Untersuchung visueller Aufmerksamkeitsparameter vor und nach einer Hepatitis C (HCV)-Therapie mit Direct Antiviral Agents bei HCV-monoinfizierten und HCV/HIV-koinfizierten Patienten

Bei Patienten mit chronischer HCV-Infektion wurden wiederholt kognitive Defizite, auch unabhängig von einer Leberzirrhose, nachgewiesen. Ebenso leidet ein Teil der HIV-Patienten trotz adäquater antiretroviraler Therapie unter kognitiven Beeinträchtigungen, den sogenannten „HIV-associated neurocognitive disorders“ (HAND). HCV/HIV-koinfizierte Patienten könnten stärker von diesen Beeinträchtigungen betroffen sein als HCV- oder HIV-Monoinfizierte. Bei beiden Patientengruppen ist der Bereich Aufmerksamkeit oft beeinträchtigt. Die frühere HCV-Standardtherapie mit Interferon-alpha verursachte häufig selbst neuropsychiatrische Nebenwirkungen. Durch die neuen, interferonfreien Therapiemöglichkeiten für Hepatitis C, die Direct Antiviral Agents (DAA), haben sich sichere und Erfolg versprechende Behandlungsoptionen hinsichtlich der HCV-Eradikation ergeben. Die Auswirkungen einer HCV-Eradikation mit DAA auf kognitive Beeinträchtigungen sind bisher nicht erforscht. In dieser Promotionsarbeit wird der Einfluss einer DAA-Therapie auf visuelle Aufmerksamkeitsparameter, basierend auf der Theorie der visuellen Aufmerksamkeit (TVA), bei HCV-monoinfizierten und HCV/HIV-koinfizierten Patienten untersucht. Fünf visuelle Aufmerksamkeitsparameter wurden in einer Gruppe von 20 HCV-infizierten Patienten, darunter 12 HCV/HIV-Koinfizierte, vor und 12 Wochen nach Ende einer DAA-basierten HCV-Therapie mittels TVA-basierter Testung untersucht. Die Ergebnisse wurden mit einer gesunden, parallelisierten Kontrollgruppe (n = 35), die in ähnlichem Zeitabstand zweimal getestet wurde, verglichen. Im Rahmen einer anderen Dissertation wurde in der gleichen Patientengruppe eine neuropsychologische Standardtestung durchgeführt, deren Ergebnisse zum Vergleich herangezogen wurden. In der Patientengruppe wurden außerdem subjektive Gesundheitszustände mittels „Patient Reported Outcomes“ (PROs) zu Fatigue, Depression und gesundheitsbezogener Lebensqualität erhoben. Beim ersten Testzeitpunkt gab es hinsichtlich der TVA-Parameter keine signifikanten Unterschiede zwischen der Patienten- und der Kontrollgruppe. Jedoch waren 40% der Patienten in mindestens einem TVA-Parameter beeinträchtigt. 95% der Patienten hatten 12 Wochen nach Therapieende keine nachweisbare Viruslast mehr („Sustained virological response“, SVR12). Hinsichtlich der Leistungsänderung zum zweiten Testzeitpunkt waren keine Unterschiede zwischen Patienten und Kontrollprobanden zu verzeichnen. Der Anteil an in mindestens einem TVA-Parameter beeinträchtigten Patienten blieb konstant. Keine Unterschiede waren zwischen HCV-Monoinfizierten und HCV/HIV-Koinfizierten zu beobachten. In der neuropsychologischen Standardtestung wurden in fünf kognitiven Bereichen signifikante Verbesserungen beobachtet, jedoch nicht im Bereich Aufmerksamkeit/Arbeitsgedächtnis. Die Fatigue-Symptomatik und die mentale Gesundheit in der Patientengruppe verbesserten sich signifikant. In dieser Studie wurden erstmals visuelle Aufmerksamkeitsparameter in einer Gruppe HCV-infizierter Patienten im Rahmen einer DAA-Therapie untersucht. Die Therapie hatte keinen signifikanten Einfluss auf die visuelle Aufmerksamkeitsleistung der Patienten. In Übereinstimmung damit wurden in einer neuropsychologischen Standardtestung keine signifikanten Verbesserungen im Bereich Aufmerksamkeit beobachtet. Fortbestehende Defizite bei einem Teil der Patienten könnten Hinweise auf eine HCV-Persistenz im ZNS darstellen. Die Therapie wirkte sich positiv auf die Fatigue-Symptomatik und die mentale Gesundheit aus.Cognitive deficits have repeatedly been demonstrated in patients with chronic HCV-infection, independently of liver cirrhosis. Likewise, HIV-infected patients can suffer from “HIV-associated neurocognitive disorders” (HAND), despite adequate antiretroviral therapy. Possibly, HCV/HIVcoinfected patients are affected more severely by these deficits compared to HCV- or HIVmonoinfected. Attention is often affected in both groups. The former interferon-alpha-based HCVtherapy frequently caused neuropsychiatric side effects itself. With the new, interferon-free therapies for hepatitis C, the Direct Antiviral Agents (DAA), arose safe and promising therapeutic options concerning HCV eradication. If an HCV eradication by DAA has positive effects on cognitive deficits has not been explored. In this dissertation, the influence of a DAA therapy on visual attention parameters, based on the Theory of Visual Attention (TVA), in HCVmonoinfected and HCV/HIV-coinfected patients is investigated. Methods In a group of 20 HCV-infected patients, including 12 HCV/HIV-coinfected, five parameters of visual attention were assessed before and 12 weeks after a DAA-based therapy, using TVA-based testing. The results were compared to a healthy, matched control group (n = 35) tested twice. In another dissertation, the same patient group underwent a standard neuropsychological testing which was used for a comparison*. Additionally, the subjective state of health was assessed in the patient group, using patient reported outcomes (PROs) for fatigue, depression and health-related quality of life. Results At baseline, there were no significant differences between patients and control subjects. However, 40% of the patients were impaired in at least one TVA parameter. 12 weeks after the end of therapy, 95% of the patients had no detectable viral load anymore (“Sustained virological response”, SVR12). Concerning performance changes at follow-up, we did not observe significant differences between patients and controls. The portion of patients who were impaired in TVA parameters remained stable. There were no significant differences between HCV-monoinfected and HCV/HIV-coinfected patients. The neuropsychological standard testing revealed improvements in five cognitive domains, but not in terms of attention/working memory. Fatigue and mental health significantly improved in the patient group. Conclusions This study represents the first investigation of visual attention parameters in a group of HCVinfected patients in the context of a DAA-based therapy. There was no significant effect on the patients’ visual attention. Consistently with this, there was no improvement in the domain of attention in a neuropsychological standard testing. Remaining deficits in some patients might reflect a CNS-persistence of HCV. The therapy had positive effects on fatigue and mental health

Proteome remodelling during development from blood to insect-form Trypanosoma brucei quantified by SILAC and mass spectrometry

Trypanosoma brucei is the causative agent of human African sleeping sickness and Nagana in cattle. In addition to being an important pathogen T. brucei has developed into a model system in cell biology

Delaying histone deacetylase response to injury accelerates conversion into repair Schwann cells and nerve regeneration

The peripheral nervous system (PNS) regenerates after injury. However, regeneration is often compromised in the case of large lesions, and the speed of axon reconnection to their target is critical for successful functional recovery. After injury, mature Schwann cells (SCs) convert into repair cells that foster axonal regrowth, and redifferentiate to rebuild myelin. These processes require the regulation of several transcription factors, but the driving mechanisms remain partially understood. Here we identify an early response to nerve injury controlled by histone deacetylase 2 (HDAC2), which coordinates the action of other chromatin-remodelling enzymes to induce the upregulation of Oct6, a key transcription factor for SC development. Inactivating this mechanism using mouse genetics allows earlier conversion into repair cells and leads to faster axonal regrowth, but impairs remyelination. Consistently, short-term HDAC1/2 inhibitor treatment early after lesion accelerates functional recovery and enhances regeneration, thereby identifying a new therapeutic strategy to improve PNS regeneration after lesion

Proteomic characterization of Toxoplasma gondii ME49 derived strains resistant to the artemisinin derivatives artemiside and artemisone implies potential mode of action independent of ROS formation.

The sesquiterpene lactone artemisinin and its amino-artemisinin derivatives artemiside (GC008) and artemisone (GC003) are potent antimalarials. The mode of action of artemisinins against Plasmodium sp is popularly ascribed to 'activation' of the peroxide group by heme-Fe(II) or labile Fe(II) to generate C-radicals that alkylate parasite proteins. An alternative postulate is that artemisinins elicit formation of reactive oxygen species by interfering with flavin disulfide reductases resposible for maintaining intraparasitic redox homeostasis. However, in contradistinction to the heme-activation mechanism, the amino-artemisinins are effective in vitro against non-heme-degrading apicomplexan parasites including T. gondii, with IC 50 values of 50-70 nM, and induce distinct ultrastructural alterations. However, T. gondii strains readily adapted to increased concentrations (2.5 μM) of these two compounds within few days. Thus, T. gondii strains that were resistant against artemisone and artemiside were generated by treating the T. gondii reference strain ME49 with stepwise increasing amounts of these compounds, yielding the artemisone resistant strain GC003R and the artemiside resistant strain GC008R. Differential analyses of the proteomes of these resistant strains compared to the wildtype ME49 revealed that 215 proteins were significantly downregulated in artemisone resistant tachyzoites and only 8 proteins in artemiside resistant tachyzoites as compared to their wildtype. Two proteins, namely a hypothetical protein encoded by ORF TGME49_236950, and the rhoptry neck protein RON2 encoded by ORF TGME49_300100 were downregulated in both resistant strains. Interestingly, eight proteins involved in ROS scavenging including catalase and superoxide dismutase were amongst the differentially downregulated proteins in the artemisone-resistant strain. In parallel, ROS formation was significantly enhanced in isolated tachyzoites from the artemisone resistant strain and - to a lesser extent - in tachyzoites from the artemiside resistant strain as compared to wildtype tachyzoites. These findings suggest that amino-artemisinin derivatives display a mechanism of action in T. gondii distinct from Plasmodium

Targeted and non-targeted proteomics to characterize the parasite proteins of Echinococcus multilocularis metacestodes.

The larval stage of the cestode Echinococcus multilocularis is the causative agent of alveolar echinococcosis. To investigate the biology of these stages and to test novel compounds, metacestode cultures represent a suitable in vitro model system. These metacestodes are vesicles surrounded by an envelope formed by the vesicle tissue (VT), which is formed by the laminated and germinal layer, and filled with vesicle fluid (VF). We analyzed the proteome of VF and VT by liquid chromatography tandem mass spectrometry (LC-MS/MS) and identified a total of 2,954 parasite proteins. The most abundant protein in VT was the expressed conserved protein encoded by EmuJ_000412500, followed by the antigen B subunit AgB8/3a encoded by EmuJ_000381500 and Endophilin B1 (protein p29). In VF, the pattern was different and dominated by AgB subunits. The most abundant protein was the AgB8/3a subunit followed by three other AgB subunits. In total, the AgB subunits detected in VF represented 62.1% of the parasite proteins. In culture media (CM), 63 E. multilocularis proteins were detected, of which AgB subunits made up 93.7% of the detected parasite proteins. All AgB subunits detected in VF (encoded by EmuJ_000381100-700, corresponding to AgB8/2, AgB8/1, AgB8/4, AgB8/3a, AgB8/3b, and AgB8/3c) were also found in CM, except the subunit encoded by EmuJ_000381800 (AgB8/5) that was very rare in VF and not detected in CM. The relative abundance of the AgB subunits in VF and CM followed the same pattern. In VT, only the subunits EmuJ_000381500 (AgB8/3a) and EmuJ_000381200 (AgB8/1) were detected among the 20 most abundant proteins. To see whether this pattern was specific to VF from in vitro cultured metacestodes, we analyzed the proteome of VF from metacestodes grown in a mouse model. Here, the AgB subunits encoded by EmuJ_000381100-700 constituted the most abundant proteins, namely, 81.9% of total protein, with the same order of abundance as in vitro. Immunofluorescence on metacestodes showed that AgB is co-localized to calcareous corpuscles of E. multilocularis. Using targeted proteomics with HA-tagged EmuJ_000381200 (AgB8/1) and EmuJ_000381100 (AgB8/2), we could show that uptake of AgB subunits from CM into VF occurs within hours

Comparative Proteomic Analysis of Toxoplasma gondii RH Wild-Type and Four SRS29B (SAG1) Knock-Out Clones Reveals Significant Differences between Individual Strains.

In T. gondii, as well as in other model organisms, gene knock-out using CRISPR-Cas9 is a suitable tool to identify the role of specific genes. The general consensus implies that only the gene of interest is affected by the knock-out. Is this really the case? In a previous study, we generated knock-out (KO) clones of TgRH88_077450 (SRS29B; SAG1) which differed in the numbers of the integrated dihydrofolate-reductase-thymidylate-synthase (MDHFR-TS) drug-selectable marker. Clones 18 and 33 had a single insertion of MDHFR-TS within SRS29B. Clone 6 was disrupted by the insertion of a short unrelated DNA-sequence, but the marker was integrated elsewhere. In clone 30, the marker was inserted into SRS29B, and several other MDHFR-TS copies were found in the genome. KO and wild-type (WT) tachyzoites had similar shapes, dimensions, and vitality. This prompted us to investigate the impact of genetic engineering on the overall proteome patterns of the four clones as compared to the respective WT. Comparative shotgun proteomics of the five strains was performed. Overall, 3236 proteins were identified. Principal component analysis of the proteomes revealed five distinct clusters corresponding to the five strains by both iTop3 and iLFQ algorithms. Detailed analysis of the differentially expressed proteins revealed that the target of the KO, srs29B, was lacking in all KO clones. In addition to this protein, 20 other proteins were differentially expressed between KO clones and WT or between different KO clones. The protein exhibiting the highest variation between the five strains was srs36D encoded by TgRH_016110. The deregulated expression of SRS36D was further validated by quantitative PCR. Moreover, the transcript levels of three other selected SRS genes, namely SRS36B, SRS46, and SRS57, exhibited significant differences between individual strains. These results indicate that knocking out a given gene may affect the expression of other genes. Therefore, care must be taken when specific phenotypes are regarded as a direct consequence of the KO of a given gene

Überblick über den aktuellen Stand der Forschungsberichterstattung: Integration, Standardisierung, verteilte Informationssysteme

Dieses Diskussionspapier gibt einen Überblick über die Entwicklung der Forschungsberichterstattung im Wissenschaftssystem. Dabei werden aktuelle Entwicklungen von integrierten Forschungsinformationssystemen und aktuelle Standardisierungsinitiativen aufgezeigt und ein Einblick in ein Umsetzungsprojekt eines verteilten Systems gegeben. Der Beitrag schließt in mit einer Diskussion von Weiterentwicklungsszenarien verteilter Forschungsinformationssysteme im Wis-senschaftssystem

Effect of Sample Transportation on the Proteome of Human Circulating Blood Extracellular Vesicles

Circulating extracellular vesicles (cEV) are released by many kinds of cells and play an important role in cellular communication, signaling, inflammation modulation, coagulation, and tumor growth. cEV are of growing interest, not only as biomarkers, but also as potential treatment targets. However, very little is known about the effect of transporting biological samples from the clinical ward to the diagnostic laboratory, notably on the protein composition. Pneumatic tube systems (PTS) and human carriers (C) are both routinely used for transport, subjecting the samples to different ranges of mechanical forces. We therefore investigated qualitatively and quantitatively the effect of transport by C and PTS on the human cEV proteome and particle size distribution. We found that samples transported by PTS were subjected to intense, irregular, and multidirectional shocks, while those that were transported by C mostly underwent oscillations at a ground frequency of approximately 4 Hz. PTS resulted in the broadening of nanoparticle size distribution in platelet-free (PFP) but not in platelet-poor plasma (PPP). Cell-type specific cEV-associated protein abundances remained largely unaffected by the transport type. Since residual material of lymphocytes, mono- cytes, and platelets seemed to dominate cEV proteomes in PPP, it was concluded that PFP should be preferred for any further analyses. Differential expression showed that the impact of the transport method on cEV-associated protein composition was heterogeneous and likely donor-specific. Correla- tion analysis was nonetheless able to detect that vibration dose, shocks, and imparted energy were associated with different terms depending on the transport, namely in C with cytoskeleton-regulated cell organization activity, and in PTS with a release of extracellular vesicles, mainly from organelle origin, and specifically from mitochondrial structures. Feature selection algorithm identified proteins which, when considered together with the correlated protein-protein interaction network, could be viewed as surrogates of network clusters

The continuity of separation. Preparatory classes for recently immigrated children and youth in the context of historical forms of segregated schooling

Der Beitrag zeigt aus organisationstheoretischer Perspektive die historischen Kontinuitäten der separierten Beschulung von migrantischen Kindern in Berlin auf. Auf Grundlage der Ergebnisse einer 2016 durchgeführten Untersuchung an Berliner Grundschulen wird rekonstruiert, wie im Falle der Willkommensklassen ein Mangel an Regeln zu einer Entwicklung von Routinen führt, die sich weitgehend auf historische Verfahrensweisen stützen und mit historisch bekannten Begründungen legitimiert werden. (DIPF/Orig.)Arguing from a perspective of organizational theory this article shows historical continuities of segregated schooling of migrant children in Berlin. Based on results of a study conducted at Berlin primary schools it is shown how the lack of regulations for Willkommensklassen ["Welcoming Classes"] leads to the development of routines, which mainly rely on historical procedures and are legitimized with historically known legitimations. (DIPF/Orig.

Differential Affinity Chromatography Coupled to Mass Spectrometry: A Suitable Tool to Identify Common Binding Proteins of a Broad-Range Antimicrobial Peptide Derived from Leucinostatin.

Leucinostatins are antimicrobial peptides with a broad range of activities against infectious agents as well as mammalian cells. The leucinostatin-derivative peptide ZHAWOC_6027 (peptide 6027) was tested in vitro and in vivo for activity against the intracellular apicomplexan parasite Toxoplasma gondii. While highly efficacious in vitro (EC50 = 2 nM), subcutaneous application of peptide 6027 (3 mg/kg/day for 5 days) in mice experimentally infected with T. gondii oocysts exacerbated the infection, caused mild clinical signs and elevated cerebral parasite load. Peptide 6027 also impaired the proliferation and viability of mouse splenocytes, most notably LPS-stimulated B cells, in vitro. To identify common potential targets in Toxoplasma and murine splenocytes, we performed differential affinity chromatography (DAC) with cell-free extracts from T. gondii tachyzoites and mouse spleens using peptide 6027 or an ineffective analogue (peptide 21,358) coupled to N-hydroxy-succinimide sepharose, followed by mass spectrometry. Proteins specifically binding to peptide 6027 were identified in eluates from the peptide 6027 column but not in peptide 21,358 nor the mock column eluates. In T. gondii eluates, 269 proteins binding specifically to peptide 6027 were identified, while in eluates from mouse spleen extracts 645 proteins specifically binding to this peptide were detected. Both datasets contained proteins involved in mitochondrial energy metabolism and in protein processing and secretion. These results suggest that peptide 6027 interacts with common targets in eukaryotes involved in essential pathways. Since this methodology can be applied to various compounds as well as target cell lines or organs, DAC combined with mass spectrometry and proteomic analysis should be considered a smart and 3R-relevant way to identify drug targets in pathogens and hosts, thereby eliminating compounds with potential side effects before performing tedious and costly safety and efficacy assessments in animals or humans