Integrating domain similarity to improve protein complexes identification in TAP-MS data

Background

Detecting protein complexes in protein-protein interaction (PPI) networks plays an important role in improving our understanding of the dynamic of cellular organisation. However, protein interaction data generated by high-throughput experiments such as yeast-two-hybrid (Y2H) and tandem affinity-purification/mass-spectrometry (TAP-MS) are characterised by the presence of a significant number of false positives and false negatives. In recent years there has been a growing trend to incorporate diverse domain knowledge to support large-scale analysis of PPI networks.

Methods

This paper presents a new algorithm, by incorporating Gene Ontology (GO) based semantic similarities, to detect protein complexes from PPI networks generated by TAP-MS. By taking co-complex relations in TAP-MS data into account, TAP-MS PPI networks are modelled as bipartite graph, where bait proteins consist of one set of nodes and prey proteins are on the other. Similarities between pairs of bait proteins are computed by considering both the topological features and GO-driven semantic similarities. Bait proteins are then grouped in to sets of clusters based on their pair-wise similarities to produce a set of 'seed' clusters. An expansion process is applied to each 'seed' cluster to recruit prey proteins which are significantly associated with the same set of bait proteins. Thus, completely identified protein complexes are then obtained.

Results

The proposed algorithm has been applied to real TAP-MS PPI networks. Fifteen quality measures have been employed to evaluate the quality of generated protein complexes. Experimental results show that the proposed algorithm has greatly improved the accuracy of identifying complexes and outperformed several state-of-the-art clustering algorithms. Moreover, by incorporating semantic similarity, the proposed algorithm is more robust to noises in the networks.

     

Determination of carbamazepine in human urine and serum samples by high‐performance liquid chromatography with post‐column Ru(bipy)‐Ce(SO4)2 chemiluminescence detection

A novel, sensitive and rapid CL method coupled with high‐performance liquid chromatography separation for the determination of carbamazepine is described. The method was based on the fact that carbamazepine could significantly enhance the chemiluminescence of the reaction of cerium sulfate and tris(2,2‐bipyridyl) ruthenium(II) in the presence of acid. The chromatographic separation was performed on a Kromasil® (Sigma‐Aldrich) TM RP‐C18 column (id: 150 mm × 4.6 mm, particle size: 5 µm, pore size: 100 Å) with a mobile phase consisting of methanol–water‐glacial acetic acid (70:29:1, v/v/v) at a flowrate of 1.0 mL/min, the total analysis time was within 650 s. Under optimal conditions, CL intensity was linear for carbamazepine in the range 2.0 × 10^?8 ~ 4.0 × 10^?5 g/mL, with a detection limit of 6.0 × 10^?9 g/mL (S/N = 3) and the relative standard detection was 2.5% for 2.0 × 10^?6 g/mL (n = 11). This method was successfully applied to the analysis of carbamazepine in human urine and serum samples. The possible mechanism of the CL reaction is also discussed briefly. Copyright © 2012 John Wiley & Sons, Ltd.     

Synthesis and interaction of bovine serum albumin with p‐hydroxybenzoic acid derivatives

Three novel p‐hydroxybenzoic acid derivatives (HSOP, HSOX, HSCP) were synthesized from p‐hydroxybenzoic acid and sulfonamides (sulfamonomethoxine sodium, sulfamethoxazole and sulfachloropyridazine sodium) and characterized by elemental analysis, HNMR and MS. Interactions between derivatives and bovine serum albumin (BSA) were studied by fluorescence quenching spectra, UV–vis absorption spectra and time‐resolved fluorescence spectra. Based on fluorescence quenching calculation and Förster's non‐radioactive energy transfer theory, the values of the binding constants, basic thermodynamic parameters and binding distances were obtained. Experimental results indicated that the three derivatives had a strong ability to quench fluorescence from BSA and that the binding reactions of the derivatives with BSA were a static quenching process. Thermodynamic parameters showed that binding reactions were spontaneous and exothermic and hydrogen bond and van der Waals force were predominant intermolecular forces between the derivatives and BSA. Synchronous fluorescence spectra suggested that HSOX and HSCP had little effect on the microenvironment and conformation of BSA in the binding reactions but the microenvironments around tyrosine residues were disturbed and polarity around tyrosine residues increased in the presence of HSOP. Copyright © 2012 John Wiley & Sons, Ltd.     

Spectrofluorimetric determination of certain biologically active phenothiazines in commercial dosage forms and human plasma

A validated simple and sensitive spectrofluorimetric method was developed for the determination of chlorpromazine hydrochloride, promethazine hydrochloride, trifluperazine hydrochloride, thioridazine hydrochloride, perazine maleate and oxomemazine. The method was based on condensation of malonic acid/acetic anhydride (MAA) under the catalytic effect of the tertiary amine moiety of the studied phenothiazines to provide a deep yellow to brown colour with green florescence. Relative fluorescence intensity of the products was measured at λ_exc 398 nm and λ_em 432 nm. Different variables affecting the reaction were studied and optimized. The method was successfully applied for the determination of the studied drugs in commercial dosage forms. The lower detection limits allowed the application of this method for the determination of the compounds in plasma as an example of a biological fluid. In addition, the method was considered specific for the determination of tertiary amines in the presence of primary and secondary amines; as a result, it was deemed suitable for the determination of the cited drugs in the presence of their degradation products resulting from N‐dealkylation or oxidation of the corresponding sulphoxides or sulphones. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid methods of detecting the target molecule in immunohistology using a bioluminescence probe

We demonstrate a novel rapid direct detection method for immunohistochemistry, using a bioluminescent probe. An anti‐CEA antibody‐fused far‐red bioluminescent protein can monitor the accumulation of this type of probe in tumour tissues. The bimodal spectrum (λ_max = 460 and 675 nm) of this bioluminescent probe is extremely stable under different conditions of pH and ion concentration. The sensitivity of our bioluminescent labelling was at the same level of enzymatic labelling, e.g. peroxidase, as an indirect system. Our novel technique is simple and can shorten the pretreatment time of paraffin sections to around 30 min. The utility of our bioluminescent labelling covers all imaging in vitro, in vivo and ex vivo, suggesting that our antibody‐fused bioluminescent probe has the potential to detect tumour antigens with a high sensitivity in routine immune histological examinations. Copyright © 2012 John Wiley & Sons, Ltd.     

Optimization and validation of spectrofluorimetric method for determination of cefadroxile and cefuroxime sodium in pharmaceutical formulations

A simple, accurate, precise and validated spectrofluorimetric method is proposed for the determination of two cephalosporins, namely, cefadroxile (cefa) and cefuroxime sodium (cefu) in pharmaceutical formulations. The method is based on a reaction between cephalosporins with 1,2‐naphthoquinone‐4‐sulfonate in alkaline medium, to form fluorescent derivatives that are extracted with chloroform and subsequently measured at 610 and 605 nm after excitation at 470 and 460 nm for cefa and cefu respectively. The optimum experimental conditions have been studied. Beer's law is obeyed over the concentrations of 20–70 ng/mL and 15–40 ng/mL for cefa and cefu, respectively. The detection limits were 4.46 ng/mL and 3.02 ng/mL with a linear regression correlation coefficient of 0.9984 and 0.998, and recoveries ranging 97.50–109.96% and 95.73–98.89% for cefa and cefu, respectively. The effects of pH, temperature, reaction time, 1,2‐naphthoquinone‐4‐sulfonic concentration and extraction solvent on the determination of cefa and cefu, have been examined. The proposed method can be applied for the determination of cefa and cefu in pharmaceutical formulations in quality control laboratories. Copyright © 2013 John Wiley & Sons, Ltd.     

Development of a dual‐label time‐resolved fluoroimmunoassay for the detection of α‐fetoprotein and hepatitis B virus surface antigen

Time‐resolved fluorometry of lanthanide chelates is one of the most useful non‐isotopic detection techniques and has been used in numerous applications in biomedical science. We developed a time‐resolved fluoroimmunoassay (TRFIA) to quantify α‐fetoprotein (AFP) and hepatitis B virus surface antigen (HBsAg) in human serum. Based on a two‐site sandwich protocol, monoclonal antibodies (McAbs) against AFP and HBsAg were co‐coated in 96 microtitration wells and tracer McAbs against HBsAg and AFP were labeled with europium (Eu) and samarium (Sm) chelates, respectively. After application of diluted serum samples, Eu³⁺‐ and Sm³⁺‐McAbs were added and fluorescence signals of Sm³⁺ and Eu³⁺ tracers were collected. Detection limits of AFP and HBsAg were 0.09 mIU/L and 0.01 µg/L, respectively. Measurement ranges of AFP‐TRFIA and HBsAg‐TRFIA were 1–1000 mIU/L and 0.2‐150 µg/L, respectively. Intra‐ and inter‐assay coefficients of variation of AFP‐TRFIA were 3.3‐4.1% and 5.7‐7.2% and for HBsAg‐TRFIA were 2.9‐3.9% and 4.9‐6.8%, respectively. Linear correlation of TRFIA and chemiluminescence immunoassay measurements resulted in a correlation coefficient of 0.9949 for AFP and 0.9940 for HBsAg. For the endurance test, Eu‐labeled McAbs were stable for at least one year at ?20°C and the results of the TRFIA with the same reagents were also reproducible after one year. The availability of a highly sensitive, reliable and convenient AFP/HBsAg TRFIA will allow the quantification of both AFP and HBsAg, thereby providing diagnostic value in various clinical conditions and could be applied for clinical use. Copyright © 2012 John Wiley & Sons, Ltd.     

Spinal cord regeneration: Lessons for mammals from non‐mammalian vertebrates

Unlike mammals, regenerative model organisms such as amphibians and fish are capable of spinal cord regeneration after injury. Certain key differences between regenerative and nonregenerative organisms have been suggested as involved in promoting this process, such as the capacity for neurogenesis and axonal regeneration, which appear to be facilitated by favorable astroglial, inflammatory and immune responses. These traits provide a regenerative‐permissive environment that the mammalian spinal cord appears to be lacking. Evidence for the regenerative nonpermissive environment in mammals is given by the fact that they possess neural stem/progenitor cells, which transplanted into permissive environments are able to give rise to new neurons, whereas in the nonpermissive spinal cord they are unable to do so. We discuss the traits that are favorable for regeneration, comparing what happens in mammals with each regenerative organism, aiming to describe and identify the key differences that allow regeneration. This comparison should lead us toward finding how to promote regeneration in organisms that are unable to do so. genesis 51:529–544. © 2013 Wiley Periodicals, Inc.