Quantification of sphingosine and sphinganine from crude lipid extracts by HPLC electrospray ionization tandem mass spectrometry

Sphingosine (SPH) comprises the backbone of sphingolipids and is known as a second messenger involved in the modulation of cell growth, differentiation, and apoptosis. The currently available methods for the quantification of SPH are, in part, complicated, time-consuming, insensitive, or unselective. Therefore, a fast and convenient methodology for the quantification of SPH and the biosynthetic intermediate sphinganine (SPA) was developed. The method is based on an HPLC separation coupled to electrospray ionization tandem mass spectrometry (MS/MS). Quantitation is achieved by the use of a constant concentration of a non-naturally occurring internal standard, 17-carbon chain SPH (C17-SPH), together with a calibration curve established by spiking different concentrations of naturally occurring sphingoid bases. SPH and SPA coeluted with C17-SPH, which allows an accurate correction of the analyte response. Interference of the SPH+2 isotope with SPA quantification was corrected by an experimentally determined factor. The limits of detection were 9 fmol for SPH and 21 fmol for SPA. The overall coefficients of variation were 8% and 13% for SPH and SPA, respectively. The developed HPLC-tandem mass spectrometry methodology, with an analysis time of 3.5 min, simple sample preparation, and automated data analysis, allows high-throughput quantification of sphingoid bases from crude lipid extracts and is a valuable tool for studies of cellular sphingolipid metabolism and signaling.     

Super pairwise alignment (SPA): an efficient approach to global alignment for homologous sequences.

Sequence analysis is the basis of bioinformatics, while sequence alignment is a fundamental task for sequence analysis. The widely used alignment algorithm, Dynamic Programming, though generating optimal alignment, takes too much time due to its high computation complexity O(N(2)). In order to reduce computation complexity without sacrificing too much accuracy, we have developed a new approach to align two homologous sequences. The new approach presented here, adopting our novel algorithm which combines the methods of probabilistic and combinatorial analysis, reduces the computation complexity to as low as O(N). The computation speed by our program is at least 15 times faster than traditional pairwise alignment algorithms without a loss of much accuracy. We hence named the algorithm Super Pairwise Alignment (SPA). The pairwise alignment execution program based on SPA and the detailed results of the aligned sequences discussed in this article are available upon request.     

Recipe for revealing informative metabolites based on model population analysis

An important application of metabolic profiles is to discover informative metabolites/biomarkers which are predictive of a clinical outcome under investigation. Therefore, there is a need to develop statistically efficient method for screening such kind of metabolites from the candidates. The most commonly used criteria to assess variable (metabolite) importance may be the P value obtained by performing t test on each metabolite alone, without considering the influence of other variables. In this work, a new strategy, called subwindow permutation analysis (SPA) coupled with partial least squares linear discriminant analysis (PLSLDA), is developed for statistical assessment of variable importance. The main contribution of SPA is that, unlike t test, it can output a conditional P value by implicitly taking into account the synergetic effect of all the other variables. In this sense, the conditional P value could to some extent help locate a good combination of informative variables. When applied to two metabolic datasets (type 2 diabetes mellitus data and childhood overweight data), it is shown that the performance of both the unsupervised principal component analysis (PCA) and the supervised PLSLDA are greatly improved when using the informative metabolites revealed by SPA. The source codes for implementing SPA in both MATLAB and R (R package for both Linux and Windows) are freely available at: .     

The SPA1-like proteins SPA3 and SPA4 repress photomorphogenesis in the light

Suppressor of phyA-105 (SPA1) is a phytochrome A-specific signaling intermediate that acts as a light-dependent repressor of photomorphogenesis in Arabidopsis seedlings. SPA1 is part of a small gene family comprising three genes: SPA1-related 2 (SPA2), SPA1-related 3 (SPA3), and SPA1-related 4 (SPA4). Here, we investigate the functions of SPA3 and SPA4, two very closely related genes coding for proteins with 74% identical amino acids. Seedlings with mutations in SPA3 or SPA4 exhibit enhanced photomorphogenesis in the light, but show no phenotype in darkness. While there are small differences between the effects of spa3 and spa4 mutations, it is apparent that SPA3 and SPA4 function to inhibit light responses in continuous far-red, red, and blue light. Phytochrome A is necessary for all aspects of the spa4 mutant phenotype, suggesting that SPA4, like SPA1, acts specifically in phytochrome A signaling. Enhanced photoresponsiveness of spa3 mutants is also fully dependent on phytochrome A in far-red and blue light, but not in red light. Hence, SPA3 function in red light may be dependent on other phytochromes in addition to phytochrome A. Using yeast two-hybrid and in vitro interaction assays, we further show that SPA3 as well as SPA4 can physically interact with the constitutive repressor of light signaling COP1. Deletion analyses suggest that SPA3 and SPA4, like SPA1, bind to the coiled-coil domain of COP1. Taken together, our results have identified two new loci coding for negative regulators that may be involved in fine tuning of light responses by interacting with COP1.     

Measuring substrate binding and affinity of purified membrane transport proteins using the scintillation proximity assay

The scintillation proximity assay (SPA) is a rapid radioligand binding assay. Upon binding of radioactively labeled ligands (here L-[(3)H]arginine or D-[(3)H]glucose) to acceptor proteins immobilized on fluoromicrospheres (containing the scintillant), a light signal is stimulated and measured. The application of SPA to purified, detergent-solubilized membrane transport proteins allows substrate-binding properties to be assessed (e.g., substrate specificity and affinity), usually within 1 d. Notably, the SPA makes it possible to study specific transporters without interference from other cellular components, such as endogenous transporters. Reconstitution of the target transporter into proteoliposomes is not required. The SPA procedure allows high sample throughput and simple sample handling without the need for washing or separation steps: components are mixed in one well and the signal is measured directly after incubation. Therefore, the SPA is an excellent tool for high-throughput screening experiments, e.g., to search for substrates and inhibitors, and it has also recently become an attractive tool for drug discovery.     

Scintillation proximity assay: A sensitive and continuous isotopic method for monitoring ligand/receptor and antigen/antibody interactions

Scintillation proximity assay (SPA) makes it possible to use radioisotopes for monitoring binding reactions continuously without the need to separate free from bound components. As a result SPA can be carried out more rapidly than most other methods used to monitor binding reactions. The methodology also lends itself to automation. The sensitivities already achieved with SPA procedures are comparable to the sensitivities of other procedures in use today. Another feature of SPA is that the key reagents (beads, ¹²⁵I labeling) are relatively inexpensive. The principles of SPA, utilizing ¹²⁵I-labeled molecules, are discussed and some applications to immunology, receptor binding, and measurement of potential across membranes are presented. SPA should also be applicable to monitoring interactions involving nucleic acids, lipids, and carbohydrates. Characteristics of some radionuclides, other than tritium and ¹²⁵I, that may be used in SPA are presented.     

Solid phase DNA amplification: a simple Monte Carlo Lattice model

Recently, a new way to amplify DNA, called solid phase amplification (SPA), has been introduced. SPA differs from the traditional polymerase chain reaction (PCR) in the use of surface-bound instead of freely-diffusing primers to amplify DNA. This limits the amplification to two-dimensional surfaces and therefore allows the easy parallelization of DNA amplification in a single system. Furthermore, SPA could provide an alternate route to DNA target implantation on DNA chips for genomic studies. Standard PCR processes are usually characterized (at least initially) by an exponential growth and a broad population distribution, and they are well described by the theory of branching processes, wherein a generating function can be used to obtain the probability distribution function for the population of offspring. This theoretical approach is not appropriate for SPA because it cannot properly take into account the many-body (steric) and geometric effects in a quenched two-dimensional environment. In this article, we propose a simple Lattice Monte Carlo technique to model SPA. We study the growth, stability, and morphology of isolated DNA colonies under various conditions. Our results indicate that, in most cases, SPA is characterized by a geometric growth and a rather sharp size distribution. Various non-ideal effects are studied, and we demonstrate that such effects do not generally change the nature of the process, except in extreme cases.     

Development of a scintillation proximity assay for histone deacetylase using a biotinylated peptide derived from histone-H4

Measurement of histone deacetylase activity is usually accomplished by incubation of the enzyme(s) with acetate-radiolabeled histones or synthetic peptides based on histone sequences, followed by extraction and quantification of released radiolabeled acetic acid. Consequently, this assay is both time consuming and extremely limiting when large numbers of samples are involved. We have now developed a simple, two-step histone deacetylase assay that is based on the scintillation proximity assay (SPA) principle. A biotinylated [3H]acetyl histone H4 peptide substrate was synthesized and shown to generate a radioactive signal upon binding to streptavidin-coated SPA beads. Incubation of biotinylated [3H]acetyl peptide with HeLa nuclear extract (source of histone deacetylase) resulted in a time- and protein-dependent decrease in the SPA signal, providing a measure of enzyme activity. The histone deacetylase-mediated decrease in SPA counts was accompanied by a proportional appearance in free 3H-labeled acetate in the assay mixture. Histone deacetylase activity measured by SPA was concordant with that determined via the traditional ethyl acetate extraction procedure. Furthermore, a broad range of histone deacetylase inhibitors was demonstrated to have comparable effects on the catalytic activity of the HeLa nuclei enzyme using both assays. The histone deacetylase SPA system described here should be readily applicable for automated high-throughput screening and therefore facilitate the discovery of new inhibitors of histone deacetylases.     

Recent advances and current trends in cryo-electron microscopy

All steps of cryogenic electron-microscopy (cryo-EM) workflows have rapidly evolved over the last decade. Advances in both single-particle analysis (SPA) cryo-EM and cryo-electron tomography (cryo-ET) have facilitated the determination of high-resolution biomolecular structures that are not tractable with other methods. However, challenges remain. For SPA, these include improved resolution in an additional dimension: time. For cryo-ET, these include accessing difficult-to-image areas of a cell and finding rare molecules. Finally, there is a need for automated and faster workflows, as many projects are limited by throughput. Here, we review current developments in SPA cryo-EM and cryo-ET that push these boundaries. Collectively, these advances are poised to propel our spatial and temporal understanding of macromolecular processes.     

Cryo-Electron Microscopy: The field of 1,000+ methods

Cryo-Electron Microscopy (CryoEM) is currently a well-established method to elucidate a biological macromolecule’s three-dimensional (3D) structure. Its success is due to technological and methodological advances in several fronts: sample preparation, electron optics and detection, image acquisition, image processing, and map interpretation. The first methods started in the late 1960s and, since then, new methods on all fronts have continuously been published, maturating the field as we know it now.In terms of publications, we can distinguish several periods, witnessing a substantial acceleration of methodological publications in recent years, pointing out to an increased interest in the domain. On the other hand, this accelerated increase of methods development may confuse practitioners about which method they should be using (and how) and highlight the importance of paying attention to establishing best practices for methods reporting and usage.In this paper, we analyze the trends identified in over 1,000 methodological papers. Our focus is primarily on computational image processing methods. However, our list also covers some aspects of sample preparation and image acquisition.Several interesting ideas stem out from this study: (1) Single Particle Analysis (SPA) has largely accelerated in the last decade and sample preparation methods in the last five years; (2) Electron Tomography is not yet in a rapidly growing phase, but it is foreseeable that it will soon be; (3) the work horses of SPA are 3D classification, 3D reconstruction, and 3D alignment, and there have been many papers on these topics, which are not considered to be solved yet, but ever improving; and (4) since the resolution revolution, atomic modelling has also caught on as a hot topic.     

Light-induced degradation of SPA2 via its N-terminal kinase domain is required for photomorphogenesis

Arabidopsis (Arabidopsis thaliana) CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and members of the SUPPRESSOR OF PHYTOCHROMEA-105 (SPA) protein family form an E3 ubiquitin ligase that suppresses light signaling in darkness by polyubiquitinating positive regulators of the light response. COP1/SPA is inactivated by light to allow photomorphogenesis to proceed. Mechanisms of inactivation include light-induced degradation of SPA1 and, in particular, SPA2, corresponding to a particularly efficient inactivation of COP1/SPA2 by light. Here, we show that SPA3 and SPA4 proteins are stable in the light, indicating that light-induced destabilization is specific to SPA1 and SPA2, possibly related to the predominant function of SPA1 and SPA2 in dark-grown etiolating seedlings. SPA2 degradation involves cullin and the COP10-DEETIOLATED-DAMAGED-DNA BINDING PROTEIN (DDB1) CDD complex, besides COP1. Consistent with this finding, light-induced SPA2 degradation required the DDB1-interacting Trp-Asp (WD)-repeat domain of SPA2. Deletion of the N-terminus of SPA2 containing the kinase domain led to strong stabilization of SPA2 in darkness and fully abolished light-induced degradation of SPA2. This prevented seedling de-etiolation even in very strong far-red and blue light and reduced de-etiolation in red light, indicating destabilization of SPA2 through its N-terminal domain is essential for light response. SPA2 is exclusively destabilized by phytochrome A in far-red and blue light. However, deletion of the N-terminal domain of SPA2 did not abolish SPA2-phytochrome A interaction in yeast nor in vivo. Our domain mapping suggests there are two SPA2-phytochrome A interacting domains, the N-terminal domain and the WD-repeat domain. Conferring a light-induced SPA2-phyA interaction only via the WD-repeat domain may thus not lead to COP1/SPA2 inactivation.

Light inactivates the COP1/SPA2 repressor of photomorphogenesis through cullin- and CDD-mediated degradation of SPA2, whereas the family members SPA3 and SPA4 are stable in the light.     

A homogeneous method to measure aminoacyl-tRNA synthetase aminoacylation activity using scintillation proximity assay technology

A new method to measure the aminoacylation of tRNA based upon the use of the scintillation proximity assay (SPA) technology has been developed. The assay detects incorporation of radiolabeled amino acids into cognate tRNA, catalyzed by a specific aminoacyl-tRNA synthetase (aaRS). Under acidic conditions, uncoated yttrium silicate SPA beads were found to bind tRNA aggregates, while the radiolabeled amino acid substrate remains in solution, resulting in good signal discrimination of these two species in the absence of any separation steps. The usefulness of this approach was demonstrated by measurement of steady-state kinetic constants and inhibitor binding constants for a range of aaRS enzymes in comparison with data from standard, trichloroacetic acid-precipitation-based assays. In all cases, the data were quantitatively comparable. Although the radioisotopic counting efficiency of the SPA method was less than that of standard liquid scintillation counting, the statistical performance (i.e., signal to background, variability, stability) of the SPA assays was at least equivalent to the separation-based methods. The assay was also shown to work well in miniaturized 384-well microtiter plate formats, resulting in considerable reagent savings. In summary, a new method to characterize aaRS activity is described that is faster and more amenable to high-throughput screening than traditional methods.     

葡萄球菌蛋白A免疫粘附法检测单纯疤疹病毒抗原

本文用葡萄球菌蛋白A(Staphylococcal Protein A,简称SPA)免疫粘附法快速检出单纯疱疹病毒抗原。试验观察了正常人角膜上皮细胞、兔角膜上皮细胞及来感染的传代细胞(Vero细胞等)对SPA的反应。同时,对单纯疱疹病毒Ⅰ型(HSV-1)实验感染性角膜炎的家兔,用SPA免疫粘附法与病毒分离培养法做了对照观察,并对临床诊断为单纯疱疹病毒性角膜炎的患者做了初步观察。结果表明,SPA免疫粘附法特异、简便,适合基层实验室应用。     

A scintillation proximity assay for human interleukin-5 (hIL-5) high-affinity binding in insect cells coexpressing hIL-5 receptor alpha and beta subunits

The high-affinity receptor for human interleukin-5 (hIL-5) is composed of alpha and beta subunits. A baculovirus expression system was established in Sf9 cells capable of expressing hIL-5 receptor alpha and beta subunits simultaneously. By using wheat germ agglutinin (WGA)-coated scintillation proximity assay (SPA) beads to capture 125I-labeled hIL-5-bound Sf9 cells, a SPA was developed and used to measure hIL-5 high-affinity binding. The hIL-5 receptors expressed in the Sf9 cells represented a single class of high-affinity binding sites with a dissociation constant (Kd) of 0. 24 nM and a density of 2.95 x 10(5) sites/cell. This is the first study in which the high-affinity Kd value similar to that for hIL-5 binding to human eosinophils was achieved using a recombinant expression system. The SPA compared favorably with the filter binding assay with regard to various binding parameters. We also found that several lectins, when coated on SPA beads, were even more effective than WGA-coated SPA beads for capturing the insect cells. We conclude that the baculovirus expression system was highly efficient in producing the high-affinity hIL-5 receptors and that the SPA was a simple and sensitive assay that could be readily adapted into a high-throughput screening format. The SPA described here could be a prototype for binding assays for other multimeric receptors.     

Methods of remote homology detection can be combined to increase coverage by 10% in the midnight zone

MOTIVATION: A recent development in sequence-based remote homologue detection is the introduction of profile-profile comparison methods. These are more powerful than previous technologies and can detect potentially homologous relationships missed by structural classifications such as CATH and SCOP. As structural classifications traditionally act as the gold standard of homology this poses a challenge in benchmarking them. RESULTS: We present a novel approach which allows an accurate benchmark of these methods against the CATH structural classification. We then apply this approach to assess the accuracy of a range of publicly available methods for remote homology detection including several profile-profile methods (COMPASS, HHSearch, PRC) from two perspectives. First, in distinguishing homologous domains from non-homologues and second, in annotating proteomes with structural domain families. PRC is shown to be the best method for distinguishing homologues. We show that SAM is the best practical method for annotating genomes, whilst using COMPASS for the most remote homologues would increase coverage. Finally, we introduce a simple approach to increase the sensitivity of remote homologue detection by up to 10%. This is achieved by combining multiple methods with a jury vote. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Collective Motion in a Network of Self-Propelled Agent Systems

Collective motions of animals that move towards the same direction is a conspicuous feature in nature. Such groups of animals are called a self-propelled agent (SPA) systems. Many studies have been focused on the synchronization of isolated SPA systems. In real scenarios, different SPA systems are coupled with each other forming a network of SPA systems. For example, a flock of birds and a school of fish show predator-prey relationships and different groups of birds may compete for food. In this work, we propose a general framework to study the collective motion of coupled self-propelled agent systems. Especially, we study how three different connections between SPA systems: symbiosis, predator-prey, and competition influence the synchronization of the network of SPA systems. We find that a network of SPA systems coupled with symbiosis relationship arrive at a complete synchronization as all its subsystems showing a complete synchronization; a network of SPA systems coupled by predator-prey relationship can not reach a complete synchronization and its subsystems converges to different synchronized directions; and the competitive relationship between SPA systems could increase the synchronization of each SPA systems, while the network of SPA systems coupled by competitive relationships shows an optimal synchronization for small coupling strength, indicating that small competition promotes the synchronization of the entire system.     

Vaccination against large Babesia species from dogs

The original observation of Sibinovic that soluble parasite antigens (SPA) of B. canis could be used to protect dogs against challenge infection formed the starting point for the development of an effective vaccine. With the advent of in vitro cultivation techniques for haemoprotozoan parasites an important tool became available for the commercial production of the vaccine antigens. A first generation vaccine was developed for dogs, but it appeared that the level of protection induced was not complete. In contrast to what was found with the SPA from serum/plasma of infected animals, protection induced with SPA from a single Babesia canis strain protected against a homologous challenge infection only. Further research led to the discovery that a combination of SPA of B. canis and SPA of B. rossi induced a broad spectrum of immunity. This improved vaccine, Nobivac Piro, not only induces protection against heterologous B. canis infection, but also against heterologous B. rossi infection.     

Src homology-2 domain binding assays by scintillation proximity and surface plasmon resonance

Various SH2 competitive binding assays, based on different techniques, have been described in the literature to identify and characterize SH2 ligands. The consideration that most reported methods show experimental limitations associated with assay parameters has prompted us to base our Src-SH2 inhibitor discovery program on the use of two different assays. In this study, two conceptually different biochemical methods designed to discover Src-SH2 inhibitors, respectively scintillation proximity assay (SPA) and surface plasmon resonance (SPR), have been evaluated and compared. For its high sensitivity and adaptability to automation SPA was chosen for high capacity screening (primary screen), whereas SPR was used for hits confirmation (secondary screening). However with the drastic improvement of inhibitor affinities, the limit of sensitivity was rapidly reached for the SPR assay based on the canonical pYEEI ligand. The substitution of the natural, monophosphorylated peptide ligand with a triphosphorylated peptide has allowed us to remarkably increase its sensitivity, so that molecules with nanomolar affinities could be easily differentiated in terms of IC(50) ranking. Such a new, improved SPR assay can be of great interest for the study of high affinity ligands of different SH2-based drug targets.     

A scintillation proximity assay amenable for screening and characterization of DNA gyrase B subunit inhibitors.

DNA gyrase is the target of coumarin and cyclothialidine antibacterials, which bind to the B subunit of the enzyme (GyrB). Currently available GyrB inhibitors have not been clinically successful, but their high in vitro potency against DNA gyrase has raised interest in the development of novel noncoumarin antibacterials acting at the same site. We report the development of a simple scintillation proximity assay (SPA) for the study of binding interactions between coumarin or noncoumarin antibacterials and GyrB, which prevents the needs of separation steps and can be run in microtiter plate formats. The assay is based on the detection of the binding of a radioligand, [3H]dihydronovobiocin, to a biotin-labeled 43-kDa fragment of GyrB (biotin-GyrB43), which is captured by streptavidin-coated SPA beads. The typical assay was conducted in 96-well microtiter plates, with final concentration of 10 nM for biotin-GyrB43, 20 nM for [3H]dihydronovobiocin, and 33 microg of SPA beads/well. From saturation experiments, an equilibrium dissociation constant (K(d)) for dihydronovobiocin of 8.10 nM was found. Displacement studies gave 50% inhibitory concentrations (IC(50)) of 42, 64, and 11 nM for novobiocin, dihydronovobiocin, and the cyclothialidine analogue GR122222X, respectively, consistent with previous findings. The assay was found to be robust to dimethyl sulfoxide up to 5% (v/v) and can be used for high-throughput screens of large chemical collections in the search of novel DNA gyrase inhibitors.