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Attention-deficit/hyperactivity disorder (ADHD) is highly heritable and the most common neurodevelopmental disorder in childhood. In recent decades, it has been appreciated that in a substantial number of cases the disorder does not remit in puberty, but persists into adulthood. Both in childhood and adulthood, ADHD is characterised by substantial comorbidity including substance use, depression, anxiety, and accidents. However, course and symptoms of the disorder and the comorbidities may fluctuate and change over time, and even age of onset in childhood has recently been questioned. Available evidence to date is poor and largely inconsistent with regard to the predictors of persistence versus remittance. Likewise, the development of comorbid disorders cannot be foreseen early on, hampering preventive measures. These facts call for a lifespan perspective on ADHD from childhood to old age. In this selective review, we summarise current knowledge of the long-term course of ADHD, with an emphasis on clinical symptom and cognitive trajectories, treatment effects over the lifespan, and the development of comorbidities. Also, we summarise current knowledge and important unresolved issues on biological factors underlying different ADHD trajectories. We conclude that a severe lack of knowledge on lifespan aspects in ADHD still exists for nearly every aspect reviewed. We encourage large-scale research efforts to overcome those knowledge gaps through appropriately granular longitudinal studies.
Prenatal and postnatal experiences associated with epigenetic changes in the adult mouse brain
(2018)
To analyze the influences of early-life history on the brain epigenome, the offspring of mouse dams kept in an enriched or standard environment were exposed postnatally to enriched, standard, or adverse conditions. The methylation patterns of 7 candidate genes (9 loci) involved in developmental programming of stress vulnerability/resilience and psychiatric disease were analyzed in 6 brain regions of adult male and female mice. Exposure to an enriched prenatal environment was associated with widespread epigenetic changes (all of small effect size), affecting 29 of 324 (9%) gene/region-specific methylation patterns. The effects of either adverse or enriched postnatal conditions were tested separately in the two prenatal cohorts. Significant changes were observed in 2 of 324 (0.6%) loci in offspring of dams in a standard environment and 6 of 324 (1.9%) loci in animals that were exposed prenatally to an enriched environment. Prenatal life experiences appear to have a bigger effect on the adult brain epigenome than postnatal experiences. Positive prenatal life experiences may increase epigenetic plasticity of the brain later in life. All observed between-group differences were sex-specific, consistent with largely different developmental trajectories of the male and female brain. Multiple changes of small effect size are consistent with a multifactorial model of developmental programming of adult behavior and disease susceptibility.
Pseudomonas aeruginosa is a human pathogen that causes health-care associated blood stream infections (BSI). Although P. aeruginosa BSI are associated with high mortality rates, the clinical relevance of pathogen-derived prognostic biomarker to identify patients at risk for unfavorable outcome remains largely unexplored. We found novel pathogen-derived prognostic biomarker candidates by applying a multi-omics approach on a multicenter sepsis patient cohort. Multi-level Cox regression was used to investigate the relation between patient characteristics and pathogen features (2298 accessory genes, 1078 core protein levels, 107 parsimony-informative variations in reported virulence factors) with 30-day mortality. Our analysis revealed that presence of the helP gene encoding a putative DEAD-box helicase was independently associated with a fatal outcome (hazard ratio 2.01, p = 0.05). helP is located within a region related to the pathogenicity island PAPI-1 in close proximity to a pil gene cluster, which has been associated with horizontal gene transfer. Besides helP, elevated protein levels of the bacterial flagellum protein FliL (hazard ratio 3.44, p < 0.001) and of a bacterioferritin-like protein (hazard ratio 1.74, p = 0.003) increased the risk of death, while high protein levels of a putative aminotransferase were associated with an improved outcome (hazard ratio 0.12, p < 0.001). The prognostic potential of biomarker candidates and clinical factors was confirmed with different machine learning approaches using training and hold-out datasets. The helP genotype appeared the most attractive biomarker for clinical risk stratification due to its relevant predictive power and ease of detection.
Rhythmic neural spiking and attentional sampling arising from cortical receptive field interactions
(2018)
Summary: Growing evidence suggests that distributed spatial attention may invoke theta (3-9 Hz) rhythmic sampling processes. The neuronal basis of such attentional sampling is however not fully understood. Here we show using array recordings in visual cortical area V4 of two awake macaques that presenting separate visual stimuli to the excitatory center and suppressive surround of neuronal receptive fields elicits rhythmic multi-unit activity (MUA) at 3-6 Hz. This neuronal rhythm did not depend on small fixational eye movements. In the context of a distributed spatial attention task, during which the monkeys detected a spatially and temporally uncertain target, reaction times (RT) exhibited similar rhythmic fluctuations. RTs were fast or slow depending on the target occurrence during high or low MUA, resulting in rhythmic MUA-RT cross-correlations at at theta frequencies. These findings suggest that theta-rhythmic neuronal activity arises from competitive receptive field interactions and that this rhythm may subserve attentional sampling.
Highlights:
* Center-surround interactions induce theta-rhythmic MUA of visual cortex neurons
* The MUA rhythm does not depend on small fixational eye movements
* Reaction time fluctuations lock to the neuronal rhythm under distributed attention
Relevance of the natural HDAC inhibitor sulforaphane as a chemopreventive agent in urologic tumors
(2018)
Due to an increased understanding of molecular biology and the genomics of cancer, new and potent agents have been approved by the Food and Drug Administration (FDA) to fight this disease. However, all of these drugs cause severe side effects and resistance inevitably develops, re-activating tumor growth and dissemination. For this reason, patients turn to natural compounds as alternative or complementary treatment options, since it has been found that natural plant products may block, inhibit, or reverse cancer development. The present review focusses on the role of the natural compound sulforaphane (SFN) as an anti-tumor agent in urologic cancer. SFN is a natural compound found in cruciferous vegetables from the Brassicaceae family such as broccoli, cauliflower and cabbage. Several epidemiologic and clinical studies have documented chemopreventive properties of SFN, making it an interesting candidate for additive cancer treatment. SFN shows remarkable anti-tumor effects in vitro and in vivo without exerting toxicity. The review summarizes the current understanding of SFN and provides insights into its molecular mode of action with particular emphasis on epigenetic tumor control.
Delayed-onset muscle soreness (DOMS) is a common symptom in people participating in exercise, sport, or recreational physical activities. Several remedies have been proposed to prevent and alleviate DOMS. In 2008 and 2015, two studies have been conducted to investigate the effects of acupuncture on symptoms and muscle function in eccentric exercise-induced DOMS of the biceps brachii muscle. In 2008 a prospective, randomized, controlled, observer and subject-blinded trial was undertaken with 22 healthy subjects (22–30 years; 12 females) being randomly assigned to three treatment groups: real acupuncture (deep needling at classic acupuncture points and tender points; n = 7), sham-acupuncture (superficial needling at non-acupuncture points; n = 8), and control (n = 7). In 2015, a five-arm randomized controlled study was conducted with 60 subjects (22 females, 23.6 ± 2.8 years). Participants were randomly allocated to needle, laser, sham needle, sham laser acupuncture, and no intervention.
In both cases treatment was applied immediately, 24 and 48 hours after DOMS induction.
The outcome measures included pain perception (visual analogue scale; VAS), mechanical pain threshold (MPT), maximum isometric voluntary force (MIVF) and pressure pain threshold (PPT).
Results: In 2008, following nonparametric testing, there were no significant differences between groups in outcome measures at baseline. After 72 hours, pain perception (VAS) was significantly lower in the acupuncture group compared to the sham acupuncture and control subjects. However, the mean MPT and MIVF scores were not significantly different between groups. This lead to the conclusion, that acupuncture seemed to have no effects on MPT and muscle function, but reduced perceived pain arising from exercise-induced DOMS.
The more recent results from 2015 indicated that neither verum nor sham interventions significantly improved outcomes within 72 hours when compared with the no treatment control (P > 0.05).
Osteocalcin, Azan and Toluidine blue staining in fibrous dysplasia and ossifying fibroma of the jaws
(2018)
Background: Fibrous dysplasia (FD) and ossifying fibroma (OF) are fibro-osseous lesions (FOLs) having several overlaps that may make final diagnosis difficult by hematoxylin and eosin (H/E) alone.
Aim: This study seeks to detect any association between Azan and Toluidine blue staining as compared with osteocalcin in FD and OF diagnosis.
Methods:Forty formalin fixed paraffin embedded (FFPE) blocks of FD and OF were prepared for Azan, Toluidine blue and osteocalcin staining. Brown staining of calcified structures was considered as positive for osteocalcin. Scoring for Azan and Toluidine blue was evaluated based on intensity and localization. Level of agreement of original and revised diagnosis was determined.
Results: Six (40%) of 15 FD were corroborated by osteocalcin. Eight cases initially diagnosed as OF were revised to FD. There were 25 OF according to H/E, and 17 (68%) were validated by osteocalcin. Measure of agreement between histology and immunohistochemistry was 0.081; p = .608. Eleven (42.3%) OF expressed strong toluidine blue staining of the intervening fibrous connective tissue stroma while only 2 (14.2%) FD showed similar staining, this difference was statistically significant [p = .001].
Conclusions: Histomorphometric analysis with Toluidine blue may reduce diagnostic errors of OF and FD.
Exposure to locusts, which belong to the arthropod phylum, is an underestimated health problem, especially among workers in research facilities exposed to laboratory animals. We describe a rare case of an occupational immediate-type reaction to locusts with a possible cross-reactivity between desert locust (Schistocerca gregaria) and migratory locust (Locusta migratoria).
Purpose: To report a case of autoimmune keratitis in a patient with mycobacterium tuberculosis (MBT).
Methods: An 84-year-old male with pulmonary tuberculosis (TB) was admitted with chronic, non-healing bilateral ulcerations of the inferior peripheral cornea associated with stromal and subconjunctival nodules.
Results: Clinical examination revealed circumscribed peripheral corneal ulceration with whitish nodules in adjacent stromal and subconjunctival tissue. Microbiological cultures of the corneal tissue were negative for MBT and other microbial pathogens; however, enzyme-linked immunosorbent assay (ELISA) of blood and corneal samples showed significantly elevated levels of IgM and IgA against MBT. In addition to systemic anti-tuberculosis therapy, the patient was treated topically with Polyspectran® eye drops, Dexamethasone eye drops, and Bepanthen® ointment, for 2 weeks. Both eyes showed dramatic improvement after 2 weeks.
Conclusion: The present report demonstrates that MBT is able to initiate delayed autoimmune response within the corneal tissue during an intensive phase of anti-tuberculosis treatment.
Introduction: Ferroptosis has recently been identified as a form of programmed cell death caused by an accumulation of lipid reactive oxygen species (ROS). However, little is yet known about the role in hepatocellular carcinoma (HCC) and its signalling mechanism as well the modulation of ROS.
Material and methods: Human HCC cell lines were treated with different concentrations of ROS modulators (Auranofin, Erastin, BSO). Cell death was determined by analysis of PI-stained nuclei using flow cytometry. ROS production and lipid peroxidation were analysed at early time points before cell death starts. For mechanistic studies we performed Western Blot and a Proteome array. Different inhibitors of cell death target proteins, ROS-scavengers as well as lipoxygenase inhibitors were used. To investigate the functional relevance of NAPDH oxidases (NOX) 1 and 4 for ROS modulation and ferroptosis we genetically silenced its genes using three distinct siRNAs and we used the NOX1/4-inhibitor GKT137831.
Results and discussions: Compared to the single treatment, Auranofin/BSO-cotreatment as well as Erastin/BSO-cotreatment acted in concert to trigger cell death and to reduce cell viability of HCC cells in a dose- and time-dependent manner. Furthermore, both cotreatments induce ROS production, lipid peroxidation and ferroptotic cell death, which could be inhibited by the use of Ferrostatin-1 (inhibitor of lipid peroxidation) and Liproxstatin-1 (specific inhibitor of ferroptosis). The broad-range caspase inhibitor zVAD.fmk failed to rescue cells from Auranofin/BSO- or Erastin/BSO-cotreatment induced cell death. No activation of caspases-3 could be seen in the proteome profiler apoptosis assay. Importantly, the selective lipoxygenase (LOX) inhibitor Baicalain and the pan-LOX inhibitor NDGA protect HCC cells from Auranofin/BSO- and Erastin/BSO-cotreatment stimulated lipid peroxidation, ROS generation and cell death, indication that the induction of ferroptosis may bypass apoptosis resistance of HCC cells. Mechanistic studies showed that Auranofin/BSO-cotreatment decreased TrxR-activity, led to Nrf2 accumulation and promoted the activation of HO-1. In contrast, NOX 1 and 4 were involved in Erastin/BSO-mediated cell death and the use of the NOX1/4-inhibitor GKT137831 rescued HCC cells from the Erastin/BSO-induced cell death.
Conclusion: By providing new insights into the molecular regulation of ROS and ferroptosis, our study contributes to the development of novel treatment strategies to reactivate programmed cell death in HCC cells.
Cancer is the leading cause of death worldwide after cardiovascular diseases. This has been the case for the last few decades despite there being an increase in the number of cancer treatments. One reason for the apparent lack of drug effectiveness might be, at least in part, due to unspecificity for tumors; which often leads to substantial side effects. One way to improve the treatment of cancer is to increase the specificity of the treatment in accordance with the concept of individualized medicine. This will help to prevent further progression of an existing cancer or even to reduce the tumor burden. Alternatively it would be much more attractive and efficient to prevent the development of cancer in the first place. Therefore, it is important to understand the risk factors and the mechanisms of carcinogenesis in detail. One such risk factor, often associated with tumorigenesis and tumor progression, is an increased abundance of reactive oxygen species (ROS) arising from an imbalance of ROS-producing and -eliminating components. A surplus of ROS can induce oxidative damage of macromolecules including proteins, lipids and DNA. In contrast, ROS are essential for an adequate signal transduction and are known to regulate crucial cellular processes like cellular quiescence, differentiation and even apoptosis. Therefore, regulated ROS-formation at physiological levels can inhibit tumor formation and progression. With this review we provide an overview on the current knowledge of redox control in cancer development and progression.
MAPK6/ERK3 is an atypical member of the MAPKs. An essential role has been suggested by the perinatal lethal phenotype of ERK3 knockout mice carrying a lacZ insertion in exon 2 due to pulmonary disfunction and by defects in function, activation and positive selection of T cells. To study the role of ERK3 in vivo, we generated mice carrying a conditional Erk3 allele with exon3 flanked by LoxP sites. Loss of ERK3 protein was validated after deletion of Erk3 in the female germ line using zona pellucida 3 (Zp3)-cre and a clear reduction of the protein kinase MK5 is detected, providing first evidence for the existence of the ERK3/MK5 signaling complex in vivo. In contrast to the previously reported Erk3 knockout phenotype, these mice are viable and fertile, do not display pulmonary hypoplasia, acute respiratory failure, abnormal T cell development, reduction of thymocyte numbers or altered T cells selection. Hence, ERK3 is dispensable for pulmonary and T-cell functions. The perinatal lethality, lung and T-cell defects of the previous ERK3 knockout mice are likely due to ERK3-unrelated effects of the inserted lacZ-neomycin-resistance-cassette. The knockout mouse of the closely related atypical MAPK ERK4/MAPK4 is also normal suggesting redundant functions of both protein kinases.
Background: MDM2 inhibitors are under investigation for the treatment of acute myeloid leukaemia (AML) patients in phase III clinical trials. To study resistance formation to MDM2 inhibitors in AML cells, we here established 45 sub-lines of the AML TP53 wild-type cell lines MV4-11 (15 sub-lines), OCI-AML-2 (10 sub-lines), OCI-AML-3 (12 sub-lines), and SIG-M5 (8 sub-lines) with resistance to the MDM2 inhibitor nutlin-3.
Methods: Nutlin-3-resistant sub-lines were established by continuous exposure to stepwise increasing drug concentrations. The TP53 status was determined by next generation sequencing, cell viability was measured by MTT assay, and p53 was depleted using lentiviral vectors encoding shRNA.
Results: All MV4-11 sub-lines harboured the same R248W mutation and all OCI-AML-2 sub-lines the same Y220C mutation, indicating the selection of pre-existing TP53-mutant subpopulations. In concordance, rare alleles harbouring the respective mutations could be detected in the parental MV4-11 and OCI-AML-2 cell lines. The OCI-AML-3 and SIG-M5 sub-lines were characterised by varying TP53 mutations or wild type TP53, indicating the induction of de novo TP53 mutations. Doxorubicin, etoposide, gemcitabine, cytarabine, and fludarabine resistance profiles revealed a noticeable heterogeneity among the sub-lines even of the same parental cell lines. Loss-of-p53 function was not generally associated with decreased sensitivity to cytotoxic drugs.
Conclusion: We introduce a substantial set of models of acquired MDM2 inhibitor resistance in AML. MDM2 inhibitors select, in dependence on the nature of a given AML cell population, pre-existing TP53-mutant subpopulations or induce de novo TP53 mutations. Although loss-of-p53 function has been associated with chemoresistance in AML, nutlin-3-adapted sub-lines displayed in the majority of experiments similar or increased drug sensitivity compared to the respective parental cells. Hence, chemotherapy may remain an option for AML patients after MDM2 inhibitor therapy failure. Even sub-lines of the same parental cancer cell line displayed considerable heterogeneity in their response to other anti-cancer drugs, indicating the need for the detailed understanding and monitoring of the evolutionary processes in cancer cell populations in response to therapy as part of future individualised treatment protocols.
The entire chemical modification repertoire of yeast ribosomal RNAs and the enzymes responsible for it have recently been identified. Nonetheless, in most cases the precise roles played by these chemical modifications in ribosome structure, function and regulation remain totally unclear. Previously, we demonstrated that yeast Rrp8 methylates m1A645 of 25S rRNA in yeast. Here, using mung bean nuclease protection assays in combination with quantitative RP-HPLC and primer extension, we report that 25S/28S rRNA of S. pombe, C. albicans and humans also contain a single m1A methylation in the helix 25.1. We characterized nucleomethylin (NML) as a human homolog of yeast Rrp8 and demonstrate that NML catalyzes the m1A1322 methylation of 28S rRNA in humans. Our in vivo structural probing of 25S rRNA, using both DMS and SHAPE, revealed that the loss of the Rrp8-catalyzed m1A modification alters the conformation of domain I of yeast 25S rRNA causing translation initiation defects detectable as halfmers formation, likely because of incompetent loading of 60S on the 43S-preinitiation complex. Quantitative proteomic analysis of the yeast Δrrp8 mutant strain using 2D-DIGE, revealed that loss of m1A645 impacts production of specific set of proteins involved in carbohydrate metabolism, translation and ribosome synthesis. In mouse, NML has been characterized as a metabolic disease-associated gene linked to obesity. Our findings in yeast also point to a role of Rrp8 in primary metabolism. In conclusion, the m1A modification is crucial for maintaining an optimal 60S conformation, which in turn is important for regulating the production of key metabolic enzymes.
Autosomal recessive Ataxia Telangiectasia (A-T) is characterized by radiosensitivity, immunodeficiency and cerebellar neurodegeneration. A-T is caused by inactivating mutations in the Ataxia-Telangiectasia-Mutated (ATM) gene, a serine-threonine protein kinase involved in DNA-damage response and excitatory neurotransmission. The selective vulnerability of cerebellar Purkinje neurons (PN) to A-T is not well understood.
Interest in time-resolved connectivity in fMRI has grown rapidly in recent years. The most widely used technique for studying connectivity changes over time utilizes a sliding windows approach. There has been some debate about the utility of shorter versus longer windows, the use of fixed versus adaptive windows, as well as whether observed resting state dynamics during wakefulness may be predominantly due to changes in sleep state and subject head motion. In this work we use an independent component analysis (ICA)-based pipeline applied to concurrent EEG/fMRI data collected during wakefulness and various sleep stages and show: 1) connectivity states obtained from clustering sliding windowed correlations of resting state functional network time courses well classify the sleep states obtained from EEG data, 2) using shorter sliding windows instead of longer non-overlapping windows improves the ability to capture transition dynamics even at windows as short as 30 seconds, 3) motion appears to be mostly associated with one of the states rather than spread across all of them 4) a fixed tapered sliding window approach outperforms an adaptive dynamic conditional correlation approach, and 5) consistent with prior EEG/fMRI work, we identify evidence of multiple states within the wakeful condition which are able to be classified with high accuracy. Classification of wakeful only states suggest the presence of time-varying changes in connectivity in fMRI data beyond sleep state or motion. Results also inform about advantageous technical choices, and the identification of different clusters within wakefulness that are separable suggest further studies in this direction.
Objectives Omeprazole was shown to improve the anti-cancer effect of the nucleoside-analogue 5-fluorouracil. Here, we investigated the effects of omeprazole on the activities of the antiviral nucleoside analogues ribavirin and acyclovir.
Methods West Nile virus-infected Vero cells and influenza A H1N1-infected MDCK cells were treated with omeprazole and/ or ribavirin. Herpes simplex virus 1 (HSV-1)- or HSV-2-infected Vero or HaCat cells were treated with omeprazole and/ or acyclovir. Antiviral effects were determined by examination of cytopathogenic effects (CPE), immune staining, and virus yield assay. Cell viability was investigated by MTT assay.
Results Omeprazole concentrations up to 80μg/mL did not affect the antiviral effects of ribavirin. In contrast, omeprazole increased the acyclovir-mediated effects on HSV-1- and HSV-2-induced CPE formation in a dose-dependent manner in Vero and HaCat cells. Addition of omeprazole 80μg/mL resulted in a 10.8-fold reduction of the acyclovir concentration that reduces CPE formation by 50% (IC50) in HSV-1-infected Vero cells and in a 47.7-fold acyclovir IC50 reduction in HSV-1-infected HaCat cells. In HSV-2-infected cells, omeprazole reduced the acyclovir IC50 by 7.3-fold (Vero cells) or by 12.9-fold (HaCat cells). Omeprazole also enhanced the acyclovir-mediated effects on viral antigen expression and virus replication in HSV-1- and HSV-2-infected cells. In HSV-1-infected HaCat cells, omeprazole 80μg/mL reduced the virus titre in the presence of acyclovir 1μg/mL by 1.6×105-fold. In HSV-2-infected HaCat cells omeprazole 80μg/mL reduced the virus titre in the presence of acyclovir 2μg/mL by 9.2×103-fold. The investigated drug concentrations did not affect cell viability, neither alone nor in combination.
Conclusions Omeprazole increases the anti-HSV activity of acyclovir. As clinically well-established and tolerated drug, it is a candidate drug for antiviral therapies in combination with acyclovir.
The genetic mutation of the coagulation factor VIII (fVIII) results in a defective or missing protein, leading to a malfunctioning blood coagulation. The resulting disease is called hemophilia A. Depending on the severity of the mutation, affected patients experience an increased risk of pathologic bleeding after minor trauma or even sudden bleeding events. Substitution therapies with extrinsic fVIII exist using plasmatic or recombinant fVIII products. Due to an insufficient immune tolerance towards substituted fVIII, about 30 % of patients develop allogenic neutralizing antibodies (inhibitors) against substituted fVIII products. The gold standard of treating inhibitors is the immune tolerance induction (ITI), where patients are given frequent, high doses of fVIII to induce an immune tolerance. ITI therapy fails in about 30 % of patients. Mechanisms of action of ITI are part of research, as insufficient knowledge about mechanisms and prognostic factors complicate treatment. For example, the development of anti-idiotypic antibodies, which occur naturally as a regulatory mechanism of the immune system, are being studied. Such anti-idiotypes have been detected in immunoglobuline preparations and in patients after successful ITI.
Inhibitors interfere with fVIII function in coagulation by binding functional epitopes within fVIII domains. Inhibitors against the A2 and C2 domain are predominantly found, however also the C1 domain has been shown to be highly immunogenic in some patients. The polyclonality of inhibitors aggravates the understanding and treatment of these. The present project therefore focusses on the selection of synthetic anti-idiotypic antibodies to target inhibitors in patients. The phage display method was applied to, for one, isolate anti-idiotypic single chain variable fragments (scFvs) specific against human polyclonal anti-fVIII antibodies and second against two C1 domain-specific inhibitory monoclonal antibodies (mAbs).
In the first project, anti-fVIII antibodies were purified from human plasma to serve as target molecules. A previous project showed that using full plasma as a target did not yield anti-idiotypic antibodies from phage display. For the purification, protein A chromatography and fVIII coupled Affi Gel® chromatography were applied. The isolated antibodies were next used as targets for the selection of anti-idiotypic scFvs. Analysis revealed that none of the selected phages solely bound the anti-fVIII antibody target. Consequently, the test protocol was modified, which resulted in a reduction of unspecific binders. Yet, no target-specific binders were isolated from phage pools. Reason for this may have been the high diversity of the polyclonal antibody target and the limited diversity of the phage libraries.
The aim of the second project, was the selection and characterization of scFvs, that target the paratopes of C1 domain-specific mAbs GMA8011 and LE2E9. From a therapeutic viewpoint, the preparation of an anti-idiotypic antibody pool, tailored to each patient’s inhibitor population, could help neutralize inhibitors in patients. Ultimately, one GMA8011-specific scFv-carrying phage clone (H2C1) and two specifics to LE2E9 (H3G7, H3F10) were isolated. In further experiments, only the GMA8011-specific scFv showed competitive behavior in presence of fVIII, pointing towards an anti-idiotypic binding to the inhibitor paratope. The LE2E9-specific scFvs did not prevent binding of the inhibitor to fVIII. Hence, no anti-idiotypic behavior could be determined. For further characterization, selected scFvs were genetically fused to Fc antibody fragments and recombinantly produced. In this antibody format, all three scFvs showed concentration dependent binding to the target and the isotype control. The binding specificity to the target, observed in phage context, could not be reproduced. Competition experiments with fVIII confirmed that none of the scFvs bound the paratope of their target inhibitor.
The selection of anti-idiotypic scFvs from phage display libraries proves to be effortful. Polyclonal anti-fVIII antibodies purified from hemophilic plasma appear to be unsuitable as a target for phage display, likely due to the high diversity of the target molecules. Furthermore, the preparation of an individualized anti-idiotypic pools for patients by selecting scFvs against single inhibitory mAbs proves to be difficult. The selection of scFvs against anti-C1 inhibitors GMA8011 and LE2E9 produced three promising scFv-carrying phages. However, analysis could not detect anti-idiotypic behavior. Further research with inhibitors, monoclonal and polyclonal, and anti-idiotypic antibodies should be performed to bring better insight into the highly complex paratope-epitope interaction.
Aims: Long non-coding RNAs (lncRNAs) have been shown to regulate numerous processes in the human genome, but the function of these transcripts in vascular aging is largely unknown. We aim to characterize the expression of lncRNAs in endothelial aging and analyse the function of the highly conserved lncRNA H19.
Methods and results: H19 was downregulated in endothelium of aged mice. In human, atherosclerotic plaques H19 was mainly expressed by endothelial cells and H19 was significantly reduced in comparison to healthy carotid artery biopsies. Loss of H19 led to an upregulation of p16 and p21, reduced proliferation and increased senescence in vitro. Depletion of H19 in aortic rings of young mice inhibited sprouting capacity. We generated endothelial-specific inducible H19 deficient mice (H19iEC-KO), resulting in increased systolic blood pressure compared with control littermates (Ctrl). These H19iEC-KO and Ctrl mice were subjected to hindlimb ischaemia, which showed reduced capillary density in H19iEC-KO mice. Mechanistically, exon array analysis revealed an involvement of H19 in IL-6 signalling. Accordingly, intercellular adhesion molecule 1 and vascular cell adhesion molecule 1 were upregulated upon H19 depletion. A luciferase reporter screen for differential transcription factor activity revealed STAT3 as being induced upon H19 depletion and repressed after H19 overexpression. Furthermore, depletion of H19 increased the phosphorylation of STAT3 at TYR705 and pharmacological inhibition of STAT3 activation abolished the effects of H19 silencing on p21 and vascular cell adhesion molecule 1 expression as well as proliferation.
Conclusion: These data reveal a pivotal role for the lncRNA H19 in controlling endothelial cell aging.
Cancer is one of the leading causes of death across all countries and its diagnosis still yields fear for the affected patient. Although treatment of cancer has made marvelous progress compared to the agents available thirty years ago, a cure for cancer, however, is still a distant prospect. Modern therapy still is a burden for many patients due to heavy side effects. With the development of agents targeting specific molecular targets on cancer cells, a new field of cancer therapy was opened and a small success story in the history of cancer began.
Aurora kinases represent a relatively new target in cancer therapy. The kinase is a essential part of mitosis and cell cycle progression and its overexpression has been shown to be related to many kinds of malignancies. Allosteric inhibition of a kinase is an increasing pre-clinical approach not yet established in the treatment of patients. In this thesis, we combine allostery with another innovative approach that is drug repurposing. If repurposed, a drug can be permitted to fast track drug admission to clinical trials.
I set up a screening of 1280 FDA approved drugs to identify small molecule compounds that affect the binding of Aurora kinase A and its main physiologic binding partner, TPX2. Further, I characterized the positive hits in vitro for their capabilities to displace TPX2 from Aurora A, to inhibit Aurora kinase activity, to thermally stabilize the protein and performed assays to determine their dissociation constant. Last but not least, I tested the compounds in cells for their effect on the cell viability and cell cycle via flow cytometry. Comparing the hit-compounds with controls I found that ATP-competitive AurA inhibitor MLN 8237 strongly displaces the interaction of Aurora A with TPX2.
Summarized, we identified eight hit compounds allosterically affecting Aurora A, but no compound proved to be active in all assays. Just one compound, PS 731, identified in another screening performed by our group and further characterized in this thesis remains interesting, especially when put in context with recent publications released in the time between the start of experiments for this thesis and its finalization.
How much we trust our own decisions, knowledge or perceptions influences our behavior in many everyday situations. Normally the confidence we have in our decisions is rather accurate, but under certain circumstances the subjective evaluation of a decision and its objective quality can differ heavily. Subjectively over- or underestimating the quality of decisions can lead to disadvantageous behavior. Little is known about how this feeling of confidence about a decision is generated. Is it computed automatically with the decision or does it arise in a different process?
This thesis is based on a publication that contributed to the investigation of this question by comparing the influence of two different forms of spatial attention on decision confidence. Visual spatial attention is a cognitive mechanism that serves to select parts of the visual field, leading to more accurate decisions about the attended items. It can be either voluntarily controlled (endogenous) or reflexively driven by external events (exogenous). In an orientation-matching task participants performed better in both attentional conditions than in a control condition without directed attention. Additionally, we found that only endogenous, but not exogenous attention led the subjects to overestimate the quality of their performance. The possible implications of this “relative overconfidence” were discussed with respect to the theoretical framework of spatial attention and decision confidence. The present findings support the idea that decision confidence is generated in a distinct metacognitive process. Possible ideas for further neurophysiological research are proposed. The thesis concludes with an attempt to integrate the discussion into a broader context of medical research on certain neuropsychiatric symptoms and conditions.
Throughout the entire life, new neurons of the granule cell type (GCs) are continu-ously generated in the mammalian hippocampal dentate gyrus (DG). As a part of the limbic system, the hippocampus is concerned with spatial and declarative memory for-mation. Increasing evidence exists, that adult born granule cells (ABGCs) play an im-portant role in this process. An especially critical period, when these ABGCs are 4-6 weeks old, has come into the focus of research. It is during this specific time-span that the ABGCs express enhanced excitability and synaptic plasticity as well as a lowered threshold for the induction of long term potentiation (LTP), a mechanism associated to learning and memory formation.
This study investigates the time course and dynamics of synaptic integration in ABGCs and mature GCs together with which differences exist between them at various cell ages. Furthermore, spine plasticity following high frequency stimulation (HFS) is analysed focusing on a critical phase of enhanced excitability in 4-5 week old ABGCs.
In this thesis, two approaches at studying the synaptic integration and structural plas-ticity of ABGCs in rats were investigated. This work was performed on fixed brain ma-terial that was provided by two laboratories that performed the in vivo labelling, stimu-lation procedures and brain fixation. In the first project, 6, 12 and 35 weeks old XdU-labelled ABGCs were studied. Adult rats were exposed to an enriched environment and received unilateral intrahippocampal delta burst stimulation (DBS) and LTP induction. The ABGCs and a control population of mature GCs were immunohistologically ana-lysed for Egr1 (early growth response 1) expression. Egr1 is an immediate early gene (IEG), expressed after LTP induction and marks neuronal excitation.
It was found, that unilateral stimulation of the perforant path of the hippocampus re-sults in an increase of Egr1 expression in ABGCs of both hemispheres. It could be shown that the enhanced expression intensity of Egr1 in ABGCs is not a usual state of young GCs but a reaction to DBS. ABGCs from unstimulated control animals and mature GCs expressed lower levels of Egr1. Interestingly, the stimulation induced a similar degree of Egr1 expression intensity in all ABGC age groups. Furthermore, it was found that young ABGC from the infrapyramidal dentate gyrus (DG) express a higher excita-bility than those from the suprapyramidal DG.
In the second project, fixed brain sections were analysed. They stemmed from rat brains containing 28 and 35 day old ABGC that had been transfected with intrahippo-campal RV-GFP (retroviral-green fluorescent protein) injections and had received uni-lateral high frequency stimulation of the medial perforant path in vivo. Nuclear Egr1 expression intensity was analysed in a cell specific manner. Dendritic spine size was measured in the inner-, middle- and outer molecular layer (IML, MML, OML). It was found that in ABGC, stimulation induced Egr1 expression increase is lower than in ma-ture GC. Following HFS, a significant homosynaptic spine enlargement was observed in the MML indicating homosynaptic LTP, while heterosynaptic spine shrinkage was found in the adjacent IML and OML. The latter corresponds to heterosynaptic long term depression (LTD). Homosynaptic plasticity describes an input-specific potentiation of synapses that received direct activation. The weakening of synapses not stimulated dur-ing homosynaptic potentiation is oppositely coined heterosynaptic plasticity1.
A positive correlation between an increase in nuclear Egr1 expression intensity and spine enlargement due to homosynaptic plasticity induced by HFS could be shown. Concomitant heterosynaptic plasticity, as indicated by spine shrinkage was observed. Spine shrinkage in the IML and OML showed a negative correlation to a decrease in Egr1 intensity.
Taken together, the results provide detailed information on the gradual integration of ABGC with ongoing maturation. Cell specific proof for homo- and heterosynaptic plas-ticity following HFS was found in the critical period of synaptic integration of ABGCs.
After entorhinal deafferentiation of the hippocampal dentate gyrus a reinnervation of the denervated neurons by axon collaterals can be observed. This process takes place in a matter of weeks. However, the overall functional effect on the hippocampal network is still unclear.
In an effort to investigate this effect of axonal sprouting on the neuronal network of the dentate gyrus we compared the electrophysiological response of the dentate gyrus after electric stimulation in wild-type mice (WT mice) with a normal post-lesion sprouting, with genetically modified mice with an overexpression of the growth-protein CAP23 (cytoskeleton-associated protein 23). CAP23 overexpressing mice (CAP23tg mice) are known to have an enhanced axonal growth and sprouting after lesion.
The mice (both the WT as well as the CAP23tg mice) were deeply anesthetized and a lesion of the perforant path was induced stereotactically with a wire knife. After that the mice were permitted to survive for 4-6 weeks for partial reinnervation of the dentate gyrus before they were again operated and evoked potentials were measured (extracellular recordings of evoked potentials in the dentate gyrus). Non-lesioned litter-mate mice were taken as reference. The sprouting and the correct position of the electrodes was confirmed histologically.
For electrophysiological investigation we assessed laminar profiles and calculated a current-source density (CSD). In lesioned CAP23tg mice compared to lesioned WT mice this CSD-analysis revealed a significant enhancement of the current sink in the area of deafferentiation (outer molecular layer) and a significant excitation in the granule-cell layer.
Our results show that axonal sprouting seems to enhance the excitability of granule-cells. Thus, even if an enhanced axonal sprouting might accelerate the reinnervation of denervated dendrites after lesion, but it also leads to posttraumatic hyperexcitability of the neuronal network. In a therapeutic approach of fascilitating axonal sprouting this hyperexcitability has to be taken into consideration.