Characterization of CD177-reactive iso- and auto-antibodies.

BACKGROUND: CD177 is a surface protein on neutrophils and a main mediator for the surface expression of proteinase 3 (PR3). Its functions are largely unknown. At least three types of antibodies have been described to target CD177: isoantibodies, which are formed in CD177-null individuals as a result of an immune reaction following transfusion or pregnancy; autoantibodies present in sera from patients with autoimmune neutropenia; and antineutrophil cytoplasmic antibodies in sera from patients with glomerulonephritis with polyangiitis. In this study, we aimed to compare the binding characteristics of auto- and iso-antibodies to optimize their detectability in the neutrophil serology laboratory. STUDY DESIGN AND METHODS: The reactivity of iso- and auto-antibodies against CD177 was studied using granulocytes, "native" CD177/PR3 complex, and recombinant CD177 or PR3. RESULTS: All iso- and auto-antibodies were reactive with CD177/PR3 when immobilized with monoclonal antibody (moab) 7D8. Seventy-five percent of autoantibodies, but none of the isoantibodies, did not react with CD177/PR3 immobilized with moab MEM166. The majority of autoantibodies did not react with recombinant CD177, whereas most isoantibodies tested positive. DISCUSSION: Our results suggest that iso- and auto-antibodies against CD177 target different epitopes. Isoantibodies mainly target CD177 alone, while the majority of autoantibodies target a native epitope present on the neutrophil surface, but absent from recombinant CD177 which lacks PR3. Moab MEM166 binds to the native epitope and hinders the binding of CD177 autoantibodies. The results may help to design diagnostic strategies, especially for the identification of autoantibodies.

Pushing product formation to its limit: metabolic engineering of Corynebacterium glutamicum for L-leucine overproduction.

Using metabolic engineering, an efficient L-leucine production strain of Corynebacterium glutamicum was developed. In the wild type of C. glutamicum, the leuA-encoded 2-isopropylmalate synthase (IPMS) is inhibited by low L-leucine concentrations with a K(i) of 0.4 mM. We identified a feedback-resistant IMPS variant, which carries two amino acid exchanges (R529H, G532D). The corresponding leuA(fbr) gene devoid of the attenuator region and under control of a strong promoter was integrated in one, two or three copies into the genome and combined with additional genomic modifications aimed at increasing L-leucine production. These modifications involved (i) deletion of the gene encoding the repressor LtbR to increase expression of leuBCD, (ii) deletion of the gene encoding the transcriptional regulator IolR to increase glucose uptake, (iii) reduction of citrate synthase activity to increase precursor supply, and (iv) introduction of a gene encoding a feedback-resistant acetohydroxyacid synthase. The production performance of the resulting strains was characterized in bioreactor cultivations. Under fed-batch conditions, the best producer strain accumulated L-leucine to levels exceeding the solubility limit of about 24 g/l. The molar product yield was 0.30 mol L-leucine per mol glucose and the volumetric productivity was 4.3 mmol l⁻¹ h⁻¹. These values were obtained in a defined minimal medium with a prototrophic and plasmid-free strain, making this process highly interesting for industrial application.

["Frühe Hilfen": what must pediatricians know?] / Frühe Hilfen: Was müssen Kinderärzte wissen?

In early childhood, essential foundations are laid for healthy growing up and long-term quality of life and health. Stressful life situations can disrupt good early childhood development, therefore early support is essential. Early childhood interventions like "Frühe Hilfen" provide support systems with coordinated services for parents and children during early childhood. They are particularly aimed at families in stressful life situations and thus counteract the long-term consequences of negative childhood experiences and promote healthy growing up. Pediatricians are important cooperation partners of "Frühe Hilfen", especially with respect to access to the services. Early life care is an interdisciplinary and integrative concept that aims to promote optimal conditions for families around the desire to have children, pregnancy, birth and early childhood, among other things, with a view to risk situations.

A manually curated compendium of expression profiles for the microbial cell factory Corynebacterium glutamicum.

Corynebacterium glutamicum is the major host for the industrial production of amino acids and has become one of the best studied model organisms in microbial biotechnology. Rational strain construction has led to an improvement of producer strains and to a variety of novel producer strains with a broad substrate and product spectrum. A key factor for the success of these approaches is detailed knowledge of transcriptional regulation in C. glutamicum. Here, we present a large compendium of 927 manually curated microarray-based transcriptional profiles for wild-type and engineered strains detecting genome-wide expression changes of the 3,047 annotated genes in response to various environmental conditions or in response to genetic modifications. The replicates within the 927 experiments were combined to 304 microarray sets ordered into six categories that were used for differential gene expression analysis. Hierarchical clustering confirmed that no outliers were present in the sets. The compendium provides a valuable resource for future fundamental and applied research with C. glutamicum and contributes to a systemic understanding of this microbial cell factory. Measurement(s) Gene Expression Analysis Technology Type(s) Two Color Microarray Factor Type(s) WT condition A vs. WT condition B • Plasmid-based gene overexpression in parental strain vs. parental strain with empty vector control • Deletion mutant vs. parental strain Sample Characteristic - Organism Corynebacterium glutamicum Sample Characteristic - Environment laboratory environment Sample Characteristic - Location Germany.

Promoting citizen science in the energy sector: Generation Solar, an open database of small-scale solar photovoltaic installations.

Citizen science is becoming an effective approach in building a new relationship between science and society, in which the desire of citizens to participate actively in knowledge production meets the needs of researchers. A citizen science initiative dealing with the development of photovoltaics (PV) is presented. To generate a "responsible" initiative, the research question has been designed collectively from the beginning, involving diverse actors in order to encourage creativity while addressing their interests and concerns. The result has been called Generation Solar. It aims at co-creating an open database of PV installations including their technical characteristics, and an online map for visualizing them. The initiative responds to a clear scientific demand; an important drawback for researchers working on energy modelling and predictions of production lays precisely in the lack of information about these installations' locations and characteristics. The initiative invites citizens, companies and public institutions with a PV installation to collaborate by providing such data. Data will follow the format of Open Power System Data in order to be fully exploitable by the scientific community and society. The success of the initiative will rely on the capacity to mobilize citizens and register the largest possible number of installations worldwide.

Production of 2-ketoisocaproate with Corynebacterium glutamicum strains devoid of plasmids and heterologous genes.

2-Ketoisocaproate (KIC), the last intermediate in l-leucine biosynthesis, has various medical and industrial applications. After deletion of the ilvE gene for transaminase B in l-leucine production strains of Corynebacterium glutamicum, KIC became the major product, however, the strains were auxotrophic for l-isoleucine. To avoid auxotrophy, reduction of IlvE activity by exchanging the ATG start codon of ilvE by GTG was tested instead of an ilvE deletion. The resulting strains were indeed able to grow in glucose minimal medium without amino acid supplementation, but at the cost of lowered growth rates and KIC production parameters. The best production performance was obtained with strain MV-KICF1, which carried besides the ilvE start codon exchange three copies of a gene for a feedback-resistant 2-isopropylmalate synthase, one copy of a gene for a feedback-resistant acetohydroxyacid synthase and deletions of ltbR and iolR encoding transcriptional regulators. In the presence of 1 mM l-isoleucine, MV-KICF1 accumulated 47 mM KIC (6.1 g l(-1)) with a yield of 0.20 mol/mol glucose and a volumetric productivity of 1.41 mmol KIC l(-1) h(-1). Since MV-KICF1 is plasmid free and lacks heterologous genes, it is an interesting strain for industrial application and as platform for the production of KIC-derived compounds, such as 3-methyl-1-butanol.

Overweight prevention in adolescents and children (behavioural and environmental prevention).

HEALTH POLITICAL BACKGROUND: In 2006, the prevalence of overweight and adiposity among children and adolescents aged three to 17 years is 15%, 6.3% (800,000) of these are obese. SCIENTIFIC BACKGROUND: Obese children and adolescents have an increased body fat ratio. The reasons for overweight are - among others - sociocultural factors, and a low social status as determined by income and educational level of the parents. The consequences of adiposity during childhood are a higher risk of metabolic and cardiovascular diseases and increased mortality in adulthood. Possible approaches to primary prevention in children and adolescents are measures taken in schools and kindergarten, as well as education and involvement of parents. Furthermore, preventive measures geared towards changing environmental and living conditions are of particular importance. RESEARCH QUESTIONS: What is the effectiveness and efficiency of different measures and programs (geared towards changing behaviour and environmental and living conditions) for primary prevention of adiposity in children and adolescents, with particular consideration of social aspects? METHODS: The systematic literature search yielded 1,649 abstracts. Following a two-part selection process with predefined criteria 31 publications were included in the assessment. RESULTS: The majority of interventions evaluated in primary studies take place in schools. As the measures are mostly multi-disciplinary and the interventions are often not described in detail, no criteria of success for the various interventions can be extrapolated from the reviews assessed. An economic model calculation for Australia, which compares the efficiency of different interventions (although on the basis of low evidence) comes to the conclusion that the intervention with the greatest impact on society is the reduction of TV-ads geared towards children for foods and drinks rich in fat and sugar. There is a significant correlation between adiposity and socioeconomic deprivation. The lack of interventions (especially preventive measures geared towards changing environmental and living conditions) and studies focusing on this population group is noticeable. DISCUSSION: There are only a few primary studies of high quality on adiposity prevention in children and adolescents. Especially studies which compare different measures are lacking. This holds also true for the economic analysis, which seems logical insofar, as the basis for economic analyses are usually primary studies (preferably randomized controlled trials (RCT)) due to their evidence level). Studies on interventions geared towards changing environmental and living conditions and towards specific population groups (i. e. the socially disadvantaged) are hardly available. CONCLUSIONS: There are hardly any primary studies of high quality on adiposity prevention in children and adolescents, especially studies which compare different measures are lacking. Interventions geared towards specific population groups (particularly for the socioeconomically disadvantaged) are specifically underrepresented. Establishing such studies is an essential requirement of adiposity prevention. Recommended are a combination of measures geared towards changing environmental and living conditions and towards specific population groups. Furthermore, it is recommended to systematically register future programs (preferably online) in order to be able to draft criteria of success.

Long term substitution treatment (maintenance treatment) of opioid dependent persons.

HEALTH POLITICAL BACKGROUND: Methadone substitution treatment in Germany is introduced in 1988 in the framework of a scientific pilot study in North Rhein Westphalia. Recent statistics show that by now a broad offer of substitution treatment exists. From 1 June 2002 to 31 December 2003 113,000 substitution treatments have been recorded as being started of which around 56,000 have been recorded as ongoing treatments by 1 December 2003. SCIENTIFIC BACKGROUND: Substitution treatment (treatment of opioid-dependent persons using substitution substances) is one part of addiction treatment. Its goals are harm reduction and the stabilisation of opioid dependent persons. Integration of opioid-dependent persons in a treatment-setting, reduction of consumption of psychoactive substances, reduction of risk behaviour (primarily related to infectious diseases), decrease of mortality and improvements concerning the social, psychic and physic situation are seen as a success of substitution treatment as maintenance therapy. RESEARCH QUESTIONS: The aim of this HTA report is to investigate which indicators can be used to evaluate the effectiveness of substitution treatment. Based on these indicators an evaluation of the medical, social and economical benefit of substitution treatment - also in relation to abstinence oriented treatment - is carried out. METHODS: A systematic literature search was performed in 31 international databases which yielded 2451 articles with publication date between 1995 and February 2005. RESULTS: After a twofold selection process 32 publications were included for assessment and 276 publications were used as background literature. Despite serious restrictions due to selection bias and dropout in most studies focusing on substitution treatment, reduction of consumption of illegal opioids, reduction of risk behaviour, criminal behaviour, mortality and incidence of HIV can be seen as an empirically proven success of substitution treatment. Concerning the improvement of life and health situation the results of the studies are contradictory. The results show that retention rate of substitution treatment is higher than retention rate of abstinence oriented treatment. Regarding economical aspects substitution treatment is efficient in avoiding secondary illnesses (infections) and decreasing criminality. From the perspective of medical ethics substitution treatment as well as medical prescription of heroin is in principle acceptable. DISCUSSION AND CONCLUSIONS: Based on these results, it can be recommended that substitution treatment in principle should be made available for all opioid dependent persons. The decision whether substitution treatment or another treatment (e. g. abstinence oriented treatment) is more promising has to take into account the individual situation of the client. In addition a combination of substitution treatment and abstinence oriented treatment might be promising although there is a lack of studies about this approach. In any case the decision concerning a certain form of treatment should leave aside pseudo-moralic concerns and should be made on the base of established medical ethic principles - like the interest of the patient - taking into account the specific situation of the client.

Characterization of the dexniguldipine binding site in the multidrug resistance-related transport protein P-glycoprotein by photoaffinity labeling and mass spectrometry.

Human P-glycoprotein (P-gp), an integral membrane transport protein, is responsible for the efflux of various drugs, including cytostatics from cancer cells leading to multidrug resistance. P-gp is composed of two homologous half domains, each carrying one nucleotide binding site. The drug extrusion is ATP-dependent and can be inhibited by chemosensitizers, such as the dihydropyridine derivative dexniguldipine-HCl, through direct interaction with P-gp. To evaluate the mechanism(s) of chemosensitization and identify the binding sites of dexniguldipine-HCl, a tritium-labeled azido analog of dexniguldipine, [(3)H]B9209-005, was used as a photoaffinity probe. Using the multidrug resistant T-lymphoblastoid cell line CCRF-ADR5000, two proteins were specifically labeled in membranes by [(3)H]B9209-005. These proteins were identified by immunoprecipitation such as P-gp and its N-terminal fragment. The membranes were solubilized and the labeled P-gp proteins first isolated by lectin-chromatography and then digested with trypsin. SDS-polyacrylamide gel electrophoresisanalysis of the digest revealed a major radioactive 7-kDa fragment. The tryptic fragments were separated by high-performance liquid chromatography and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The MS results, corroborated by MALDI-MS of peptides after one step of Edman analysis, identified the radioactive 7-kDa band as the dexniguldipine-bound, tryptic P-gp peptide, 468-527. This sequence region is flanked by the Walker motifs A and B of the N-terminal ATP-binding cassette suggesting direct interaction of the chemosensitizer with the nucleotide binding site is involved in the mechanism of chemosensitization.