Numerous features distinguish prokaryotes from eukaryotes, chief among which are the distinctive internal membrane systems of eukaryotic cells. These membrane systems form elaborate compartments and vesicular trafficking pathways, and sequester the chromatin within the nuclear envelope. The nuclear pore complex is the portal that specifically mediates macromolecular trafficking across the nuclear envelope. Although it is generally understood that these internal membrane systems evolved from specialized invaginations of the prokaryotic plasma membrane, it is not clear how the nuclear pore complex could have evolved from organisms with no analogous transport system. Here we use computational and biochemical methods to perform a structural analysis of the seven proteins comprising the yNup84/vNup107–160 subcomplex, a core building block of the nuclear pore complex. Our analysis indicates that all seven proteins contain either a β-propeller fold, an α-solenoid fold, or a distinctive arrangement of both, revealing close similarities between the structures comprising the yNup84/vNup107–160 subcomplex and those comprising the major types of vesicle coating complexes that maintain vesicular trafficking pathways. These similarities suggest a common evolutionary origin for nuclear pore complexes and coated vesicles in an early membrane-curving module that led to the formation of the internal membrane systems in modern eukaryotes. Structural similarities between the proteins of a nuclear pore subcomplex and proteins comprising vesicle coating complexes indicate a common origin for nuclear pore complexes and coated vesicles The ability to sharply curve membranes was a defining event in the evolution of early eukaryotes, allowing the formation of endomembrane systems . In mode(Saccharomyces) , which form pores between the inner and outer NE membranes by stabilizing a sharply curved section of connecting pore membrane. NPCs are approximately 50-MDa octagonally symmetric cylinders that function as the only known mediators of nucleocytoplasmic exchange; while permitting the free flow of small molecules, they restrict macromolecular trafficking to selected cargoes that are recognized by cognate transport factors. NPCs are found in all eukaryotic cells and are composed of a broadly conserved set of proteins, termed nups (reviewed in romyces)  and vertromyces) , there iThe yNup84/vNup107–160 subcomplex has a molecular weight of approximately 600 kDa and has been shown in yeast to be flexible , presentWe support our fold assignments using four considerations . First, Finally, we provide direct biochemical evidence in support of our fold assignments, using proteolytic mapping of domain boundaries and loop locations in the seven nups see . Tagged Arabidopsis) homologs. All seven nups from both human and Arabidopsis yielded identical domain fold assignments to their yeast counterparts  and plant  is as components either of coated vesicles or of the yNup84/vNup107–160 subcomplex. Thus, the clathrin heavy chain, a major component of clathrin-coated vesicles, appears remarkably similar in domain architecture  to both In clathrin-coated vesicles, clathrin is attached via its amino-terminal domain to an adaptin complex. There are four types of adaptin complexes, all made of two large subunits that wrap around two small subunits. The bulk of each large subunit is made of an α-solenoid trunk  Collins. SimilarOur analyses showed that the yNup84/vNup107–160 subcomplex and all three major classes of vesicle coating complexes can be linked together through their common architecture. As summarized in The common architecture of the yNup84/vNup107–160 subcomplex and all three major classes of vesicle-coating complexes suggests that all of these complexes have common function in curving membranes. There is, in fact, circumstantial evidence for a role of the yNup84/vNup107–160 subcomplex in the establishment and maintenance of pore membrane curvature. Members of this complex, when disrupted in yeast, cause the uniformly distributed NPCs to cluster into patches in the plane of the NE , suggestAs shown here, protein structure modeling is particularly useful in uncovering potential evolutionary and functional relationships that are refractory to classical approaches based on comparison of protein sequences alone. Our results show that clathrin/adaptin complexes, COPI complexes, COPII complexes, and the yNup84/vNup107–160 subcomplex all share a common molecular architecture. This commonality could have arisen by either convergent or divergent evolutionary pathways.In a convergent pathway, β-propeller and α-solenoid folds could have been independently utilized by both NPCs and vesicle-coating complexes at different stages of eukaryotic evolution. This possibility is supported by the high abundance of both fold types in eukaryotic genomes   and the In a divergent pathway, NPCs and vesicle-coating complexes share these folds because both complex types could have originated from a common ancestor. In this scenario, a single “protocoatomer” would have been the progenitor for numerous vesicle coating complexes, as well as the yNup84/vNup107–160 subcomplex. Several lines of evidence support this latter hypothesis. First, the most confident models of the yNup84/vNup107–160 subcomplex proteins are based on structures of coated vesicle proteins . Second,The lack of detectable sequence similarity between the proteins in the yNup84/vNup107–160 subcomplex and the coated vesicles is not surprising. Sequence comparisons of α-solenoid- and β-propeller-containing proteins suggest that these folds arose just before or around the time of the origin of eukaryotes, then rapidly duplicated and diversified . Both foThe current protocoatomer hypothesis posits that a simple coating module containing minimal copies of the two conserved folds evolved in protoeukaryotes as a mechanism to bend membranes into sharply curved sheets and invaginated tubules . The abiIn conclusion, we suggest that the progenitor of the NPC arose from a membrane-coating module that wrapped extensions of an early ER around the cell's chromatin. In this primitive NE, the coating modules would have originally formed the sharply curved membrane, creating large and freely permeable pores . These pOnly two domains in the seven nups have their folds assigned by sequence comparison to proteins of known structure . TherefoFor each of the seven yeast nups and representative homologs, potentially related known structures were detected by the mGenThreader  and FUGUThe matches obtained in the previous step provided an operational definition of a domain. They were either accepted or refined by manual and automated alignment. Manual realignment relied on sequence conservation and secondary structure predictions by PROF  and PSI-For each alignment, an all-atom model was built by comparative modeling based on satisfaction of spatial restraints as implemented in MODELLER .The fold assignment, alignment, and model building were repeated by varying the domain boundaries, target sequences for modeling, template structures, and their alignments. The aim was to improve model assessment by statistical potentials of ProsaII  and DFIRTo search for proteins that resemble the domain architecture of clathrin, we queried MODBASE , our rel2, 1 mM DTT, 1:500 protease inhibitor cocktail [#P-8340; Sigma, St. Louis, Missouri, United States]) were added to cells and homogenized at 4 °C with a Polytron (Kinematica, Littau-Luzerne, Switzerland). The cell lysate was clarified by centrifugation (2,000 g for 5 min at 4 °C). The magnetic beads were added to the extract to a ratio of about 8 × 109 beads per g of cells. After incubation for 1 h at 4 °C, the beads were magnetically recovered. The beads were washed, resuspended in 50 μl of reaction buffer (according to the manufacturer's specifications), and Asp-N  or Lys-C  proteinase was added to give a weight ratio of 1:200 of proteinase to the tagged nup. After incubation at different time points at 37 °C, bead aliquots were removed and washed, and tagged fragments were eluted with 0.5 M NH4OH containing 0.5 mM EDTA. The eluant was vacuum-dried, resuspended in SDS-PAGE sample buffer, and separated on a 4%–12% bis-Tris gel . Proteins were then either transferred electrophoretically to nitrocellulose or PVDF and probed with HRP-rabbit IgG , or analyzed by amino-terminal Edman sequencing  were conjugated to rabbit IgG  according to the manufacturer's instructions. Yeast cells carrying PrA-tagged versions of nups were grown and harvested as described previously . Cell pequencing .Figure S1The graphs plot the assessment score of the model (Melo Z-score)  versus t(87 KB DOC).Click here for additional data file.Figure S2http://salilab.org/damien/NPC/).The secondary structure predicted from sequence by PROF  and PSI-(47 KB DOC).Click here for additional data file.Protocol S1(42 KB DOC).Click here for additional data file.Table S1(491 KB DOC).Click here for additional data file.Table S2(101 KB DOC).Click here for additional data file.Table S3(115 KB DOC).Click here for additional data file.Table S4(132 KB DOC).Click here for additional data file.Table S5(124 KB DOC).Click here for additional data file.Table S6(93 KB DOC).Click here for additional data file.Table S7(144 KB DOC).Click here for additional data file.http://www.pir.uniprot.org) for proteins discussed in this paper are as follows. Yeast: ySeh1 (P53011), ySec13 (Q04491), yNup84 (P52891), yNup85 (P46673), yNup120 (P35729), yNup133 (P36161), and yNup145C (P49687). Human: vSec13 l (Q96EE3), vSec13R (P55735), vNup107 (P57740), vNup75 (Q9BW27), vNup160 (Q12769), vNup133 (Q8WUM0), and vNup96 (P52948).Uniprot  accessio


How does the brain convert sounds and phonemes into comprehensible speech? In the present magnetoencephalographic study we examined the hypothesis that the coherence of electromagnetic oscillatory activity within and across brain areas indicates neurophysiological processes linked to speech comprehension.Amplitude-modulated  auditory verbal and nonverbal stimuli served to drive steady-state oscillations in neural networks involved in speech comprehension. Stimuli were presented to 12 subjects in the following conditions (a) an incomprehensible string of words, (b) the same string of words after being introduced as a comprehensible sentence by proper articulation, and (c) nonverbal stimulations that included a 600-Hz tone, a scale, and a melody. Coherence, defined as correlated activation of magnetic steady state fields across brain areas and measured as simultaneous activation of current dipoles in source space (Minimum-Norm-Estimates), increased within left- temporal-posterior areas when the sound string was perceived as a comprehensible sentence. Intra-hemispheric coherence was larger within the left than the right hemisphere for the sentence (condition (b) relative to all other conditions), and tended to be larger within the right than the left hemisphere for nonverbal stimuli (condition (c), tone and melody relative to the other conditions), leading to a more pronounced hemispheric asymmetry for nonverbal than verbal material.We conclude that coherent neuronal network activity may index encoding of verbal information on the sentence level and can be used as a tool to investigate auditory speech comprehension. One key function of the cerebral cortex involves the integration of elements into a percept that separates them from the background. In this process, changes in cortical networks are formed and modified by experience through the simultaneous excitation of groups of neurons -3. TheseEvent-related brain responses, traditionally used in the study of cognitive processes, have been found to result from regional perturbations in ongoing brain activities in a self-organizing system rather than constituting a response set from an otherwise silent system. For example, Makeig and coworkers -7 showedOn a macroscopic level, oscillatory coupling between large neuronal populations can be examined by externally driving the nervous system using oscillatory stimulation and then measuring the regional coherence of the electromagnetic activity . Amplitucomprehension of speech was related to the activation of neuronal assemblies in the left hemisphere, then we should see increased coherence in this region with the recognition of a meaningful sentence as compared to an incomprehensible string of sounds. We further hypothesized that meaningful verbal stimuli should be processed differently from musical melodies. That is to say, verbal material should affect the coherence of electromagnetic signals more in the left than in the right hemisphere whereas listening to a nonverbal complement of a meaningful sentence like a melody will activate more right- than left-hemispheric neuronal networks and influence coherence patterns involving the right hemisphere. Given that language and music share components, we assumed only a relative dominance in the interconnection of networks toward left- or right-hemispheric activity.The present study investigated coherence patterns of the auditory evoked magnetic Steady-State-Field (SSF), specifically coherence among SSF-generators within and across hemispheres, as a measure of neural networks involved in speech comprehension. If, as we hypothesized, the The present study studied co-activated cortical networks involved in speech comprehension by using auditory steady-state (41.5-Hz amplitude modulated) stimuli and measuring the coherence of generator activity of the magnetic steady state response. Steady-state stimulus modulation were used for a sentence, which – following a German play-of-words – was first presented as an incomprehensible string of sounds, but became a comprehensible sentence after the sentence's meaning was explained to the subjects and was properly articulated. In addition to verbal stimuli, non-verbal stimuli were also studied which included a 600-Hz tone, a scale, and a melody-like combination of the scales' tones. The present analysis of SSF coherence in the source space (see methods) extended previous approaches , which eFigure MNE amplitudes were most pronounced in auditory areas of both hemispheres, with a varying degree of laterality. For the Laterality Index  = 3.72, p < 0.05, ε = 0.46): As illustrated in Fig. Intra-hemispheric Given that the major goal was to depict network signatures specifically involved in sentence comprehension, we applied an ANOVA to compare the coherence measure of the two verbal conditions. These were identical with respect to the physical stimulation, but differed in meaningful comprehension. For intra-hemispheric coherence a significant interaction involving CONDITION × HEMISPHERE × GRADIENT (F(1, 11)= 7.37, p < 0.05) reflected a relatively higher coherence in the left-posterior area after the string of words had been made comprehensible by explaining the sentence's meaning as opposed to the higher coherence in the right-posterior area for the incomprehensible word string. Profiles of intra- and inter-hemispheric coherence were similar, thereby resulting in similar statistical power for the CONDITION effect. This cannot be explained simply by a reduced signal-to-noise ratio in the verbal conditions, because normalized values show the same effect. We rather assume that increased laterality varies with decreased inter-hemispheric communication (coherence).Inter-hemispheric coherence between dipoles located in the left  and right  auditory cortex and the remaining dipole sites are characterized Fig.  by largeConsidering coherent activity, i.e., synchronized oscillations between spatially distributed maps, as the representation of a percept, we followed Makeig et al. ,7 who diWhereas Scott et al.  reportedFinally, there is the possibility that the order of stimulus presentations may have affected the results. While counterbalancing was not possible for the specific verbal stimulus condition (see methods), we would not have expected order effects to be large since similar temporal dynamics were not observed for the nonverbal conditions. However, an effect of time cannot be ruled out as steady state responses and their generator activity were largest for a simple 600-Hz tone which was presented first.SSF were larger for the nonverbal conditions  than for the verbal material, particularly in the right hemisphere. While right-hemispheric processing of tonal perception has frequently been reported -31, the Still, greater right- over left-hemispheric generator activity asymmetry was found in the nonverbal conditions and less asymmetry found in the verbal conditions. Moreover, intra-hemispheric coherence patterns showed distinct, hemisphere-specific patterns for verbal (more pronounced left-hemispheric) and nonverbal (more pronounced right-hemispheric coherence) processing. When lateralized coherence patterns were examined by a laterality index, the clearest left-hemispheric coherence focus emerged for the comprehensible sentence and the clearest right-hemispheric coherence focus emerged for the tone. While we had expected a melody induced dominant right-hemispheric activation, a more bilateral activation was found for the melody-like tone sequence. For the scale, there was a shift towards left-hemispheric asymmetry of coherence. An explanation for this finding might be that the 'melody' was constructed to include the tones of the scale which may have resulted in a melody-like tone sequence even though it did not resemble common melodies or songs. This processing of an unfamiliar 'melody' might have activated temporal (left) and spectral (right) processing, as suggested by ,29, resuIn sum, the present study demonstrates that the analysis of the synchronization of evoked magnetic steady-state fields in the source space can map neuronal networks (co-)activated during speech comprehension. Our techniques add spatial information to evidence on left-hemispheric areas involved in language processing, and support co-activation or synchronization within complex neuronal networks as a cortical substrate of integration in perception – like speech comprehension.Data of twelve German native speaking subjects  were included in the analysis. (From the 14 subjects, who had participated in the study, data from one subject had to be discarded because of frequent movement artifacts and from another one, who recognized the play-of-words, see below.) It was ascertained by interview that the subjects did not suffer from any language, audiological or neurological dysfunction. Right-handedness was assessed by a modified version of the Edinburgh handedness questionnaire  to be 97All stimuli were amplitude modulated at 41.5 Hz  with a modulation depth of 90%. Verbal stimuli consisted of words composing a sentence. Nonverbal stimuli consisted of tones forming a scale or a tune or a simple tone. A German play-on-words served as the template for the two verbal conditions. In the first case a sentence is spoken without spacing between words and without accents which creates an incomprehensible word string. The German sentence 'Mähn Äbte Heu? Heu mähn Äbte nie! Äbte mähn Gras' means in English 'Do abbots cut hay? Abbots never cut hay, abbots mow lawns'. If pronounced as a string 'MähnÄbteHeuHeumähnÄbtenieÄbtemähnGras' this utterance, due to a lack of non-phonetic context , sounds In each subject, the hearing threshold was assessed by presenting short 600 Hz beeps with ascending and descending intensity. For each subject and ear the mean hearing threshold was determined from the ascending and descending sequence.The three nonverbal conditions comprised of a 600 Hz sinusoidal tone, a descending major scale (C6 B5 A5 G5 F5 E5 D5 C5, 1034 – 517 Hz), and an arrangement of the same tones (C5 E5 G5 C6 A5 F5 D5 B5). All stimuli of all conditions were adjusted to the length of the sentence and lasted for 4419 ms (sample-rate of 16 kHz/16 bit, mono), and each of the five conditions comprised 15 repetitions that were separated by inter-stimulus intervals of 4419 ms. This long inter-stimulus interval allowed the same signal-to-noise ratio for the baseline and the stimulus conditions which should prevent habituation effects on the SSF. Stimuli were adjusted to have the same average loudness by normalizing to root-mean square (RMS) and were presented at 50 dB above the individually assessed hearing threshold balanced for both ears. During the experiment, which lasted about 45 minutes, the subject was seated in a supine position. Subjects were asked to listen carefully to the stimuli, while fixating a point at the ceiling of the chamber in order to avoid head and eye movements. They were further informed that they would be asked questions about the stimuli during the experimental session, and that they should reply by saying 'yes' or 'no'.th presentation, whether he/she understood the meaning of the speech (None of them could).All stimuli were presented in blocks with 15 repetitions. Conditions were separated by breaks of about 5 min each. For every subject the experimental session started with the 600-Hz sinus tone (15 repetitions, condition 1), followed by the word string (condition 2). After 5 repetitions, the subject was asked whether he/she understood what he/she was hearing and could reproduce the meaning of the speech. (None of the subjects could.) Subsequently, the stimulus presentation was continued, and the subject was asked again after the 10th and the 15Then, the experimenter entered the room and pronounced the sentence properly and slowly, so that its meaning became clear. Each subject was asked to reproduce the sentence, in order to ascertain that it was properly understood. After the experimenter had left the subject chamber, the experiment continued with condition 3, which comprised the identical physical stimulation as condition 2 differing only in that the subject now listened to the string of words knowing its meaning, Again, the subjects were asked after 5 repetitions, if they could reproduce the meaning of the sentence, which now they all could. Given that once the sentence's meaning is obvious, one can easily grasp the sentence, the sequence of condition 2 and 3 could not be reversed and thus, the sequence of presentation could not be randomized across subjects.Condition 4  and 5 (melody-like tone sequence) were arranged in a similar way, in that the subject was asked after 5 repetitions each, whether or not s/he perceived the sequence of tones as a melody. Eleven of the twelve subjects indicated that the tone sequence sounded like a melody and one was not sure about it. None of them perceived the scale or the simple tone as melodic.® 2500WH, 4D Neuroimaging, San Diego, USA) installed in a magnetically shielded room (Vaccumschmelze, Hanau, Germany). Data were recorded continuously with a sampling-rate of 1017.25 Hz and a 0.1–100 Hz band-pass filter. The electrooculogram (EOG) and the electrocardiogram (EKG) were recorded and stored together with the MEG-data for offline artifact control. Silver-silverchloride electrodes were placed on the outer canthi for the monitoring of horizontal eye movements, and above and below the right eye for vertical eye movements. EKG electrodes were placed on the right collarbone and below left costal arch.The magnetoencephalogram (MEG) was recorded with a 148-channel whole head system  and averaged across epochs separately for each condition (epoch-length: 8838 ms, 4419 ms pre-stimulus baseline). Epochs were visually inspected for EOG and EKG artifacts and epochs with magnetic fields greater than 5 pT were rejected. A minimum 13  epochs per subject were available for further analyses.The steady state field (SSF) in response to the 41.5-Hz amplitude modulated stimuli was extracted using a moving average procedure. A window of 5 cycles (120.5 ms) of the 41.5 Hz Steady-State signal was shifted 179 times cycle-by-cycle (24.5 ms) across averaged epochs (separately for the 4419-ms baseline and the 4419-ms stimulus duration, the moving average procedure starting 144.5 ms post stimulus). The resulting moving-average epoch was detrended. Figure The generators of the SSF were determined in the source space for each epoch using the minimum norm estimate, MNE -37 using2 = min). This solution is obtained by multiplying the pseudo-inverse of the leadfield matrix L with the data. Given the high number of sensors and the presence of noise, spatial regularization is performed with the factor λ. This algorithm allows sources to be omitted, if they do not contribute to the measured magnetic field. A priori information about the number or locations of cortical sources is not required. Following Hauk et al. [2, was determined as mean vector length of both tangentially orientated dipole-components across 5 cycles.The MNE is an inverse method reconstructing the primary current that underlies an extracranially recorded time-locked magnetic field. The procedure is based on the assumption that the data vector d, which contains the recorded magnetic activity at given sensor sites, can be described as the product of the leadfield matrix L, which specifies the sensor's sensitivity to the sources, the source current vector j  and a nok et al. ,36, who Co-activation of generators was evaluated by all possible pair-wise combinations of the MNE at all dipole locations, according to the algorithm (Matlab, Mathworks):Spectral coherence is a function of frequency with values between 0 and 1 that indicate how well the input x (in the present study MNE at dipole location x) corresponds to the output y (MNE at dipole location y) as a function of frequency (in the present study 41.5 Hz). This algorithm estimates the coherence of two vectors x and y by computing the ratio of the squared cross power spectra (Pxy), divided by the product of the power spectra for each vector (Pxx.*Pyy), where Pxy(f) is the cross power spectrum estimate, Pxx(f) is the power spectrum of the time series at location x, Pyy(f) is the power spectrum estimate of time series at location y and f is the frequency index.x -dpy). As a measure of co-activation or coherence, the first order coherence between a region of interest (ROI) covering 7 locations over Heschl's gyrus and all other 77 locations was determined.The vectors of 4495 points in length were subdivided into 8 overlapping segments of 613 points (603 ms), each of which was submitted to Hanning windowing. For each vector, the power spectra were obtained as the product of the Discrete Fourier Transforms and its complex conjugate, scaled by the number of points used for x and y, and averaged across segments . For croEffects of the five conditions on the distribution of MNE amplitudes and on the coherence measure were evaluated by means of repeated measurement analyses of variance (ANOVA) with the factors CONDITION, HEMISPHERE , and GRADIENT (comparing left- and right-anterior versus left- and right- posterior dipole-locations, excluding midline locations). For inspection of the hemispheric asymmetry of MNE, the ANOVA was performed on the Laterality Index (LI: left- minus right-hemispheric MNE divided by their sum, resulting in an index without units). For the evaluation of intra-hemispheric coherence, the first order coherence between the respective left- or right-hemispheric ROI and the other 34 locations of the respective hemisphere entered the ANOVA (comparing conditions), for the evaluation of inter-hemispheric coherence, coherence between the left-hemispheric ROI and all other 34 locations of the right hemisphere and between the right-hemispheric ROI and all other 34 locations of the left hemisphere was submitted to the ANOVA comparing conditions. A separate ANOVA of the two verbal conditions with the factors CONDITION, HEMISPHERE and GRADIENT probed the hypothesis of a change in coherence-topography induced by sentence comprehension. Where appropriate, significance levels are reported with Greenhouse-Geisser correction adjusted degrees of freedom. Interactions were verified by planned posthoc comparisons (two-tailed paired t-tests), and displayed in t-maps without additional alpha correction.ANOVA: Analysis of varianceEEG: ElectroencephalogramMEG: MagnetoencephalogramMNE: Minimum Norm EstimateRMS: Root-mean squareSSF: Steady-State- (magnetic) FieldSSR: Steady-State-ResponseMH developed the experimental design, carried out the experimental study and developed and accomplished the data analyses, BR supervised the study and composed the paper, AK advised and assisted the SS-design and SSF analysis, CW supervised the MEG measurements and advised the coherence analyses, TE provided the experimental idea, advised the experimental design, the MEG methods and analyses.


Certain conditions characterised by blood vessel occlusion or vascular spasm have been found to cluster together in epidemiological studies. However the biological causes for these associations remain controversial. This study used a classical twin design to examine whether these conditions are linked through shared environmental exposures or by a common underlying genetic propensity to vasospasm.We investigated the association between hypertension, migraine, Raynaud's phenomenon and coronary artery disease in twins from a national register. Phenotype status was determined using a questionnaire and the genetic and environmental association between phenotypes was estimated through variance components analysis.Responses were obtained from 2,204 individuals comprising 525 monozygotic and 577 dizygotic pairs. There was a significant genetic contribution to all four traits with heritabilities ranging from 0.34 to 0.64. Multivariate model-fitting demonstrated that a single common genetic factor underlies the four conditions.We have confirmed an association between hypertension, migraine, Raynaud's phenomenon and coronary artery disease, and shown that a single genetic factor underlies them. The demonstration of a shared genetic factor explains the association between them and adds weight to the theory of an inherited predisposition to vasospasm. A number of conditions characterised by blood vessel occlusion and/or vascular spasm have been found to be associated in both clinical and epidemiological studies. These include hypertension (HPT), Raynaud's phenomenon (RP), migraine (MIG) and coronary artery disease (CAD) -5. WhethSubjects for this study were twins enlisted with the St Thomas' UK Adult Twin Registry . These mClassification of the traits HPT, RP, MIG and CAD was based on standard, validated criteria where possible. HPT was classified by asking about a doctor's diagnosis of "high blood pressure" when not pregnant. Questions to determine presence of migraine were based on the UCSD Migraine Questionnaire   corrected to take into account the paired nature of the data. The odds ratios were adjusted for possible confounding by age, smoking and body mass index (BMI). Evidence for a genetic contribution to each trait was examined by estimating casewise concordance . This meWhere data suggested a genetic influence, a quantitative measure of genetic contribution was estimated using structural equation modelling (Mx software ). This sModeling was then extended to consider the genetic and environmental associations between all four variables simultaneously . Three m1) the Cholesky decomposition  the independent pathway model  the common pathway model  with that of the fullest model, the Cholesky. The AIC represents the balance between model fit and the number of parameters (parsimony), with lower values of AIC indicating the most suitable model. Parameter estimates from the most appropriate model were used to calculate the genetic and environmental correlation between variables.Complete data for analysis were available from 1,102 pairs of female twins, comprising 525 MZ and 577 DZ pairs. The mean (± SD) age of the twins was 48.5 (± 8.2) years. The prevalence of each trait is shown in Table Four of the six combinations of traits showed significant phenotypic association after adjusting for age, smoking and BMI  declare that they have no competing interests.FMKW analysed the data and drafted the manuscript. LFC designed the questionnaires, analysed the data and assisted with the manuscript. TDS collected the twins on the Twin Register and assisted with the manuscript. AJM conceived of the study, collected the twins on the Twin Register, analysed the data and co-drafted the manuscript.The pre-publication history for this paper can be accessed here:


Estrogens are known to regulate the proliferation of breast cancer cells and to modify their phenotypic properties. Identification of estrogen-regulated genes in human breast tumors is an essential step toward understanding the molecular mechanisms of estrogen action in cancer. To this end we generated and compared the Serial Analysis of Gene Expression (SAGE) profiles of 26 human breast carcinomas based on their estrogen receptor α (ER) status. Thus, producing a breast cancer SAGE database of almost 2.5 million tags, representing over 50,000 transcripts.Estrogen Responsive Elements (EREs) distributed on the promoter regions of 163 out of the 473 up-modulated genes in ERα (+) breast tumors. In brief, we observed predominantly up-regulation of cell growth related genes, DNA binding and transcription factor activity related genes based on Gene Ontology (GO) biological functional annotation. GO terms over-representation analysis showed a statistically significant enrichment of various transcript families including: metal ion binding related transcripts (p = 0.011), calcium ion binding related transcripts (p = 0.033) and steroid hormone receptor activity related transcripts (p = 0.031). SAGE data associated with ERα status was compared with reported information from breast cancer DNA microarrays studies. A significant proportion of ERα associated gene expression changes was validated by this cross-platform comparison. However, our SAGE study also identified novel sets of genes as highly expressed in ERα (+) invasive breast tumors not previously reported. These observations were further validated in an independent set of human breast tumors by means of real time RT-PCR.We identified 520 transcripts differentially expressed between ERα-positive (+) and ERα-negative (-) primary breast tumors . Furthermore, we identified 220 high-affinity The integration of the breast cancer comparative transcriptome analysis based on ERα status coupled to the genome-wide identification of high-affinity EREs and GO over-representation analysis, provide useful information for validation and discovery of signaling networks related to estrogen response in this malignancy. Estrogen plays essential roles in the development, growth control and differentiation of the normal mammary gland. However, it is well documented that endogenous estrogens are powerful mitogens critical for the initiation and progression of human breast and gynecological cancers . This ce2) is mediated by two main mechanisms. In one, the E2-ER complex binds to a specific DNA sequence called the estrogen response element (ERE), this receptor-ligand DNA bounded complex interacts with co-regulatory proteins, promoting chromatin remodeling and bridging with the general gene transcription machinery thus resulting in transcription initiation [2 also exerts rapid, non-genomic effects attributed to cell membrane-initiated signaling [Transcriptional regulation of target genes in response to 17β-estradiol  at the time of diagnosis and expression of this receptor is determinant of a tumor phenotype that is associated with hormone-responsiveness. Patients with tumors that express ERα have a longer disease-free interval and overall survival than patients with tumors that lack ERα expression . However2 induced proliferative response predominantly via the regulation of estradiol-responsive genes. Nevertheless, the expression of additional subsets of genes not necessarily directly regulated by estrogen may also be fundamental in defining the breast cancer hormone-responsive phenotype.Several studies have been carried out using cDNA and oligonucleotide microarrays identifying breast cancer subclasses possessing distinct biological and clinical properties -13. AmonTo further elucidate the molecular basis of estrogen-dependent breast carcinogenesis, we here report a comparative transcriptome profiling of invasive breast tumors based on ERα status obtained by SAGE. The SAGE method provides a statistical description of the mRNA population present in a cell without prior selection of the genes to be studied, and this constitutes a major advantage . The brei.e. ERα (+) vs. ERα (-) breast tumors.The primary goal of our study was to identify the most commonly deregulated genes in invasive breast carcinomas related to ERα status. To this end SAGE data was obtained from a set of primary breast carcinomas. Thus, a breast cancer SAGE database of almost 2.5 million tags was analyzed, representing over 50,000 tag species. We performed a comprehensive evaluation and comparison of gene expression profiles using a recently developed supervised method , to idenThis statistical analysis revealed 520 genes differentially expressed  between ERα (+) and ERα (-) primary breast carcinomas see . Among ttrefoil factor 1 (TFF1/pS2), synaptotagmin-like 4 (SYTL4), regulating synaptic membrane exocytosis 4 (RIMS4), dual specificity phosphatase 4 (DUSP4), chromosome 1 open reading frame 34 (C1orf34), necdin homolog (NDN), n-acetyltransferase 1 (NAT1) and caspase recruitment domain family 10 (CARD10) (Table The most commonly over-expressed transcripts in ERα (+) tumors were: 0) Table .signal peptide CUB domain EGF-like 2 (SCUBE2) (p = 0.0001), SYTL4 (p = 0.0005), KIAA0882 protein (p = 0.0005), tetraspan 1 (TSPAN1) (p = 0.001), myeloblastosis viral oncogene homolog (C-MYB) (p = 0.002), epidermal growth factor-like 2 (CELSR2) (p = 0.011), nuclear receptor subfamily 4 (NR4A1) (p = 0.029), and enolase 2 (ENO2) (p = 0.033)  (p = 0.079) transcript  breast tumors including: p = 0.000, SYTL4  encodes a secreted and cell-surface protein containing EGF and CUB domains that defines a novel gene family [SCUBE2 has been detected in vascular endothelium and may play important roles in development, inflammation and perhaps carcinogenesis [e family . The epie family . Expressogenesis .CELSR2 gene  encodes a protein member of the nonclassic-type cadherins (flamingo subfamily). These 7-pass transmembrane proteins have nine cadherin domains, seven-epidermal growth factor-like repeats and two laminin A G-type repeats [The  repeats . It is p repeats  playing  repeats ,23.SYTL4  contains an N-terminal Slp homology domain (SHD) than can specifically and directly bind the GTP-bound form of Rab27A, a small GTP-binding protein involved in granule exocytosis in cytotoxic T lymphocytes [SYTL4 is associated with ERα (+) tumors  encodes one of three enolase isoenzymes found in mammals. This isoenzyme was described to be expressed in cells of neuronal origin. Interestingly, in a recent report Hao et al. (2004) showed high expression of ENO2 transcripts in breast cancer lymph node metastases when compared with primary breast tumors [t tumors .TSPAN1 gene  encodes a cell-surface protein member of the transmembrane 4 superfamily (TM4SF), involved in the regulation of cell development, activation, growth and motility. A number of tetraspanins were described as tumor-specific antigens, and it was suggested that the function of some TM4SF proteins may be particularly relevant to tumor cell metastasis [TSPAN1 protein complexes may control the invasive migration of tumor cells and contribute to ECM-induced production of MMP2 in breast cancer cell line [The tastasis . Sugiuraell line .NR4A1, a nuclear receptor subfamily 4, group A gene  encodes an orphan member of the steroid-thyroid hormone-retinoid receptor superfamily whose members mainly act as transcriptional factors to positively or negatively regulate gene expression and play roles in regulating growth and apoptosis [NR4A1 in cell proliferation has been previously reported. It was shown that its expression is rapidly induced by various mitogenic stimuli such as: serum growth factor, epidermal growth factor and fibroblast growth factor [poptosis ,29. A roh factor .Taken together, the genes that we identified and validated appear to be involved in signaling pathways related to cell proliferation, invasion and metastatic processes, but their exact role in breast carcinogenesis remains to be elucidated.Expression Analysis Systematic Explorer software (EASE) [Classification of genes based on Gene Ontology (GO) terms is a powerful bioinformatics tool suited for the analysis of DNA microarray and SAGE data. Analysis of GO annotation allows one to identify families of genes that may play significant roles related to specific molecular or biological processes in expression profiles . We usede (EASE)  to annote (EASE) . The GO cell growth and/or maintenance, 21% are related to cell communication, and 16% are related to regulation of transcription. Approximately 16% of these deregulated genes are related to molecular functions associated with DNA binding and more specifically with transcription factor activity (10%) , calcium ion binding proteins  and steroid hormone receptor activity related proteins  (steroid hormone receptor activity proteins includes: estrogen receptor 1 (ESR1, i.e. ERα), androgen receptor (AR), hydroxysteroid 17-β dehydrogenase 4 (HSD17β4), glucocorticoid receptor (NR3C1), oxysterol binding protein (OSBP), and retinoic acid receptor α (RARA). The observation of functionally related groups of genes identified in the SAGE dataset via GO over representation analysis allows the identification of distinct biological pathways directly or indirectly associated to estrogen response related processes and provides the basis for future mechanistic studies.We observed that 31% of ERα associated transcripts are involved in biological processes related to = 0.031) . The GO et al. [We used a recently reported genome-wide high-affinity ERE database  to identet al. .The observed frequency of these elements in our study was 220 EREs in 3260 kb (considering a DNA window of 20 kb for each one of the 163 up-modulated genes with EREs). Compared with the expected frequency from random distribution of high-affinity EREs found in the genome (732 EREs in 3,069334 kb 0.8 ERE in 3260 kb)  [et al. [2-ER binding sites related to transcriptional activation.Fifty percent (110 out of 220) of the detected EREs mapped within a 10 kb region 5' of the TSS, while the rest mapped to 3' regions Figure . Approxiet al. . HoweverSYTL4 (-8384 bp from the TSS: tggacatcatgacct), TSPAN1 (+974 bp and +9384 bp from the TSS: tggtctgaatgaccc and aggtcatttccacct respectively), CELSR2 (+173 bp and +3607 bp from the TSS: tgctcagggtgaccc and aggtcaccatgaccg respectively), and NR4A1 (-3478 bp and +4217 bp from the TSS: tgttcactctgacct).Of the validated transcripts previously discussed Figure , we deteovalbumin  and c-fos ; two transcripts detected as over-expressed in ERα (+) breast tumors by SAGE (retinoic acid receptor α (RARA) , vascular endothelial growth factor (VEGFC) , insulin-like growth factor binding protein-4 (IGFBP4)  and heat shock protein 27 (HSPB1) ; four transcripts detected as over-expressed in ERα (+) tumors in our study (It is interesting to note that we were unable to identify high-affinity EREs on the majority of deregulated genes 65%) associated with a positive ER α status. The possibility exists that many of these genes are transcriptionally regulated by non-ERE mediated mechanisms such as those involving ER binding to the AP1 or SP1 transcription factors . The AP15% associ by SAGE . Similarur study .nuclear orphan receptors with significant homology to ERs, which do not bind estrogen and have unknown physiological ligands. ERRs are known to bind to the steroidogenic factor 1 response element (SFRE) and also bind to classic EREs, by means of which they exert constitutive transcriptional activity [NR4A1 by SAGE and subsequently validated this observation by real time RT-PCR , activity . We deteR Figure . Interes by ERRs .As described, ERα can mediate estrogenic response through multiple genomic and non-genomic mechanisms, many of which affect proteins and pathways not necessarily directly or exclusively associated with ERα. Thus it is worth stressing that it will the totality of deregulated proteins the ones that ultimately define the phenotype of ERα (+) breast carcinomas regardless of whether a "direct association" with ER transcriptional regulation exists or not.2-responsive genes in MCF-7 cell line, previously reported by us [et al, we observed a general up-regulation cell cycle progression-related genes including: CCT2, CCND1, PES1, RAN/TC4, CALM1, CALM2; and tumor-associated genes such as: RFP, D52L1, TFF1/PS2, CAV1, and NDKA among others [2-ER transcriptional regulation.The SAGE profiles for Eed by us , was comg others . These tin vitro  and in vivo  gene expression profiles, to our surprise we detect that only few transcripts: TFF1, CCND1, H19, SREBF1 and WWP1 behaved similarly (i.e. up-regulation) in both studies. This is similar to observations made previously by Meltzer and co-workers whom showed that the majority of genes regulated in cell culture do not predict ER status in breast carcinomas [in vitro response of a single cell line to E2, in this particular case the widely used MCF-7 cells, may not faithfully reproduce the physiological effects of ER signaling in vivo.By comparing the rcinomas ,37. Thiset al. [et al. [In order to identify and validate the most reliable set of genes able to discriminate breast carcinomas based on their ERα status, we performed a cross-platform comparison between the described SAGE dataset with two previously reported breast cancer studies based on DNA microarray methods ,13. van'et al.  reportedestrogen receptor 1 (ESR1), GATA-binding protein 3 (GATA3), mucin 1 (MUC1), v-myb-myeloblastosis viral oncogene homolog (C-MYB) , X-box-binding protein 1 (XBP1), hydroxysteroid 17-β dehydrogenase 4 (HSD17B4), BTG family member 2 (BTG2), transforming growth factor β-3 (TGFB3), member RAS oncogene family (RAB31), START domain containing 10 (STARD10), and KIAA0089  breast tumors. One hundred and eighty-three genes were identified by more than one method Figure . Eleven One hundred and fourteen genes were identified as over-expressed by oligonucleotide microarrays  and SAGEInterestingly, we found a higher number of overlapping genes between the oligonucleotide microarray and SAGE platforms 114 genes), while only 66 genes were observed overlapping when comparing both microarray platforms. It is worth noting that 96% of the 470 genes  breast tumors. More importantly this analysis identifies the most promising biomarkers for further evaluation as ERα associated genes in breast cancer. Furthermore, the identified proteins may be of value as breast cancer prognostic indicators analyzed either as a group or individually. It is also likely that groups of co-regulated genes in ERα (+) breast cancers may be associated to the hormonal control of mammary epithelial cells growth and differentiation. Finally, a better understanding of the signaling networks controlled or associated with the estrogen response may lead to the identification of novel breast cancer therapeutic targets.. For the generation of our SAGE libraries, snap frozen samples were obtained from the M.D. Anderson breast cancer tumor bank, and SAGE analysis was performed as previously described [To perform the comparative breast cancer SAGE analysis based on ERα status, we analyzed 26 Stage I – Stage II invasive breast carcinomas (8 ERα-negative tumors and 18 ERα-positive tumors). To this end, we generated and sequenced 24 breast cancer SAGE libraries at an approximate resolution of 100,000 tags per library, combined with 2 additional breast cancer libraries (ERα-negative tumors) downloaded from the Cancer Genome Anatomy Project – SAGE Genie database (SAGE_Breast_Carcinoma_B_95-259 and B_IDC_4) escribed ,38.. In our analyses we only considered tags with single tag-to-gene reliable matches. To compare these SAGE libraries, we utilized a modified t-test recently developed by us [SAGE tag extraction from sequencing files was performed by using the SAGE2000 software version 4.0 (a kind gift of Dr. K. Kinzler, John Hopkins University). SAGE data management, tag to gene matching as well as additional gene annotations and links to publicly available resources such as GO, UniGene, LocusLink, were performed using a suite of web-based SAGE library tools developed by us ed by us . This teed by us ..All raw SAGE data reported as Supplementary tables in this manuscript is publicly available at ® 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Experiments were performed in duplicate for each data point and 18s rRNA was used as control. Results were expressed as mean ± 2 Standard Error based on Log2 transformation of normalized real time RT-PCR values of the assayed genes. We used t-test to compare the gene expression levels of validated genes between ERα (+) and ERα (-) breast tumors (p < 0.05).Template cDNAs were synthesized on mRNAs isolated from an independent set of 36 Stage I – Stage II human breast carcinomas (13 ERα-negative tumors and 23 ERα-positive tumors) obtained from our tumor bank. Primers and probes were obtained from the TaqMan Assays-on-Demand™ Gene Expression Products (Applied Biosystems, Foster City, CA, USA). All the PCR reactions were performed using the TaqMan PCR Core Reagents kit and the ABI Prism2O2 in methanol for 10 min. After pretreatment with Tris-EDTA buffer, in order to block non-specific antibody binding, the slides were incubated with 10% goat serum in PBS for 30 min. Primary monoclonal ERα antibody (ER-6F11, Novocastra, Newcastle, UK) was used at 1:50 dilution and detected following standard immunohistochemical techniques. DAB was used as chromogen and Mayers hematoxylin is used as counterstain. Scoring was performed by breast pathologist (AS). Cuttoff for positivity was determined at 5% of tumor cells staining positively for ER (i.e. < 5% of cells the tumor was considered negative for ERα).IHC staining and ER status determination was performed by the Pathology Department, MDACC following routine immunohistochemical procedures. Briefly, five micrometer sections of invasive breast carcinomas paraffin embedded tissues were used. Endogenous peroxidase activity was blocked with 3% HEASE [Database for Annotation, Visualization and Integrated Discovery (DAVID) at [EASE software calculates over-representation of specific GO terms with respect to the total number of genes assayed and annotated. Statistical measures of specific enrichment of GO terms are determined by means of an EASE score (p < 0.05). The EASE score is a conservative adjustment of the Fisher exact probability that weights significance in favor of biological themes supported by more genes and is calculated using the Gaussian hypergeometric probability distribution that describes sampling without replacement from a finite population [EASE analyzed genes.For automated functional annotation and classification of genes of interest based on GO terms, we used the EASE  availablAVID) at . The EASpulation . This al[11 = 732 high-affinity EREs) [To identify the occurrence of EREs within the promoter regions of up-modulated genes in ERα (+) breast tumors, we used a human genome-wide high-affinity ERE database . This puERα status associated genes identified in previous breast cancer studies ,13 using and ) [All over-expressed genes in ERα (+) breast tumors obtained from these studies were downloaded from the corresponding web sites ( and ) ,13.EASE software [e.g. SAGE study vs. DNA microarray) was determined. We used the normal approximation to the binomial distribution as previously described [These datasets were annotated by LocusLink ID using the software , and theescribed  to calcuM.C.A. conceived the study idea and carried out the real time RT-PCR validations, the biostatistical/ bioinformatics analysis and writing the manuscript. Y.H., H.S. and J.A.D. carried out the breast cancer SAGE libraries and provided practical feedback on aspects of the manuscript. K.B. and S.G. developed the biostatistical and web-page base methodology. A.S. provides the tissue samples and clinical information. C.M.A. is the principal investigator and was involved in the conceptualization, design and writing of the manuscript. All authors read and approved the final manuscript.The author(s) declare that they have no competing interests.Differentially expressed genes between ERα (+) vs. ER α (-) breast carcinomas .Click here for fileGene Ontology overrepresentation analysis.Click here for fileHigh-affinity EREs identified in ERα (+) up-modulated genes.Click here for fileCross-platform comparison of the up-modulated transcripts in ERα (+) breast carcinomas.Click here for file


This review article is aimed at comparing the results of histopathological and clinical imaging studies to assess coronary collateral circulation in humans. The role of collaterals, as emerging from morphological studies in both normal and atherosclerotic coronary vessels, is described; in addition, present role and future perpectives of echocardiographic techniques in assessing collateral circulation are briefly summarized. In the past 25 years, the concept of a compensatory function of the coronary collaterals (or anastomoses) – i.e. vessels that join different coronary arteries or branches – has been practically cancelled from the mind of cardiologists since cineangiography shows that the onset of coronary heart disease (CHD) occurs independently of their presence. The assumption, therefore, was and is that they have no compensatory meaning  and corohomocoronary (between branches of the same coronary artery) and intercoronary (between different coronary arteries) anastomoses were present everywhere joining at any level the intramural branches. Only in two of more than 600 hearts, superficial collaterals between extramural coronary arteries were seen and sampled for histology. The diameter of the innumerable normal collaterals ranged from 20  to 350 microns, frequently assuming a corkscrew aspect, possible adaptation to the contraction cycle of the myocardium ; when more than one severe stenoses exist each one had its own satellite collaterals. However, an identical obstruction located at the same level of an artery might show relatively few highly enlarged collaterals (the only ones visible by cineangiography), or numerous relatively small collaterals  in apparently healthy people acting their normal life, 89% with a fatal AMI had one or more (47%) severe atherosclerotic stenosis greater than 70% ;65% of sudden and unexpected death (SUD) showed the same finding in one or more (35%) vessels; 66% of non cardiac patients dead from other diseases and 39% of normal subjects dying from accident had the same severe atherosclerotic stenosis in one or more (40% and 16% respectively) coronary arteries  does not correspond to that observed in pathological studies in which the mean figure is 50% for AMI and 29% for SUD patients. Nevertheless, different selection of material, divergent definition and an absence of a correct correlation of all pertinent variables give reason of dissimilar conclusions. In 200 selected AMIs and 208 SUD cases the unique cause of occlusion was a thrombus found in 41% and 29% respectively. In AMI group it correlated significantly with a lumen reduction greater than 70% (93%), length of plaque more than 6 millimeters (95%), its concentric shape (100%), prevailing atheroma (84%), medial neuritis (92%) infarct size greater than 50% (86%). SUD cases showed a similar behavior.In reality, both clinicians and pathologists observe a phenomenon which started before, missing its onset and sequence of events to distinguish whether primary or secondary. In only one case reported in literature , this seOne case is only one case but when for the first time shows how the events developed, it becomes a precious mile stone for our knowledge demonstrating that the cineangio occlusion was a pseudocclusion namely a blood flow stasis in LAD secondary to an increased intramyocardial resistance. The first main question is how many of the 87% cineangio occlusion are pseudocclusion and whether the "layered" thrombus recovered at bypass surgery was a true thrombus or a coagulum which frequently show a layering of blood elements not seen in thrombus formation."Red" thrombus, namely a coagulum, is frequently and erroneously considered as thrombus. The second question concerns the nature of increased intramyocardial resistance: spasm of intramural arterial vessels or their extravascular compression by an asynergic myocardium? The first sign of CHD is hypokinesis of a myocardial zone which particularly in systole may compress vessels. Any time there is an increase of the intraventricular pressure with bulging of hypokinetic myocardium such a compression may abolish blood flow with subsequent infarction. In the reported patient location and infarct size corresponded to the hypokinetic area observed before the infarct onset.A last comment deserves the supposition that small atherosclerotic plaques undetectable at cineangio, may rupture causing an infarct. A supposition based on the cineangio finding of a non critical stenosis observed in a vessel tributary of a territory in which an infarct will develop. Since, when the latter occurred, stenoses in other non supplying vessels did not show a further lumen reduction, the conclusion was that even the plaque related to infarction had a non critical lumen reduction . A conclIn the past years, clinical methods available to measure collateral flow have been too crude and showed major limitations, thus contributing to debate and confusion about the functional relevance of collateral circulation in the human myocardium. Coronary angiography allows visualization of collateral vessels having a diameter ≥100 μm, that actually prevents the majority of them from being detectable with this technique ,17. On tFinally, intravenous MCE has been recently reported to provide qualitative and quantitative evaluation of collateral blood flow in the presence of an occluded infarct-related artery, and to emerge as the only predictor of true collateral blood flow among other markers .caveat, however, have to be taken into account. First, although the peak contrast pixel intensity has been reported as the most accurate of the variables obtained to measure collateral flow, there is a remarkable scatter in the correlation between peak pixel intensity and true collateral flow [All these reports as a whole support the concept that MCE provides important information on collateral flow and represents a promising mean for evaluating the status of coronary collateral circulation in clinical practice. Some important ral flow . Second,ral flow . FinallyBesides the attempt to obtain direct echocardiographic assessment, coronary collateral circulation can indirectly affect the result of diagnostic stress testing with the use of echocardiographic technique. Increased vulnerability to myocardial ischemia induced by pharmacological coronary vasodilation was reported consistently with the hypothesis of a facilitated steal phenomenon in the presence of good collateral circulation . On the The application of low-frequency ultrasound to intravascular microbubble contrast agents has been receiving attention in the last few years due to its potential therapeutic application, primarily as targeted gene delivery systems . FurtherAny hypothesis on the pathogenic role of a plaque and its activity and vulnerability should consider all interrelated variables for a correct interpretation of findings. When only one or few variables are investigated erroneous conclusions can be reached. An atherosclerotic plaque is always an active structure since its progression depends on a sequence of events due to a variety of correlated phenomena; while vulnerability is just an hypothesis which believe that some findings indicate a risk of plaque rupture.The known variables are: degree of lumen reduction, shape, length, satellite collaterals, tunica media changes, inflammatory reaction per se and associated with media nerves , survival Table  macrophaMost studies analized few variables mainly observed in animals after hypercholesterol diet or in familial hypercholesterolemia. A pattern ,47 totalThe presence of functioning collaterals induces a particular hemodynamic condition within the residual lumen at the plaque level with proximal flow reduction counterbalanced by distal collateral flow. Any time there is a regional asynergy Figure  with incProf. Giorgio Baroldi contributed to the conception and organization of this review and to the final comments. Dr. Riccardo Bigi and Dr. Lauro Cortigiani summarized the use of ultrasound techniques in atherosclerotic plaque imaging.


The aim of this study was to describe practice activity trends among oral and maxillofacial surgeons in Australia over time.All registered oral and maxillofacial surgeons in Australia were surveyed in 1990 and 2000 using mailed self-complete questionnaires.Data were available from 79 surgeons from 1990 (response rate = 73.8%) and 116 surgeons from 2000 (response rate = 65.1%). The rate of provision of services per visit changed over time with increased rates observed overall (from 1.43 ± 0.05 services per visit in 1990 to 1.66 ± 0.06 services per visit in 2000), reflecting increases in pathology and reconstructive surgery. No change over time was observed in the provision of services per year (4,521 ± 286 services per year in 1990 and 4,503 ± 367 services per year in 2000). Time devoted to work showed no significant change over time (1,682 ± 75 hours per year in 1990 and 1,681 ± 94 hours per year in 2000), while the number of visits per week declined (70 ± 4 visits per week in 1990 to 58 ± 4 visits per week in 2000).The apparent stability in the volume of services provided per year reflected a counterbalancing of increased services provided per visit and a decrease in the number of visits supplied. In Australia the majority of dentists work in private general practice . RelativIdentifying trends over time in the practice activity of oral and maxillofacial surgeons is a key element in planning by informing debate on issues relevant to the speciality such as the future supply of services and training needs. Previous Australian data have shown the distribution of oral and maxillofacial surgery was dominated by dentoalveolar services . HoweverConsidering the trend towards oral and maxillofacial surgeons gaining medical qualifications and the potential impact that this may have on practice activity, the aims of this study are to describe practice activity trends among oral and maxillofacial surgeons in Australia in 1990 and 2000 in terms of time worked, visits supplied and services provided.All registered oral and maxillofacial surgeons in Australia were surveyed in both 1990 and 2000. A surgeon must be registered with a dental board in the state/territory in which they practice. For the purposes of this analysis trainees were excluded. Although some of the same surgeons may have responded to the survey at both points in time for this study the design and analysis is treated as two sequential cross-sectional surveys.Data were collected using mailed self-complete questionnaires with a primary approach letter sent initially to each surgeon, followed a week later by the survey materials, with a reminder card two weeks later, and up to four follow-up mailings of survey materials to surgeons who had not yet responded in order to ensure higher response rates . SurgeonThe questionnaire was designed to provide comprehensive coverage of a range of workforce issues and the analysis presented here is limited to a subset of the total number of variables that were collected. The questionnaire included surgeon demographic and background variables , practice details (e.g., level of activity in private and public practice, type of practice, level of practice activity), and a log of services provided in a typical week. Surgeons recorded details of the patients they treated over a one-week period. Main areas of service were classified as dentoalveolar, trauma, pathology, orthognathic, reconstructive surgery and other/major medical compromise. An outline of the key variables collected and how measures of time worked, services provided and visits supplied were derived is presented in Figure Data were available from 79 surgeons from 1990 (response rate = 73.8%) and 116 from 2000 (response rate = 65.1%). Service provision data were available for 4,847 patients from 1990 and 3,292 patients from 2000. Table The rate of provision of services is presented in Table The number of hours per year devoted to work by surgeons did not change significantly between 1990 and 2000, reflecting stable levels of hours per day, days per week and weeks per year worked. However, the number of visits per week supplied by surgeons decreased between 1990 and 2000. The number of visits per year supplied by surgeons also showed a trend towards a decreased number of visits over time, but the change (P = 0.0508) was not significant at the P < 0.05 level. The relationship between the service and visit measures is presented in Figure While the findings of this study are based on a small sample size this primarily reflects the size of the population studied. Oral and maxillofacial surgeons comprise a small percentage of the dental workforce , hence aThe practice patterns of oral and maxillofacial surgeons have been reported to be largely stable, showing no change between 1990 and 2000 in their age and sex distribution, place of birth, practice activity level, referral sources, mix of cases and perceptions of work . The disThe decline in patient visits supplied by oral and maxillofacial surgeons, while statistically significant for visits supplied per week, was not statistically significant at the conventional level of P < 0.05 for visits supplied per year. This partly reflects the stability in weeks per year worked, one of the component measures used to derive visits per year. However, the number of visits per year was at the borderline of statistical significance (P = 0.0508) and considerations other than a reliance on P values is recommended in the literature , with moThe trends observed in practice activity among oral and maxillofacial surgeons show parallels with private general practice dentists in Australia. Trends in private general practice have also shown a counterbalancing effect of declining numbers of visits supplied with increased rates of services per visit resulting in a stable level of provision of services per year. Medical general practitioners have shown a trend towards longer consultations , consistPossible explanations for the lower levels of patient visits per hour among private general practice dentists include increased numbers of older patients , who mayHistorical records have indicated that average length of dental appointments changed little over the period 1960–61 to 1974–75, but there was an increase since 1974–75 that was quite marked across the 1977–78 to 1982–83 period -29, and Estimates of the capacity to supply services and projections of labour force requirements need to consider that while the rate of services per visit has increased this has been counterbalanced by decreases in the number of visits supplied. This has resulted in a stable volume of services provided by oral and maxillofacial surgeons per year over the study period.The author(s) declare that they have no competing interests.DSB performed data analyses and drafting of the manuscript. AJS provided overall supervision of the project. KAS performed data processing and preliminary analyses. DNT was involved in data collection. ANG provided specialist advice on oral and maxillofacial surgery. All authors contributed to and approved the manuscript.The pre-publication history for this paper can be accessed here:


Studies in animal models of AD, in which brain complement activation can be experimentally altered, should be of value for clarifying this issue. However, surprisingly little is known about complement activation in the transgenic animal models that are popular for studying this disorder. An optimal animal model for studying the significance of complement activation on Alzheimer's – related neuropathology should have complete complement activation associated with senile plaques, neurofibrillary tangles (if present), and dystrophic neurites. Other desirable features include both classical and alternative pathway activation, increased neuronal synthesis of native complement proteins, and evidence for an increase in complement activation prior to the development of extensive pathology. In order to determine the suitability of different animal models for studying the role of complement activation in AD, the extent of complement activation and its association with neuropathology in these models must be understood.Complement inflammation is a major inflammatory mechanism whose function is to promote the removal of microorganisms and the processing of immune complexes. Numerous studies have provided evidence for an increase in this process in areas of pathology in the Alzheimer's disease (AD) brain. Because complement activation proteins have been demonstrated  A variety of inflammatory processes are increased in regions of pathology in the Alzheimer's disease (AD) brain -4. Therein vitro protection against excitotoxicity [The significance of complement activation, a major inflammatory mechanism, in AD is particularly problematic. The complement system is composed of more than 30 plasma and membrane-associated proteins which function as an inflammatory cascade. Complement activation promotes the removal of microorganisms and the processing of immune complexes. The liver is the main source of these proteins in peripheral blood, but they are also synthesized in other organs including the brain . Proteintoxicity ,52 and Atoxicity , as welltoxicity ,55. Furttoxicity , althougtoxicity . Understtoxicity , and Mortoxicity ). Condittoxicity , organ ttoxicity , glomerutoxicity , ischemitoxicity , and hertoxicity . ModulatWhile animal models of human disease generally have similar pathological findings to the human disorders, distinct differences remain. These models may be appropriate for studying some aspects of a disease process, while less suitable for others. To determine the significance of complement activation in the development of AD-type pathology, for example, some animal models may be of value primarily for investigating the relationship between early complement activation and SP and NFT formation, whereas others may be more relevant for studying the role of the MAC in neuronal loss.Investigators at the Academic Hospital Free University in Amsterdam first reported the presence of early activation proteins in the classical complement cascade in the AD brain -34,36,37in vitro studies indicating that Aβ and tau protein, the major components in SPs and NFTs, can fully activate human complement [Complement proteins are detectable on or closely associated with SPs, NFTs, and dystrophic neurites in the AD brain. These findings are in agreement with mplement ,68-71. Amplement . In contmplement ,73,74. Cmplement . The aboHow the increased complement activation in AD relates to the development of SPs and NFTs, and to neuronal loss, is unclear. Immunocytochemical staining for complement activation proteins in the aged normal human brain is generally faint, and may be below the level of detection ,69,73; oBoth mRNA expression and protein synthesis of native complement proteins are increased in the AD brain -80.  in transgenic mice expressing mutations in the human amyloid precursor protein (hAPP) gene. The APP mutations expressed in these mice have been associated with early-onset, familial AD . The TGF for C3a , and the for C3a  reported for C3a  found on for C3a  and many for C3a , and (b) for C3a .In addition to APP, mutations in the gene encoding for presenilin-1 (PS-1) have also been associated with familial AD . The PS/- and APP/PS1/C1q- animals, lack of C1q did not alter either plaque density or the time course of plaque deposition. Neuronal cell numbers (NeuN+ cells), assessed only in the Tg2576 (APP) mouse, were not changed by the absence of C1q; however, immunoreactivity to MAP-2  and synaptophysin  in the hippocampus (region CA3) was increased 2-fold in the APP/C1q- animals, compared with APP mice. Microglial and astrocytic activation was significantly reduced in the APP/C1q- animals. These results were interpreted to suggest that in these animal models of AD, (1) early complement activation (as indicated by C1q deposition) in response to fibrillar Aβ deposition might be responsible for the chemotactic attraction of activated glial cells, and (2) the activated microglia, while unable to clear fibrillar Aβ, may have contributed to the loss of neuronal integrity indicated by reduced MAP-2 and synaptophysin staining in the APP mice. By recruiting activated microglia, complement activation could potentially contribute to neuronal injury even if full activation (MAC formation) does not occur.Fonseca et al.  investigCoimbra and colleagues  describeAlterations in native complement mRNA and protein levels have been evaluated in the rat hippocampus following experimental induction of acute neuronal injury. These surgical and pharmacological procedures result in neuronal loss in the entorhinal cortex, and deafferentation of hippocampal neurons, similar to that which occurs in AD . Selectiin vitro study of Webster et al. [Frautschy et al.  examinedr et al. , in whicr et al.  in whichswe mice (Tg2576) with mice expressing the transgene for a human tau mutation (JNPL3 mice) to generate a double mutant tau/APP mouse (the "TAPP mouse"). These mice develop SPs similar to APP mice , and older TAPP mice have NFTs, in association with increased astrocyte proliferation, in limbic areas. The plaques contain both Aβ40 and Aβ42. Oddo et al. [42) detected in the frontal cortex by 6 months of age, and in other cortical regions and hippocampus by 12 months. Many of the extracellular Aβ deposits are thioflavin-S-positive and are associated with reactive astrocytes. Phosphorylated tau initially appears in the hippocampus and subsequently in cortical regions; it is detected within neurons by 12–15 months and within dystrophic neurites at 18 months. Though Aβ immunoreactivity precedes that of tau, these proteins co-localize to the same neurons. The presence of NFTs as well as SPs suggests that the 3xTg-AD and TAPP models may be more relevant than APP or APP/PS-1 mice for studying the significance of complement activation in the development of AD-type pathology. Potential drawbacks for using these models for complement-related studies include, as discussed earlier, functional deficiencies in activation of mouse complement [Mutations in the gene encoding for human tau protein have been linked to the development of frontotemporal dementia with parkinsonism . By combo et al.  injectedmplement , decreasmplement , and themplement . It is nRuberti et al.  developeC. pneumoniae is an intracellular, gram-negative or gram-variable bacterium long identified as a respiratory pathogen. It has more recently been demonstrated to be a causative agent in reactive arthritis [C. pneumoniae infection in BALB/c mice could produce AD-like pathology. Intranasal inoculation with C. pneumoniae resulted in deposition of Aβ1–42 in the hippocampus, amygdala, entorhinal cortex, perirhinal cortex, and thalamus by 3 months post-inoculation. The majority of these Aβ deposits appeared similar to diffuse plaques, though a small number of them were thioflavin-S-reactive. NFTs were not detected. The authors suggested that soluble factors such as lipopolysaccharides, which are present in the cell wall of all Chlamydiae [C. pneumoniae-infected mouse may offer a novel infectious model for studying the relationship of complement activation to the development of Aβ-containing plaques.rthritis  and to brthritis  and atherthritis . Some larthritis -127, altrthritis -131. A rrthritis  examinedlamydiae , may havlamydiae ,135, the42 is the predominant type of Aβ deposited in plaques [Old dogs, in particular the beagle, have been extensively investigated as a model for CNS Aβ deposition and associated age-related cognitive dysfunction. Aβ deposits are detectable in the brains of most older dogs . The reg plaques . Canine  plaques ,139. Age plaques ,141. The40 [Age-related formation of SPs has been reported in a variety of non-human primates including the cynomolgus monkey , rhesus 40 . NFTs ap40 ,148, and40 -151. In 42-containing plaques, and NFTs are not detected [Scattered reports of AD-type pathology in other species have also appeared. Adding trace amounts of copper to the water supply of cholesterol-fed rabbits results in Aβ deposition within SP-like structures in the hippocampus and temporal cortex, with associated learning deficits . The neudetected . A repordetected  describedetected . While t1. Complement activation has been extensively studied in the AD brain. There is convincing evidence for activation of both the classical and alternative pathways, resulting in full activation as indicated by the presence of the MAC. Both aggregated Aβ (in SPs) and phosphorylated tau (in NFTs) are likely to be responsible for this activation.2. Because complement activation generates both both neuroprotective and neurotoxic effects, the significance of increased complement activation in the development and progression of AD is unclear.3. An optimal animal model for studying the significance of complement activation in the development of AD-type pathology would have complete activation of this process, with co-localization of complement activation proteins with SPs and with NFTs (if present). Other desirable features include early complement activation prior to the development of extensive neuropathology, increased CNS production of native complement proteins, and both classical and alternative pathway activation.4. Surprisingly little is known about the extent of complement activation in animal models of AD. The postischemic hyperthermic rat  is the o5. The extent of complement activation and its association with neuropathology must be determined in animal models of AD to clarify the relevance of these models for investigating the significance of complement activation in the development of AD-type pathology.Aβ, amyloid beta; AD, Alzheimer's disease; APP, amyloid precursor protein; CNS, central nervous system; MAC, membrane attack complex; mRNA, messenger ribonucleic acid; NFTs, neurofibrillary tangles; NGF, nerve growth factor; PS-1, presenilin-1; sCrry, soluble complement receptor-related protein y; SPs, senile plaque; TGF-β1, transforming growth factor beta1.The author declares that he has no competing interests.


The aim of this study was firstly, to improve and validate a cerebrospinal fluid (CSF) prefractionation method followed by two-dimensional electrophoresis (2-DE) and secondly, using this strategy to investigate differences between the CSF proteome of frontotemporal dementia (FTD) patients and controls. From each subject three ml of CSF was prefractionated using liquid phase isoelectric focusing prior to 2-DE.With respect to protein recovery and purification potential, ethanol precipitation of the prefractionated CSF sample was found superior, after testing several sample preparation methods.The reproducibility of prefractionated CSF analyzed on 2-D gels was comparable to direct 2-DE analysis of CSF. The protein spots on the prefractionated 2-D gels had an increased intensity, indicating a higher protein concentration, compared to direct 2-D gels. Prefractionated 2-DE analysis of FTD and control CSF showed that 26 protein spots were changed at least two fold. Using mass spectrometry, 13 of these protein spots were identified, including retinol-binding protein, Zn-α-2-glycoprotein, proapolipoproteinA1, β-2-microglobulin, transthyretin, albumin and alloalbumin.The results suggest that the prefractionated 2-DE method can be useful for enrichment of CSF proteins and may provide a new tool to investigate the pathology of neurodegenerative diseases. This study confirmed reduced levels of retinol-binding protein and revealed some new biomarker candidates for FTD. Frontotemporal dementia (FTD) accounts for up to 20% of presenil dementia cases  and is, The diagnosis of FTD is often difficult and would be greatly enhanced by the use of disease specific neurochemical markers . SeveralSome abundant proteins, for example albumin and immunoglobulins, limit the total amount of CSF proteins that can be loaded on the 2-D gel, resulting in difficulties detecting low abundant proteins of CSF. By using liquid phase isoelectric focusing (LP-IEF) as a prefractionation step prior to 2-DE we have previously shown that less abundant CSF proteins can be enriched, thus making them more easily detected and identified by mass spectrometry (MS) . The advThe aim of the present study was to improve the prefractionation procedure for individual CSF samples and to determine its reproducibility. Moreover, the second aim of this study was to apply the method and further explore disease-influenced proteins in CSF from FTD patients compared to controls.Several strategies to reduce impurities, i.e. salt and ampholytes, of the prefractionated CSF samples were performed including, trichloroacetic acid (TCA)-acetone precipitation, ethanol precipitation, chloroform/methanol/water precipitation and, micro Bio-Spin desalting column. The protein pattern on the 2-D gels and protein recovery from the different sample preparation methods were compared to that of acetone precipitation, previously used prior to prefractionated 2-DE of CSF . We founBoth TCA-acetone and chloroform/methanol/water precipitation substantially reduced the number of spots and resulted in streaky 2-D gels (data not shown). The use of biospin columns gave well-focused 2-D gels but several protein spots were reduced or lost (data not shown).Four identical CSF samples were individually prefractionated by LP-IEF. Fractions 6–9 (having a pH of 4.5–6.0) were pooled and analyzed on pH 4–7 strips in four replicates. Coefficients of variation (CVs) were calculated for the protein spots quantities, determined by the PD-Quest software, and a selection of 20 spots had CVs ranging from 1–33% (15.4% ± 8.3%, mean ± SD) Figure . The spoComparison of direct and prefractionated 2-DE showed that the prefractionated gels contained more spots with a higher protein concentration Figure . The spoThe prefractionated 2-DE method was used to screen for changes in the CSF proteome of five FTD patients compared to that of five non-dementia controls in the pH intervals 3–6, 4–7, and 5–8 Figure .Comparing the protein densities of the gels, 10 spots were up regulated and 16 spots down regulated, at least two fold, in FTD patients compared to non-dementia controls. Increased proteins are marked with a square and decreased with a circle Figure . ThirteeIn this study we present an improved method for increased detection of CSF proteins by a combination of LP-IEF and 2-DE, followed by SYPRO Ruby protein staining and protein identification by mass spectrometry, for investigation of protein differences in CSF of FTD patients compared to controls. To our knowledge no other prefractionation method combined with 2-DE has so far been developed and evaluated for CSF proteins.The study showed that the reproducibility of prefractionated 2-D gels could be compared to that of direct 2-D gels, indicating that the extra prefractionation step did not introduce additional variation and could be reproduced from sample to sample. The protein detection and quantitative reproducibility of Coomassie Brilliant Blue , silver,In addition to the high salt concentration of CSF (> 150 mmol/L), ampholytes are also introduced into the sample in the prefractionation step, LP-IEF. We previously reported that the focusing time in the first dimension of prefractionated 2-DE had to be increased  probablyWhen comparing direct and prefractionated 2-DE it is evident that the prefractionated gels contain more spots, with higher protein quantities. Thus, the CSF proteins are enriched in the prefractionation step, simplifying their identification by MS, as shown in our previous study . CSF anaIn order to widen our search for protein differences in the CSF of FTD patients the improved prefractionated 2-DE procedure was applied to CSF from five FTD patients and five control subjects. 26 protein spots were changed at least two fold and 13 of these protein spots, representing seven different proteins, were identified as ZAG, ProapoA1, β-2-m, TTR, RBP, serum albumin and alloalbumin. Our previous direct 2-DE study  of the FA reduced level of RBP was consistent between our two studies, and in this case RBP was found reduced in both the pH 4–7 and the pH 5–8 gels. In contrast, CSF analysis of AD showed increased levels of one isoform of RBP , indicatBrain TTR is exclusively produced, secreted and regulated by the choroid plexus -34. TTR One isoform of β-2-m was found increased in this study. Other studies have shown that CSF β-2-m is elevated in patients with various neurological diseases including AD , infectiThe function of ZAG is unknown but studies have shown that it is present in several body fluids, including CSF, sweat, saliva, seminal fluid, plasma, milk, amniotic fluid and urine, suggesting a fairly widespread exocrine function of the protein . In thisThe level of ProapoA1 was also increased in this study. Our previous study of the FTD proteome  did not The reason that several protein changes were inconsistent between this and our previous study of the FTD proteome may be explained by the fact that a smaller sample size and a different population of FTD patients, which is a rather heterogeneous disease, were used in this study. Due to the small sample size it must also be emphasized that the protein changes found in this study are preliminary. Moreover, direct and prefractionated 2-DE are still two different proteomic approaches and a somewhat different analytical window was not unexpected. Indeed, direct and prefractionated 2-D gels show different protein patterns, for example, the apolipoprotein E and apolipoprotein J isoforms seem to be missing in the prefractionated 2-D gels, which may be explained by the fact that lipoproteins tend to adhere to plastics  and coulAs shown in the present study, the levels of specific isoforms are altered and these are unlikely to be detected using methods measuring the total concentration of a protein. Therefore, determination of posttranslational modifications is of importance for understanding the neuropathology in FTD, and 2-DE is a useful method for sensitive detection of different protein isoforms.We have shown that the prefractionated 2-DE method is reproducible to the same extent as traditional 2-DE and can enrich CSF proteins in the gel. This approach may offer new perspectives on the pathology of neurodegenerative diseases. Prefractionated 2-DE analysis of FTD CSF proteins confirmed some of the proteins previously detected by direct 2-DE and revealed some new biomarker candidates. The protein changes should be further validated on a larger patient material, preferably also with complementary methods, in order to assess any of the proteins potential as biomarkers for FTD.CSF samples were obtained from the Clinical Neurochemical Laboratory . All CSF samples had a normal white-cell count, normal blood-brain barrier function and absence of intrathecal IgG and IgM production. The CSF samples for the proteomic study, described in table The study was approved by the Ethical Committee of Göteborg University. All participants or their relatives gave their informed consent to participation in the study, which was performed in accordance with the Declaration of Helsinki.To find a method for effective reduction of impurities and maximal protein recovery, 300 μL of prefractionated pooled CSF samples (fraction 6–9, pH 4.5–6.0) were purified using each of the following methods:Ice cold acetone precipitation; acetone: sample (4:1, v/v) precipitated at -20°C for 2 hours.1. Ice cold acetone-TCA precipitation; 2a) acetone: TCA: sample (4:10%:1, v/w/v) at -20°C for 45 min. 2b) acetone: TCA: sample (4:20%:1, v/w/v) at -20°C for 45 min. The protein pellet was washed 2 times with acetone after centrifugation.2. Chloroform/methanol/water precipitation, chloroform: methanol: sample (4:8:3, v/v/v) at room temperature for 2 hours.3. Ice cold ethanol precipitation; final concentrations of 60%, 70% and 80% ethanol was added to the sample and precipitated for 2 hours at -20°C.4. Purification using micro Bio-Spin column (Bio-Rad, Hercules, CA, USA) with a MW cut off of 6 kDa. The purification procedure w two-dimensional electrophoresis (2-DE) as performed according to the manufacturer's instructions.5. After precipitation all samples were centrifuged and the protein pellet analyzed on 2-D gels, described below.The protein recovery of acetone and ethanol treated samples was measured using the RC DC protein assay (Bio-Rad) according to the manufacturer's instructions.The CSF samples from individual patients were prefractionated using LP-IEF in the Rotofor cell (Bio-Rad). Three mL CSF sample was mixed with 9 mL millipore water, 1% ampholytes , 20 mM dithiothreitol (DTT) and 1 × Complete antiprotease solution (Roche Diagnostics, Mannheim, Germany). The focusing was performed at 4°C and at 12 W constant power for 2.5 hours. Then the 20 Rotofor fractions were harvested and fraction 2–5 corresponding to pH 1.5–4.5, fraction 6–9 corresponding to pH 4.5–6.0 and fraction 10–14 corresponding to pH 6.0–7.5 were pooled and concentrated to 300 μL in a vacuum centrifuge prior to 2-DE.In the FTD-study, the prefractionated pooled protein fractions were precipitated using 900 μL 95 % ice-cold ethanol  for two hours at -20°C. The mixture was centrifuged at 10,000 × g for 10 min at 4°C. The protein pellets were air-dried and then resolved in a buffer containing 9 M urea, 35 mM tris, 42 mM DTT, 2% 3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate (CHAPS), 0.66% sodium dodecyl sulfate (SDS), 2% IPG buffer and bromophenol blue.The first dimension was carried out using immobilized pH gradient (IPG) strips (Bio-Rad), 7 cm, pH 3–6 for Rotofor fractions 2–5, pH 4–7 for Rotofor fractions 6–9 or pH 5–8 for Rotofor fractions 10–14. The IPG-strips were actively rehydrated in the CSF-protein sample for 12 h at 50 V followed by protein focusing for 20,000 Vh using the Protean IEF Cell (Bio-Rad). The IPG strips were placed in 5 ml equilibration solution (50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS, bromophenol blue) containing 1% DTT, and 2.5% iodoacetamide in the second equilibrium step for 2 × 15 min.The second dimension was performed using the Nu-PAGE gel system (NOVEX, San Diego, CA, USA) with (2-(N-morpholino) ethane sulfonic acid (MES) buffer: 50 mM MES, 50 mM tris, 3.5 mM SDS, 1 mM EDTA), for 35 min at 200 V.In the direct 2-DE procedure, 300 μL CSF proteins were precipitated using 900 μL ice-cold acetone and stored for two hours at -20°C. The mixture was then centrifuged at 10,000 × g for 10 min at 4°C. The 2-DE procedure was performed as described above for prefractionated 2-DE.The gels were stained using SYPRO Ruby Protein Stain (Molecular-Probes, Eugene, Oregon, USA) according to the manufacturer's instructions. Image acquisition and analysis were performed on a Fluor-S MultiImager (Bio-Rad). The protein spots were detected, quantified and matched with the PD-Quest 2-D gel analysis software, v.7.0 (Bio-Rad). The gels were normalized according to the total quantity in valid spots . Protein levels increased or decreased two fold were taken into account.The protein digestion method has been previously described in detail . BrieflyMatrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis was performed using an upgraded Reflex II MALDI-TOF MS  equipped with a two-stage electrostatic reflectron, a delayed extraction (time-lag-focusing) ion source, a high resolution reflector detector and a 2 GHz digitizer. The spectra were acquired in the reflection mode at an accelerating voltage of 20 kV. The mass spectra, acquired and analyzed using Bruker software, were initially calibrated by external calibration using a mixture of known peptides and later recalibrated using two auto digestion products of porcine trypsin as internal calibrants. The protein database search tool "MASCOT Peptide Mass Fingerprint" on the Matrix Science web site  was usedCoefficient of variation (CV) was calculated (standard deviation (SD)/ Mean × 100) of the normalized protein spot densities from four replicate 2-D gels.In the proteomic study a 2-fold increase or decrease of normalized protein quantities was taken into account.The authors declare that they have no competing interests.SFH carried out all the 2D gel experiments, mass spectrometry analysis, participated in results evaluation and drafted the manuscript.MP participated in the 2D gel experiments, results evaluation and participated to the manuscript writing.KB contributed with material and critical reading of the manuscript.MJ contributed with material and participated in the design of the study.PD conceived the study, participated in its design and coordination, results analysis and supervised the manuscript writing.