oligometastatic prostate cancer with distant (extrapelvic) nodal recurrences, SABR alone can be performed in selected cases. Application of additional systemic treatments should be based on current guidelines, with ADT as first-line treatment for hormone-sensitive prostate cancer. Only in carefully selected patients can radiotherapy be initially used without additional ADT outside of the current standard recommendations. Results of (randomized) prospective studies are needed for definitive recommendations.
The mason wasp Odynerus spinipes shows an exceptional case of intrasexual cuticular hydrocarbon (CHC) profile dimorphism. Females of this species display one of two CHC profiles (chemotypes) that differ qualitatively and quantitatively from each other. The ratio of the two chemotypes was previously shown to be close to 1:1 at three sites in Southern Germany, which might not be representative given the Palearctic distribution of the species. To infer the frequency of the two chemotypes across the entire distributional range of the species, we analyzed with GC–MS the CHC profile of 1042 dry-mounted specimens stored in private and museum collections. We complemented our sampling by including 324 samples collected and preserved specifically for studying their CHCs. We were capable of reliably identifying the chemotypes in 91% of dry-mounted samples, some of which collected almost 200 years ago. We found both chemotypes to occur in the Far East, the presumed glacial refuge of the species, and their frequency to differ considerably between sites and geographic regions. The geographic structure in the chemotype frequencies could be the result of differential selection regimes and/or different dispersal routes during the colonization of the Western Palearctic. The presented data pave the route for disentangling these factors by providing information where to geographically sample O. spinipes for population genetic analyses. They also form the much-needed basis for future studies aiming to understand the evolutionary and geographic origin as well as the genetics of the astounding CHC profile dimorphism that O. spinipes females exhibit.
Bacteriophage AR9 is a recently sequenced jumbo phage that encodes two multisubunit RNA polymerases. Here we investigated the AR9 transcription strategy and the effect of AR9 infection on the transcription of its host, Bacillus subtilis. Analysis of whole-genome transcription revealed early, late, and continuously expressed AR9 genes. Alignment of sequences upstream of the 5′ ends of AR9 transcripts revealed consensus sequences that define early and late phage promoters. Continuously expressed AR9 genes have both early and late promoters in front of them. Early AR9 transcription is independent of protein synthesis and must be determined by virion RNA polymerase injected together with viral DNA. During infection, the overall amount of host mRNAs is significantly decreased. Analysis of relative amounts of host transcripts revealed notable differences in the levels of some mRNAs. The physiological significance of up- or downregulation of host genes for AR9 phage infection remains to be established. AR9 infection is significantly affected by rifampin, an inhibitor of host RNA polymerase transcription. The effect is likely caused by the antibiotic-induced killing of host cells, while phage genome transcription is solely performed by viral RNA polymerases.
In recent history, normalized digital surface models (nDSMs) have been constantly gaining importance as a means to solve large-scale geographic problems. High-resolution surface models are precious, as they can provide detailed information for a specific area. However, measurements with a high resolution are time consuming and costly. Only a few approaches exist to create high-resolution nDSMs for extensive areas. This article explores approaches to extract high-resolution nDSMs from low-resolution Sentinel-2 data, allowing us to derive large-scale models. We thereby utilize the advantages of Sentinel 2 being open access, having global coverage, and providing steady updates through a high repetition rate. Several deep learning models are trained to overcome the gap in producing high-resolution surface maps from low-resolution input data. With U-Net as a base architecture, we extend the capabilities of our model by integrating tailored multiscale encoders with differently sized kernels in the convolution as well as conformed self-attention inside the skip connection gates. Using pixelwise regression, our U-Net base models can achieve a mean height error of approximately 2 m. Moreover, through our enhancements to the model architecture, we reduce the model error by more than 7%.
Background: Due to the importance of radiologic examinations, such as X-rays or computed tomography scans, for many clinical diagnoses, the optimal use of the radiology department is 1 of the primary goals of many hospitals.
Objective: This study aims to calculate the key metrics of this use by creating a radiology data warehouse solution, where data from radiology information systems (RISs) can be imported and then queried using a query language as well as a graphical user interface (GUI).
Methods: Using a simple configuration file, the developed system allowed for the processing of radiology data exported from any kind of RIS into a Microsoft Excel, comma-separated value (CSV), or JavaScript Object Notation (JSON) file. These data were then imported into a clinical data warehouse. Additional values based on the radiology data were calculated during this import process by implementing 1 of several provided interfaces. Afterward, the query language and GUI of the data warehouse were used to configure and calculate reports on these data. For the most common types of requested reports, a web interface was created to view their numbers as graphics.
Results: The tool was successfully tested with the data of 4 different German hospitals from 2018 to 2021, with a total of 1,436,111 examinations. The user feedback was good, since all their queries could be answered if the available data were sufficient. The initial processing of the radiology data for using them with the clinical data warehouse took (depending on the amount of data provided by each hospital) between 7 minutes and 1 hour 11 minutes. Calculating 3 reports of different complexities on the data of each hospital was possible in 1-3 seconds for reports with up to 200 individual calculations and in up to 1.5 minutes for reports with up to 8200 individual calculations.
Conclusions: A system was developed with the main advantage of being generic concerning the export of different RISs as well as concerning the configuration of queries for various reports. The queries could be configured easily using the GUI of the data warehouse, and their results could be exported into the standard formats Excel and CSV for further processing.
Group-based communication is a highly popular communication paradigm, which is especially prominent in mobile instant messaging (MIM) applications, such as WhatsApp. Chat groups in MIM applications facilitate the sharing of various types of messages (e.g., text, voice, image, video) among a large number of participants. As each message has to be transmitted to every other member of the group, which multiplies the traffic, this has a massive impact on the underlying communication networks. However, most chat groups are private and network operators cannot obtain deep insights into MIM communication via network measurements due to end-to-end encryption. Thus, the generation of traffic is not well understood, given that it depends on sizes of communication groups, speed of communication, and exchanged message types. In this work, we provide a huge data set of 5,956 private WhatsApp chat histories, which contains over 76 million messages from more than 117,000 users. We describe and model the properties of chat groups and users, and the communication within these chat groups, which gives unprecedented insights into private MIM communication. In addition, we conduct exemplary measurements for the most popular message types, which empower the provided models to estimate the traffic over time in a chat group.
ABSTRACT
The highly conserved heterotrimeric protein kinase SNF1 is important for metabolic adaptations in the pathogenic yeast Candida albicans. A key function of SNF1 is to inactivate the repressor protein Mig1 and thereby allow the expression of genes that are required for the utilization of alternative carbon sources when the preferred carbon source, glucose, is absent or becomes limiting. However, how SNF1 controls Mig1 activity in C. albicans has remained elusive. Using a phosphoproteomics approach, we found that Mig1 is phosphorylated at multiple serine residues. Replacement of these serine residues by nonphosphorylatable alanine residues strongly increased the repressor activity of Mig1 in cells lacking a functional SNF1 complex, indicating that additional protein kinases are involved in the regulation of Mig1. Unlike wild-type Mig1, whose levels strongly decreased when the cells were grown on sucrose or glycerol instead of glucose, the levels of a mutant Mig1 protein lacking nine phosphorylation sites remained high under these conditions. Despite the increased protein levels and the absence of multiple phosphorylation sites, cells with a functional SNF1 complex could still sufficiently inhibit the hyperactive Mig1 to enable wild-type growth on alternative carbon sources. In line with this, phosphorylated forms of the mutant Mig1 were still detected in the presence and absence of a functional SNF1, demonstrating that Mig1 contains additional, unidentified phosphorylation sites and that downstream protein kinases are involved in the control of Mig1 activity by SNF1.
IMPORTANCE
The SNF1 protein kinase signaling pathway, which is highly conserved in eukaryotic cells, is important for metabolic adaptations in the pathogenic yeast Candida albicans. However, so far, it has remained elusive how SNF1 controls the activity of one of its main effectors, the repressor protein Mig1 that inhibits the expression of genes required for the utilization of alternative carbon sources when glucose is available. In this study, we have identified multiple phosphorylation sites in Mig1 that contribute to its inactivation. Mutation of these sites strongly increased Mig1 repressor activity in the absence of SNF1, but SNF1 could still sufficiently inhibit the hyperactive Mig1 to enable growth on alternative carbon sources. These findings reveal features of Mig1 that are important for controlling its repressor activity. Furthermore, they demonstrate that both SNF1 and additional protein kinases regulate Mig1 in this pathogenic yeast.
Abstract
Protein kinases are central components of almost all signaling pathways that control cellular activities. In the model organism Saccharomyces cerevisiae, the paralogous protein kinases Ypk1 and Ypk2, which control membrane lipid homeostasis, are essential for viability, and previous studies strongly indicated that this is also the case for their single ortholog Ypk1 in the pathogenic yeast Candida albicans. Here, using FLP-mediated inducible gene deletion, we reveal that C. albicans ypk1Δ mutants are viable but slow-growing, explaining prior failures to obtain null mutants. Phenotypic analyses of the mutants showed that the functions of Ypk1 in regulating sphingolipid biosynthesis and cell membrane lipid asymmetry are conserved, but the consequences of YPK1 deletion are milder than in S. cerevisiae. Mutational studies demonstrated that the highly conserved PDK1 phosphorylation site T548 in its activation loop is essential for Ypk1 function, whereas the TORC2 phosphorylation sites S687 and T705 at the C-terminus are important for Ypk1-dependent resistance to membrane stress. Unexpectedly, Pkh1, the single C. albicans orthologue of Pkh1/Pkh2, which mediate Ypk1 phosphorylation at the PDK1 site in S. cerevisiae, was not required for normal growth of C. albicans under nonstressed conditions, and Ypk1 phosphorylation at T548 was only slightly reduced in pkh1Δ mutants. We found that another protein kinase, Pkh3, whose ortholog in S. cerevisiae cannot substitute Pkh1/2, acts redundantly with Pkh1 to activate Ypk1 in C. albicans. No phenotypic effects were observed in cells lacking Pkh3 alone, but pkh1Δ pkh3Δ double mutants had a severe growth defect and Ypk1 phosphorylation at T548 was completely abolished. These results establish that Ypk1 is not essential for viability in C. albicans and that, despite its generally conserved function, the Ypk1 signaling pathway is rewired in this pathogenic yeast and includes a novel upstream kinase to activate Ypk1 by phosphorylation at the PDK1 site.
Author summary
Protein kinases are key components of cellular signaling pathways, and elucidating the specific roles of individual kinases is important to understand how organisms adapt to changes in their environment. The protein kinase Ypk1 is highly conserved in eukaryotic organisms and crucial for the maintenance of cell membrane homeostasis. It was previously thought that Ypk1 is essential for viability in the pathogenic yeast Candida albicans, as in the model organism Saccharomyces cerevisiae. Here, by using forced, inducible gene deletion, we reveal that C. albicans mutants lacking Ypk1 are viable but have a strong growth defect. The phenotypes of the mutants indicate that the known functions of Ypk1 are conserved in C. albicans, but loss of this kinase has less severe consequences than in S. cerevisiae. We also unravel the puzzling previous observation that C. albicans mutants lacking the Ypk1-activating kinase Pkh1, which is essential in S. cerevisiae, have no obvious growth defects. We show that the protein kinase Pkh3, which has not previously been implicated in the Ypk1 signaling pathway, can substitute Pkh1 and activate Ypk1 in C. albicans. These findings provide novel insights into this conserved signaling pathway and how it is rewired in a human-pathogenic fungus.
Bulk RNA sequencing technologies have provided invaluable insights into host and bacterial gene expression and associated regulatory networks. Nevertheless, the majority of these approaches report average expression across cell populations, hiding the true underlying expression patterns that are often heterogeneous in nature. Due to technical advances, single-cell transcriptomics in bacteria has recently become reality, allowing exploration of these heterogeneous populations, which are often the result of environmental changes and stressors. In this work, we have improved our previously published bacterial single-cell RNA sequencing (scRNA-seq) protocol that is based on multiple annealing and deoxycytidine (dC) tailing-based quantitative scRNA-seq (MATQ-seq), achieving a higher throughput through the integration of automation. We also selected a more efficient reverse transcriptase, which led to reduced cell loss and higher workflow robustness. Moreover, we successfully implemented a Cas9-based rRNA depletion protocol into the MATQ-seq workflow. Applying our improved protocol on a large set of single Salmonella cells sampled over different growth conditions revealed improved gene coverage and a higher gene detection limit compared to our original protocol and allowed us to detect the expression of small regulatory RNAs, such as GcvB or CsrB at a single-cell level. In addition, we confirmed previously described phenotypic heterogeneity in Salmonella in regard to expression of pathogenicity-associated genes. Overall, the low percentage of cell loss and high gene detection limit makes the improved MATQ-seq protocol particularly well suited for studies with limited input material, such as analysis of small bacterial populations in host niches or intracellular bacteria.
IMPORTANCE: Gene expression heterogeneity among isogenic bacteria is linked to clinically relevant scenarios, like biofilm formation and antibiotic tolerance. The recent development of bacterial single-cell RNA sequencing (scRNA-seq) enables the study of cell-to-cell variability in bacterial populations and the mechanisms underlying these phenomena. Here, we report a scRNA-seq workflow based on MATQ-seq with increased robustness, reduced cell loss, and improved transcript capture rate and gene coverage. Use of a more efficient reverse transcriptase and the integration of an rRNA depletion step, which can be adapted to other bacterial single-cell workflows, was instrumental for these improvements. Applying the protocol to the foodborne pathogen Salmonella, we confirmed transcriptional heterogeneity across and within different growth phases and demonstrated that our workflow captures small regulatory RNAs at a single-cell level. Due to low cell loss and high transcript capture rates, this protocol is uniquely suited for experimental settings in which the starting material is limited, such as infected tissues.
The development of retrogressive thaw slumps (RTS) is known to be strongly influenced by relief-related parameters, permafrost characteristics, and climatic triggers. To deepen the understanding of RTS, this study examines the subsurface characteristics in the vicinity of an active thaw slump, located in the Richardson Mountains (Western Canadian Arctic). The investigations aim to identify relationships between the spatiotemporal slump development and the influence of subsurface structures. Information on these were gained by means of electrical resistivity tomography (ERT) and ground-penetrating radar (GPR). The spatiotemporal development of the slump was revealed by high-resolution satellite imagery and unmanned aerial vehicle–based digital elevation models (DEMs). The analysis indicated an acceleration of slump expansion, especially since 2018. The comparison of the DEMs enabled the detailed balancing of erosion and accumulation within the slump area between August 2018 and August 2019. In addition, manual frost probing and GPR revealed a strong relationship between the active layer thickness, surface morphology, and hydrology. Detected furrows in permafrost table topography seem to affect the active layer hydrology and cause a canalization of runoff toward the slump. The three-dimensional ERT data revealed a partly unfrozen layer underlying a heterogeneous permafrost body. This may influence the local hydrology and affect the development of the RTS. The results highlight the complex relationships between slump development, subsurface structure, and hydrology and indicate a distinct research need for other RTSs.
Early-onset torsion dystonia (DYT-TOR1A, DYT1) is an inherited hyperkinetic movement disorder caused by a mutation of the TOR1A gene encoding the torsinA protein. DYT-TOR1A is characterized as a network disorder of the central nervous system (CNS), including predominantly the cortico-basal ganglia-thalamo-cortical loop resulting in a severe generalized dystonic phenotype. The pathophysiology of DYTTOR1A is not fully understood. Molecular levels up to large-scale network levels of the CNS are suggested to be affected in the pathophysiology of DYT-TOR1A. The reduced penetrance of 30% - 40% indicates a gene-environmental interaction, hypothesized as “second hit”. The lack of appropriate and phenotypic DYT-TOR1A animal models encouraged us to verify the “second hit” hypothesis through a unilateral peripheral nerve trauma of the sciatic nerve in a transgenic asymptomatic DYT-TOR1A rat model (∆ETorA), overexpressing the human mutated torsinA protein. In a multiscale approach, this animal model was characterized phenotypically and pathophysiologically.
Nerve-injured ∆ETorA rats revealed dystonia-like movements (DLM) with a partially generalized phenotype. A physiomarker of human dystonia, describing increased theta oscillation in the globus pallidus internus (GPi), was found in the entopeduncular nucleus (EP), the rodent equivalent to the human GPi, of nerve-injured ∆ETorA rats. Altered oscillation patterns were also observed in the primary motor cortex. Highfrequency stimulation (HFS) of the EP reduced DLM and modulated altered oscillatory activity in the EP and primary motor cortex in nerve-injured ∆ETorA rats. Moreover, the dopaminergic system in ∆ETorA rats demonstrated a significant increased striatal dopamine release and dopamine turnover. Whole transcriptome analysis revealed differentially expressed genes of the circadian clock and the energy metabolism, thereby pointing towards novel, putative pathways in the pathophysiology of DYTTOR1A dystonia.
In summary, peripheral nerve trauma can trigger DLM in genetically predisposed asymptomatic ΔETorA rats leading to neurobiological alteration in the central motor network on multiple levels and thereby supporting the “second hit” hypothesis. This novel symptomatic DYT-TOR1A rat model, based on a DYT-TOR1A genetic background, may prove as a valuable chance for DYT-TOR1A dystonia, to further investigate the pathomechanism in more detail and to establish new treatment strategies.
Young grapevines (Vitis vinifera) suffer and eventually can die from the crown gall disease caused by the plant pathogen Allorhizobium vitis (Rhizobiaceae). Virulent members of A. vitis harbor a tumor-inducing plasmid and induce formation of crown galls due to the oncogenes encoded on the transfer DNA. The expression of oncogenes in transformed host cells induces unregulated cell proliferation and metabolic and physiological changes. The crown gall produces opines uncommon to plants, which provide an important nutrient source for A. vitis harboring opine catabolism enzymes. Crown galls host a distinct bacterial community, and the mechanisms establishing a crown gall–specific bacterial community are currently unknown. Thus, we were interested in whether genes homologous to those of the tumor-inducing plasmid coexist in the genomes of the microbial species coexisting in crown galls. We isolated 8 bacterial strains from grapevine crown galls, sequenced their genomes, and tested their virulence and opine utilization ability in bioassays. In addition, the 8 genome sequences were compared with 34 published bacterial genomes, including closely related plant-associated bacteria not from crown galls. Homologous genes for virulence and opine anabolism were only present in the virulent Rhizobiaceae. In contrast, homologs of the opine catabolism genes were present in all strains including the nonvirulent members of the Rhizobiaceae and non-Rhizobiaceae. Gene neighborhood and sequence identity of the opine degradation cluster of virulent and nonvirulent strains together with the results of the opine utilization assay support the important role of opine utilization for cocolonization in crown galls, thereby shaping the crown gall community.
Abstract
Cell lineage decisions occur in three-dimensional spatial patterns that are difficult to identify by eye. There is an ongoing effort to replicate such patterns using mathematical modeling. One approach uses long ranging cell-cell communication to replicate common spatial arrangements like checkerboard and engulfing patterns. In this model, the cell-cell communication has been implemented as a signal that disperses throughout the tissue. On the other hand, machine learning models have been developed for pattern recognition and pattern reconstruction tasks. We combined synthetic data generated by the mathematical model with spatial summary statistics and deep learning algorithms to recognize and reconstruct cell fate patterns in organoids of mouse embryonic stem cells. Application of Moran’s index and pair correlation functions for in vitro and synthetic data from the model showed local clustering and radial segregation. To assess the patterns as a whole, a graph neural network was developed and trained on synthetic data from the model. Application to in vitro data predicted a low signal dispersion value. To test this result, we implemented a multilayer perceptron for the prediction of a given cell fate based on the fates of the neighboring cells. The results show a 70% accuracy of cell fate imputation based on the nine nearest neighbors of a cell. Overall, our approach combines deep learning with mathematical modeling to link cell fate patterns with potential underlying mechanisms.
Author summary
Mammalian embryo development relies on organized differentiation of stem cells into different lineages. Particularly at the early stages of embryogenesis, cells of different fates form three-dimensional spatial patterns that are difficult to identify by eye. Pattern quantification and mathematical modeling have produced first insights into potential mechanisms for the cell fate arrangements. However, these approaches have relied on classifications of the patterns such as inside-out or random, or used summary statistics such as pair correlation functions or cluster radii. Deep neural networks allow characterizing patterns directly. Since the tissue context can be readily reproduced by a graph, we implemented a graph neural network to characterize the patterns of embryonic stem cell organoids as a whole. In addition, we implemented a multilayer perceptron model to reconstruct the fate of a given cell based on its neighbors. To train and test the models, we used synthetic data generated by our mathematical model for cell-cell communication. This interplay of deep learning and mathematical modeling in combination with summary statistics allowed us to identify a potential mechanism for cell fate determination in mouse embryonic stem cells. Our results agree with a mechanism with a dispersion of the intercellular signal that links a cell’s fate to those of the local neighborhood.
The ANTARES neutrino telescope has an energy threshold of a few tens of GeV. This allows to study the phenomenon of atmospheric muon neutrino disappearance due to neutrino oscillations. In a similar way, constraints on the 3+1 neutrino model, which foresees the existence of one sterile neutrino, can be inferred. Using data collected by the ANTARES neutrino telescope from 2007 to 2016, a new measurement of m 2 and (23) has been performed which is consistent with world best-fit values and constraints on the 3+1 neutrino model have been derived.
Background
Neoadjuvant chemotherapy (NACT) for early breast cancer can make breast-conserving surgery more feasible and might be more likely to eradicate micrometastatic disease than might the same chemotherapy given after surgery. We investigated the long-term benefits and risks of NACT and the influence of tumour characteristics on outcome with a collaborative meta-analysis of individual patient data from relevant randomised trials.
Methods
We obtained information about prerandomisation tumour characteristics, clinical tumour response, surgery, recurrence, and mortality for 4756 women in ten randomised trials in early breast cancer that began before 2005 and compared NACT with the same chemotherapy given postoperatively. Primary outcomes were tumour response, extent of local therapy, local and distant recurrence, breast cancer death, and overall mortality. Analyses by intention-to-treat used standard regression (for response and frequency of breast-conserving therapy) and log-rank methods (for recurrence and mortality).
Findings
Patients entered the trials from 1983 to 2002 and median follow-up was 9 years (IQR 5-14), with the last follow-up in 2013. Most chemotherapy was anthracycline based (3838 [81%] of 4756 women). More than two thirds (1349 [69%] of 1947) of women allocated NACT had a complete or partial clinical response. Patients allocated NACT had an increased frequency of breast-conserving therapy (1504 [65%] of 2320 treated with NACT vs 1135 [49%] of 2318 treated with adjuvant chemotherapy). NACT was associated with more frequent local recurrence than was adjuvant chemotherapy: the 15 year local recurrence was 21.4% for NACT versus 15.9% for adjuvant chemotherapy (5.5% increase [95% CI 2.4-8.6]; rate ratio 1.37 [95% CI 1.17-1.61]; p = 0.0001). No significant difference between NACT and adjuvant chemotherapy was noted for distant recurrence (15 year risk 38.2% for NACT vs 38.0% for adjuvant chemotherapy; rate ratio 1.02 [95% CI 0.92-1.14]; p = 0.66), breast cancer mortality (34.4% vs 33.7%; 1.06 [0.95-1.18]; p = 0.31), or death from any cause (40.9% vs 41.2%; 1.04 [0.94-1.15]; p = 0.45).
Interpretation
Tumours downsized by NACT might have higher local recurrence after breast-conserving therapy than might tumours of the same dimensions in women who have not received NACT. Strategies to mitigate the increased local recurrence after breast-conserving therapy in tumours downsized by NACT should be considered-eg, careful tumour localisation, detailed pathological assessment, and appropriate radiotherapy. Copyright (c) The Author(s). Published by Elsevier Ltd.
We consider the process of muon-electron elastic scattering, which has been proposed as an ideal framework to measure the running of the electromagnetic coupling constant at space-like momenta and determine the leading-order hadronic contribution to the muon g-2 (MUonE experiment). We compute the next-to-leading (NLO) contributions due to QED and purely weak corrections and implement them into a fully differential Monte Carlo event generator, which is available for first experimental studies. We show representative phenomenological results of interest for the MUonE experiment and examine in detail the impact of the various sources of radiative corrections under different selection criteria, in order to study the dependence of the NLO contributions on the applied cuts. The study represents the first step towards the realisation of a high-precision Monte Carlo code necessary for data analysis.
The Best for the Most Important: Maintaining a Pristine Proteome in Stem and Progenitor Cells
(2019)
Pluripotent stem cells give rise to reproductively enabled offsprings by generating progressively lineage-restricted multipotent stem cells that would differentiate into lineage-committed stem and progenitor cells. These lineage-committed stem and progenitor cells give rise to all adult tissues and organs. Adult stem and progenitor cells are generated as part of the developmental program and play critical roles in tissue and organ maintenance and/or regeneration. The ability of pluripotent stem cells to self-renew, maintain pluripotency, and differentiate into a multicellular organism is highly dependent on sensing and integrating extracellular and extraorganismal cues. Proteins perform and integrate almost all cellular functions including signal transduction, regulation of gene expression, metabolism, and cell division and death. Therefore, maintenance of an appropriate mix of correctly folded proteins, a pristine proteome, is essential for proper stem cell function. The stem cells' proteome must be pristine because unfolded, misfolded, or otherwise damaged proteins would interfere with unlimited self-renewal, maintenance of pluripotency, differentiation into downstream lineages, and consequently with the development of properly functioning tissue and organs. Understanding how various stem cells generate and maintain a pristine proteome is therefore essential for exploiting their potential in regenerative medicine and possibly for the discovery of novel approaches for maintaining, propagating, and differentiating pluripotent, multipotent, and adult stem cells as well as induced pluripotent stem cells. In this review, we will summarize cellular networks used by various stem cells for generation and maintenance of a pristine proteome. We will also explore the coordination of these networks with one another and their integration with the gene regulatory and signaling networks.
Summary
Embryos develop in a concerted sequence of spatiotemporal arrangements of cells. In the preimplantation mouse embryo, the distribution of the cells in the inner cell mass evolves from a salt-and-pepper pattern to spatial segregation of two distinct cell types. The exact properties of the salt-and-pepper pattern have not been analyzed so far. We investigate the spatiotemporal distribution of NANOG- and GATA6-expressing cells in the ICM of the mouse blastocysts with quantitative three-dimensional single-cell-based neighborhood analyses. A combination of spatial statistics and agent-based modeling reveals that the cell fate distribution follows a local clustering pattern. Using ordinary differential equations modeling, we show that this pattern can be established by a distance-based signaling mechanism enabling cells to integrate information from the whole inner cell mass into their cell fate decision. Our work highlights the importance of longer-range signaling to ensure coordinated decisions in groups of cells to successfully build embryos.
Highlights
• The local cell neighborhood and global ICM population composition correlate
• ICM cells show characteristics of local clustering in early and mid mouse blastocysts
• ICM patterning requires integration of signals from cells beyond the first neighbors
Background:
Cancer patients often suffer from psychological symptoms and need psychological support. Especially during the COVID-19 pandemic, eHealth interventions might be helpful to overcome the obstacles of the pandemic. This study evaluates the effectiveness of a video sequence-based eHealth intervention on anxiety, fatigue, and depression in cancer patients.
Methods:
Patients (N = 157) with different tumor entities were randomly assigned to the video intervention group (IG) and the waiting control group (CG). Patients in the IG received a video intervention comprising 8 video sequences over 4 weeks. The videos included psychoeducation on distress and psychological symptoms, Acceptance and Commitment Therapy elements, and Yoga and Qigong exercises. Patients’ anxiety and fear of progression (primary outcomes) and secondary outcomes were assessed before randomization (T1) and after the end of the intervention for IG or the waiting period for CG (T2) using self-reported questionnaires (GAD-7, PA-F-KF, EORTC QLQ-FA12, PHQ-8).
Results:
Patients of the IG showed no significant improvement in anxiety (GAD-7; P = .75), fear of progression (FoP-Q-SF; P = .29), fatigue (EORTC QLQ-FA12; P = .72), and depression (PHQ-8; P = .95) compared to patients in the waiting CG. However, symptoms of anxiety, fatigue, and depression decreased in both groups. Exploratory subgroup analysis regarding sex, therapy status, therapy goal, and tumor entity showed no effects. Overall, the intervention had a high level of acceptance.
Conclusions:
The video intervention was ineffective in reducing the psychological burden compared to a waiting CG. The findings support prior observations of the value of therapeutic guidance and promoting self-management for improving patients’ psychological burdens. Further studies are required to evaluate the effectiveness of psycho-oncological eHealth delivered through video sequences.
RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54\(^{nrb}\) marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA–RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair.
The transcription factor SPT5 physically interacts with MYC oncoproteins and is essential for efficient transcriptional activation of MYC targets in cultured cells. Here, we use Drosophila to address the relevance of this interaction in a living organism. Spt5 displays moderate synergy with Myc in fast proliferating young imaginal disc cells. During later development, Spt5-knockdown has no detectable consequences on its own, but strongly enhances eye defects caused by Myc overexpression. Similarly, Spt5-knockdown in larval type 2 neuroblasts has only mild effects on brain development and survival of control flies, but dramatically shrinks the volumes of experimentally induced neuroblast tumors and significantly extends the lifespan of tumor-bearing animals. This beneficial effect is still observed when Spt5 is knocked down systemically and after tumor initiation, highlighting SPT5 as a potential drug target in human oncology.
Chapter I: Introduction
Temperature is a major driver of biodiversity and abundance patterns on our planet, which becomes particularly relevant facing the entanglement of an imminent biodiversity and climate crisis. Climate shapes the composition of species assemblages either directly via abiotic filtering mechanisms or indirectly through alterations in biotic interactions. Insects - integral elements of Earth’s ecosystems - are affected by climatic variation such as warming, yet responses vary among species. While species’ traits, antagonistic biotic interactions, and even species’ microbial mutualists may determine temperature-dependent assembly processes, the lion’s share of these complex relationships remains poorly understood due to methodological constraints. Mountains, recognized as hotspots of diversity and threatened by rapidly changing climatic conditions, can serve as natural experimental settings to study the response of insect assemblages and their trophic interactions to temperature variation, instrumentalizing the high regional heterogeneity of micro- and macroclimate. With this thesis, we aim to enhance our mechanistic understanding of temperature-driven assembly processes within insect communities, exemplified by Orthoptera, that are significant herbivores in temperate mountain grassland ecosystems. Therefore, we combined field surveys of Orthoptera assemblages on grassland sites with molecular tools for foodweb reconstruction, primarily leveraging the elevational gradients offered by the complex topography within the Berchtesgaden Alpine region (Bavaria, Germany) as surrogate for temperature variation (space-for-time substitution approach). In this framework, we studied the effects of temperature variation on (1) species richness, abundance, community composition, and interspecific as well as intraspecific trait patterns, (2) ecological feeding specialisation, and (3) previously neglected links to microbial associates found in the faeces.
Chapter II: Temperature-driven assembly processes
Climate varies at multiple scales. Since microclimate is often overlooked, we assessed effects of local temperature deviations on species and trait compositions of insect communities along macroclimatic temperature gradients in Chapter II. Therefore, we employed joint species distribution modelling to explore how traits drive variation in the climatic niches of Orthoptera species at grassland sites characterized by contrasting micro- and macroclimatic conditions. Our findings revealed two key insights: (1) additive effects of micro- and macroclimate on the diversity, but (2) interactive effects on the abundance of several species, resulting in turnover and indicating that species possess narrower climatic niches than their elevational distributions might imply. This chapter suggests positive effects of warming on Orthoptera, but also highlights that the interplay of macro- and microclimate plays a pivotal role in structuring insect communities. Thus, it underscores the importance of considering both elements when predicting the responses of species to climate change. Additionally, this chapter revealed inter- and intraspecific effects of traits on the niches and distribution of species.
Chapter III: Dietary specialisation along climatic gradients
A crucial trait linked to the position of climatic niches is dietary specialisation. According to the ‘altitudinal niche-breadth hypothesis’, species of high-elevation habitats should be less specialized compared to their low-elevation counterparts. However, empirical evidence on shifts in specialization is scarce for generalist insect herbivores and existing studies often fail to control for the phylogeny and abundance of interaction partners. In Chapter III, we used a combination of field observations and amplicon sequencing to reconstruct dietary relationships between Orthoptera and plants along an extensive temperature gradient. We did not find close but flexible links between individual grasshopper and plant taxa in space. While interaction network specialisation increased with temperature, the corrected dietary specialisation pattern peaked at intermediate elevations on assemblage level. These nuanced findings demonstrate that (1) resource availability, (2) phylogenetic relationships, and (3) climate can affect empirical foodwebs intra- and interspecifically and, hence, the dietary specialisation of herbivorous insects. In this context, we discuss that the underlying mechanisms involved in shaping the specialisation of herbivore assemblages may switch along temperature clines.
Chapter IV: Links between faecal microbe communities, feeding habits, and climate
Since gut microbes affect the fitness and digestion of insects, studying their diversity could provide novel insights into specialisation patterns. However, their association with insect hosts that differ in feeding habits and specialisation has never been investigated along elevational climatic gradients. In Chapter IV, we utilized the dietary information gathered in Chapter III to characterize links between insects with distinct feeding behaviour and the microbial communities present in their faeces, using amplicon sequencing. Both, feeding and climate affected the bacterial communities. However, the large overlap of microbes at site level suggests that common bacteria are acquired from the shared feeding environment, such as the plants consumed by the insects. These findings emphasize the influence of a broader environmental context on the composition of insect gut microbial communities.
Chapter V: Discussion & Conclusions
Cumulatively, the sections of this dissertation provide support for the hypothesis that climatic conditions play a role in shaping plant–herbivore systems. The detected variation of taxonomic and functional compositions contributes to our understanding of assembly processes and resulting diversity patterns within Orthoptera communities, shedding light on the mechanisms that structure their trophic interactions in diverse climates. The combined results presented suggest that a warmer climate could foster an increase of Orthoptera species richness in Central European semi-natural grasslands, also because the weak links observed between insect herbivores and plants are unlikely to limit decoupled range shifts. However, the restructuring of Orthoptera communities in response to warmer temperatures depends on species' traits such as moisture preferences or phenology. Notably, we were able to demonstrate a crucial role of microclimate for many species, partly unravelling narrower climatic niches than their elevational ranges suggest. We found evidence that not only Orthoptera community composition, specialisation, and traits varied along elevational gradients, but even microbial communities in the faeces of Orthoptera changed, which is a novel finding. This complex restructuring and reassembly of communities, coupled with the nonlinear specialisation of trophic interactions and a high diversity of associated bacteria, emphasize our currently incomplete comprehension of how ecosystems will develop under future climatic conditions, demanding caution in making simplified predictions for biodiversity change under climate warming. Since these predictions may benefit from including biotic interactions and both, micro- and macroclimate based on our findings, conservation authorities and practitioners must not neglect improving microclimatic conditions to ensure local survival of a diverse set of threatened and demanding species. In this context, mountains can play a pivotal role for biodiversity conservation since these offer heterogeneous microclimatic conditions in proximity that can be utilized by species with distinct niches.
KM3NeT will be a network of deep-sea neutrino telescopes in the Mediterranean Sea. The KM3NeT/ARCA detector, to be installed at the Capo Passero site (Italy), is optimised for the detection of high-energy neutrinos of cosmic origin. Thanks to its geographical location on the Northern hemisphere, KM3NeT/ARCA can observe upgoing neutrinos from most of the Galactic Plane, including the Galactic Centre. Given its effective area and excellent pointing resolution, KM3NeT/ARCA will measure or significantly constrain the neutrino flux from potential astrophysical neutrino sources. At the same time, it will test flux predictions based on gamma-ray measurements and the assumption that the gamma-ray flux is of hadronic origin. Assuming this scenario, discovery potentials and sensitivities for a selected list of Galactic sources and to generic point sources with an E-2 spectrum are presented. These spectra are assumed to be time independent. The results indicate that an observation with 3 sigma significance is possible in about six years of operation for the most intense sources, such as Supernovae Remnants RX J1713.7-3946 and Vela Jr. If no signal will be found during this time, the fraction of the gamma-ray flux coming from hadronic processes can be constrained to be below 50% for these two objects. (C) 2019 The Authors. Published by Elsevier B.V.
Cooperative, connected and automated mobility (CCAM) systems depend on a reliable communication to provide their service and more crucially to ensure the safety of users. One way to ensure the reliability of a data transmission is to use multiple transmission technologies in combination with redundant flows. In this paper, we describe a system requiring multipath communication in the context of CCAM. To this end, we introduce a data plane-based scheduler that uses replication and integration modules to provide redundant and transparent multipath communication. We provide an analytical model for the full replication module of the system and give an overview of how and where the data-plane scheduler components can be realized.
The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97–cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.
YAP, the key protein effector of the Hippo pathway, is a transcriptional co-activator that controls the expression of cell cycle genes, promotes cell growth and proliferation and regulates organ size. YAP modulates gene transcription by binding to distal enhancers, but the mechanisms of gene regulation by YAP-bound enhancers remain poorly understood. Here we show that constitutive active YAP5SA leads to widespread changes in chromatin accessibility in untransformed MCF10A cells. Newly accessible regions include YAP-bound enhancers that mediate activation of cycle genes regulated by the Myb-MuvB (MMB) complex. By CRISPR-interference we identify a role for YAP-bound enhancers in phosphorylation of Pol II at Ser5 at MMB-regulated promoters, extending previously published studies that suggested YAP primarily regulates the pause-release step and transcriptional elongation. YAP5SA also leads to less accessible ‘closed’ chromatin regions, which are not directly YAP-bound but which contain binding motifs for the p53 family of transcription factors. Diminished accessibility at these regions is, at least in part, a consequence of reduced expression and chromatin-binding of the p53 family member ΔNp63 resulting in downregulation of ΔNp63-target genes and promoting YAP-mediated cell migration. In summary, our studies uncover changes in chromatin accessibility and activity that contribute to the oncogenic activities of YAP.
Addition of heparin binding sites strongly increases the bone forming capabilities of BMP9 in vivo
(2023)
Highlights
• Despite not being crucial for bone development BMP9 can induce bone growth in vivo.
• BMP9 induced bone formation is strongly enhanced by introduced heparin binding sites.
• BMP9s bone forming capabilities are triggered by extracellular matrix binding.
• Heparin binding BMP9 (BMP9 HB) can improve the current therapies in treating bone fractures.
Abstract
Bone Morphogenetic proteins (BMPs) like BMP2 and BMP7 have shown great potential in the treatment of severe bone defects. In recent in vitro studies, BMP9 revealed the highest osteogenic potential compared to other BMPs, possibly due to its unique signaling pathways that differs from other osteogenic BMPs. However, in vivo the bone forming capacity of BMP9-adsorbed scaffolds is not superior to BMP2 or BMP7. In silico analysis of the BMP9 protein sequence revealed that BMP9, in contrast to other osteogenic BMPs such as BMP2, completely lacks so-called heparin binding motifs that enable extracellular matrix (ECM) interactions which in general might be essential for the BMPs' osteogenic function. Therefore, we genetically engineered a new BMP9 variant by adding BMP2-derived heparin binding motifs to the N-terminal segment of BMP9′s mature part. The resulting protein (BMP9 HB) showed higher heparin binding affinity than BMP2, similar osteogenic activity in vitro and comparable binding affinities to BMPR-II and ALK1 compared to BMP9. However, remarkable differences were observed when BMP9 HB was adsorbed to collagen scaffolds and implanted subcutaneously in the dorsum of rats, showing a consistent and significant increase in bone volume and density compared to BMP2 and BMP9. Even at 10-fold lower BMP9 HB doses bone tissue formation was observed. This innovative approach of significantly enhancing the osteogenic properties of BMP9 simply by addition of ECM binding motifs, could constitute a valuable replacement to the commonly used BMPs. The possibility to use lower protein doses demonstrates BMP9 HB's high translational potential.
The detection of smallest mechanical loads plays an increasingly important role in many areas of advancing automation and manufacturing technology, but also in everyday life. In this doctoral thesis, various microparticle systems were developed that are able to indicate mechanical shear stress via simple mechanisms. Using a toolbox approach, these systems can be spray-dried from various nanoscale primary particles (silica and iron oxide) to micrometer-sized units, so-called supraparticles. By varying the different building blocks and in combination with different dyes, a new class of mechanochromic shear stress indicators was developed by constructing hierarchically structured core-shell supraparticles that can indicate mechanical stress via an easily detectable color change. Three different mechanisms can be distinguished. If a signal becomes visible only by a mechanical load, it is a turn-on indicator. In the opposite case, the turn-off indicator, the signal is switched off by a mechanical load. In the third mechanism, the color-change indicator, the color changes as a result of a mechanical load. In principle, these indicators can be used in two different ways. First, they can be incorporated into a coating as an additive. These coatings can be applied to a wide range of products, including food packaging, medical devices, and generally any sensitive surface where mechanical stress, such as scratches, is difficult to detect but can have serious consequences. Second, these shear stress indicators can also be used directly in powder form and for example then applied in 3D-printing or in ball mills. A total of six different shear stress indicators were developed, three of which were used as additives in coatings and three were applied in powder form. Depending on their composition, these indicators were readout by fluorescence, UV-Vis or Magnetic Particle Spectroscopy. The development of these novel shear stress indicator supraparticles were successfully combined molecular chemistry with the world of nano-objects to develop macroscopic systems that can enable smart and communicating materials to indicate mechanical stress in a variety of applications.
The Hamamatsu R12199-023-inch photomultiplier tube is the photodetector chosen for the first phase of the KM3NeT neutrino telescope. About 7000 photomultipliers have been characterised for dark count rate, timing spread and spurious pulses. The quantum efficiency, the gain and the peak-to-valley ratio have also been measured for a sub-sample in order to determine parameter values needed as input to numerical simulations of the detector.
Lung cancer is the main cause of cancer-related deaths worldwide. Despite the availability of several targeted therapies and immunotherapies in the clinics, the prognosis for lung cancer remains poor. A major problem for the low benefit of these therapies is intrinsic and acquired resistance, asking for pre-clinical models for closer investigation of predictive biomarkers for refined personalized medicine and testing of possible combination therapies as well as novel therapeutic approaches to break resistances.
One third of all lung adenocarcinoma harbor mutations in the KRAS gene, of which 39 % are transitions from glycine to cysteine in codon 12 (KRASG12C). Being considered “undruggable” in previous decades, KRASG12C-inhibitors now paved the way into the standard-of-care for lung adenocarcinoma treatment in the clinics. Still, the overall response rates as well as overall survival of patients treated with KRASG12C-inhibitors are sobering. Therefore, 3D KRASG12C-biomarker in vitro models were developed based on a decellularized porcine jejunum (SISmuc) using commercial and PDX-derived cell lines and characterized in regards of epithelial-mesenchymal-transition (EMT), stemness, proliferation, invasion and c-MYC expression as well as the sensitivity towards KRASG12C-inhibiton. The phenotype of lung tumors harboring KRAS mutations together with a c-MYC overexpression described in the literature regarding invasion and proliferation for in vivo models was well represented in the SISmuc models. A higher resistance towards targeted therapies was validated in the 3D models compared to 2D cultures, while reduced viability after treatment with combination therapies were exclusively observed in the 3D models. In the test system neither EMT, stemness nor the c-MYC expression were directly predictive for drug sensitivity. Testing of a panel of combination therapies, a sensitizing effect of the aurora kinase A (AURKA) inhibitor alisertib for the KRASG12C-inhibitor ARS-1620 directly correlating with the level of c-MYC expression in the corresponding 3D models was observed. Thereby, the capability of SISmuc tumor models as an in vitro test system for patient stratification was demonstrated, holding the possibility to reduce animal experiments.
Besides targeted therapies the treatment of NSCLC with oncolytic viruses (OVs) is a promising approach. However, a lack of in vitro models to test novel OVs limits the transfer from bench to bedside. In this study, 3D NSCLC models based on the SISmuc were evaluated for their capability to perform efficacy and risk assessment of oncolytic viruses (OVs) in a pre-clinical setting. Hereby, the infection of cocultures of tumor cells and fibroblasts on the SISmuc with provided viruses demonstrated that in contrast to a wildtype herpes simplex virus 1 (HSV-1) based OV, the attenuated version of the OV exhibited specificity for NSCLC cells with a more advanced and highly proliferative phenotype, while fibroblasts were no longer permissive for infection. This approach introduced SISmuc tumor models as novel test system for in vitro validation of OVs.
Finally, a workflow for validating the efficacy of anti-cancer therapies in 3D tumor spheroids was established for the transfer to an automated platform based on a two-arm-robot system. In a proof-of-concept process, H358 spheroids were characterized and treated with the KRASG12C-inhibitor ARS-1620. A time- and dose-dependent reduction of the spheroid area after treatment was defined together with a live/dead-staining as easy-to-perform and cost-effective assays for automated drug testing that can be readily performed in situ in an automated system.
In the present report, well-defined WO3 nanorods (NRs) and a rGO–WO\(_3\) composite were successfully synthesized using a one-pot hydrothermal method. The crystal phase, structural morphology, shape, and size of the as-synthesized samples were studied using X-ray diffraction (XRD) and transmission electron microscopy (TEM) measurements. The optical properties of the synthesized samples were investigated by Raman, ultraviolet-visible (UV-Vis) and photoluminescence (PL) spectroscopy. Raman spectroscopy and TEM results validate the formation of WO\(_3\) (NRs) on the rGO sheet. The value of the dielectric constant (ε′) of WO3 NRs and rGO–WO\(_3\) composite is decreased with an increase in frequency. At low frequency (2.5 to 3.5 Hz), the value of ε′ for the rGO–WO3 composite is greater than that of pure WO\(_3\) NRs. This could be due to the fact that the induced charges follow the ac signal. However, at higher frequency (3.4 to 6.0), the value of ε′ for the rGO–WO\(_3\) composite is less compared to that of the pure WO3 NRs. The overall decrease in the value of ε′ could be due to the occurrence of a polarization process at the interface of the rGO sheet and WO3 NRs. Enhanced interfacial polarization in the rGO–WO\(_3\) composite is observed, which may be attributed to the presence of polar functional groups on the rGO sheet. These functional groups trap charge carriers at the interface, resulting in an enhancement of the interfacial polarization. The value of the dielectric modulus is also calculated to further confirm this enhancement. The values of the ac conductivity of the WO\(_3\) NRs and rGO–WO\(_3\) composite were calculated as a function of the frequency. The greater value of the ac conductivity in the rGO–WO\(_3\) composite compared to that of the WO\(_3\) NRs confirms the restoration of the sp:\(^{++}\) network during the in situ synthesis of the rGO–WO\(_3\) composite, which is well supported by the results obtained by Raman spectroscopy.
Infection research largely relies on classical cell culture or mouse models. Despite having delivered invaluable insights into host-pathogen interactions, both have limitations in translating mechanistic principles to human pathologies. Alternatives can be derived from modern Tissue Engineering approaches, allowing the reconstruction of functional tissue models in vitro. Here, we combined a biological extracellular matrix with primary tissue-derived enteroids to establish an in vitro model of the human small intestinal epithelium exhibiting in vivo-like characteristics. Using the foodborne pathogen Salmonella enterica serovar Typhimurium, we demonstrated the applicability of our model to enteric infection research in the human context. Infection assays coupled to spatio-temporal readouts recapitulated the established key steps of epithelial infection by this pathogen in our model. Besides, we detected the upregulation of olfactomedin 4 in infected cells, a hitherto unrecognized aspect of the host response to Salmonella infection. Together, this primary human small intestinal tissue model fills the gap between simplistic cell culture and animal models of infection, and shall prove valuable in uncovering human-specific features of host-pathogen interplay.
In the fast-evolving landscape of biomedical research, the emergence of big data has presented researchers with extraordinary opportunities to explore biological complexities. In biomedical research, big data imply also a big responsibility. This is not only due to genomics data being sensitive information but also due to genomics data being shared and re-analysed among the scientific community. This saves valuable resources and can even help to find new insights in silico. To fully use these opportunities, detailed and correct metadata are imperative. This includes not only the availability of metadata but also their correctness. Metadata integrity serves as a fundamental determinant of research credibility, supporting the reliability and reproducibility of data-driven findings. Ensuring metadata availability, curation, and accuracy are therefore essential for bioinformatic research. Not only must metadata be readily available, but they must also be meticulously curated and ideally error-free. Motivated by an accidental discovery of a critical metadata error in patient data published in two high-impact journals, we aim to raise awareness for the need of correct, complete, and curated metadata. We describe how the metadata error was found, addressed, and present examples for metadata-related challenges in omics research, along with supporting measures, including tools for checking metadata and software to facilitate various steps from data analysis to published research.
Highlights
• Data awareness and data integrity underpins the trustworthiness of results and subsequent further analysis.
• Big data and bioinformatics enable efficient resource use by repurposing publicly available RNA-Sequencing data.
• Manual checks of data quality and integrity are insufficient due to the overwhelming volume and rapidly growing data.
• Automation and artificial intelligence provide cost-effective and efficient solutions for data integrity and quality checks.
• FAIR data management, various software solutions and analysis tools assist metadata maintenance.
Machine learning techniques are excellent to analyze expression data from single cells. These techniques impact all fields ranging from cell annotation and clustering to signature identification. The presented framework evaluates gene selection sets how far they optimally separate defined phenotypes or cell groups. This innovation overcomes the present limitation to objectively and correctly identify a small gene set of high information content regarding separating phenotypes for which corresponding code scripts are provided. The small but meaningful subset of the original genes (or feature space) facilitates human interpretability of the differences of the phenotypes including those found by machine learning results and may even turn correlations between genes and phenotypes into a causal explanation. For the feature selection task, the principal feature analysis is utilized which reduces redundant information while selecting genes that carry the information for separating the phenotypes. In this context, the presented framework shows explainability of unsupervised learning as it reveals cell-type specific signatures. Apart from a Seurat preprocessing tool and the PFA script, the pipeline uses mutual information to balance accuracy and size of the gene set if desired. A validation part to evaluate the gene selection for their information content regarding the separation of the phenotypes is provided as well, binary and multiclass classification of 3 or 4 groups are studied. Results from different single-cell data are presented. In each, only about ten out of more than 30000 genes are identified as carrying the relevant information. The code is provided in a GitHub repository at https://github.com/AC-PHD/Seurat_PFA_pipeline.
CRISPR/Cas9 gene editing has revolutionised loss-of-function experiments in Leishmania, the causative agent of leishmaniasis. As Leishmania lack a functional non-homologous DNA end joining pathway however, obtaining null mutants typically requires additional donor DNA, selection of drug resistance-associated edits or time-consuming isolation of clones. Genome-wide loss-of-function screens across different conditions and across multiple Leishmania species are therefore unfeasible at present. Here, we report a CRISPR/Cas9 cytosine base editor (CBE) toolbox that overcomes these limitations. We employed CBEs in Leishmania to introduce STOP codons by converting cytosine into thymine and created http://www.leishbaseedit.net/ for CBE primer design in kinetoplastids. Through reporter assays and by targeting single- and multi-copy genes in L. mexicana, L. major, L. donovani, and L. infantum, we demonstrate how this tool can efficiently generate functional null mutants by expressing just one single-guide RNA, reaching up to 100% editing rate in non-clonal populations. We then generated a Leishmania-optimised CBE and successfully targeted an essential gene in a plasmid library delivered loss-of-function screen in L. mexicana. Since our method does not require DNA double-strand breaks, homologous recombination, donor DNA, or isolation of clones, we believe that this enables for the first time functional genetic screens in Leishmania via delivery of plasmid libraries.
Increased intestinal permeability and inflammation, both fueled by dysbiosis, appear to contribute to rheumatoid arthritis (RA) pathogenesis. This single-center pilot study aimed to investigate zonulin, a marker of intestinal permeability, and calprotectin, a marker of intestinal inflammation, measured in serum and fecal samples of RA patients using commercially available kits. We also analyzed plasma lipopolysaccharide (LPS) levels, a marker of intestinal permeability and inflammation. Furthermore, univariate, and multivariate regression analyses were carried out to determine whether or not there were associations of zonulin and calprotectin with LPS, BMI, gender, age, RA-specific parameters, fiber intake, and short-chain fatty acids in the gut. Serum zonulin levels were more likely to be abnormal with a longer disease duration and fecal zonulin levels were inversely associated with age. A strong association between fecal and serum calprotectin and between fecal calprotectin and LPS were found in males, but not in females, independent of other biomarkers, suggesting that fecal calprotectin may be a more specific biomarker than serum calprotectin is of intestinal inflammation in RA. Since this was a proof-of-principle study without a healthy control group, further research is needed to validate fecal and serum zonulin as valid biomarkers of RA in comparison with other promising biomarkers.
Making judgments of learning (JOLs) after studying can directly improve learning. This JOL reactivity has been shown for simple materials but has scarcely been investigated with educationally relevant materials such as expository texts. The few existing studies have not yet reported any consistent gains in text comprehension due to providing JOLs. In the present study, we hypothesized that increasing the chances of covert retrieval attempts when making JOLs after each of five to-be-studied text passages would produce comprehension benefits at 1 week compared to restudy. In a between-subjects design, we manipulated both whether participants (N = 210) were instructed to covertly retrieve the texts, and whether they made delayed target-absent JOLs. The results indicated that delayed, target-absent JOLs did not improve text comprehension after 1 week, regardless of whether prior instructions to engage in covert retrieval were provided. Based on the two-stage model of JOLs, we reasoned that participants’ retrieval attempts during metacomprehension judgments were either insufficient (i.e., due to a quick familiarity assessment) or were ineffective (e.g., due to low retrieval success).
Background: The COVID-19 pandemic has led to a flood of — often contradictory — evidence. HCWs had to develop strategies to locate information that supported their work. We investigated the information-seeking of different HCW groups in Germany. Methods: In December 2020, we conducted online surveys on COVID-19 information sources, strategies, assigned trustworthiness, and barriers — and in February 2021, on COVID-19 vaccination information sources. Results were analyzed descriptively; group comparisons were performed using χ\(^2\)-tests. Results: For general COVID-19-related medical information (413 participants), non-physicians most often selected official websites (57%), TV (57%), and e-mail/newsletters (46%) as preferred information sources — physicians chose official websites (63%), e-mail/newsletters (56%), and professional journals (55%). Non-physician HCWs used Facebook/YouTube more frequently. The main barriers were insufficient time and access issues. Non-physicians chose abstracts (66%), videos (45%), and webinars (40%) as preferred information strategy; physicians: overviews with algorithms (66%), abstracts (62%), webinars (48%). Information seeking on COVID-19 vaccination (2700 participants) was quite similar, however, with newspapers being more often used by non-physicians (63%) vs. physician HCWs (70%). Conclusion: Non-physician HCWs more often consulted public information sources. Employers/institutions should ensure the supply of professional, targeted COVID-19 information for different HCW groups.
PRO-Simat is a simulation tool for analysing protein interaction networks, their dynamic change and pathway engineering. It provides GO enrichment, KEGG pathway analyses, and network visualisation from an integrated database of more than 8 million protein-protein interactions across 32 model organisms and the human proteome. We integrated dynamical network simulation using the Jimena framework, which quickly and efficiently simulates Boolean genetic regulatory networks. It enables simulation outputs with in-depth analysis of the type, strength, duration and pathway of the protein interactions on the website. Furthermore, the user can efficiently edit and analyse the effect of network modifications and engineering experiments. In case studies, applications of PRO-Simat are demonstrated: (i) understanding mutually exclusive differentiation pathways in Bacillus subtilis, (ii) making Vaccinia virus oncolytic by switching on its viral replication mainly in cancer cells and triggering cancer cell apoptosis and (iii) optogenetic control of nucleotide processing protein networks to operate DNA storage. Multilevel communication between components is critical for efficient network switching, as demonstrated by a general census on prokaryotic and eukaryotic networks and comparing design with synthetic networks using PRO-Simat. The tool is available at https://prosimat.heinzelab.de/ as a web-based query server.
In recent years, various forms of caloric restriction (CR) and amino acid or protein restriction (AAR or PR) have shown not only success in preventing age-associated diseases, such as type II diabetes and cardiovascular diseases, but also potential for cancer therapy. These strategies not only reprogram metabolism to low-energy metabolism (LEM), which is disadvantageous for neoplastic cells, but also significantly inhibit proliferation. Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumour types, with over 600,000 new cases diagnosed annually worldwide. With a 5-year survival rate of approximately 55%, the poor prognosis has not improved despite extensive research and new adjuvant therapies. Therefore, for the first time, we analysed the potential of methionine restriction (MetR) in selected HNSCC cell lines. We investigated the influence of MetR on cell proliferation and vitality, the compensation for MetR by homocysteine, the gene regulation of different amino acid transporters, and the influence of cisplatin on cell proliferation in different HNSCC cell lines.
Alveolar (AE) and cystic (CE) echinococcosis are two parasitic diseases caused by the tapeworms Echinococcus multilocularis and E. granulosus sensu lato (s. l.), respectively. Currently, AE and CE are mainly diagnosed by means of imaging techniques, serology, and clinical and epidemiological data. However, no viability markers that indicate parasite state during infection are available. Extracellular small RNAs (sRNAs) are short non-coding RNAs that can be secreted by cells through association with extracellular vesicles, proteins, or lipoproteins. Circulating sRNAs can show altered expression in pathological states; hence, they are intensively studied as biomarkers for several diseases. Here, we profiled the sRNA transcriptomes of AE and CE patients to identify novel biomarkers to aid in medical decisions when current diagnostic procedures are inconclusive. For this, endogenous and parasitic sRNAs were analyzed by sRNA sequencing in serum from disease negative, positive, and treated patients and patients harboring a non-parasitic lesion. Consequently, 20 differentially expressed sRNAs associated with AE, CE, and/or non-parasitic lesion were identified. Our results represent an in-depth characterization of the effect E. multilocularis and E. granulosus s. l. exert on the extracellular sRNA landscape in human infections and provide a set of novel candidate biomarkers for both AE and CE detection.
Grading, immunohistochemistry and c-kit mutation status are criteria for assessing the prognosis and therapeutic options of canine cutaneous mast cell tumours (MCTs). As a subset, canine digital MCTs have rarely been explored in this context. Therefore, in this retrospective study, 68 paraffin-embedded canine digital MCTs were analysed, and histological grading was assessed according to Patnaik and Kiupel. The immunohistochemical markers KIT and Ki67 were used, as well as polymerase chain reaction (PCR) for mutational screening in c-kit exons 8, 9, 11 and 14. Patnaik grading resulted in 22.1% grade I, 67.6% grade II and 10.3% grade III tumours. Some 86.8% of the digital MCTs were Kiupel low-grade. Aberrant KIT staining patterns II and III were found in 58.8%, and a count of more than 23 Ki67-positive cells in 52.3% of the cases. Both parameters were significantly associated with an internal tandem duplication (ITD) in c-kit exon 11 (12.7%). French Bulldogs, which tend to form well-differentiated cutaneous MCTs, had a higher proportion of digital high-grade MCTs and ITD in c-kit exon 11 compared with mongrels. Due to its retrospective nature, this study did not allow for an analysis of survival data. Nevertheless, it may contribute to the targeted characterisation of digital MCTs.
Improved wall temperature prediction for the LUMEN rocket combustion chamber with neural networks
(2023)
Accurate calculations of the heat transfer and the resulting maximum wall temperature are essential for the optimal design of reliable and efficient regenerative cooling systems. However, predicting the heat transfer of supercritical methane flowing in cooling channels of a regeneratively cooled rocket combustor presents a significant challenge. High-fidelity CFD calculations provide sufficient accuracy but are computationally too expensive to be used within elaborate design optimization routines. In a previous work it has been shown that a surrogate model based on neural networks is able to predict the maximum wall temperature along straight cooling channels with convincing precision when trained with data from CFD simulations for simple cooling channel segments. In this paper, the methodology is extended to cooling channels with curvature. The predictions of the extended model are tested against CFD simulations with different boundary conditions for the representative LUMEN combustor contour with varying geometries and heat flux densities. The high accuracy of the extended model’s predictions, suggests that it will be a valuable tool for designing and analyzing regenerative cooling systems with greater efficiency and effectiveness.
After priming in Peyer's patches (PPs) and mesenteric lymph nodes (mLN) T- cells infiltrate the intestine through lymphatic draining and homing through the bloodstream. However, we found that in mouse models of acute graft-versus-host disease (GvHD), a subset of alloreactive T-cells directly migrates from PPs to the adjacent intestinal lamina propria (LP), bypassing the normal lymphatic drainage and vascular trafficking routes. Notably, this direct migration occurred in irradiated and unirradiated GvHD models, indicating that irradiation is not a prerequisite for this observed behavior.
Next, we established a method termed serial intravascular staining (SIVS) in mouse models to systematically investigate the trafficking and migration of donor T- cells in the early stages of acute GvHD initiation. We found that the direct migration of T-cells from PPs to LP resulted in faster recruitment of cells after allogeneic hematopoietic cell transplantation (allo-HCT). These directly migrating T-cells were found to be in an activated and proliferative state, exhibiting a TH1/TH17-like phenotype and producing cytokines such as IFN-γ and TNF-α. Furthermore, we observed that the directly migrating alloreactive T-cells expressed specific integrins (α4+, αE+) and chemokine receptors (CxCR3+, CCR5+, and CCR9+). Surprisingly, blocking these integrins and chemokine-coupled receptors did not hinder the direct migration of T- cells from PPs to LP, suggesting the involvement of alternative mechanisms. Previous experiments ruled out the involvement of S1PR1 and topographical features of macrophages, leading us to hypothesize that mediators of cytoskeleton reorganization, such as Coro1a, Dock2, or Cdc42, may play a role in this unique migration process.
Additionally, we observed that directly migrating T-cells created a local inflammatory microenvironment, which attracts circulating T-cells. Histological analysis confirmed that alloreactive PPs-derived T-cells and bloodborne T-cells colocalized. We employed two experimental approaches, including either photoconversion of T-cells in PPs or direct transfer of activated T-cells into the vasculature, to demonstrate this colocalization. We hypothesize that cytokines released by migrating T-cells, such as IFN-γ and TNF-α, may play a role in recruiting T-cells from the vasculature, as inhibiting chemokine-coupled receptors did not impair recruitment.
Gastroesophageal junction (GEJ), demarcating the region where the distal esophagus meets with the proximal stomach region, is known for developing pathological conditions, including metaplasia and esophageal adenocarcinoma (EAC). It is essential to understand the mechanisms of developmental stages which lead to EAC since the incidence rate of EAC increased over 7-fold during the past four decades, and the overall five years survival rate is 18.4%. In most cases, patients are diagnosed in the advanced stage without prior symptoms. The main precursor for the development of EAC is a pre-malignant condition called Barrett's esophagus (BE). BE is the metaplastic condition where the multilayered squamous epithelium of the native esophagus is replaced by specialized single-layered columnar epithelium, which shows the molecular characteristics of the gastric as well as intestinal epithelium. The main risk factors for BE development include chronic gastro-esophageal acid reflux disease (GERD), altered microbiota, and altered retinoic acid signaling (RA). The cell of origin of BE is under debate due to a lack of clear evidence demonstrating the process of BE initiation. Here, I investigated how GEJ homeostasis is maintained in healthy tissue by stem cell regulatory morphogens, the role of vitamin A (RA signaling), and how its alteration contributes to BE development.
In the first part of my thesis, I showed the presence of two types of epithelial cells, the squamous type in the esophagus and the columnar type in the stomach region in the GEJ, using single-molecule RNA in situ hybridization (smRNA-ISH) and immunohistochemistry. Employing lineage tracing in the mouse model, I have demonstrated that the esophageal epithelial and stomach epithelial cells derived from two distinct epithelial stem cell lineages in the GEJ. The border between squamous and columnar epithelial cells in the Squamo-columnar junction (SCJ) of GEJ is regulated by opposing Wnt microenvironments. The regeneration of stomach columnar epithelial stem cells is maintained by Wnt activating signal from the stromal compartment while squamous epithelial stem cells of the esophagus are maintained by the Wnt inhibitory signals. I recapitulated the in vivo GEJ epithelial stem cell maintenance by using in vitro epithelial 3D organoid culture model. The growth and propagation of stomach columnar epithelial organoids depend on Wnt growth factors, while squamous epithelial organoids' development needs Wnt-deficient culture conditions.
Further, single-cell RNA sequence (scRNA-seq) analysis of organoid-derived epithelial cells revealed the non-canonical Wnt/ planar cell polarity (PCP) pathway involvement in regulating the squamous epithelial cells. In contrast, columnar stomach epithelial cells are regulated by the canonical Wnt/ beta-catenin and non-canonical Wnt/Ca2+ pathways. My data indicate that the SCJ epithelial cells that merge at the GEJ are regulated by opposing stromal Wnt factors and distinct Wnt pathway signaling in the epithelial cells.
In the second part of the thesis, I investigated the role of Vitamin A-derived bioactive compound RA on esophageal and stomach epithelial stem cells. In vitro treatment of esophageal and stomach, epithelial organoids with RA or its pharmacological inhibitor BMS 493 revealed that each cell type was regulated distinctly. I observed that enhanced RA promoted esophageal stem cell differentiation and loss of stratification, while RA inhibition led to enhanced stemness and regeneration of the esophagus stratified epithelium. As opposed to the esophagus, RA signaling is active in the stomach organoids, and inhibition of RA reduces the growth of stomach organoids. Global transcriptomic data and scRNA-seq data revealed that RA signaling induces dormancy phenotype in the esophageal cells. In contrast, the absence of RA in stomach epithelial cells induces the expression of genes associated with BE. Thus, spatially defined regulation of Wnt and RA signaling at GEJ is critical for healthy homeostasis, and its perturbation leads to disease development.
The unicellular pathogen Trypanosoma brucei is the causative agent of African
trypanosomiasis, an endemic disease prevalent in sub-Saharan Africa. Trypanosoma brucei alternates between a mammalian host and the tsetse fly vector. The extracellular parasite survives in the mammalian bloodstream by periodically exchanging their ˈvariant surface glycoproteinˈ (VSG) coat to evade the host immune response. This antigenic variation is achieved through monoallelic expression of one VSG variant from subtelomeric ˈbloodstream
form expression sitesˈ (BES) at a given timepoint. During the differentiation from the bloodstream form (BSF) to the procyclic form (PCF) in the tsetse fly midgut, the stage specific surface protein is transcriptionally silenced and replaced by procyclins. Due to their subtelomeric localization on the chromosomes, VSG transcription and silencing is partly regulated by homologues of the mammalian telomere complex such as TbTRF, TbTIF2 and TbRAP1 as well as by ˈtelomere-associated proteinsˈ (TelAPs) like TelAP1. To gain more insights into transcription regulation of VSG genes, the identification and characterization of other TelAPs is critical and has not yet been achieved. In a previous study, two biochemical approaches were used to identify other novel TelAPs. By using ˈco-immunoprecipitationˈ (co-IP) to enrich possible interaction partners of TbTRF and by affinity chromatography using telomeric repeat oligonucleotides, a listing of TelAP candidates has been conducted. With this approach TelAP1 was identified as a novel component of the telomere complex, involved in the kinetics of transcriptional BES silencing during BSF to PCF differentiation. To gain further insights into the telomere complex composition, other previously enriched proteins were characterized through a screening process using RNA interference to deplete potential candidates. VSG expression profile changes and overall proteomic changes after depletion were analyzed by mass spectrometry. With this method, one can gain insights into the functions of the proteins and their involvement in VSG expression site regulation. To validate the interaction of proteins enriched by co-IP with TbTRF and TelAP1 and to identify novel interaction proteins, I performed reciprocal affinity purifications of the four most promising candidates (TelAP2, TelAP3, PPL2 and PolIE) and additionally confirmed colocalization of two candidates with TbTRF via immunofluorescence (TelAP2, TelAP3). TelAP3 colocalizes with TbTRF and potentially interacts with TbTRF, TbTIF2, TelAP1 and TelAP2, as well as with two translesion polymerases PPL2 and PolIE in BSF. PPL2 and PolIE seem to be in close contact to each other at the telomeric ends and fulfill different roles as only PolIE is involved in VSG regulation while PPL2 is not. TelAP2 was previously characterized to be associated with telomeres by partially colocalizing with TbTRF and cells show a VSG derepression phenotype when the protein was depleted. Here I show that TelAP2 interacts with the telomere-binding proteins TbTRF and TbTIF2 as well as with the telomere-associated protein TelAP1 in BSF and that TelAP2 depletion results in a loss of TelAP1 colocalization with TbTRF in BSF.
In conclusion, this study demonstrates that characterizing potential TelAPs is effective in gaining insights into the telomeric complex's composition and its role in VSG regulation in Trypanosoma brucei. Understanding these interactions could potentially lead to new therapeutic targets for combatting African trypanosomiasis.
The current study presents a new a group of Demotic ostraca in the belongings of the Cairo Museum. A large part of this group stem from Medinet Habu in the western bank of modern Luxor in Upper Egypt and was discovered in the beginning of the thirties of the last century by the Chicago Oriental Institute (recently renamed as Institute for the Study of Ancient Cultures ‘ISAC’). A small portion of the collection under consideration come from other Upper Egyptian provenances including Gebelein, Edfu, Kom Ombo, and possibly elsewhere in Thebes. The main goal of the present dissertation is to decipher, translate, and provide a philological, paleographical, and cultural analysis of the group of texts in question. The results of this study are spread over two main parts, the first of which is dedicated to the main and largest part of the collection, i.e. ostraca from Medinet Habu, while the second is concerned with ostraca from other places. The first part comprises of five sections beginning with receipts of money and in-kind payments including some receipts for the payments of the different capitation charges in the Ptolemaic and Roman Periods, a few for land-related payments, as well as others related to different Ptolemaic monopolies or trades such as a receipt for the price of oil, one for the linen tax, in addition to a unique receipt for the rarely attested fish tax. The second section includes accounts and lists of different kinds be it monetary, in-kind, agriculture, or any other type of lists or accounts that record different everyday transactions. The following section presents a relatively different type of lists, namely lists of personal names. The fourth section incorporates a variety of texts of different concerns, e.g. texts of religious nature, letters, temples oaths, or other private documents. Unidentified texts occupy the fifth and final section of the first part. The second part of the study, which comprises texts that originate from different Upper Egyptian localities, includes three sections, i.e. receipts, accounts, and lists of names.
The mold Aspergillus fumigatus (A. fumigatus) is known as human pathogen and can cause life-threatening infections in humans with a weakened immune system. This is a known complication in patients receiving glucocorticoids, e.g. after hematopoietic stem cell transplantation or solid organ transplantation. Although research in the field of immune cell/fungus interaction has discovered key strategies how immune cells fight against infectious fungi, our knowledge is still incomplete. In order to develop effective treatment options against fungal infections, a detailed understanding of their interactions is crucial. Thus, visualization of immune cell and fungus is an excellent approach to gain further knowledge. For a detailed view of such interaction processes, a high optical resolution on nanometer scale is required. There is a variety of super resolution microscopy techniques, enabling fluorescence imaging beyond the diffraction limit. This work combines the use of three complementary super resolution microscopy techniques, in order to study immune cell/fungus interaction from different points of view.
Aim of this work is the introduction of the recently invented imaging technique named expansion microscopy (ExM) for the study of immune cell/fungus interactions. The core aspect of this method is the physical magnification of the specimen, which increases the distance between protein structures that are close to each other and which can therefore be imaged separately.
The simultaneous magnification of primary human natural killer (NK) cells and A. fumigatus hyphae was established in this work using ExM. Reorganization of cytoskeletal components of interacting NK cells was demonstrated here, by expansion of the immunological synapse (IS), formed between NK cells and A. fumigatus. In addition, reorganization of the microtubule-organizing center (MTOC) towards fungal hyphae and an accumulation of actin at the IS has been observed. Furthermore, ExM has been used to visualize lytic granules of NK cells after degranulation. After magnification of the specimen, lysosome associated protein 1 (LAMP1) was shown to surround perforin. In absence of the plasma membrane-exposed degranulation marker LAMP1, a “ring-shaped” structure was often observed for fluorescently labeled perforin. Volume calculation of lytic granules demonstrated the benefit of ExM. Compared to pre-expansion images, analyses of post-expansion images showed two volume distributions for degranulated and non-degranulated NK cells. In addition, this work emphasizes the importance of determining the expansion factor for a structure in each species, as variations of expansion factors have been observed. This factor, as well as possible sample distortions should be considered, when ExM is used in order to analyze the interaction between two species.
A second focus of this work is the visualization of a chimeric antigen receptor (CAR), targeting an epitope on the cell wall of A. fumigatus. Structured illumination microscopy (SIM) revealed that the CAR is part of the immunological synapse of primary human CAR T cells and CAR-NK-92 cells. At the interaction site, an accumulation of the CAR was observed, as well as the presence of perforin. CAR accumulation at fungal hyphae was further demonstrated by automated live cell imaging of interacting CAR-NK-92 cells, expressing a fluorescent fusion protein.
Additionally, the use of direct stochastic optical reconstruction microscopy (dSTORM) gave first insights in CAR expression levels on the basal membrane of CAR-NK-92 cells, with single molecule sensitivity. CAR cluster analyses displayed a heterogeneous CAR density on the basal membrane of transfected NK 92 cells.
In summary, this work provides insights into the application of ExM for studying the interaction of primary human NK cells and A. fumigatus for the first time. Furthermore, this thesis presents first insights regarding the characterization of an A. fumigatus-targeting CAR, by applying super-resolution fluorescence microscopy, like SIM and dSTORM.
Theory and simulation of ultrafast autodetachment dynamics and nonradiative relaxation in molecules
(2024)
In this thesis, theoretical approaches for the simulation of electron detachment processes in molecules following vibrational or electronic excitation are developed and applied. These approaches are based on the quantum-classical surface-hopping methodology, in which nuclear motion is treated classically as an ensemble of trajectories in the potential of quantum-mechanically described electronic degrees of freedom.
This compilation focuses on adolescent mental disorders and their prevention. It comprises three distinct studies, each contributing to a deeper understanding of this critical topic. This work addresses a critical gap in the understanding of, and approach to, adolescent mental health, and as a result reveals a critically important and urgently needed policy implication for action. The thematic structure of these studies begins with an examination of the epidemiology of child and adolescent mental disorders. Baseline data were collected from N = 877 adolescents with a mean age of 12.43 years (SD = 0.65). Mental health problems, such as depressive symptoms, non-suicidal self-injury, suicidal ideation, symptoms of eating disorders, and gender differences, are thoroughly examined. Results revealed a significant portion of our sample displaying mental health problems as early as the 6th and 7th grades, with girls generally being more affected than boys. The findings underscore the importance of early adolescence in the emergence of mental health problems and thereby emphasize the need for preventive measures. Moving beyond prevalence estimates, the compilation delves into the etiology of these disorders, exploring their potential correlation with a COVID-19 infection. Understanding the early signs and risk factors is crucial for timely support. While numerous studies have investigated potential risk and protective factors during the pandemic, our focus shifts to adolescents’ coping when an infection with the virus was involved (N = 2,154, M = 12.31, SD = 0.67). We hypothesized that students infected or with close family members infected, would exhibit an increased psychopathology and a decreased functioning of protective factors such as self-efficacy or self-esteem. We found no connection between infection and the mental health status within our sample, but protective factors and mental well-being were positively associated. Thus, universal primary prevention appears to be the preferred approach for promoting mental health. Lastly, the compilation introduces LessStress, a noteworthy contribution to more evidence-based prevention programs. This universal approach is designed to reduce stress in schools, accompanied by a cluster-randomized trial to evaluate its effectiveness (estimated sample size N = 1,894). Existing studies have demonstrated the effectiveness of stress prevention, leading us to introduce a short and easy-to-implement prevention program. There is positive evidence for one-lesson interventions in schools for promoting well-being and health behaviors among adolescents. LessStress is designed based on a life skills approach that not only imparts psychoeducational content but also teaches skills relevant to everyday life and directly applicable. Throughout these studies, a common thread emerges: the pressing need to address mental disorders during childhood and adolescence. These formative years play a pivotal role in the development of mental health problems. These formative years play a crucial role in the development of mental health problems. They highlight the importance of epidemiological data collection and analysis based on the latest models to develop prevention interventions that are not only effective but also reach young people on a global level.
Dipolar merocyanines are very attractive supramolecular building blocks, as they combine interesting functional properties with strong, directional intermolecular interactions. The pyridine dioxocyano-pyridine (PYOP) chromophore (Chapter 2.2), used in this thesis, stands out because of its exceptionally high ground state dipole moment (g ~ 17 D), in combination with the option to retain good solubility also in unpolar solvents, by decoration with solubilizing groups.
The reliable binding motif of anti-parallel -stacking due to dipole-dipole interactions has allowed the design of molecular building blocks that form assemblies of predictable geometry. The intense unstructured charge transfer UV/Vis absorption band (eg ~ 10.7 D) is a result of the dominant contribution of the zwitterionic resonance structure which brings the PYOP chromophore just beyond the cyanine limit in solvents of low polarity (c2 = 0.60, 1,4 dioxane). The high sensitivity of the S0 – S1 UV/Vis absorption band to the environment manifests itself in a pronounced negative solvatochromism and strong H-type exciton coupling within -stacked PYOP assemblies. In accordance with the classical molecular exciton theory, an increasing hypsochromic shift of the dominant absorption band of these H aggregates can be observed as the stack size increases up to about six chromophores, where it levels out at about max ~ 440 nm (CHCl3). This allows a uniquely simple estimation of the number of interacting chromophores within the self-assembled structure from a single UV/Vis absorption spectrum of an aggregate.
The defined and well investigated PYOP dimer formation was employed in this thesis to probe the applicability and limitations of concentration-, temperature-, and solvent-dependent self-assembly studies (Chapter 3). Straightforward theoretical models to evaluate datasets of concentration-, temperature-, and solvent-dependent UV/Vis absorption by nonlinear regression analysis were derived for the case of dimer formation (Chapter 2.1). Although the dimer model is well known and widely applied in literature, this detailed derivation is helpful to understand assumptions and potential problems of the different approaches for the determination of thermodynamic parameters. This helps to decide on the most appropriate method to analyse a system of interest. In this regard it should be noted that covering a large portion of the self-assembly process with the experimental data is a prerequisite for the accuracy of the analysis. Additionally, many of the insights can also be transferred to other self-assembly systems like supramolecular polymerization or host-guest interactions.
The concentration-dependent analysis is the most straightforward method to investigate self-assembly equilibria. No additional assumptions, besides mass balance and mass action law, are required. Since it includes the least number of parameters (only K, if M/D are known), it is the most, or even only, reliable method, to elucidate the self-assembly mechanism of an unknown system by model comparison. To cover a large concentration range, however, the compound must be soluble enough and generally sample amounts at least in the low mg scale must be available.
The temperature-dependent analysis has the advantage that all thermodynamic parameters G0, H0 and S0 can be obtained from a single sample in one automated measurement. However, the accessible temperature-range is experimentally often quite limited and dependent on the solvent. For systems which do not show the transition from monomer to aggregate in a narrow temperature range, as given for, e.g., cooperative aggregation or processes with a high entropy contribution, often not the entire self-assembly process can be monitored. Furthermore, the assumptions of temperature-independent extinction coefficients of the individual species as well as temperature-independent H0 and S0 must be met. Monte Carlo simulations of data sets demonstrated that even minor changes in experimental data can significantly impact the optimized values for H0 and S0. This is due to the redundancy of these two parameters within the model framework and even small thermochromic effects can significantly influence the results. The G0 value, calculated from H0 and S0, is, however, still rather reliable.
Solvent-dependent studies can often cover the entire self-assembly process from monomeric (agg = 0) to the fully aggregated state (agg = 1). However, for dyes with strong solvatochromic effects, such as the dipolar merocyanines investigated in this thesis, the results are affected. Also, the assumption of a linear relation of the binding energy G0 and the fraction of denaturating solvent f, which is based on linear free energy relationships between G0 and the solvent polarity, can lead to errors. Especially when specific solvent effects are involved.
For the evaluation of experimental data by nonlinear regression, general data analysis software can be used, where user-defined fit models and known parameters can be implemented as desired. Alternatively, multiple specialized programs for analysing self-assembly data are available online. While the latter programs are usually more user-friendly, they have the disadvantage of being a “black box” where only pre-implemented models can be used without the option for the user to adapt models or parameters for a specific system.
In Chapter 3 comprehensive UV/Vis absorption datasets are presented for the dimerization of merocyanine derivative 1 in 1,4-dioxane, which allowed for the first time a direct comparison of the results derived from concentration-, temperature-, and solvent-dependent self-assembly studies.
The results for the binding constant K and corresponding G0 from the concentration- and temperature-dependent analysis were in very good agreement, also in comparison to the results from ITC. For the temperature-dependent analysis, though, multiple datasets of samples with different concentration had to be evaluated simultaneously to cover a meaningful part of the self-assembly process. Furthermore, a significant dependence of the optimized parameters H0 and S0 on the wavelength chosen for the analysis was observed. This can be rationalized by the small thermochromic shifts of both the monomer and the dimer UV/Vis absorption band. The results from the solvent-dependent evaluation showed the largest deviation, as expected for the highly solvatochromic merocyanine dye.
However, even here by evaluation at 491 and 549 nm the deviation for G0 was only 2.5 kJ mol1 (9%) with respect to the results from the concentration-dependent analysis (G0 = 29.1 kJ mol1). Thus, despite the strong solvatochromism of the dipolar chromophore, it can still be considered a reliable method for estimating the binding strength. Furthermore, multiple repetitions of the concentration-, temperature-, and solvent-dependent studies provided insight into the reproducibility of the results and possible sources of experimental errors. In all cases, the deviations of the results were small (G0 < 0.4 kJ mol1) and within the same range as the fit error from the nonlinear regression analysis.
The insights from these studies were an important basis for the in-depth investigation of a more complex supramolecular system in Chapter 4, as a single method is often not enough to capture the full picture of a more complicated self-assembly process. To elucidate the anti-cooperative self-assembly of the chiral merocyanine 2, a combination of multiple techniques had to be applied.
Solvent-dependent UV/Vis absorption studies in CH2Cl2/MCH mixtures showed the step-wise assembly of the merocyanine monomer (max(M) = 549 nm, CH2Cl2) to first a dimer (max(D) = 498 nm, CH2Cl2/MCH 15:85) by dipole-dipole interactions, and then a -stacked higher aggregate (max(H) = 477 nm, MCH), with pronounced H-type coupling.
The thermodynamic evaluation of this data, however, suffered from the severe solvatochromism, especially of the monomeric species (max(M, CH2Cl2) = 549 nm, max(M, MCH) = 596 nm). Therefore, concentration-dependent studies were performed at three different temperatures (298, 323, 353 K) to elucidate the self-assembly mechanism and determine reliable thermodynamic parameters. The studies at elevated temperatures were hereby necessary, to obtain experimental data over a larger agg--range. Due to the pronounced difference in the thermodynamic driving force for dimerization and higher aggregate formation (KD/K5 = 6500) a concentration range exists in MCH where almost exclusively the dimer species of 2 is present, before further self-assembly by dispersion interactions occurs. Therefore, the data could be evaluated independently for the two self-assembly steps. The self-assembly of dimers into the higher aggregate could not be described by the isodesmic model but was fitted satisfactorily to a pentamer model. This rather small size of about ten -stacked PYOP chromophores was, furthermore, consistently indicated by AFM, VPO and DOSY NMR measurements. Based on 1D and 2D NMR data as well as the strong bisignate CD signal of the higher aggregate in combination with TD-DFT calculations, a P-helical stack is proposed as its structure. The small size can be rationalized by the anti-cooperative self-assembly mechanism and the sterical demand of the solubilizing trialkoxyphenyl and the chiral tetralin substituents. Additionally, the aliphatic shell formed by the solubilizing chains around the polar chromophore stack, can account for the exceptionally high solubility of 2 in MCH (> 15 mg mL1). These combined studies of the self-assembly process enabled the identification of suitable conditions for the investigation of fluorescence properties of the individual aggregate species. Aggregation-induced emission enhancement was observed for the almost non-emissive monomer (Fl(M) = 0.23%), which can be rationalized by the increasing rigidification within the dimer (Fl(D) = 2.3%) and the higher aggregate (Fl(H) = 4.5%). The helical chirality of the PYOP decamer stack, furthermore, gave rise to a strong CPL signal with a large glum value of 0.011.
The important conclusion of this thesis is that the temperature- and solvent-dependent analyses are valid alternatives to the classical concentration-dependent analysis to determine thermodynamic parameters of self-assembly equilibria. Although, for a specific supramolecular system, one approach might be favourable over the others for a variety of reasons. The experimental limitations often demand a combination of techniques to fully elucidate a self-assembly process and to gain insights in the aggregate structure. The anti-cooperative merocyanine self-assembly, which was described here for the first time for the PYOP merocyanine 2, is no exception. Besides the interest in the merocyanine assemblies from a structural and functional point of view, the insights gained from the presented studies can also be transferred to other self-assembly systems and be a guide to find the most appropriate analysis technique.
This work aims at elucidating chemical processes involving homogeneous catalysis and photo–physical relaxation of excited molecules in the solid state. Furthermore, compounds with supposedly small singlet–triplet gaps and therefore biradicaloid character are investigated with respect to their electro–chemical behavior. The work on hydroboration catalysis via a reduced 9,10–diboraanthracene (DBA) was preformed in collaboration with the Wagner group in Frankfurt, more specifically Dr. Sven Prey, who performed all laboratory experiments. The investigation of delayed luminescence properties in arylboronic esters in their solid state was conducted in collaboration with the Marder group in Würzburg. The author of this work took part in the synthesis of the investigated compounds while being supervised by Dr. Zhu Wu. The final project was a collaboration with the group of Anukul Jana from Hyderabad, India who provided the experimental data.
In this thesis, we apply the information-theoretic approach in the context of quantum dynamics and wave packet motion: Information-theoretic measures are calculated from position and momentum densities, which are obtained from time-dependent quantum wave functions. The aim of this thesis is to benchmark, analyze and interpret these quantities and relate their features to the wave packet dynamics. Firstly, this is done for the harmonic oscillator (HO) with and without static disorder. In the unperturbed HO, the analytical study of coherent and squeezed states reveals time-dependent entropy expressions related to the localization of the wave function. In the disordered HO, entropies from classical and quantum dynamics are compared for short and long times. In the quantum case, imprints of wave packet revivals are found in the entropy. Then, the energy dependence of the entropy for very long times is discussed. Secondly, this is donefor correlated electron-nuclear motion. Here, entropies derived from the total, electronic and nuclear density, respectively, are calculated in position and momentum space for weak and strong adiabatic electronic coupling. The correlation between electron and nucleus is investigated using different correlation measures, where some of these functions are sensitive to the nodal structure of the wave function. An analytic ansatz to interpret the information-theoretical quantities is applied as well.
N\(^6\)-methyladenosine (m\(^6\)A) is an important modified nucleoside in cellular RNA associated with multiple cellular processes and is implicated in diseases. The enzymes associated with the dynamic installation and removal of m\(^6\)A are heavily investigated targets for drug research, which requires detailed knowledge of the recognition modes of m\(^6\)A by proteins. Here, we use atomic mutagenesis of m\(^6\)A to systematically investigate the mechanisms of the two human m\(^6\)A demethylase enzymes FTO and ALKBH5 and the binding modes of YTH reader proteins YTHDF2/DC1/DC2. Atomic mutagenesis refers to atom-specific changes that are introduced by chemical synthesis, such as the replacement of nitrogen by carbon atoms. Synthetic RNA oligonucleotides containing site-specifically incorporated 1-deaza-, 3-deaza-, and 7-deaza-m\(^6\)A nucleosides were prepared by solid-phase synthesis and their RNA binding and demethylation by recombinant proteins were evaluated. We found distinct differences in substrate recognition and transformation and revealed structural preferences for the enzymatic activity. The deaza m\(^6\)A analogues introduced in this work will be useful probes for other proteins in m\(^6\)A research.