Acquisition of structure-guiding and structure-forming properties during maturation from the pro-silicatein to the silicatein form

Silicateins are the key enzymes involved in the enzymatic polycondensation of the inorganic scaffold of the skeletal elements of the siliceous sponges, the spicules. The gene encoding pro-silicatein is inserted into the pCold TF vector, comprising the gene for the bacterial trigger factor. This hybrid gene is expressed in Escherichia coli and the synthesized fusion protein is purified. The fusion protein is split into the single proteins with thrombin by cleavage of the linker sequence present between the two proteins. At 23 °C, the 87 kDa trigger factor-pro-silicatein fusion protein is cleaved to the 51 kDa trigger factor and the 35 kDa pro-silicatein. The cleavage process proceeds and results in the release of the 23 kDa mature silicatein, a process which very likely proceeds by autocatalysis. Almost in parallel with its formation, the mature enzyme precipitates as pure 23 kDa protein. When the precipitate is dissolved in an urea buffer, the solubilized protein displays its full enzymatic activity which is enhanced multi-fold in the presence of the silicatein interactor silintaphin-1 or of poly(ethylene glycol) (PEG). The biosilica product formed increases its compactness if silicatein is supplemented with silintaphin-1 or PEG. The elastic modulus of the silicatein-mediated biosilica product increases in parallel with the addition of silintaphin-1 and/or PEG from 17 MPa (silicatein) via 61 MPa (silicatein:silintaphin-1) to 101 MPa (silicatein:silintaphin-1 and PEG). These data show that the maturation process from the pro-silicatein state to the mature form is the crucial step during which silicatein acquires its structure-guiding and structure-forming properties.     

Ly6Chigh Monocytes Become Alternatively Activated Macrophages in Schistosome Granulomas with Help from CD4+ Cells

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1^GFP/+ mice. CX₃CR1-GFP⁺ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX₃CR1-GFP⁺ Ly6C^low monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX₃CR1-GFP⁺ cells in the blood and the tissue showed CD4⁺ T cell dependent accumulation of PD-L2⁺ CX₃CR1-GFP⁺ AAM in the tissues as granulomas form. By adoptive transfer of Ly6C^high and Ly6C^low monocytes into infected mice, we found that AAM originate primarily from transferred Ly6C^high monocytes, but that these cells may transition through a Ly6C^low state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6C^high monocytes via help from CD4⁺ T cells.     

eGender—from e-Learning to e-Research: a web-based interactive knowledge-sharing platform for sex- and gender-specific medical education

Background

Sex and Gender Medicine is a novel discipline that provides equitable medical care for society and improves outcomes for both male and female patients. The integration of sex- and gender-specific knowledge into medical curricula is limited due to adequate learning material, systematic teacher training and an innovative communication strategy. We aimed at initiating an e-learning and knowledge-sharing platform for Sex and Gender Medicine, the eGender platform (http://egender.charite.de), to ensure that future doctors and health professionals will have adequate knowledge and communication skills on sex and gender differences in order to make informed decisions for their patients.

Methods

The web-based eGender knowledge-sharing platform was designed to support the blended learning pedagogical teaching concept and follows the didactic concept of constructivism. Learning materials developed by Sex and Gender Medicine experts of seven universities have been used as the basis for the new learning tools. The content of these tools is patient-centered and provides add-on information on gender-sensitive aspects of diseases. The structural part of eGender was designed and developed using the open source e-learning platform Moodle. The eGender platform comprises an English and a German version of e-learning modules: one focusing on basic knowledge and seven on specific medical disciplines. Each module consists of several courses corresponding to a disease or symptom complex. Self-organized learning has to be managed by using different learning tools, e.g., texts and audiovisual material, tools for online communication and collaborative work.

Results

More than 90 users from Europe registered for the eGender Medicine learning modules. The most frequently accessed module was “Gender Medicine—Basics” and the users favored discussion forums. These e-learning modules fulfill the quality criteria for higher education and are used within the elective Master Module “Gender Medicine—Basics” implemented into the accredited Master of Public Health at Charité—Berlin.

Conclusions

The eGender platform is a flexible and user-friendly electronical knowledge-sharing platform providing evidence-based high-quality learning material used by a growing number of registered users. The eGender Medicine learning modules could be key in the reform of medical curricula to integrate Sex and Gender Medicine into the education of health professionals.

     

An infrared sensor analysing label‐free the secondary structure of the Abeta peptide in presence of complex fluids

The secondary structure change of the Abeta peptide to beta‐sheet was proposed as an early event in Alzheimer's disease. The transition may be used for diagnostics of this disease in an early state. We present an Attenuated Total Reflection (ATR) sensor modified with a specific antibody to extract minute amounts of Abeta peptide out of a complex fluid. Thereby, the Abeta peptide secondary structure was determined in its physiological aqueous environment by FTIR‐difference‐spectroscopy. The presented results open the door for label‐free Alzheimer diagnostics in cerebrospinal fluid or blood. It can be extended to further neurodegenerative diseases.

An immunologic ATR‐FTIR sensor for Abeta peptide secondary structure analysis in complex fluids is presented.     

Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli

The periplasmic murein (peptidoglycan) sacculus is a giant macromolecule made of glycan strands cross-linked by short peptides completely surrounding the cytoplasmic membrane to protect the cell from lysis due to its internal osmotic pressure. More than 50 different muropeptides are released from the sacculus by treatment with a muramidase. Escherichia coli has six murein synthases which enlarge the sacculus by transglycosylation and transpeptidation of lipid II precursor. A set of twelve periplasmic murein hydrolases (autolysins) release murein fragments during cell growth and division. Recent data on the in vitro murein synthesis activities of the murein synthases and on the interactions between murein synthases, hydrolases and cell cycle related proteins are being summarized. There are different models for the architecture of murein and for the incorporation of new precursor into the sacculus. We present a model in which morphogenesis of the rod-shaped E. coli is driven by cytoskeleton elements competing for the control over the murein synthesis multi-enzyme complexes.     

Bansformation of tobacco with a mutated cyanobacterial phytoene desaturase gene confers resistance to bleaching herbicides

Carotenoids are constituents of the photosynthetic apparatus and essential for plant survival because of their involvement in protection of chlorophylls against photooxidation. Certain classes of herbicides are interfering with carotenoid biosynthesis leading to pigment destruction and a bleached plant phenotype. One important target site for bleaching herbicides is the enzyme phytoene desaturase catalysing the desaturation of phytoene in zeta-carotene. This enzymatic reaction can be inhibited by norflurazon or fluridone. We have transformed tobacco with a mutated cyanobacterial phytoene desaturase gene (pds) derived from the Synechococcus PCC 7942 mutant NFZ4. Characterization of the resulting transformants revealed an up to 58 fold higher norflurazon resistance in comparison to wild type controls. The tolerance for fluridone was also increased 3 fold in the transgenics. Furthermore, the transformed tobacco maintained a higher level of D1 protein of photosystem II indicating a lower susceptibility to photooxidative damage in the presence of norflurazon. In contrast, the genetic manipulation did not confer herbicide resistance against zeta-carotene desaturase inhibitors.     

Constitutive plant toxins and their role in defense against herbivores and pathogens

Most recent investigations have focused on induced, rather than constitutive, plant defenses. Yet significant research has helped to illuminate some of the principal characteristics of constitutive defenses, including mechanisms of action and synergistic effects, as well as strategies used by herbivores and pathogens to circumvent them.