Klauselrhythmus in den direkten Reden des Corpus Caesarianum

Comparing the prose-rhythm in the direct speeches of the Corpus Caesarianum produces another proof for Caesar being not the author of the writings Bellum Alexandrinum, Bellum Africum and Bellum Hispaniense

A single-use chromatographic purification platform for viral gene transfer vectors & viral vaccines

In steric exclusion chromatography (SXC), a crude sample is mixed with polyethylene glycol (PEG) and fed onto a single-use cellulose column. In this operation, selectivity is influenced strongly by the target species’ size, so SXC is well suited for purification of virus particles. The purified product is recovered at physiological pH and conductivity. We have observed recoveries above 95% for several cell culture-based virus particles used as viral gene transfer vectors or as viral vaccines, including: adeno-associated virus (AAV), Modified Vaccinia Ankara (MVA) virus, influenza virus, and yellow fever virus. Preliminary data for purification of lentiviruses suggests recoveries exceeding 60%. Host cell DNA and protein depletion are typically above 90% and infectivity is not compromised thanks to the inert character of PEG towards biomolecules and the mild elution conditions. Several AAV serotypes and display mutants were produced using HEK cells and purified with up to 95% recovery. Elution fractions had ≤2×1014 viral genomes·L−1 and, depending on the specific AVV particle, the purified viruses successfully transduced or induced gene knockdown in vitro. Elution pools from MVA virus produced in continuous bioreactors with an avian cell line contained about 3.7×109 infectious virions measured by TCID50. For influenza virus, four strains were produced in MDCK cells. Full recovery of all strains was observed using identical SXC conditions for both infectious and chemically inactivated viruses. The column capacity in terms of the viral hemagglutinin antigen was \u3e 50 mg·m−2. In the case of yellow fever virus, two attenuated strains were produced in Vero cells. Here, full recovery of infective titers was also achieved: the elution fraction was concentrated more than 100-fold to a titer of \u3e6×109 plaque forming units (≈100 000 doses). In summary, SXC capture with PEG and unmodified cellulose membranes seems to perform very well for a broad range of viruses from different production processes. Thanks to the high degree of success in a relatively narrow operational range, SXC can drastically reduce process development. The high recoveries obtained so far, enable subsequent polishing operations with minimum risk to low overall process yields

Proteome analysis of the HIV-1 Gag interactome

AbstractHuman immunodeficiency virus Gag drives assembly of virions in infected cells and interacts with host factors which facilitate or restrict viral replication. Although several Gag-binding proteins have been characterized, understanding of virus–host interactions remains incomplete. In a series of six affinity purification screens, we have identified protein candidates for interaction with HIV-1 Gag. Proteins previously found in virions or identified in siRNA screens for host factors influencing HIV-1 replication were recovered. Helicases, translation factors, cytoskeletal and motor proteins, factors involved in RNA degradation and RNA interference were enriched in the interaction data. Cellular networks of cytoskeleton, SR proteins and tRNA synthetases were identified. Most prominently, components of cytoplasmic RNA transport granules were co-purified with Gag. This study provides a survey of known Gag–host interactions and identifies novel Gag binding candidates. These factors are associated with distinct molecular functions and cellular pathways relevant in host–pathogen interactions

MAP3K7 is recurrently deleted in pediatric T-lymphoblastic leukemia and affects cell proliferation independently of NF-κB

Background: Deletions of 6q15–16.1 are recurrently found in pediatric T-cell acute lymphoblastic leukemia (T-ALL). This chromosomal region includes the mitogen-activated protein kinase kinase kinase 7 (MAP3K7) gene which has a crucial role in innate immune signaling and was observed to be functionally and prognostically relevant in different cancer entities. Therefore, we correlated the presence of MAP3K7 deletions with clinical parameters in a cohort of 327 pediatric T-ALL patients and investigated the function of MAP3K7 in the T-ALL cell lines CCRF-CEM, Jurkat and MOLT-4. Methods: MAP3K7 deletions were detected by multiplex ligation-dependent probe amplification (MLPA). T-ALL cell lines were transduced with adeno-associated virus (AAV) vectors expressing anti-MAP3K7 shRNA or a non-silencing shRNA together with a GFP reporter. Transduction efficiency was measured by flow cytometry and depletion efficiency by RT-PCR and Western blots. Induction of apoptosis was measured by flow cytometry after staining with PE-conjugated Annexin V. In order to assess the contribution of NF-κB signaling to the effects of MAP3K7 depletion, cells were treated with TNF-α and cell lysates analyzed for components of the NF-κB pathway by Western blotting and for expression of the NF-κB target genes BCL2, CMYC, FAS, PTEN and TNF-α by RT-PCR. Results: MAP3K7 is deleted in approximately 10% and point-mutated in approximately 1% of children with T-ALL. In 32 of 33 leukemias the deletion of MAP3K7 also included the adjacent CASP8AP2 gene. MAP3K7 deletions were associated with the occurrence of SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and outcome. Depletion of MAP3K7 expression in T-ALL cell lines by shRNAs slowed down proliferation and induced apoptosis, but neither changed protein levels of components of NF-κB signaling nor NF-κB target gene expression after stimulation with TNF-α. Conclusions: This study revealed that the recurrent deletion of MAP3K7/CASP8AP2 is associated with SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and risk of relapse. Homozygous deletions of MAP3K7 were not observed, and efficient depletion of MAP3K7 interfered with viability of T-ALL cells, indicating that a residual expression of MAP3K7 is indispensable for T-lymphoblasts

MAP3K7 is recurrently deleted in pediatric T-lymphoblastic leukemia and affects cell proliferation independently of NF-κB

Background: Deletions of 6q15–16.1 are recurrently found in pediatric T-cell acute lymphoblastic leukemia (T-ALL). This chromosomal region includes the mitogen-activated protein kinase kinase kinase 7 (MAP3K7) gene which has a crucial role in innate immune signaling and was observed to be functionally and prognostically relevant in different cancer entities. Therefore, we correlated the presence of MAP3K7 deletions with clinical parameters in a cohort of 327 pediatric T-ALL patients and investigated the function of MAP3K7 in the T-ALL cell lines CCRF-CEM, Jurkat and MOLT-4. Methods: MAP3K7 deletions were detected by multiplex ligation-dependent probe amplification (MLPA). T-ALL cell lines were transduced with adeno-associated virus (AAV) vectors expressing anti-MAP3K7 shRNA or a non-silencing shRNA together with a GFP reporter. Transduction efficiency was measured by flow cytometry and depletion efficiency by RT-PCR and Western blots. Induction of apoptosis was measured by flow cytometry after staining with PE-conjugated Annexin V. In order to assess the contribution of NF-κB signaling to the effects of MAP3K7 depletion, cells were treated with TNF-α and cell lysates analyzed for components of the NF-κB pathway by Western blotting and for expression of the NF-κB target genes BCL2, CMYC, FAS, PTEN and TNF-α by RT-PCR. Results: MAP3K7 is deleted in approximately 10% and point-mutated in approximately 1% of children with T-ALL. In 32 of 33 leukemias the deletion of MAP3K7 also included the adjacent CASP8AP2 gene. MAP3K7 deletions were associated with the occurrence of SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and outcome. Depletion of MAP3K7 expression in T-ALL cell lines by shRNAs slowed down proliferation and induced apoptosis, but neither changed protein levels of components of NF-κB signaling nor NF-κB target gene expression after stimulation with TNF-α. Conclusions: This study revealed that the recurrent deletion of MAP3K7/CASP8AP2 is associated with SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and risk of relapse. Homozygous deletions of MAP3K7 were not observed, and efficient depletion of MAP3K7 interfered with viability of T-ALL cells, indicating that a residual expression of MAP3K7 is indispensable for T-lymphoblasts

Lipidomimetic Compounds Act as HIV-1 Entry Inhibitors by Altering Viral Membrane Structure

The envelope of Human Immunodeficiency Virus type 1 (HIV-1) consists of a liquid-ordered membrane enriched in raft lipids and containing the viral glycoproteins. Previous studies demonstrated that changes in viral membrane lipid composition affecting membrane structure or curvature can impair infectivity. Here, we describe novel antiviral compounds that were identified by screening compound libraries based on raft lipid-like scaffolds. Three distinct molecular structures were chosen for mode-of-action studies, a sterol derivative (J391B), a sphingosine derivative (J582C) and a long aliphatic chain derivative (IBS70). All three target the viral membrane and inhibit virus infectivity at the stage of fusion without perturbing virus stability or affecting virion-associated envelope glycoproteins. Their effect did not depend on the expressed envelope glycoproteins or a specific entry route, being equally strong in HIV pseudotypes carrying VSV-G or MLV-Env glycoproteins. Labeling with laurdan, a reporter of membrane order, revealed different membrane structure alterations upon compound treatment of HIV-1, which correlated with loss of infectivity. J582C and IBS70 decreased membrane order in distinctive ways, whereas J391B increased membrane order. The compounds' effects on membrane order were reproduced in liposomes generated from extracted HIV lipids and thus independent both of virion proteins and of membrane leaflet asymmetry. Remarkably, increase of membrane order by J391B required phosphatidylserine, a lipid enriched in the HIV envelope. Counterintuitively, mixtures of two compounds with opposite effects on membrane order, J582C and J391B, did not neutralize each other but synergistically inhibited HIV infection. Thus, altering membrane order, which can occur by different mechanisms, constitutes a novel antiviral mode of action that may be of general relevance for enveloped viruses and difficult to overcome by resistance development

Detailed Characterization of Early HIV-1 Replication Dynamics in Primary Human Macrophages

Macrophages are natural target cells of human immunodeficiency virus type 1 (HIV-1). Viral replication appears to be delayed in these cells compared to lymphocytes; however, little is known about the kinetics of early post-entry events. Time-of-addition experiments using several HIV-1 inhibitors and the detection of reverse transcriptase (RT) products with droplet digital PCR (ddPCR) revealed that early replication was delayed in primary human monocyte-derived macrophages of several donors and peaked late after infection. Direct imaging of reverse-transcription and pre-integration complexes (RTC/PIC) by click-labeling of newly synthesized DNA further confirmed our findings and showed a concomitant shift to the nuclear stage over time. Altering the entry pathway enhanced infectivity but did not affect kinetics of viral replication. The addition of viral protein X (Vpx) enhanced productive infection and accelerated completion of reverse transcription and nuclear entry. We propose that sterile alpha motif (SAM) and histidine/aspartate (HD) domain-containing protein 1 (SAMHD1) activity lowering deoxyribonucleotide triphosphate (dNTP) pools is the principal factor delaying early HIV-1 replication in macrophages

Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8

Preclinical studies on gene delivery into mouse lymphocytes are often hampered by insufficient activity of lentiviral (LV) and adeno-associated vectors (AAVs) as well as missing tools for cell type selectivity when considering in vivo gene therapy. Here, we selected designed ankyrin repeat proteins (DARPins) binding to murine CD8. The top-performing DARPin was displayed as targeting ligand on both vector systems. When used on engineered measles virus (MV) glycoproteins, the resulting mCD8-LV transduced CD8+ mouse lymphocytes with near-absolute (>99%) selectivity. Despite its lower functional titer, mCD8-LV achieved 4-fold higher gene delivery to CD8+ cells than conventional VSV-LV when added to whole mouse blood. Addition of mCD8-LV encoding a chimeric antigen receptor (CAR) specific for mouse CD19 to splenocytes resulted in elimination of B lymphocytes and lymphoma cells. For display on AAV, the DARPin was inserted into the GH2-GH3 loop of the AAV2 capsid protein VP1, resulting in a DARPin-targeted AAV we termed DART-AAV. Stocks of mCD8-AAV contained similar genome copies as AAV2 but were >20-fold more active in gene delivery in mouse splenocytes, while exhibiting >99% specificity for CD8+ cells. These results suggest that receptor targeting can overcome blocks in transduction of mouse splenocytes

Detailed Characterization of Early HIV-1 Replication Dynamics in Primary Human Macrophages

Macrophages are natural target cells of human immunodeficiency virus type 1 (HIV-1). Viral replication appears to be delayed in these cells compared to lymphocytes; however, little is known about the kinetics of early post-entry events. Time-of-addition experiments using several HIV-1 inhibitors and the detection of reverse transcriptase (RT) products with droplet digital PCR (ddPCR) revealed that early replication was delayed in primary human monocyte-derived macrophages of several donors and peaked late after infection. Direct imaging of reverse-transcription and pre-integration complexes (RTC/PIC) by click-labeling of newly synthesized DNA further confirmed our findings and showed a concomitant shift to the nuclear stage over time. Altering the entry pathway enhanced infectivity but did not affect kinetics of viral replication. The addition of viral protein X (Vpx) enhanced productive infection and accelerated completion of reverse transcription and nuclear entry. We propose that sterile alpha motif (SAM) and histidine/aspartate (HD) domain-containing protein 1 (SAMHD1) activity lowering deoxyribonucleotide triphosphate (dNTP) pools is the principal factor delaying early HIV-1 replication in macrophages

Secrets of Success in a Landscape of Fear: Urban Wild Boar Adjust Risk Perception and Tolerate Disturbance

In urban areas with a high level of human disturbance, wildlife has to adjust its behavior to deal with the so called “landscape of fear.” This can be studied in risk perception during movement in relation to specific habitat types, whereby individuals trade-off between foraging and disturbance. Due to its high behavioral plasticity and increasing occurrence in urban environments, wild boar (Sus scrofa) is an excellent model organism to study adjustment to urbanization. With the help of GPS tracking, space use of 11 wild boar from Berlin's metropolitan region was analyzed: we aimed at understanding how animals adjust space use to deal with the landscape of fear in urban areas compared to rural areas. We compared use vs. availability with help of generalized linear mixed models. First, we studied landscape types selected by rural vs. urban wild boar, second, we analyzed distances of wild boar locations to each of the landscape types. Finally, we mapped the resulting habitat selection probability to predict hotspots of human-wildlife conflicts. A higher tolerance to disturbance in urban wild boar was shown by a one third shorter flight distance and by an increased re-use of areas close to the trap. Urban wild boar had a strong preference for natural landscapes such as swamp areas, green areas and deciduous forests, and areas with high primary productivity, as indicated by high NDVI (normalized difference vegetation index) values. The areas selected by urban wild boar were often located closely to roads and houses. The spatial distribution maps show that a large area of Berlin would be suitable for urban wild boar but not their rural conspecifics, with the most likely reason being a different perception of anthropogenic disturbance. Wild boar therefore showed considerable behavioral plasticity suitable to adjust to human-dominated environments in a potentially evolutionarily adaptive manner