Detection of amplified DNA sequences by reverse chromosome painting using genomic tumor DNA as probe

A modification of reverse chromosome painting was carried out using genomic DNA from tumor cells as a complex probe for chromosomal in situ suppression hybridization to normal metaphase chromsome spreads. Amplified DNA sequences contained in such probes showed specific signals, revealing the normal chromosome positions from which these sequences were derived. As a model system, genomic DNAs were analyzed from three tumor cell lines with amplification units including the proto-oncogene c-myc. The smallest amplification unit was about 90 kb and was present in 16–24 copies; the largest unit was bigger than 600 kb and was present in 16–32 copies. Specific signals that co-localized with a differently labeled c-myc probe on chromosome band 8q24 were obtained with genomic DNA from each cell line. In further experiments, genomic DNA derived from primary tumor material was used in the case of a male patient with glioblastoma multiforme (GBM). Southern blot analysis using an epidermal growth factor receptor gene (EGFR) probe that maps to 7p13 indicated the amplification of sequences from this gene. Using reverse chromosome painting, signals were found both on band 7p13 and bands 12q13–q15. Notably, the signal on 12q13–q15 was consistently stronger. The weaker 7p13 signal showed co-localization with the major signal of the differently labeled EGFR probe. A minor signal of this probe was seen on 12q13, suggesting cross-hybridization to ERB3 sequences homologous to EGFR. The results indicate co-amplification of sequences from bands 12q13–q15, in addition to sequences from band 7p13. Several oncogenes map to 12q13–q15 providing candidate genes for a tumor-associated proto-oncogene amplification. Although the nature of the amplified sequences needs to be clarified, this experiment demonstrates the potential of reverse chromosome painting with genomic tumor DNA for rapidly mapping the normal chromosomal localization of the DNA from which the amplified sequences were derived. In addition, a weaker staining of chromosomes 10 and X was consistently observed indicating that these chromosomes were present in only one copy in the GBM genome. This rapid approach can be used to analyze cases where no metaphase spreads from the tumor material are available. It does not require any preknowledge of amplified sequences and can be applied to screen large numbers of tumors

A simple method for isolation of PCR fragments from silver-stained polyacrylamide gels by scratching with a fine needle

This project investigated how NoSQL databases can be used together with a logical layer, instead of a relational database with separated backend logic, to search for products with customer specific constraints in an e-commerce scenario. The motivation behind moving from a relational database was the scalability issues and increased read latencies experienced as the data increased. The work resulted in a framework called Cauldron that uses pipelines a sequence of execution steps to expose its data stored in an in-memory key-value store and a document database. Cauldron uses write replication between distributed instances to increase read throughput at the cost of write latency. A product database with customer specific constraints was implemented using Cauldron to compare it against an existing solution based on a relational database. The new product database can serve search queries 10 times faster in the general case and up to 25 times faster in extreme cases compared to the existing solution.Projektet undersökte hur NoSQL databaser tillsammans med ett logiskt lager, istället för en relationsdatabas med separat backend logik, kan användas för att söka på produkter med kundunika restriktioner. Motivationen till att byta ut relationsdatabasen berodde på skalbarhetsproblem och långsammare svarstider när datamängden ökade. Arbetet resulterade i ett ramverk vid namn Cauldron som använder pipelines sammankopplade logiska steg för att exponera sin data från en minnesbunden nyckel-värde-databas och en dokumentdatabas. Cauldron använder replikering mellan distribuerade instanser för att öka läsgenomstömmningen på bekostnad av högre skrivlatenser. En produktdatabas med kundunika restriktioner implementerades med hjälp av Cauldron för att jämföra den mot en befintlig lösning baserad på en relationsdatabas. Den nya databasen kan besvara sökförfrågningar 10 gånger snabbare i normalfallen och upp till 25 gånger snabbare i extremfallen jämfört med den befintliga lösningen

Enhanced sensitivity to ALDH1A3-dependent ferroptosis in TMZ-resistant glioblastoma cells

Temozolomide (TMZ) is standard treatment for glioblastoma (GBM); nonetheless, resistance and tumor recurrence are still major problems. In addition to its association with recurrent GBM and TMZ resistance, ALDH1A3 has a role in autophagy-dependent ferroptosis activation. In this study, we treated TMZ-resistant LN229 human GBM cells with the ferroptosis inducer RSL3. Remarkably, TMZ-resistant LN229 clones were also resistant to ferroptosis induction, although lipid peroxidation was induced by RSL3. By using Western blotting, we were able to determine that ALDH1A3 was down-regulated in TMZ-resistant LN229 cells. Most intriguingly, the cell viability results showed that only those clones that up-regulated ALDH1A3 after TMZ withdrawal became re-sensitized to ferroptosis induction. The recovery of ALDH1A3 expression appeared to be regulated by EGFR-dependent PI3K pathway activation since Akt was activated only in ALDH1A3 high clones. Blocking the EGFR signaling pathway with the EGFR inhibitor AG1498 decreased the expression of ALDH1A3. These findings shed light on the potential application of RSL3 in the treatment of glioblastoma relapse

YB-1 dependent oncolytic adenovirus efficiently inhibits tumor growth of glioma cancer stem like cells

Background: The brain cancer stem cell (CSC) model describes a small subset of glioma cells as being responsible for tumor initiation, conferring therapy resistance and tumor recurrence. In brain CSC, the PI3-K/AKT and the RAS/mitogen activated protein kinase (MAPK) pathways are found to be activated. In consequence, the human transcription factor YB-1, knowing to be responsible for the emergence of drug resistance and driving adenoviral replication, is phosphorylated and activated. With this knowledge, YB-1 was established in the past as a biomarker for disease progression and prognosis. This study determines the expression of YB-1 in glioblastoma (GBM) specimen in vivo and in brain CSC lines. In addition, the capacity of Ad-Delo3-RGD, an YB-1 dependent oncolytic adenovirus, to eradicate CSC was evaluated both in vitro and in vivo. Methods: YB-1 expression was investigated by immunoblot and immuno-histochemistry. In vitro, viral replication as well as the capacity of Ad-Delo3-RGD to replicate in and, in consequence, to kill CSC was determined by real-time PCR and clonogenic dilution assays. In vivo, Ad-Delo3-RGD-mediated tumor growth inhibition was evaluated in an orthotopic mouse GBM model. Safety and specificity of Ad-Delo3-RGD were investigated in immortalized human astrocytes and by siRNA-mediated downregulation of YB-1. Results: YB-1 is highly expressed in brain CSC lines and in GBM specimen. Efficient viral replication in and virus-mediated lysis of CSC was observed in vitro. Experiments addressing safety aspects of Ad-Delo3-RGD showed that (i) virus production in human astrocytes was significantly reduced compared to wild type adenovirus (Ad-WT) and (ii) knockdown of YB-1 significantly reduced virus replication. Mice harboring othotopic GBM developed from a temozolomide (TMZ)-resistant GBM derived CSC line which was intratumorally injected with Ad-Delo3-RGD survived significantly longer than mice receiving PBS-injections or TMZ treatment. Conclusion: The results of this study supported YB-1 based virotherapy as an attractive therapeutic strategy for GBM treatment which will be exploited further in multimodal treatment concepts

Restoration of Sensitivity in Chemo

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. Despite multimodal treatments including surgery, chemotherapy and radiotherapy the prognosis remains poor and relapse occurs regularly. The alkylating agent temozolomide (TMZ) has been shown to improve the overall survival in patients with malignant gliomas, especially in tumors with methylated promoter of the O6-methylguanine-DNA-methyltransferase (MGMT) gene. However, intrinsic and acquired resistance towards TMZ makes it crucial to find new therapeutic strategies aimed at improving the prognosis of patients suffering from malignant gliomas. Cold atmospheric plasma is a new auspicious candidate in cancer treatment. In the present study we demonstrate the anti-cancer properties of different dosages of cold atmospheric plasma (CAP) both in TMZ-sensitive and TMZ-resistant cells by proliferation assay, immunoblotting, cell cycle analysis, and clonogenicity assay. Importantly, CAP treatment restored the responsiveness of resistant glioma cells towards TMZ therapy. Concomitant treatment with CAP and TMZ led to inhibition of cell growth and cell cycle arrest, thus CAP might be a promising candidate for combination therapy especially for patients suffering from GBMs showing an unfavorable MGMT status and TMZ resistance

Progressive multifocal leukoencephalopathy associated with chemotherapy induced lymphocytopenia in solid tumors – case report of an underestimated complication

Background JC virus reactivation causing progressive multifocal leukoencephalopathy (PML) occurs preferentially in human immunodeficiency virus (HIV) positive individuals or patients suffering from hematologic neoplasms due to impaired viral control. Reactivation in patients suffering from solid malignancies is rarely described in published literature. Case Presentation Here we describe a case of PML in a male patient suffering from esophageal cancer who underwent neoadjuvant radiochemotherapy and surgical resection in curative intent resulting in complete tumor remission. The radiochemotherapy regimen contained carboplatin and paclitaxel (CROSS protocol). Since therapy onset, the patient presented with persistent and progredient leukopenia and lymphopenia in absence of otherwise known risk factors for PML. Symptom onset, which comprised aphasia, word finding disorder, and paresis, was apparent 7 months after therapy initiation. There was no relief in symptoms despite standard of care PML directed supportive therapy. The patient died two months after therapy onset. Conclusion PML is a very rare event in solid tumors without obvious states of immununosuppression and thus harbors the risk of unawareness. The reported patient suffered from lymphopenia, associated with systemic therapy, but was an otherwise immunocompetent individual. In case of neurologic impairment in patients suffering from leukopenia, PML must be considered – even in the absence of hematologic neoplasia or HIV infection

Bericht der Arbeitsgruppe "Open-Access-Publikationsplattformen"

Die Open-Access-Strategie des Landes Berlin von 2015 formuliert den Anspruch: "Als langfristiges Ziel wird der Aufbau einer landesweiten Open-Access- Infrastruktur für Monografien, Sammelbände und Zeitschriften in Kooperation mit an wissenschaftlichen Einrichtungen agierenden Infrastrukturdienstleistern, sowie mit in Berlin ansässigen Verlagen und Publikationsdienstleistern angestrebt." Eine landesweite Berliner Open-Access- Publikationsinfrastruktur soll dabei von den vorhandenen infrastrukturellen und wissenschaftlichen Expertisen mehrerer, kooperativ agierender Einrichtungen in Berlin profitieren und qualitativ hochwertige Publikationen zu fairen Kosten umsetzen. Die Arbeitsgruppe "Open-Access- Publikationsplattformen" mit Vertreter/innen mehrerer Berliner Forschungseinrichtungen wurde beauftragt, dafür notwendige Vorarbeiten zu leisten. So wurden im ersten Teil des Berichtes die an Berliner wissenschaftlichen Einrichtungen bereits bestehenden Publikationsinfrastrukturen erfasst. Im zweiten Teil wurden drei Modelle für den Aufbau einer landesweiten Open-Access-Publikationsinfrastruktur für Monografien, Sammelbände und Zeitschriften beschrieben. The Berlin Open Access Strategy claims: "It is a long-term goal to establish a Berlin-wide open access infrastructure for monographs, anthologies and periodicals – as a cooperation of infrastructure service providers located at research institutions as well as Berlin-based publishers and publishing service providers." A Berlin-wide open access publishing infrastructure should be based on the existing infrastructural and scholarly expertise of several, cooperating institutions in Berlin and result in high-quality publications at fair costs. A working group "Open Access Publishing Platforms" consisting of representatives of several Berlin research institutions was set up and commissioned to carry out the necessary preliminary work. The first part reports on existing publication infrastructures run by Berlin research institutions. The second part describes three possible model for re- structuring and setting up a Berlin-wide open access publishing infrastructure for monographs, anthologies and journals