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The investigation of complex molecular systems by molecular dynamics simulations has been successfully established and proven as a standard method during the last decades. The use of highly optimized algorithms and steadily increasing, generally available computing resources enables even larger and longer simulations. However, the dynamics of the system itself is not accelerated, and it can be trapped in low energy minima that can only be overcome slowly. A number of methods have therefore been developed to address this problem.
Within the context of this dissertation, a novel algorithm based on replica exchange was developed to solve problems with existing methods, which can now be used for large molecular systems with a low resource consumption. Parameter dependence was systematically evaluated and optimized to define guidelines for correct application. This algorithm was successfully applied to various pharmaceutical and biochemical problems, such as protein folding or protein-protein interactions.
The soluble blood protein beta2-glycoprotein I (beta2GPI; 326 aa, MW: 48 kDa, 5 domains) is one of the most abundant proteins in human serum and exhibits two main conformational states: the circular or closed conformation, where the first domain (DI) is bound to the last domain (DV) of the protein, and the linear or open conformation. The defined physiological function of beta2GPI is still unknown, though several roles in pro- and anticoagulation as well as oxidative stress protection were discovered. The open form is considered to play a crucial role in the systemic autoimmune disease antiphospholipid syndrome (APS), which is an acquired thrombophilia characterized by recurring thrombotic events and pregnancy morbidity. Beta2GPI constitutes the main antigen for APS autoantibodies which are supposed to bind a cryptic epitope within DI after a conformational change from closed to open form. However, the pathophysiological mechanism of APS is poorly understood. Therefore, investigating the structural dynamics of this protein in relation to its antigenicity is of high interest.
Post-translational modifications (PTM) of a target protein often show an impact on the formation of neoantigens, for instance in the autoimmune-mediated diseases type 1 diabetes mellitus, rheumatoid arthritis, or multiple sclerosis. Such modified antigens may lead to immune tolerance breakdown as they are unknown to the immune system, which therefore could mistakes self for non-self proteins. In this thesis, two frequently occurring PTM were introduced to beta2GPI and their impact on the protein conformation was studied by biophysical tools (i.e. atomic force microscopy (AFM) imaging, transmission electron microscopy (TEM) imaging, dynamic light scattering (DLS), and circular dichroism (CD) spectroscopy). In order to examine immunopathophysiological relevance of these PTM, additional insights were gained from ELISA which was used to examine binding of anti-DI autoantibodies purified from the blood of APS patients to the modified beta2GPI species.
A characteristic feature of beta2GPI is the high content of lysine residues. Previously, opening of beta2GPI was found to be triggered by a drastic shift in pH and salt concentration (pH 11.5 and 1.15 M NaCl), which results in reversible uncharging of the lysine residues. The aim of this study was to investigate the beta2GPI conformation after lysine acetylation as a model system, to elucidate the role of lysine residues on the conformational dynamics of this protein, and to examine anti-DI autoantibody binding to both the untreated as well as acetylated species.
A strategy to permanently open up the closed form under physiological conditions by chemical acetylation of lysine residues utilizing the sensitive acetylation agent acetic acid N-hydroxysuccinimide ester (NHS-Ac) was established. Complete and specific lysine acetylation was verified by quantification of primary amines exerting a fluoraldehyde o-phthaldialdehyde (OPA) reagent assay, as well as by native PAGE and western blot analysis with an anti-acetylated lysine antibody. Beta2GPI acetylation revealed a partial opening of beta2GPI molecules. Compared to untreated, i.e. native beta2GPI which exhibited 93% of the molecules in closed and 7% in open form, complete lysine residue acetylation generated 39% of beta2GPI in closed and 61% in open conformation as shown by AFM high-resolution imaging. pH 11.5-treated beta2GPI was used as a reference in the applied methods and revealed 38% of the protein in closed and 62% in open conformation. Thus, a significant shift in beta2GPI conformation occurred upon lysine residue acetylation as well as basic pH-treatment. The data indicate that lysine residue acetylation destabilizes the closed form, leading to a facilitated opening of the structure. The closed conformation might be predominantly stabilized by electrostatic interactions of lysine residues, which potentially control the conformational dynamics of this glycoprotein. ELISA confirmed that anti-DI autoantibodies do not bind to untreated (closed) beta2GPI. Although acetylated beta2GPI was shown to have a substantial portion of open proteins, no binding of anti-DI autoantibodies to the acetylated species was found either. Hence, acetylated lysine residues may disrupt the immunorelevant epitope in DI which prevents antibody binding. This finding reveals a new hint for epitope organization. However, further detailed epitope mapping has to be performed.
Beta2GPI carries two structural disulfide bonds per domain, whereas an additional disulfide bond Cys288/Cys326 is located at the C-terminus of DV near the putative contact interface of DI and DV in the closed conformation. It was previously shown that beta2GPI is a substrate of thiol oxidoreductases, including human thioredoxin-1 (Trx-1) generating different redox states of disulfide bond Cys288/Cys326, which might serve as a scavenger in oxidative stress protection in the blood stream. In APS patients, anti-DI antibody titers as well as an enhanced risk for thrombotic events are associated with an increase in the oxidized state of the protein. Hitherto, no structural study has been performed in order to prove a correlation of the redox state and the conformation of beta2GPI. Therefore, investigations of beta2GPI conformation in different redox states of disulfide bond Cys288/Cys326 were carried out. In addition, binding of anti-DI autoantibodies to the untreated (native) as well as reduced protein should be explored.
At first, cysteine residues of untreated, i.e. native beta2GPI were confirmed to be completely in oxidized state using Ellman’s reagent assay and the absence of binding of a thiol-specific agent. Statistical analyses of AFM images revealed that untreated beta2GPI was mainly in closed conformation (80% in closed and 20% in open conformation) in the respective system. In this study, an optimized protocol for enzymatic reduction of disulfide bond Cys288/Cys326 was established. The agent TCEP was used to reduce human Trx-1, which in turn enzymatically reduced beta2GPI. To block reoxidation of free thiols and to facilitate product analysis, cysteine residues of reduced beta2GPI were subsequently labeled with the sensitive and thiol-specific reagent 3-(N-maleimidopropionyl) biocytin (MPB), which carries a biotin function. During protocol establishment, complete and specific reduction of disulfide bond Cys288/Cys326 was confirmed utilizing SDS-PAGE, streptavidin western blot, mass spectrometry (MS) analyses, and a biotin quantification assay. Protocol improvements constituted a homogenous system with remarkable decrease of unspecifically reduced beta2GPI. Upon beta2GPI reduction, AFM imaging revealed no significant shift in protein conformation (75% in closed and 25% in open conformation). These results were qualitatively confirmed by TEM imaging. Therefore, reduction of beta2GPI disulfide bond Cys288/Cys326 did not result in a major conformational change of the protein. Upon in vitro reduction, the closed form is still the main conformation and a direct correlation of beta2GPI redox state and conformation must be refused. Furthermore, beta2GPI reduction led to a strong and statistically highly significant increase in anti-DI autoantibody binding compared to untreated beta2GPI. Thus, the reduced form might be the antigenic form of the protein. In contrast to previous knowledge, these findings suggest that anti-DI autoantibodies may also bind to the closed conformation under certain conditions. Hypothetically, reduction of beta2GPI could induce a minor structural change in DV that might facilitate the binding of APS autoantibodies.
Overall, this study reveals that PTM of beta2GPI may lead to a critical level of destabilization of the closed conformation (as in the case of acetylated beta2GPI) or significantly increase the binding of APS autoantibodies (as in the case of reduced beta2GPI), both of which could have a large impact on APS disease. However, further investigations are necessary to put these new findings in the context of APS immunopathophysiology.
Haloalkanes are serious environmental pollutants commonly employed as pesticides, herbicides, and chemical warfare agents. Although haloalkane production is performed mostly in the chemical industry, it also occurs naturally, mostly enzymatically (halide methyltransferases and haloperoxidases). Elimination of toxic haloalkanes is very important and using haloalkane dehalogenases is a promising and environmentally friendly way to achieve this.[53] Therefore, assays are needed for detecting dehalogenase activity either to find new enzymes or to generate laboratory-evolved variants. In this thesis, a new assay for dehalogenase activity was developed based on halide detection. In this assay halides, as dehalogenase products, are oxidized under mild conditions using the vanadium-dependent chloroperoxidase from Curvularia inaequalis, forming hypohalous acids that are detected using aminophenyl fluorescein.[53] This new halide oxidation assay is much more sensitive than previously known assays, with detection limits of 20 nM for bromide and 1 μM for chloride and iodide. Validation of the assay was done by comparison to a well-established GC-MS method in terms of determining the specific activities of two dehalogenases towards five common substrates (Figure 5).
The HOX assay was modified for iodide-specific detection by using two other dyes, o-phenylenediamine (OPD) and 3,3′,5,5′-tetramethylbenzidine (TBM), instead of APF. Also, selective bromide detection in the presence of the common contaminant chloride was achieved by using a bromoperoxidase. Since the assay relies on halide detection, it is possible to use it for other halide-producing enzymes (Section 8.1). For example, the TMB-modified version was used for screening of halide methyltransferase libraries towards various alkyl iodides.[166] Furthermore, the HOX assay was used to identify promiscuous dehalogenase activity of the epoxide hydrolase CorEH from Corynebacterium sp. C12.[105]
Moreover, studies showed that the HOX assay could be used with in-vitro synthesized protein. Selected dehalogenases, DhlA, DhaA, and DmmA, were synthesized in vitro and used in the assay; the product formation was also validated using GC-MS. In conclusion, the HOX assay can be used with purified protein, whole cells, or in vitro synthesized proteins.
The HOX assay application in microfluidic droplets was investigated since an ultra-high-throughput assay for haloalkane dehalogenases is needed. This investigation showed no leakage of reaction components and products in the short term (~24 h), based on tests done on water-in-oil droplets generated by microfluidic chips. Even though 20 μM droplets were not working, 70 μM droplets were successful for assay implementation. Since the Damborsky group in Brno (CZ) and the deMello group in Zürich (CH), have large dehalogenase libraries and more experience in microfluidics, respectively, we collaborated with these groups to finalize implementation of the assay in an ultrahigh-throughput format. Since the studies are ongoing, final results could not yet be shown in this thesis. However, it can be noted that the issue with 20 μm droplets has been sorted out since our collaborators in Brno noticed that the low fluorescence of the droplets is actually caused by excessive accumulation of fluorescein, which is self-quenching, resulting in low fluorescence once the concentration exceeds 1 μM. By lowering the APF concentration they could optimize the maximum amount of fluorescein formed, and a mutant library has now been successfully screened by our collaborators at the ETH. The last topic of the thesis was an investigation of converting an epoxide hydrolase into a haloalkane dehalogenase. These studies focused on increasing the minor dehalogenase activity of two previously identified epoxide hydrolase (Cif) variants. These Cif variants hardly led to soluble proteins, the PROSS algorithm was used to increase soluble expression. New variants of Cif were generated using a 3DM analysis and the PROSS[164] design. The activities of these variants were determined with the newly developed HOX assay in a whole-cell format. Cif23 E153N-H269D and the PROSS D7 E153N-H269D variant, were found being active against 1,2-dibromoethane. Since the determination of enzyme concentration was hard to measure due to the expression/purification problem, specific activities could not be determined. To solve this problem, a HiBiT-tag was added to the selected variants for determining soluble expression. However, the planned studies could not be completed because of a lack of time and will form the basis for a future study.
This thesis focuses on the establishment of biocatalytic cascade reactions for the production and detection of industrially relevant flavor and fragrance compounds for food and cosmetic products. To meet the consumer’s demand for those products to be natural, environmentally friendly biocatalytic manufacturing processes that operate GMO-free must be established. Thus, this thesis presents such pathways for the production of an industrially relevant long-chain hydroxy fatty acid and the important flavor and aroma compound raspberry ketone. Furthermore, a biosensor for aldehyde detection was implemented to facilitate screening for suitable biocatalysts that produce industrially relevant aldehydes that are widely applied in the flavor and fragrance industry.
Frequency of biopsies after the disclosure of incidental findings from whole-body research MRI
(2021)
Large-scale, population-based cohort studies gather a range of data from participants over extended periods of time with the goal of providing researchers with information regarding the health status, prevalence of disease, and risk factors in a regional population. Examinations conducted in the context of population-based studies include imaging and laboratory testing and may yield abnormal results, also called incidental findings. According to predetermined disclosure policies, incidental findings may be disclosed to study participants. Evidence shows that the disclosure of incidental findings results in medical follow-up as research participants and their physicians seek to clarify the significance of findings.
This work examined the effect of disclosing incidental findings from whole-body MRI (wb-MRI) on the frequency and organ system of biopsies in participants in the Study of Health in Pomerania (SHIP), a population-based cohort study in Mecklenburg-West Pomerania. As most of the incidental wb-MRI findings involved unclear masses and lesions, we hypothesized that the disclosure of wb-MRI findings would lead to an increase in diagnostic biopsies. Based on current data showing that the outcomes of incidental imaging findings are frequently clinically irrelevant, we further hypothesized that an increase in biopsies would not translate to a clinically relevant increase in diagnoses of malignancies. We also took disclosed laboratory findings into account, as they were disclosed to all SHIP participants and may play a role in the decision to pursue a biopsy.
We found that the rate of biopsies increased after participation in SHIP and disclosure of incidental MRI and laboratory findings. Overall, most biopsies showed nonmalignant findings, indicating likely overdiagnosis and overtesting resulting from the disclosure of incidental findings in our cohort. However, subgroups of participants with disclosed MRI findings had a higher proportion of biopsies revealing premalignant or malignant diagnoses after SHIP, indicating that the applied decision rules for disclosure of MRI findings led to the identification of individuals with an elevated risk for premalignant or malignant diagnoses. The clinical relevance of these diagnoses is unclear and overdiagnosis cannot be ruled out.
In summary, we recommend more restrictive disclosure policies for incidental imaging findings in research to protect research participants from overtesting and to reduce bias. Further studies regarding the long-term morbidity and mortality of participants are needed to better understand the therapeutic impact of the disclosure of incidental wb-MRI findings in the research setting.
Objectives: Clear guidelines on when to remove an implant are missing. This study aimed to evaluate the amount of peri-implant bone loss at explantation by specialists.
Material and Methods: Implantology specialists were asked to provide implants explanted due to peri-implantitis with related clinical information. Questionnaires inquired age, sex, smoking habit, implant location, usage of bone substitutes, and implant brand. Early failures (survival time <12 months) were analysed separately. Explants were measured and bone loss and type of bone loss were assessed using radiographs. Bivariate analysis was used for the type of bone loss, and covariate-adjusted mixed-effects models were evaluated for the amount of bone loss and survival time.
Results: Twelve dental offices provided 192 explants from 161 patients with 99 related radiographs. Most implants were affected by vertical bone loss (51.1%), followed by combined horizontal and vertical bone loss (22.3%), peri-implant gap (11.7%), horizontal bone loss (10.6%), and only a few by apical inflammation (4.3%). Thirty-three (17.2%) explants were early failures. Type of bone loss was significantly associated with survival time and implant brand. Implant brand also showed a significant correlation with early/late implant failure. Excluding early failures, combined horizontal and vertical bone loss was additionally significantly associated with smoking, and the location when grouped to incisor, canine, premolar, and molar showed a significant association with the type of bone loss. Further, the average survival time was 9.5 ± 5.8 years with absolute and relative bone loss of 7.0 ± 2.7 mm and 66.2 ± 23.7%, respectively. Late failures were removed at a mean bone loss of 50.0% with 5.44 mm residual alveolar bone in the posterior maxilla and 73.8% with 2.89 mm residual alveolar bone in other locations. In fully adjusted mixed-effects models, only the age at implantation (B=-0.19; 95% CI: -0.27 to -0.10) remained a significant factor for survival time. Implants exhibited significantly more relative bone loss if they were positioned in the mandible (B=17.3; 95% CI: 3.91 to 30.72) or if they were shorter (B=-2.79; 95% CI: -5.50 to -0.08).
Conclusions: Though the mean bone loss (66.2%) at which implants were explanted was in accordance with the literature, its wide variation and differentiation between the posterior maxilla and other locations showed that the profession has no universally accepted threshold beyond which an implant cannot be preserved.
This thesis discusses three publications in the field of dusty plasmas.
In the first section, measurements of the ir absorption of silica nanoparticles confined in an argon radiofrequency plasma discharge using a Fourier transform infrared spectrometer have been performed. By varying the gas pressure of the discharge and duty cycle of the applied radiofrequency voltage, a shift of the absorption peak of silica is observed. This shift is attributed to charge-dependent absorption features of silica. The charge-dependent shift has been calculated for silica particles, and from comparisons with the experiment the particle charge has been retrieved using the infrared phonon resonance shift method. With the two different approaches of changing the gas pressure and altering the duty cycle, one is able to deduce a relative change of the particle charge with pressure variations and an absolute estimate of the charge with the duty cycle.
In the second part, infrared (IR) absorption spectra of melamine-formaldehyde (MF) microparticles confined in an rf plasma are studied at different plasma conditions. Several absorption peaks have been analysed in dependence of plasma power and their temporal evolution. For comparison, the IR absorption spectra of heated MF microparticles without plasma exposition are used to determine the general influence of the temperature on the IR spectra. Measuring the temperature of the particles inside the plasma shows that the temperature is not the only process changing the particles' IR spectra. Chemical changes of the MF particles with increasing plasma power influence the absorption peak structure.
Finally, experiments on dust clusters trapped in the sheath of a radio frequency discharge have been performed for different magnetic field strengths ranging from a few milliteslas to 5.8 T. The dynamics of the dust clusters are analyzed in terms of their normal modes. From that, various dust properties such as the kinetic temperature, the dust charge, and the screening length are derived. It is found that the kinetic temperature of the cluster rises with the magnetic field, whereas the dust charge nearly remains constant. The screening length increases slightly at intermediate magnetic field strengths. Generally, the dust properties seem to correlate with magnetization parameters of the plasma electrons and ions, however only to a small degree.
The human innate response plays a pivotal role in detection of pathogen- or damage-associated molecular patterns (PAMPs and DAMPs) and contributes to a crucial inflammatory response. PAMPs or DAMPs are recognized by the host immune system via pattern recognition receptors (PRRs). NLR family pyrin domain-containing 3 (NLRP3) inflammasome is one of these PRRs. NLRP3 is a cytoplasmic immune sensor that upon activation produce pro-inflammatory cytokines such as IL-1β and IL-18. These cytokines induce a diverse range of protective host pathways aiming to eradicate the pathogen. However, excessive or chronic inflammasome activation are implicated in the pathogenesis of several autoimmune and auto-inflammatory disorders. Pharmacologic inhibitors of IL-1 are commonly used to combat these disorders. In paper I, we explore the currently available IL-1β inhibiting therapies and how patients undergoing these treatments are at a disproportionate risk to experience invasive bacterial infections. We also summarize the limited knowledge on the role of NLRP3 inflammasome in pneumococcal pathogenesis.
Hydrogen peroxide (H2O2) is a physiological metabolite and an important virulence determinant produced by pneumococci. It is highly cytotoxic to host cells. However, not much is known about its impact on host cell death pathways such as NLRP3 inflammasome mediated pyroptosis. In Paper II, we examined the effect of pneumococci-derived H2O2 on epithelial cells by analyzing the interplay between two key cell death pathways, namely apoptosis and pyroptosis. We show that H2O2 can prime as well as activate the NLRP3 inflammasome. Furthermore, we demonstrate that pneumococcal H2O2 initiates cell death via the activation of both apoptotic as well as pyroptotic pathways, mediated by the activation of caspase-3/7 and caspase-1, respectively. H2O2 mediated inflammasome activation results in caspase-1 dependent IL 1β production. However, we show that the final IL-1β release is independent of gasdermin-D (GSDMD) and mainly dependent on the apoptotic cell lysis.
In paper III, we focused on understanding the host metabolic responses to infections with pathogens which cause respiratory diseases. We performed metabolome profiling of in vitro single bacterial and viral as well as co-infections of bronchial epithelial cells with Influenza A virus (IAV), Streptococcus pneumoniae, and Staphylococcus aureus. We show that IAV and S. aureus use the host resources for survival and multiplication and have minimal effects on the host metabolome. In contrast, pneumococci significantly alter various host metabolome pathways, including glycolysis, tricarboxylic acid (TCA) cycle and amino-acid metabolism. A hallmark of pneumococcal infections was the intracellular citrate accumulation, which was directly attributed to the action of pneumococci-derived H2O2.
Host cell death during an infection results in the release of pro-inflammatory cytokines and danger signals such as ATP. Released ATP can induce neutrophil chemotaxis mediated via purinergic signaling. Neutrophils are typically the first leukocytes to be recruited to the site of infection and are key players in bacterial clearance. However, excessive neutrophil activation is associated with further tissue injury. In paper IV, we investigated the role of ATP in neutrophil response to pneumococcal infections. We show that pneumolysin (Ply), a highly effective pore-forming toxin produced by pneumococci, is a potent activator of neutrophils. Microscale Thermophoresis analysis revealed that Ply and ATP bind to each other. Subsequently, ATP binding neutralizes Ply-mediated neutrophil degranulation, suggesting that Ply-ATP interactions are potentially beneficial during the course of the infection as this could limit the lung injury resulting from excessive Ply-mediated neutrophil activation.
Due to demographic changes, medical and nursing care in Germany faces new challenges. Combined with the aging of the population, an increase in age-associated diseases, including dementia, is to be expected. In addition to the increase in the number of persons with certain age-specific diseases, the aging of the German population also results in an increase in the number of persons with multiple diseases. The coexistence of dementia and comorbidity in people with dementia creates complex challenges for ambulatory and clinical care. The existence of comorbidity also leads to significantly higher medical costs.
Implementing new collaborative care programs and redistributing the responsibilities among outpatient care providers in the ambulatory care of patients may be one approach to ensure and improve the life and care situation of people with dementia. Collaborative Dementia Care Management, with the concept of support of general practitioners by specific qualified nurses demonstrated an adequate and effective approach for the compensation of supply deficits of PwD in the primary care sector. The aim of the dissertation is the health economic analysis of comorbidities in dementia and the evaluated Dementia Care Management of the DelpHi-MV study as an innovative approach for care and treatment of comorbidities in people with dementia. It is assumed that the cost of care for PwD varies depending on comorbidity and socio-demographic and clinical characteristics. Therefore, the health care costs of people with dementia are calculated and the association between these care costs and comorbidity and socio-demographic and clinical factors of PwD was analyzed. In addition, we aimed to detect important subgroups (e.g. PwD with low, high or very high comorbidity) who benefit most from the DCM intervention and for whom a significant effect on costs, Quality-adjusted Life Years (QALY) and on the individual cost-effectiveness could be achieved, considering different sociodemographic and clinical characteristics like comorbidity.
In the sample of PwD comorbidity was highly prevalent. 47% of PwD had a very high, 37% a high and only 16% a low comorbidity in addition to dementia. The most prevalent co-existing comorbidity were diabetes mellitus (42%), peripheral vascular disease (28%) and cerebrovascular disease (25%). Total costs significantly increased by 528 € (SE=214, CI 95%=109-947, p=0.014) with each further comorbidity, especially due to significantly higher cost for medication and medical aids. Compared with a low comorbidity, a very high comorbidity was significantly associated with 818 € (SE=168, CI 95%= 489-1147, p<0.001) higher medication costs and 336 € (SE=161, CI 95%=20-652, p=0.037) higher cost for medical aids. There was no significant association between a higher comorbidity and cost for formal care services. The probability of DCM being cost-effective at a willingness-to-pay of 40,000€/QALY was higher especially in PwD having a high comorbidity (96% vs. 26% for patients with a low comorbidity), in females (96% vs. 16% for males), in those living alone (96% vs. 26% for those living not alone) and in those being moderately to severely cognitively (100% vs. 3% for patients without cognitive impairment) and functionally impaired (97% vs. 16% for patients without functional impairment).
Comorbidity in PwD represents a substantial financial burden on healthcare payer’s and is a challenge for patients, healthcare providers and the health system. Innovative approaches are needed to achieve a patient-oriented management of treatment and care in comorbid PwD to reduce long-term costs. Collaborative dementia care management is one approach to solve these problems in dementia care. Thereby, patients characteristics significantly affect the cost-effectiveness of collaborative care. Female patients, patients living alone, and those with a high comorbidity as well as those being moderately cognitively and functionally impaired benefit most from DCM. For those subgroups of patients, healthcare payers could gain the highest cost savings and the highest effects on QALYs when the DCM approach will be implemented.
Adaptation mechanisms within the B cell composition for successful human and murine pregnancies.
(2021)
Introduction
A well-balanced immune maternal status is essential for favourable outcome of pregnancy. Due to their complexities, not all immune adaptations that promote tolerance during pregnancy are known. To understand the adaptation of the B cell compartment, we analysed and compared B cell lymphopoiesis in different lymphoid tissues in a number of murine models.
Furthermore, we focused on the humoral immune response during pregnancy. We analysed immunoglobulin profiles in human subjects and mice during pregnancy.
These cellular alterations are subject to the influence of chemokines, among others. Therefore, we assessed serum levels of B cell activation factor to clarify its effects during pregnancy.
Methods
For analysis of the human peripheral B cell compartment, peripheral blood samples from age-matched non-pregnant and pregnant women without pregnancy complications, immunological disease or acute/chronic inflammation were collected and sub-classified into four different groups: non-pregnant, and first, second, or third trimester of pregnancy. The experiments, based on a mouse model, were performed with 8-week-old female mice: clinically healthy non-pregnant (CBA/J (H2k)), pregnant mice with normal gestation (BALB/c (H2d) x CBA/J (H2k)), and mice with pregnancy loss (DBA/2J (H2d) x CBA/J (H2k)). Subsequently, peripheral blood mononuclear cells from blood and lymphatic organs were isolated following standard protocols. The B cell analysis was performed by flow cytometry. The immunoglobulin serum levels of the human and murine subgroups were quantitated using Bio-Plex isotyping assay and analysed by a Bio-Plex reader. To quantify B cell activating factor (BAFF) in serum of pregnant and non-pregnant mice a BAFF enzyme-linked immunosorbent assay was used. The concentrations were determined by using a FLUOstar OPTIMA microplate reader. All statistical analyses were performed using the Kruskal–Wallis test with Dunn’s post-test in GraphPad Prism software. P values of < 0.05 were considered statistically significant.
Results
We were able to demonstrate B cell lymphopenia in mice bone marrow downstream of pre-pro B cells, irrespective of pregnancy outcome. The mature bone marrow B cells did not show this adjustment mechanism during normal gestation.
Closer inspection of the splenic tissue revealed expansion and activation of marginal zone B cells in mice with a normal pregnancy. However, this was not observed in mice suffering from pregnancy disturbances. Natural antibodies secreted from marginal zone B cells were also present at higher concentrations in serum of pregnant mice, compared to non-pregnant animals.
We also found significantly higher levels of natural antibodies in serum of pregnant women compared to non-pregnant age-matched controls. Analysis showed significantly lower levels of BAFF in mice with normal pregnancy as compared to non-pregnant mice.
Conclusions
We are able to show mechanisms within the B cell compartment as well as the change within the natural antibodies that might be crucial for successful pregnancy in both humans and mice. Furthermore, BAFF seems to play a central role as a mediator of peripheral B cell compartment and B cell lymphopoiesis in the bone marrow for successful pregnancy.
Objective: Extracellular vesicles (EV) are sub-1 μm bilayer lipid coated particles and have been shown play a role in long-term cardiovascular outcome after ischemic stroke. However, the dynamic change of EV after stroke and their implications for functional outcome have not yet been elucidated.
Methods: Serial blood samples from 110 subacute ischemic stroke patients enrolled in the prospective BAPTISe study were analyzed. All patients participated in the PHYS-STROKE trial and received 4-week aerobic training or relaxation sessions. Levels of endothelial-derived (EnV: Annexin V+, CD45–, CD41–, CD31+/CD144+/CD146+), leukocyte-derived (LV: Annexin V+, CD45+, CD41–), monocytic-derived (MoV: Annexin V+, CD41–, CD14+), neuronal-derived (NV: Annexin V+, CD41–, CD45–, CD31–, CD144–, CD146–, CD56+/CD171+/CD271+), and platelet-derived (PV: Annexin V+, CD41+) EV were assessed via fluorescence-activated cell sorting before and after the trial intervention. The levels of EV at baseline were dichotomized at the 75th percentile, with the EV levels at baseline above the 75th percentile classified as “high” otherwise as “low.” The dynamic of EV was classified based on the difference between baseline and post intervention, defining increases above the 75th percentile as “high increase” otherwise as “low increase.” Associations of baseline levels and change in EV concentrations with Barthel Index (BI) and cardiovascular events in the first 6 months post-stroke were analyzed using mixed model regression analyses and cox regression.
Results: Both before and after intervention PV formed the largest population of vesicles followed by NV and EnV. In mixed-model regression analyses, low NV [−8.57 (95% CI −15.53 to −1.57)] and low PV [−6.97 (95% CI −13.92 to −0.01)] at baseline were associated with lower BI in the first 6 months post-stroke. Patients with low increase in NV [8.69 (95% CI 2.08–15.34)] and LV [6.82 (95% CI 0.25–13.4)] were associated with reduced BI in the first 6 months post-stroke. Neither baseline vesicles nor their dynamic were associated with recurrent cardiovascular events.
Conclusion: This is the first report analyzing the concentration and the dynamic of EV regarding associations with functional outcome in patients with subacute stroke. Lower levels of PV and NV at baseline were associated with a worse functional outcome in the first 6 months post-stroke. Furthermore, an increase in NV and LV over time was associated with worse BI in the first 6 months post-stroke. Further investigation of the relationship between EV and their dynamic with functional outcome post-stroke are warranted.
Clinical Trial Registration: clinicaltrials.gov/, identifier: NCT01954797.
Midges are small mosquitoes that can transmit pathogens to susceptible hosts through their blood-sucking act. They are known as biological vectors that can transmit the bluetongue virus (BTV) and the Schmallenberg virus (SBV) to ruminants, among others. Various vector control measures can be used to curtail the spread of the virus during an epidemic. However, for effective vector measures, it is essential to have profound knowledge of the role of biting midges as vectors, as well as their biology and phenology. For several years, midges were not in the focus of research and there are still considerable gaps in knowledge. Therefore, the present work examines various aspects of biting midges of the genus Culicoides, whose function as vectors of the Schmallenberg virus was already proven at the beginning of the project.
The aim of the first part of this work was to determine the percentage of infected midges in various German areas in order to determine the influence of Culicoides midges within the virus epidemic. For this purpose, samples, collected during 2011 and 2012 as part of monitoring projects, were analysed. Additionally, in early 2013, various farms in southern and eastern regions of Germany, where SBV was considered to be largely absent, were equipped with UV traps. The small number of virus-positive samples did not allow a more precise assessment of the viral spread in culicoid midges. Instead, it revealed the importance to conduct targeted samplings of its vectors during an acute outbreak. Additionally, the presented results and statements made by several animal owners, gave reason to believe, that SBV must have affected the southern and eastern parts of Germany earlier than actually assumed. This would consequently have led to an increased immunity in host animals, which provides a reasonable explanation for the low positive values and is in agrement with the statements made by various farmers.
The second part of this work identifies the conditions and surrounding factors under which acute SBV diseases emerged in ruminants in the cold winter months of 2012/2013. After the diagnosis of several acute SBV infections of sheep in a sheep pen in Mecklenburg-Western Pomerania, culicoid midge activity could be proven. This demonstrates that, suitable conditions for its vectors given, an infection of SBV can also take place during wintertime. A more detailed analysis of the surrounding conditions revealed, that the outdoor temperatures during infection were consistently at values of at least 5-9 ° C for several consecutive days, which enabled the flight and blood-sucking activity of the midges within
the shelter.
Midge activity during wintertime represents a crucial component in understanding how the virus can outlast the cold season. A constant midge presence could lead to a low but permanent infection rate throughout the cold months, enabling a recurrence of the pathogen the following year. Instead, a longer vector-free timeperiod would point to other mechanisms that allow the virus to re-occur in Germany on a yearly basis. Thus, the acute cases of SBV infections in sheep rose the question of critical threshold temperatures, representing the beginning of midge activity. The investigation of several stables sheltering cattle, horse or sheep addressed potential differences between indoor and outdoor activity and whether the type of host animal has an influence on the beginning of the flight. In the third part of this work, a long vector-free period and several differences in the onset of midge activity between different types of host animals could be detected. It could also be illustrated that the progression of the flight began differently depending on the present type of host animal/type of stable. For all cattle stables and the sheep barn the first midge activity was measured indoors, whereas for horses, culicoid midges were found to become active either at the same time or almost simultaneously inside and outside the animal shelters. This suggests that the horse stables do not represent good breeding sites for midges, which might be attributed to husbandry practices. In addition, it was possible to determine specific threshold temperatures for the different types of host animals and for various midge species. Altogether, the late beginning of flight, measured at the beginning of March, was surprising. This raises more questions of alternative mechanisms enabling the virus to outlast the winter months. The documentation of species-specific threshold temperatures can be a useful tool f.i. within automated large stables to keep indoor temperatures under the threshold value in order to postpone the onset of culicoid activity of various vector species. This may help to prevent virus transmissions during winter or to evoke a delay in spring, making it more difficult for the virus to overwinter.
To be able to start instant defense measures during an ongoing virus epidemic, which is transmitted by Culicoides midges, reducing the ground-living midge larvae offers a promising option during the warm season. For targeted vector control measures, it is important to know the breeding sites of culicoid midge species. Therefore, four agriculturally used biotopes were sampled and compared to four biotopes of a forest-dominated area. The results clearly show that meadows per se are not suitable breeding habitats for Culicoides
spp. Only the influence of livestock animals induces their potential as developmental sites. The various biotopes of the forest-dominated region were less subject to anthropogenic influences. Although fewer individual midges were found here, it displayed a higher biodiversity than the agricultural habitats. These results demonstrate once more the potential of forests in regards to the preservation of biodiversity. In Particular, the alder on fen site revealed most midge species and also the highest number of collected specimens among the studied biotopes. That illustrates the high impact of this specific humid type of habitat in respect to species diversity and the need of its perpetuation.
As part of this work, new breeding sites for a variety of culicoid species were identified and assigned to the usually rather short profiles of known Culicoides species. For one part, previous observations of chosen substrates could be consolidated. Furthermore, new breeding substrates were identified. Additionally, information of abiotic factors such as ph-value, soil moisture or organic compound of all sampled breeding substrates obtained from a soil analysis, extended the knowledge about the species-specific choice of breeding habitats and their characteristical traits. The additional knowledge about potential breeding substrates and their soil factors might be useful for future epidemiological modelling approaches. It can also raise the effectiveness and accuracy of targeted vector control measurements during an epidemic outbreak. Therefore, it may indirectly contribute to the preservation of endangered rare species. However, there is still an enormous need for more research before this goal can be fully achieved.
Hypoxia is common in marine environments and a major stressor for marine organisms inhabiting benthic and intertidal zones. Several studies have explored the responses of these organisms to hypoxic stress at the whole organism level with a focus on energy metabolism and mitochondrial response, but the instrinsic mitochondrial responses that support the organelle’s function under hypoxia and reoxygenation (H/R) stress are not well understood. We studied the effects of acute H/R stress (10 min anoxia followed by 15 min reoxygenation) on mitochondrial respiration, production of reactive oxygen species (ROS) and posttranslational modifications (PTM) of the proteome in a marine facultative anaerobe, the blue mussel Mytilus edulis. The mussels’ mitochondria showed increased OXPHOS respiration and suppressed proton leak resulting in a higher coupling efficiency after H/R stress. ROS production decreased in both the resting (LEAK) and phosphorylating (OXPHOS) state indicating that M. edulis was able to prevent oxidative stress and mitochondrial damage during reoxygenation. Hypoxia did not lead to rearrangement of the mitochondrial supercomplexes but impacted the mitochondrial phosphoproteome including the proteins involved in OXPHOS, amino acid- and fatty acid catabolism, and protein quality control. This study indicates that mussels’ mitochondria possess intrinsic mechanisms (including regulation via reversible protein phosphorylation) that ensure high respiratory flux and mitigate oxidative damage during H/R stress and contribute to the hypoxia-tolerant mitochondrial phenotype of this metabolically plastic species.
The recent developments in artificial intelligence have the potential to facilitate new research methods in ecology. Especially Deep Convolutional Neural Networks (DCNNs) have been shown to outperform other approaches in automatic image analyses. Here we apply a DCNN to facilitate quantitative wood anatomical (QWA) analyses, where the main challenges reside in the detection of a high number of cells, in the intrinsic variability of wood anatomical features, and in the sample quality. To properly classify and interpret features within the images, DCNNs need to undergo a training stage. We performed the training with images from transversal wood anatomical sections, together with manually created optimal outputs of the target cell areas. The target species included an example for the most common wood anatomical structures: four conifer species; a diffuse-porous species, black alder (Alnus glutinosa L.); a diffuse to semi-diffuse-porous species, European beech (Fagus sylvatica L.); and a ring-porous species, sessile oak (Quercus petraea Liebl.). The DCNN was created in Python with Pytorch, and relies on a Mask-RCNN architecture. The developed algorithm detects and segments cells, and provides information on the measurement accuracy. To evaluate the performance of this tool we compared our Mask-RCNN outputs with U-Net, a model architecture employed in a similar study, and with ROXAS, a program based on traditional image analysis techniques. First, we evaluated how many target cells were correctly recognized. Next, we assessed the cell measurement accuracy by evaluating the number of pixels that were correctly assigned to each target cell. Overall, the “learning process” defining artificial intelligence plays a key role in overcoming the issues that are usually manually solved in QWA analyses. Mask-RCNN is the model that better detects which are the features characterizing a target cell when these issues occur. In general, U-Net did not attain the other algorithms’ performance, while ROXAS performed best for conifers, and Mask-RCNN showed the highest accuracy in detecting target cells and segmenting lumen areas of angiosperms. Our research demonstrates that future software tools for QWA analyses would greatly benefit from using DCNNs, saving time during the analysis phase, and providing a flexible approach that allows model retraining.
Quality of Life in Young Adults With Cerebral Palsy: A Longitudinal Analysis of the SPARCLE Study
(2021)
Introduction: While most people with cerebral palsy (CP) will have a life expectancy similar to that of the general population, international research has primarily focused on childhood and adolescence; and knowledge about the quality of life (QoL) of young adults with CP, its trajectories, and associated factors remains scarce.
Methods: This longitudinal study included young adults with CP living in five European regions and who had previously participated in the SPARCLE cohort as children and/or adolescents. Their QoL in the psychological well-being and social relationships domains was estimated using age-appropriate validated instruments (KIDSCREEN-52 in childhood/adolescence and WHOQOL-Bref in young adulthood). We used generalized linear mixed-effect models with random intercept to estimate long-term trajectories of QoL in both domains and to investigate whether severity of impairment, pain, and seizure influenced these trajectories. We sought to identify potentially different trajectories of QoL from childhood to adulthood using a shape-based clustering method.
Results: In total, 164 young adults with CP aged 22–27 years participated in the study. Psychological well-being linearly decreased by 0.78 points (scale 0–100) per year (95% confidence interval (CI) −0.99 to −0.56) from childhood to young adulthood, whereas QoL in the social relationships domain increased (β coefficient 1.24, 95% CI 0.92–1.55). Severity of impairment was associated with reduced QoL in all life periods of the study (childhood, adolescence, and young adulthood): motor impairment with social relationships, and more nuancedly intellectual disability with psychological well-being and social relationships. At all periods, frequent pain significantly reduced psychological well-being, and seizures were associated with lower QoL in the social relationships domain. In both domains, we identified a group of individuals with CP who presented a reverse trajectory compared with the general QoL trajectory.
Conclusion: Identification of QoL trajectories and their associated factors yields improved knowledge about the experience of individuals with CP until young adulthood. Further studies are needed to better understand the determinants that have the greatest influence on the different shapes of long-term trajectories of QoL.
The pathobiont Streptococcus pneumoniae causes life-threatening diseases, including pneumonia, sepsis, meningitis, or non-invasive infections such as otitis media. Serine proteases are enzymes that have been emerged during evolution as one of the most abundant and functionally diverse group of proteins in eukaryotic and prokaryotic organisms. S. pneumoniae expresses up to four extracellular serine proteases belonging to the category of trypsin-like or subtilisin-like family proteins: HtrA, SFP, PrtA, and CbpG. These serine proteases have recently received increasing attention because of their immunogenicity and pivotal role in the interaction with host proteins. This review is summarizing and focusing on the molecular and functional analysis of pneumococcal serine proteases, thereby discussing their contribution to pathogenesis.
This thesis describes how the data of the Langmuir probes in the Wendelstein 7-X (W7X) Test Divertor Unit (TDU) were evaluated, checked for consistency with other diagnostics and used to analyse plasma detachment.
Langmuir probes are an electronic diagnostic, and were among the first to be used in plasma physics to determine particle fluxes, potentials, temperatures and densities.
W7X is a large, advanced stellarator, magnetic confinement fusion experiment, operated at the Max-Planck-Institut for Plasma Physics(IPP) in Greifswald, Germany.
Its TDU is an uncooled graphite component, shaped and positioned to intercept the convective heat load of the plasma.
Detachment describes a desirable operation state of strongly reduced loads on this component.
The evaluation of Langmuir probe data relies heavily on models of the sheath, formed at the interface between plasma and a solid surface, to infer plasma parameters from the directly measured quantities.
Multiple such models are analysed, generalised, and adapted to our use case.
A detailed comparison is made to determine the most suitable model, as this choice strongly affects the predicted parameters.
Special attention is paid to uncertainties on the parameters, which are determined using a Bayesian framework.
From the inferred parameters, heat and particle fluxes are calculated.
These are also indirectly measured by two other, camera-based diagnostic systems.
Observations are compared to test the validity of assumptions and calculations in the evaluation of all three diagnostics by checking their results for consistency.
The first comparison, with the infrared emission camera system, shows good agreement with theoretical predictions and reported measurements of the sheath transmission factor, for which we derive and measure a value in W7X.
Parameter dependencies in the quality of this agreement hint at remaining issues.
The second comparison, with the Hydrogen alpha photon flux camera system, shows significant discrepancy with expectations.
These are argued to originate from systematic differences in the measurement locations, which are quantified and related to the magnetic topology.
Langmuir probe observations of individual discharges are analysed to discuss conditions under which detachment occurs, transition into that state and fluctuations observed prior to and during it.
A spatial parametrisation of the data is developed and used to facilitate this.
These observations contribute to the larger aim of understanding particle balance control and fusion plasma edge processes.
Three-dimensionally extended dusty plasmas containing mixtures of two particle species of different size have been investigated on parabolic flights. To distinguish the species even at small size disparities, one of the species is marked with a fluorescent dye, and a two-camera video microscopy setup is used for position determination and tracking. Phase separation is found even when the size disparity is below 5%. Particles are tracked to obtain the diffusion flux, and resulting diffusion coefficients are in the expected range for a phase separation process driven by plasma forces. Additionally, a measure for the strength of the phase separation is presented that allows to quickly characterize measurements. There is a clear correlation between size disparity and phase separation strength.
Molecular dynamics simulations of binary dusty plasmas have been performed and their behavior with respect to the phase separation process has been analyzed. Here as well, it is found that even the smallest size disparities lead to phase separation. The separation is due to the force imbalance on the two species and the separation becomes weaker with increasing mean particle size.
In the second part of the thesis, Experiments on self-excited dust-density waves under various magnetic fields have been performed. For that purpose, different dust clouds of micrometer-sized dust particles were trapped in the sheath of a radio frequency discharge. The self-excited dust-density waves were studied for magnetic field strengths ranging from 0 mT to about 2 T. It was observed that the waves are very coherent at the lowest fields (B < 20 mT). At medium fields (20 mT < B < 300 mT), the waves seem to feature a complex competition between different wave modes before, at even higher fields, the waves become more coherent again. At the highest fields (B > 1 T), the wave activity is diminished. The corresponding wave frequencies and wavenumbers have been derived. From the comparison of the measured wave properties and a model dispersion relation, the ion density and the dust charge are extracted. Both quantities show only little variation with magnetic field strength.
The development of the two main types of diabetes mellitus, type 1 and type 2 (T1D, T2D), is closely associated with the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in insulin-secreting pancreatic β-cells. In T1D, β-cell death
is triggered by proinflammatory cytokines, which mainly lead to the formation of ROS
in mitochondria and RNS in the cytosol. Pancreatic β-cells are extraordinarily sensitive
to oxidative stress due to their low glutathione peroxidase and catalase expression.
Thus, hydrogen peroxide (H2O2) cannot be detoxified, neither sufficiently, nor rapidly.
H2O2 itself is a rather weakly reactive ROS but can react in the Fenton reaction to form
highly reactive hydroxyl radicals (●OH), that can damage cells in a variety of ways and
induce cell death. The cell and its organelles are bounded by biological membranes
that differ in their permeability to H2O2. Aquaporins (AQPs) are water-transporting
transmembrane proteins, and some isoforms have been shown to facilitate a bidirectional transport of H2O2 across cellular membranes in addition to water. The role of
AQP8 was investigated in an insulin-producing cell model by stably overexpressing
AQP8 (AQP8↑) and by a CRISPR/Cas9-mediated AQP8 knockout. However, AQP8
proved to be an essential protein for the viability of the insulin-producing RINm5F cells, and so we established a tet-on-regulated AQP8 knockdown (AQP8 KD). Our results highlight that AQP8 is involved in H2O2 transport across the plasma and mitochondrial membranes, and that AQP8 expression gets upregulated by proinflammatory cytokines (in vitro) and in an acutely diabetic rat model (in vivo). Furthermore, it was shown that the increased proinflammatory cytokine toxicity is due to enhanced mitochondrial oxidative stress, because H2O2 cannot be efficiently transported in AQP8 KD cells and ●OH
are increasingly generated. Caspase activity then raises, and apoptosis is increasingly
induced coupled with a proportion of ferroptosis-mediated cell death because of a concomitant decrease in nitric oxide (NO●) concentration. In conclusion, AQP8 is localized in the plasma and mitochondrial membrane of insulin-producing RINm5F cells, where it is involved in H2O2 transport. In T1D, AQP8 plays an important role in the transport of H2O2 from the mitochondrial matrix to the cytosol so that the concentration is lowered in the mitochondria. This wider distribution of H2O2 may ease the inactivation of H2O2.
Allicin (diallyl thiosulfinate) is the major thiol-reactive organosulfur compound produced by garlic plants (Allium sativum) upon tissue damage. Allicin exerts its strong antimicrobial activity against bacteria and fungi via S-thioallylation of protein thiols and low molecular weight thiols. Here, we investigated the effect of allicin on SARS-CoV-2 infected Vero E6 and Calu-3 cells. Toxicity tests revealed that Calu-3 cells showed greater allicin tolerance, probably due to >4-fold higher GSH levels compared to the very sensitive Vero E6 cells. Exposure of infected Vero E6 and Calu-3 cells to biocompatible allicin doses led to a ∼60–70% decrease of viral RNA and infectious viral particles. Label-free quantitative proteomics was used to investigate the changes in the Calu-3 proteome after SARS-CoV-2 infection and the effect of allicin on the host-virus proteome. SARS-CoV-2 infection of Calu-3 cells caused a strong induction of the antiviral interferon-stimulated gene (ISG) signature, including several antiviral effectors, such as cGAS, Mx1, IFIT, IFIH, IFI16, IFI44, OAS, and ISG15, pathways of vesicular transport, tight junctions (KIF5A/B/C, OSBPL2, CLTCL1, and ARHGAP17) and ubiquitin modification (UBE2L3/5), as well as reprogramming of host metabolism, transcription and translation. Allicin treatment of infected Calu-3 cells reduced the expression of IFN signaling pathways and ISG effectors and reverted several host pathways to levels of uninfected cells. Allicin further reduced the abundance of the structural viral proteins N, M, S and ORF3 in the host-virus proteome. In conclusion, our data demonstrate the antiviral and immunomodulatory activity of biocompatible doses of allicin in SARS-CoV-2-infected cell cultures. Future drug research should be directed to exploit the thiol-reactivity of allicin derivatives with increased stability and lower human cell toxicity as antiviral lead compounds.
Modern space missions depend more and more on electric propulsion devices for in-space
flights. The superior efficiency by ionizing the feedgas and propelling them using electric
fields with regard to conventional chemical thrusters makes them a great alternative. To
find optimized thruster designs is of high importance for industrial applications. Building
new prototypes is very expensive and takes a lot of time. A cheaper alternative is to rely
on computer simulations to get a deeper understanding of the underlying physics. In order
to gain a realistic simulation the whole system has to be taken into account including the
channel and the plume region. Because numerical models have to resolve the smallest time
and spatial scales, simulations take up an unfeasible amount of time. Usually a self-similarity
scaling scheme is used to greatly speed up these simulations. Until now the limits of this
method have not been thoroughly discussed. Therefore, this thesis investigates the limits
and the influence of the self-similarity scheme on simulations of ion thrusters. The aim
is to validate the self-similarity scaling and to look for application oriented tools to use
for thruster design optimization. As a test system the High-Efficiency-Multistage-Plasma
thruster (HEMP-T) is considered.
To simulate the HEMP-T a fully kinetic method is necessary. For low-temperature plasmas,
as found in the HEMP-T, the Particle-in-Cell (PIC) method has proven to be the best
choice. Unfortunately, PIC requires high spatial and temporal resolution and is hence
computationally costly. This limits the size of the devices PIC is able to simulate as well
as limiting the exploration of a wider design space of different thrusters. The whole system
is physically described using the Boltzmann and Maxwell equations. Using these system
of equations invariants can be derived. In the past, these invariants were used to derive a
self-similarity scaling law, maintaining the exact solution for the plasma volume, which is
applicable to ion thrusters and other plasmas. With the aid of the self-similarity scaling
scheme the computation cost can be reduced drastically. The drawback of the geometrical
scaling of the system is, that the plasma density and therefore the Debye length does not
scale. This expands the length at which charge separation occurs in respect to the system
size. In this thesis the limits of this scaling are investigated and the influence of the scaling
at higher scaling factors is studied. The specific HEMP-T design chosen for these studies is
the DP1.
Because the application of scaling laws is limited by the increasing influence of charge separation with increased scaling, PIC simulations still are computationally costly. Another approach to explore a wider design space is given using Multi-Objective-Design-Optimization
(MDO). MDO uses different tools to generate optimized thruster designs in a comparatively
short amount of time. This new approach is validated using the PIC method. During this
validation the drawback of the MDO surfaces. The MDO calculations are not self-consistent
and are based on empirical values of old thruster designs as input parameters, which not
necessarily match the new optimized thruster design. By simulating the optimized thruster
design with PIC and recalculate the former input parameters, a more realistic thruster design is achieved. This process can be repeated iteratively. The combination of self-consistent
PIC simulations with the performance of MDO is a great way to generate optimized thruster
designs in a comparatively short amount of time. The proof of concept of such a combination
is the pinnacle of this thesis.
ABSTRACT
In the present study, we performed gastropod analyses on loess–palaeosol sequences from northeast Armenia (Southern Caucasia) covering at least three glacial–interglacial cycles. The elaborated ecostratigraphy shows significant patterns of species composition related to the succession of pedocomplexes and loess, respectively. Pedocomplexes included species that can be associated with high‐grass to forest‐steppe biomes, indicating increased humidity for these sections compared to the loess layers. In contrast, loess layers that relate to glacial periods are associated with gastropod species of semidesert environments with shrub‐ and shortgrass‐steppes, indicating semiarid to arid conditions. Furthermore, the loess deposits do not show any evidence for cold‐adapted gastropod species. Therefore, we suggest that average July temperatures in the study area were above 10 °C, even during periods of loess deposition. Consequently, we propose that the limiting factor for tree growth during glacial periods was aridity, rather than temperature. In addition, we observe environmental differences between the various glacial times, with our results indicating a trend towards steadily increasing aridity in Southern Caucasia across the Middle to Late Pleistocene.
Direct and Indirect Effects of Environmental Limitations on White Spruce Xylem Anatomy at Treeline
(2021)
Treeline ecosystems are of great scientific interest to study the effects of limiting environmental conditions on tree growth. However, tree growth is multidimensional, with complex interactions between height and radial growth. In this study, we aimed to disentangle effects of height and climate on xylem anatomy of white spruce [Picea glauca (Moench) Voss] at three treeline sites in Alaska; i.e., one warm and drought-limited, and two cold, temperature-limited. To analyze general growth differences between trees from different sites, we used data on annual ring width, diameter at breast height (DBH), and tree height. A representative subset of the samples was used to investigate xylem anatomical traits. We then used linear mixed-effects models to estimate the effects of height and climatic variables on our study traits. Our study showed that xylem anatomical traits in white spruce can be directly and indirectly controlled by environmental conditions: hydraulic-related traits seem to be mainly influenced by tree height, especially in the earlywood. Thus, they are indirectly driven by environmental conditions, through the environment’s effects on tree height. Traits related to mechanical support show a direct response to environmental conditions, mainly temperature, especially in the latewood. These results highlight the importance of assessing tree growth in a multidimensional way by considering both direct and indirect effects of environmental forcing to better understand the complexity of tree growth responses to the environment.
Trametes spec. laccase (EC 1.10.3.2.) mediates the oxidative coupling of antibiotics with sulfonamide or sulfone structures with 2,5-dihydroxybenzene derivatives to form new heterodimers and heterotrimers. These heteromolecular hybrid products are formed by nuclear amination of the p-hydroquinones with the primary amino group of the sulfonamide or sulfone antibiotics, and they inhibited in vitro the growth of Staphylococcus species, including multidrug-resistant strains.
Objective: In the rat, the pancreatic islet transplantation model is an established method to induce hepatocellular carcinomas (HCC), due to insulin-mediated metabolic and molecular alterations like increased glycolysis and de novo lipogenesis and the oncogenic AKT/mTOR pathway including upregulation of the transcription factor Carbohydrate-response element-binding protein (ChREBP). ChREBP could therefore represent an essential oncogenic co-factor during hormonally induced hepatocarcinogenesis. Methods: Pancreatic islet transplantation was implemented in diabetic C57Bl/6J (wild type, WT) and ChREBP-knockout (KO) mice for 6 and 12 months. Liver tissue was examined using histology, immunohistochemistry, electron microscopy and Western blot analysis. Finally, we performed NGS-based transcriptome analysis between WT and KO liver tumor tissues. Results: Three hepatocellular carcinomas were detectable after 6 and 12 months in diabetic transplanted WT mice, but only one in a KO mouse after 12 months. Pre-neoplastic clear cell foci (CCF) were also present in liver acini downstream of the islets in WT and KO mice. In KO tumors, glycolysis, de novo lipogenesis and AKT/mTOR signalling were strongly downregulated compared to WT lesions. Extrafocal liver tissue of diabetic, transplanted KO mice revealed less glycogen storage and proliferative activity than WT mice. From transcriptome analysis, we identified a set of transcripts pertaining to metabolic, oncogenic and immunogenic pathways that are differentially expressed between tumors of WT and KO mice. Of 315 metabolism-associated genes, we observed 199 genes that displayed upregulation in the tumor of WT mice, whereas 116 transcripts showed their downregulated expression in KO mice tumor. Conclusions: The pancreatic islet transplantation model is a suitable method to study hormonally induced hepatocarcinogenesis also in mice, allowing combination with gene knockout models. Our data indicate that deletion of ChREBP delays insulin-induced hepatocarcinogenesis, suggesting a combined oncogenic and lipogenic function of ChREBP along AKT/mTOR-mediated proliferation of hepatocytes and induction of hepatocellular carcinoma.
The objectives of this study were to ascertain the fecal ESBL/AmpC-E. coli prevalence and to detect risk factors for their occurrence in young pre-weaned calves and their dams on large dairy farms in Germany. From 2018–2019 we investigated 2816 individual fecal samples from pre-weaned dairy calves and their dams, representing seventy-two farms (mean = 667 milking cows) from eight German federal states. To assess possible risk factors associated with ESBL/AmpC-E. coli prevalence in calves and dams, a questionnaire was performed, collecting management data. We observed an ESBL/AmpC-E. coli prevalence of 63.5% (95% CI: 57.4–69.5) among the sampled calves and 18.0% (95% CI: 12.5–23.5) among the dams. On all farms, at least one positive sample was obtained. To date, this is the highest ESBL/AmpC-E. coli prevalence observed in dairy herds in Europe. Feeding with waste milk was identified as a significant risk factor for a high prevalence of ESBL/AmpC-E. coli in calves. Many calves at large dairies in Germany are fed with waste milk due to the large amounts generated as a result of antibiotic dry-off routines and mastitis treatment with antibiotics. Other notable risk factors for high ESBL/AmpC-E. coli in calves were the general fitness/health of dams and calves, and the quality of farm hygiene. Taken together, these findings suggest that new or improved approaches to animal health management, for example, antibiotic dry cow management (selective dry cow therapy) and mastitis treatment (high self-recovery), as well as farm hygiene, should be researched and implemented.
Background: The management of degenerative hip diseases in young patients remains a challenge. Despite the improvement of hip-preserving procedures, total hip arthroplasty (THA) may be required in some instances. In addition, young patients undergoing THA have high expectations concerning their postoperative level of activity. Purpose: (1) to define the sports activity level and the return to sports after THA, (2) to describe the modification or initiation of new sports disciplines, and (3) to report the clinically meaningful outcomes after THA in patients younger than 40 years. Methods: A total of 36 patients (40 hips) were prospectively analyzed at a midterm follow-up of 3.9 years. The modified Harris Hip Score (mHHS); the Visual Analog Scale (VAS) for pain; the University of California, Los Angeles (UCLA) activity scale; and sports and recreational activity levels were assessed via questionnaire. The minimal clinically important difference (MCID) was determined by calculating half of the standard deviation, and the substantial clinical benefit (SCB) as well as patient acceptable symptomatic state (PASS), were calculated by the anchor method for the mHHS. Results: At the final follow-up, there was a significant improvement in mHHS (34.1 to 92.6; p < 0.0001), UCLA (3.2 to 7.6; p < 0.0001), and VAS for pain (8 to 1; p < 0.0001). More patients were active in sports at follow-up than before surgery (44% to 92%, p < 0.0001). In addition, the duration and frequency of sports activities showed a significant increase (p < 0.0001). The MCID, SCB and PASS for mHHS were 89% and 58%, respectively. No revision surgery had to be performed. Conclusion: This study showed that a large proportion of patients under 40 years of age who underwent THA increased their physical activity. Eighty-six percent of the patients were highly active, with a UCLA score ≥ 7. Furthermore, the reported MCID, SCB, and PASS for mHHS were achieved by more than 80% of patients.
Abstract
The article focuses on whether and to which extent heritage bilinguals make use of their heritage language while developing receptive skills in unknown languages which are either related to the majority language or the heritage language. Thirty four adolescent heritage speakers of Russian and Polish and a control group of thirty three German monolinguals were first exposed to a text in Swedish. The monolingual control group was matched with regard to age, educational background, foreign languages learned at school as well as proficiency in English. All participants had to determine the parts of speech of ten items from the text, translate them into German, and extract the main pieces of information from the text. In a second step, the heritage speakers completed a similar task with an unknown Slavic target language (Serbian). The results revealed no bilingual advantage of the heritage speakers over the monolinguals in the Swedish task. Furthermore, they scored lower in the Serbian trial. We treat this as evidence that access to the heritage language as a resource for solving these tasks is limited compared to the majority language and English which might be due to lesser metalinguistic knowledge about structures of the heritage language.
Non-invasive physical plasma (NIPP) achieves biomedical effects primarily through the formation of reactive oxygen and nitrogen species. In clinical use, these species interact with cells of the treated tissue, affecting the cytoplasmic membrane first. The present study investigated the permeability of the cytoplasmic membrane of breast cancer cells with different fluorescent dyes after NIPP treatment and determined the subsequent effects on cell viability. After NIPP treatment and the associated formation of reactive oxygen species, low molecular weight compounds were able to pass through the cytoplasmic membrane in both directions to a higher extent. Consequently, a loss of cellular ATP into the extracellular space was induced. Due to these limitations in cell physiology, apoptosis was induced in the cancer cells and the entire cell population exhibited decreased cell growth. It can be concluded that NIPP treatment disturbs the biochemical functionality of the cytoplasmic membrane of cancer cells, which massively impairs their viability. This observation opens a vast application horizon of NIPP therapy to treat precancerous and malignant diseases beyond breast cancer therapy.
Background: Gastrointestinal hormones (GIHs) are crucial for the regulation of a variety of physiological functions and have been linked to hunger, satiety, and appetite control. Thus, they might constitute meaningful biomarkers in longitudinal and interventional studies on eating behavior and body weight control. However, little is known about the physiological levels of GIHs, their intra-individual stability over time, and their interaction with other metabolic and lifestyle-related parameters. Therefore, the aim of this pilot study is to investigate the intra-individual stability of GIHs in normal-weight adults over time. Methods: Plasma concentrations of ghrelin, leptin, GLP-1 (glucagon-like-peptide), and PP (pancreatic polypeptide) were assessed by enzyme-linked immunosorbent assay (ELISA) in 17 normal-weight, healthy adults in a longitudinal design at baseline and at follow-up six months later. The reliability of the measurements was estimated using intra-class correlation (ICC). In a second step, we considered the stability of GIH levels after controlling for changes in blood glucose and hemoglobin A1 (HbA1c) as well as self-reported physical activity and dietary habits. Results: We found excellent reliability for ghrelin, good reliability for GLP1 and PP, and moderate reliability for leptin. After considering glucose, HbA1c, physical activity, and dietary habits as co-variates, the reliability of ghrelin, GLP1, and PP did not change significantly; the reliability of leptin changed to poor reliability. Conclusions: The GIHs ghrelin, GLP1, and PP demonstrated good to excellent test–retest reliability in healthy individuals, a finding that was not modified after adjusting for glucose control, physical activity, or dietary habits. Leptin showed only moderate to poor reliability, which might be linked to weight fluctuations, albeit small, between baseline and follow-up assessment in our study sample. Together, these findings support that ghrelin, GLP1, and PP might be further examined as biomarkers in studies on weight control, with GLP1 and PP serving as anorexic markers and ghrelin as an orexigenic marker. Additional reliability studies in obese individuals are necessary to verify or refute our findings for this cohort.
Simple Summary
Hepatocellular carcinoma (HCC) ranks second among the leading causes of cancer-related death. Since current therapeutic options are very limited, a deeper understanding of the molecular mechanisms underlying the tumor onset and progression of HCC holds great potential for improved therapeutic options. Although it has been shown that deleted in liver cancer 1 (DLC1) acts as a tumor suppressor whose allele is lost in 50% of liver cancers, alterations in gene expression initiated by DLC1 loss have not yet been the primary focus of liver cancer research. To identify novel gene targets that allow for a personalized medicine approach for HCC therapy, we performed gene expression profiling for HepG2 cells stably expressing DLC1shRNA. We provide evidence that TSPAN5 is required for HCC growth, migration and invasion, and dissected the underlying molecular mechanisms involving myocardin-related transcription factors. Thus, TSPAN5 represents a novel therapeutic target for the treatment of HCC characterized by DLC1 loss.
Abstract
Human hepatocellular carcinoma (HCC) is among the most lethal and common cancers in the human population, and new molecular targets for therapeutic intervention are urgently needed. Deleted in liver cancer 1 (DLC1) was originally identified as a tumor suppressor gene in human HCC. DLC1 is a Rho-GTPase-activating protein (RhoGAP) which accelerates the return of RhoGTPases to an inactive state. We recently described that the restoration of DLC1 expression induces cellular senescence. However, this principle is not amenable to direct therapeutic targeting. We therefore performed gene expression profiling for HepG2 cells depleted of DLC1 to identify druggable gene targets mediating the effects of DLC1 on senescence induction. This approach revealed that versican (VCAN), tetraspanin 5 (TSPAN5) and N-cadherin (CDH2) were strongly upregulated upon DLC1 depletion in HCC cells, but only TSPAN5 affected the proliferation of HCC cells and human HCC. The depletion of TSPAN5 induced oncogene-induced senescence (OIS), mediated by the p16INK4a/pRb pathways. Mechanistically, silencing TSPAN5 reduced actin polymerization and thereby myocardin-related transcription factor A- filamin A (MRTF-A-FLNA) complex formation, resulting in decreased expression of MRTF/SRF-dependent target genes and senescence induction in vitro and in vivo. Our results identify TSPAN5 as a novel druggable target for HCC.
Anaplasma phagocytophilum and Anaplasma ovis–Emerging Pathogens in the German Sheep Population
(2021)
Knowledge on the occurrence of pathogenic tick-borne bacteria Anaplasma phagocytophilum and Anaplasma ovis is scarce in sheep from Germany. In 2020, owners from five flocks reported ill thrift lambs and ewes with tick infestation. Out of 67 affected sheep, 55 animals were clinically examined and hematological values, blood chemistry and fecal examinations were performed to investigate the underlying disease causes. Serological tests (cELISA, IFAT) and qPCR were applied to all affected sheep to rule out A. phagocytophilum and A. ovis as a differential diagnosis. Ticks were collected from selected pastures and tested by qPCR. Most animals (n = 43) suffered from selenium deficiency and endoparasites were detected in each flock. Anaplasma spp. antibodies were determined in 59% of examined sheep. Seventeen animals tested positive for A. phagocytophilum by qPCR from all flocks and A. phagocytophilum was also detected in eight pools of Ixodes ricinus. Anaplasma phagocytophilum isolates from sheep and ticks were genotyped using three genes (16S rRNA, msp4 and groEL). Anaplasma ovis DNA was identified in six animals from one flock. Clinical, hematological and biochemical changes were not significantly associated with Anaplasma spp. infection. The 16S rRNA analysis revealed known variants of A. phagocytophilum, whereas the msp4 and groEL showed new genotypes. Further investigations are necessary to evaluate the dissemination and health impact of both pathogens in the German sheep population particularly in case of comorbidities.
Quality of life (QoL) is a core patient-reported outcome in healthcare research, alongside primary clinical outcomes. A conceptual, operational, and psychometric elaboration of QoL in the context of TM is needed, because standardized instruments to assess QoL do not sufficiently represent essential aspects of intended outcomes of telemedical applications (TM). The overall aim is to develop an instrument that can adequately capture QoL in TM. For that purpose, an extended working model of QoL will be derived. Subsequently, an instrument will be developed and validated that captures those aspects of QoL that are influenced by TM. The initial exploratory study section includes (a) a systematic literature review, (b) a qualitative survey for concept elicitation, and (c) pre-testings using cognitive debriefings with patients and an expert workshop. The second quantitative section consists of an online expert survey and two patient surveys for piloting and validation of the newly developed instrument. The resulting questionnaire will assess central experiences of patients regarding telemedical applications and its impact on QoL more sensitively. Its use as adjunct instrument will lead to a more appropriate evaluation of TM and contribute to the improvement of care tailored to patients’ individual needs.
Background and Purpose: In the setting of acute ischemic stroke, increased blood-brain barrier permeability (BBBP) as a sign of injury is believed to be associated with increased risk of poor outcome. Pre-clinical studies show that selected serum biomarkers including C-reactive protein (CRP), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNFα), matrix metallopeptidases (MMP), and vascular endothelial growth factors (VEGFs) may play a role in BBBP post-stroke. In the subacute phase of stroke, increased BBBP may also be caused by regenerative mechanisms such as vascular remodeling and therefore may improve functional recovery. Our aim was to investigate the evolution of BBBP in ischemic stroke using contrast-enhanced (CE) magnetic resonance imaging (MRI) and to analyze potential associations with blood-derived biomarkers as well as functional recovery in subacute ischemic stroke patients.
Methods: This is an exploratory analysis of subacute ischemic stroke patients enrolled in the BAPTISe study nested within the randomized controlled PHYS-STROKE trial (interventions: 4 weeks of aerobic fitness training vs. relaxation). Patients with at least one CE-MRI before (v1) or after (v2) the intervention were eligible for this analysis. The prevalence of increased BBBP was visually assessed on T1-weighted MR-images based on extent of contrast-agent enhancement within the ischemic lesion. The intensity of increased BBBP was assessed semi-quantitatively by normalizing the mean voxel intensity within the region of interest (ROI) to the contralateral hemisphere (“normalized CE-ROI”). Selected serum biomarkers (high-sensitive CRP, IL-6, TNF-α, MMP-9, and VEGF) at v1 (before intervention) were analyzed as continuous and dichotomized variables defined by laboratory cut-off levels. Functional outcome was assessed at 6 months after stroke using the modified Rankin Scale (mRS).
Results: Ninety-three patients with a median baseline NIHSS of 9 [IQR 6–12] were included into the analysis. The median time to v1 MRI was 30 days [IQR 18–37], and the median lesion volume on v1 MRI was 4 ml [IQR 1.2–23.4]. Seventy patients (80%) had increased BBBP visible on v1 MRI. After the trial intervention, increased BBBP was still detectable in 52 patients (74%) on v2 MRI. The median time to v2 MRI was 56 days [IQR 46–67]. The presence of increased BBBP on v1 MRI was associated with larger lesion volumes and more severe strokes. Aerobic fitness training did not influence the increase of BBBP evaluated at v2. In linear mixed models, the time from stroke onset to MRI was inversely associated with normalized CE-ROI (coefficient −0.002, Standard Error 0.007, p < 0.01). Selected serum biomarkers were not associated with the presence or evolution of increased BBBP. Multivariable regression analysis did not identify the occurrence or evolution of increased BBBP as an independent predictor of favorable functional outcome post-stroke.
Conclusion: In patients with moderate-to-severe subacute stroke, three out of four patients demonstrated increased BBB permeability, which decreased over time. The presence of increased BBBP was associated with larger lesion volumes and more severe strokes. We could not detect an association between selected serum biomarkers of inflammation and an increased BBBP in this cohort. No clear association with favorable functional outcome was observed.
Trial registration: NCT01954797.
Dynamics of Vascular Protective and Immune Supportive Sphingosine-1-Phosphate During Cardiac Surgery
(2021)
Introduction
Sphingosine-1-phosphate (S1P) is a signaling lipid and crucial in vascular protection and immune response. S1P mediated processes involve regulation of the endothelial barrier, blood pressure and S1P is the only known inducer of lymphocyte migration. Low levels of circulatory S1P correlate with severe systemic inflammatory syndromes such as sepsis and shock states, which are associated with endothelial barrier breakdown and immunosuppression. We investigated whether S1P levels are affected by sterile inflammation induced by cardiac surgery.
Materials and Methods
In this prospective observational study we included 46 cardiac surgery patients, with cardiopulmonary bypass (CPB, n=31) and without CPB (off-pump, n=15). Serum-S1P, S1P-sources and carriers, von-Willebrand factor (vWF), C-reactive protein (CRP), procalcitonin (PCT) and interleukin-6 (IL-6) were measured at baseline, post-surgery and at day 1 (POD 1) and day 4 (POD 4) after surgical stimulus.
Results
Median S1P levels at baseline were 0.77 nmol/mL (IQR 0.61-0.99) and dropped significantly post-surgery. S1P was lowest post-surgery with median levels of 0.37 nmol/mL (IQR 0.31-0.47) after CPB and 0.46 nmol/mL (IQR 0.36-0.51) after off-pump procedures (P<0.001). The decrease of S1P was independent of surgical technique and observed in all individuals. In patients, in which S1P levels did not recover to preoperative baseline ICU stay was longer and postoperative inflammation was more severe. S1P levels are associated with its sources and carriers and vWF, as a more specific endothelial injury marker, in different phases of the postoperative course. Determination of S1P levels during surgery suggested that also the anticoagulative effect of heparin might influence systemic S1P.
Discussion
In summary, serum-S1P levels are disrupted by major cardiac surgery. Low S1P levels post-surgery may play a role as a new marker for severity of cardiac surgery induced inflammation. Due to well-known protective effects of S1P, low S1P levels may further contribute to the observed prolonged ICU stay and worse clinical status. Moreover, we cannot exclude a potential inhibitory effect on circulating S1P levels by heparin anticoagulation during surgery, which would be a new pro-inflammatory pleiotropic effect of high dose heparin in patients undergoing cardiac surgery.
Quality of life (QoL) is a core patient-reported outcome in healthcare research, alongside primary clinical outcomes. A conceptual, operational, and psychometric elaboration of QoL in the context of TM is needed, because standardized instruments to assess QoL do not sufficiently represent essential aspects of intended outcomes of telemedical applications (TM). The overall aim is to develop an instrument that can adequately capture QoL in TM. For that purpose, an extended working model of QoL will be derived. Subsequently, an instrument will be developed and validated that captures those aspects of QoL that are influenced by TM. The initial exploratory study section includes (a) a systematic literature review, (b) a qualitative survey for concept elicitation, and (c) pre-testings using cognitive debriefings with patients and an expert workshop. The second quantitative section consists of an online expert survey and two patient surveys for piloting and validation of the newly developed instrument. The resulting questionnaire will assess central experiences of patients regarding telemedical applications and its impact on QoL more sensitively. Its use as adjunct instrument will lead to a more appropriate evaluation of TM and contribute to the improvement of care tailored to patients’ individual needs.
Ac(et)ylation is a post-translational modification present in all domains of life. First identified in mammals in histones to regulate RNA synthesis, today it is known that is regulates fundamental cellular processes also in bacteria: transcription, translation, metabolism, cell motility. Ac(et)ylation can occur at the ε-amino group of lysine side chains or at the α-amino group of a protein. Furthermore small molecules such as polyamines and antibiotics can be acetylated and deacetylated enzymatically at amino groups. While much research focused on N-(ε)-ac(et)ylation of lysine side chains, much less is known about the occurrence, the regulation and the physiological roles on N-(α)-ac(et)ylation of protein amino termini in bacteria. Lysine ac(et)ylation was shown to affect protein function by various mechanisms ranging from quenching of the positive charge, increasing the lysine side chains’ size affecting the protein surface complementarity, increasing the hydrophobicity and by interfering with other post-translational modifications. While N-(ε)-lysine ac(et)ylation was shown to be reversible, dynamically regulated by lysine acetyltransferases and lysine deacetylases, for N-(α)-ac(et)ylation only N-terminal acetyltransferases were identified and so far no deacetylases were discovered neither in bacteria nor in mammals. To this end, N-terminal ac(et)ylation is regarded as being irreversible. Besides enzymatic ac(et)ylation, recent data showed that ac(et)ylation of lysine side chains and of the proteins N-termini can also occur non-enzymatically by the high-energy molecules acetyl-coenzyme A and acetyl-phosphate. Acetyl-phosphate is supposed to be the key molecule that drives non-enzymatic ac(et)ylation in bacteria. Non-enzymatic ac(et)ylation can occur site-specifically with both, the protein primary sequence and the three dimensional structure affecting its efficiency. Ac(et)ylation is tightly controlled by the cellular metabolic state as acetyltransferases use ac(et)yl-CoA as donor molecule for the ac(et)ylation and sirtuin deacetylases use NAD+ as co-substrate for the deac(et)ylation. Moreover, the accumulation of ac(et)yl-CoA and acetyl-phosphate is dependent on the cellular metabolic state. This constitutes a feedback control mechanism as activities of many metabolic enzymes were shown to be regulated by lysine ac(et)ylation. Our knowledge on lysine ac(et)ylation significantly increased in the last decade predominantly due to the huge methodological advances that were made in fields such as mass-spectrometry, structural biology and synthetic biology. This also includes the identification of additional acylations occurring on lysine side chains with supposedly different regulatory potential. This review highlights recent advances in the research field. Our knowledge on enzymatic regulation of lysine ac(et)ylation will be summarized with a special focus on structural and mechanistic characterization of the enzymes, the mechanisms underlying non-enzymatic/chemical ac(et)ylation are explained, recent technological progress in the field are presented and selected examples highlighting the important physiological roles of lysine ac(et)ylation are summarized.
Terrestrial surface waters and submarine ground water discharge (SGD) act as a source of dissolved substances for coastal systems. Solute fluxes of SGD depend on the ground water composition and the water-solid-microbe interactions close to the sediment-water interface. Thus, this study aims to characterize and evaluate the hydrogeochemical gradients developing in the fresh-salt water mixing zone of the Wismar Bay (WB), southern Baltic Sea, Germany. Sampling campaigns covering the WB, the fresh-salt water mixing zone at the beach of the WB shoreline, terrestrial surface and ground waters near the WB as well sediments pore water were carried out. In these different waters, the distribution of dissolved inorganic carbon, nutrients, major ions, trace elements, stable isotopes (H, O, C, S), and radium isotopes have been investigated. Enhanced concentrations of radium isotopes together with dissolved manganese, barium in the surface waters of the eastern WB indicated benthic-pelagic coupling via the exchange between pore water and the water column. Salinity, stable isotopes, and major ions in sediment pore water profiles identified the presence of fresh ground water below about 40 cmbsf in the central part of the bay. Geophysical acoustic techniques revealed the local impact of anthropogenic sediment excavation, which reduced the thickness of a sediment layer between the coastal aquifer and the bottom water, causing, therefore, a ground water upward flow close to the top sediments. The fresh impacted pore water stable isotope composition (δ18O, δ2H) plot close to the regional meteoric water line indicating a relatively modern ground water source. The calculated organic matter mineralization rates and the dissolved inorganic carbon sediment-water fluxes were much higher at the fresh impacted site when compared to other unimpacted sediments. Therefore, this study reveals that different fresh water sources contribute to the water balance of WB including a SGD source.
Objective: To compare the effectiveness and complications of intraligamentary anesthesia
(ILA) with conventional inferior alveolar nerve block (IANB) during injection and dental
treatment of mandibular posterior teeth.
Materials and Methods: In this randomized, prospective clinical trial, 72 patients (39 males, 33
females) patients scheduled for dental treatment of mandibular posterior teeth, were randomly
allocated to ILA group (n=35) received ILA injection or IANB group (n=37) received the
conventional IANB. Our primary outcome was to assess pain and stress (discomfort) during the
injection and dental treatment, using the Numeric Rating Scale (NRS) from 0 to 10 (0 = no
pain, 10= the worst pain imaginable). Whereas; recording 24 hours postoperative complications
were our Secondary outcomes.
Results: Patients in ILA group reported significantly less pain during injection when compared
with IANB group (p=0.03). While pain during dental treatment was similar in both groups
(p=0.2). Patients in both groups also reported similar low values of discomfort during treatment
(p= 0.7). Although no signs of nerve contact or any other postoperative complications were
observed, five patients in IANB group (none in ILA group) reported temporary irritations
Conclusion: This study showed equivalent effectiveness of both intraligamentary anesthesia
and conventional inferior alveolar nerve block, for pain control during routine dental treatment
of mandibular posterior teeth. Nevertheless, ILA showed significantly less pain during
injection. No major postoperative complications in both groups were observed.
Clinical Relevance: ILA could be considered as an effective alternative for routine dental
treatment.
Background: Annual transfusion rates in many European countries range between 25 and 35 red blood cell concentrates (RBCs)/1,000 population.It is unclear why transfusion rates in Germany are considerably higher (approx. 50–55 RBCs/1,000 population). Methods: We assessed the characteristics of transfusion recipients at all hospitals of the German federal state Mecklenburg-Western Pomerania during a 10-year longitudinal study. Results: Although 75% of patients received ≤4 RBCs/patient in 2015 (1 RBC: 11.3%; 2 RBCs: 42.6%; 3 RBCs: 6.3%; 4 RBCs: 15.0%), the mean transfusion index was 4.6 RBCs due to a minority of patients with a high transfusion demand. Two thirds of all RBCs were transfused to only 25% of RBC recipients. Consistently, male patients received a higher number of RBCs (2005: 54.2%; 2015: 56.8%) and had a higher mean transfusion index than female patients (mean 5.1 ± 7.2; median 2; inter-quartile range [IQR] 2–4 vs. mean 4.0 ± 5.8; median 2; IQR 2–4). The absolute transfusion demand decreased between 2005 and 2015 by 13.5% due to a composite of active reduction (clinical practice change) and population decline in the 65- to 75-year age group (lower birth rate cohort 1940–1950); however, with major differences between hospitals (range from –61.0 to +41.4%). Conclusion: Transfusion demand in a population could largely be driven by patients with high transfusion demand. Different treatment practices in this group of patients probably add to the major differences in transfusion demand per 1,000 individuals between countries. The available data cannot prove this hypothesis. Implementation of a diagnosis-related group-based monitoring system is urgently needed to allow informative monitoring on the population level and meaningful comparisons between transfusion practices.
Results on gray matter alterations in complex regional pain syndrome (CRPS) showed heterogeneous findings. Since CRPS is a rare disease, most studies included only small and heterogeneous samples resulting in a low reliability of findings between studies. We investigated 24 CRPS patients with right upper limb affection in the chronic stage of disease using structural MRI and clinical testing. We focused on gray matter volume (GMV) alterations of the brain in comparison to 33 age matched healthy controls, their association to clinical characteristics (duration of pain syndrome and pain intensity ratings) and sensorimotor performance (finger dexterity and spatiotactile resolution). When applying an explorative whole brain analysis CRPS patients showed lower GMV in the bilateral medial thalamus. No other areas showed a relevant GMV difference for the group comparisons. When applying a region of interest driven approach using anatomical masks of the thalamus, ACC/mPFC, putamen, and insula we found relevant associations of clinical and behavioral data in ACC and insula. Whereas, the GMV in ACC showed negative associations with pain intensity and CRPS duration, the GMV of the left posterior insula was negatively associated with sensorimotor performance of the affected hand side. Overall, our results are in accordance to results of others describing a thalamic reduction of GMV in patients with neuropathic pain and are also in accordance with associations of pain intensity and duration with reduced ACC in general in patients with chronic pain syndromes. Sensorimotor performance seems to be related to posterior insula GMV reduction, which has not been described yet for other patient groups.
Background: Native breast milk composition displays significant inter- and intra-individual variation which persists after standard fortification with fixed doses and challenges target fortification. This study aims to analyze the macronutrient composition of different commercially available fortifiers and the effect of different fortification strategies on nutritional intake of preterm infants.
Methods: In 103 preterm infants, native breast milk samples were collected from 24-h feeding batches (n = 3,338) and fat, protein and carbohydrate contents were analyzed. Nutrient content was compared for breast milk that had undergone either (i) standard fortification, (ii) targeted fortification, (iii) selective batching according to breast milk composition, or (iv) partial lyophilization. For (i) eight commercially available standard fortifiers were tested. Targeted fortification (ii) involved the addition of single component modulars of either protein, fat or carbohydrates to standard fortified breast milk. Using a mathematical growth model, the combined effect of protein, fat and carbohydrate intake on growth was assessed. The best composition of standard fortifiers as the initial step for target fortification was explored assuming three clinical scenarios for milk analysis.
Results: Macronutrient content was highly variable between native breast milk samples, and this variation was still present after standard fortification, however at elevated macronutrient levels. Standard fortification, breast milk batching, as well as partial lyophilization of human milk resulted in deficient and imbalanced enteral intakes in a significant proportion of infants. Target fortification reduced this variation in a, respectively, higher percentage of samples. The effect size was dependent on the number of measurements per week. The optimum composition of standard fortifiers was dependent on the clinical scenario (measurement frequency) for target fortification.
Conclusions: To provide precise and accurate intakes of macronutrients, breast milk should be target fortified. Standard fortified breast milk can result in excess above recommended intakes of some macronutrients which limits the efficiency of target fortification. Standard fortifiers with improved composition are needed for target fortification.
Discrimination of Streptococcus pneumoniae from other Streptococcus mitis group (SMG) species is still challenging but very important due to their different pathogenic potential. In this study, we aimed to develop a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based optochin susceptibility test with an objective read-out. Optimal test performance was established and evaluated by testing consecutively collected respiratory isolates. Optochin in different concentrations as a potential breakpoint concentration was added to a standardized inoculum. Droplets of 6 µL with optochin and, as growth control, without optochin were spotted onto a MALDI target. Targets were incubated in a humidity chamber, followed by medium removal and on-target protein extraction with formic acid before adding matrix with an internal standard. Spectra were acquired, and results were interpreted as S. pneumoniae in the case of optochin susceptibility (no growth), or as non-S. pneumoniae in the case of optochin non-susceptibility (growth). Highest test accuracy was achieved after 20 h incubation time (95.7%). Rapid testing after 12 h incubation time (optochin breakpoint 2 µg/mL; correct classification 100%, validity 62.5%) requires improvement by optimization of assay conditions. The feasibility of the MALDI-TOF MS-based optochin susceptibility test was demonstrated in this proof-of-principle study; however, confirmation and further improvements are warranted.
The loss of skin integrity is inevitable in life. Wound healing is a necessary sequence of events to reconstitute the body’s integrity against potentially harmful environmental agents and restore homeostasis. Attempts to improve cutaneous wound healing are therefore as old as humanity itself. Furthermore, nowadays, targeting defective wound healing is of utmost importance in an aging society with underlying diseases such as diabetes and vascular insufficiencies being on the rise. Because chronic wounds’ etiology and specific traits differ, there is widespread polypragmasia in targeting non-healing conditions. Reactive oxygen and nitrogen species (ROS/RNS) are an overarching theme accompanying wound healing and its biological stages. ROS are signaling agents generated by phagocytes to inactivate pathogens. Although ROS/RNS’s central role in the biology of wound healing has long been appreciated, it was only until the recent decade that these agents were explicitly used to target defective wound healing using gas plasma technology. Gas plasma is a physical state of matter and is a partially ionized gas operated at body temperature which generates a plethora of ROS/RNS simultaneously in a spatiotemporally controlled manner. Animal models of wound healing have been vital in driving the development of these wound healing-promoting technologies, and this review summarizes the current knowledge and identifies open ends derived from in vivo wound models under gas plasma therapy. While gas plasma-assisted wound healing in humans has become well established in Europe, veterinary medicine is an emerging field with great potential to improve the lives of suffering animals.
Pilot sites are currently used to test the performance of bentonite barriers for sealing high-level radioactive waste repositories, but the degree of mineral stability under enhanced thermal conditions remains a topic of debate. This study focuses on the SKB ABM5 experiment, which ran for 5 years (2012 to 2017) and locally reached a maximum temperature of 250 °C. Five bentonites were investigated using XRD with Rietveld refinement, SEM-EDX and by measuring pH, CEC and EC. Samples extracted from bentonite blocks at 0.1, 1, 4 and 7 cm away from the heating pipe showed various stages of alteration related to the horizontal thermal gradient. Bentonites close to the contact with lower CEC values showed smectite alterations in the form of tetrahedral substitution of Si4+ by Al3+ and some octahedral metal substitutions, probably related to ferric/ferrous iron derived from corrosion of the heater during oxidative boiling, with pyrite dissolution and acidity occurring in some bentonite layers. This alteration was furthermore associated with higher amounts of hematite and minor calcite dissolution. However, as none of the bentonites showed any smectite loss and only displayed stronger alterations at the heater–bentonite contact, the sealants are considered to have remained largely intact.
CFTR encodes for a chloride and bicarbonate channel expressed at the apical membrane of polarized epithelial cells. Transepithelial sodium transport mediated by the amiloride-sensitive sodium channel ENaC is thought to contribute to the manifestation of CF disease. Thus, ENaC is a therapeutic target in CF and a valid cystic fibrosis modifier gene. We have characterized SCNN1B as a genetic modifier in the three independent patient cohorts of F508del-CFTR homozygotes. We could identify a regulatory element at SCNN1B to the genomic segment rs168748-rs2303153-rs4968000 by fine-mapping (Pbest = 0.0177), consistently observing the risk allele rs2303153-C and the contrasting benign allele rs2303153-G in all three patient cohorts. Furthermore, our results show that expression levels of SCNN1B are associated with rs2303153 genotype in intestinal epithelia (P = 0.003). Our data confirm that the well-established biological role of SCNN1B can be recognized by an association study on informative endophenotypes in the rare disease cystic fibrosis and calls attention to reproducible results in association studies obtained from small, albeit carefully characterized patient populations.
Unlike the native surface of the implant material (Ti6Al4V), oxidation with H2O2 leads to increased binding of the effective antimicrobial agent poly(hexamethylene) biguanide [PHMB]. However, treating with NaOH instead results in an even higher PHMB mass coverage. After oxidation with H2O2, strong differences in the PHMB adsorption capability between polished and corundum-blasted surfaces appear, indicating a roughness dependence. After NaOH treatment, no such effect was observed. The wetting properties of specimens treated with either H2O2 or NaOH prior to PHMB exposure clearly varied. To unravel the nature of this interaction, widespread in silico and in vitro experiments were performed. Methods: By X-ray photoelectron spectroscopy, scanning electron microscopy, water contact angle measurements and MD simulations, we characterized the interplay between the polycationic antimicrobial agent and the implant surface. A theoretical model for PHMB micelles is tested for its wetting properties and compared to carbon contaminated TiO2. In addition, quantitation of anionic functional group equivalents, the binding properties of PHMB with blocked amino end-group, and the ability to bind chlorhexidine digluconate (CHG) were investigated. Ultimately, the capability of osteoblasts to build calcium apatite, and the activity of alkaline phosphatase on PHMB coated specimens, were determined. Results: Simulated water contact angles on carbon contaminated TiO2 surfaces and PHMB micelle models reveal little influence of PHMB on the wetting properties and point out the major influence of remaining and recovering contamination from ambient air. Testing PHMB adsorption beyond the critical micelle concentration and subsequent staining reveals an island-like pattern with H2O2 as compared to an evenly modified surface with NaOH. Both CHG and PHMB, with blocked amino end groups, were adsorbed on the treated surfaces, thus negating the significant influence of PHMB’s terminal groups. The ability of osteoblasts to produce calcium apatite and alkaline phosphatase is not negatively impaired for PHMB mass coverages up to 8 μg/specimen. Conclusion: Differences in PHMB adsorption are triggered by the number of anionic groups and carbon contaminants, both of which depend on the specimen pre-treatment. With more PHMB covering, the implant surface is protected against the capture of new contamination from the ambient air, thus building a robust antimicrobial and biocompatible surface coating.
Permafrost-affected soil stores a significant amount of organic carbon. Identifying the biological constraints of soil organic matter transformation, e.g., the interaction of major soil microbial soil organic matter decomposers, is crucial for predicting carbon vulnerability in permafrost-affected soil. Fungi are important players in the decomposition of soil organic matter and often interact in various mutualistic relationships during this process. We investigated four different soil horizon types (including specific horizons of cryoturbated soil organic matter (cryoOM)) across different types of permafrost-affected soil in the Western Canadian Arctic, determined the composition of fungal communities by sequencing (Illumina MPS) the fungal internal transcribed spacer region, assigned fungal lifestyles, and by determining the co-occurrence of fungal network properties, identified the topological role of keystone fungal taxa. Compositional analysis revealed a significantly higher relative proportion of the litter saprotroph Lachnum and root-associated saprotroph Phialocephala in the topsoil and the ectomycorrhizal close-contact exploring Russula in cryoOM, whereas Sites 1 and 2 had a significantly higher mean proportion of plant pathogens and lichenized trophic modes. Co-occurrence network analysis revealed the lowest modularity and average path length, and highest clustering coefficient in cryoOM, which suggested a lower network resistance to environmental perturbation. Zi-Pi plot analysis suggested that some keystone taxa changed their role from generalist to specialist, depending on the specific horizon concerned, Cladophialophora in topsoil, saprotrophic Mortierella in cryoOM, and Penicillium in subsoil were classified as generalists for the respective horizons but specialists elsewhere. The litter saprotrophic taxon Cadophora finlandica played a role as a generalist in Site 1 and specialist in the rest of the sites. Overall, these results suggested that fungal communities within cryoOM were more susceptible to environmental change and some taxa may shift their role, which may lead to changes in carbon storage in permafrost-affected soil.
The first Therapeutic ROS and Immunity in Cancer (TRIC) meeting was organized by the excellence research center ZIK plasmatis (with its previous Frontiers in Redox Biochemistry and Medicine (FiRBaM) and Young Professionals’ Workshop in Plasma Medicine (YPWPM) workshop series in Northern Germany) and the excellence research program ONKOTHER-H (Rostock/Greifswald, Germany). The meeting showcased cutting-edge research and liberated discussions on the application of therapeutic ROS and immunology in cancer treatment, primarily focusing on gas plasma technology. The 2-day hybrid meeting took place in Greifswald and online from 15–16 July 2021, facilitating a wide range of participants totaling 66 scientists from 12 countries and 5 continents. The meeting aimed at bringing together researchers from a variety of disciplines, including chemists, biochemists, biologists, engineers, immunologists, physicists, and physicians for interdisciplinary discussions on using therapeutic ROS and medical gas plasma technology in cancer therapy with the four main sessions: “Plasma, Cancer, Immunity”, “Plasma combination therapies”, “Plasma risk assessment and patients studies”, and “Plasma mechanisms and treated liquids in cancer”. This conference report outlines the abstracts of attending scientists submitted to this meeting.
Out of Control: The Role of the Ubiquitin Proteasome System in Skeletal Muscle during Inflammation
(2021)
The majority of critically ill intensive care unit (ICU) patients with severe sepsis develop ICU-acquired weakness (ICUAW) characterized by loss of muscle mass, reduction in myofiber size and decreased muscle strength leading to persisting physical impairment. This phenotype results from a dysregulated protein homeostasis with increased protein degradation and decreased protein synthesis, eventually causing a decrease in muscle structural proteins. The ubiquitin proteasome system (UPS) is the predominant protein-degrading system in muscle that is activated during diverse muscle atrophy conditions, e.g., inflammation. The specificity of UPS-mediated protein degradation is assured by E3 ubiquitin ligases, such as atrogin-1 and MuRF1, which target structural and contractile proteins, proteins involved in energy metabolism and transcription factors for UPS-dependent degradation. Although the regulation of activity and function of E3 ubiquitin ligases in inflammation-induced muscle atrophy is well perceived, the contribution of the proteasome to muscle atrophy during inflammation is still elusive. During inflammation, a shift from standard- to immunoproteasome was described; however, to which extent this contributes to muscle wasting and whether this changes targeting of specific muscular proteins is not well described. This review summarizes the function of the main proinflammatory cytokines and acute phase response proteins and their signaling pathways in inflammation-induced muscle atrophy with a focus on UPS-mediated protein degradation in muscle during sepsis. The regulation and target-specificity of the main E3 ubiquitin ligases in muscle atrophy and their mode of action on myofibrillar proteins will be reported. The function of the standard- and immunoproteasome in inflammation-induced muscle atrophy will be described and the effects of proteasome-inhibitors as treatment strategies will be discussed.
The EyeFlowCell: Development of a 3D-Printed Dissolution Test Setup for Intravitreal Dosage Forms
(2021)
An in vitro dissolution model, the so-called EyeFlowCell (EFC), was developed to test intravitreal dosage forms, simulating parameters such as the gel-like consistency of the vitreous body. The developed model consists of a stereolithography 3D-printed flow-through cell with a polyacrylamide (PAA) gel as its core. This gel needed to be coated with an agarose sheath because of its low viscosity. Drug release from hydroxypropyl methylcellulose-based implants containing either triamcinolone acetonide or fluorescein sodium was studied in the EFC using a schematic eye movement by the EyeMovementSystem (EyeMoS). For comparison, studies were performed in USP apparatus 4 and USP apparatus 7. Significantly slower drug release was observed in the PAA gel for both model drugs compared with the compendial methods. Drug release from fluorescein sodium-containing model implants was completed after 40 min in USP apparatus 4, whereas drug release in the gel-based EFC lasted 72 h. Drug release from triamcinolone acetonide-containing model implants was completed after 35 min in USP apparatus 4 and after 150 min in USP apparatus 7, whereas this was delayed until 96 h in the EFC. These results suggest that compendial release methods may overestimate the drug release rate in the human vitreous body. Using a gel-based in vitro release system such as the EFC may better predict drug release.
Re-Establishment Techniques and Transplantations of Charophytes to Support Threatened Species
(2021)
Re-establishment of submerged macrophytes and especially charophyte vegetation is a common aim in lake management. If revegetation does not happen spontaneously, transplantations may be a suitable option. Only rarely have transplantations been used as a tool to support threatened submerged macrophytes and, to a much lesser extent, charophytes. Such actions have to consider species-specific life strategies. K-strategists mainly inhabit permanent habitats, are perennial, have low fertility and poor dispersal ability, but are strong competitors and often form dense vegetation. R-strategists are annual species, inhabit shallow water and/or temporary habitats, and are richly fertile. They disperse easily but are weak competitors. While K-strategists easily can be planted as green biomass taken from another site, rare R-strategists often must be reproduced in cultures before they can be planted on-site. In Sweden, several charophyte species are extremely rare and fail to (re)establish, though apparently suitable habitats are available. Limited dispersal and/or lack of diaspore reservoirs are probable explanations. Transplantations are planned to secure the occurrences of these species in the country. This contribution reviews the knowledge on life forms, dispersal, establishment, and transplantations of submerged macrophytes with focus on charophytes and gives recommendations for the Swedish project.
Analysis and Reduction of Cellular Heterogeneity in Strain Optimization of Bacillus licheniformis
(2021)
Bacillus species invest substantial resources in inherent cellular processes for pre-adaptation to environmental changes, many of which are dispensable in the controlled environment of industrial bioprocesses. The underlying physiological mechanisms are well characterized in B. subtilis, but only little is known about these processes in the closely related B. licheniformis. Moreover, experimental conditions in previous studies differ from industrial settings in most parameters, foremost in batch cultures or plate-based analysis over fed-batch processes. In this thesis, cellular heterogeneity was analyzed in B. licheniformis in optimized, nutrient-rich media in batch and fed-batch cultivations. Systematic inactivation of genes involved in biofilm formation and synthesis of the flagellar apparatus or global regulators thereof resulted in higher protein production and provided new insights into biofilm formation and cellular heterogeneity in this strain.
High Na+ Environments Impair Phagocyte Oxidase-Dependent Antibacterial Activity of Neutrophils
(2021)
Infection and inflammation can augment local Na+ abundance. These increases in local Na+ levels boost proinflammatory and antimicrobial macrophage activity and can favor polarization of T cells towards a proinflammatory Th17 phenotype. Although neutrophils play an important role in fighting intruding invaders, the impact of increased Na+ on the antimicrobial activity of neutrophils remains elusive. Here we show that, in neutrophils, increases in Na+ (high salt, HS) impair the ability of human and murine neutrophils to eliminate Escherichia coli and Staphylococcus aureus. High salt caused reduced spontaneous movement, degranulation and impaired production of reactive oxygen species (ROS) while leaving neutrophil viability unchanged. High salt enhanced the activity of the p38 mitogen-activated protein kinase (p38/MAPK) and increased the interleukin (IL)-8 release in a p38/MAPK-dependent manner. Whereas inhibition of p38/MAPK did not result in improved neutrophil defense, pharmacological blockade of the phagocyte oxidase (PHOX) or its genetic ablation mimicked the impaired antimicrobial activity detected under high salt conditions. Stimulation of neutrophils with phorbol-12-myristate-13-acetate (PMA) overcame high salt-induced impairment in ROS production and restored antimicrobial activity of neutrophils. Hence, we conclude that high salt-impaired PHOX activity results in diminished antimicrobial activity. Our findings suggest that increases in local Na+ represent an ionic checkpoint that prevents excessive ROS production of neutrophils, which decreases their antimicrobial potential and could potentially curtail ROS-mediated tissue damage.
Over thirty years have passed since the first description of ubiquitin-positive structures in the brain of patients suffering from Alzheimer’s disease. Meanwhile, the intracellular accumulation of ubiquitin-modified insoluble protein aggregates has become an indisputable hallmark of neurodegeneration. However, the role of ubiquitin and a fortiori the ubiquitin-proteasome system (UPS) in the pathogenesis of neurodevelopmental disorders (NDD) is much less described. In this article, we review all reported monogenic forms of NDD caused by lesions in genes coding for any component of the UPS including ubiquitin-activating (E1), -conjugating (E2) enzymes, ubiquitin ligases (E3), ubiquitin hydrolases, and ubiquitin-like modifiers as well as proteasome subunits. Strikingly, our analysis revealed that a vast majority of these proteins have a described function in the negative regulation of the innate immune response. In this work, we hypothesize a possible involvement of autoinflammation in NDD pathogenesis. Herein, we discuss the parallels between immune dysregulation and neurodevelopment with the aim at improving our understanding the biology of NDD and providing knowledge required for the design of novel therapeutic strategies.
Moral rules are a cornerstone of many societies. Most moral rules are concerned with the welfare of other individuals, reflecting individuals’ innate aversion against harming other individuals. Harming others is associated with aversive experiences, implying that individuals who are sensitive to the aversiveness of these experiences are more likely to follow moral rules than individuals who are insensitive to the aversiveness of these experiences. Individuals’ sensitivity for aversive experiences depends on individuals’ ability to integrate the underlying neural and physiological processes: Individuals who are more efficient in integrating these processes are more sensitive to the aversiveness that is associated with moral rule violations than individuals who are less efficient in integrating these processes. Individuals who differ in their ability to integrate these processes may, thus, also differ in their inclination to follow moral rules. We tested this assumption in a sample of healthy individuals (67 males) who completed measures of moral rule adherence and integration abilities. Moral rule adherence was assessed with self-report measure and integration abilities were assessed with a resting state measure of heart rate variability (HRV), which reflects prefrontal–(para-)limbic engagement during the integration of physical and neural processes. We found a positive association between individuals’ HRV and individuals’ moral rule adherence, implying that individuals with efficient integration abilities were more inclined to follow moral rules than individuals with inefficient integration abilities. Our findings support the assumption that individuals with different integration abilities also differ in moral rule adherence, presumably because of differences in aversiveness sensitivity.
Background: There is an urgent need for effective follow-up treatments after acute electroconvulsive therapy (ECT) in depressed patients. Preliminary evidence suggests psychotherapeutic interventions to be a feasible and efficacious follow-up treatment. However, there is a need for research on the long-term usefulness of such psychotherapeutic offers in a naturalistic setting that is more representative of routine clinical practice. Therefore, the aim of the current pilot study was to investigate the effects of a half-open continuous group cognitive behavioral therapy (CBT) with cognitive behavioral analysis system of psychotherapy elements as a follow-up treatment for all ECT patients, regardless of response status after ECT, on reducing depressive symptoms and promoting psychosocial functioning.
Method: Group CBT was designed to support patients during the often-difficult transition from inpatient to outpatient treatment. In a non-controlled pilot trial, patients were offered 15weekly sessions of manualized group CBT (called EffECTiv 2.0). The Montgomery-Åsberg Depression Rating Scale was assessed as primary outcome; the Beck Depression Inventory, WHO Quality of Life Questionnaire–BREF, and the Cognitive Emotion Regulation Questionnaire were assessed as secondary outcomes. Measurements took place before individual group start, after individual group end, and 6months after individual group end.
Results: During group CBT, Post-ECT symptom reduction was not only maintained but there was a tendency toward a further decrease in depression severity. This reduction could be sustained 6months after end of the group, regardless of response status after ECT treatment. Aspects of quality of life and emotion regulation strategies improved during group CBT, and these improvements were maintained 6months after the end of the group.
Conclusion: Even though the interpretability of the results is limited by the small sample and the non-controlled design, they indicate that manualized group CBT with cognitive behavioral analysis system of psychotherapy elements might pose a recommendable follow-up treatment option after acute ECT for depressed patients, regardless of response status after ECT. This approach might not only help to further reduce depressive symptoms and prevent relapse, but also promote long-term psychosocial functioning by improving emotion regulation strategies and psychological quality of life and thus could be considered as a valuable addition to clinical routine after future validation.
An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum
(2021)
Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF.
Background: Inflammatory markers, such as C-reactive Protein (CRP), Interleukin-6 (IL-6), tumor necrosis factor (TNF)-alpha and fibrinogen, are upregulated following acute stroke. Studies have shown associations of these biomarkers with increased mortality, recurrent vascular risk, and poor functional outcome. It is suggested that physical fitness training may play a role in decreasing long-term inflammatory activity and supports tissue recovery.
Aim: We investigated the dynamics of selected inflammatory markers in the subacute phase following stroke and determined if fluctuations are associated with functional recovery up to 6 months. Further, we examined whether exposure to aerobic physical fitness training in the subacute phase influenced serum inflammatory markers over time.
Methods: This is an exploratory analysis of patients enrolled in the multicenter randomized-controlled PHYS-STROKE trial. Patients within 45 days of stroke onset were randomized to receive either four weeks of aerobic physical fitness training or relaxation sessions. Generalized estimating equation models were used to investigate the dynamics of inflammatory markers and the associations of exposure to fitness training with serum inflammatory markers over time. Multiple logistic regression models were used to explore associations between inflammatory marker levels at baseline and three months after stroke and outcome at 3- or 6-months.
Results: Irrespective of the intervention group, high sensitive CRP (hs-CRP), IL-6, and fibrinogen (but not TNF-alpha) were significantly lower at follow-up visits when compared to baseline (p all ≤ 0.01). In our cohort, exposure to aerobic physical fitness training did not influence levels of inflammatory markers over time. In multivariate logistic regression analyses, increased baseline IL-6 and fibrinogen levels were inversely associated with worse outcome at 3 and 6 months. Increased levels of hs-CRP at 3 months after stroke were associated with impaired outcome at 6 months. We found no independent associations of TNF-alpha levels with investigated outcome parameters.
Conclusion: Serum markers of inflammation were elevated after stroke and decreased within 6 months. In our cohort, exposure to aerobic physical fitness training did not modify the dynamics of inflammatory markers over time. Elevated IL-6 and fibrinogen levels in early subacute stroke were associated with worse outcome up to 6-months after stroke.
Clinical Trial Registration: ClinicalTrials.gov, NCT01953549.
Organisms often employ ecophysiological strategies to exploit environmental conditions and ensure bio-energetic success. However, the many complexities involved in the differential expression and flexibility of these strategies are rarely fully understood. Therefore, for the first time, using a three-part cross-disciplinary laboratory experimental analysis, we investigated the diversity and plasticity of photoresponsive traits employed by one family of environmentally contrasting, ecologically important phytoflagellates. The results demonstrated an extensive inter-species phenotypic diversity of behavioural, physiological, and compositional photoresponse across the Chlamydomonadaceae, and a multifaceted intra-species phenotypic plasticity, involving a broad range of beneficial photoacclimation strategies, often attributable to environmental predisposition and phylogenetic differentiation. Deceptively diverse and sophisticated strong (population and individual cell) behavioural photoresponses were observed, with divergence from a general preference for low light (and flexibility) dictated by intra-familial differences in typical habitat (salinity and trophy) and phylogeny. Notably, contrasting lower, narrow, and flexible compared with higher, broad, and stable preferences were observed in freshwater vs. brackish and marine species. Complex diversity and plasticity in physiological and compositional photoresponses were also discovered. Metabolic characteristics (such as growth rates, respiratory costs and photosynthetic capacity, efficiency, compensation and saturation points) varied elaborately with species, typical habitat (often varying more in eutrophic species, such as Chlamydomonas reinhardtii), and culture irradiance (adjusting to optimise energy acquisition and suggesting some propensity for low light). Considerable variations in intracellular pigment and biochemical composition were also recorded. Photosynthetic and accessory pigments (such as chlorophyll a, xanthophyll-cycle components, chlorophyll a:b and chlorophyll a:carotenoid ratios, fatty acid content and saturation ratios) varied with phylogeny and typical habitat (to attune photosystem ratios in different trophic conditions and to optimise shade adaptation, photoprotection, and thylakoid architecture, particularly in freshwater environments), and changed with irradiance (as reaction and harvesting centres adjusted to modulate absorption and quantum yield). The complex, concomitant nature of the results also advocated an integrative approach in future investigations. Overall, these nuanced, diverse, and flexible photoresponsive traits will greatly contribute to the functional ecology of these organisms, addressing environmental heterogeneity and potentially shaping individual fitness, spatial and temporal distribution, prevalence, and ecosystem dynamics.
Introduction: Multiparametric MRI (mpMRI) and MRI targeted biopsies (MRtb) are a new standard in prostate cancer (PCa) screening and diagnosis. Guidelines already include this approach for patients at risk. We aimed to gather information from German urologists about their knowledge, routine use, and attitude toward mpMRI and consecutive biopsy methods. Materials and Methods: An anonymous online questionnaire was sent via Survey Monkey to the members of the German Society of Urology (DGU). Statistical analyses were performed using SPSS version 25.0. Results: 496 members with a median age of 48.6 years (±11.7) participated in the survey. The majority rated mpMRI of the prostate as a very useful diagnostic tool (72.7%). MRtb of the prostate was considered as very advantageous (71.5%). MpMRI was used by 95.9%, and 83.2% also recommended MRtb predominantly in clinical institutions. For targeted biopsy, MRI-ultrasound fusion biopsy was clearly favored (75.8%). MpMRI was mostly used in patients with previously negative biopsy (90.9%) and in patients under active surveillance (60.9%). Arguments against the use of prostate mpMRI are costs (84.9%) and/or lack of sufficient radiological infrastructure (17.4%). Conclusion: Our data illustrate the meanwhile high acceptance and clinical use of the prostate mpMRI and MRtb in Germany.
Ebolaviruses are zoonotic pathogens causing severe hemorrhagic fevers in humans
and non-human primates with high case fatality rates. In recent years, the number and
scope of outbreaks has increased, highlighting the importance of better understanding
the molecular aspects of ebolaviral infection and host cell interactions in order to be able to better control this virus.
To facilitate virus genome replication, transcription and protein expression,
ebolaviruses recruit and interact with specific host factors. These interactions play a key role in viral infection and influence virus survival and disease outcome. Based on a genome-wide siRNA screen, the three host factors CAD, NXF1 and UAP56 were
recently identified to be involved in ebolavirus genome replication and/or transcription
and/or mRNA-translation. However, mechanistical details of how these host factors
affect the ebolavirus lifecycle remained elusive.
In this thesis I analyzed the functional interactions between EBOV and these newly
identified host proteins in order to better understand the virus-host interface. To this
end I used siRNA knockdown as well as overexpression of these host proteins in
combination with different reverse-genetics based lifecycle modelling assays to
investigate the influence of CAD, NXF1 and UAP56 on individual aspects of the EBOV
lifecycle. Using these systems in relation with a host factor knockdown I was able to
show that the provision of pyrimidines by CAD plays an important role for both EBOV
genome replication and transcription, whereas NXF1 is predominantly required for
mRNA transport. I furthermore used immunofluorescence analysis to examine whether
these host factors are recruited by one or more EBOV proteins to inclusion bodies,
which represent physical sites of ebolavirus genome replication. During these
experiments, I was able to show that CAD and NXF1, and possibly also UAP56, are
recruited to EBOV inclusion bodies in order to fulfill their individual function for EBOV RNA synthesis or later steps in protein expression. Additionally, I was able to show that the uptake of NXF1 into NP-induced inclusion bodies is most likely mediated via the C-terminal domain of NP, and that the FG-repeat interaction domains of NXF1 are sufficient for recruitment. Further, my data indicate that RNA interaction of both NXF1 and NP is not required for this process, but rather important for exit of NXF1 from inclusion bodies. I therefore suggest that the viral mRNA is transferred in inclusionbodies from NP to NXF1, which leads to a rapid export of the NXF1 packed viral mRNA into the cytosol for mRNA translation.
The exact mechanism of how these host factors are recruited into inclusion bodies and whether they have similar functions in the lifecycle of other negative-sense RNA viruses still needs to be investigated. Nevertheless, this study increases our understanding of virus-host interaction of ebolaviruses, and thus helps to identify targets for the development of novel therapeutics against these viruses.
Gene Expression and Protein Abundance of Hepatic Drug Metabolizing Enzymes in Liver Pathology
(2021)
LPAIV H9N2 and HPAIV H5N8 clade 2.3.4.4 viruses have been frequently isolated from domestic and wild birds in Germany and they are endemic in poultry worldwide. H9N2 is known to donate gene segments to other AIV with high case fatality rate in humans (e.g. H5N1, H7N9). Similarly, H5N8 devastated poultry worldwide since 2014 and has been recently isolated from humans. Therefore, it is important to understand the genetic predisposition for adaptation of H9N2 and H5N8 AIV in poultry and mammals. In the first publication, we focused on the variable hemagglutinin cleavage site (HACS) of European and Non-European H9N2 viruses, since the HACS is a main virulence determinant of AIV in birds. We found a preferential substitution of non-basic amino acids (G, A, N, S, D, K) in the HACS at position 319 of European H9N2 viruses compared to non-European H9N2 viruses. Recombinant viruses carrying different non-basic amino acids in the HACS modulated replication in vitro. While these non-basic amino acids did not affect virulence or transmission in chickens, they modulated virulence and replication in turkeys. Moreover, H9N2 viruses with non-basic amino acids in the HACS were able to replicate in mammalian brain cells for multiple cycles even without trypsin. In the second publication, we addressed the question whether reassortment between two recent German H9N2 and H5N8 clade 2.3.4.4. B viruses is possible and analysed the impact on virus fitness in mammals and birds. We found that H9N2 PB1 and NP segments were not compatible to generate infectious H5N8 viruses and this incompatibility was due to mutations outside the packaging region. However, H9N2 NS alone or in combination with PB2 and PA significantly increased replication of H5N8 in human cells. Moreover, H9N2 PB2, PA and/or NS segments increased virulence of H5N8 in mice. Interestingly, in chickens, reassortment with H9N2 gene segments, particularly NS, partially or fully impaired chicken-to-chicken transmission. These results indicate that the evolution of H9N2/H5N8 reassortants showing high virulence for mammals is unlikely to occur in chickens. In the third publication, we focused on the NS1 protein of different HPAIV H5N8 clade 2.3.4.4 viruses from 2013 to 2019 and studied the impact of its C-terminus (CTE) variation on virus fitness in chickens and ducks. Our findings revealed a preferential selection for a certain NS1 CTE length in 2.3.4.4. H5N8 clade A (237 aa) and B (217 aa) viruses over the common length of 230 aa. Indeed, the NS1 CTE can affect virus virulence and pathogenesis in a species and virus clade dependent manner. In chickens, although there was no impact on virulence, NS1 CTE of H5N8-A and H5N8-B, regardless of the length, have evolved towards higher efficiency to block the IFN response. In ducks, NS1 CTE contributed to efficient transmission, replication and high virulence of H5N8-B. In the fourth publication, we assessed the impact of variable length of NS1 on H5N8 virus replication in human cells and virulence in mice. We showed that NS1 of H5N8-B virus unlike the vast majority of NS1 of AIV, shared preferences for short NS1 similar to human and zoonotic influenza viruses. This virus (i) was able to efficiently block IFN and apoptosis induction which might be the first steps for efficient adaptation to human cells and (ii) without prior adaptation replicated at higher levels and was more virulent in mice than H5N8-A. The virulence of the latter virus increased after shortening the NS1 similar to H5N8-B virus. Therefore, it is conceivable that truncation in NS1 is a determinant for adaptation of H5N8 in mammals irrespective of its impact on virus fitness in poultry. Findings in this dissertation indicated that HA mutations in the European H9N2 and NS1 variations in H5N8 viruses play a role in virus fitness in poultry and/or mammals. These results improve our current understanding for AIV adaptation and are useful to assess the potential of these viruses to infect mammals.
Humans are exposed to a plethora of microorganisms that reside on outer and inner body surfaces. These are collectively referred to as the human microbiome. The evolutionary relationship between humans and their microbiome is very complex. It is now widely accepted that these microorganisms are not just passive spectators but play an important role in health. The presence or absence of certain microbes is also linked to various diseases, including inflammatory bowel disease, cardiovascular disease, obesity, cancer, and allergies.
Allergies are several conditions caused by a misguided immune response to foreign antigens that are typically harmless. Common allergic diseases include atopic dermatitis (AD), allergic asthma, hay fever, and anaphylaxis. The incidences of allergic diseases are continuously rising, with up to 40% of the human population thought to be sensitised to environmental antigens. This increased incidence is not simply the result of societies becoming more aware and better at diagnosing these diseases. It is believed that the increases in allergies and sensitisation have environmental causes and are related to Western lifestyles. It is known that the rate of allergies is less frequent in developing countries. They are also more likely to occur in urban than rural areas. The prevailing view of the involvement of bacteria in allergies is described by the hygiene hypothesis. The hypothesis claims that decreased exposure to diverse microbial communities early in life increases the risk of developing allergic diseases. There are numerous examples to support this claim. For example, children born and raised in close contact to farm animals or in the presence of pets, and who are thus in direct and constant contact with a complex microbial environment, are protected from allergic diseases. On the other hand, colonisation or infection with certain bacteria increases allergic disease risks. This seems to contradict the hygiene hypothesis.
It appears that the members of the microbiome have different effects on allergy, and the hygiene hypothesis may not apply to every player in the complex microbial diversity that humans are in contact with. Therefore, a better understanding of the host bacterial interaction is required on the level of bacterial species.
This work studies the interplay between bacteria and the immune system to identify and characterise bacterial components with allergenic properties. In this quest, Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) were investigated for their allergenic properties and involvement in different allergic diseases. In the case of S. aureus, evidence is presented on allergic implications for two different components; serine protease-like proteins (Spls) and superantigens (SAg). Furthermore, experimental support is provided on the allergenic properties of the extracellular serine protease (Esp) from S. epidermidis. We argue that stimulating allergic reactions by staphylococci is an immune evasion mechanism that increases the survival chances of the bacteria within the host.
In chapter 1, an introduction is given to both S. aureus and S. epidermidis and their interactions with the immune system. Also, the bacterial components with allergenic properties and allergic diseases with known bacterial involvement are presented. Finally, the question of why bacteria cause allergy is discussed.
Chapter 2 describes allergic reactions to the Spls of S. aureus in a cohort of cystic fibrosis patients. Chapter 3 focuses on the SAgs of S. aureus. SAgs were discovered more than 30 years ago, but their physiological function is still under discussion. In this chapter, the allergenic properties of SAgs and their possible immunological mechanisms are reviewed, and a possible link between SAgs and allergic diseases is discussed. In chapter 4, the focus shifts to S. epidermidis and its involvement in AD. The human immune response to the Esp from S. epidermidis is characterised in healthy and AD individuals. The allergenic properties of Esp imply a detrimental role of S. epidermidis in AD. Finally, chapter 5 summarises and discusses the results of this thesis. In this section, the pieces are put together, and attention is brought back to the question of why bacteria cause allergies.
The genus Capripoxvirus of the family Poxviridae consists of the species lumpy skin disease virus, sheeppox virus and goatpox virus that affect cattle, sheep and goats, respectively. Whereas lumpy skin disease virus (LSDV) is transmitted mainly mechanically via blood-feeding insects and possibly hard ticks, the major transmission routes of sheeppox virus (SPPV) and goatpox virus (GTPV) are via direct contact and aerosols. Affected animals develop fever and display clinical signs such as ocular and nasal discharge, lymphadenopathy and characteristic lesions of the skin. Severe clinical course, especially in combination with respiratory signs, can result in the death of the affected animals. In endemic regions, mortality of capripox virus-induced diseases is low (1-10%). However, mortalities of up to 75% have been reported for LSDV and up to 100% for SPPV and GTPV in exotic breeds and high-producing dairy or beef animals. The loss of quality of the leather, reduced weight gain and milk yield as well as complete loss of affected animals have severe impact on national and global economies. Therefore, capripox virus-induced diseases have significant impact on both the affected individual animal as well as on the existence of small-scale farmers and large agricultural enterprises. However, until now, only live attenuated vaccines are commercially available. These attenuated vaccines are not authorized in the European Union and their administration would comprise the disease-free status of the respective country. Thus, reliable diagnostic tools for the detection and characterization of capripox viruses as well as safe and efficient control measures are of high importance.
The objectives of the present thesis were the development, validation and comparison of diagnostic tools, the establishment of challenge infection models and the performance of pathogenesis studies for all three capripox virus species, and the development and testing of different inactivated prototype vaccine candidates against LSDV.
First, new real-time quantitative polymerase chain reaction (qPCR) assays for robust detection and differentiation of LSDV field strains, LSDV vaccine strains, SPPV and GTPV were developed and extensively validated. In the following, two single assays were combined to duplex assays, one for the differentiation between LSDV field strains and LSDV vaccine strains, and the second for discrimination of SPPV and GTPV. Finally, a diagnostic workflow based on these new duplex assays in combination with already published methods was established. This workflow enables time-saving, robust and reliable detection, species-specific identification and genetic and phylogenetic characterization of all three capripox virus species. In addition, already existing serological examination methods (serum neutralization assay and commercial enzyme-linked immunosorbent assay) were compared regarding their sensitivity and specificity. Furthermore, pathogenesis studies with different capripox virus isolates were performed in the respective target species, and the suitability of selected virus isolates as challenge viruses for future vaccine studies was analyzed. Pathogenesis studies with isolates GTPV-“V/103” and LSDV-“Macedonia2016” revealed that both are proper candidates for challenge models. Finally, three different SPPV isolates (SPPV-“V/104”, SPPV-“India/2013/Surankote” and SPPV-“Egypt/2018”) were tested in sheep regarding their virulence to find a suitable challenge model for SPPV, and SPPV-“India/2013/Surankote” was chosen for future vaccine studies.
Once appropriate challenge models were established, different inactivated prototype vaccines against LSDV were developed, and vaccine safety as well as vaccine efficacy were tested in cattle. Eventually, a Polygen-adjuvanted inactivated LSDV-vaccine candidate was selected that is able to fully prevent cattle from any LSDV-related clinical signs after severe challenge infection. Furthermore, molecular and serological data indicate that this inactivated prototype vaccine is even able to induce a kind of “sterile immunity” against LSDV in those cattle. It has to be mentioned that a commercially available vaccine similar to this prototype vaccine would be a great advance for the control of LSDV.
In the future, additional studies addressing diagnostics and optimized control of capripox viruses should be performed. Firstly, probe-based real-time qPCR assays for the differentiation of SPPV and GTPV vaccine strains from their respective virulent field strains should be developed and included into the diagnostic workflow. Secondly, further tests of the inactivated prototype vaccine, e.g. determination of the minimum protective dose and the possibility of cross-protection in sheep and goats against SPPV and GTPV, respectively, should be performed.
Primary producers, alongside heterotrophic bacteria and viruses, modulate the essential global carbon cycle. About half of the Earth’s net primary production originates in the marine environment. By effecting these systems and the burial of carbon, bacteria play a significant role in the world’s climate, especially with regard to rising temperatures and increasing anthropogenic carbon dioxide production.
Particles present substrate-rich niches for particle-associated bacteria, but are rare in the marine system. Particle-associated bacteria, comprising of chemotactic motile free-living and particle-attached bacteria, were shown to have higher respiration rates, were larger in cell and genome size and showed a higher hydrolytic activity of extracellular enzymes compared to the free-living fraction.
Understanding the contribution of particle-associated bacteria to the degradation of algal biomass is essential to understand the marine carbon cycle. However, the identification of this group is difficult and required refinement.
Sequential filtration, the most commonly used technique for the separation of bacterial fractions, provides only access to a part of the particle-associated microbiome, and includes with large and clustered bacteria undesired false-positives. To overcome these limitations, separation by gravity in Imhoff sedimentation cones was explored in this thesis to access, identify and define particle-associated microbiomes, in comparison and conjunction with the established separation techniques like sequential filtration and centrifugation.
The cultivability on agar plates was assessed, aiming at the question which portion of the colony-forming bacteria belong to free-living non-motile or motile bacteria or to particle-attached bacteria. As continuous cultivation on plates often involves loss of cultures, colonies of the original plate were used to obtain partial 16S rRNA sequences of individual colonies and of plate microbiomes.
For future studies on particle-associated bacteria, a representative strain collection was established from particle-attached bacteria retained on 3 μm filters and from particle-associated bacteria collected together with settled algae in sedimentation cones.
To understand the contribution of top-down selection to a yearly recurring bacterioplankton bloom at our sampling site Helgoland, particle-associated strains were included in isolation experiments for flavophages, since Flavobacteriia are among the most important responder to the yearly observed blooms.
Overall, this thesis provides new insights into the isolation and cultivation of particle-associated bacteria – an important, but currently not fully understood fraction of organisms within the marine system.
Insect migration redistributes enormous quantities of biomass, nutrients and species globally. A subset of insect migrants perform extreme long-distance journeys, requiring specialized morphological, physiological and behavioral adaptations. The migratory globe skimmer dragonfly (Pantala flavescens) is hypothesized to migrate from India across the Indian Ocean to East Africa in the autumn, with a subsequent generation thought to return to India from East Africa the following spring. Using an energetic flight model and wind trajectory analysis, we evaluate the dynamics of this proposed transoceanic migration, which is considered to be the longest regular non-stop migratory flight when accounting for body size. The energetic flight model suggests that a mixed strategy of gliding and active flapping would allow a globe skimmer to stay airborne for up to 230–286 h, assuming that the metabolic rate of gliding flight is close to that of resting. If engaged in continuous active flapping flight only, the flight time is severely reduced to ∼4 h. Relying only on self-powered flight (combining active flapping and gliding), a globe skimmer could cross the Indian Ocean, but the migration would have to occur where the ocean crossing is shortest, at an exceptionally fast gliding speed and with little headwind. Consequently, we deem this scenario unlikely and suggest that wind assistance is essential for the crossing. The wind trajectory analysis reveals intra- and inter-seasonal differences in availability of favorable tailwinds, with only 15.2% of simulated migration trajectories successfully reaching land in autumn but 40.9% in spring, taking on average 127 and 55 h respectively. Thus, there is a pronounced requirement on dragonflies to be able to select favorable winds, especially in autumn. In conclusion, a multi-generational, migratory circuit of the Indian Ocean by the globe skimmer is shown to be achievable, provided that advanced adaptations in physiological endurance, behavior and wind selection ability are present. Given that migration over the Indian Ocean would be heavily dependent on the assistance of favorable winds, occurring during a relatively narrow time window, the proposed flyway is potentially susceptible to disruption, if wind system patterns were to be affected by climatic change.
Background: Interpersonal skills deficits and dysfunctional metacognitive beliefs have been implicated in the etiology and maintenance of depression. This study aimed to investigate the association between changes in these skills deficits and change in depressive symptoms over the course of treatment with Cognitive Behavioral Analysis System of Psychotherapy (CBASP) and Metacognitive Therapy (MCT).
Methods: In this prospective, parallel group observational study, data was collected at baseline and after 8 weeks of an intensive day clinic psychotherapy program. Based on a shared decision between patients and clinicians, patients received either CBASP or MCT. Ninety patients were included in the analyses (CBASP: age M = 38.7, 40.5% female, MCT: age M = 44.7, 43.3% female). Interpersonal deficits were assessed with the short-form of the Luebeck Questionnaire for Recording Preoperational Thinking (LQPT-SF) and the Impact Message Inventory (IMI-R). Metacognitive beliefs were assessed with the Metacognition Questionnaire-30 (MCQ-30). The Quick Inventory of Depressive Symptomatology (QIDS-SR16) was utilized to assess depressive symptoms. A regression analysis was conducted to assess variables associated with outcome. ANCOVAs were utilized to investigate whether improvement in skills deficits is dependent on type of treatment received.
Results: Improvements in preoperational thinking and increases in friendly-dominant behavior were associated with change in depressive symptoms. There was no association between reductions in dysfunctional metacognitive beliefs and a decrease in depressive symptoms. While both treatment groups showed significant improvements in interpersonal and metacognitive skills, there was no significant between-group difference in the change scores for either of these skills.
Conclusion: Our findings suggest that changes in interpersonal skills seem to be of particular relevance in the treatment of depression. These results have to be replicated in a randomized-controlled design before firm conclusions can be drawn.
Objective: To develop an instrument for the observation of therapeutic communication interactions during rehabilitation sessions and test its inter-rater reliability.
Methods: The new instrument THER-I-ACT (THERapy–related Inter-ACTion) has been designed to assess both the frequency and timing of therapeutic interactions in the thematic fields information provision, feedback, other motivational interaction, and bonding. For this inter-rater reliability study, a sample of stroke survivors received arm rehabilitation as either arm ability training, arm basis training, or mirror therapy, or neglect training as individually indicated. Therapy sessions were video-recorded (one for each participant) and therapeutic interactions rated by two independent raters using THER-I-ACT.
Results: With regard to the instrument's comprehensiveness to document therapeutic interactions with pre-defined categories the data from 29 sessions suggested almost complete coverage. Inter-rater reliability was very high both for individual categories of therapeutic interaction (frequency and time used for interaction) (intraclass correlation coefficient, ICC 0.91–1.00) and summary scores for the thematic fields of interaction (again for frequency and time used for interaction) (ICC 0.98–1.00).
The inter-rater reliability for rating engagement and being focussed for both the therapist and patient was substantial (ICC 0.71 and 0.86).
Conclusions: The observational study documented that by use of the newly designed THER-I-ACT various types of therapy-related communication interactions performed by therapists can be assessed with a very high inter-rater reliability. In addition, the thematic fields and categories of therapeutic interaction as defined by the instrument comprehensively covered the type of interaction that occurred in the therapeutic sessions observed.
For an Organisation for Caries Research/European Federation of Conservative Dentistry consensus, this systematic review is aimed to assess the question of how to manage the caries process in the case of early childhood caries (ECC). Medline via PubMed was searched systematically regarding management of ECC. First priority was existing systematic reviews or randomized clinical trials otherwise cohort studies dealing with management of ECC, primarily with carious anterior teeth. After data extraction, the potential risk of bias was estimated depending on the study types, and the level of evidence was evaluated. Regarding management of ECC, results are presented for silver diamine fluoride (SDF, n = 5), nonoperative caries management (NOCM, n = 10), and restorative approaches (RA, n = 8) separately, as different kinds of studies with different levels of evidence were found for the different aspects in the management of ECC. The 5 systematic reviews on SDF showed a high potential for arrest of ECC on a high level of evidence. In NOCM, a low level of evidence for a moderate effect of fluoride varnish in arresting or remineralizing, especially non-cavitated lesions, was assessed. For RA in carious anterior upper primary teeth, a low level of evidence was found for higher failure rates of glass ionomer cement and composite fillings than composite strip crowns even if placed under general anaesthesia and especially compared to other crowns (stainless steel and zirconia). In conclusions, ECC may be managed successfully with nonoperative (SDF, regular fluoride application) and moderately well with operative approaches, but the decision is affected by many other variables such as pulpal involvement, the child’s cooperation, or a general anaesthesia setting.
Despite effective treatment approaches within the cognitive behavioral framework general treatment effects for chronic pain are rather small to very small. Translation from efficacy trials to naturalistic settings is questionable. There is an urgent need to improve the effectiveness of well-established treatments, such as cognitive-behavior therapy (CBT) and the investigation of mechanisms of change is a promising opportunity. We performed secondary data analysis from routine data of 1,440 chronic pain patients. Patients received CBT in a multidisciplinary setting in two inpatient clinics. Effect sizes and reliable change indices were computed for pain-related disability and depression. The associations between changes in the use of different pain coping skills (cognitive restructuring, activity despite pain, relaxation techniques and mental distraction) and changes in clinical outcomes were analyzed in structural equation models. Pre–post effect sizes range from g = 0.47 (disability) to g = 0.89 (depression). Changes in the use of cognitive restructuring, relaxation and to a lesser degree mental distraction were associated with changes in disability and depression. Effects from randomized trials can be translated to naturalistic settings. The results complement experimental research on mechanisms of change in the treatment of chronic pain and indicate an important role of cognitive change and relaxation as mechanisms of change. Our findings cautiously suggest that clinicians should optimize these processes in chronic pain patients to reduce their physical and emotional disability.
Abstract
Macroalgae species are fast growing and their polysaccharides are already used as food ingredient due to their properties as hydrocolloids or they have potential high value bioactivity. The degradation of these valuable polysaccharides to access the sugar components has remained mostly unexplored so far. One reason is the high structural complexity of algal polysaccharides, but also the need for suitable enzyme cocktails to obtain oligo‐ and monosaccharides. Among them, there are several rare sugars with high value. Recently, considerable progress was made in the discovery of highly specific carbohydrate‐active enzymes able to decompose complex marine carbohydrates such as carrageenan, laminarin, agar, porphyran and ulvan. This minireview summarizes these achievements and highlights potential applications of the now accessible abundant renewable resource of marine polysaccharides.
Abstract
The known Schiff base compound, (E)1‐benzyl‐3‐((4‐methoxyphenyl)imino)‐5‐methylindolin‐2‐one, was prepared as before by reacting 1‐benzyl‐5‐methylindoline‐2,3‐dione with 4‐methoxyaniline. The product was unambiguously characterized using elemental analysis, 1H and 13C‐NMR spectroscopy, and its new single‐crystal X‐ray structural analysis. Molecular orbital calculations were conducted in order to investigate the structures and relative stabilities of the (E) and (Z) isomers of 1‐benzyl‐3‐([4 methoxyphenyl]‐imino)‐5‐methylindolin‐2‐one. Specific attention was paid to the (E) isomer. The available crystallographic experimental data for the latter ensured also validation of the model structures computationally derived at the theoretical B3LYP/6‐31G(d,p) level.
Subjective cognitive decline (SCD) is considered an early risk stage for dementia due to Alzheimer's disease (AD) and the development of pathological brain changes, such as the aggregation of amyloid-beta (amyloid-β) plaques. This study evaluates the association between specific features of SCD and cerebral amyloid-β load measured by positron emission tomography (PET) with 18F-florbetaben in 40 cognitively normal older individuals. Global amyloid-β, as well as regional amyloid-β load for the frontal, temporal, parietal, and cingulate cortex, was quantified. Specific features of SCD, such as subjective cognitive complaints and worry, were assessed using the 39-item Everyday Cognition Scales and the 16-item Penn State Worry Questionnaire. Spearman's rank partial correlation analyses, adjusted for age and apolipoprotein E ε4 status, were conducted to test the associations between specific features of SCD and cerebral amyloid-β load. The severity of subjective cognitive complaints in everyday memory and organization was positively correlated with amyloid-β load in the frontal cortex. In addition, the severity of subjective cognitive complaints in everyday planning was positively correlated with amyloid-β load in the parietal cortex. Higher levels of worry were associated with higher amyloid-β load in the frontal cortex. After correction of the PET data for partial volume effects, these associations were reduced to trend level. In conclusion, the severity of subjective cognitive complaints and the level of trait worry were positively associated with cortical amyloid-β burden, particularly in the frontal and parietal cortex. Further studies are required to elucidate the direction of these associations in order to develop strategies to prevent amyloid deposition and cognitive decline.
Abstract
Aim
Distribution ranges of temperate tree species are shifting poleward and upslope into cooler environments due to global warming. Successful regeneration is crucial for population persistence and range expansion. Thus, we aimed to identify environmental variables that affect germination and seedling establishment of Europe's dominant forest tree, to compare the importance of plasticity and genetic variation for regeneration, and to evaluate the regeneration potential at and beyond the southern and northern distribution margins.
Location
Europe.
Time period
2016–2018.
Major taxa studied
European beech (Fagus sylvatica (L.)).
Methods
We investigated how germination, establishment and juvenile survival change across a reciprocal transplantation experiment using over 9,000 seeds of beech from 7 populations from its southern to its northern distribution range margins.
Results
Germination and establishment at the seedling stage were highly plastic in response to environmental conditions. Germination success increased with warmer and declined with colder air temperature, whereas establishment and survival were hampered under warmer and drier conditions. Germination differed among populations and was positively influenced by seed weight. However, there was no evidence of local adaptation in any trait.
Main conclusions
The high plasticity in the early life‐history traits found irrespective of seed origin may allow for short‐term acclimatization. However, our results also indicate that this plasticity might not be sufficient to ensure the regeneration of beech in the future due to the low survival found under dry and hot conditions. The future climatic conditions in parts of the distribution centre and at the rear edge might thus become limiting for natural regeneration, as the likelihood of extreme heat and drought events will increase. By contrast, at the cold distribution margin, the high plasticity in the early life‐history traits may allow for increasing germination success with increasing temperatures and may thus facilitate natural regeneration in the future.
Summary
Outer membrane extensions are common in many marine bacteria. However, the function of these surface enlargements or extracellular compartments is poorly understood. Using a combined approach of microscopy and subproteome analyses, we therefore examined Pseudoalteromonas distincta ANT/505, an Antarctic polysaccharide degrading gamma‐proteobacterium. P. distincta produced outer membrane vesicles (MV) and vesicle chains (VC) on polysaccharide and non‐polysaccharide carbon sources during the exponential and stationary growth phase. Surface structures of carbohydrate‐grown cells were equipped with increased levels of highly substrate‐specific proteins. At the same time, proteins encoded in all other polysaccharide degradation‐related genomic regions were also detected in MV and VC samples under all growth conditions, indicating a basal expression. In addition, two alkaline phosphatases were highly abundant under non‐limiting phosphate conditions. Surface structures may thus allow rapid sensing and fast responses in nutritionally deprived environments. It may also facilitate efficient carbohydrate processing and reduce loss of substrates and enzymes by diffusion as important adaptions to the aquatic ecosystem.
ABSTRACT
The small terrestrial gastropod Vertigo pseudosubstriata Ložek, 1954 is one of the rarest glacial indicator species in the Pleistocene of Central and Eastern Europe. In all, this species has been found at only about 15 sites in Europe. V. pseudosubstriata was initially described as a fossil in Central Europe and was discovered only later alive in Central Asia. With regard to its modern distribution, 25 habitats with V. pseudosubstriata have been examined in Tien Shan and in the central and southern Altai. These findings seem to capture the contemporary distribution of the species and provide information on the boundaries of its ecological requirements. These data are of great significance for the interpretation of the fossil assemblages. Since the few fossil specimens in Europe date from very different glacial periods in the Elsterian, Saalian Complex and Weichselian, it can be concluded that V. pseudosubstriata apparently immigrated in at least three distinct waves. Most of the Pleistocene specimens in eastern Central Europe and Eastern Europe are reported from archaeological sites of the Upper Middle Weichselian (Gravettian), roughly between 33 and 29 ka cal
bp. In this paper, we review all reported modern and fossil occurrences and discuss the species' ecological range.
The here presented dissertation investigated the molecular mechanisms, by which the food industry model bacteria Pseudomonas fluorescens and Listeria monocytogenes, grown either as planktonic cultures, were inhibited by plasma treated water (PTW) produced by a microwave-induced plasma source (MidiPLexc). As a starting point, optimal operating parameters were determined with 5 standard liters per minutes(slm)compressed air during the treatment of 10 ml deionized water within a treatment time of up to 15 min (pre-treatment time). Treatment times of 1, 3 and 5 min were selected (post-treatment time). In addition to physical parameters, i.e. temperature measurements at different spots at the plasma source during the production of the PTW, the chemical composition of PTW was determined by pH measurements, chronoamperometry (determination of the H2O2 concentration), ion chromatography (determination of the NO2-, NO3- and ONOO- concentrations) and mass spectrometry (qualitative determination of the molecules). In addition, concentration changes of reactive species over a period of 3 h indicated a decrease of the NO2- concentration as well as an increase of the NO3- and ONOO- concentration in the PTW. Microbiological assays, i.e. quantification of colony-forming units (CFU), fluorescence and XTT assays, revealed a significant reduction of the proliferation ability of the cells, membrane damages and metabolic activity have been demonstrated for planktonic cultures as well as mono- and multispecies biofilms. PTW effects on biofilm structures were investigated using microscopic methods such as fluorescence microscopy, confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM), and scanning electron microscopy (SEM), as well as physical methods such as contact angle measurements. Significant changes in the biofilm structure have been shown, which indicate an ablation of the biofilm mass from top to bottom by approximately 2/3 of the biofilm mass and a destruction of the extracellular matrix (ECM) by the reactive species within the PTW. Subsequently, fresh-cut lettuce has been treated with PTW produced by up-scaled plasma sources. Apart from qualitative parameters of the lettuce after PTW treatment such as texture and color, the concentration of PTW reactive species have been determined. These experiments showed that the composition of the reactive species were slightly different from that of the laboratory-scaled plasma source MidiPLexc. Notably, the PTW treatment did not cause significant changes in texture and color of the fresh-cut lettuce. Finally, a synergistic effect of PTW treatment followed by plasma-processed air (PPA) drying was demonstrated application-specific.
Streptococcus pneumoniae colonizes asymptomatically the upper respiratory tract as a commensal, but has also a high virulence potential and can leave this ecological niche, thereby spreading to the lungs and blood. During this process, pneumococci must adapt to changing external environmental conditions and parameters such as nutrient availability, temperature, or oxygen levels. The transmission of these signals into the bacterial cell interior occurs via the process of signal transduction, which ultimately results in controlled differential gene expression. The most commonly strategy for signal transduction is the use of two-component regulatory systems (TCS), consisting of a membrane-bound histidine kinase as a sensor and a cytoplasmic response regulator that binds to the promoter region of its target genes and interferes with gene expression.
In this study the regulatory impact and influence of the TCS08 and TCS09 on the phenotype and pathophysiology of S. pneumoniae were investigated using two different serotypes
(serotype 2: D39 and serotype 4: TIGR4). For all functional assays, single (Δrr08/Δrr09 or Δhk08/Δhk09) and double (Δtcs08 or Δtcs09) mutants that were constructed by insertion-deletion mutagenesis, were applied.
In the first study a comparative transcriptome analysis using RNA-sequencing was conducted with our tcs09-mutants and the parental wild-type D39. The data indicated upregulation of the aga operon, which is related to galactose metabolism, and downregulation of the regulator AgaR, particularly in the absence of HK09. Interestingly, encapsulated and nonencapsulated hk09-mutants in D39 showed significant growth defects when galactose was used as sole carbohydrate source. Electron microscopy revealed morphological changes such as an increased number of membrane vesicles and cell wall degradation for the nonencapsulated hk09- and tcs09-mutants of strain D39. An increased capsule production was indicated for the encapsulated hk09- and tcs09-mutants in D39. The latter two mutants as well as the encapsulated rr09-mutant also showed altered colony morphology. While D39Δhk09 formed only opaque colonies, the mutants D39Δrr09 and D39Δtcs09 showed increased numbers of transparent colonies. In a Triton X-100 induced autolysis assay and in the presence of oxidative stress, a negative effect of the morphological changes of D39ΔcpsΔhk09 and D39ΔcpsΔtcs09 on their survivability was demonstrated. In conclusion, we observed that TCS09 in S. pneumoniae D39 is important for its fitness through regulation of carbohydrate metabolism. This indirectly influences cell wall integrity and capsular polysaccharide amount via other regulatory mechanisms, which ultimately affects stress tolerance.
In a second study, we investigated the virulence potential of TCS09 in pneumococcal strain TIGR4. In vitro growth analyses in complex medium showed no effect after loss of function of TCS09 on pneumococcal fitness. In contrast, using the disaccharides lactose and sucrose in chemically defined medium, an extended lag phase of tcs09-mutants was monitored. To assess changes of virulence factor expression, immunoblots were applied to demonstrate the abundance of various essential virulence factors of S. pneumoniae. The results revealed a decreased amount for RrgB, which is the backbone pilus component of type 1 pili, in the hk09-mutant. Field emission scanning electron microscopy and transmission electron microscopy images were applied to study alterations of the bacterial cell shape. The illustrations by FESEM and TEM showed no effect of TCS09-deletion on pneumococcal cell morphology. Cell culture-based infection analyses revealed a similar adhesion capacity of the parental strain and isogenic mutants to lung epithelial cells. However, phagocytosis assays indicated a significantly increased killing rate of intracellular TIGR4ΔcpsΔtcs09, when compared to the isogenic parental strain. In experimental mouse infection models of acute pneumonia and systemic infection the tcs09-mutants were not attenuated. However, to decipher in more detail differences between the wild-type and tcs09-mutants, in vivo co-infection were performed, which highlighted a significantly lower bacterial load of TIGR4luxΔhk09 and TIGR4luxΔtcs09 especially in the lungs, blood, and brain after 48 h. In conclusion, the TCS09 in TIGR4 is necessary for maintaining metabolic fitness, which in turn contributes to dissemination in the host.
In the third study, the influence of TCS08 on gene expression and metabolic and pathophysiological processes of S. pneumoniae was analyzed. In particular, differential gene expression in the hk08-mutant of TIGR4 was detected using microarray and qPCR. The transcriptome analysis revealed a downregulation of cellobiose specific phosphotransferase systems as well as an upregulation of the fab operon, arc operon, and psa operon. These operons encode proteins involved in fatty acid biosynthesis, arginine catabolism, and manganese uptake, respectively. Furthermore, we measured a downregulation of pilus 1 genes in TIGR4ΔcpsΔtcs08 and an increased expression of pavB in TIGR4ΔcpsΔhk08. These data were confirmed by immunoblotting and surface localization studies. Using in silico analysis, a SaeR-like binding motif was identified in the promoter region of pavB. Furthermore, the impact of TCS08 on pneumococcal virulence was investigated in vivo using the acute pneumonia and sepsis models. These models showed a strain-dependent effect of the single TCS08 component deletions between D39 and TIGR4 pneumococci. Whereas loss of HK08 or TCS08 in D39 attenuated the mutants in the pneumonia model, loss of RR08 in TIGR4 was responsible for a similar effect. In contrast, loss of HK08 in TIGR4 promoted increased virulence in the pneumonia and sepsis model. Overall, these data indicate that TCS08 is involved as key player in bacterial fitness during host colonization.
In this study we investigated the synergistic antimicrobial effect of a dual protocol combining cold atmospheric pressure plasma (CAP) and antimicrobial photodynamic therapy (aPDT) against different planktonic bacterial and yeast species including methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Escherichia coli, extended-spectrum β-lactamase-positive Escherichia coli and Candida albicans. A DBD plasma device was used for CAP treatment while for aPDT, toluidine blue O (TBO) was the photosensitizer (PS) of choice and a radiator emitting visible and water-filtered-Infrared A light (VIS-wIRA) was used as irradiation source.
Microbial suspensions were either exposed to CAP treatment alone, aPDT treatment alone or aPDT followed by CAP exposure in a dual treatment protocol. Aliquots from each suspension were plated on agar plates and the number of colonies surviving after each treatment was counted. Under the experimental conditions conducted in this study, combining sub-lethal exposure doses of CAP and aPDT treatment showed significantly higher antimicrobial efficacy (P<0.0001) compared to single treatments against all tested microorganisms suggesting a synergistic effect which yielded at least 3.3 log microbial reduction corresponding to 99.6 % microbial death. In the dual CAP-aPDT approach, aPDT did not interfere with CAP-induced acidification of solution, a crucial feature for CAP antimicrobial efficiency, which further confirms the promising clinical potential of this combination regime.
We believe that the CAP-aPDT dual approach described in this study holds great potential as a successful novel antimicrobial and healing-supporting strategy especially when directed for the management of acute and chronic wounds and possibly other skin and soft tissue infections. The use of a VIS-wIRA light source in treating skin infections is preferential, due to the additional therapeutic effects of wIRA in wound healing. Furthermore, the enhanced antimicrobial effects of aPDT when combined with CAP as shown in this study may grant for a reduction in treatment times and costs as well as improving patient compliance.
Social arthropods such as termites, ants, and bees are among others the most successful animal groups on earth. However, social arthropods face an elevated risk of infections due to the dense colony structure, which facilitates pathogen transmission. An interesting hypothesis is that social arthropods are protected by chemical compounds produced by the arthropods themselves, microbial symbionts, or plants they associate with. Stegodyphus dumicola is an African social spider species, inhabiting communal silk nests. Because of the complex three-dimensional structure of the spider nest antimicrobial volatile organic compounds (VOCs) are a promising protection against pathogens, because of their ability to diffuse through air-filled pores. We analyzed the volatilomes of S. dumicola, their nests, and capture webs in three locations in Namibia and assessed their antimicrobial potential. Volatilomes were collected using polydimethylsiloxane (PDMS) tubes and analyzed using GC/Q-TOF. We showed the presence of 199 VOCs and tentatively identified 53 VOCs. More than 40% of the tentatively identified VOCs are known for their antimicrobial activity. Here, six VOCs were confirmed by analyzing pure compounds namely acetophenone, 1,3-benzothiazole, 1-decanal, 2-decanone, 1-tetradecene, and docosane and for five of these compounds the antimicrobial activity were proven. The nest and web volatilomes had many VOCs in common, whereas the spider volatilomes were more differentiated. Clear differences were identified between the volatilomes from the different sampling sites which is likely justified by differences in the microbiomes of the spiders and nests, the plants, and the different climatic conditions. The results indicate the potential relevance of the volatilomes for the ecological success of S. dumicola.
Scholz et al. developed an electrochemical assay to study the impact of reactive species on self-assembled monolayer (SAM). The aim of this thesis is to use this electrochemical assay with gold supported lipid bilayers instead of SAM to study the effect of reactive species on model membranes that mimic oxidative damage to the biological cell membrane. Here, three questions will be addressed: I) how specific substances such as lipophilic and hydrophilic antioxidants protect a membrane from oxidative damage, II) what are the lipid oxidation products after oxidative damage of the model membrane, and III) whether oxidative damage of the model membranes causes pore formation on lipid bilayer. Electrochemistry was first used to measure the oxidative damage over the entire lipid membrane. Then, mass spectroscopy was used to characterize how lipids as the molecular building blocks of the membrane, change when exposed to reactive species. Imaging the membrane with AFM showed how oxidative damage in the model membrane alters lipid self-assembly within the supported lipid bilayer in nanometer scale. In addition, cold physical plasma (CPP) was used to produce the biological relevant reactive species. This fundamental research demonstrates the great potential of supported lipid bilayers as model membranes and cold physical plasma as a source for the production of biologically relevant reactive species to study the effect of oxidative stress on cell membranes.
Lyssaviruses, the causative agents of rabies, are a long-known threat for animals and humans. To date, terrestrial rabies still accounts for tens of thousands of human deaths annually, notwithstanding ambitious vaccination campaigns targeting susceptible dog and wildlife populations that act as reservoirs for the prototypic rabies virus. Moreover, the continuing discovery of newly emerging virus species in hitherto unconcerned chiropteran hosts and geographic regions drive the expansion of the Lyssavirus genus by unveiling its actual variety, host range and distribution.In this work, the genetic diversity of three distinct lyssaviruses, namely EBLV-1, KBLV and RABV, was elucidated by in-depth genomic analyses to provide further insight into lyssavirus evolution. The generation of full-genome sequences from primarily bat-associated Danish EBLV-1 samples significantly increased the number of available Danish EBLV-1 genome sequences while phylogenetic and phylogeographic analysis revealed a stronger phylogeographic structure for the cluster A1 of the sublineage EBLV-1a than it was postulated in previous studies. In addition, the acquisition of a nearly complete genome sequence for the Kotalahti bat lyssavirus provided the basis for the classification of this putative new lyssavirus species as a recognized member of the genus. Furthermore, phylogenetic analysis revealed the affiliation of KBLV to a group of Myotis-associated lyssaviruses giving a deeper insight into the shared evolutionary history of lyssaviruses co-evolving with particular bat species. Moreover, a deep-sequencing approach was utilized to assess the high genetic diversity of vaccine virus populations, uncovering three independent patterns of single nucleotide variants (SNVs) that became selected in ERA-related vaccine-induced cases. However, no apparent influence of the genetic diversity of vaccine viruses on microevolutionary processes like a potential reversion to virulence or a species-specific adaptation of the vaccine virus strains could be detected, leaving the question for the cause of rabies induction in the affected animals unanswered. Lastly, the successful implementation of a hybridization capturing system for the generation of full-genome sequences and deep-sequencing variant analyses of RABV and KBLV samples was demonstrated for a diagnostic bait set, highlighting the versatility and consistency of this approach to assess the genetic spectrum of known and novel lyssavirus species while setting the basis for its application and optimization in upcoming projects.In conclusion, as shown by the studies in this work, the investigation of lyssavirus genomes at the sub-consensus, full-genome and population level remains crucial to assess the complexity of lyssavirus evolution, as it provides an indispensable source of information to cover the diversity of the genus and understand evolutionary dynamics on a long-term and microevolutionary scale.
Marine bacteria represent the most diverse organisms in the marine environment. The majority of these microbes is unknown and unculturable. Algae represent the main nutrient source for bacteria. Macro- and microalgae can consist to 70% of polysaccharides. The metabolic degradation of marine polysaccharides is underexplored and thus these mechanisms have to be investigated. These mechanisms are of high importance to generate defined oligosaccharides for the medical and pharmaceutical applications. The specific structure of marine poly- and oligosaccharides show antiviral activities, e.g. carrageenans from red algae are used for the inhibition of human papillomavirus. Another alginate derived marine polysaccharide show inhibition of the replication of the human immunodeficiency virus (HIV). The degradation mechanisms of marine CAZymes and the structure of marine polysaccharides should be further investigated for their high potential of antiviral activities and the creation of new marine drugs.
Many marine bacteria produce membrane extension like membrane vesicles or appendages but the function of these is poorly understood. In order to investigate their function, especially concerning polysaccharide utilization, proteomic analyses of subcellular compartments were performed. Microscopy analyses revealed that, beside MV, P. distincta forms different appendages, vesicle chains (VC) and thin filaments which were dedicated to extracellular polymeric substance. The formation of MV and VC was independent of growth phase or carbon source. The proteomic data showed that transporters end enzymes for the initial degradation of pectin and alginate were highly abundant in these membrane extensions and that there could be a kind of sorting for proteins in the membrane extensions. Additionally, two PUL encoded alkaline phosphatases and other phosphate acquiring enzymes were abundant in the MV and VC fractions. This indicates, that P. distincta constitutively produces enzymes for phosphate uptake, which would be necessary in the phosphate-limiting environment of the Southern Ocean. On the one hand marine bacteria produce membrane extensions in order to create a larger surface in the nutrient limiting marine environment for an increased chance to get in contact to nutrients and on the other hand the results indicate an accumulation of enzymes responsible for uptake and degradation of carbohydrates and phosphates in the MV and VC. Therefore, the membrane extensions act as nutrient traps and this might be beneficial for the bacteria in the diffuse aquatic environment.
The microbial community structure and the metabolism of bacteria in the Southern Ocean are very poorly investigated. The SO is a harsh environment for all organism but nevertheless, the SO is of high importance for the climate in the world due to the high carbon dioxide uptake. In this study water samples from two different sampling sites (S1 and S2) in the SO were investigated. With a metagenomic and metaproteomic approach the key players and the metabolic activity were analyzed. Additionally, the surface water was inoculated with pectin and incubated for several days in order to analyze polysaccharide utilization loci for pectin degradation and to isolate new pectin degraders. 16S-rDNA analyses revealed the bacterial community from the genomic data. Bacteria were separated in particle-associated and free-living bacteria. The overall particle associated bacterial community at both sampling sites was comparable, with Bacteroidetes and Gammaproteobacteria as the abundant phylum. Within the Gammaproteobacteria the Alteromonadaceae and Colwelliaceae were more abundant at S2 than at S1. The free-living bacteria at S1 were dominated by the Alphaproteobacteria, especially the SAR11 clade I. Metagenomic analyses showed that both sampling sites had comparable PUL composition, but taxonomical classification of PULs was differently. The metaproteome data revealed that PUL encoded enzymes were not highly abundant. Only few CAZymes were found, mostly TonB-dependent transporters belonged to the detected PUL proteins. Taxonomical classification of proteins showed differences between the sampling sites. At S2 the genus Colwellia and Arcobacter were highly increased compared to S1. At this location Candidatus Pelagibacter, Planktomarina and Polaribacter were the abundant taxa. The functional classification at both sampling sites was comparable. The only difference was the high abundance of Epsilonproteobacteria at S2 referable to the Arcobacter species. Nevertheless, the notably taxonomical differences could not be explained by the proteomic data and the functional classification, because no specific metabolic function could be highly addressed to these bacteria. These results assumed that different abundance of the key players could be explained by different environmental conditions. The pectin enriched cultured at both sampling sites were investigated for the functional potential of pectin degrading enzymes. No metaproteomic approach could be performed due to less sampling material. Only one PUL for the degradation of rhamnogalacturonan, a component of pectin, was found at S1. In contrast, bacteria grown on pectin could be isolated from these samples. Genome sequencing of five isolates showed that functional potential of pectin degradation is available. Due to the limitations of sequence alignments, it was not possible to detect a PUL responsible for pectin utilization in the metagenomic data. The results show that the polysaccharide degradation mechanism in the Southern Ocean has to be more investigated to get knowledge about the bacterial activity in the ocean’s surface and the carbon turnover in this underexplored environment.
Haploidentical stem cell transplantation (haplo SCT) in Stage IV neuroblastoma relapsed patients has been proven efficacious, while immunotherapy utilizing the anti-GD2 antibody dinutuximab beta has become a standard treatment for neuroblastoma. The combinatorial therapy of haplo SCT and dinutuximab may potentiate the efficacy of the immunotherapy. To gain further understanding of the synergistic effects, functional immunomonitoring was assessed during the clinical trial CH14.18 1021 Antibody and IL2 After haplo SCT in Children with Relapsed Neuroblastoma (NCT02258815). Rapid immune reconstitution of the lymphoid compartment was confirmed, with clinically relevant dinutuximab serum levels found in all patients over the course of treatment. Only one patient developed human anti-chimeric antibodies (HACAs). In-patient monitoring revealed highly functional NK cell posttransplant capable of antibody-dependent cellular cytotoxicity (ADCC). Degranulation of NK cell subsets revealed a significant response increased by dinutuximab. This was irrespective of the KIR receptor–ligand constellation within the NK subsets, defined by the major KIR receptors CD158a, CD158b, and CD158e. Moreover, complement-dependent cytotoxicity (CDC) was shown to be an extremely potent effector-cell independent mechanism of tumor cell lysis, with a clear positive correlation to GD2 expression on the cancer cells as well as to the dinutuximab concentrations. The ex vivo testing of patient-derived effector cells and the sera collected during dinutuximab therapy demonstrated both high functionality of the newly established lymphoid immune compartment and provided confidence that the antibody dosing regimen was sufficient over the duration of the dinutuximab therapy (up to nine cycles in a 9-month period). During the course of the dinutuximab therapy, proinflammatory cytokines and markers (sIL2R, TNFa, IL6, and C reactive protein) were significantly elevated indicating a strong anti-GD2 immune response. No impact of FcGR polymorphism on event-free and overall survival was found. Collectively, this study has shown that in-patient functional immunomonitoring is feasible and valuable in contributing to the understanding of anti-cancer combinatorial treatments such as haplo SCT and antibody immunotherapy.