Multiplexed detection of protein-peptide interaction and inhibition using capillary electrophoresis

High-speed capillary electrophoresis (CE) was employed to detect binding and inhibition of SH2 domain proteins using fluorescently labeled phosphopeptides as affinity probes. Single SH2 protein-phosphopeptide complexes were detected and confirmed by competition and fluorescence anisotropy. The assay was then extended to a multiplexed system involving separation of three SH2 domain proteins: Src, SH2-Bbeta, and Fyn. The selectivity of the separation was improved by altering the charge of the peptide binding partners used, thus demonstrating a convenient way to control resolution for the multiplexed assay. The separation was completed within 6 s, allowing rapidly dissociating complexes to be detected. Two low molecular weight inhibitors were tested for inhibition selectivity and efficacy. One inhibitor interrupted binding interaction of all three proteins, while the other selectively inhibited Src only leaving SH2-Bbeta and Fyn complex barely affected. IC(50) of both selective and nonselective inhibitors were determined and compared for different proteins. The IC(50) of the nonselective inhibitor was 49 +/- 9, 323 +/- 42, and 228 +/- 19 microM (n = 3) for Src, SH2-Bbeta, and Fyn, respectively, indicating different efficacy of the nonselective inhibitor for different SH2 domain protein. It is concluded that high-speed CE has the potential for multiplexed screening of drugs that disrupt protein-protein interactions.     

Metabolism of long-chain fatty acids,alcohols and alkylglycerols in the fish parasiteParatenuisentis ambiguus (Acanthocephala)

Specific differences between the acyl composition of lipids on the helminthParatenuisentis ambiguus and its host eel, as shown previously, prompted us to study the lipid metabolism in this intestinal fish parasite. Adults and larvae ofP. ambiguus were fed various lipid precursors, e.g., fatty acids, long-chain alcohols and 1-O-alkylglycerols, which may occur as common nutrients of intestinal parasites. Incorporation of [1-¹⁴C]palmitic acid into neutral and polar lipids was found to be similar under aerobic and near-anaerobic conditions. In adult parasites maintained in culture medium supplemented with glucose, [1-¹⁴C]palmitic acid was incorporated mainly into triacylglycerols and phosphatidylcholines, whereas [1-¹⁴C]oleic acid was incorporated preferentially into triacylglycerols. In fasted adults, as well as in larvae, [1-¹⁴C]oleic acid was mainly transferred to phosphatidylcholines. Lipolytic activity was detected in adult parasites that had been incubated with radioactive trioleoylglycerol. [1-¹⁴C]Hexadecan-1-ol was oxidized inP. ambiguus at a high rate to labeled palmitic acid, which was incorporated into various lipid classes ofP. ambiguus. Small but significant proportions of radioactivity from hexadecan-1-ol were incorporated into ether glycerolipids of the parasite. A more direct precursor in ether glycerolipid metabolism, i.e.,rac-1-O-[1′-¹⁴C] hexadecylglycerol, was incorporated into alkyl and 1′-alkenyl moieties of choline and etha-nolamine etherglycerophospholipids ofP. ambiguus in high yield. High proportions of labeled diacylglycerols, triacylglycerols and steryl esters were detected in surface lipids as well as lipid extracts of the culture media after incubation ofP. ambiguus with [1-¹⁴C]palmitic or [1-¹⁴C]oleic acids. The results suggest that palmitic acid and oleic acid are incorporated into neutral and polar lipids ofP. ambiguus maintained in glucose medium quite differently with oleic acid showing a strong preference for triacylglycerols. However, the incorporation of palmitic acid in glucose-fed parasites was similar to that of oleic acid in fasted parasites, as well as in larvae. This may be explained by partial fatty acid depletion in fasted worms and rapid cell division in larvae, respectively.     

Active Targeting of Dendritic Polyglycerols for Diagnostic Cancer Imaging

Active tumor targeting involves the decoration of nanomaterials (NMs) with oncotropic vector biomolecules that selectively recognize certain antigens on malignant cells or in the tumor microenvironment. This strategy can facilitate intracellular uptake of NM through specific interactions such as receptor‐mediated endocytosis and can lead to prolonged retention in the malignant tissues by preventing rapid efflux from the tumor. Here, the design of actively targeting, renally excretible bimodal dendritic polyglycerols (dPGs) for diagnostic cancer imaging is described. Single‐domain antibodies (sdAbs) specifically binding to the epidermal growth factor receptor (EGFR) are employed herein as targeting warheads owing to their small size and high affinity for their corresponding antigen. The dPGs equipped with EGFR‐targeting feature are compared head‐to‐head with their nontargeting counterparts in terms of interaction with EGFR‐overexpressing cells in vitro as well as accumulation at receptor‐positive tumors in vivo. Experimental results reveal a higher specificity and preferential tumor accumulation for the α‐EGFR dPGs, resulting from the introduction of active targeting capabilities on their backbone. These results highlight the potential for improving the tumor uptake properties of dPGs by strategic use of sdAb functionalization, which can ultimately prove useful to the development of ultrasmall NM with highly specific tumor accumulation.     

A lightweight privacy preserving SMS-based recommendation system for mobile users

In this paper, we propose a fully decentralized approach for recommending new contacts in the social network of mobile phone users. With respect to existing solutions, our approach is characterized by some distinguishing features. In particular, the application we propose does not assume any centralized coordination: It transparently collects and processes user information that is accessible in any mobile phone, such as the log of calls, the list of contacts or the inbox/outbox of short messages and exchanges it with other users. This information is used to recommend new friendships to other users. Furthermore, the information needed to perform recommendation is collected and exchanged between users in a privacy preserving way. Finally, information necessary to implement the application is exchanged transparently and opportunistically, by using the residual space in standard short messages occasionally exchanged between users. As a consequence, we do not ask users to change their habits in using SMS.     

Reengineering component-based software systems with Archimetrix

Many software development, planning, or analysis tasks require an up-to-date software architecture documentation. However, this documentation is often outdated, unavailable, or at least not available as a formal model which analysis tools could use. Reverse engineering methods try to fill this gap. However, as they process the system’s source code, they are easily misled by design deficiencies (e.g., violations of component encapsulation) which leaked into the code during the system’s evolution. Despite the high impact of design deficiencies on the quality of the resulting software architecture models, none of the surveyed related works is able to cope with them during the reverse engineering process. Therefore, we have developed the Archimetrix approach which semiautomatically recovers the system’s concrete architecture in a formal model while simultaneously detecting and removing design deficiencies. We have validated Archimetrix on a case study system and two implementation variants of the CoCoME benchmark system. Results show that the removal of relevant design deficiencies leads to an architecture model which more closely matches the system’s conceptual architecture.     

Plant Sterol-Poor Diet Is Associated with Pro-Inflammatory Lipid Mediators in the Murine Brain

Plant sterols (PSs) cannot be synthesized in mammals and are exclusively diet-derived. PSs cross the blood-brain barrier and may have anti-neuroinflammatory effects. Obesity is linked to lower intestinal uptake and blood levels of PSs, but its effects in terms of neuroinflammation—if any—remain unknown. We investigated the effect of high-fat diet-induced obesity on PSs in the brain and the effects of the PSs campesterol and β-sitosterol on in vitro microglia activation. Sterols (cholesterol, precursors, PSs) and polyunsaturated fatty acid-derived lipid mediators were measured in the food, blood, liver and brain of C57BL/6J mice. Under a PSs-poor high-fat diet, PSs levels decreased in the blood, liver and brain (>50%). This effect was reversible after 2 weeks upon changing back to a chow diet. Inflammatory thromboxane B2 and prostaglandin D2 were inversely correlated to campesterol and β-sitosterol levels in all brain regions. PSs content was determined post mortem in human cortex samples as well. In vitro, PSs accumulate in lipid rafts isolated from SIM-A9 microglia cell membranes. In summary, PSs levels in the blood, liver and brain were associated directly with PSs food content and inversely with BMI. PSs dampen pro-inflammatory lipid mediators in the brain. The identification of PSs in the human cortex in comparable concentration ranges implies the relevance of our findings for humans.     

Joint building ventures as a new instrument for urban development: a qualitative analysis of Baugruppen in Freiburg,Germany

Abstract

This article reviews a special type of collaborative housing that has emerged in the German housing market in response to the growing need for urban housing and increasing focus on active and networking housing communities and stable neighbourhoods. Joint building ventures are projects in which private individuals jointly establish residential property. However, the academic literature on this issue is underdeveloped. To foster a better understanding of the topic, this article focuses specifically on detecting the strengths and weaknesses of this type of joint building venture and its contribution to a higher homeownership rate. The qualitative interview data were collected between December 2015 and March 2016 in Freiburg, Germany. The findings reveal perceived advantages in terms of supportive networks, customised solutions and potential cost savings, whereas the identified disadvantages are high financial risks, mutual dependencies and personal efforts. Practical implications and avenues for future research are derived.     

Synthesis of Galactooligosaccharides in Milk and Whey: A Review

Galactooligosaccharides (GOS) are synthesized by the enzyme β‐galactosidase during the hydrolysis of lactose. In this so‐called transgalactosylation reaction the galactosyl moiety is transferred to another sugar molecule instead of water resulting in oligosaccharides of different chain lengths and glycosidic linkages. Because their structures are similar to oligosaccharides present in human breast milk, they act as prebiotics, which has been shown for infants and adults to be alike. While so far most of the research to maximize GOS yield has been carried out using buffered lactose solution as a starting material, more and more work is now conducted with dairy by‐products such as whey and whey permeate, or even milk, for direct GOS synthesis in order to develop new GOS‐enriched dairy products. This review aims to summarize the results obtained with various dairy liquids, and it rates their suitabilities to act as raw material for GOS production. Most of the studies using whey or milk have been carried out with enzymes from Aspergillus oryzae, Kluyveromyces lactis, Bacillus circulans, Streptococcus thermophilus, and several Lactobacillus species. As the initial lactose concentration (ILC) is known to be a crucial factor for high GOS yield, most of the research has been done with concentrated or supplemented milk and whey. However, a clear dependency on ILC could only be observed for the A. oryzae lactase, indicating a strong influence of milk components like minerals and proteins on the transfer activities of most enzymes.     

PMEL Amyloid Fibril Formation: The Bright Steps of Pigmentation

In pigment cells, melanin synthesis takes place in specialized organelles, called melanosomes. The biogenesis and maturation of melanosomes is initiated by an unpigmented step that takes place prior to the initiation of melanin synthesis and leads to the formation of luminal fibrils deriving from the pigment cell-specific pre-melanosomal protein (PMEL). In the lumen of melanosomes, PMEL fibrils optimize sequestration and condensation of the pigment melanin. Interestingly, PMEL fibrils have been described to adopt a typical amyloid-like structure. In contrast to pathological amyloids often associated with neurodegenerative diseases, PMEL fibrils represent an emergent category of physiological amyloids due to their beneficial cellular functions. The formation of PMEL fibrils within melanosomes is tightly regulated by diverse mechanisms, such as PMEL traffic, cleavage and sorting. These mechanisms revealed increasing analogies between the formation of physiological PMEL fibrils and pathological amyloid fibrils. In this review we summarize the known mechanisms of PMEL fibrillation and discuss how the recent understanding of physiological PMEL amyloid formation may help to shed light on processes involved in pathological amyloid formation.     

Enzyme-Assisted Extraction of Alginate from Beach Wrack Fucus vesiculosus

Beach wrack constitutes an unutilized, abundant renewable source of marine macro algae being rich in valuable biopolymers. This work investigates the revalorization of the brown algae Fucus vesiculosus collected from beach wrack as a source of alginate, the main polysaccharide in brown algae which constitutes a potential ingredient for the development of biopolymer films. Enzyme-assisted extraction has been investigated for cell disruption with commercial cellulase blends and proteases. The effect of enzyme type, enzyme activity, and extraction time on alginate yields, molecular weight distribution, functional groups, and purity has been studied.