Plant species have several mechanisms to avoid selfing such as dichogamy or a self-incompatibility response. Dichogamy in a single flower may reduce autogamy but, to avoid geitonogamy, plants must show flowering synchronization among all their flowers (i.e. synchronous dichogamy). It is hypothesized that one species would not simultaneously show synchronous dichogamy and self-incompatibility because they are redundant mechanisms to reduce selfing; however, this has not been accurately assessed.Euphorbia boetica and E. nicaeensis, which completely avoid autogamy by protogyny at the cyathia level. Both spurges showed a high population synchrony (Z<79), and their inflorescences flower synchronously. In E. nicaeensis, there was no overlap among the cyathia in anthesis of successive inflorescence levels and the overlap between sexual phases of cyathia of the same inflorescence level was uncommon (4–16%). In contrast, E. boetica showed a high overlap among consecutive inflorescence levels (74–93%) and between sexual phases of cyathia of the same inflorescence level (48–80%). The flowering pattern of both spurges was consistent in the two populations and over the two successive years. A hand-pollination experiment demonstrated that E. nicaeensis was strictly self-compatible whereas E. boetica was partially self-incompatible.This expectation was tested over two years in two natural populations of the closely related Mediterranean spurges E. nicaeensis prevents geitonogamous crosses and, consequently, avoids selfing and inbreeding depression. In E. boetica, a high probability of geitonogamous crosses may occur but, alternatively, this plant escapes selfing through a self-incompatibility response. We posit that synchronous dichogamy and physiological self-incompatibility do not co-occur in the same species because each process is sufficiently effective in avoiding self-fertilization.We propose that the complex pattern of synchronized protogyny in  Angiosperms with hermaphroditic flowers have the capacity to self-fertilize. Although self-fertilization may be advantageous in some circumstances, in general, it has negative consequences for the individuals because they are more likely to express recessive or partially recessive deleterious mutations. Therefore, the fitness of the offspring produced by selfing can be lower than that of offspring produced by outcrossing (i.e. inbreeding depression) There exists considerable literature about the functional significance and the evolutionary implications of dichogamy . Depending on the criteria used, various types of dichogamy have been proposed Plants with complete intrafloral dichogamy can totally avoid autogamy; moreover, if dichogamy is synchronous, geitonogamy is avoided as well Euphorbia boetica Boiss. as well as two natural populations of E. nicaeensis All. are investigated, both of whom are Mediterranean perennial spurges which are phylogenetically very close Thus, a fundamental aspect to determine if dichogamy and SI are two mutually exclusive mechanisms is to analyze the effectiveness of both mechanisms to avoid selfing. To test the effectiveness of dichogamy as an anti-selfing mechanism, we need to study the dichogamy of a flower and its synchronization with all the flowers on the plant Euphorbia boetica Boiss. is endemic to the southern half of the Iberian Peninsula and grows in coastal pinewoods on sandy soils at altitudes of 0–100 m Euphorbia nicaeensis All. subsp. nicaeensis has a circum-Mediterranean distribution and prefers calcareous soils and grows in dry, sunny places at altitudes above 600 m in southern Spain Euphorbia boetica and E. nicaeensis are perennial herbaceous shrubs that branch at the base and produce numerous floral stems that bear a terminal inflorescence. Inflorescences present cyathia arranged in dichasia or pleiochasia . The inflorescence is compound with several levels of branching, hereafter inflorescence levels, which bloom sequentially  and two of E. nicaeensis (La Camilla and Aracena) were studied in southern Spain. The Hinojos population  is within a mixed woodland of Pinus pinea L. and Quercus suber L. The Gandul population  is located on an abandoned farmland without tree cover. The La Camilla population  is in the Sierra de Grazalema Natural Park on sparse forest of Quercus ilex L. and Ceratonia siliqua L. The Aracena population  is situated on a cultivated woodland of Castanea sativa Mill.Two contrasting populations of E. boetica (27 from each Hinojos and El Gandul populations) and from 51 plants of E. nicaeensis . The study was conducted in 1999 and 2000 during the whole flowering period. Unfortunately, in 1999, plants of the Aracena population were eaten by goats in the middle of the blooming period and data were not available. Plants were revisited every 7–10 days throughout the flowering period  to assess the number of blooming male and hermaphrodite cyathia.Flowering phenology was studied in three inflorescences from each of 54 plants of Euphorbia boetica and E. nicaeensis present a complex flowering phenology because cyathia show intra- and interfloral dichogamy and the inflorescences have several levels of branching. For these reasons, we employed the widely used Z index je is the number of days during which both individuals i and j flower synchronously (j ≠ i); fi is the number of days individual i is in flower, and n is the number of individuals in the population. Xi may vary between 0 and 1; when Xi = 0 no synchrony occurs, and when Xi = 1 perfect synchrony occurs. The index of population synchrony (Z) is the average of the Xi of all plants of the population.Several indices to measure flowering synchrony within populations have been developed [35–36 and see references therein]. E. boetica (Hinojos and El Gandul) and E. nicaeensis (La Camilla) along a linear transect. The censuses were carried out in 1999 (flowering peak in La Camilla) and in 2000 .To determine whether the inflorescences of a plant flower synchronously, we utilized a method modified from Thomson and Barrett The synchrony among successive inflorescence levels (i.e. flowering overlap) on the same inflorescence  was estiEuphorbia species, generally all cyathia of the same inflorescence level bloom at the same time; however, synchrony among them may be not perfect. The asynchrony of flowering was estimated by considering the overlap among different sexual phases of the cyathia of the same inflorescence level (In both ce level . We asseE. boetica) and at La Camilla (E. nicaeensis). Prior to anthesis, inflorescences were bagged with polyester mesh bags (pore size ca. 2×2 mm) and tightened around the base of the peduncle. Only one inflorescence was used in each plant. In all experimental hand-pollinations, we used only cyathia that belonged to the inflorescence levels 5 and 3 in E. boetica and E. nicaeensis, respectively, because are the levels with the maximum cyathia production The breeding system of both species was assessed experimentally in 1999 in the El Gandul population  for each species by dividing the number of fruits or seeds of the self-pollination (geitonogamy) treatment by those of the cross-pollination (xenogamy) treatment To determine whether fruits or seeds produced by the geitonogamous crosses were due to an incomplete SI system or as the result of inbreeding depression in early stages Differences in the duration of flowering and population synchrony between species and years were tested by means of Generalized Linear Models (GLM), assuming a log link function with a Poisson error distribution and a logit link function with a binomial error distribution n is compared to inflorescence n+1 of the same plant. The levels within each dimension depends on the numbers of states of development of the cyathia  in each population on the census day. In all contingency tables, some rows and columns were pooled to avoid bias of chi-square goodness of fit when at least one of the expected frequencies was less than five To test for intraindividual synchrony between inflorescences of a plant, we utilized contingency tables to test the hypothesis that the bloom  of an inflorescence is independent of those of other inflorescences of the plant. Contingency tables have two dimensions: phenological state of inflorescence Differences between species in the percentage of overlap among different sexual phases of the cyathia of the same inflorescence level were analyzed by means of a binomial test to compare two proportions t tests based on the standard errors calculated from the specific model.Fruit and seed set of experimental crosses were evaluated by means of GLMs assuming binomial error distribution and a probit link function. When the GLMs showed significant differences, the means of treatments were compared using For each response variable in the GLMs, we tested the link functions and error distributions that generated the smaller deviance in the model. Model selection was carried out using the Akaike's Information Criterion E. boetica and E. nicaeensis took place from March to July in E. boetica and from May to August in E. nicaeensis  and 66 days  to 42 days ; differences between years were not significant .The mean flowering duration of the individuals of the populations of E. boetica, flowering phenology of each plant was continuous, i.e. there were some cyathia in anthesis during the entire flowering period of a plant. In contrast, in E. nicaeensis, flowering phenology of each plant was discontinuous and alternated between periods of flowering and no flowering.In E. boetica and E. nicaeensis showed a high population synchrony , but between years differences were significant .Individuals of E. boetica), the inflorescences within each plant displayed the same phenological state both at the beginning of flowering  and at the end . In El Gandul, the same results were observed in the only census that was carried out . In the La Camilla population (E. nicaeensis), the inflorescences within each plant displayed the same phenological state both in 1999  and in 2000 .Results of contingency tests showed that the flowering phenology of an inflorescence was not independent of the rest of the analyzed inflorescences of a plant; thus, plants of both spurges presented synchronized flowering among inflorescences. In the Hinojos population  and 80%  to 16% . The same was true considering the number of censuses in which overlapping between different sexual phases occurred .In E. boetica and E. nicaeensis bore fruit; thus, apomixis (except pseudogamy) was discounted , and again the seed set of selfed cyathia was lower than those of the other two treatments  there were only male cyathia with no ovaries to fertilize in the entire population  In both spurges, male cyathia were more numerous than hermaphrodites at the beginning of the blooming period. This pattern was due both to the exclusive presence of male cyathia in the first levels of the inflorescence pulation . This raE. boetica and E. nicaeensis rely entirely on pollinators for reproduction because bagged cyathia did not produce fruits. Both spurges can produce fruits and seeds after geitonogamous crosses, but based on fruit and seed SCI, E. nicaeensis is a strictly self-compatible species, whereas E. boetica is a partially self-incompatible species E. boetica most plants in the population did not develop fruits after selfing (68%), and that callose deposit were found around the sites where selfed pollen tubes penetrated the stigma, support the idea of the presence of an incomplete SI system Euphorbia esulaE. boetica (El Gandul), the cross-pollination treatment produced ca. 20% more fruit set than the open pollination treatment, suggesting that reproduction is pollen limited in this population Our results demonstrate that E. nicaeensis, there was no overlap among cyathia in anthesis of successive inflorescence levels and moreover, any overlap between sexual phases of cyathia of the same inflorescence level was markedly rare. This flowering pattern can be considered as a form of synchronous protogyny, as most of the inflorescences of a plant are also synchronized. Under these circumstances, the probability of natural geitonogamous fertilization in E. nicaeensis is extremely rare E. nicaeensis has only been found in species of the Araliaceae and Umbelliferae E. nicaeensis are effective mechanisms to reduce geitonogamous crosses and, consequently, to avoid selfing and inbreeding depression.In E. boetica showed overlap among two or more inflorescence levels. Although the frequency of overlapping decreased from the first to the last levels, it was relatively high in the intermediate levels, which bore the highest number of cyathia E. boetica typically visit all the cyathia in anthesis of an inflorescence E. boeticaE. boetica, geitonogamous fertilizations are highly limited due to its partial-SI system.In contrast, E. boetica and E. nicaeensis. Barrett Euphorbia as two different ways to avoid selfing It is noteworthy that synchronous dichogamy and SI were not simultaneously found in both Euphorbia, has been mainly considered a self-compatible group Euphorbia species with reported SI belong to different subgenera and sections E. boetica may have evolved to avoid inbreeding depression, which is not excluded by their pre-pollination anti-selfing mechanism. On the other hand, synchronous dichogamy may have originated in E. nicaeensis as a modification of the floral and flowering characteristics shared by all species of the Euphorbia subgenus Esula: intrafloral protogyny and cyathia arranged in compound pleiochasial inflorescences Euphorbia, specifically in section Paralias, which include the reconstruction of the evolutionary history of synchronous dichogamy and SI, and their phylogenetic associations Euphorbiaceae, and specifically Figure S1Frequency histograms showing the number of times that two inflorescence levels in anthesis of the same inflorescence overlapped in two populations over two years in E. boetica. Total number of censuses is shown in (TIF)Click here for additional data file.Figure S2Frequency histograms showing in which inflorescence levels the overlapping between two inflorescence levels occurs in two populations over two years in E. boetica. Total number of censuses is shown in (TIF)Click here for additional data file.Figure S3Pollen germination in stigmas of E. boetica and E. nicaeensis. Styles were fixed 24 h after pollination and stained with aniline blue. A, pollen germination after geitonogamous crosses in E. boetica (×2500). B, pollen germination after xenogamous crosses in E. boetica (×2500). C, pollen germination after geitonogamous crosses in E. nicaeensis (×1600). D, pollen germination after xenogamous crosses in E. nicaeensis (×1600). Bar  = 5 µm. Yellow arrows show fluorescent accumulations of callose on stigma cells around the pollen tube penetration.(TIF)Click here for additional data file.


Epidemiological and molecular findings suggest a relationship between Alzheimer’s disease (AD) and dyslipidemia, although the nature of this association is not well understood.1–42 and occurred irrespective of phospho-tau181p status.Using linear mixed effects models, we investigated the relationship between CSF levels of heart fatty acid binding protein (HFABP), a lipid binding protein involved with fatty acid metabolism and lipid transport, amyloid-β (Aβ), phospho-tau, and longitudinal MRI-based measures of brain atrophy among 295 non-demented and demented older individuals. Across all participants, we found a significant association of CSF HFABP with longitudinal atrophy of the entorhinal cortex and other AD-vulnerable neuroanatomic regions. However, we found that the relationship between CSF HABP and brain atrophy was significant only among those with low CSF AβOur findings indicate that Aβ-associated volume loss occurs in the presence of elevated HFABP irrespective of phospho-tau. This implicates a potentially important role for fatty acid binding proteins in Alzheimer’s disease neurodegeneration. A growing number of epidemiological and experimental studies suggest an association between Alzheimer’s disease (AD) and dyslipidemia. In observational studies, high serum cholesterol levels have been associated with increased risk of AD ,2. Genetin vivo. Structural MRI provides measures of brain atrophy, which reflect loss of dendrites, synapses [1–42 and high CSF HFABP and high CSF p-tau181p are associated with brain atrophy over time. We also evaluated the relationship between CSF HFABP and other lipid binding proteins including Apolipoprotein (Apo) C III, Apo D, and Apo E.In humans, structural MRI and CSF biomarkers allow for the indirect assessment of the cellular changes underlying AD synapses  and neursynapses . Low CSFsynapses ,14. Recesynapses  may havesynapses -20. HereIn our initial analyses, we used linear mixed effects models, co-varying for baseline age, sex, presence ("carriers") or absence ("non-carriers") of at least one ϵ4 allele of apolipoprotein E (APOE ϵ4), diagnostic status (AD vs mild cognitive impairment (MCI) vs healthy elderly controls (HC)), and disease severity (CDR-Sum of Boxes score at baseline) to examine whether CSF HFABP levels are associated with longitudinal atrophy of the entorhinal cortex and other AD-vulnerable regions of interest  Figure . We foun1–42 and between CSF HFABP and CSF p-tau181p are associated with brain atrophy over time. These linear mixed effects models included the main and interactive effects of CSF HFABP, CSF Aβ1–42, status and CSF p-tau181 status, and co-varied for the demographic and clinical variables mentioned previously (see Methods for further details on the model). Key results from these models are shown in Table Next, we asked whether statistical interactions between CSF HFABP and CSF Aβ1–42 status x time and CSF HFABP x CSF p-tau181p status x time on atrophy of both the entorhinal cortex and the AD-vulnerable ROI. The only CSF biomarker to show a significant association with atrophy over time in the entorhinal cortex and AD-vulnerable ROI was CSF p-tau181p. Of the co-variates, the interaction of time by diagnostic status and APOE ϵ4 carrier status were significant for both entorhinal cortex atrophy  and AD-vulnerable ROI atrophy . None of the other variables showed significant main or interactive effects.As illustrated in Table 1–42 status (i.e. low and high status) and CSF p-tau181p status (i.e. high and low status). We found a significant CSF HFABP by time interaction on entorhinal cortex and AD-vulnerable ROI atrophy rate only among individuals with low CSF Aβ1–42 levels . In these analyses, neither the interaction between CSF HFABP, CSF Aβ1–42 status and time nor the interaction between CSF HFABP, CSF p-tau181p status and time was significantly associated with longitudinal change in ADAS-cog  C III, Apo D, and Apo E at baseline. The relation of each of these apolipoproteins with CSF HFABP was assessed in separate models; all models controlled for age, sex, APOE ϵ4 carrier status, diagnostic status, and CDR-Sum of Boxes score.We next used generalized linear models to investigate the relationship between CSF HFABP and CSF p-tau181p , CSF levels of Apo C III , ApoD , and ApoE  . None of the other lipid binding proteins demonstrated a significant interaction with CSF HFABP on atrophy rates of the entorhinal cortex and the AD-vulnerable ROI .1–42, and occurs irrespective of phospho-tau levels. Considered together, these results suggest a potentially important role for fatty acid binding proteins in early Alzheimer’s disease pathobiology.Here, we demonstrate that in non-demented older individuals at risk for AD and in mild AD participants, HFABP is associated with volume loss in brain areas selectively affected in the earliest stages of AD. However, we found that the relationship between HFABP and brain atrophy is present only among individuals with low CSF AβOur findings also indicate that rather than simply representing a generalized marker of neuronal degeneration, elevated CSF levels of HFABP may reflect central nervous system lipid dyshomeostasis. Consistent with prior studies ,18, we fOur results suggest that the relationship between neuronal lipid biology and neurodegeneration may be influenced by amyloid pathology even after controlling for the effects of APOE ϵ4. An important aspect of our findings is the specific relationship between HFABP and Aβ deposition, where volume loss occurs only in the presence of elevated HFABP and decreased Aβ (i.e. increased Aβ deposition in the brain). A growing body of experimental evidence from various model systems indicates that phospholipids play an integral role in regulating amyloidogenesis. Enriched cholesterol and lipid microenvironments ('lipid rafts’) within the plasma membrane and the mitochondria-associated endoplasmic reticulum membrane promote γ-secretase activity resulting in increased Aβ production ,21. In cOne potential concern is that our current findings do not explain the previously noted relationships observed between HFABP and non-AD neurodegenerative diseases such as vascular dementia, Creutzfeldt-Jakob disease, Parkinson’s disease, and dementia with Lewy bodies ,24-27. TA limitation of our study is its observational nature, which precludes conclusions regarding causation. Our results cannot differentiate whether elevated HFABP causes, results from, or is simply correlated with amyloid deposition and volume loss. Additionally, our findings require further validation on a larger, independent population-based cohort.In summary, our results suggest that CSF HFABP reflects intra-cranial lipid biology and associates with Aβ-associated neurodegeneration irrespective of tau. Clinically, our findings suggest that HFABP may represent an important modifier of progression from amyloid deposition to neurodegeneration. Considered together with our prior findings ,34, thisWe evaluated cognitively normal older adults (HC = 90), individuals diagnosed with amnestic MCI n = 139), and probable AD (n = 66) from the Alzheimer’s Disease Neuroimaging Initiative . Using previously proposed CSF cutoffs 19 we classified participants based on low (<192 pg/ml) and high (>192 pg/ml) Aβ1–42 levels and high (>23 pg/ml) and low (<23 pg/ml) p-tau181p levels.We evaluated baseline CSF HFABP levels analyzed using a multiplex-based immunoassay panel. This immunoassay panel, based on Luminex xMAP immunoassay technology and developed by Rules Based Medicine (MyriadMBM), measures a range of lipid, inflammatory, metabolic, and other AD-relevant indices . We also examined baseline CSF Aβ1-weighted MRI scans. We performed quantitative volume and surface-based analysis of all baseline MRI scans using automated region-of-interest (ROI) labeling techniques [We examined 1205 Tchniques ,36, primchniques   or absence ("non-carriers") of at least one ϵ4 allele of apolipoprotein E (APOE ϵ4), diagnostic status (AD vs MCI vs HC), and disease severity (CDR-Sum of Boxes score at baseline).For the primary analyses, we used linear mixed effects models to examine the associations of CSF HFABP, CSF AβSpecifically, our initial model was:3) and Δt is the change in time from baseline MRI scan (years).Here, Δv is the entorhinal cortex or AD-vulnerable ROI thickness change from baseline . Dr. Brewer also receives research support from General Electric and Janssen Alzheimer Immunotherapy.Dr. Kaj Blennow has served on the advisory boards for Innogenetics, Lilly, Pfizer and Roche.Drs. D, McE, H and D were involved with conception, design, and interpretation of data. Drs. D, McE, H and D provided general overall supervision of the study. Drs. D and T were involved with data analysis. Drs. D, B, McE, A, and D acquired funding. All authors contributed to drafting and critical revision of the manuscript and have given final approval of the version to be published.Methods and results.Click here for file


Veb1 is commonly inserted into the variable region of different class 1 integrons in which it is always associated with a downstream-located aadB gene cassette encoding an aminoglycoside adenylyltransferase. In Pseudomonas aeruginosa, the majority of veb1-containing integrons also carry an insertion sequence, IS1999 that is inserted upstream of the veb1 gene cassette and disrupts the integron specific recombination site, attI1. Investigation of the recombination properties of the sites surrounding veb1 revealed that insertion of IS1999 reduces significantly the recombination frequency of attI1 and that veb1 attC is not efficient for recombination in contrast to aadB attC. Subsequent sequence optimisation of veb1 attC by mutagenesis, into a more consensual attC site resembling aadB attC, successfully improved recombination efficiency. Overall, this work gives some insights into the organisation of veb1-containing integrons. We propose that IS1999 and the nature of veb1 attC stabilize the veb1 gene cassette environment likely by impairing recombination events upstream or downstream of veb1, respectively.The  Anruginosa  [28]. Moveb1 environment was puzzling given the fact that the variable region of an integron is normally in constant evolution since it is subject to gene cassette rearrangement, loss and acquisition veb1 vicinity . This work revealed that veb1 attC and the disrupted attI1 site of IS1999-containing integrons are not efficient for recombination and consequently might preserve the associations veb1-aadB and IS1999-veb1 from being disrupted, respectively.The stability of the attC and either attI1 or another attC site from another gene cassette Integrase-mediated recombination involving integrons located on multicopy plasmids can generate different recombination products including: (i) free circular DNA molecules comprising one or more gene cassettes, resulting from recombination between the gene cassette veb1-containing constructs was tested .The relative recombination efficiency of the sites present within different ic probe . Our expattI1 site, the veb1 cassette and a truncated aadB cassette, while pAttI.veb.aadB contains, in addition to attI1 and veb1, a full-length aadB cassette. While attI1 and veb1 attC sites are theoretically the only sites available for recombination in pAttI.veb, pAttI.veb.aadB offers an additional recombination site, aadB attC. Recombination between attI1 and veb1 attC leading to either veb1 cassette excision, duplication or plasmid cointegration should produce, after BspE1 digestion, a veb1-containing DNA fragment of 1.1-kb. . However, no such product was observed with pAttI.veb and pAttI.veb.aadB, suggesting that veb1 attC is not efficient for recombination (E. coli (pAttI.veb.aadB) (Plasmid pAttI.veb contains a full eb.aadB)  lane 2. blaVEB-1 specific primers. These primers are located on each side of the BspE1 restriction site and specifically allow for amplification of veb1-containing DNA fragments that were linearized (excised gene cassettes) or released upon BspE1 restriction from cointegrates and plasmids with veb1 duplication .A more sensitive approach using PCR amplification was used. Circular DNA molecules were purified, digested and separated on an agarose gel as before but agarose gel slices were cut at the expected migration of the 1.1-kb and 1.6-kb DNA fragments. Gel extracted DNAs were then subjected to PCR using outward-directed E. coli (pAttI.veb) and E. coli (pAttI.veb.aadB) yielded a 0.7-kb PCR product  yielded a 1.3-kb fragment  at the junction with the veb1 gene cassette were tested for recombination (E. coli (pVeb.aadB) albeit at low levels (aadB attC and a secondary site (1α) instead of the expected GTTAGCG motif of the veb1 gene cassette  found in the most easily excisable attC sites aadB attC worked efficiently in our recombination assays. Folding of the bottom strand of veb1 attC revealed a characteristic secondary structure with the three structural elements common to attC sites veb1 attC presents two striking differences as compared to aadB attC: (i) the veb1 attC site (133-bp) has a longer variable terminal structure (VTS) and (ii) the two putative integrase binding sites 2L and 2R diverge from the attC site consensus sequence   . In thisr levels  lane 6 sttC site .veb attC is not optimal for recombination. The veb1 attC site was further modified to determine whether the long intermediate region located between the 2L and 2R integrase binding sites or the 2L and 2R sequences that diverged from an attC consensus sequence was responsible for the low recombination efficiency of veb attC. Two different veb1 attC sites named veb1 attCΔ and veb1 attC' were generated. The DNA segment located between the 2L and 2R binding sites from veb1 attC was reduced to 20-bp as found in aadB attC giving rise to veb1 attCΔ  remain adjacent to veb1 (E. coli (pAttI.IS.veb.aadB), veb1-containing recombination products that resulted from recombination between the disrupted attI1 and aadB attC were detected by hybridization  followed by an open reading frame, orfA, of unknown function.Activities of the sites present within different Veb1-containing constructs  were introduced into a rifampin sensitive E. coli containing the integrase expressing plasmid and plasmid R388. Upon induction of integrase expression, cointegrates resulting from recombination between the plasmid-located veb-1 integrons and the recipient integron In3 located on plasmid R388 were predominantly formed. Plasmid R388 and cointegrates were transferred by conjugation into E. coli DH10B-Rif (rifampin resistant) and cointegration frequencies were measured as ratios of cointegrates (CazR-RifR) to total R388 transconjugants (TmpR-RifR) (pR-RifR) .veb1 attC is the only site available for recombination with either attI1 or an attC site from In3. Cointegrates (pVeb::R388) were selected at a very low frequency (4.41×10−5) close to the limit of detection of our assay, thus reflecting the inefficiency of veb1 attC for recombination. As compared to pVeb, the presence of aadB attC in pVeb.aadB, led to a 50-fold increase in the cointegration frequency, confirming that aadB attC is more efficient for recombination than veb1 attC. Comparison between (pVeb::R388) and (pAttI.IS.veb::R388) revealed that the disrupted attI1 site is 3-times more efficient for recombination than the 7-bp core site from pVeb. However, insertion of IS1999 into attI1 (pAttI.IS.veb) led to a 6-fold decrease in the cointegration frequency, as compared to plasmid pAttI.veb. Sequence optimisation of veb1 attC into veb1 attC* led to an almost 20-fold increase in the cointegration frequency of pAttI.veb* as compared to pAttI.veb. Similar cointegration frequencies were obtained with plasmids pVeb.aadB, pAttI.veb.aadB, pAttI.IS.veb.aadB and pAttI.veb*.aadB. All these constructs contain the highly efficient recombination site aadB attC that was likely mainly involved in cointegration.Plasmid cointegration was IntI1 mediated since CazR-RifR cointegrates were only recovered from strains over-expressing the integrase. In plasmid pVeb, attI1, veb1 attC, veb1 attC* or aadB attC from the veb1-containing plasmids) were involved in the recombination. As expected, all the pVeb::R388 and pVeb.aadB::R388 cointegrates involved veb1 attC and aadB attC, respectively. Cointegrates pAttI.veb::R388 and pAttI.IS.veb::R388 were formed by recombination using attI1 or veb1 attC. All the pAttI.veb.aadB::R388 and pAttI.IS.veb.aadB::R388 cointegrates involved aadB attC and retained the aadB cassette in association with veb1. All the pAttI.veb::R388 cointegrates involved veb1 attC* and the pAttI.veb.aadB::R388 cointegrates had systematically lost the aadB cassette. Since aadB cassette excision could occur before or after cointegration, we could not determine whether pAttI.veb.aadB::R388 arose by cointegration involving veb1 attC* or aadB attC.For each plasmid tested, several cointegrates were analyzed by PCR to determine which sites  and occurs at a lower frequency. Despite most colonies (>95%) were resistant to tetracycline, few CazR-RifR-TetS transconjugants were identified. CazR-RifR-TetS transconjugants were recovered only from donor cells containing pAttI.veb.aadB, pAttI.veb* and pAttI.veb*.aadB plasmids with similar frequencies  excluding recombination between homologous sequences. Thus, despite plasmids share some sequence identity, cointegration is unlikely to have occurred by homologous recombination but is truly the result of site-specific recombination mediated by the integrase.Recombination activities of the sites surrounding the attCveb1 attC found within the veb1 gene cassette, is not efficient for recombination implying that veb1 alone is not highly mobilizable. Our cointegration experiments also revealed that aadB attC is favored over veb1 attC as it is almost exclusively involved in recombination when both gene cassettes are present on a plasmid. Despite no selection, aadB remained associated to wild type veb1 in all the cointegrates tested, while gene cassettes that are unnecessary for bacterial growth, are often unstable and lost from the variable region when IntI1 is overexpressed aadB would be rarely excised since aadB excision require the involvement of veb1 attC. Stability of the association veb1-aadB within integrons is likely preserved by the nature of veb1 attC, which impairs not only aadB excision but also new insertions of gene cassettes in between them. Our cassette integration assay in plasmid R388 showed that the association veb1-aadB is to the benefit of veb1, which is mobilized along with aadB thanks to the high recombination efficiency of aadB attC.Dissemination and acquisition of a single gene cassette within the variable region of integrons is the result of integrase-mediated recombination between two different sites, one of which is the gene cassette aadB attC site is 50 times more efficient for recombination than veb1 attC. Sequence optimisation of veb1 attC into a more consensual attC site resembling aadB attC (veb1 attC*) improved drastically the recombination efficiency and allowed independent excision of aadB from veb1-containing integrons. This experiment revealed also that the features responsible for the weak activity of veb1 attC reside within the DNA sequence located between the 1L and 1R integrase binding sites. Two major differences between veb1 attC and aadB attC were found in this region and concerned: (i) the 2L and 2R integrase binding sites, which sequences in veb1 attC diverged from the consensus and (ii) the length of the variable terminal structure (VTS). However, modification of one or the other in veb1 attC was not sufficient to improve recombination activity. While VTS seem to have a minor role in the attC recombination efficiency, other structural features including the unpaired central spacer (UCS) bulge shape and two extrahelical bases (EHBs) appear to be important for recognition and recombination rate aadB attC, veb1 attC presents slightly larger UCS. Also, while the EHBs in aadB attC are 6 nucleotides apart (T-N6-G) as found in the attC sites flanking cassettes that are efficiently excised by IntI1 veb1 attC contains only one EHB (G). It has been recently demonstrated that the identity and spacing of the EHBs in the attC sites has a pronounced effect on the efficiency of cassette excision veb1 attC remains to be established. It is also possible that the recombination efficiency of veb1 attC is not affected by only one of these features but by several of them.The attI1 site containing the four integrase binding sites was required for high efficiency recombination with attC and that progressive 5′ deletions reaching closer to the 7-bp core site of attI1 leads to decreased recombination rates 1999 into attI1 displaces only the weak integrase binding site located in DR2. Accordingly, the disrupted attI1 is still sufficient to support recombination albeit at a lower frequency than a full attI1 site. Analysis of the bases from IS1999 replacing part of attI1 did not reveal any obvious 7-bp core site sequence, which could have compensated for the loss of the fourth integrase binding site. Also, by reducing the recombination rate of attI1, IS1999 likely increases the stability of veb1 and consequently aadB at the first positions within the variable region by impairing integration of new gene cassettes upstream of veb1 and excision of veb1-aadB. However, since the disrupted attI1 still supports recombination, new gene cassettes could be inserted at the first position and benefit from the additional promoter Pout located in IS1999 for their expression Several studies have shown that a full attI1 or attC) and non-specific secondary sites conforming to the consensus GNT at a very low frequency attI1 to the 7-bp core site is insufficient for determining recombination specificity alone and leads to the formation of cointegrates owing to recombination with secondary sites aadB attC is able to recombine with the secondary site (GTTAAGT) located within the blaVEB-1 gene. This event resulting in a truncated veb1 gene cassette is extremely rare since it has been detected only once in our repeats. The region that is truncated in blaVEB-1 encodes the first eleven amino acids of the pre-enzyme VEB-1 and includes the translation initiation codon and most of the signal peptide. Even upon insertion in an environment providing transcription and translation signals, it is unlikely that the protein encoded by the truncated veb1 cassette would be functional in vivo since it would not be properly targeted to the periplasm.Several studies have demonstrated that integrase is also able to catalyse recombination between one specific site  was used as bacterial host in electroporation experiments. The conjugative plasmid R388  includes an integron (In3) that contains the dfrB2 cassette conferring resistance to trimethoprim (Tmp) followed by an open reading frame, orfA, of unknown function intI1 gene under the control of an IPTG-inducible synthetic Ptrc promoter. E. coli DH10B harboring various plasmids and E. coli DH10B-Rif (rifampin resistant) were used for conjugation experiments. The low-copy number cloning vector, pBBR1MCS.3 was used for cloning experiments Bacterial strains and plasmids used in this study are listed in The antimicrobial agents and their sources were as follows: ceftazidime, GlaxoSmithKline ; rifampin, Aventis ; trimethoprim and tetracycline, Sigma ; kanamycin, Euromedex .Circular DNA molecules were extracted using Plasmid Maxi Kits  according to the instructions of the manufacturer. Extractions of whole-cell DNAs were done as described elsewhere Taq and Pfu DNA polymerases were from Roche Diagnostics  and Promega Corporation , respectively. PCR experiments veb1 cassette or derivatives into the In3 integron, plasmids from independent E. coli DH10B-Rif (R388::veb1) transconjugants were extracted and amplifications using combination of primers VEBINV2-TMPB, VEBINV2-ORFAB, VEBINV2-QACEB, and TMPA-VEBINV3 were performed. The aadB cassette was detected by PCR amplification using the AADBF and AADBB primers.veb1-containing plasmids were analyzed. Amplifications using combination of primers T3-5′CS, T7 promoter-3′CS and VEBINV2-3′CS were performed in order to determine which recombination sites were involved in the formation of the cointegrates.For each construct tested, five cointegrates resulting from cointegration between R388 and aph(3′)-IIa) flanked by transcriptional and translational termination sequences, into the HindIII site from the multiple cloning site of p112 plasmid (pTRC99A::intI1) intI1 gene from p112.Kan, filling in its ends with Pfu DNA polymerase and followed by self ligation. The inserts of the recombinant plasmids pVeb and pVeb.aadB, corresponded to fragments of 1.6-kb (containing the veb1 cassette) and 1.7-kb (containing the veb1 and aadB cassettes) that were amplified with the pairs of primers VEBCASF/AADBB and VEBCASF/3′CS, respectively and genomic DNA of P. aeruginosa 14 as template . The inserts of the recombinant plasmids pAttI.veb and pAttI.veb.aadB corresponded to fragments of 2-kb (containing an entire attI1 site and the veb1 cassette) and 2.1-kb (containing an entire attI1 site and the veb1 and aadB cassettes) that were amplified with the pairs of primers INTIN/AADBB and INTIN/3′CS, respectively and genomic DNA of P. aeruginosa 14 as template . The inserts of the recombinant plasmids pAttI.IS.veb and pAttI.IS.veb.aadB corresponded to fragments of 3.4-kb  and 3.5-kb  that were amplified with the pairs of primers INTIN/AADBB and INTIN/3′CS, respectively and genomic DNA of P. aeruginosa 15 as template . PCR products were purified prior to cloning into the SmaI-restricted pBBR1MCS.3 vector.T4 DNA ligase, and restriction endonucleases were used according to the recommendations of the manufacturer . The plasmid p112.Kan was constructed by inserting a HindIII-digested omega fragment (ΩKm) from plasmid pHP45Ω-Km veb1 attC site  were generated. Creation of the veb1 attC* site was performed by using the attCVEB1 and attCVEB2 primers that anneal to the beginning of the veb1 attC site up to the inverse core site and to the beginning of the aadB gene cassette (core site), respectively. These primers have floating 5′ ends, each corresponding to a half of the aadB attC site, and have in common the portion containing the BsaHI restriction site . The recombinant plasmid pAttI.veb* containing veb1 attC* site was constructed as follows: a 1.5-kb fragment amplified with the pair of primers T7 promoter-attCVEB1 and pAttI.veb as template was digested with SpeI-BsaHI. A 0.7-kb fragment was amplified with the primers attCVEB2-T3 and pAttI.veb as template and was digested with BsaHI-PstI. Then, the digested inserts were purified and mixed in a three-way ligation with the PstI-SpeI-restricted pBBR1MCS.3 vector generating pAttI.veb* . The pAttI.veb.aadB and pAttI.veb* recombinant plasmids were digested with SacI endonucleases. The SacI insert of pAttI.veb* was cloned into the SacI-restricted pAttI.veb.aadB plasmid generating pAttI.veb*.aadB .Three different modifications of the Δ (containing veb1 attCΔ) was constructed as follows: a 1.5-kb fragment amplified with the pair of primers T7 promoter-Shortattc1 and pAttI.veb as template was digested with SacI. A 0.6-kb fragment was amplified with the primers Shortattc2-T3 and pAttI.veb as template and was digested with KpnI. Then, the digested inserts were purified and mixed in a three-way ligation with the SacI-KpnI-restricted pBBR1MCS.3 vector generating pAttI.vebΔ . The recombinant plasmid pAttI.veb' (containing veb1 attC') was constructed as follows: a 1.6-kb fragment amplified with the pair of primers T7 promoter-attc2L and pAttI.veb as template was digested with NdeI. The resulting 0.9-kb and 0.7-kb fragments were purified and subsequently digested with AvaI and SacI, respectively. A 0.3-kb fragment was amplified with the primers attc2R-T3 and pAttI.veb as template and was digested with AvaI-XhoI. Then, the digested inserts were purified and mixed before ligation with the SacI-XhoI-restricted pAttI.veb plasmid generating pAttI.veb' .Similarly, pAttI.vebE. coli DH10B as previously described Δ, and pAttI.veb' were retained for further experiments .Ligation products were electroporated first into Sequencing was performed on both strands using laboratory-designed primers on an ABI PRISM 3100 automated sequencer .Δ, and pAttI.veb' were freshly electroporated into E. coli DH10B (p112.Kan); the plasmid pAttI.veb.aadB was also freshly electroporated into E. coli DH10B (pTRC99A.Kan) before each experiment. Strains were grown to stationary phase in TS-broth containing tetracycline 15 µg/ml and kanamycin 30 µg/ml. The cultures were then diluted 1000-fold into 200 ml TS-broth containing tetracycline 15 µg/ml, kanamycin 30 µg/ml and grown to exponential phase (OD 600 nm: 0.5). Integrase expression was then induced for 2 h by adding IPTG at a final concentration of 0.6 M.The recombinant plasmids pVeb, pVeb.aadB, pAttI.veb, pAttI.veb.aadB, pAttI.IS.veb, pAttI.IS.veb.aadB, pAttI.veb*, pAttI.veb*.aadB, pAttI.vebveb-1 containing molecules that are the result of a recombination event between attI1 (or disrupted attI1) and an attC site and gives an insight into their recombination efficiency. Total circular DNA content was extracted from E. coli DH10B (p112.Kan) strains harboring recombinant plasmids pVeb, pVeb.aadB, pAttI.veb, pAttI.veb.aadB, pAttI.IS.veb, pAttI.IS.veb.aadB, pAttI.veb*, pAttI.veb*.aadB, pAttI.vebΔ, and pAttI.veb' and E. coli DH10B (pTRC99A.Kan) harboring pAttI.veb.aadB after IPTG-induction. Five µg of circular DNA extracts were digested in duplicate with 10 units of BspEI, which cleaves at a unique site located in the veb1 cassettes . Digested DNA samples were then loaded on two agarose gels  and electrophoresed at 45 V for 16 h using Tris-borate-EDTA running buffer This assay allows for the detection of + Hybond nylon membrane (Amersham Biosciences). Southern blot hybridizations blaVEB-1 that was amplified using primers VEB1A/VEB1B and whole-cell DNA of P. aeruginosa 14 as template.The first agarose gel was used for hybridization experiments as follows: The bottom section of the gel that contained small BspEI excision products (<2-kb), was cut and transferred onto a Nveb1-containing fragments. Since DNA amounts were very low and could not be detected visually after ethidium bromide staining, DNA location was spotted by superposing the autoradiography film obtained from the first gel after hybridization. Gel slices were cut with a separate disposable scalpel to avoid sample cross-contamination. DNA was extracted using Qiaquick Gel extraction kit (Qiagen) and subjected to PCR amplification using the VEBINV3-VEBINV2 outward-directed primers. These primers are located on each side of the BspE1 restriction site, and thus allowed amplification of the recombinant junction that was created upon recombination .The second agarose gel was used to extract BspEI digested veb1 gene cassettes into the class 1 integron In3 carried by the conjugative plasmid R388 or cointegration between veb1-containing plasmids and R388 was investigated using a mating-out assay as follows. The recombinant plasmids pVeb, pVeb.aadB, pAttI.veb, pAttI.veb.aadB, pAttI.IS.veb, pAttI.IS.veb.aadB, pAttI.veb*, pAttI.veb*.aadB and pBBR1MCS.3 cloning vector were freshly electroporated into E. coli DH10B ; the plasmid pAttI.veb.aadB was freshly electroporated into E. coli DH10B . Twenty-four hours after electroporation, three single colonies (for each plasmid tested) were independently cultured overnight at 37°C in 10 ml TS broth containing tetracycline, 15 µg/ml and kanamycin, 30 µg/ml. The overnight cultures were diluted 10-fold in fresh TS broth without antibiotic and were cultured under low agitation at 37°C for 1 h 30, prior to a 2 h IPTG-induction (induction of intI1 expression). Mating was performed by incubating 800 µl of recipient E. coli DH10B-Rif and 200 µl of the tested strain under low agitation at 37°C for 3 h. Subsequently, the mating mixture was vigorously vortexed, placed on ice, and plated. One hundred microliters aliquots of serial ten fold dilutions were plated onto trimethoprim- (25 µg/ml) and rifampin- (200 µg/ml) containing plates and ceftazidime- (15 µg/ml) and rifampin- (200 µg/ml) containing plates. For E. coli DH10B  aliquots were plated onto trimethoprim- (25 µg/ml) and rifampin- (200 µg/ml) containing plates and tetracycline- (15 µg/ml) and rifampin- (200 µg/ml) containing plates. The cointegration frequency was calculated by dividing the number of ceftazidime and rifampin resistant (CazR-RifR) transconjugants by the number of trimethoprim and rifampin resistant (TmpR-RifR) transconjugants. For each plasmid tested, up to 484 CazR-RifR colonies were further screened for tetracycline susceptibility in order to discriminate cointegrates from precise veb1 cassettes integration into In3.Precise integration of E. coli DH10B  the cointegration frequency was calculated by dividing the number of tetracycline and rifampin resistant (TetR-RifR) transconjugants by the number of TmpR-RifR transconjugants.For Figure S1Schematic representation of the plasmid constructs used in this study. All constructs were cloned into the multiple cloning site of the pBBR1MCS.3 shuttle-vector represented with a solid line. The coding regions are shown as boxes with an arrow indicating the orientation of their transcription. Dashed lines indicate truncated genes. The black diamond, white, grey, white with black dots, and black with white dots circles represent attI1, veb1 attC, aadB attC, veb1 attCΔ, and veb1 attC', respectively. The veb1 attC* site is highly similar to the aadB attC site and is also represented by a grey circle. The IS1999 inverted repeats are shown by empty triangles. The broken arrows indicate the Pc, Pout and Plac promoters. Restriction sites used for cloning are indicated on the pAttI.veb.aadB representation. Small arrows (1 and 2) located on each side of the BspEI restriction site represent the positions of the VEBINV3 and VEBINV2 primers, respectively.(TIF)Click here for additional data file.Figure S2Recombination products obtained with pAttI.veb. A) Schematic representation of plasmid pAttI.veb. Construct was made in the pBBR1MCS.3 shuttle-vector represented with a solid line. The coding regions are shown as boxes with an arrow indicating the orientation of their transcription. Dashed lines indicate truncated genes. The black diamond and white circle represent attI1 and veb1 attC, respectively. The broken arrow indicates the Pc promoter. Small arrows (1 and 2) located on each side of the BspEI restriction site represent the positions of the VEBINV3 and VEBINV2 primers, respectively. B–D) Recombination products. Each possible recombination product is shown. The sites involved in the recombination and the size of the relevant BspEI digestion products are indicated. B) Veb1 cassette excision product. C) Cointegrates. Double lines represent scale breaks. D) Gene duplications. Veb1 duplication can arise from integration (attI x veb1 attC or veb1 attC x veb1 attC) of an excised veb1 gene cassette, or by resolution (veb1 attC x attI1 or attI1 x attI1) of cointegrates. In any case, the 1.1-kb fragment is only recovered after BspEI digestion when recombination between attI1 and veb1 attC has occurred.(TIF)Click here for additional data file.Figure S3Recombination products obtained with pAttI.veb.aadB. A) Schematic representation of plasmid pAttI.veb.aadB. Construct was made in the pBBR1MCS.3 shuttle-vector represented with a solid line. The coding regions are shown as boxes with an arrow indicating the orientation of their transcription. Dashed lines indicate truncated genes. The black diamond, white and grey circles represent attI1, veb1 attC and aadB attC, respectively. The broken arrow indicates the Pc promoter. Small arrows (1 and 2) located on each side of the BspEI restriction site represent the positions of the VEBINV3 and VEBINV2 primers, respectively. B–D) Recombination products. The different veb1-containing recombination products are represented. The sites involved in the recombination and the size of the relevant BspEI digestion products are indicated. B) Veb1-containing excision product. C) Cointegrates. Double lines represent scale breaks. D) Gene duplications. Veb1 duplications can arise from integration of an excised veb1-containing gene cassette or by resolution of cointegrates. In any case, the 1.1-kb and 1.6-kb fragments are only recovered after BspEI digestion when recombinations attI1 x veb1 attC and attI1 x aadB attC have occurred, respectively.(TIF)Click here for additional data file.Table S1Sequence of primers used in this study.a Nucleotides that are complementary to the aadB attC site are underlined; b Restriction sites are bolded; c Nucleotides that are mutated are boxed in grey. Supplemental references are as follows: (1). Naas T, Coignard B, Carbonne A, Blanckaert K, Bajolet O, et al. (2006) VEB-1 Extended-spectrum beta-lactamase-producing Acinetobacter baumannii, France. Emerg Infect Dis 12: 1214–1222. (2). Girlich D, Naas T, Leelaporn A, Poirel L, Fennewald M, et al. (2002) Nosocomial spread of the integron-located veb-1-like cassette encoding an extended-pectrum beta-lactamase in Pseudomonas aeruginosa in Thailand. Clin Infect Dis 34: 603–611. (3). Martinez E, de la Cruz F (1990) Genetic elements involved in Tn21 site-specific integration, a novel mechanism for the dissemination of antibiotic resistance genes. EMBO J 9: 1275–1281.(DOCX)Click here for additional data file.


This study aimed to establish whether PC12 cells were responsive to fetal CSF and therefore whether they might be used to investigate CSF physiology in a stable cell line lacking the time-specific response patterns of primary cells previously described.Fetal cerebrospinal fluid (CSF) contains many neurotrophic and growth factors and has been shown to be capable of supporting viability, proliferation and differentiation of primary cortical progenitor cells. Rat pheochromocytoma PC12 cells have been widely used as an In vitro assays of viability, proliferation and differentiation were carried out after incubation of PC12 cells in media with and without addition of fetal rat CSF. An MTT tetrazolium assay was used to assess cell viability and/or cell proliferation. Expression of neural differentiation markers (MAP-2 and β-III tubulin) was determined by immunocytochemistry. Formation and growth of neurites was measured by image analysis.PC12 cells differentiate into neuronal cell types when exposed to bFGF. Viability and cell proliferation of PC12 cells cultured in CSF-supplemented medium from E18 rat fetuses were significantly elevated relative to the control group. Neuronal-like outgrowths from cells appeared following the application of bFGF or CSF from E17 and E19 fetuses but not E18 or E20 CSF. Beta-III tubulin was expressed in PC12 cells cultured in any media except that supplemented with E18 CSF. MAP-2 expression was found in control cultures and in those with E17 and E19 CSF. MAP2 was located in neurites except in E17 CSF when the whole cell was positive.in vivo for the promotion of normal brain development. There were significant differences in the effects on PC12 cells compared to primary cortical cells. This suggests there is an interaction in vivo between developmental stage of cells and the composition of CSF. The data presented here support an important, perhaps driving role for CSF composition, specifically neurotrophic factors, in neuronal survival, proliferation and differentiation. The effects of CSF on PC12 cells can thus be used to further investigate the role of CSF in driving development without the confounding issues of using primary cells.Fetal rat CSF supports viability and stimulates proliferation and neurogenic differentiation of PC12 cells in an age-dependent way, suggesting that CSF composition changes with age. This feature may be important  The central nervous system develops around a fluid filled tube, the neural tube. Initially the tube forms around amniotic fluid which is then modified by secretions from a structure in the mesencephalon which transports blood components into the neural tube fluid . This hain vitro cultures of the PC12 cell line.CSF is the major element forming the external environment for germinal matrix, stem and progenitor cells of the developing cortex, containing a high concentration of cytokines, growth factors and other proteins secreted by the choroid plexus, and which acts as a growth medium for brain development ,12,27-29in vitro model for neuronal differentiation. These cells differentiate into dopaminergic neurons when cultured with certain growth factors, including nerve growth factor (NGF), basic fibroblastic growth factor (b-FGF), insulin growth factor-I (IGF-I), glial cell line-derived neurotrophic factor (GDNF), epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) [PC12 cells are frequently used as an  (TGF-α) -44. It sWistar rats were bred in house in the research facility of the Department of Biology, Tarbiat Moallem University following ethical review of the project by the animal use committees of both The University of Manchester and Tarbiat Moallem University. They were kept in large rat boxes at constant temperature and 12hour light/dark cycle with free access to food and water. Individual male and female rats were paired in mating cages and checked regularly for the presence of a vaginal plug which was taken as an indication of successful mating and the day noted as embryonic day 0 (E0). Embryonic age was calculated from that day. At a particular time point pregnant dams were euthanized by cervical dislocation, the uterus rapidly removed onto ice and fetuses dissected out onto ice. Each pregnant dam usually produced between 10–15 fetuses.CSF was collected from the cisterna magna of rat fetuses at E17, E18, E19, and E20 using glass micropipettes and capillary action without aspiration. Aspiration invariably resulted in bleeding and contamination of the samples. Fetuses were positioned with heads flexed down onto the chest to allow penetration into the cisternal cavity through the skin and underlying muscle. Samples containing undesirable blood contamination, visualised as a pink colour in the fluid, caused by damaging a blood vessel within the cisternal cavity, were discarded. All samples were collected into sterile microtubes and centrifuged at 14,000 rpm to remove cells or debris from the fluid, and the supernatant was transferred into another sterile tube. These samples were stored at −80°C until use. The volume of CSF collected from each fetus by this method was between 5 and 50 μl and samples were pooled for each experiment. At least three litters provided six independent pooled samples  for each age of CSF tested.Total protein concentration in each pooled CSF sample was determined by the Bio-Rad protein assay , based on the Bradford dye procedure. The absorbance of samples was measured at 595 nm wavelength. Each pooled sample was analysed for each age tested.2 and maintained at 37°C. PC12 culture medium was refreshed in a 2/3 ratio every 2 days. For tests, 100 μl of cells at 4 × 104 cells/ml (4000 cells/well) in RPMI1640 medium were plated into poly-D-lysine coated 96-well plates and cultured in a RPMI1640 medium without serum for 24 hours and then supplemented with CSF (E17-20) (10% v/v) or bFGF (10 ng/ml) for 7 days. bFGF was used as a positive promoter of PC12 cell proliferation and differentiation into neuronal phenotypes to compare to the effects of CSF. After one week, cells were photographed and then prepared for morphological examination and immunocytochemical staining. Three wells were used for each pooled CSF sample giving a total of at least nine wells per CSF age tested. An additional three wells per pooled sample were used for the MTT assay. Cells were photographed at the end of each experiment using phase contrast optics. Cells were then fixed in 4% paraformaldehyde and immunostained for β-tubulin and MAP2 using monoclonal antibodies , visualised with FITC conjugated goat-anti mouse secondary antibodies and photographed using fluorescence microscopy. Controls were stained with vehicle solution without the primary antibody. At least six wells were stained for both β-tubulin and MAP2 expression and at least three wells were used as negative controls.PC12 cells were cultured in RPMI1640 medium  containing 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin. The cultures were kept in a humidified incubator with 5% COFor each culture condition, cells in individual wells were photographed with phase-contrast optics  to visualize outgrowths from the cells. Measurements were made using ImageJ software (NIH). A neurite was counted when a cellular process was longer than the diameter of the cell body. The average length of neurites was calculated from measurements of 10 cells in each of 6 wells for each age of CSF tested.Cell viability and/or proliferation was quantitatively determined by the MTT method using a colorimetric assay at the end of 7 days in control medium or medium containing CSF or bFGF. MTT -2,5-diphenyltetrazolium bromide), is a yellow tetrazolium dye that responds to metabolic activity. Reductase enzymes in living cells reduce MTT from a pale yellow colour to dark blue formazan crystals. Cells were plated at 4000 cells per well as described above. In separate experiments cells were plated at 2000 cells per well with no difference in proliferation or differentiation results (data not shown). At 24 h, prior to addition of CSF or bFGF control plates were analysed for starting number of cells. Experimental and control media plates were left for a further 7 days and then analysed for proliferation. Wells were incubated with MTT (5 mg/mL in PBS) for 3 h at 37°C. In order to make formazan crystals soluble, 0.04 N HCl, prepared in isopropranolol, was added. The absorbance of the formazan product was determined at a wavelength of 570 nm using a plate reader.For immunocytochemistry, after three washes with PBS for 5 min, cells were fixed in 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 for 30 min at room temperature and subsequently blocked with 5% BSA in TPBS (Tween 20 in PBS) for 1 h at room temperature. Cells were incubated at 4°C overnight in the presence of either anti-beta III tubulin mAb (1:50 dilution) or anti-MAP2 mAb (1:50 dilution). The following day, after three washes with TPBS, FITC-conjugated goat anti-mouse IgG  was added at room temperature for 1 hr. Cells were then washed and cellular nuclei were counterstained with propidium iodide (Sigma-Aldrich). Photomicrographs were taken with a florescence microscope .p values of <0.05.All values are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using the one-way ANOVA and Kruskal-Wallis test, and significance was accepted for p < 0.05, Figure CSF of rat fetuses aged E17 had a mean total protein concentration of 3.65 ± 0.26 mg/ml, which was significantly higher than that of E20 CSF  was obtained when cells were cultured with the medium supplemented with CSF of any age indicating greater viability of cells compared with that in media alone. E18 CSF gave a significant increase in viability over that seen in media alone which fits with our previous data from primary cortical cells where E18 and E19 CSF gave increased proliferation of E20 cells [Figure 20 cells . For thePhase-contrast images of cultured PC12 cells are shown in Figure We found beta III tubulin expression in PC12 cells grown in control media as well as in media supplemented with E17, E19 and E20 CSF but not in E18 CSF Figure  By contrp < 0.001), or CSF from E17 (p < 0.01) and E19 (p < 0.001) for 7 days  has a high protein concentration of around 3 mg/ml that declined to 2 mg/ml by E20. CSF when added to cultures of PC12 cells had different effects on survival, proliferation and neuronal differentiation depending on age. In contrast to the control groups, samples of CSF at all ages tested, gave a greater formazan absorbance reading indicating improved cell survival and/or proliferation. This was only significant with E18 CSF indicating a greater effect of CSF at this age. Interestingly we previously showed a greater proliferation of primary rat cortical cells in both E19 and E18 CSF over both controls and E17 or E20 CSF ,46. The versus the contributions made by the changes in CSF composition with age.In our experiments, we used cisternal CSF which is likely to contain both proliferation, differentiation and migration signals as it contains all the additions to CSF that are made as the fluid passes through the ventricles. It would of great value to test ventricular CSF and compare its effects since our previous arguments suggest that this may only contain proliferation and possibly differentiation signals but not migration signals ,7,23,25.in vivo by the CP located in the lateral ventricles with an additional 10% and 5% secreted by the CP in the third and fourth ventricles respectively [in vitro survival, proliferation and neuronal differentiation of PC12 cells are dependent on certain growth factors which must also be present in the CSF to elicit the effects observed in this study. The evidence from previous studies indicates that understanding the detailed role of CSF in development, function and pathophysiology of the brain will be one key productive area to promote normal development and to develop strategies and treatments to prevent abnormal development and neuropathological conditions. Isolating CSF components responsible for these different effects can be achieved using the parallel approach we propose. In our recent work we have described a unique folate handling system serving the developing cerebral cortex that operates through the CSF [Almost 75% of CSF is secreted ectively ,48. Addiectively -54. Receectively ,12,19,55ectively ,49,56-61ectively -66. Abouectively ,67. The ectively ,67. Thesectively . The utiectively , while tectively ,71. The  the CSF . Obstruc the CSF ,24,26. F the CSF ,61,72-81in vivo programming.This study has shown that CSF from fetal rat brains of different gestational ages can promote the survival, proliferation and differentiation of PC12 cells in an age dependent manner. Significant differences exist between the response of PC12 cells compared to primary fetal cortical cells perhaps indicating an interaction between the programming of primary cells and age-dependent composition differences in CSF in the latter case. The use of PC12 cells in future studies of CSF physiology may therefore allow the identification of CSF factors effecting cell physiology in isolation of primary cell, ANOVA: analysis of variance; BSA: Bovine serum albumin; CNS: Central Nervous System; CP: choroid plexus; CSF: cerebrospinal fluid; ECSF: Embryonic cerebrospinal Fluid; FITC: fluorescein isothiocyanate; IgG: Immunoglobulin G; MAP: mitogen-activated protein; MTT: 3--2,5-diphenyltetrazolium bromide; PBS: phosphate buffered saline; PC: pheochromocytoma.None of the authors have any competing interests.MN and JAM conceived and designed the experiments. JR, KP, HMK and SI carried out the experiments. All authors were involved in writing and editing the manuscript and preparation of figures. All authors have read and approved the final version of the manuscript.


Elevated plasma fibrinogen levels are a well known finding in acute infectious diseases, acute stroke and myocardial infarction. However its role in the cerebrospinal fluid (CSF) of acute and chronic central (CNS) and peripheral nervous system (PNS) diseases is unclear.We analyzed CSF and plasma fibrinogen levels together with routine parameters in patients with multiple sclerosis (MS), acute inflammatory diseases of the CNS  and PNS , as well as in non-inflammatory neurological controls (OND) in a total of 103 patients. Additionally, MS patients underwent cerebral MRI scans at time of lumbar puncture.p < 0.001) which strongly suggests passive transfer of fibrinogen through the blood-CSF-barrier during acute inflammation. Hence, in MS, the prototype of chronic neuroinflammation, CSF fibrinogen levels were not elevated and could not be correlated to clinical and neuroradiological outcome parameters.CSF and plasma fibrinogen levels were significantly lower in patients with MS and OND patients as compared to patients with BM, VM and GBS. There was a close correlation between fibrinogen levels and albumin quotient (rho = 0.769, Although previous work has shown clear evidence of the involvement of fibrinogen in MS pathogenesis, this is not accompanied by increased fibrinogen in the CSF compartment. Fibrinogen is a soluble 340 kDa dimeric glycoprotein that is synthesized in the liver, secreted into the plasma and able to signal via a number of receptors expressed on cells of the hematopoietic, immune and nervous systems ,2. ApartAccumulations of fibrinogen and its degradation products have been demonstrated in the central nervous system (CNS) tissue of stroke, bacterial meningitis (BM), HIV-encephalitis, Alzheimer's disease and multiple sclerosis (MS) patients . In MS, The aim of this study was to determine the value of fibrinogen as biomarker in plasma and CSF of patients with MS compared to other neurological diseases, and to analyze potential clinical and neuroradiological correlations.A total of 103 patients were recruited between 2004 and 2009 at the Clinical Department of Neurology, Innsbruck Medical University, Austria, with approval of the local ethical committee . All patients gave written informed consent and were assigned to the following groups:1. Clinically definite MS patients (n = 40), 27 with relapsing-remitting (RR) and 13 with chronic progressive (CP) disease course. Acute relapse was present at time of CSF analysis in 30% of MS patients. All MS patients were examined by a standardized brain MRI protocol including Gadolinium-DTPA and scanned using a 1.5 T MR scanner  at the time of diagnostic lumbar puncture, again prior to the application of methylprednisolone.2. Patients with BM (n = 15).3. Patients with viral meningoencephalitis . These two groups (2 and 3) were included as controls, reflecting an acute inflammatory CNS disease. The neurological outcome of these patients was evaluated at discharge from clinics by chart review using the Glasgow Outcome Scale (GOS) ,12.4. Patients with GBS n = 14) comprised a group with acute inflammatory disease of the peripheral nervous system (PNS). All GBS patients were assessed using standard nerve conduction studies within 6.2 ± 7.7 (mean ± STD) days after disease onset and classified according to common criteria  into dem comprise5. Patients with other, non-inflammatory neurological diseases  consisted of patients with chronic back pain (n = 6), chronic headache (n = 5), ischemic transverse myelopathy (n = 4) and Bell's palsy (n = 5). To exclude any biasing inflammatory conditions, patients with OND were only included if they had a normal CSF evaluation.Plasma, serum and CSF samples were obtained during standard diagnostic lumbar and peripheral vein puncture, and immediately analyzed for CSF white cell count (WCC), CSF/serum glucose ratio (GluR), CSF/serum albumin quotient , IgG index, total protein levels, plasma fibrinogen and serum C-reactive protein (CRP) levels using standard methods: photometric measurement for GluR and total CSF protein, nephelometric measurement for serum and CSF IgG, ELISA for plasma fibrinogen and immunturbidimetric measurement for CRP . CSF fibPatient characteristics were compared between diagnostic groups by Kruskal-Wallis test or chi-square test when appropriate. Spearman's rho was calculated and rho > 0.4 was considered as a relevant correlation. GOS at hospital discharge of patients with BM and VM were grouped into favorable (GOS = 5) and unfavorable outcome (GOS < 5). Disabilities of GBS patients at the nadir were scored according to the HGS and dichotomized into two groups (< 4 and > = 4). CSF data were compared between groups by Kruskal-Wallis test, post-hoc analysis was done by Dunn's Multiple Comparison Test. Calculations were done using PASW 18 , and GraphPad Prism 5.00 .p < 0.001), but significantly less T2 hyperintense lesions .Groups differed significantly in age and sex with an older age and female predominance in the GBS group Table . As expep < 0.001), VM  and GBS (both p < 0.01). Additionally, OND patients had lower fibrinogen levels in CSF and plasma compared to patients with BM  and also lower than in GBS (p < 0.05), whereas there was no difference to OND  and plasma fibrinogen . Fibrinogen was independent of age for all investigated groups. Fibrinogen levels did not differ for different clinical disease courses or during acute relapses in MS patients when compared to times of remission or chronic progression. Furthermore, CSF, plasma fibrinogen and fibrinogen quotient were independent of the number of T2-hyperintense lesions and the number of CEL on cerebral MRI scans. Patients with BM and VM did not differ in clinical outcome using GOS with respect to their fibrinogen levels. In the GBS group, the fibrinogen status was independent of the electrophysiological form of the disease and the severity of the disease at nadir (data not shown).Significant correlations were found between serum CRP and CSF fibrinogen (rho = 0.483, This study was designed to investigate a possible involvement of CSF fibrinogen in MS pathogenesis during times of acute inflammation, since vascular abnormalities and accumulation of perivascular fibrin are key findings in MS . Thus, tHowever, our study found highly increased fibrinogen levels in the acute inflammatory conditions that are associated with a diffuse alteration in the state of the BCSFB, where it is a highly sensitive marker for measuring acute neuroinflammation. The subsequent altering of the extracellular matrix of the brain parenchyma by fibrinogen has been shown to inhibit neuroregeneration and potentiate inflammation in experimental animal models ,17. The Due to the relatively small number of patients investigated and the retrospective design, our study was not powered to detect other influencing factors on the plasma fibrinogen status  and the BBB: blood-brain barrier; BCSFB: blood-CSF barrier; BM: bacterial meningoencephalitis; CEL: contrast enhancing lesions; CSF: cerebrospinal fluid; CNS: central nervous system; CP: chronic progressive; EAE: experimental allergic encephalomyelitis; GBS: Guillain-Barré syndrome; GluR: CSF/serum glucose ratio; GOS: Glascow Outcome Scale; HGS: Hughes Functional Grading Scale; MS: multiple sclerosis; OND: other neurological controls; PNS: peripheral nervous system; Qalb: CSF/serum albumin quotient; RR: relapsing-remitting; VM: viral meningitis; WCC: white cell count.The authors declare that they have no competing interests.Conceived and designed the study: RE BK MR. Patient recruitment: RE BK FdP PL AL CG HH TB. Analyzed the data: RE PL WS MS FD MR. Wrote the paper: RE MR. All authors read and approved the final manuscript.


B.C. . One widely accepted proposal is that the plaques are ancient mnemonic devices that record genealogies. The analysis reported here demonstrates that this is not the case, even when the most supportive data and techniques are used. Rather, we suspect there was a common ideological background to the use of plaques that overlay the southwestern Iberian Peninsula, with little or no geographic patterning. This would entail a cultural system in which plaque design was based on a fundamental core idea, with a number of mutable and variable elements surrounding it.Prehistoric material culture proposed to be symbolic in nature has been the object of considerable archaeological work from diverse theoretical perspectives, yet rarely are methodological tools used to test the interpretations. The lack of testing is often justified by invoking the opinion that the slippery nature of past human symbolism cannot easily be tackled by the scientific method. One such case, from the southwestern Iberian Peninsula, involves engraved stone plaques from megalithic funerary monuments dating ca. 3,500–2,750  B.C.    are founted age) . In addiInterpretations of the intended function(s) of the stone plaques extend back to the last quarter of the nineteenth century and includea kind of ideographic writing system prestige objects symbolic items used by groups within a social hierarchy heraldic objects amulets or cult objects apotropaic images of the deceased to ward off evil idols symbolic expressions related to different specific geographical regions and units of cultural identity More recently, Katina Lillios combined two of those functions—ideographic writing and heraldic items—hypothesizing that the majority of the plaques codify genealogical information Lillios created a sequence of types based on expected chronological changes in various features of the plaques, with emphasis on decorative motifs on the lower portion of the plaque. She anchored the sequence with plaques containing vertical bands, herringbone designs, or checkerboard patterning , followeLillios , but as maximize the expectations of Lillios's hypothesis. This meant that we weighted every experimental protocol and analytical decision in favor of her hypothesis, our rationale being that if we tried every way possible to meet the expectations but could not, then the hypothesis should be rethought.Despite these problems, Lillios's hypothesis has gained considerable weight among archaeologists working on the Iberian Peninsula . To testnot support Lillios's hypothesis that the plaques are genealogical mnemonic recording systems. We should say that her hypothesis is not supported in its current form, meaning that it is her proposed sequence of plaque designs that is unsupported. Our analysis does not negate the possibility that the plaques served as mnemonic devices or some other function tied to “external symbolic storage” In summary, our analysis did http://research2.its.uiowa.edu/iberian/index.php) created by Lillios and collaborators Our data were derived from the online Esprit (Engraved Stone Plaque Registry and Inquiry Tool) database . Those 349 specimens were spread across 81 classes. By excluding specimens that were broken, showed evidence of re-engraving, or were not illustrated in the online database, we ended up with a population of 735 plaques. We judged the population to be too large to analyze because of the number of unique classes involved (see below), so we reduced it to 349 specimens using a 5% error and a 99% confidence interval  of evolutionary change among the states of each character. One method for doing this is outgroup analysis homoplasy. This problem is overcome through the fourth step:Construction of a branching diagram of relationships for each character by joining the two most derived taxa—those at the branch tips of a tree—and then successively connecting each of the other taxa according to how derived they are. Ideally, the distribution of character states among the taxa will be such that all the character trees imply relationships among the taxa that are congruent with one another. Normally, however, a number of the character trees will suggest relationships that are incompatible—a phenomenon known as hypothetical statements of relatedness, “given the model and parameters used” , the retention index (RI), and the rescaled consistency index (RC) being the most commonly used. The CI measures the relative amount of homoplasy in a data set but is dependent on the number of taxa. Thus, the expected CI for a given tree must be assessed relative to the number of taxa used in the analysis Our phylogenetic analysis consisted of four exercises , each ofweight can be placed on them. As mentioned, our goal was to stack the deck in favor of Lillios's hypothesis, in which character DM plays a crucial role, so for exercises 2 and 4  and herringbone (DM6) designs are ancestral to plaques with other decorative designs, given that the former are more limited to her suspected core area of plaque manufacture and use .Character states can also be 6) to zigzag (DM3)—have the lowest cost (1), meaning that they have evolved in the manner Lillios suggested. At the other end of the spectrum, a transformation from a derived state to the original ancestral state—say, from triangle (DM2) to herringbone (DM6) or from unipartite (ST0) to bipartite (ST2)—has a cost of 3. Any transformation between a derived state and one immediately preceding it (an intermediate state)—say, from triangles (DM2) to zigzag (DM3)—has a cost of 2, as does any transformation between derived states of character H. Here, Lillios proposed that the inverted triangular head (H1) was immediately ancestral to all three other states , meaning she identified no intermediate states between the ancestral state and the three derived states. We find it difficult to believe, however, that there are no intermediate states and view the situation as a polytomy—an unresolved (nondichotomous) branching episode. If so, there are more possible reversals than there are nonreversals and thus we gave all changes among H0, H2, and H3 a cost of 2 For exercises 3 and 4 , we assiTwo exercises were carried out with outgroups ROOT in  and two The RI scores from all the exercises indicate that the data set has some consistency and phylogenetic structure. The sharp contrast observed for CI and RC values between the first two and the last two exercises indicates a significant difference related to the methodological parameters applied, specifically the switch from parameter US (unordered states) to parameter OS (ordered-states). In contrast, parameter WC, which implements different weights for a couple of characters, has no influence on the values. This indicates that the suspected order of character states in the hypothesis is inaccurate.best fit the expectations of Lillios's hypothesis  and a bootstrap tree . The respothesis . Those tpothesis —and exerpothesis . Both arpothesis  was rootpothesis , the claIn general, the two trees have large sections that exhibit poor structural and topological resolution. Both have some large polytomies (unresolved branches) close to their roots. In the 2/3 tree , the bas1→0 and NK1→0 at the base of the tree; DM6→3 at the next node up; and DM3→2 in the seven-class red clade). The character with a balance between nonhomoplastic (not necessarily synapomorphic) and homoplastic change is DM, which has four instances of the former and three of the latter. There are several reversals to ancestral states as a result of convergence. In the 4/2 tree . All characters exhibit at least one instance of homoplasy.Given the overall lack of deep structure, it is not surprising that no character is free of homoplasy. In the 2/3 tree , only 9 4/2 tree , only siSynapomorphies help resolve trees, but here our interest is primarily in the polarity of specific character states: How well does polarity meet the expectations of Lillios's hypothesis? We identified the following expectations:4 and DM6—the presumed ancestral states  is ancestral to ST1  and ST0 (unipartite); andST1 (inverted triangles) is presumed to be ancestral to H0 (no head), H2 (triangles and appendages), and H3 (appendages and figurative features).H0→1 in the latter (three instances of convergence). With respect to character TT, there is no clear, nonrandom patterning.To assess how well the expectations are met, we created the trees shown in How do the expectations fare in the 2/3 tree, which has weighted characters and unordered character states ? In gene3→4 (zigzags→checkerboard) in class 21. The majority of the changes (five out of seven) are located on terminal branches and thus contribute nothing to the tree structure. Only two synapomorphies are consistent with the expected polarity: DM6→3 (herringbone→zigzags) in the large polytomy and then DM3→2 (zigzags→triangles), which creates the clade of seven classes.Only one of the seven changes in character DM has an unexpected direction (three are homoplastic changes), the transition DM2) is highly conserved, appearing in 15 of the 20 classes.Character ST has four changes that fit the expected polarity (three are homoplastic), and none is a synapomorphy. The ancestral state (ST2→0 (triangle & appendage→absence) and H2→1 (triangle & appendage→triangle). In addition, the two H2→1 reversals are located in unexpected places along the tree. Instead of appearing close to the basal node , they are at the branch tips of classes 32 and 189. Also, the ancestral trait (H1) is highly conserved, appearing in 15 of the 20 classes.Character H exhibits eight changes , four of them in the hypothesized direction and another four in the opposite direction, with repeated transitions HHow do the expectations fare in the 4/2 tree, which has weighted characters and ordered character states ? At firs4→3 (checkerboard→zigzags) in the basal node and DM3→2 (zigzags→triangles), which creates the green clade.Five of the seven changes in character DM exhibit the expected polarity, but two of those are homoplastic. The only two synapomorphies that are consistent with the expected polarity are DM2) is very conserved, remaining in 12 of the 20 classes.All seven changes in character ST have the expected polarity, but all of them are homoplastic. Also, the ancestral state (ST1→2 (triangle→triangle & appendage)—sorts only classes 12 and 442. The ancestral state (H1) is also highly conserved, remaining in 13 of the 20 classes.The three changes in character H meet the expected polarity, but two are homoplastic. The one synapomorphy—H3→4 [zigzags→checkerboard] and DM3→6 [zigzags→herringbone]) in both trees, which undermine the suspected relative late position of plaques containing zigzag decoration. Also, most changes in character DM occur at the branch tips in both trees, which reduces the consistency of this presumptive positive result .What does the combined topology of two trees tell us about Lillios's hypothesis, especially in combination with radiocarbon dates and stratigraphic information? With respect to character DM, the data might appear at first glance to support Lillios's prediction that plaques with herringbone and checkerboard decoration at the bottom may be the oldest forms. However, there are several reversals (DM2), as, for example, at Cova das Lapas I, in the district of Leiria, with a radiocarbon date of 4550±60 B.P. (3238–3108 B.C. [1 sigma]) B.P. (3332–3214 B.C. [1 sigma]) 4), supposedly the most ancestral in the suggested sequence of character states, is a late occurrence at Olival da Pega 2b, in the district of Évora, with three calibrated dates B.C. (2) but so do those from later levels. There also are late plaques that exhibit zigzag motif (DM3), when, according to the hypothesis, they should be older than plaques with triangles.Radiocarbon dates and stratigraphic information also call into serious question the proposed sequence, with several of the oldest dated plaques exhibiting the triangle motif at their base (DMtes B.C. . With re2 (bipartite) appears to be ancestral to ST1  and ST0 (unipartite)—in line with predictions. In fact, the bipartite structure (ST2) is highly conserved. The few relevant dates and available stratigraphic evidence support the conservatism in ST2 as well as the derived nature of ST0. Whereas dated bipartite (ST2) plaques occur  at least throughout the period 4550±60 B.P. to 4140±110 B.P. , dated unipartite (ST0) plaques occur only in the early part of the third millennium (from 4270±40 B.P. [2917–2877 B.C. 1 sigma] in Anta de STAM-3). At Anta da Horta, all of the plaques in the oldest level are bipartite (ST2), and the majority of plaques in the later levels are unipartite (ST0).With respect to character ST, character state ST1) is the ancestral character state, and appendages (H2) and plain (H0) are the derived states. H1 is highly conserved in the 4/2 tree, and radiocarbon and stratigraphic data bear this out. At Anta da Horta 1 and the other H2. Plaques from the latest levels exhibit states H0 and H3. At Olival da Pega 2b 2 lasts throughout the sequence, becoming associated with other character states in the later levels.Recall that predictions relative to character H are not well met in the 2/3 tree , but theThe overall implications of Lillios's hypothesis with respect to the evolutionary history of stone plaques on the Iberian Peninsula are not met by a phylogenetic model, even when the two best trees—one with weighted characters (the 2/3 tree) and the other with weighted characters and ordered character states (the 4/2 tree)—are considered. There are at least three possible causes for the poor and arbitrary topology of large sections of both trees, with numerous polytomies and instances of homoplasy:The people who created the plaques were free to use any of the possible states in the design palette at any time and any place. If this were the case, however, we would expect to see unlimited and totally random character-state reversals, which is not the case.There was a high rate of cultural borrowing or horizontal transfer of information among populations scattered across the southwestern Iberian Peninsula, which tended to swamp most of the phylogenetic signal. This would mean that different genealogical and heraldic clans (according to Lillios) shared and transferred much of the information reflected in this material culture, an assumption that would run counter to the hypothesis that the plaques were linked to specific lineages and/or clans.There was a common ideological background  to the use of plaques that overlay the southwestern Iberian Peninsula. This would entail a cultural system in which plaque design was based on a fundamental core idea, with a number of mutable and variable elements surrounding it.We suspect number 3 was the case, at least in part. It seems reasonable to conclude that most cultures have a conservative “core tradition”—similar to Swadesh's some structure in the data. In fact, given the manner in which we stacked the analysis in favor of Lillios's hypothesis, one might have expected more structure in the plaques from the southwestern Iberian Peninsula, irrespective of whether they served the purpose(s) assigned to them. Certainly the available stratigraphic evidence and radiocarbon dates do not impart clear chronological structure to the plaques, which is yet another strike against Lillios's hypothesis that they served as genealogical mnemonic recording systems.Nor should we be surprised that within the broad trees comprising classes of Neolithic slate plaques from the southwestern Iberian Peninsula, several character-state polarities suggested by Lillios seem broadly successful. After all, people learn from those with whom they are culturally related and/or with those with whom they are in contact, and ideas as well as people move across the landscape. Thus we should expect


Prolonged central circulation transit time (TT) has long been associated with heart failure and left ventricular (LV) dysfunction. However, it is not known which portion of the central circulation is associated with this prolongation. Moreover, the relation of global and regional TT to LV end diastolic pressure (LVEDP) is not defined. In this study we assessed the global and regional TT in each portion of the central circulation and investigated their relations to LVEDP in patients undergoing cardiac catheterization.All patients were prospectively recruited and underwent cardiac MRI within 5 hours of cardiac catheterization. Dynamic MR images were acquired during the infusion of gadopentetate dimeglumine (0.01 mmol/kg) at 6 ml/s. Areas of interest were drawn and propagated through phases in the right atrium (RA), right ventricle (RV), main pulmonary artery (PA), left and right lung, left atrium (LA), LV and ascending aorta to construct time-intensity curves. TT was defined as the time between the peaks of time-intensity curves. All TTs were normalized to RR intervals. Central circulation TT was defined as TT from RA to aorta, while regional TT was defined as RA to RV, RV to PA, PA to lungs , lungs to LA, LA to LV, and LV to aorta. LVEDP was assessed during cardiac catheterization.Of the 48 subjects studied the mean age was 59 years and 32 (67%) were male. Average LV ejection fraction (LVEF) was 48±15% and the LVEDP 15±8 mmHg. Central TT was significantly prolonged in patients with elevated LVEDP (>12 mmHg) 15.7±5.8 cardiac cycles vs. 10.5±2.2 cardiac cycles (p=0.002) in those with normal LVEDP (≤12 mmHg). Regional analysis demonstrated that TT prolongation was present in PA to lungs, lungs to LA, LA to LV and LV to aorta but not in RA to RV and RV to PA. The multivariate regression model which included all regional TTs as covariables showed that TT from lung to LA had the strongest association with LVEDP  followed by TT from LA to LV  and TT from PA to lungs . TTs from other regions were not significantly associated with LVEDP.Central and regional circulation TT can be assessed by dynamic MR imaging. Central TT was significantly prolonged in patients with elevated LVEDP largely due to regional TT prolongation in lungs to LA, LA to LV and PA to lungs circulation.None.