Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment

A prerequisite for the enrichment of antibodies screened fromphage display libraries is their stable expression on a phageduring multiple selection rounds. Thus, if stringent panningprocedures are employed, selection is simultaneously drivenby antigen affinity, stability and solubility. To take advantageof robust pre-selected scaffolds of such molecules, we graftedsingle-chain Fv (scFv) antibodies, previously isolated froma human phage display library after multiple rounds of in vitropanning on tumor cells, with the specificity of the clinicallyestablished murine monoclonal anti-CD22 antibody RFB4. We showthat a panel of grafted scFvs retained the specificity of themurine monoclonal antibody, bound to the target antigen withhigh affinity (6.4–9.6 nM), and exhibited exceptionalbiophysical stability with retention of 89–93% of theinitial binding activity after 6 days of incubation in humanserum at 37°C. Selection of stable human scaffolds withhigh sequence identity to both the human germline and the rodentframeworks required only a small number of murine residues tobe retained within the human frameworks in order to maintainthe structural integrity of the antigen binding site. We expectthis approach may be applicable for the rapid generation ofhighly stable humanized antibodies with low immunogenic potential. Received June 10, 2003; accepted August 27, 2003.     

Energy policy scenarios to reach challenging climate protection targets in the German electricity sector until 2050

In this article we demonstrate how challenging greenhouse gas reduction targets of up to 95% until 2050 can be achieved in the German electricity sector. In the analysis, we focus on the main requirements to reach such challenging targets. To account for interdependencies between the electricity market and the rest of the economy, different models were used to account for feedback loops with all other sectors. We include scenarios with different runtimes and retrofit costs for existing nuclear plants to determine the effects of a prolongation of nuclear power plants in Germany. Key findings for the electricity sector include the importance of a European-wide coordinated electricity grid extension and the exploitation of regional comparative cost effects for renewable sites. Due to political restrictions, nuclear energy will not be available in Germany in 2050. However, the nuclear life-time extension has a positive impact on end consumer electricity prices as well as economic growth in the medium term, if retrofit costs do not exceed certain limits.     

Oxidative Damage in Sporadic Colorectal Cancer: Molecular Mapping of Base Excision Repair Glycosylases MUTYH and hOGG1 in Colorectal Cancer Patients

Oxidative stress, oxidative DNA damage and resulting mutations play a role in colorectal carcinogenesis. Impaired equilibrium between DNA damage formation, antioxidant status, and DNA repair capacity is responsible for the accumulation of genetic mutations and genomic instability. The lesion-specific DNA glycosylases, e.g., hOGG1 and MUTYH, initiate the repair of oxidative DNA damage. Hereditary syndromes (MUTYH-associated polyposis, NTHL1-associated tumor syndrome) with germline mutations causing a loss-of-function in base excision repair glycosylases, serve as straight forward evidence on the role of oxidative DNA damage and its repair. Altered or inhibited function of above glycosylases result in an accumulation of oxidative DNA damage and contribute to the adenoma-adenocarcinoma transition. Oxidative DNA damage, unless repaired, often gives rise G:C > T:A mutations in tumor suppressor genes and proto-oncogenes with subsequent occurrence of chromosomal copy-neutral loss of heterozygosity. For instance, G>T transversions in position c.34 of a KRAS gene serves as a pre-screening tool for MUTYH-associated polyposis diagnosis. Since sporadic colorectal cancer represents more complex and heterogenous disease, the situation is more complicated. In the present study we focused on the roles of base excision repair glycosylases (hOGG1, MUTYH) in colorectal cancer patients by investigating tumor and adjacent mucosa tissues. Although we found downregulation of both glycosylases and significantly lower expression of hOGG1 in tumor tissues, accompanied with G>T mutations in KRAS gene, oxidative DNA damage and its repair cannot solely explain the onset of sporadic colorectal cancer. In this respect, other factors (especially microenvironment) per se or in combination with oxidative DNA damage warrant further attention. Base excision repair characteristics determined in colorectal cancer tissues and their association with disease prognosis have been discussed as well.     

Autogenous healing and reinforcement corrosion of water-penetrated separation cracks in reinforced concrete

Depending on the crack width, the thickness of the structure, the water pressure, and the degree of acid of the water, long-term investigations have been performed over a period of 2 years with respect to the autogenous healing and reinforcement corrosion of water-penetrated separation cracks in reinforced concrete by the University of Kaiserslautern, supported by the Deutsche Forschungsgemeinschaft (DFG). For the waters penetrating the cracks deionised water (neutral, pH=7.0), and boric acid treated deionised water with a pH-value of 6.1 and 5.2 (weakly acid waters) were used. A complete autogenous healing could not be observed. The water penetrating the cracks could hardly be measured with a pH-value of 7.0 at the end of the test. While naturally at the beginning of the test, no influence of the water-chemical degree of the acids could be determined, the existing flow-through quantities towards the end of the test period depended clearly on the crack width and the pH-value. With an increasing crack width and an increasing acid-degree larger flow-through quantities were measured. Depending on the pH-value and the crack width it was determined whether and to which extent corrosion developed at the reinforcing steel bars crossing the cracks. With a crack width of 0.1 mm, corrosion was not to be observed in any case. For the test specimens with a crack width of 0.2 mm a start of the corrosion was found depending on the pH-value. With an increasing width of the crack, an increasing corrosion development is to be expected for test specimens penetrated by acid water. For a crack width of 0.4 mm and a pH-value of 5.2, the highest corrosion development was to be observed, however, there were weakenings of the cross section not worth being mentioned even after a 2-year test period.     

Amyloid Beta-Mediated Changes in Synaptic Function and Spine Number of Neocortical Neurons Depend on NMDA Receptors

Onset and progression of Alzheimer’s disease (AD) pathophysiology differs between brain regions. The neocortex, for example, is a brain region that is affected very early during AD. NMDA receptors (NMDARs) are involved in mediating amyloid beta (Aβ) toxicity. NMDAR expression, on the other hand, can be affected by Aβ. We tested whether the high vulnerability of neocortical neurons for Aβ-toxicity may result from specific NMDAR expression profiles or from a particular regulation of NMDAR expression by Aβ. Electrophysiological analyses suggested that pyramidal cells of 6-months-old wildtype mice express mostly GluN1/GluN2A NMDARs. While synaptic NMDAR-mediated currents are unaltered in 5xFAD mice, extrasynaptic NMDARs seem to contain GluN1/GluN2A and GluN1/GluN2A/GluN2B. We used conditional GluN1 and GluN2B knockout mice to investigate whether NMDARs contribute to Aβ-toxicity. Spine number was decreased in pyramidal cells of 5xFAD mice and increased in neurons with 3-week virus-mediated Aβ-overexpression. NMDARs were required for both Aβ-mediated changes in spine number and functional synapses. Thus, our study gives novel insights into the Aβ-mediated regulation of NMDAR expression and the role of NMDARs in Aβ pathophysiology in the somatosensory cortex.     

On-line monitoring and identification of bioaerosols

Fast analysis of bioaerosols in clean room environments is necessary in order to prevent contamination of pharmaceutical products, minimize machine downtimes, or both. The detection and identification of microbes will be carried out in several steps: After impaction of the aerosol on a surface, the particles are presorted with glancing light illumination and fluorescence imaging in order to distinguish between abiotic and biotic particles. Since only the biotic particles are of interest, the analysis time can be minimized due to reduction of the data set. The biotic particles are then analyzed further with Raman spectroscopy and identified with a support vector machine.     

Material‐Auswahlbox zur Herstellung fortgeschrittener Polymermembranen für die Wasseraufbereitung

Within the framework of the MABMEM research project, new high‐performance membranes are being developed for sustainable water management. The performance of the membranes will be evaluated in comparative and standardized fouling tests as well as in terms of the removal of trace impurities on a laboratory scale. Seven candidates are currently being tested in demonstrator trials with real‐water matrix in a waterworks for the direct treatment of dam water without prior coagulation over a period of 6 months. Subsequently, the new membrane materials will be operated with the effluent of a wastewater treatment plant.     

2-Aminobenzothiazoles Inhibit Virulence Gene Expression and Block Polymyxin Resistance in Salmonella enterica

One promising strategy to combat antibiotic-resistant bacteria is to develop compounds that block bacterial defenses against antibacterial conditions produced by the innate immune system. Salmonella enterica, which causes food-borne gastroenteritis and typhoid fever, requires histidine kinases (HKs) to resist innate immune defenses such as cationic antimicrobial peptides (CAMPs). Herein, we report that 2-aminobenzothiazoles block histidine kinase-dependent phenotypes in Salmonella enterica serotype Typhimurium. We found that 2-aminobenzothiazoles inhibited growth under low Mg²⁺, a stressful condition that requires histidine kinase-mediated responses, and decreased expression of the virulence genes pagC and pagK. Furthermore, we discovered that 2-aminobenzothiazoles weaken Salmonella’s resistance to polymyxin B and polymyxin E, which are last-line antibiotics and models for host defense CAMPs. These findings raise the possibilities that 2-aminobenzothiazoles can block HK-mediated bacterial defenses and can be used in combination with polymyxins to treat infections caused by Salmonella.     

Mesenchymal Stromal Cell Therapy in Novel Porcine Model of Diffuse Liver Damage Induced by Repeated Biliary Obstruction

In liver surgery, biliary obstruction can lead to secondary biliary cirrhosis, a life-threatening disease with liver transplantation as the only curative treatment option. Mesenchymal stromal cells (MSC) have been shown to improve liver function in both acute and chronic liver disease models. This study evaluated the effect of allogenic MSC transplantation in a large animal model of repeated biliary obstruction followed by partial hepatectomy. MSC transplantation supported the growth of regenerated liver tissue after 14 days (MSC group, n = 10: from 1087 ± 108 (0 h) to 1243 ± 92 mL (14 days); control group, n = 11: from 1080 ± 95 (0 h) to 1100 ± 105 mL (14 days), p = 0.016), with a lower volume fraction of hepatocytes in regenerated liver tissue compared to resected liver tissue (59.5 ± 10.2% vs. 70.2 ± 5.6%, p < 0.05). Volume fraction of connective tissue, blood vessels and bile vessels in regenerated liver tissue, serum levels of liver enzymes (AST, ALT, ALP and GGT) and liver metabolites (albumin, bilirubin, urea and creatinine), as well as plasma levels of IL-6, IL-8, TNF-α and TGF-β, were not affected by MSC transplantation. In our novel, large animal (pig) model of repeated biliary obstruction followed by partial hepatectomy, MSC transplantation promoted growth of liver tissue without any effect on liver function. This study underscores the importance of translating results between small and large animal models as well as the careful translation of results from animal model into human medicine.     

“Quinone Millipedes” Reconsidered: Evidence for a Mosaic-Like Taxonomic Distribution of Phenol-Based Secretions across the Julidae

The defensive chemistry of juliformian millipedes is characterized mainly by benzoquinones (”quinone millipedes”), whereas the secretions of the putative close outgroup Callipodida are considered to be exclusively phenolic. We conducted a chemical screening of julid secretions for phenolic content. Most species from tribes Cylindroiulini (15 species examined), Brachyiulini (5 species examined), Leptoiulini (15 species examined), Uncigerini (2 species examined), Pachyiulini (3 species examined), and Ommatoiulini (2 species examined) had non-phenolic, in most cases exclusively benzoquinonic secretions. In contrast, tribes Cylindroiulini, Brachyiulini, and Leptoiulini also contained representatives with predominantly phenol-based exudates. In detail, p-cresol was a major compound in the secretions of the cylindroiulines Styrioiulus pelidnus and S. styricus (p-cresol content 93 %) and an undetermined Cylindroiulus species (p-cresol content 51 %), in the brachyiulines Brachyiulus lusitanus (p-cresol content 21 %) and Megaphyllum fagorum (p-cresol content 92 %), as well as in an undescribed Typhloiulus species (p-cresol content 32 %, Leptoiulini). In all species, p-cresol was accompanied by small amounts of phenol. The secretion of M. fagorum was exclusively phenolic, whereas phenols were accompanied by benzoquinones in all other species. This is the first incidence of clearly phenol-dominated secretions in the Julidae. We hypothesize a shared biosynthetic route to phenols and benzoquinones, with benzoquinones being produced from phenolic precursors. The patchy taxonomic distribution of phenols documented herein supports multiple independent regression events in a common pathway of benzoquinone synthesis rather than multiple independent incidences of phenol biosynthesis.