Entwicklung von zellulären Indikatorsystemen zur Identifikation von Modulatoren der HIV-1 Genexpression

Es wurde ein Assay-System auf Basis von Reporter-Konstrukten entwickelt, dass eine Detektion der HIV1- Tat und Rev-Funktion auf Einzelzell-Ebene ermöglichte. Dieses fluoreszenzbasierte System wurde hinsichtlich seiner Eigenschaften untersucht und charakterisiert. Es wurde gezeigt, dass es sich dazu eignet sowohl die Tat- als auch die Rev-Funktion konzentrationsabhängig darzustellen. Des Weiteren konnten bereits beschriebene Inhibitoren der Rev-Funktion durch den Assay bestätigt werden, was die Zuverlässigkeit des Testsystems belegt. Außerdem ermöglichte dieses System in durchgeführten Versuchen eine Quantifizierung der RNA-destabilisierenden Aktivität von INS-Elementen des HIV-Genoms. Durch die Herstellung einer stabilen Zelllinie mit einem entwickelten Reporter-Konstrukt und die Etablierung einer Fluoreszenzmikroskopie- und FACS-basierten Auswertung der Daten wurde die Grundlage für eine umfangreiche Suche nach Inhibitoren der HIV-1 Genexpression gelegt. Im Laufe erster „Screenings“ nach zellulären Inhibitoren der HIV-Genregulation wurden in einer cDNA-Bank aus fötalem Hirn mehrere inhibitorisch wirksame Kandidaten-cDNAs identifiziert. Damit konnte gezeigt werden, dass zelluläre Faktoren potentiell dazu in der Lage sind regulierend bzw. inhibierend in die HIV-1 Genexpression einzugreifen

Activation of a HERV-H LTR induces expression of an aberrant calbindin protein in human prostate carcinoma cells

The human genome contains around 500 000 LTR retrotransposons including endogenous retroviruses (HERVs). Most of these LTR elements are silenced by epigenetic conditions, but may be reactivated by environmental factors such as chemicals, radiation or exogenous viruses. We have analyzed a HERV-H element located upstream of the human calbindin gene (CALB-1) that leads to expression of a truncated calbindin protein by alternative splicing in a human prostate carcinoma cell line (PC3). PC3 cells are polyploid with one or two complete alleles of chromosome 8, and four to five pieces of the 8q-arm, all containing the CALB-1 locus. Analysis of these loci did not reveal substantial alterations on DNA sequence level, such as modification of splice sites, suggesting an epigenetic activation of the HERV-H LTR. Therefore, we compared the DNA methylation status of this LTR in PC3 cells and in three prostate carcinoma cell lines not expressing the truncated calbindin protein. We found that the HERV-H LTR is hypomethylated in two cell lines including PC3. Chromatin immunoprecipitation (ChIP) analysis, however, revealed that the chromatin is associated with active marks (acetylated histone H4 and tri-methylated lysine4 at histone H3) at this locus only in PC3 cells. This data suggests that reactivation of the HERV-H LTR requires at least two steps: demethylation of DNA and modification of histones. There is evidence that expression of the truncated calbindin prevents apoptosis and may thus contribute to the malignant phenotype of PC3 cells

Identification of a novel Rev-interacting cellular protein

BACKGROUND: Human cell types respond differently to infection by human immunodeficiency virus (HIV). Defining specific interactions between host cells and viral proteins is essential in understanding how viruses exploit cellular functions and the innate strategies underlying cellular control of HIV replication. The HIV Rev protein is a post-transcriptional inducer of HIV gene expression and an important target for interaction with cellular proteins. Identification of Rev-modulating cellular factors may eventually contribute to the design of novel antiviral therapies. RESULTS: Yeast-two hybrid screening of a T-cell cDNA library with Rev as bait led to isolation of a novel human cDNA product (16.4.1). 16.4.1-containing fusion proteins showed predominant cytoplasmic localization, which was dependent on CRM1-mediated export from the nucleus. Nuclear export activity of 16.4.1 was mapped to a 60 amino acid region and a novel transport signal identified. Interaction of 16.4.1 with Rev in human cells was shown in a mammalian two-hybrid assay and by colocalization of Rev and 16.4.1 in nucleoli, indicating that Rev can recruit 16.4.1 to the nucleus/nucleoli. Rev-dependent reporter expression was inhibited by overexpressing 16.4.1 and stimulated by siRNAs targeted to 16.4.1 sequences, demonstrating that 16.4.1 expression influences the transactivation function of Rev. CONCLUSION: These results suggest that 16.4.1 may act as a modulator of Rev activity. The experimental strategies outlined in this study are applicable to the identification and biological characterization of further novel Rev-interacting cellular factors

Monitoring the US ATLAS Network Infrastructure with perfSONAR-PS

Global scientific collaborations, such as ATLAS, continue to push the network requirements envelope. Data movement in this collaboration is routinely including the regular exchange of petabytes of datasets between the collection and analysis facilities in the coming years. These requirements place a high emphasis on networks functioning at peak efficiency and availability; the lack thereof could mean critical delays in the overall scientific progress of distributed data-intensive experiments like ATLAS. Network operations staff routinely must deal with problems deep in the infrastructure; this may be as benign as replacing a failing piece of equipment, or as complex as dealing with a multi-domain path that is experiencing data loss. In either case, it is crucial that effective monitoring and performance analysis tools are available to ease the burden of management. We will report on our experiences deploying and using the perfSONAR-PS Performance Toolkit at ATLAS sites in the United States. This software creates a dedicated monitoring server, capable of collecting and performing a wide range of passive and active network measurements. Each independent instance is managed locally, but able to federate on a global scale; enabling a full view of the network infrastructure that spans domain boundaries. This information, available through web service interfaces, can easily be retrieved to create customized applications. The US ATLAS collaboration has developed a centralized “dashboard” offering network administrators, users, and decision makers the ability to see the performance of the network at a glance. The dashboard framework includes the ability to notify users (alarm) when problems are found, thus allowing rapid response to potential problems and making perfSONAR-PS crucial to the operation of our distributed computing infrastructure.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98635/1/1742-6596_396_4_042038.pd

A suicide gene approach using the human pro-apoptotic protein tBid inhibits HIV-1 replication

<p>Abstract</p> <p>Background</p> <p>Regulated expression of suicide genes is a powerful tool to eliminate specific subsets of cells and will find widespread usage in both basic and applied science. A promising example is the specific elimination of human immunodeficiency virus type 1 (HIV-1) infected cells by LTR-driven suicide genes. The success of this approach, however, depends on a fast and effective suicide gene, which is expressed exclusively in HIV-1 infected cells. These preconditions have not yet been completely fulfilled and, thus, success of suicide approaches has been limited so far. We tested truncated Bid (tBid), a human pro-apoptotic protein that induces apoptosis very rapidly and efficiently, as suicide gene for gene therapy against HIV-1 infection.</p> <p>Results</p> <p>When tBid was introduced into the HIV-1 LTR-based, Tat- and Rev-dependent transgene expression vector pLRed(INS)₂R, very efficient induction of apoptosis was observed within 24 hours, but only in the presence of both HIV-1 regulatory proteins Tat and Rev. Induction of apoptosis was not observed in their absence. Cells containing this vector rapidly died when transfected with plasmids containing full-length viral genomic DNA, completely eliminating the chance for HIV-1 replication. Viral replication was also strongly reduced when cells were infected with HIV-1 particles.</p> <p>Conclusions</p> <p>This suicide vector has the potential to establish a safe and effective gene therapy approach to exclusively eliminate HIV-1 infected cells before infectious virus particles are released.</p

Language and memory for object location

In three experiments, we investigated the influence of two types of language on memory for object location: demonstratives (this, that) and possessives (my, your). Participants first read instructions containing demonstratives/possessives to place objects at different locations, and then had to recall those object locations (following object removal). Experiments 1 and 2 tested contrasting predictions of two possible accounts of language on object location memory: the Expectation Model (Coventry, Griffiths, & Hamilton, 2014) and the congruence account (Bonfiglioli, Finocchiaro, Gesierich, Rositani, & Vescovi, 2009). In Experiment 3, the role of attention allocation as a possible mechanism was investigated. Results across all three experiments show striking effects of language on object location memory, with the pattern of data supporting the Expectation Model. In this model, the expected location cued by language and the actual location are concatenated leading to (mis)memory for object location, consistent with models of predictive coding (Bar, 2009; Friston, 2003)

Neutronenradiographie an bentonitgebundenem Formstoff

Die Wiederverwendbarkeit von bentonitgebundenem Formstoff ist begrenzt. Die Ursachen sind vielfältig. Eine Hauptursache liegt in der Abnahme der Bindefähigkeit der Tonminerale. Wird diese Abnahme ausschließlich durch Bedingungen in der industriellen Anwendung ausgelöst, wie zum Beispiel durch Totbrand oder Versalzung, oder könnte sie auch Folge einer inhärenten Eigenschaft der Tonminerale sein, nämlich einer Minderung der Reversibilität des Wasseraustausches schon bei Temperaturen unter 200 °C? Mit der Neutronenradiographie kann Wasser im Formstoff während einer Hitzeeinwirkung quantitativ und in Echtzeit detektiert werden. Untersuchungen mit reinen Quarz-Bentonit-Wasser-Mischungen zeigten nach vier Umläufen keine signifikante Abweichung der Wasserkinematik während der Hitzeeinwirkung. In Formstoffbereichen, die Temperaturen über 100 °C ausgesetzt waren, verlief die Wassergabe in den verschiedenen Proben vergleichbar. In den Bereichen, die unter 100 °C blieben, hing jedoch der momentane lokale Wassergehalt deutlich von der anfänglichen Feuchte des Formstoffs und von der Qualität der Durchmischung ab. Letztere beeinflusste wiederum deutlich die Ausprägung der Kondensationszone im Formstoff

Retinal Involvement in a Patient with Cerebral Manifestation of Chronic Graft-Versus-Host-Disease

Background: We report a 35-year-old female patient with cerebral manifestations of chronic graft-versus-host disease (cGvHD) and putative retinal involvement after allogeneic peripheral blood stem cell transplantation (alloHSCT). Patient and Methods: The patient experienced recurrent episodes of fever and encephalitic signs 7 months after alloHSCT during taper of immunosuppression (IS). Results: Cerebral magnetic resonance imaging (MRI) showed non-gadolinium-enhancing confluent periventricular lesions and cerebrospinal fluid inflammation. After exclusion of infectious causes, treatment with steroids and antiepileptics improved cognitive deficits. Steroid reduction provoked a relapse responding to IS. 2 years later, she complained of right-sided blurred vision and floaters; both eyes showed whitish circumscribed retinal infiltrations, cellular infiltration of the vitreous and mild bilateral optic disc edema. Oncological and neurological work-up ruled out infectious diseases and other GvHD manifestations. Symptoms and signs resolved under continued systemic IS, leaving pigmented retinal scars. After IS withdrawal, classical cutaneous cGvHD developed, resolving on systemic IS. 94 months after transplantation, she is doing well. Conclusion: To our knowledge, this is the first observation of retinal involvement of cerebral cGvHD manifestations with retinal infiltrations documented in the absence of other causes and in parallel to periventricular lesions in cerebral MRI. Based on bone marrow histology, we discuss a small vessel pathophysiology of cGvHD

Caenorhabditis elegans HCF-1 Functions in Longevity Maintenance as a DAF-16 Regulator

The transcription factor DAF-16/forkhead box O (FOXO) is a critical longevity determinant in diverse organisms, however the molecular basis of how its transcriptional activity is regulated remains largely unknown. We report that the Caenorhabditis elegans homolog of host cell factor 1 (HCF-1) represents a new longevity modulator and functions as a negative regulator of DAF-16. In C. elegans, hcf-1 inactivation caused a daf-16-dependent lifespan extension of up to 40% and heightened resistance to specific stress stimuli. HCF-1 showed ubiquitous nuclear localization and physically associated with DAF-16. Furthermore, loss of hcf-1 resulted in elevated DAF-16 recruitment to the promoters of its target genes and altered expression of a subset of DAF-16-regulated genes. We propose that HCF-1 modulates C. elegans longevity and stress response by forming a complex with DAF-16 and limiting a fraction of DAF-16 from accessing its target gene promoters, and thereby regulates DAF-16-mediated transcription of selective target genes. As HCF-1 is highly conserved, our findings have important implications for aging and FOXO regulation in mammals