Comparing and Combining Capillary Electrophoresis Electrospray Ionization Mass Spectrometry and Nano–Liquid Chromatography Electrospray Ionization Mass Spectrometry for the Characterization of Post-translationally Modified Histones

We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano–liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N^α-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example.Histones are the most intensively studied group of basic nuclear proteins and are of great importance with regard to the organization of chromatin structure and control of gene activity. They are highly conserved during evolution, binding to and condensing eukaryotic chromosomal DNA to form chromatin. The fundamental chromatin subunit is the nucleosome, in which 166 bp of DNA are wrapped around a core histone octamer and a further ∼40 bp constitute the linker between one nucleosome core and the next. The histone octamer contains two molecules of each of the core histones H2A, H2B, H3, and H4. A fifth type of histone, referred to as linker histone (H1, H5), binds to both the DNA on the outer surface of nucleosomes and the linker DNA.There are numerous microsequence variants of linker and core histones (except H4) differing only slightly in primary sequence. In rat testis, for example, six somatic H1 subtypes, designated as H1a, H1b, H1c, H1d, H1e, and H1.0, as well as germ cell specific subtypes (i.e. H1t, H1T2, and HILS1), have been identified (1–3). Under various biological conditions, all histone proteins, for both linker and core histones, are subjected to post-translational modifications, including phosphorylation, acetylation, methylation, ubiquitination, deamidation, glycosylation, and ADP-ribosylation, which have a great influence on the epigenetic control of gene expression (4–6). The multitude of histone proteins resulting from closely related sequence variants and post-translational modifications, as well as their highly basic nature combined with hydrophobic properties, provides a major analytical challenge in current proteomics research. Over the past several years, considerable efforts have been expended to develop methods to identify the specific sites of histone modifications. Mass spectrometry (MS) coupled to liquid chromatography (LC) is the dominant technique for their characterization (7–14). However, because histone proteins contain up to nearly 35% basic amino acids, the analysis of histone peptides is still problematic, as digestion with many commonly used enzymes (e.g. trypsin, Lys-C, etc.) causes the formation of many short and polar peptides that poorly interact with the reverse-phase (RP)¹ material and go undetected by conventional liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). To overcome this problem, chemical derivatization such as propionylation is often applied (15, 16).Capillary electrophoresis (CE) overcomes this disadvantage; this technique allows separations based on the mass-to-charge ratio of peptides and does not utilize their hydrophobic nature as a separation principle. The methods of electrophoresis and LC and their applicability for histone analysis have been reviewed in detail by Lindner (17). CE has proven to be a remarkably powerful method for separating individual histones and their modified forms based on their different electrophoretic mobilities. Using a bare fused silica capillary and hydroxypropylmethyl cellulose (HPMC) as a buffer additive in order to avoid undesired protein adsorption, different core and linker histones and their multiply phosphorylated and acetylated forms were successfully separated via capillary zone electrophoresis (CZE) (18–22). So far, no data have been published about the identification of histone modifications by means of capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS). LC is given preference over CE because of the difficulty of achieving on-line interfacing of CE with MS that allows stable electrospray processes without compromising the quality of separation or the detection sensitivity. However, CE-MS is a promising technique with constantly increasing importance, as documented by numerous articles (23–26).Various interfaces have been constructed to improve CESI-MS coupling (27, 28). Sheathflow interfaces are the most widely used, and although the drawback of having to dilute the analyte is inherent in this kind of interface, they offer stable electrophoretic separations and allow greater versatility in the choice of background electrolyte (BGE) and the range of flow rates (29–32). Sheathless interfaces have generated interest because no sheath liquid is added, which leads to enhanced detection sensitivity (33, 34). However, they have not been used frequently because of their limited robustness and lack of well-established interfaces and routine analysis protocols. The most widely used method for establishing the terminating electrical contact is coating the outer surface of the CE capillary tip with a conductive material (35–37). Unfortunately, the lifetimes of such coatings are generally very limited, as they suffer from deterioration under the influence of the high voltages applied.A recently published concept of a sheathless interface based on a separation capillary with a porous tip acting as a nanospray emitter overcomes these disadvantages (38). The capillary tip is etched using hydrofluoric acid until the capillary wall becomes so thin and porous that an electric contact can be established. The performance of this methodology, which combines the low-flow characteristics of CE with an integrated ESI source, is described in Refs. 39–41. Applications such as the analysis of intact proteins (42), protein–protein and protein–metal complexes (43), and ribosomal protein digests from E. coli (44) have been published. Method-inherent advantages of CESI-MS are highly efficient separations, low flow rates leading to reduced ion suppression, and greater sensitivity (40). In contrast to nano-LC, no column equilibration is needed, there are no gradient effects, and the instrumentation is less maintenance-intensive.Our group recently described important features of CESI-MS and reported the comparison of this method with LC-ESI-MS for the analysis of a 5% perchloric acid extraction of rat testis consisting mainly of different histone H1 subtypes (39). The performance of both techniques was evaluated regarding analysis time, protein sequence coverage, and number and molecular mass distribution of the identified peptides. The CESI-MS method provided shorter analysis times, narrower peaks yielding high signals, and the identification of a greater number of low molecular mass range peptides than LC-ESI-MS (39).In the current study, we investigated the analysis of post-translationally modified peptides, particularly phosphopeptides, obtained from endoproteinase Arg-C digested histones from rat testis; this organ contains the whole set of somatic and germ cell specific H1 histones, as well as numerous modified core histone proteins. CESI-MS and LC-ESI-MS were compared regarding the number and type of identified modified peptides. Without any pre-separation of the perchloric acid extraction, we found numerous known and novel modification sites in linker histones. In addition, immobilized metal-affinity chromatography (IMAC) experiments were utilized to enrich phosphopeptides prior to MS analysis. CESI-MS was also used for the rapid identification of post-translational modifications (PTMs) of rat testis core histones, which were pre-fractionated via RP-HPLC and digested with Arg-C. Using core histones from butyrate-treated mouse erythroleukemia cells, we further demonstrated that our method achieves excellent separations of intact histone subtypes and their multiply modified forms and enables the detection of the extent of PTMs in a fast and reproducible way. Our work represents the first detailed characterization of modified linker and core histone peptides and clearly demonstrates that CESI-MS is a promising alternative tool for epigenetic studies.     

Simulations of the Ion Current to a Probe in Plasma with Allowance for Ionization and Ion–Neutral Collisions: II. Cylindrical Probe

The ion current to a cylindrical probe is considered with allowance for volume ionization, ion–neutral collisions, and the ion orbital moment. A model based on the molecular dynamics method and applicable in a wide range of plasma parameters (r_p/λ_D= 0.01–100, r_i/λ_D= 0.002–200, ν_i/ω_0i= 0.01–0.05, and T_i/T_e = 0?0.01) is proposed A convenient representation of the dependence of the relative ion current density on the Langmuir coefficient β² and a technique for determining the plasma density from simulation results are offered.     

Nonorthogonal tight-binding model with H–C–N–O parameterisation

A parametric nonorthogonal tight-binding model (NTBM1) with the set of parameters for H–C–N–O systems is presented. This model compares well with widely used semi-empirical AM1 and PM3/PM7 models but contains less fitting parameters per atom. All NTBM1 parameters are derived based on a criterion of the best agreement between the calculated and experimental values of bond lengths, valence angles and binding energies for various H–C–N–O molecules. Results for more than 200 chemical compounds are reported. Parameters are currently available for hydrogen, carbon, nitrogen, oxygen atoms and corresponding interatomic interactions. The model has a good transferability and can be used for both relaxation of large molecular systems (e.g., high-molecular compounds or covalent cluster complexes) and long-timescale molecular dynamics simulation (e.g., modelling of thermal decomposition processes). The program package based on this model is available for download at no cost from http://ntbm.info.     

Invaders interfere with native parasite–host interactions

The introduction of species is of increasing concern as invaders often reduce the abundance of native species due to a variety of interactions like habitat engineering, predation and competition. A more subtle and not recognized effect of invaders on their recipient biota is their potential interference with native parasite–host interactions. Here, we experimentally demonstrate that two invasive molluscan filter-feeders of European coastal waters interfere with the transmission of free-living infective trematode larval stages and hereby mitigate the parasite burden of native mussels (Mytilus edulis). In laboratory mesocosm experiments, the presence of Pacific oysters (Crassostrea gigas) and American slipper limpets (Crepidula fornicata) reduced the parasite load in mussels by 65–77% and 89% in single and mixed species treatments, respectively. Both introduced species acted as decoys for the trematodes thus reducing the risk of hosts to become infected. This dilution effect was density-dependent with higher reductions at higher invader densities. Similar effects in a field experiment with artificial oyster beds suggest the observed dilution effect to be relevant in the field. As parasite infections have detrimental effects on the mussel hosts, the presence of the two invaders may elicit a beneficial effect on mussels. Our experiments indicate that introduced species alter native parasite–hosts systems thus extending the potential impacts of invaders beyond the usually perceived mechanisms.     

Metallic Slit–Loaded Ring Resonator–Based Plasmonic Demultiplexer with Large Crosstalk

Plasmonics - Photonics provides a key solution to limit the myriads of challenges offered by existing silicon technology. Here, we propose a simple 2-channel Plasmonic demultiplexer with...     

Solving the decentralised gathering problem with a reaction–diffusion–chemotaxis scheme

The decentralised gathering problem consists in grouping in a compact cluster agents that are initially randomly scattered. We propose a bio-inspired algorithm, the Reaction–Diffusion–Chemotaxis aggregation scheme, to group agents that have limited abilities. The agents and their environment are described with a stochastic model inspired by the aggregation of the Dictyostelium discoideum cellular slime mold. The environment is an active lattice, whose cells transmit information according to a reaction–diffusion mechanism. The agents are virtual amoebae; they trigger excitations randomly and move by following reaction–diffusion waves. We demonstrate that despite its simplicity, this model exhibits interesting properties of self-organisation and is efficient for gathering agents. Moreover, observations show that the system is robust to various perturbations, such as the presence of obstacles on the lattice or noise in the movements of the agents.     

Bayesian area–age–period–cohort model with carcinogenesis age effects in estimating cancer mortality

Objective: Area–age–period–cohort (AAPC) model has been widely used in studying the spatial and temporal pattern of disease incidence and mortality rates. However, lack of biological plausibility and ease of interpretability on temporal components especially for age effects are generally the weakness of AAPC models. We develop a Bayesian AAPC model where carcinogenesis age effect is incorporated to explain age effects from the underlying disease process. An autoregressive prior structure and an arbitrary linear constraint are used to solve the nonidentifiability issues. Methods: Two multistage carcinogenesis models are employed to derive the hazard functions to substitute the age effects in the AAPC models. The Iowa county-wide lung cancer mortality data are used for the model fitting and Deviance Information Criteria (DIC) is used for model comparison. Results: Our study shows that conventional AAPC model (DIC = 19,231.30), AAPC model with Armitage–Doll age effect (DIC = 19,233.00) and with two-stage clonal expansion (TSCE) age effect (DIC = 19,234.70) achieved the similar DIC values which indicated consistent model fitting among three models. The spatial pattern shows that the high spatial effects are clustered in the south of Iowa and also in largely populated areas. The lung cancer mortality rate is continuously declining by birth cohorts while increasing by the calendar period until 2000–2004. The age effects show an increasing pattern over time which can be easily explained by Armitage–Doll carcinogenesis model since we assume a log-linear relationship between age and hazard function. Conclusions: Our finding suggests that the proposed Bayesian AAPC model can be used to replace the conventional AAPC model without affecting model performance while providing a more biological sound approach from the underlining disease process.     

Modeling spatial–temporal operations with context-dependent associative memories

We organize our behavior and store structured information with many procedures that require the coding of spatial and temporal order in specific neural modules. In the simplest cases, spatial and temporal relations are condensed in prepositions like “below” and “above”, “behind” and “in front of”, or “before” and “after”, etc. Neural operators lie beneath these words, sharing some similarities with logical gates that compute spatial and temporal asymmetric relations. We show how these operators can be modeled by means of neural matrix memories acting on Kronecker tensor products of vectors. The complexity of these memories is further enhanced by their ability to store episodes unfolding in space and time. How does the brain scale up from the raw plasticity of contingent episodic memories to the apparent stable connectivity of large neural networks? We clarify this transition by analyzing a model that flexibly codes episodic spatial and temporal structures into contextual markers capable of linking different memory modules.     

Simulating Fluid–Solid Equilibrium with the Gibbs Ensemble

The Gibbs ensemble is employed to simulate fluid–solid equilibrium for a shifted-force Lennard-Jones system. This is achieved by generating an accurate canonical Helmholtz free-energy model of the (defect-free) solid phase. This free-energy model is easily generated, with accuracy limited only by finite-size effects, by a single isothermal–isobaric simulation at a pressure not too far from coexistence for which the chemical potential is known. We choose to illustrate this method at the known triple-point because the chemical potential is easily calculated from the coexisting gas. Alternatively, our methods can be used to locate fluid–solid coexistence and the triple-point of pure systems if the chemical potential of the solid phase can be efficiently calculated at a pressure not too far from the actual coexistence pressure. Efficient calculation of the chemical potential of solids would also enable the Gibbs ensemble simulation of bulk solid–solid equilibrium and the grand-canonical ensemble simulation of bulk solids.     

Dissecting intercellular signaling with mass spectrometry–based proteomics

Physiological functions depend on a coordinated interplay of numerous different cell types. Proteins serve as major signaling molecules between cells; however, their comprehensive investigation in physiologically relevant settings has remained challenging. Mass spectrometry (MS)–based shotgun proteomics is emerging as a powerful technology for the systematic analysis of protein-mediated intercellular signaling and regulated post-translational modifications. Here, we discuss recent advancements in cell biological, chemical, and biochemical MS-based approaches for the profiling of cellular messengers released by sending cells, receptors expressed on the cell surface, and their interactions. We highlight methods tailored toward the mapping of dynamic signal transduction mechanisms at cellular interfaces and approaches to dissect communication cell specifically in heterocellular systems. Thereby, MS-based proteomics contributes a unique systems biology perspective for the identification of intercellular signaling pathways deregulated in disease.     

A Lotka–Volterra competition model with seasonal succession

A complete classification for the global dynamics of a Lotka–Volterra two species competition model with seasonal succession is obtained via the stability analysis of equilibria and the theory of monotone dynamical systems. The effects of two death rates in the bad season and the proportion of the good season on the competition outcomes are also discussed.     

Interaction of Tetrahydrocortisol–Apolipoprotein A-I Complex with Eukaryotic DNA and with Single-Stranded Oligonucleotides

The complex formed by tetrahydrocortisol (THC) and apolipoprotein A-I (ApoAI) specifically interacts with eukaryotic DNA from rat liver. Taken together, physical and chemical data and the results of small-angle X-ray scattering analysis show that interaction of the THC–ApoAI complex with eukaryotic DNA results in deformation of the DNA double helix. Single-stranded fragments were demonstrated to cause deformation of the double helix. In this state DNA forms complexes with DNA-dependent RNA polymerase. This interaction is cooperative and of saturating type; up to six enzyme molecules bind with one DNA molecule. The putative site of complex binding with DNA is the sequence CC(GCC)_n found in many genes including the human ApoAI gene. An oligonucleotide of this type was synthesized. Its association constant (K _a) was 1.66·10⁶ M^–1. Substitution of THC with cortisol considerably decreases the K _a. We suggest that THC interacting with GC pairs of the binding site forms hydrogen bonds with cytosine, inducing rupture of the bonds within the complementary nucleic base pair.     

Lyapunov functions for Lotka–Volterra predator–prey models with optimal foraging behavior

 The theory of optimal foraging predicts abrupt changes in consumer behavior which lead to discontinuities in the functional response. Therefore population dynamical models with optimal foraging behavior can be appropriately described by differential equations with discontinuous right-hand sides. In this paper we analyze the behavior of three different Lotka–Volterra predator–prey systems with optimal foraging behavior. We examine a predator–prey model with alternative food, a two-patch model with mobile predators and resident prey, and a two-patch model with both predators and prey mobile. We show that in the studied examples, optimal foraging behavior changes the neutral stability intrinsic to Lotka–Volterra systems to the existence of a bounded global attractor. The analysis is based on the construction and use of appropriate Lyapunov functions for models described by discontinuous differential equations. Received: 23 March 1999     

Heterozygosity–behavior and heterozygosity–fitness correlations in a salamander with limited dispersal

Inbreeding and genetic drift can decrease genetic heterozygosity, and this low heterozygosity can depress fitness, resulting in heterozygosity–fitness correlations (HFCs). HFCs are typically small in magnitude, a result often attributed to power of the analyses. Animal behaviors often affect fitness and are often heritable to some degree. We hypothesized that heterozygosity influences behavior, which, in turn, potentially influences fitness. Specifically, in red-backed salamanders, Plethodon cinereus, which have limited dispersal and the potential for inbreeding, we tested whether heterozygosity, as estimated from six microsatellite loci, affected home range size, juvenile growth, and survival. We found that salamanders with higher heterozygosity had larger home ranges and grew faster, which is indicative of reproductive success. However, we found no effects of heterozygosity on survival. We conclude that, because activity in P. cinereus is tightly linked to food uptake and mass gain, heterozygosity influences growth via effects on foraging behavior. Future research should investigate how the relationship between heterozygosity, behavior, and fitness may be affected or mediated by endocrine or immune systems.     

Possible antioxidant role of SPA therapy with chlorine–sulphur–bicarbonate mineral water

The aim of our research was to analyze the antioxidant role and efficacy of thermal or salus per aquam (spa) therapy with chlorine-sulphur-bicarbonate mineral water. The study has been performed on 30 rats. The animals were randomized in three groups, each of them composed by ten animals, denominated A, B and C. The A group was the control group and was not subjected to any specific treatment (placebo); the B group has been treated with a standard cycle of hydropinics treatment with mineral water of Therme of Stabia in Castellammare (Naples, Italy) denominated STABIA; the C group was treated with a standard cycle of hydropinic treatment with mineral water of Therme of Stabia in Castellammare (Naples, Italy) denominated SULFUREA. After two weeks of treatment all the rats were sacrificed and blood was collected for the plasmatic determination of reactive oxygen species (ROS). The results demonstrated a significant (P < 0.05) reduction of ROS in B (374 Carr. U. +/-73) and C group (399 carr. U. +/-62) treated with mineral waters if compared with control group (571 + 69 Carr. U.). In conclusion this study suggests a possible antioxidant effect of chlorine-sulphur-bicarbonate spa hydropinic treatment with a consequent suitable intestinal physiology, with reduction of the functional and organic modifications that can lead to pathological disorders of the gastroenteric diseases in whose pathogenesis the oxidative stress can develop an important role.     

A host–microparasite model with a resistant host

The theory of heterozygote advantage is often used to explain the genetic variation found in natural populations. If a large population randomly mates and the various genotypes have the same growth and death rates, the evolution of the genotypes follows Hardy–Weinberg proportions and polymorphism results. When other environmental stresses, like predators, prey and diseases, are present, polymorphism may or may not occur depending on how the various genotypes are affected by the stress. In this paper, we use a basic host–microparasite model to demonstrate that polymorphism can occur even if one genotype suffers a higher death rate than the others in the absence of the parasite if the heterozygote has resistance or immunity to the parasite.     

Dynamics of variable-yield nutrient–phytoplankton–zooplankton models with nutrient recycling and self-shading

 Several nutrient–phytoplankton–zooplankton models with internal nutrient storage by phytoplankton are derived and analyzed. It is shown that there are thresholds beyond which the system is uniformly persistent. Variable-yield models with self-shading of phytoplankton are also considered. With respect to uniform persistence, our result demonstrates that the global dynamics of the system with shading are the same as those for which the self-shading mechanism is ignored. Received: 16 March 1999