Construction of a dual-tag system for gene expression, protein affinity purification and fusion protein processing

An E. coli vector system was constructed which allows the expression of fusion genes via a l-rhamnose-inducible promotor. The corresponding fusion proteins consist of the maltose-binding protein and a His-tag sequence for affinity purification, the Saccharomyces cerevisiae Smt3 protein for protein processing by proteolytic cleavage and the protein of interest. The Smt3 gene was codon-optimized for expression in E. coli. In a second rhamnose-inducible vector, the S. cerevisiae Ulp1 protease gene for processing Smt3 fusion proteins was fused in the same way to maltose-binding protein and His-tag sequence but without the Smt3 gene. The enhanced green fluorescent protein (eGFP) was used as reporter and protein of interest. Both fusion proteins (MalE-6xHis-Smt3-eGFP and MalE-6xHis-Ulp1) were efficiently produced in E. coli and separately purified by amylose resin. After proteolytic cleavage the products were applied to a Ni-NTA column to remove protease and tags. Pure eGFP protein was obtained in the flow-through of the column in a yield of around 35% of the crude cell extract.     

The Network of Knowledge approach: improving the science and society dialogue on biodiversity and ecosystem services in Europe

The absence of a good interface between scientific and other knowledge holders and decision-makers in the area of biodiversity and ecosystem services has been recognised for a long time. Despite recent advancements, e.g. with the Intergovernmental Platform on Biodiversity and Ecosystem Services (IPBES), challenges remain, particularly concerning the timely provision of consolidated views from different knowledge domains. To address this challenge, a strong and flexible networking approach is needed across knowledge domains and institutions. Here, we report on a broad consultation process across Europe to develop a Network of Knowledge on biodiversity and ecosystem services (NoK), an approach aiming at (1) organising institutions and knowledge holders in an adaptable and responsive framework and (2) informing decision-makers with timely and accurate biodiversity knowledge. The consultation provided a critical analysis of the needs that should be addressed by a NoK and how it could complement existing European initiatives and institutions at the interface between policy and science. Among other functions, the NoK provides consolidated scientific views on contested topics, identification of research gaps to support relevant policies, and horizon scanning activities to anticipate emerging issues. The NoK includes a capacity building component on interfacing activities and contains mechanisms to ensure its credibility, relevance and legitimacy. Such a network would need to ensure credibility, relevance and legitimacy of its work by maximizing transparency and flexibility of processes, quality of outputs, the link to data and knowledge provision, the motivation of experts for getting involved and sound communication and capacity building.     

Iron chelation and 2‐oxoglutarate‐dependent dioxygenase inhibition suppress mantle cell lymphoma's cyclin D1

The patients with mantle cell lymphoma (MCL) have translocation t(11;14) associated with cyclin D1 overexpression. We observed that iron (an essential cofactor of dioxygenases including prolyl hydroxylases [PHDs]) depletion by deferoxamine blocked MCL cells’ proliferation, increased expression of DNA damage marker γH2AX, induced cell cycle arrest and decreased cyclin D1 level. Treatment of MCL cell lines with dimethyloxalylglycine, which blocks dioxygenases involving PHDs by competing with their substrate 2‐oxoglutarate, leads to their decreased proliferation and the decrease of cyclin D1 level. We then postulated that loss of EGLN2/PHD1 in MCL cells may lead to down‐regulation of cyclin D1 by blocking the degradation of FOXO3A, a cyclin D1 suppressor. However, the CRISPR/Cas9‐based loss‐of‐function of EGLN2/PHD1 did not affect cyclin D1 expression and the loss of FOXO3A did not restore cyclin D1 levels after iron chelation. These data suggest that expression of cyclin D1 in MCL is not controlled by ENGL2/PHD1‐FOXO3A pathway and that chelation‐ and 2‐oxoglutarate competition‐mediated down‐regulation of cyclin D1 in MCL cells is driven by yet unknown mechanism involving iron‐ and 2‐oxoglutarate‐dependent dioxygenases other than PHD1. These data support further exploration of the use of iron chelation and 2‐oxoglutarate‐dependent dioxygenase inhibitors as a novel therapy of MCL.     

Gold nanorods targeted to delta opioid receptor: plasmon-resonant contrast and photothermal agents

Molecularly targeted gold nanorods were investigated for applications in both diagnostic imaging and disease treatment with cellular resolution. The nanorods were tested in two genetically engineered cell lines derived from the human colon carcinoma HCT-116, a model for studying ligand-receptor interactions. One of these lines was modified to express delta opioid receptor (deltaOR) and green fluorescent protein, whereas the other was receptor free and expressed a red fluorescent protein, to serve as the control. Deltorphin, a high-affinity ligand for deltaOR, was stably attached to the gold nanorods through a thiol-terminated linker. In a mixed population of cells, we demonstrated selective imaging and destruction of receptor-expressing cells while sparing those cells that did not express the receptor. The molecularly targeted nanorods can be used as an in vitro ligand-binding and cytotoxic treatment assay platform and could potentially be applied in vivo for diagnostic and therapeutic purposes with endoscopic technology.     

Preserved functional specialization for spatial processing in the middle occipital gyrus of the early blind

The occipital cortex (OC) of early-blind humans is activated during various nonvisual perceptual and cognitive tasks, but little is known about its modular organization. Using functional MRI we tested whether processing of auditory versus tactile and spatial versus nonspatial information was dissociated in the OC of the early blind. No modality-specific OC activation was observed. However, the right middle occipital gyrus (MOG) showed a preference for spatial over nonspatial processing of both auditory and tactile stimuli. Furthermore, MOG activity was correlated with accuracy of individual sound localization performance. In sighted controls, most of extrastriate OC, including the MOG, was deactivated during auditory and tactile conditions, but the right MOG was more activated during spatial than nonspatial visual tasks. Thus, although the sensory modalities driving the neurons in the reorganized OC of blind individuals are altered, the functional specialization of extrastriate cortex is retained regardless of visual experience.     

Free radical scavengers vitamins A, C, and E plus magnesium reduce noise trauma

Free radical formation in the cochlea plays a key role in the development of noise-induced hearing loss (NIHL). The amount, distribution, and time course of free radical formation have been defined, including a clinically significant formation of both reactive oxygen species and reactive nitrogen species 7-10 days after noise exposure. Reduction in cochlear blood flow as a result of free radical formation has also been described. Here we report that the antioxidant agents vitamins A, C, and E act in synergy with magnesium to effectively prevent noise-induced trauma. Neither the antioxidant agents nor the magnesium reliably reduced NIHL or sensory cell death with the doses we used when these agents were delivered alone. In combination, however, they were highly effective in reducing both hearing loss and cell death even with treatment initiated just 1 h before noise exposure. This study supports roles for both free radical formation and noise-induced vasoconstriction in the onset and progression of NIHL. Identification of this safe and effective antioxidant intervention that attenuates NIHL provides a compelling rationale for human trials in which free radical scavengers are used to eliminate this single major cause of acquired hearing loss.     

Mikrospektrographische Untersuchungen zur vitalen Akridinorange-Fluorochromierung vonAllium cepa-Epidermen

Ohne ZusammenfassungIhrem hochverehrten Lehrer, Univ.-Prof. Dr. Karl Höfler, zum 70. Geburtstag in aufrichtiger Dankbarkeit gewidmet.     

Untersuchungen zur Systematik der Gattung Phytophthora de Bary

Ohne ZusammenfassungAuszug aus einer gleichnamigen Dissertation der Mathematisch-Naturwissen-schaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn, 1958.