A versatile new tool derived from a bacterial deubiquitylase to detect and purify ubiquitylated substrates and their interacting proteins

Protein ubiquitylation is an important posttranslational modification affecting a wide range of cellular processes. Due to the low abundance of ubiquitylated species in biological samples, considerable effort has been spent on methods to purify and detect ubiquitylated proteins. We have developed and characterized a novel tool for ubiquitin detection and purification based on OtUBD, a high-affinity ubiquitin-binding domain (UBD) derived from an Orientia tsutsugamushi deubiquitylase (DUB). We demonstrate that OtUBD can be used to purify both monoubiquitylated and polyubiquitylated substrates from yeast and human tissue culture samples and compare their performance with existing methods. Importantly, we found conditions for either selective purification of covalently ubiquitylated proteins or co-isolation of both ubiquitylated proteins and their interacting proteins. As proof of principle for these newly developed methods, we profiled the ubiquitylome and ubiquitin-associated proteome of the budding yeast Saccharomyces cerevisiae. Combining OtUBD affinity purification with quantitative proteomics, we identified potential substrates for the E3 ligases Bre1 and Pib1. OtUBD provides a versatile, efficient, and economical tool for ubiquitin research with specific advantages over certain other methods, such as in efficiently detecting monoubiquitylation or ubiquitin linkages to noncanonical sites.

This study presents OtUBD, a new tool derived from a bacterial deubiquitylase, for the purification and analysis of a broad range of endogenous ubiquitylated proteins, including monoubiquitylation, polyubiquitylation, non-lysine ubiquitylation and potentially other macromolecules.     

A novel yeast-based tool to detect mutagenic and recombinogenic effects simultaneously

In this work, we describe a new yeast-based assay to allow efficient detection of a comprehensive spectrum of genotoxicity events. The constructed diploid Saccharomyces cerevisiae strain allows the simultaneous monitoring of forward mutations, mitotic recombination events and chromosome loss or non-disjunction by direct selection in an easy and highly reproducible approach. The strain contains a DNA module consisting of a single functional copy of the URA3 gene and the kanMX4 gene inserted at the ADE2 locus on the right arm of chromosome XV. The changes of the genotype within the marker region were primarily selected on 5-fluoroorotic acid (5-FOA) agar plates. Further simple phenotypic tests of the 5-FOA-resistant ura3 clones make it possible to analyze the genetic configuration in detail (e.g. point mutations in URA3, gene conversion, crossing-over and chromosome loss). We demonstrate the successful application of our test system by studying the effects of well-known genotoxic agents (UV radiation, N-methyl-N'-nitro-N-nitrosoguanidine, aniline and benomyl). We found that the various agents induced mutations and recombination events with different relative frequencies. The integration of the module has generated a hot spot region of mutation and recombination at the borders of the artificially integrated URA3 kanMX4 cassette, which makes the system more sensitive towards DNA-damaging agents. Unlike other test systems, our S. cerevisiae strain is capable to detect a mutagenic effect caused by aniline.     

A computational tool to detect and avoid redundancy in selected reaction monitoring

Selected reaction monitoring (SRM), also called multiple reaction monitoring, has become an invaluable tool for targeted quantitative proteomic analyses, but its application can be compromised by nonoptimal selection of transitions. In particular, complex backgrounds may cause ambiguities in SRM measurement results because peptides with interfering transitions similar to those of the target peptide may be present in the sample. Here, we developed a computer program, the SRMCollider, that calculates nonredundant theoretical SRM assays, also known as unique ion signatures (UIS), for a given proteomic background. We show theoretically that UIS of three transitions suffice to conclusively identify 90% of all yeast peptides and 85% of all human peptides. Using predicted retention times, the SRMCollider also simulates time-scheduled SRM acquisition, which reduces the number of interferences to consider and leads to fewer transitions necessary to construct an assay. By integrating experimental fragment ion intensities from large scale proteome synthesis efforts (SRMAtlas) with the information content-based UIS, we combine two orthogonal approaches to create high quality SRM assays ready to be deployed. We provide a user friendly, open source implementation of an algorithm to calculate UIS of any order that can be accessed online at http://www.srmcollider.org to find interfering transitions. Finally, our tool can also simulate the specificity of novel data-independent MS acquisition methods in Q1-Q3 space. This allows us to predict parameters for these methods that deliver a specificity comparable with that of SRM. Using SRM interference information in addition to other sources of information can increase the confidence in an SRM measurement. We expect that the consideration of information content will become a standard step in SRM assay design and analysis, facilitated by the SRMCollider.     

A new method to detect cadmium uptake in protoplasts

The mechanism for cadmium (Cd²⁺) uptake into the cytosol of protoplasts from 5- to 7-day-old wheat seedlings (Triticum aestivum L. cv. Kadett) was investigated by a new method, using fluorescence microscopy and the heavy metal-specific fluorescent dye, 5-nitrobenzothiazole coumarin, BTC-5N. Cadmium fluorescence gradually increased in the cytosol of shoot and root protoplasts upon repeated additions of CdCl₂ to the external medium, reflecting an uptake of Cd²⁺. The uptake was inhibited by calcium and potassium chloride, as well as by Verapamil and tetraethylammonium (TEA), inhibitors of calcium and potassium channels, respectively. Calcium competitively inhibited the cadmium uptake. The metabolic inhibitors vanadate and dinitrophenol partly inhibited the uptake, suggesting it was dependent on membrane potential. The results indicate that cadmium is taken up by channels permeable to both calcium and potassium. The total uptake of cadmium into the protoplasts was also detected by unidirectional flux analyses using ¹⁰⁹Cd²⁺, and showed approximately the same maximal concentration of Cd²⁺ as the fluorescence measurements. By combining the two methods it is possible to detect both uptake into the cytosol and into the vacuole.Abbreviations BTC-5N, AM Acetoxymethyl ester of 5-nitrobenzothiazole coumarin - DNP 2,4-Dinitrophenol - TEA Tetraethylammonium     

SIC: A tool to detect short inverted segments in a biological sequence

The web software SIC provides a tool to search for short inverted segments (length 3-5000 bp) in a DNA sequence. The sequence is assumed to follow a Markov model. A statistic which is sensitive to inversion is presented. Searching inverted segments is done by a scanning approach after the user specifies the size of the scanning window and the order of the Markov chain. A list of the highest score segments is given with an assessment of the randomness of the result. SIC can be accessed via the URL: http://stat.genopole.cnrs.fr/SIC/.     

MetaMine – A tool to detect and analyse gene patterns in their environmental context

Background  

Modern sequencing technologies allow rapid sequencing and bioinformatic analysis of genomes and metagenomes. With every new sequencing project a vast number of new proteins become available with many genes remaining functionally unclassified based on evidences from sequence similarities alone. Extending similarity searches with gene pattern approaches, defined as genes sharing a distinct genomic neighbourhood, have shown to significantly improve the number of functional assignments. Further functional evidences can be gained by correlating these gene patterns with prevailing environmental parameters. MetaMine was developed to approach the large pool of unclassified proteins by searching for recurrent gene patterns across habitats based on key genes.     

A new effective assay to detect antimicrobial activity of filamentous fungi

The search for new antimicrobial compounds and the optimization of production methods turn the use of antimicrobial susceptibility tests a routine. The most frequently used methods are based on agar diffusion assays or on dilution in agar or broth. For filamentous fungi, the most common antimicrobial activity detection methods comprise the co-culture of two filamentous fungal strains or the use of fungal extracts to test against single-cell microorganisms. Here we report a rapid, effective and reproducible assay to detect fungal antimicrobial activity against single-cell microorganisms. This method allows an easy way of performing a fast antimicrobial screening of actively growing fungi directly against yeast. Because it makes use of an actively growing mycelium, this bioassay also provides a way for studying the production dynamics of antimicrobial compounds by filamentous fungi. The proposed assay is less time consuming and introduces the innovation of allowing the direct detection of fungal antimicrobial properties against single cell microorganisms without the prior isolation of the active substance(s). This is particularly useful when performing large screenings for fungal antimicrobial activity. With this bioassay, antimicrobial activity of Hypholoma fasciculare against yeast species was observed for the first time.     

Ultrasound, a new separation technique to harvest microalgae

In this article it is proven that ultrasound can be used to harvest microalgae. The separation process is based on gentle acoustically induced aggregation followed by enhanced sedimentation. In this paper, the efficiency of harvesting and the concentration factor of the ingoing biomass concentration are optimized and the relevance of this process compared to other harvesting processes is determined. For the optimisation, five parameters were modeled simultaneously by the use of an experimental design. An experimental design was chosen, because of possible interaction effects between the different parameters. The efficiency of the process was modeled with a R-squared of 0.88. The ingoing flow rate and the biomass concentration had a lot of influence on the efficiency of the process. Efficiencies higher than 90% were reached at high biomass concentrations and flow rates of 4–6 L day^–1. At most, 92% of the organisms could be harvested and a concentration factor of 11 could be achieved at these settings. It was not possible to harvest this microalga with higher efficiencies due to its small size and its small density difference with water. The concentration factor of the process was modeled with a R-squared of 0.75. The ingoing flow rate, biomass concentration and ratio between harvest flow and ingoing flow rate had a significant effect on the concentration factor. Highest concentration factors, up to 20, could be reached at low biomass concentrations and low harvest flows. On industrial scale, centrifuges can better be used to harvest microalgae, because of lower power consumption, better efficiencies and higher concentration factors. On lab- or pilot-plant scale, an ultrasonic harvesting process has the advantages that it can be operated continuously, it evokes no shear stress and the occupation space is very small. Also, when the algae excrete a soluble high valued product this system can be used as a biofilter.     

The use of machine learning to detect foraging behaviour in whale sharks: a new tool in conservation

In this study we present the first attempt at modelling the feeding behaviour of whale sharks using a machine learning analytical method. A total of eight sharks were monitored with tri-axial accelerometers and their foraging behaviours were visually observed. Our results highlight that the random forest model is a valid and robust approach to predict the feeding behaviour of the whale shark. In conclusion this novel approach exposes the practicality of this method to serve as a conservation tool and the capability it offers in monitoring potential disturbances of the species.     

Adaptation of the AFLP technique as a new tool to detect genetic instability and transposition in interspecific hybrids

An adapted amplified fragment length polymorphism (AFLP) protocol is presented for detection of hybrid instability in the genome of interspecific hybrids between Drosophila buzzatii and D. koepferae species. Analyses of 15 AFLP instability markers (new bands detected in hybrids) show that up to 81% are the result of transposable element (TE) activity. Twenty TEs associated with AFLP instability markers have been detected by this method in backcross hybrids and segmental hybrids, demonstrating its validity in detecting transposition events occurring during the hybridization process. New insertions of Helena TE have been observed in the hybrid genome after hybridization of the TGTCG22 instability marker by FISH. The AFLP marker technique proved to be an efficient method that improves upon traditional and bioinformatic tools previously used to detect TE mobilization. This newly adapted AFLP protocol may also be applied to a large number of organisms outside the Drosophila genus, making it of interest to evolutionary and population genetic researchers working with species where the knowledge of the genome is scarce.     

Evaluating new isolates of microalgae from Kazakhstan for biodiesel production

New microalgal strains that are native to South-East Kazakhstan were isolated and characterized with a view to identifying suitable candidates for biodiesel production. Six strains of chlorophyte algae (named K1–K6) were recovered from environmental samples as axenic cultures, and molecular analysis revealed that five (K1–K5) are strains of Parachlorella kessleri, whereas K6 is a strain of Chlorella vulgaris. A third isolate from Uzbekistan (termed UZ) was also identified as a separate strain of P. kessleri. All strains show high growth rates and an ability to utilize acetate as an exogenous source of fixed carbon. Furthermore, under conditions of nitrogen depletion, all three strains showed a significant accumulation of neutral lipids (triacylglycerides). P. kessleri K5 and C. vulgaris K6 therefore represent promising autochthon strains for large-scale cultivation and biodiesel production in Kazakhstan.     

A process for high yield and scaleable recovery of high purity eicosapentaenoic acid esters from microalgae and fish oil

A low expense process is developed for recovering esterified eicosapentaenoic acid (EPA) from microalgae and fish oil. Over 70% of the EPA content in the esterified crude extract of microalgae were recovered at purities exceeding 90%. The recovery scheme utilizes either wet or freeze-dried algal biomass. The process consists of only three main steps: 1) simultaneous extraction and transesterification of the algal biomass; 2) argentated silica gel column chromatography of the crude extract; and 3) removal of pigments by a second column chromatographic step. Argentated silica gel chromatography recovered about 70% of the EPA ester present in the crude fatty ester mixture of fish oil, but at a reduced purity ( approximately 83% pure) compared to the microalgal derived EPA. The optimal loading of the fatty ester mixture on the chromatographic support was about 3% (w/w) but loadings up to 4% did not affect the resolution significantly. The process was scaled up by a factor of nearly 320 by increasing the diameter of the chromatography columns. The elution velocity remained constant. Compared to the green alga Monodus subterraneus, the diatom Phaeodactylum tricornutum had important advantages as a potential commercial producer of EPA. For a microalgal EPA process to be competitive with fish oil derived EPA, P. tricornutum biomass (2.5% w/w EPA) needs to be obtained at less than $4/kg. If the EPA content in the alga are increased to 3.5%, the biomass may command a somewhat higher price. The quality of microalgal EPA compares favorably with that of the fish oil product. Compared to free fatty acid, EPA ester is more stable in storage. Shelf-life is extended by storing in hexane. The silver contamination in the final purified EPA was negligibly small (<210 ppb).     

BODIPY-modified 2'-deoxyguanosine as a novel tool to detect DNA damages

BODIPY-modified 2′-deoxyguanosine was synthesized for use as a detection reagent for genotoxic compounds. BODIPY-FL is a well known fluorescence reagent whose fluorescent light emission diminishes near a guanine base by a photo-induced electron transfer process. We attached BODIPY-Fl to the 5′ position of the deoxyribose moiety of 2′-deoxyguanosine. Although this compound has low fluorescence activity, when depurination by the action of alkylating reagents and dG oxidation by singlet oxygen occurred, the emission of strong fluorescence was observed. BODIPY-dG was found, therefore, to be a very useful tool for selectively detecting DNA damaging activity particularly in natural environmental extracts.     

一种检测新型生物高分子材料聚γ-谷氨酸的新方法

通过聚γ-谷氨酸（γ-PGA）与氯化十六烷基吡啶（CPC）的乙酸钠水溶液反应形成混悬液,采用分光光度计于波长680nm处比浊,研究γ-PGA浓度与吸收度之间的线性关系,并研究本方法测定γ-PGA的稳定性、重现性和回收率。在一定pH值和离子强度下,γ-PGA在12.5~50μg/ml范围内与CPC的乙酸钠水溶液生成的混悬液在波长680nm处的吸收度与其浓度呈线性关系,R2=0.9939.本方法在2h内吸收度保持稳定(RSD=0.154%,n=10 ),CPC法测定浓度为5,10和 40μg/ml时的平均回收率分别为86%,77%和99.75%,RSD分别为0.14%,0.23%和0.025%应用比浊法测定γ-PGA的含量方便、简洁、重现性好,可用于γ-PGA浓度的检测。     

ProteoMod: A new tool to quantitate protein post-translational modifications

Post-translational modifications (PTMs) are known to regulate biological processes by controlling protein function. The effect of a PTM on protein function depends critically on the position and the number of modifications. While there are convenient methods available to qualitatively examine modifications like phosphorylation, glycosylation, acetylation and methylation, methods available for their quantitative assessment are cumbersome. We have developed a new tool that allows quantitation of the number of phosphorylation events in proteins with ease. The "ProteoMod" tool depends on shifts in the isoelectric points of proteins upon post-translational change. The extent of shift exhibited upon phosphorylation is algorithmically converted into the number of phosphorylations conferred. The validity of ProteoMod was confirmed by examining proteins with previously known number of phosphorylations. The list of proteins examined included HSP27, HSP70 and tumor suppressor p53. The approach can also be applied to estimate modifications like acetylation, methylation and sialylation in proteins. We analyzed shifts in isoelectric points due to sialylation events in N-glycoproteins. Using influenza hemagglutinin we show that shifts in isoelectric points correlate with intracellular distribution of this model membrane protein. In addition to extending the application of two dimensional gel electrophoresis to quantitate modifications, our study also highlights its potential use in cell biology.     

A new tubular reactor for mass production of microalgae outdoors

A novel reactor for outdoor production of microalgae is described. Air-lift is used for circulation of the culture in transparent tubes lying on the ground and interconnected by a manifold. Dissolved O₂ is removed through a gas-separator placed 2.0 m above the tubes and water-spray is used for cooling. The manifold permits short-run durations between leaving the gas separator and re-entering it, preventing thereby damaging accumulation of dissolved oxygen. Day temperature control in summer is attained using water-spray. In winter, temperature in the tubes rises rapidly in the morning, as compared to an open raceway even if placed in a greenhouse. The number of hours along which optimal temperature prevails in the culture throughout the year increased significantly. Very high daily productivity computed on a volumetric basis (e.g. 550 mg dry wt l^–1 culture) was obtained and preliminary observations indicate that a significantly higher output, e.g. 1500 mg dry wt l^–1 d^–1 is attainable. Much more research is required to assess the year-round, sustained productivity attainable in this reactor.     

Aptamers as a sensitive tool to detect subtle modifications in therapeutic proteins

Therapeutic proteins are derived from complex expression/production systems, which can result in minor conformational changes due to preferential codon usage in different organisms, post-translational modifications, etc. Subtle conformational differences are often undetectable by bioanalytical methods but can sometimes profoundly impact the safety, efficacy and stability of products. Numerous bioanalytical methods exist to characterize the primary structure of proteins, post translational modifications; protein-substrate/protein/protein interactions and functional bioassays are available for most proteins that are developed as products. There are however few analytical techniques to detect changes in the tertiary structure of proteins suitable for use during drug development and quality control. For example, x-ray crystallography and NMR are impractical for routine use and do not capture the heterogeneity of the product. Conformation-sensitive antibodies can be used to map proteins. However the development of antibodies to represent sufficient epitopes can be challenging. Other limitations of antibodies include limited supply, high costs, heterogeneity and batch to batch variations in titer. Here we provide proof-of-principle that DNA aptamers to thrombin can be used as surrogate antibodies to characterize conformational changes. We show that aptamers can be used in assays using either an ELISA or a label-free platform to characterize different thrombin products. In addition we replicated a heat-treatment procedure that has previously been shown to not affect protein activity but can result in conformational changes that have serious adverse consequences. We demonstrate that a panel of aptamers (but not an antibody) can detect changes in the proteins even when specific activity is unaffected. Our results indicate a novel approach to monitor even small changes in the conformation of proteins which can be used in a routine drug-development and quality control setting. The technique can provide an early warning of structural changes during the manufacturing process that could have consequential outcomes downstream.