570 Biowissenschaften; Biologie
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During the last decades the ground-breeding Montagu’s harrier ( Circus pygargus , Linnaeus, 1758) has changed its breeding habitats in Europe to agricultural areas in which many local populations would be close to extinction without a special nest protection regime. Although Montagu’s harrier is a well-studied species in terms of ecology and breeding biology, its genetic structure and population genetics are almost unknown. As there is a lack of good genetic markers we developed a set of 19 microsatellite markers comprising 16 new STR markers which were identified by next-generation sequencing (NGS) using 454 shot-gun pyrosequencing of genomic DNA. The STR markers were arranged into three multiplex PCR sets for high throughput genotyping and characterised. The marker set provides a powerful tool for kinship analysis. The combined non-exclusion probability for parent pairs was 1.13* 10-11. Only three loci showed PIC values < 0.50. In total, 121 known family relationships were compared with genetically calculated ones to test the markers suitability for parentage analysis. In 97.5% of all cases full-sibships were accurately determined and 97.6% of all mothers were assigned correctly to their chicks. The present multiplex PCR panels can be used to investigate several hypotheses concerning breeding behaviour, kinship, exchange rates between populations and phylogeography.
Visceral leishmaniasis (VL), caused by the members of the Leishmania donovani complex, has been responsible for devastating VL epidemics in the Sudan. Multilocus microsatellite and sequence typing studies can provide valuable insights into the molecular epidemiology of leishmaniasis, when applied at local scales. Here we present population genetic data for a large panel of strains and clones collected in endemic Sudan between 1993 and 2001.
Ethanol production via genetically engineered cyanobacteria is a promising solution for the production of biofuels. Through the introduction of a pyruvate decarboxylase and alcohol dehydrogenase direct ethanol production becomes possible within the cells. However, during cultivation genetic instability can lead to mutations and thus loss of ethanol production. Cells then revert back to the wild type phenotype.
A method for a rapid and simple detection of these non-producing revertant cells in an ethanol producing cell population is an important quality control measure in order to predict genetic stability and the longevity of a producing culture. Several comparable cultivation experiments revealed a difference in the pigmentation for non-producing and producing cells: the accessory pigment phycocyanin (PC) is reduced in case of the ethanol producer, resulting in a yellowish appearance of the culture. Microarray and western blot studies of Synechocystis sp. PCC6803 and Synechococcus sp. PCC7002 confirmed this PC reduction on the level of RNA and protein.
Prostate cancer (PCa) is one amongst the most common cancersin western men. Incidence rate ofPCa is on the rise worldwide. The present study deals with theserum lipidome profiling of patients diagnosed with PCa to identify potential new biomarkers. We employed ESI-MS/MS and GC-MS for identification of significantly altered lipids in cancer patient’s serum compared to controls. Lipidomic data revealed 24 lipids are significantly altered in cancer patinet’s serum (n = 18) compared to normal (n = 18) with no history of PCa. By using hierarchical clustering and principal component analysis (PCA) we could clearly separate cancer patients from control group. Correlation and partition analysis along with Formal Concept Analysis (FCA) have identified that PC (39:6) and FA (22:3) could classify samples with higher certainty. Both the lipids, PC (39:6) and FA (22:3) could influence the cataloging of patients with 100% sensitivity (all 18 control samples are classified correctly) and 77.7% specificity (of 18 tumor samples 4 samples are misclassified) with p-value of 1.612×10−6 in Fischer’s exact test. Further, we performed GC-MS to denote fatty acids altered in PCa patients and found that alpha-linolenic acid (ALA) levels are altered in PCa. We also performed an in vitro proliferation assay to determine the effect of ALA in survival of classical human PCa cell lines LNCaP and PC3. We hereby report that the altered lipids PC (39:6) and FA (22:3) offer a new set of biomarkers in addition to the existing diagnostic tests that could significantly improve sensitivity and specificity in PCa diagnosis.
Electrospinning is known as a fabrication technique for electrode architectures that serve as immobilization matrices for biomolecules. The current work demonstrates a novel approach to construct a conductive polymeric platform, capable not only of immobilization, but also of electrical connection of the biomolecule with the electrode. It is produced upon electrospinning from mixtures of three different highly conductive sulfonated polyanilines and polyacrylonitrile on ITO electrodes. The resulting fiber mats are with a well-retained conductivity. After coupling the enzyme pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) to polymeric structures and addition of the substrate glucose an efficient bioelectrocatalysis is demonstrated. Depending on the choice of the sulfonated polyanilline mediatorless bioelectrocatalysis starts at low potentials; no large overpotential is needed to drive the reaction. Thus, the electrospun conductive immobilization matrix acts here as a transducing element, representing a promising strategy to use 3D polymeric scaffolds as wiring agents for active enzymes. In addition, the mild and well reproducible fabrication process and the active role of the polymer film in withdrawing electrons from the reduced PQQ-GDH lead to a system with high stability. This could provide access to a larger group of enzymes for bioelectrochemical applications including biosensors and biofuel cells.
Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope mapping and surface plasmon resonance measurements. All antibodies were found to bind with high affinity to two epitopes at the heparin binding site of A27, toward either the N- or C-terminal of the crucial KKEP-segment of A27. Two antibodies recognizing different epitopes were implemented in an antigen capture ELISA. Validation showed robust detection of virus particles from 11 different orthopoxvirus isolates pathogenic to humans, with the exception of MVA, which is apathogenic to humans. Most orthopoxviruses could be detected reliably for viral loads above 1 × 103 PFU/mL. To our knowledge, this is the first solely monoclonal and therefore reproducible antibody-based antigen capture ELISA able to detect all human pathogenic orthopoxviruses including monkeypox virus, except variola virus which was not included. Therefore, the newly developed antibody-based assay represents important progress towards feasible particle detection of this important genus of viruses.
Polymer-multiwalled carbon nanotube (MWCNT) nanohybrids, which differ in surface charge have been synthesized to study the bioelectrocatalysis of adsorbed cellobiose dehydrogenase (CDH) from Phanerochaete sordida on gold electrodes. To obtain negatively charged nanohybrids, poly(3-amino-4-methoxybenzoic acid-co-aniline) (P(AMB-A)) was covalently linked to the surface of MWCNTs while modification with p-phenylenediamine (PDA) converted the COOH-groups to positively charged amino groups. Fourier transform infrared spectroscopy (FTIR) measurements verified the p-phenylenediamine (PDA) modification of the polymer-CNT nanohybrids. The positively charged nanohybrid MWCNT-P(AMB-A)-PDA promoted direct electron transfer (DET) of CDH to the electrode and bioelectrocatalysis of lactose was observed. Amperometric measurements gave an electrochemical response with KMapp = 8.89 mM and a current density of 410 nA/cm2 (15 mM lactose). The catalytic response was tested at pH 3.5 and 4.5. Interference by ascorbic acid was not observed. The study proves that DET between the MWCNT-P(AMB-A)-PDA nanohybrids and CDH is efficient and allows the sensorial detection of lactose.