Parallel Force Assay for Protein-Protein Interactions

Quantitative proteome research is greatly promoted by high-resolution parallel format assays. A characterization of protein complexes based on binding forces offers an unparalleled dynamic range and allows for the effective discrimination of non-specific interactions. Here we present a DNA-based Molecular Force Assay to quantify protein-protein interactions, namely the bond between different variants of GFP and GFP-binding nanobodies. We present different strategies to adjust the maximum sensitivity window of the assay by influencing the binding strength of the DNA reference duplexes. The binding of the nanobody Enhancer to the different GFP constructs is compared at high sensitivity of the assay. Whereas the binding strength to wild type and enhanced GFP are equal within experimental error, stronger binding to superfolder GFP is observed. This difference in binding strength is attributed to alterations in the amino acids that form contacts according to the crystal structure of the initial wild type GFP-Enhancer complex. Moreover, we outline the potential for large-scale parallelization of the assay.     

An Integrated Computational/Experimental Model of Lymphoma Growth

Non-Hodgkin''s lymphoma is a disseminated, highly malignant cancer, with resistance to drug treatment based on molecular- and tissue-scale characteristics that are intricately linked. A critical element of molecular resistance has been traced to the loss of functionality in proteins such as the tumor suppressor p53. We investigate the tissue-scale physiologic effects of this loss by integrating in vivo and immunohistological data with computational modeling to study the spatiotemporal physical dynamics of lymphoma growth. We compare between drug-sensitive Eμ-myc Arf-/- and drug-resistant Eμ-myc p53-/- lymphoma cell tumors grown in live mice. Initial values for the model parameters are obtained in part by extracting values from the cellular-scale from whole-tumor histological staining of the tumor-infiltrated inguinal lymph node in vivo. We compare model-predicted tumor growth with that observed from intravital microscopy and macroscopic imaging in vivo, finding that the model is able to accurately predict lymphoma growth. A critical physical mechanism underlying drug-resistant phenotypes may be that the Eμ-myc p53-/- cells seem to pack more closely within the tumor than the Eμ-myc Arf-/- cells, thus possibly exacerbating diffusion gradients of oxygen, leading to cell quiescence and hence resistance to cell-cycle specific drugs. Tighter cell packing could also maintain steeper gradients of drug and lead to insufficient toxicity. The transport phenomena within the lymphoma may thus contribute in nontrivial, complex ways to the difference in drug sensitivity between Eμ-myc Arf-/- and Eμ-myc p53-/- tumors, beyond what might be solely expected from loss of functionality at the molecular scale. We conclude that computational modeling tightly integrated with experimental data gives insight into the dynamics of Non-Hodgkin''s lymphoma and provides a platform to generate confirmable predictions of tumor growth.     

ATP Impedes the Inhibitory Effect of Hsp90 on Aβ40 Fibrillation

Heat shock protein 90 (Hsp90) is a molecular chaperone that assists protein folding in an Adenosine triphosphate (ATP)-dependent way. Hsp90 has been reported to interact with Alzheime?s disease associated amyloid-β (Aβ) peptides and to suppress toxic oligomer- and fibril formation. However, the mechanism remains largely unclear. Here we use a combination of atomic force microscopy (AFM) imaging, circular dichroism (CD) spectroscopy and biochemical analysis to quantify this interaction and put forward a microscopic picture including rate constants for the different transitions towards fibrillation. We show that Hsp90 binds to Aβ₄₀ monomers weakly but inhibits Aβ₄₀ from growing into fibrils at substoichiometric concentrations. ATP impedes this interaction, presumably by modulating Hsp90’s conformational dynamics and reducing its hydrophobic surface. Altogether, these results might indicate alternative ways to prevent Aβ₄₀ fibrillation by manipulating chaperones that are already abundant in the brain.     

Ecosystem based modeling and indication of ecological integrity in the German North Sea—Case study offshore wind parks

Human exploitation and use of marine and coastal areas are apparent and growing in many regions of the world. For instance, fishery, shipping, military, raw material exploitation, nature protection and the rapidly expanding offshore wind power technology are competing for limited resources and space. The development and implementation of Integrated Coastal Zone Management (ICZM) strategies could help to solve these problems. Therefore, suitable spatial assessment, modeling, planning and management tools are urgently needed. These tools have to deal with data that include complex information on different spatial and temporal scales. A systematic approach based on the development of future scenarios which are assessed by combining different simulation models, GIS methods and an integrating set of ecological integrity indicators, was applied in a case study in the German North Sea. Here, the installation of huge offshore wind parks within the near future is planned. The aim was to model environmental effects of altered sea-use patterns on marine biota. Indicators of ecological integrity were used to assess altering conditions and possible ecosystem shifts ranging from systems’ degradations to the development of highly productive and diverse artificial reef systems. The results showed that some ecosystem processes and properties and related indicators are sensitive to changes generated by offshore wind park installations while others did not react as hypothesized.     

Five challenges to bringing single-molecule force spectroscopy into living cells

In recent years, single-molecule force spectroscopy techniques have been used to study how inter- and intramolecular interactions control the assembly and functional state of biomolecular machinery in vitro. Here we discuss the problems and challenges that need to be addressed to bring these technologies into living cells and to learn how cellular machinery is controlled in vivo.     

Parental risk factors and anorectal malformations: systematic review and meta-analysis

Background

Anderson's disease (AD) or chylomicron retention disease (CMRD) is a very rare hereditary lipid malabsorption syndrome. In order to discover novel mutations in the SAR1B gene and to evaluate the expression, as compared to healthy subjects, of the Sar1 gene and protein paralogues in the intestine, we investigated three previously undescribed individuals with the disease.

Methods

The SAR1B, SAR1A and PCSK9 genes were sequenced. The expression of the SAR1B and SAR1A genes in intestinal biopsies of both normal individuals and patients was measured by RTqPCR. Immunohistochemistry using antibodies to recombinant Sar1 protein was used to evaluate the expression and localization of the Sar1 paralogues in the duodenal biopsies.

Results

Two patients had a novel SAR1B mutation (p.Asp48ThrfsX17). The third patient, who had a previously described SAR1B mutation (p.Leu28ArgfsX7), also had a p.Leu21dup variant of the PCSK9 gene. The expression of the SAR1B gene in duodenal biopsies from an AD/CMRD patient was significantly decreased whereas the expression of the SAR1A gene was significantly increased, as compared to healthy individuals. The Sar1 proteins were present in decreased amounts in enterocytes in duodenal biopsies from the patients as compared to those from healthy subjects.

Conclusions

Although the proteins encoded by the SAR1A and SAR1B genes are 90% identical, the increased expression of the SAR1A gene in AD/CMRD does not appear to compensate for the lack of the SAR1B protein. The PCSK9 variant, although reported to be associated with low levels of cholesterol, does not appear to exert any additional effect in this patient. The results provide further insight into the tissue-specific nature of AD/CMRD.     

Anaerobic benzene degradation by bacteria

Benzene is a widespread and toxic contaminant. The fate of benzene in contaminated aquifers seems to be primarily controlled by the abundance of oxygen: benzene is aerobically degraded at high rates by ubiquitous microorganisms, and the oxygen‐dependent pathways for its breakdown were elucidated more than 50 years ago. In contrast, benzene was thought to be persistent under anoxic conditions until 25 years ago. Nevertheless, within the last 15 years, several benzene‐degrading cultures have been enriched under varying electron acceptor conditions in laboratories around the world, and organisms involved in anaerobic benzene degradation have been identified, indicating that anaerobic benzene degradation is a relevant environmental process. However, only a few benzene degraders have been isolated in pure culture so far, and they all use nitrate as an electron acceptor. In some highly enriched strictly anaerobic cultures, benzene has been described to be mineralized cooperatively by two or more different organisms. Despite great efforts, the biochemical mechanism by which the aromatic ring of benzene is activated in the absence of oxygen is still not fully elucidated; methylation, hydroxylation and carboxylation are discussed as likely reactions. This review summarizes the current knowledge about the ‘key players’ of anaerobic benzene degradation under different electron acceptor conditions and the possible pathway(s) of anaerobic benzene degradation.     

DNA barcoding of oomycetes with cytochrome c oxidase subunit I and internal transcribed spacer

Oomycete species occupy many different environments and many ecological niches. The genera Phytophthora and Pythium for example, contain many plant pathogens which cause enormous damage to a wide range of plant species. Proper identification to the species level is a critical first step in any investigation of oomycetes, whether it is research driven or compelled by the need for rapid and accurate diagnostics during a pathogen outbreak. The use of DNA for oomycete species identification is well established, but DNA barcoding with cytochrome c oxidase subunit I (COI) is a relatively new approach that has yet to be assessed over a significant sample of oomycete genera. In this study we have sequenced COI, from 1205 isolates representing 23 genera. A comparison to internal transcribed spacer (ITS) sequences from the same isolates showed that COI identification is a practical option; complementary because it uses the mitochondrial genome instead of nuclear DNA. In some cases COI was more discriminative than ITS at the species level. This is in contrast to the large ribosomal subunit, which showed poor species resolution when sequenced from a subset of the isolates used in this study. The results described in this paper indicate that COI sequencing and the dataset generated are a valuable addition to the currently available oomycete taxonomy resources, and that both COI, the default DNA barcode supported by GenBank, and ITS, the de facto barcode accepted by the oomycete and mycology community, are acceptable and complementary DNA barcodes to be used for identification of oomycetes.     

Analysis of particle strain in stirred bioreactors by drop breakage investigations

Understanding of particle strain and drop breakage is relevant for various technical applications. To analyze it, single drop experiments in a breakage cell and evolving drop size distributions in an agitated system are studied. The mechanisms for particle strain and drop breakage are assumed to be comparable for the investigated turbulent flow regime. The agitation process is simulated using a population balance model. This model provides transient prediction capacities at different scales and can be used for scale-up/down projects. The number and the size distributions of daughter fragments for single drops have been studied. The results clearly support the assumption of binary breakage. The most common assumption of a Gaussian distribution for the daughter drop size distribution could not be supported. The evolution of a breakage-dominated toluene/water system was then simulated using different daughter drop size distributions from literature. The computational results were compared with experimental values. All simulations were able to predict the transient Sauter mean diameter excellently but varied strongly in the results on the shape of the distribution. In agreement with the experimental single drop results, the use of a bimodal or a very broad bell-shaped distribution of the daughter drops is proposed for the simulations. Although these results were obtained in a particular vessel for a specific phase system, it can be applied to simulate transient multiphase systems at different scales. We would expect that the general trends observed in this study are comparable to various applications in multiphase bioreactors.