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.     

Genetic and morphological variation in Viola suavis s.l. (Violaceae) in the western Balkan Peninsula: two endemic subspecies revealed

The Balkan Peninsula, with many endemic species, is known as one of the most important speciation and diversification centres in Europe. Here, we present a study of the western Balkan populations of the polymorphic European species, V. suavis s.l., which have been reported under the name V. adriatica, but their taxonomic status and position within the genus have remained uncertain. Viola suavis s.l. and nine close relatives sampled across Europe were subjected to molecular (sequencing of nuclear ribosomal internal transcribed spacers and amplified fragment length polymorphism), karyological and morphometric analyses. Our results revealed the presence of four allopatric, genetically and morphologically differentiated lineages within V. suavis s.l. in Europe, which are suggested here to be recognized at the subspecific rank. Populations from the western Balkans were segregated into two distinct entities: (1) those from north-western Croatia correspond to the previously recognized taxon, V. suavis subsp. adriatica and (2) those from southern Dalmatia (southern Croatia, southern Bosnia and Herzegovina, and south-western Montenegro) are described here as V. suavis subsp. austrodalmatica subsp. nov. The other two lineages of V. suavis s.l., which both harbour blue- and white-flowered morphotypes, occur in central and eastern Europe (V. suavis subsp. suavis) and in north-eastern Spain (plants provisionally treated as V. suavis ‘Spain’). The AFLP and morphological data indicate gene flow between the nominate subspecies and V. suavis subsp. adriatica in a few localities. The distribution of the two western Balkan subspecies is discussed and an identification key to the V. suavis subspecies in Europe is presented.     

HIBCH mutations can cause Leigh-like disease with combined deficiency of multiple mitochondrial respiratory chain enzymes and pyruvate dehydrogenase

Background

Deficiency of 3-hydroxy-isobutyryl-CoA hydrolase (HIBCH) caused by HIBCH mutations is a rare cerebral organic aciduria caused by disturbance of valine catabolism. Multiple mitochondrial respiratory chain (RC) enzyme deficiencies can arise from a number of mechanisms, including defective maintenance or expression of mitochondrial DNA. Impaired biosynthesis of iron-sulphur clusters and lipoic acid can lead to pyruvate dehydrogenase complex (PDHc) deficiency in addition to multiple RC deficiencies, known as the multiple mitochondrial dysfunctions syndrome.

Methods

Two brothers born to distantly related Pakistani parents presenting in early infancy with a progressive neurodegenerative disorder, associated with basal ganglia changes on brain magnetic resonance imaging, were investigated for suspected Leigh-like mitochondrial disease. The index case had deficiencies of multiple RC enzymes and PDHc in skeletal muscle and fibroblasts respectively, but these were normal in his younger brother. The observation of persistently elevated hydroxy-C4-carnitine levels in the younger brother led to suspicion of HIBCH deficiency, which was investigated by biochemical assay in cultured skin fibroblasts and molecular genetic analysis.

Results

Specific spectrophotometric enzyme assay revealed HIBCH activity to be below detectable limits in cultured skin fibroblasts from both brothers. Direct Sanger sequence analysis demonstrated a novel homozygous pathogenic missense mutation c.950G <A; p.Gly317Glu in the HIBCH gene, which segregated with infantile-onset neurodegeneration within the family.

Conclusions

HIBCH deficiency, a disorder of valine catabolism, is a novel cause of the multiple mitochondrial dysfunctions syndrome, and should be considered in the differential diagnosis of patients presenting with multiple RC deficiencies and/or pyruvate dehydrogenase deficiency.

     

Characterization of the Link between Ornithine,Arginine, Polyamine and Siderophore Metabolism in Aspergillus fumigatus

The opportunistic fungal pathogen Aspergillus fumigatus produces siderophores for uptake and storage of iron, which is essential for its virulence. The main precursor of siderophore biosynthesis (SB), ornithine, can be produced from glutamate in the mitochondria or by cytosolic hydrolysis of ornithine-derived arginine. Here, we studied the impact of mitochondrial versus cytosolic ornithine biosynthesis on SB by comparison of the arginine auxotrophic mutants ΔargEF and ΔargB, which lack and possess mitochondrial ornithine production, respectively. Deficiency in argEF (encoding acetylglutamate kinase and acetylglutamyl-phosphate-reductase), but not argB (encoding ornithine transcarbamoyl transferase) decreased (i) the cellular ornithine content, (ii) extra- and intracellular SB, (iii) growth under harsh iron starvation, (iv) resistance to the ornithine decarboxylase inhibitor eflornithine, and (v) virulence in the Galleria mellonella larvae model. These lines of evidence indicate that SB is mainly fueled by mitochondrial rather than cytosolic ornithine production and underline the role of SB in virulence. Ornithine content and SB of ΔargB increased with declining arginine supplementation indicating feedback-inhibition of mitochondrial ornithine biosynthesis by arginine. In contrast to SB, the arginine and polyamine contents were only mildly affected in ΔargEF, indicating prioritization of the latter two ornithine-consuming pathways over SB. These data highlight the metabolic differences between the two arginine auxotrophic mutants ΔargEF and ΔargB and demonstrate that supplementation of an auxotrophic mutant does not restore the wild type metabolism at the molecular level, a fact to be considered when working with auxotrophic mutants. Moreover, cross pathway control-mediating CpcA was found to influence the ornithine pool as well as biosynthesis of siderophores and polyamines.     

Fetal Eye Movements on Magnetic Resonance Imaging

Objectives

Eye movements are the physical expression of upper fetal brainstem function. Our aim was to identify and differentiate specific types of fetal eye movement patterns using dynamic MRI sequences. Their occurrence as well as the presence of conjugated eyeball motion and consistently parallel eyeball position was systematically analyzed.

Methods

Dynamic SSFP sequences were acquired in 72 singleton fetuses (17–40 GW, three age groups [17–23 GW, 24–32 GW, 33–40 GW]). Fetal eye movements were evaluated according to a modified classification originally published by Birnholz (1981): Type 0: no eye movements; Type I: single transient deviations; Type Ia: fast deviation, slower reposition; Type Ib: fast deviation, fast reposition; Type II: single prolonged eye movements; Type III: complex sequences; and Type IV: nystagmoid.

Results

In 95.8% of fetuses, the evaluation of eye movements was possible using MRI, with a mean acquisition time of 70 seconds. Due to head motion, 4.2% of the fetuses and 20.1% of all dynamic SSFP sequences were excluded.Eye movements were observed in 45 fetuses (65.2%). Significant differences between the age groups were found for Type I (p = 0.03), Type Ia (p = 0.031), and Type IV eye movements (p = 0.033). Consistently parallel bulbs were found in 27.3–45%.

Conclusions

In human fetuses, different eye movement patterns can be identified and described by MRI in utero. In addition to the originally classified eye movement patterns, a novel subtype has been observed, which apparently characterizes an important step in fetal brainstem development. We evaluated, for the first time, eyeball position in fetuses. Ultimately, the assessment of fetal eye movements by MRI yields the potential to identify early signs of brainstem dysfunction, as encountered in brain malformations such as Chiari II or molar tooth malformations.     

Multimorbidity in Adult Asylum Seekers: A First Overview

Principals

Over the last two decades, the total annual number of applications for asylum in the countries of the European Union has increased from 15,000 to more than 300,000 people. The aim of this study was to give a first overview on multimorbidity of adult asylum seekers.

Methods

Our retrospective Swiss single center data analysis examined multimorbidity of adult asylums seekers admitted to our ED between 1 January 2000 and 31 December 2012.

Results

A total of 3170 patients were eligible for the study; they were predominantly male (2392 male, 75.5% versus 778 female, 24.5). The median age of the patients was 28 years (range 28–82). The most common region of origin was Africa (1544, 48.7%), followed by the Middle East (736, 23.6%). 2144 (67.6%) of all patients were not multimorbid. A total of 1183 (37.7%) of our patients were multimorbid. The mean Charlson comorbidity index was 0.25 (SD 1.1, range 0–12). 634 (20%) of all patients sufferem from psychiatric diseases, followed by chronic medical conditions (12.6%, 399) and infectious diseases (4.7%, 150). Overall, 11% (349) of our patients presented as a direct consequence of prior violence. Patients from Sri Lanka/India most often suffered from addictions problems (50/240, 20.8%, p<0.0001). Infectious diseases were most frequent in patients from Africa (6.6%), followed by the Balkans and Eastern Europe/Russia (each 3.8%).

Conclusion

The health care problems of asylum seekers are manifold. More than 60% of the study population assessed in our study did not suffer from more than one disease. Nevertheless a significant percentage of asylum seekers is multimorbid and exhibits underlying psychiatric, infectious or chronic medical conditions despite their young age.     

MR Molecular Imaging of Prostate Cancer with a Small Molecular CLT1 Peptide Targeted Contrast Agent

Tumor extracellular matrix has abundance of cancer related proteins that can be used as biomarkers for cancer molecular imaging. In this work, we demonstrated effective MR cancer molecular imaging with a small molecular peptide targeted Gd-DOTA monoamide complex as a targeted MRI contrast agent specific to clotted plasma proteins in tumor stroma. We performed the experiment of evaluating the effectiveness of the agent for non-invasive detection of prostate tumor with MRI in a mouse orthotopic PC-3 prostate cancer model. The targeted contrast agent was effective to produce significant tumor contrast enhancement at a low dose of 0.03 mmol Gd/kg. The peptide targeted MRI contrast agent is promising for MR molecular imaging of prostate tumor.     

Chromatin-modifying complex component Nurf55/p55 associates with histones H3 and H4 and polycomb repressive complex 2 subunit Su(z)12 through partially overlapping binding sites

Drosophila Nurf55 is a component of different chromatin-modifying complexes, including the PRC2 (Polycomb repressive complex 2). Based on the 1.75-Å crystal structure of Nurf55 bound to histone H4 helix 1, we analyzed interactions of Nurf55 (Nurf55 or p55 in fly and RbAp48/46 in human) with the N-terminal tail of histone H3, the first helix of histone H4, and an N-terminal fragment of the PRC2 subunit Su(z)12 using isothermal calorimetry and pulldown experiments. Site-directed mutagenesis identified the binding site of histone H3 at the top of the Nurf55 WD40 propeller. Unmodified or K9me3- or K27me3-containing H3 peptides were bound with similar affinities, whereas the affinity for K4me3-containing H3 peptides was reduced. Helix 1 of histone H4 and Su(z)12 bound to the edge of the β-propeller using overlapping binding sites. Our results show similarities in the recognition of histone H4 and Su(z)12 and identify Nurf55 as a versatile interactor that simultaneously contacts multiple partners.     

Evaluation of a double centrifugation technique for the detection of Anoplocephala eggs in horse faeces

Faecal samples of 250 horses from farms with a known history of tapeworm infection were examined comparatively for cestode eggs using a double centrifugation/combined sedimentation-floatation technique. From each faecal sample, three 5?g and three 15?g subsamples were processed, each using either saturated NaCl solution, specific gravity (sp. g.) 1.2 [NaCl]; concentrated sugar solution, sp. g. 1.26 [sugar]; or concentrated ZnSO4 solution, sp. g. 1.3 [ZnSO4] for floatation. In total, faeces from 187 horses (?=?74.8%) tested 'positive' for Anoplocephala eggs. Percentages of samples testing 'positive' for Anoplocephala ova were: 57.2% for 5?g faeces/NaCl, 66% for 15?g faeces/NaCl, 66% for 5?g faeces/sugar, 72.8% for 15?g faeces/sugar, 55.6% for 5?g faeces/ZnSO4, and 61.2% for 15?g faeces/ZnSO4, respectively. Processing of 15?g faecal samples resulted in a significant (P?相似文献