Micro-RNA expression in cisplatin resistant germ cell tumor cell lines

<p>Abstract</p> <p>Background</p> <p>We compared microRNA expression patterns in three cisplatin resistant sublines derived from paternal cisplatin sensitive germ cell tumor cell lines in order to improve our understanding of the mechanisms of cisplatin resistance.</p> <p>Methods</p> <p>Three cisplatin resistant sublines (NTERA-2-R, NCCIT-R, 2102EP-R) showing 2.7-11.3-fold increase in drug resistance after intermittent exposure to increasing doses of cisplatin were compared to their parental counterparts, three well established relatively cisplatin sensitive germ cell tumor cell lines (NTERA-2, NCCIT, 2102EP). Cells were cultured and total RNA was isolated from all 6 cell lines in three independent experiments. RNA was converted into cDNA and quantitative RT-PCR was run using 384 well low density arrays covering almost all (738) known microRNA species of human origin.</p> <p>Results</p> <p>Altogether 72 of 738 (9.8%) microRNAs appeared differentially expressed between sensitive and resistant cell line pairs (NTERA-2R/NTERA-2 = 43, NCCIT-R/NCCIT = 53, 2102EP-R/2102EP = 15) of which 46.7-95.3% were up-regulated (NTERA-2R/NTERA-2 = 95.3%, NCCIT-R/NCCIT = 62.3%, 2102EP-R/2102EP = 46.7%). The number of genes showing differential expression in more than one of the cell line pairs was 34 between NTERA-2R/NTERA-2 (79%) and NCCIT-R/NCCIT (64%), and 3 and 4, respectively, between these two cell lines and 2102EP-R/2102EP (about 27%). Only the has-miR-10b involved in breast cancer invasion and metastasis and has-miR-512-3p appeared to be up-regulated (2-3-fold) in all three cell lines. The hsa-miR-371-373 cluster (counteracting cellular senescence and linked with differentiation potency), as well as hsa-miR-520c/-520h (inhibiting the tumor suppressor p21) were 3.9-16.3 fold up-regulated in two of the three cisplatin resistant cell lines. Several new micro-RNA species missing an annotation towards cisplatin resistance could be identified. These were hsa-miR-512-3p/-515/-517/-518/-525 (up to 8.1-fold up-regulated) and hsa-miR-99a/-100/-145 (up to 10-fold down-regulated).</p> <p>Conclusion</p> <p>Examining almost all known human micro-RNA species confirmed the miR-371-373 cluster as a promising target for explaining cisplatin resistance, potentially by counteracting wild-type P53 induced senescence or linking it with the potency to differentiate. Moreover, we describe for the first time an association of the up-regulation of micro-RNA species such as hsa-miR-512-3p/-515/-517/-518/-525 and down-regulation of hsa-miR-99a/-100/-145 with a cisplatin resistant phenotype in human germ cell tumors. Further functional analyses are warranted to gain insight into their role in drug resistance.</p

Potential sources of interference with the highly sensitive detection and quantification of alpha‐synuclein seeds by qRT‐QuIC

Parkinson’s disease (PD) is a progressive neurodegenerative disease which is histologically characterized by loss of dopaminergic neurons in the substantia nigra and deposition of aggregated alpha‐synuclein (aSyn) in the brain. The detection of aSyn in well accessible fluids has been one of the central approaches in the development of biomarkers for PD. Recently, real‐time quaking‐induced conversion (RT‐QuIC) has been successfully adapted for use with aSyn seeds. Here, we systematically analysed parameters potentially impacting the reliability of this assay by using quantitative real‐time quaking‐induced conversion (qRT‐QuIC) with in vitro‐formed aSyn seeds. Seeds diluted in cerebrospinal fluid (CSF) accelerated the seeding reaction and slightly increased the sensitivity without affecting specificity. Repeated freeze–thaw cycles decreased the apparent lag times of seeds diluted in ddH2O but did not alter the seeding activity of seeds diluted in CSF. High levels of artificial contamination with blood resulted in prolonged apparent lag times, while sensitivity and specificity were unaffected. Altogether, qRT‐QuIC with aSyn seems to be robust concerning sensitivity and specificity in our model system, but quantitative interpretation might be limited under certain conditions

Extended stereotactic brain biopsy in suspected primary central nervous system angiitis: good diagnostic accuracy and high safety

OBJECTIVE To evaluate the diagnostic accuracy and safety of extended stereotactic brain biopsy (ESBB) in a single center cohort with suspected primary angiitis of the central nervous system (PACNS). METHODS A standardized stereotactic biopsy targeting MRI-positive lesions and collecting samples from the meninges and the cortex as well as from the white matter was performed in 23 patients with clinically suspected PACNS between 2010 and 2017. The relationship between biopsy yield and clinical characteristics, cerebrospinal fluid parameters, MR-imaging, time point of biopsy and exact localization of biopsy as well as number of tissue samples were examined. RESULTS PACNS was confirmed in 7 of 23 patients (30.4%). Alternative diagnoses were identified in 7 patients (30%). A shorter time period between the onset or worsening of symptoms (p = 0.018) and ESBB significantly increased the diagnostic yield. We observed only minor and transient postoperative complications in 3 patients (13.0%). ESBB led to a direct change of the therapeutic regime in 13 of 23 patients (56.5%). Careful neuropathological analysis furthermore revealed that cortical samples were crucial in obtaining a diagnosis. CONCLUSION ESBB is a safe approach with good feasibility, even in critically ill patients, and high diagnostic accuracy in patients with suspected PACNS changing future therapies in 13 of 23 patients (56.5%). Early biopsy after symptom onset/worsening is crucial and (sub)acute MRI-lesions should be targeted with a particular need for biopsy samples from the cortical layer

Neurofilaments in blood and CSF for diagnosis and prediction of onset in Creutzfeldt-Jakob disease

While cerebrospinal fluid (CSF) biomarkers for Creutzfeldt-Jakob disease (CJD) are established and partly included in the diagnostic criteria, no blood biomarkers are available. Here, we assessed the utility of serum neurofilament light chain (NF-L) and tau protein in comparison to CSF markers (NF-L and phosphorylated NF heavy chain (pNF-H), tau, S100B, 14-3-3) and prion conversion assay (realtime quaking induced conversion (RT-QuIC)) for sporadic and genetic CJD. Importantly, a Gerstmann-Straussler-Scheinker mutation carrier in the asymptomatic phase and at disease onset was included as well. Both NF-L and tau were markedly increased in CJD serum, reaching similar or even better performance as in CSF (sensitivity and specificity for serum NF-L 100% and 85.5%, and for serum tau 84.6% and 96.2%, respectively). Serum S100B showed high sensitivity as well (84.2%), but lower specificity (63%). CSF neurofilaments were increased before symptom onset, while prion seeding assay was negative. Just before a clinical diagnosis could be made, all CSF markers and NF-L in the serum were increased and CSF prion conversion assay was positive. The data suggest that neurofilaments are sensitive and specific blood markers for the diagnosis of genetic and sporadic CJD and might represent promising tools to predict disease onset

Challenges and Rewards in Medicinal Chemistry Targeting Cardiovascular and Metabolic Diseases

Medicinal chemistry has been transformed by major technological and conceptual innovations over the last three decades: structural biology and bioinformatics, structure and property based molecular design, the concepts of multidimensional optimization (MDO), in silico and experimental high-throughput molecular property analysis. The novel technologies advanced gradually and in synergy with biology and Roche has been at the forefront. Applications in drug discovery programs towards new medicines in cardiovascular and metabolic diseases are highlighted to show impact and advancement: the early discovery of endothelin antagonists for endothelial dysfunction (Bosentan), 11-beta hydroxysteroid dehydrogenase (11?-HSD1) inhibitors for dysregulated cellular glucocorticoid tonus (type 2 diabetes and metabolic syndrome) and non-covalent hormone sensitive lipase (HSL) inhibitors to study the scope of direct inhibition of lipolysis in the conceptual frame of lipotoxicity and type 2 diabetes

Travel-associated neurological disease terminated in a postmortem diagnosed atypical HSV-1 encephalitis after high-dose steroid therapy - a case report

Background: Human encephalitis can originate from a variety of different aetiologies, of which infection is the most common one. The diagnostic work-up is specifically challenging in patients with travel history since a broader spectrum of unfamiliar additional infectious agents, e. g. tropical disease pathogens, needs to be considered. Here we present a case of encephalitis of unclear aetiology in a female traveller returning from Africa, who in addition developed an atypical herpes simplex virus (HSV) encephalitis in close temporal relation with high-dose steroid treatment. Case presentation: A previously healthy 48-year-old female presented with confusion syndrome and impaired vigilance which had developed during a six-day trip to The Gambia. The condition rapidly worsened to a comatose state. Extensive search for infectious agents including a variety of tropical disease pathogens was unsuccessful. As encephalitic signs persisted despite of calculated antimicrobial and antiviral therapy, high-dose corticosteroids were applied intravenously based on the working diagnosis of an autoimmune encephalitis. The treatment did, however, not improve the patient's condition. Four days later, bihemispheric signal amplification in the insular and frontobasal cortex was observed on magnetic resonance imaging (MRI). The intracranial pressure rapidly increased and could not be controlled by conservative treatment. The patient died due to tonsillar herniation 21 days after onset of symptoms. Histological examination of postmortem brain tissue demonstrated a generalized lymphocytic meningoencephalitis. Immunohistochemical reactions against HSV-1/2 indicated an atypical manifestation of herpesviral encephalitis in brain tissue. Moreover, HSV-1 DNA was detected by a next-generation sequencing (NGS) metagenomics approach. Retrospective analysis of cerebrospinal fluid (CSF) and serum samples revealed HSV-1 DNA only in specimens one day ante mortem. Conclusions: This case shows that standard high-dose steroid therapy can contribute to or possibly even trigger fulminant cerebral HSV reactivation in a critically ill patient. Thus, even if extensive laboratory diagnostics including wide-ranging search for infectious pathogens has been performed before and remained without results, continuous re-evaluation of potential differential diagnoses especially regarding opportunistic infections or reactivation of latent infections is of utmost importance, particularly if new symptoms occur

Prophetical talking - economic thinking - deaconry acting: Parish day care centre as a profiling living environment

In der Vergangenheit haben die Kirchen viel in die Frage der religiösen Erziehung investiert. Der Sinn kirchlicher Kindergärten wird bis heute damit erklärt. Es sind aber auch weitere nicht weniger christliche Begründungen möglich: Kinder sollen mit anderen zusammen aufwachsen, damit neben der Familie Freundschaften entstehen können und (Pfarr)-Gemeinde früh erlebbar wird. Kindergärten können als Zentren gemeindlichen Lebens verstanden werden, an denen soziale Unterschiede zum Ausgleich kommen. Kindergärten können Orte sein, zu denen Kinder unabhängig von der Situation ihrer Eltern Zugang haben und kirchliches Engagement erfahren. Der diakonische Aspekt eines Engagements für die Kindertagesstätte steht im Kontext der sozialen Problemlagen von Familien, einer zunehmenden Entsolidarisierung in der Gesellschaft und einer sich verschärfenden Sinnkrise heutiger familiärer Strukturen. Eine an systemischen Zusammenhängen sich orientierende 'Kindergartenarbeit' anzuregen ist das Ziel der vorliegenden Forschungsarbeit. Solche (systemorientierte) Seelsorgekonzepte für Pfarrgemeinden und Kindertagesstätten wurden bisher nicht konsequent entwickelt. Diese Veränderung der Kinderstättenarbeit ergänzt die kirchliche Familienarbeit und fordert die Pfarrei auf, als Träger Profil zu zeigen. Zu den Basiskompetenzen der Individualseelsorge als dialogischer Begegnung mit einzelnen Menschen (Echtheit, Empathie und Wertschätzung) werden relationale Vernetzung als Verständigungsschlüssel, Selbstdifferenzierung in Beziehungssystemen und die Kunst des sich Einfühlens in unterschiedliche Lebensmodelle als wichtige pastorale Fähigkeiten aufgezeigt. Die Lebenssituation von Familien mit kleinen Kindern bietet eine Vielzahl von Anknüpfungspunkten. Hierbei spielt die Kindertagesstätte als institutionalisiertes Angebot und als familiärer Nahraum eine herausragende Rolle. Der Titel dieser Arbeit soll dafür Programm sein: Prophetisch reden, ökonomisch denken, diakonisch Handeln. Dieser Dreischritt ist notwendig, dass systemische Seelsorgekonzepte mit ihrer vernetzten Sichtweise dem Kindergarten als pastoralem Raum ein eigenes diakonisch geprägtes Profil geben können. Prophetisch reden wird dabei zum Zeichen einer Pfarrgemeinde, die den Erwartungsdruck, der auf heutigen Familien lastet, artikuliert. Familien werden als der Ort definiert, an welchem die religiöse wie gesellschaftliche Beheimatung gewährleistet werden soll. Betriebswirtschaftliches und damit ökonomisches Denken beinhaltet die Suche nach dem Platz der Pastoral in der Führung eines Betriebes unter kirchlicher Trägerschaft. Wertorientierte Qualitätsprozesse für eine diakonisch handelnde Pfarrgemeinde sind der dritte Schwerpunkt dieser Arbeit. Die Arbeit will den Blick für vernetzt denkende, systemisch orientierte Seelsorgerinnen und Seelsorger öffnen. Neu kann dann der Dreiklang von Gottes-, Nächsten- und Selbstliebe durchbuchstabiert werden.In the past the churches have invested a lot into the question of religious education. The purpose of church kindergartens has been explained like that till this day. But there are also further, not less Christian explanations possible: children should grow up together with others, so that friendships can develop next to family and the (parish) community can be experienced at an early age. Kindergarten can be understood as centres of parish life, where social differences become balanced. Kindergarten can be places to which children, independent of the situation of their parents, have access and can experience church commitment. The deaconry aspect of commitment for a day-care centre stands in the context of social problem areas of families, a decreasing solidarity of society and an increasing identity crisis of today's familial structures. To inspire to one towards systemic connections orientated "kindergarten work" is the purpose of the present research work. Such (system orientated) concepts of pastoral care for parish communities and day-care centres have not been consequently developed yet. This modification of day-care centre work supplements the churchly family work and requests the parish to show profile within the sponsorship function. In addition to the basic competences of individual pastoral care as dialogic encounter with several people (authenticity, empathy, appreciation) are relational networking as key to understanding, self-differentiation in relation systems and the art to emphasize with different life models shown as important parish skills. The life situation of families with little children offers a variety of points of contact. At this the day-care centre has an outstanding role as institutional offer and familial surrounding. The title of this thesis shall be the program for this: Prophetical talking, economic thinking, deaconry acting. This three-step approach is necessary, thereby systemic pastoral care concepts with their networked view can give the kindergarten as pastoral space an own diocesan characterized profile. In the process, prophetical talking becomes the sign of a parish community, which articulates the pressure of expectations that weighs on today's families. Families are defines as place, where religious as well as social feeling of home shall be ensured. Business and therefore economic thinking contains the search for a place of pastoral in the management of a company under church sponsorship. Value-oriented quality processes for a deaconry acting parish community are the third focus of this research work. The thesis wants to open the view for systemically oriented pastoral workers with networked thinking. The triad of God-, brotherly- and self-love can then be spelled in a new way