Ictalurus punctatus, is invested with a high density of cutaneous taste receptors, particularly on the barbel appendages. Many of these receptors are sensitive to selected amino acids, one of these being a receptor for L-arginine (L-Arg). Previous neurophysiological and biophysical studies suggested that this taste receptor is coupled directly to a cation channel and behaves as a ligand-gated ion channel receptor (LGICR). Earlier studies demonstrated that two lectins, Ricinus communis agglutinin I (RCA-I) and Phaseolus vulgaris Erythroagglutinin (PHA-E), inhibited the binding of L-Arg to its presumed receptor sites, and that PHA-E inhibited the L-Arg-stimulated ion conductance of barbel membranes reconstituted into lipid bilayers.The channel catfish, Both PHA-E and RCA-I almost exclusively labeled an 82–84 kDa protein band of an SDS-PAGE of solubilized barbel taste epithelial membranes. Further, both rhodamine-conjugated RCA-I and polyclonal antibodies raised to the 82–84 kDa electroeluted peptides labeled the apical region of catfish taste buds. Because of the specificity shown by RCA-I, lectin affinity was chosen as the first of a three-step procedure designed to enrich the presumed LGICR for L-Arg. Purified and CHAPS-solubilized taste epithelial membrane proteins were subjected successively to (1), lectin (RCA-I) affinity; (2), gel filtration (Sephacryl S-300HR); and (3), ion exchange chromatography. All fractions from each chromatography step were evaluated for L-Arg-induced ion channel activity by reconstituting each fraction into a lipid bilayer. Active fractions demonstrated L-Arg-induced channel activity that was inhibited by D-arginine (D-Arg) with kinetics nearly identical to those reported earlier for L-Arg-stimulated ion channels of native barbel membranes reconstituted into lipid bilayers. After the final enrichment step, SDS-PAGE of the active ion channel protein fraction revealed a single band at 82–84 kDa which may be interpreted as a component of a multimeric receptor/channel complex.The data are consistent with the supposition that the L-Arg receptor is a LGICR. This taste receptor remains active during biochemical enrichment procedures. This is the first report of enrichment of an active LGICR from the taste system of vertebrata. The initial event in taste transduction involves recognition of taste stimuli by plasma membrane-associated receptor proteins. These proteins are concentrated at the apical end of specialized neuro-epithelial cells (taste cells) found within multicellular end-organs known as taste buds ,2. The rTo date, 7-transmembrane G protein coupled receptors (7TM-GPCR's) for three taste modalities have been identified by both molecular cloning and through searches of the human and mouse genome. Sweet taste stimuli appear to be recognized by at least one heterodimer T1R2/T1R3) of the three member family of 7TM-GPCR's, the T1R's  of the t,8-10. TwIctalurus punctatus -1-propanesulfonate (98%) (CHAPS), polyoxyethylenesorbitan monolaurate (TWEEN 20), phenylmethanesulfonyl fluoride (PMSF) and pepstatin A were purchased from Sigma. n-Octyl-β-D-gluco-pyranoside (n-octylglucoside) was purchased from Calbiochem . The absolute enantiomer, L-arginine HCl, was purchased from Sigma, and the absolute enantiomer, D-arginine HCl, was a gift of the Ajinomoto Co., Tokyo, Japan. Sephacryl S-300 HR, High Range Gel Filtration Calibration Kits, and 1.0 ml Hitrap Q columns were purchased from Pharmacia Biotech . Agarose-bound lectins, Maxillary and mandibular barbels from approximately 50 euthanized channel catfish, ~25–40 cm long, were removed and placed in a beaker containing 100 ml of 50 mM Tris-HCl buffer (pH = 7.8), 100 mM NaCl, 1 mM EDTA ("TRIS Wash"). After one exchange of buffer, barbels were removed to a 50 ml polypropylene screw cap tube filled with TRIS Wash buffer and stored at -80°C until used.The preparation of a solubilized, plasma membrane-enriched fraction from catfish barbels was adapted from Kalinoski . A typicTo solubilize the pellets from the 21,500 × g spin each pellet was recovered by two, 1 ml rinses of 50 mM TrisHCl (pH 7.8), 50 mM NaCl ("Low Osmolar Buffer")  and transferred to a 15 ml Teflon/glass homogenizer. Twenty milligrams of CHAPS and 40 μl of protease inhibitor-mix  were added to the ~4 ml suspension in the homogenizer. The suspension was homogenized by ten slow strokes of the Teflon pestle using a rotating motor drive at moderate speed. The homogenate was transferred to a 15 ml capped tube, diluted to 10 ml with Low Osmolar Buffer, and placed on a Clay Adams Nutator rocker/shaker overnight at 4°C to solubilize the proteins. After the overnight agitation, the suspension was centrifuged for 1 h at 100,000 × g. The supernatant was recovered and is referred to as "Sp." This Sp was then used in the subsequent lectin affinity chromatography step.All steps in the lectin affinity procedure were performed at 4°C. Agarose-bound RCA I affinity resin was pre-washed by adding 8 ml of gel slurry to a 125 ml conical glass tube and bringing the final volume to 14 ml with TRIS Wash. The tube was inverted several times, then centrifuged at 500 × g for 1 min. The supernatant was discarded and the wash step repeated three additional times with TRIS Wash buffer, then two more times with Low Osmolar buffer. The resulting agarose-bound RCA I gel, with a bed volume of 4 ml, was used for affinity chromatography.The 4 ml agarose-RCA gel in the 15 ml tube was combined with ~7 ml of Sp, and the mixture equilibrated on the Nutator tube rocker for 30 min. The mixture was poured into a 1 cm × 10 cm column and the effluent collected as 1 ml fractions at a flow rate of 6.0 ml/h. Effluent from the column was monitored by absorbance at 230 nm and/or 280 nm.To remove unbound protein, the column was washed with a sufficient volume (~12 ml) of Low Osmolar Buffer until no detectable protein eluted from the column. Proteins bound to the RCA resin were then eluted from the column with 10 ml of 50 mM Tris-HCl (pH = 7.8), 200 mM NaCl, 1 mM EDTA, 20 mM D-galactose. Protein from both the Low Osmolar elution step and the D-galactose elution step was reconstituted into lipid bilayers and assayed for L-Arg-stimulated ion channel activity (See ahead).The galactose-eluted protein fractions showing L-Arg-stimulated ion channel activity were pooled and dialyzed over night against 2000 ml Milli-Q water containing 0.05% CHAPS. Dialyzed samples were lyophilized and stored at -80°C.All steps in the gel filtration procedure were performed at 4°C. Lyophilized proteins from the galactose elution of the lectin column were prepared for further enrichment using size exclusion chromatography by dissolution in 300 μl of TRIS Wash with the addition of 0.2% CHAPS and 10% sucrose. This preparation was loaded onto a Sephacryl S-300 HR column (1 cm × 40 cm). Fractions of 0.4 ml were eluted from the column with a buffer composed of TRIS Wash plus 0.2% CHAPS. The column was run at 2.4 ml/hr, and the effluent absorbance monitored at 230 nm and 280 nm. Eluted fractions containing measurable protein were evaluated for L-Arg-stimulated ion channel activity by incorporation into a lipid bilayer (See ahead). In addition, SDS-PAGE (See ahead) was performed on each eluted fraction. Those fractions exhibiting L-Arg-stimulated ion channel activity were dialyzed overnight against 2000 ml of Milli-Q water containing 0.05% CHAPS, lyophilized and stored at -80°C.The column was calibrated with a Gel Filtration Calibration Kit with protein standards from 158 to 669 kDa.All steps in the ion exchange procedure were performed at 4°C. Further enrichment of the fractions exhibiting L-Arg-stimulated ion activity was achieved using an anion exchange column (Hitrap Q). The column was prepared by washing with 5.0 ml of Start Buffer , followed by 2.0 ml of Regeneration Buffer (Start Buffer plus 1.0 M NaCl) and then 5.0 ml of Start Buffer. Lyophilized proteins of the active fraction from the gel filtration column were dissolved in 300 μl of Start Buffer and applied to the column. Prior to elution, the loaded column was washed with 2.0 ml Start Buffer. Elution was at 12 ml/h in 400 μl fractions. First, 1.0 ml of First Elution Buffer  was applied to the column followed by (second), 2.0 ml of Start Buffer (no NaCl), followed by (third) 4.0 ml of Second Elution Buffer . Effluent from the column was monitored with in line UV (230 and 280 nm) and conductivity detectors. Each fraction was assayed for L-Arg-stimulated activity (See ahead.). The active fractions were pooled, dialyzed overnight against 2000 ml of Milli-Q water containing 0.05% CHAPS, lyophilized, and stored at -80°C.The procedure for development of antibodies against the catfish barbel peptides labelled by the lectins, RCA-I and PHAE has been described previously . BrieflyThe IgG fraction of GP1 and GP2 antisera was purified using an E-Z-SEP antibody purification kit (Pharmacia). To reduce non-specific binding in the immunohistochemical studies and Western blots, the GP1 and GP2 antibodies were incubated with powder derived from an acetone precipitation of a catfish brain homogenate. One ml of E-Z-SEP-purified antibody was incubated with 10 mg of powder for 40 min at 4°C. The powder was removed by centrifugation and the procedure was repeated once with fresh powder. The resulting pretreated antibodies are called simply, "GP1" and "GP2."Tris-glycine gels  were used for SDS-PAGE. Protein of fractions before any chromatography , as well as those from each chromatography step, were denatured by mixing 10 μl from each fraction, 1:1, with sample buffer containing 125 mM Tris-HCl (pH = 8.0), 20% glycerol, 4% SDS, 4% β-mercaptoethanol and 50 μg/ml bromophenol blue, and placing the mix in a boiling water bath for 5 min. Gels were run at a constant 20–25 mA for about 1 h, using prestained broad range molecular weight markers (Bio-Rad) in 1 or more lanes. Proteins were stained with Bio-Rad Silver Stain Plus kit.For lectin blots, proteins were electrophoretically transferred to nitrocellulose sheets . The sheets were incubated in a blocking solution  ] and 0.05% Tween 20) for 2 h at room temperature, and then incubated with biotinylated RCA-I or PHA-E (10 μg/ml) overnight at 4°C. Following exposure to biotinylated lectin, nitrocellulose sheets were washed extensively with blocking solution. Lectin-bound protein bands were visualized using a Vectastain peroxidase ABC kit with a 4 CN Membrane Peroxidase Substrate system. Development was stopped after one hour using 5% glacial acetic acid.For Western blots, proteins were transferred to nitrocellulose and incubated in blocking solution  for 2 h at ambient temperature with constant slow rocking (Nutator). The nitrocellulose was incubated overnight at 4°C (rocking) with primary antibody  in blocking solution. The nitrocellulose was washed with blocking solution and incubated with biotinylated secondary antibody (1:250) for 1 h with slow rocking. Bands were visualized as above.Rhodamine-conjugated RCA-I (Vector Labs) was used to estimate the specificity of lectin interaction with glycoproteins of catfish barbel. To assess the localization of the antigen contained in the 82–84 kDa band from the SDS-PAGE of a membrane fraction of catfish barbel , immunohistochemistry was performed using the GP1 and GP2 antibodies pretreated as described above.I. punctatus) (the fish being 5 – 7 cm in length) and immediately placed in 4% buffered paraformaldehyde (PFA)  for eight hours at 4°C. After washing out the PFA with several rinses of excess buffer, the barbels were placed successively in 10%, 20%, and 30% sucrose (in buffer) for 24 hr, all at 4°C. After the final cryoprotect sucrose step, barbels were cut into pieces of less than 1 cm and mounted with M-1 Embedding Matrix . The tissue was sectioned at 10 microns on a Microm HM500OM cryostat.Barbels were removed from euthanized albino channel catfish , diluted 1/200 for 2 to 3 hr at ambient temperature. The sections were then washed quickly with Dulbecco's PBS (GIBCO/Invitrogen Corp), followed by three incubation washes of 10 min each.For immunohistochemistry, 10 micron barbel sections, pre-washed 3 times for 10 min each in Dulbecco's PBS, were first incubated at ambient temperature for 3 to 5 hr in blocking buffer consisting of 3% bovine serum albumin, 2% goat serum, 0.3% TritonX100, and 0.1% sodium azide in Dulbecco's PBS at pH 7.1. The sections were then incubated with primary antibody, GP1 or GP2, in blocking buffer for 18 hr at 4°C. The primary antibody solution was removed and the sections were then washed once quickly with Dulbecco's PBS, followed by three incubation washes with PBS of 10 min each. The sections were then incubated in the dark with second antibody, Cy3-conjugated goat anti-guinea pig IgG  at 1:1000 dilution for 60 min. at ambient temperature. The secondary antibody was removed and the sections washed once quickly with Dulbecco's PBS, followed by three incubation washes with PBS of 10 min each. Excess fluid was removed from the slides and the sections mounted under cover slips with VectaShield (Vector Labs). Sections were observed with a Nikon Microphot FXA fluorescence microscope, photographed, and images sized and enhanced using the GNU Image Manipulation Program software .Immuno-specificity was verified by running negative controls where the primary antibody was omitted from the procedure. In all cases, this control step showed no taste bud labelling Fig. . The scaReconstitution of protein fractions containing likely L-Arg-stimulated channel activity was carried out with material from the low osmolar and galactose-eluted fractions of the lectin affinity procedure, from the protein-containing fractions off the Sephacryl S-300 gel filtration column and from each fraction of the ion exchange column. Lipid vesicles were prepared by sonication of 5 mg of DOPE:DOPC (2:1) and 0.5 ml of 5 mM Tris-HCl (pH = 7.2), 300 mM NaCl and 500 mM sucrose, to which 10 μg n-octylglucoside was added. To prepare the liposome-detergent mixture for incorporation into a lipid bilayer, approximately 0.2 to 0.5 μg of presumed receptor protein was added to the liposome in a cassette dialysis unit, and the mixture dialyzed overnight against 2000 ml of the Tris/NaCl/sucrose buffer at 4°C.trans side of the bilayer.Virtually solvent-free lipid bilayer membranes were prepared as described . The mem2, 100 mM NaCl (unless otherwise stated). Fifty to 100 μl of the dialyzed liposome vesicles containing presumed receptor was added to the cis-side of the membrane. Fusion of the vesicles was initiated mechanically by gently mixing the membrane bathing solution from the cis-side using a micro-pipette. L-Arg was added to the cis side approximately 20 min after addition of vesicles. Unless otherwise stated all other additions of reagents also were made from the cis side. Channel sidedness was determined by sensitivity of the bilayer to L-Arg. The orientation of the channels was such that the L-Arg sensitive side was normally in the cis compartment. All bilayer experiments were performed at room temperature.The bilayer was bathed symmetrically with 5 mM MOPS (pH 7.2), 1 mM CaClThe current was amplified by a Dagan 3900 integrating patch-clamp amplifier  in the voltage clamp mode. Single channel data were digitized at 15 kHz  and analyzed using pClamp6 (Axon Instruments) and Origin 5.1  software on an IBM compatible computer.The calculated success rate of incorporation of vesicular proteoliposomes into lipid bilayers was about 25%. Success rate is defined as the ratio of the number of successful incorporation attempts to the total number of incorporation attempts (an "incorporation attempt" refers here to the formation of a new bilayer and application of putative channel protein). Over the course of these studies, the number of incorporation attempts for each fraction tested as indicated above was about ten.WG developed the enrichment procedures and drafted the manuscript. YK developed protein stabilization procedures and performed and interpreted the bilayer studies. DB performed the lectin- and immunohistochemistry and refined many of the enrichment procedures. AS developed the antibodies and performed pilot chromatography procedures. DLK initially designed the lectin procedure and carried out preliminary work on solubilization that made subsequent procedures possible. JT performed initial bilayer studies and advised YK on a continuing basis. JGB, the corresponding author and head of the laboratory, conceived the study, developed tissue preparations, designed and coordinated all phases of the study, and finalized the manuscript. All authors read and approved the final manuscript.


The clear dominance of two-gender sex in recent species is a notorious puzzle of evolutionary theory. It has at least two layers: besides the most fundamental and challenging question why sex exists at all, the other part of the problem is equally perplexing but much less studied. Why do most sexual organisms use a binary mating system? Even if sex confers an evolutionary advantage (through whatever genetic mechanism), why does it manifest that advantage in two, and exactly two, genders (or mating types)? Why not just one, and why not more than two?Assuming that sex carries an inherent fitness advantage over pure clonal multiplication, we attempt to give a feasible solution to the problem of the evolution of dimorphic sexual asymmetry as opposed to monomorphic symmetry by using a spatial (cellular automaton) model and its non-spatial (mean-field) approximation. Based on a comparison of the spatial model to the mean-field approximation we suggest that spatial population structure must have played a significant role in the evolution of mating types, due to the largely clonal (self-aggregated) spatial distribution of gamete types, which is plausible in aquatic habitats for physical reasons, and appears to facilitate the evolution of a binary mating system.Under broad ecological and genetic conditions the cellular automaton predicts selective removal from the population of supposedly primitive gametes that are able to mate with their own type, whereas the non-spatial model admits coexistence of the primitive type and the mating types. Thus we offer a basically ecological solution to a theoretical problem that earlier models based on random gamete encounters had failed to resolve. One of the most general rules in biology seems to be that sex involves the fusion of gametes  of different type. In most taxa this sexual asymmetry is reflected in the male / female distinction between mating partners and/or between mating sex cells. This paper aims to help understand why sex is asymmetric.The primary difference between male and female is anisogamy, the differential size and mobility of gametes. Anisogamy is thought to have evolved from a more primitive condition of isogamy  and fungi (e.g. yeast), the asymmetry in sexual fusion and subsequent development are regulated by a binary mating type system. Mating is only possible between cells of different mating type. Molecular analysis has revealed a remarkable and complex genetic mating type structure [Aspergillus nidulans, or may consist of individuals that are heterokaryotic for mating type (carry nuclei of both mating types) such as Podospora anserina. In the latter case sexual fusion is between different mating types at the nuclear level, but can occur within a single individual mycelium.In isogamous species without apparent male-female differentiation, like unicellular green algae  approximation. Such a comparison allows precise consideration of the kinetics of gamete encounters in the model system and emphasizes the role that spatial aspects of the kinetics might have played in mating type evolution. For detailed descriptions of both models see the The specific questions we address with both the mean-field model and the cellular automaton are the following:a) Are there reasonable parameter values that allow the coexistence of the mating types and the pan-sexual type?b) Under what (if any) circumstances is it possible that the mating types exclude the pan-sexual type?c) Does spatial structure play an important role in the outcome of the mating type competition system?r, R, R', the vegetative death rates d, D, D', the germination rate g, the sex rate σ and the finess erosion rate φ in the mean-field, and the corresponding probability parameters in the cellular automaton model. Having explored a broad range of the parameter space – with straightforward constraints on the fitness parameters (birth and death rates), i.e., with D ≤ D' ≤ d <r ≤ R' ≤ R – we found that it is the strength of the inbreeding effect (the difference of D and D' and that of R' and R) and the rate of fitness erosion φ that has the most interesting effects on coexistence. Changing the remaining parameters – the sex rate and the germination rate – within reasonable limits  does not affect the results in a qualitative sense.Numerical solutions to the mean-field model and simulations with the cellular automaton reveal that the system admits a single stable equilibrium state both in the non-spatial and in the spatial setting , and ξ > 0 means a fitness difference in favour of outbred offspring.ξ – φ projection of the parameter space, for both the mean-field model . At φ = 0 (no fitness loss during vegetative multiplications), on the other hand, it is the mating types who win for any ξ > 0. At ξ = 0 = φ, the mating types and the pan-sexual type coexist, and the same applies to any parameter combination satisfying ξ ≠ 0 ≠ φ. Thus we can say that the non-spatial (mean-field) model allows coexistence for almost any parameter combination, except for the biologically less feasible margins of the parameter plane. It predicts in general that both the mating types and the pan-sexual type should have persisted, even if at variable relative frequencies. The cellular automaton model yields a different prediction, admitting the exclusion of the pan-sexual type, i.e., the victory of the two mating types on a considerable section of the parameter plane, including the ξ = 0 = φ point and its close  neighbourhood  every vegetative cell is in the post-zygote state. For the same reason the birth rates are also equal for all the vegetative cells, so the only factor that can make a difference between the cell types is the availability of empty sites: the limiting "resource" for reproduction. In the mean-field model the empty sites are equally available to any cell, so the growth rates of the pan-sexual and the mating type strains are identical in the long run, hence their coexistence. In the cellular automaton, however, each strain develops patches. The mating type strains do not have sex at all within their own patch, only at the interface with the patches of other strains. The pan-sexual strain has sex all the time everywhere in the habitat, therefore a larger part of its population is in the dormant zygote state. It is for this reason that at the interface with the mating type patches the pan-sexual strain has a smaller supply of vegetative invaders and thus a smaller chance to capture an empty site there. This results in a travelling front between a mating type patch and a pan-sexual patch and ultimately in the demise of the pan-sexual population altogether. This effect can even overcompensate a small disadvantage for the mating types arising from increasing the rate of fitness erosion φ slightly above 0, therefore the close neighbourhood of the ξ = 0 = φ point on the parameter plane belongs to the mating types as well. We think that it is exactly this mechanism that makes the mating types victorious in the spatial model at many parameter combinations that allowed for coexistence in the mean-field approximation. The elementary events at the interfaces between patches of different gamete types have a profound effect on the ultimate outcome of their competition at the larger spatial scale of the whole habitat.In the mean-field model the coexistence of mating types and the pan-sexual type at see Fig. . The detξ = 0 = φ for example (or anywhere else where the mating types take over) in the two models proves that it is not stochastic drift but a real dynamical trend that eliminates the pan-sexual strain in the cellular automaton . If the populations are viable, i.e., if they have a vegetative growth rate higher than 0, then less frequent mating (dormancy) is beneficial in terms of the average fitness of the pan-sexual population. This effect dominates at low values of φ, where the fitness advantage of sex does not vanish too fast. A comparison of Figs. φ. In all, it is quite obvious that sex rate reduction is not an efficient strategy for the pan-sexual strain to avoid exclusion by the mating types.In order to explain the net effect of sex rate reduction on the fitness, and thus on the survival chances of the pan-sexual population one has to consider two different aspects. On the one hand, sex rate reduction decreases the relative fitness of the pan-sexual strain, because it decreases the frequency of both its inbred and outbred matings, the means of keeping fitness high. This negative fitness effect is most pronounced at high rates of fitness erosion There is a logical possibility that asymmetric cell fusion has evolved for other reasons than and prior to sex and has subsequently been incorporated in the evolution of a full sexual cycle . In that case sex would have been asymmetric from the start. This speculative idea has been analysed theoretically by   our spatial (cellular automaton) model shows the evolution towards exclusively two mating types, whereas the non-spatial model of the same system with the same parameters predicts the coexistence of the mating types and the pan-sexuals. Thus, taking for granted that sex is profitable in evolutionary terms, we offer a basically ecological answer to the question why two mating types can be better than just one. This is, however, only a solution to half of the problem of the optimal number of mating types. Could a third, a fourth, a fifth etc. mating type invade the same system? These questions arise on a very general level in relation to the origin of sexual asymmetry, and they call for a more extended theoretical approach in the future.Assuming that sexual reproduction confers some average fitness advantage compared to simple clonal multiplication, and also supposing that the more genetically different the fusing gametes are the bigger the fitness benefit of the offspring can be, we show that a population consisting of two mating types can displace a pan-sexual population which is otherwise similar to the mating types in all other respects. In the most realistic domain of its parameter space  that of its parent. Sexual fusion produces a dormant zygote which upon germination gives rise to haploid vegetative cells through meiotic division, in which the parental gamete types segregate as if determined by a mendelian pair of alleles. To these meiotic products – "post-zygote" vegetative cells -a higher fitness, i.e., a higher division rate and/or a lower death rate, is attributed than to "pre-zygote" vegetative cells not having gone through a sexual cycle in the near past. That is, we assume that sexual offspring have an immediate short-term fitness advantage over asexually derived daughter cells. The actual advantage may be dependent on whether the zygote has been produced by "outbreeding" (with at least one of the gametes involved belonging to one of the two mating types) or "inbreeding" . In general we may, but need not, assume that inbred zygotes yield vegetative cells of somewhat less (but still positive) fitness advantage than outbred zygotes. Note that here "outbreeding" and "inbreeding" mean mating between different and identical gamete types, respectively, i.e., we assume – without specifying the precise nature of this outbreeding advantage – that mating between different gamete types may result in fitter offspring on average than mating between cells of the same  gamete type. The simplest possible genetic mechanism with this effect might be the production of recombinant offspring carrying fewer (slightly) deleterious alleles than both parental genotypes. This mechanism will be operative more often in heterotypic than in homotypic matings, because among the latter a larger proportion will involve selfing  identical genotypes). The fitness advantage of sexually derived vegetative cells fades away in time during successive rounds of vegetative reproduction (fitness erosion due to the accumulation of harmful mutations), but it can be re-gained through another sexual event. This means that post-zygote cells return to the pre-zygote state when they are not involved in a new sexual cycle for a sufficiently long time.Wpre-zygote <Wpost-zygote,inbred ≤ Wpost-zygote,outbred.As for the ecology of the system, we assume that the habitat consists of a limited amount of sites that cells can occupy, and that the three cell types are competing for these sites. Death events leave empty sites behind, which can be occupied later by new offspring. The chance of a newborn cell to settle is proportional to its division rate and the number of empty sites available. In accordance with what has been said earlier about the fitness advantages of sex, three different division rates and death rates are possible: one for pre-zygote, the second for inbred post-zygote, and the third for outbred post-zygote vegetative cells. The straightforward fitness order of these three types is: The fusion of two gametes produces a zygote of double size compared to a gamete, and the zygote enters a dormant state with zero rates of division and death. Zygotes leave dormancy at a constant rate, giving rise to post-zygote vegetative cells which inherit the mating type of the gametes they are produced by, and gain fitness according to whether the mating was of the inbreeding or the outbreeding type.Fig. Based on Fig. x, y and p are the numbers of sites occupied by pre-zygote vegetative cells , Zxy, Zxp, Zyp are the sites of outbred, and Zpp are those of inbred zygotes. Similarly, X, Y and P are sites of outbred, Q are those of inbred post-zygote vegetative cells. E is the number of empty sites within the habitat. The parameters of the model are listed and described in Table where x, y and p) has three terms. The first defines the vegetative fitness of the corresponding cell type , the second is the outflow from the pre-zygote vegetative state due to sex, and the third is the inflow due to the fitness erosion of post-zygote vegetative cells. Zygotes have no vegetative fitness; the first term in their differential equations is the inflow due to sex, the second is the outflow due to germination. Post-zygote vegetative cells have a vegetative fitness different from that of pre-zygotes (first term); they form zygotes fusing  with both pre- and post-zygote cells matching in mating type (second term); their fitness advantage erodes at a constant rate resulting in an outflow into the pre-zygote state (third term), and the germination of dormant zygotes maintains an inflow from the zygote states (fourth term). The number of empty sites is increased by the deaths of vegetative cells (first three terms) and decreased by the number of sites taken by newborn vegetative offspring (fourth term). The total number of sites does not change in time, so the 12 time derivatives sum up to zero.The right-hand side of the differential equations for the sites occupied by pre-zygote vegetative cells . In all numerical calculations the initial populations were 10 pre-zygote vegetative cells of both mating types and the pan-sexual type, all other states had 0 initial abundances.With assumptions as similar to the nonspatial system as possible, we have implemented a site-based (cf. ), spatiaThe pattern is updated one randomly chosen site at a time, i.e., we use an asynchronous random updating algorithm. Any site chosen for update can be empty, occupied by a vegetative cell, or occupied by a zygote. We specify the algorithm for each of these cases in turn. A schematic diagram of a single step of updating is given in Fig. i has a chance pi to put a daughter cell into the empty site. pi depends on the vegetative reproduction parameter βI (0 ≤ βI ≤ 1)of neighbour i. βI is the spatial analogon of ri in the mean-field model, and it takes one of three possible values depending on whether i is in the pre-zygote, the inbred or the outbred post-zygote state.After updating, an empty site can be occupied by one (and only one) of the vegetatively produced offspring of the cells in the 8 neighbouring sites , or it remains empty. Each vegetative neighbour Specifically, the chance of the empty site to remain empty isi takes the site isso the probability that the offspring of neighbour βi, but only one of the candidate offspring survives. The chance of survival is proportional to the reproduction parameter of the mother cell.The rationale behind this formalism is that each neighbour attempts putting an offspring into the empty site with a probability δ of the cell occupying the site, which in turn depends on its vegetative status . With a mating partner in one of the neighbouring sites, a surviving vegetative cell may enter the sexual cycle with probability s turning itself and a randomly chosen, suitable neighbour into gametes, and mate. The result is a dormant zygote occupying the two neighbouring sites of the fused gametes. A survivor skipping sex may keep its original fitness, or – if it was a post-zygote cell – it can lose its fitness advantage with a probability f .Updating a site occupied by a vegetative cell may result in four possible outcomes: turn the site into the empty state (death), leave it as it was , change the vegetative status of the resident cell from post-zygote to pre-zygote , or produce a zygote (sex). The probability of a death event depends on the death probability γ) or germinate (with probability γ). A germinated zygote yields two vegetative cells, the mating types of which are the same as those of the gametes which produced the zygote. The vegetative status of the cells thus obtained is post-zygote, and they can be either inbred or outbred, depending on the parental gamete type combination. The daughter cells are positioned at random into the two sites the zygote had occupied.A zygote can do two things: remain dormant (with probability 1 – At time 0 we have populated 2% of the sites by pre-zygote vegetative individuals of both mating types and the pan-sexual type, assigning individuals to sites at random. All other sites were empty at time 0. The simulations were run for 10.000 generations.RH organized and coordinated the project, collected most of the literature and drafted parts of the manuscript. TC designed and built the models, drafted parts of the manuscript and the figures.


The present study GREECS was conducted in order to evaluate the annual incidence of acute coronary syndromes (ACS) and to delineate the role of clinical, biochemical, lifestyle and behavioral characteristics on the severity of disease. In this work we present the design, methodology of the study and various baseline characteristics of people with ACS.A sample of 6 hospitals located in Greek urban and rural regions was selected. In these hospitals we recorded almost all admissions due to ACS, from October 2003 to September 2004. Socio-demographic, clinical, dietary, psychological and other lifestyle characteristics were recorded. 2172 patients were included in the study (76% were men and 24% women). The crude annual incidence rate was 22.6 per 10,000 people and the highest frequency of events was observed in winter. The in-hospital mortality rate was 4.3%. The most common discharged diagnosis for men was Q-wave MI, while for women it was unstable angina.This study aims to demonstrate current information about the epidemiology of patients who suffer from ACS, in Greece. During the past decades epidemiological investigations have provided a portrait of the potential candidate for acute coronary syndromes, in the US, as well as in many other parts of the world, especially in European countries -9. HowevThe profile of cardiovascular disease patients in Greece has been, mainly, investigated from few large scale, population-based studies, like the Seven Countries Study in the early 1960s , the HelThe aim of the GREEk study of acute Coronary Syndromes (GREECS) is to evaluate the prevalence and annual incidence of acute coronary syndromes (ACS), as well as the characteristics, and management of these patients, in a sample of six Greek urban and rural regions. Secondary goals are to examine the role of adoption to the Mediterranean diet and other lifestyle habits, as well as various clinical, biochemical, psychological and personal characteristics of these patients on the severity and the short (30 days) – long  – term prognosis. In this work we present the methodology used, and the baseline characteristics of the studied population.Between October 1, 2003 and September 30, 2004 (12 months) we enrolled almost all consecutive patients (participation rate = 98%) that entered in the cardiology clinics or the emergency units of six major General Hospitals, in Greece . By the exception of Athens – where there are several other hospitals -, all the other hospitals cover the whole population of the aforementioned regions, including urban and rural areas. The studied regions were randomly selected from all Greek regions in order to cover a wide range of the county  of them were men and 523 (24%) were women. Power analysis showed that the number of enrolled participants is adequate to evaluate two – sided differences between groups of the study and the investigated parameters greater than 20% (± 5%), achieving statistical power greater than 0.80 at 5% probability level .At entry a 12-lead electrocardiogram was performed and clinical symptoms were evaluated in all patients, by a cardiologist of the Study. Based on the electrocardiographic findings patients were classified as having ST-segment elevations, non-ST segment elevations or other electrocardiographic abnormalities. Moreover, blood tests were performed to detect evidence of myocardial cell death. We measured troponin I levels and the MB fraction of total creatinine posphokinase (CPK). According to the Joint European Society of Cardiology and American College of Cardiology Committee, blood samples were obtained on hospital admission, at 6 to 9 h, and again at 12 to 24 h if earlier samples were negative and the clinical index of suspicion was high . We inclThe study was approved by the Medical Research Ethics Committee of our Institution and was carried out in accordance with the Declaration of Helsinki (1989) of the World Medical Association.In all patients a detailed medical history was recorded, including previous hospitalization for cardiovascular disease , presence and management of hypertension, hypercholesterolemia, renal failure and diabetes mellitus. Moreover, we recorded patients' medical family history. In particular, we asked information concerning first-degree relatives  about presence of coronary heart disease, hypertension, dyslipidemias and diabetes. Premature history  of myocardial infarction, sudden death, coronary arteries bypass grafting procedure or percutaneous coronary angioplasty in first-degree relatives classified the participants in the positive family history group for coronary heart disease . PrematuSocio-demographic characteristics included: age, sex, marital status and number of children, years of school, type of occupation and occupational skills, which they were evaluated through a ten-point scale from unskilled – hand workers  to executive – skilled workers  that has been developed for the purposes of the Study; and mean annual income of the family (through self reports) during the last three years. Regarding people in the family who were not working, we used the average family income, while for unemployed individuals we used the basic monthly allowance they take from the Social Service Office.2, while obesity as BMI greater than 29.9 kg / m2.Height and weight was measured, to the nearest 0.5 cm and 100 g respectively. Body mass index (BMI) was then calculated as weight (in kilograms) divided by height (in meters) squared. Based on the World Health Organization , overweiTo evaluate physical activity status of the patients during the past year we used a modified version of a self-reported questionnaire provided by the American College of Sports Medicine . Based oCurrent smokers were defined as those who smoked at least one cigarette per day or have stopped cigarette smoking during the past 12 months. Former smokers were defined as those who had stopped smoking more than one year previously. The rest of them were defined as never smokers or rare smokers. Exposure to environmental cigarette smoke for at least 30 minutes per day and three days per week at workplace, home or other public places was recorded in all patients, too.The evaluation of the nutritional habits was based on a validated semi-quantitative food frequency questionnaire . The conDepressive symptomatology was assessed using a translated and validated version of the Center of Epidemiological Studies Depression Scale (CES-D) . The CESClinical symptomatology of occupational stress was determined by a special self-reported questionnaire based on the survey obtainable by the Job Stress Help Line . By thisIn this, brief, baseline report continuous variables are presented as mean values ± standard deviation. The categorical variables are presented as absolute and relative (%) frequencies. Future analyses will follow with appropriate statistical techniques. Associations between continuous variables and group of patients will be evaluated through the analysis of variance, after controlling for equality of variances (homoscedacity) using the F-test. Due to multiple comparisons we will apply the Bonferroni rule to correct for the inflation of type – I error. Associations between categorical variables will be tested by the use of the chi-squared test, without the correction of continuity. Correlations between continuous variables will be tested by the use of Pearson's correlation coefficient for the normally distributed, and by the use of Sperman's rho coefficient for the ordinal or skewed variables. The association between the investigated socio-demographic, clinical and biochemical characteristics on the short term outcome (i.e. 1 month) will be tested by the development of multiple logistic regression models, while the association of the aforementioned characteristics on the long term outcome (6 and 12 months) will be tested by the use of Cox proportional hazards models. Appropriate tests for goodness-of-fit  will be applied in all models. All statistical calculations will be performed on the SPSS version 12.0 software .From October 2003 to September 2004, 2172 patients with discharge diagnosis of ACS were enrolled into the study . The men-to-women ratio was 3- to -1. The annual incidence of ACS was 22.6 per 10000 of people . The annual incidence was calculated with the exception of events at Hippokration Hospital in Athens, because it was very difficult to define the referent population since there are many hospitals in the area. The mean number of daily admissions was 6 ± 3 persons per day, averaging 5 ± 4 men and 2 ± 1 women.Table Of the 2172 patients enrolled into the study, 38% had ST segment elevations, 27% had non-ST segment elevations and the rest of them, i.e. 35% patients, had other electrocardiographic findings. According to the discharge diagnosis, 764 (35%) patients were diagnosed as having unstable angina, 699 (32%) patients as having non-Q-wave MI and 709 (33%) patients as having Q-wave MI.Table Table Table th, 75th percentiles) time between the overt of symptoms and the time medical care was sought, was 4  hours. Based on the discharge diagnosis the duration of hospitalization was 7  days for patients with Q-wave MI, 6  days for non-Q-wave patients and 5  days for UA patients. Furthermore, 60% of patients with ST-elevation received trombolytic therapy.The median . The in-hospital mortality rate of patients with ST segment elevation was 74 deaths per 1000 patients, for non-ST segment elevation was 34 deaths per 1000 and for undetermined electrocardiographic findings was 2 deaths per 1000 patients.In this brief report we presented the design, and the aims of an epidemiological study of ACS, that has been conducted in six major general hospitals, in Greece (i.e. the GREECS). We also presented the baseline characteristics of almost all patients who hospitalized in these hospitals for ACS during the study period (i.e. October 2003 to September 2004).The overall incidence rate of ACS observed in our survey was 22.6 events per 10000 of population. Based on this figure it could be speculated that the prevalence of ACS in the investigated Greek areas is 2.6%. In a similar recent study located only in northwestern Greece, which also included sudden cardiac death before hospital admission, the incidence rate of ACS was much higher (i.e. 39 events per 10000 people) . Based oWe also observed that the annual incidence for men was 34 per 10,000 of people, while the incidence for women was significantly lower, i.e. 11 per 10,000 of people. This finding will be tested whether can be attribute to the increased smoking habits observed in women during the past decades, as well as to various other lifestyle habits and work-related conditions, like physical inactivity, unhealthy diet, social insecurity and job stress, observed in female population from late 1960s to present .Furthermore, we observed that the in-hospital mortality rate in was 4.3%, which was similar to the in-hospital mortality rate that has been calculated based on a sample of 25 European countries, i.e. 4.9% [In this work we have also presented various baseline clinical and lifestyle characteristics of the enrolled patients. Future analyses will evaluate the role of the investigated clinical characteristics as well as family history of the common cardiovascular risk factors on the severity and prognosis of the ACS patients. Regarding dietary and other lifestyle related habits, we aim to evaluate whether the adherence to a Mediterranean dietary pattern, the adoption a physically active lifestyle, the abstinence of smoking and the control of psychological factors is associated with less severe disease and a better short and long term outcome. We anticipate that the completion of the follow up and the analysis of the results will provide current, novel and valuable information about the epidemiology of ACS, in Greece, as well as the role of clinical, psychosocial and lifestyle characteristics on the prognosis of cardiac patients.ACS = acute coronary syndromesCPK = creatinine posphokinaseMI = acute myocardial infarctionUA = unstable anginaECG = electrocardiographicCES-D = Center of Epidemiological Studies depressionBMI = body mass indexThe author(s) declare that they have no competing interests.CP, DBP = are the principal investigators of the study, had the concept and the design of the study, and wrote the paper, AA, YK, YM, SZ, PS, CS = contributed to the design of the study, GK = contributed to the data management and analysisThe pre-publication history for this paper can be accessed here:


Locus heterogeneity poses a major difficulty in mapping genes that influence complex genetic traits. A widely used approach to deal with this problem involves modeling linkage data in terms of finite mixture distributions. In its simplest setup, also known as the admixture approach, a single parameter is used to model the probability that the disease-causing gene of a family is linked to a reference marker. This parameter is usually interpreted as the overall proportion of linked families.In this article, we address two issues regarding the admixture approach. First, we tackle the question of whether the single parameter of linked proportion is well defined in general. By formulating the likelihood under a classification scheme based on distributions, we show that such a parameter is meaningful only when a certain well-characterized condition is met. Second, we study a condition given in the literature for validating the admixture approach. A counter example is constructed to illustrate that the condition does not necessarily lead to valid estimates.Estimators from the admixture approach may be inconsistent. This holds even if a condition given in the literature to validate the approach is satisfied. The heterogeneity is modeled by a single parameter (α) that denotes the probability that a family is linked. This parameter is interpreted by many researchers as the overall proportion of linked families.Mapping disease genes influencing complex genetic traits is far more difficult than mapping genes underlying Mendelian traits. One of the difficulties is due to locus heterogeneity, in which the disease in different families  is caused by different loci or non-hereditary factors. Finite mixture distributions have been proposed to model linkage data in the presence of locus heterogeneity , also knα does not even measure the proportion of linked families within the sample, contrary to popular belief. Similar conclusions were obtained in the simulation study by Pal and Greenberg )n et al.  showed, In this article, we consider the same problems discussed in Janssen et al.  and WhitT types such that families in the same type follow the same distribution. That is, families are classified into the same type based on their "structures" that lead to the specification of their distributions. Here we use the term structure in a broad sense that includes not only the pedigree structure but also any other available information about the pedigree (such as knowledge of phase) that contributes to the distribution from which the family is generated. Note that structure does not include any phenotypic or genotypic data of a family. Families with the same structure but different phenotypic and/or genotypic data are simply different realizations from the same distribution and hence are iid, and are classified into the same type. In the most general setup, with g potential linkage scenarios, e.g., linked or unlinked (g = 2) in the simplest setting, the probability distribution, ft*, for a realization (yt) from type t, t = 1,…,T, can be expressed in terms of its component distributions, fti, i = 1,…,g [Suppose that data from independent families in the sample are classified into  = 1,…,g :αti ≤ 1 , , and θ is the linkage parameter vector, including various recombination fractions.where the mixing parameters satisfy 0 ≤ θ or by a locus unlinked to the reference marker. Further, we assume that the genetic (trait) models at the linked and unlinked loci are the same. So, the linkage parameter vector is θ = {θ, 1/2}, and the probability distribution is simplified toTo simplify our presentation without compromising our objectives, we assume that the disease under study is caused either by a locus linked to the reference marker with a recombination fraction ft* = αtft + (1 - αt)ft,αt is the probability that a type t family is linked. Note that the two distributions under the mixture have the same parametric form but differ in their linkage parameter values, being θ or 1/2. Also, note that a single α parameter is used for all families in the same type.where a denote the true proportion of linked families in the entire sample. Let pt, t = 1,…, T, be the true proportion of type t families in the linked group. Thus p =  is the distribution of types among the linked families. Recall that all families in a given type have the same distribution, i.e., same likelihood for any value of the recombination fraction. Similarly, define q =  to be the distribution of family types among the unlinked group. Then the probability that a family is of type t is st = apt + (1 - a)qt, and the probability that a type t family is linked is thus . Furthermore, let r denote the true common recombination fraction for the linked families. The likelihood contribution for estimating the parameters {αt, θ} by a type t family with data yt isLet Lt = αtft + (1 - αt)ft.     (1)t can be expressed asThen the expected contribution to the log-likelihood from a family of type r and  are the true underlying quantities, whereas θ and α =  are the parameters to be estimated from the data. Combining the expected log-likelihoods, ELLt, t = 1,…, T, we can form the total expected log-likelihood from all family types as follows:Note that α0, r) ≥ ELL, for any , following Stuart and Ord [α, θ). Note that α is a vector of nuisance parameters; only θ is the true parameter of interest.Since the above formulation of likelihood is correct under the stated assumptions, it can be shown that ELL families with the same structure (and hence their data coming from the same distribution as elucidated at the beginning of this section) share a common α parameter. Since the issue addressed by Vieland and Logue [We note that the above likelihood, although similar to the one presented in Vieland and Logue , is diffnd Logue  is diffep = q, that is, the distribution of family types among the linked families is the same as that among the unlinked families. In this setup, all αt's are set to be equal to a common parameter, α, the overall proportion of linked families. Hence, the total expected log-likelihood is with respect to the recombination fraction θ and a single proportion parameter α:In this subsection, we show that the two-parameter setup leads to correct likelihood formulation only when a, r) and , isThe expected log-likelihood ratio between the two sets of parameter values,  ≥ ELL, for any , implying that  = a and  = r are the maximum likelihood estimates of α and θ. However, as shown in the examples in the next section, when p ≠ q, ELL may be smaller than ELL for some parameter values . That is, the maximum likelihood estimates of α and θ may not be consistent because the likelihood model is incorrectly specified (under-parameterized). In order for the two-parameter formulation to be correct, the following constraints should be placed on the makeup of the families:In other words, when p = q given the additional constraints that  and . Notice that p = q is stronger than the LUFED condition. Equal distributions of types in the linked and unlinked families according to the classification scheme based on distributions of the data implies equal distributions of informativeness in the same two groups. Therefore, LUFED is a necessary condition for the admixture approach. We also note that p = q is not necessarily satisfied for every dataset (see Discussion).which implies that α, the relationship between α and αt is as follows:In general, in a parameterization that demands an estimate for the overall proportion of linked families, ut and vt are also unknown parameters (with constraints ∑ut = 1 and ∑vt = 1) that need to be estimated. Note that ut and vt are the parameters to be estimated from the data and are not necessarily the same as pt and qt, respectively, as the latter are the true values of the former. We have shown earlier that the maximum likelihood estimates for αt exist. However, one cannot solve the equations in (3) to obtain a corresponding estimate for α. This is because there are only T + 2 equations (including the two constraints) but 2T + 1 unknown parameters, and thus not all parameters are identifiable when T ≥ 2. Hence, one may not be able to find a meaningful estimate of the overall proportion of linked families, contrary to popular desire for such an estimate. This is consistent with the finding of Whittemore and Halpern [where  Halpern , althougWe have already shown that LUFED is a necessary condition for obtaining consistent estimators using the admixture approach. In this section, we investigate, through a contrived dataset, whether LUFED is also a sufficient condition for achieving satisfactory results, as contended by Janssen et al. . We use p1, and Phase Unknown (PU) families in proportion p2, where p1 + p2 = 1. The unlinked group is also composed of PK and PU families, but in proportions q1 and q2, respectively, where q1 + q2 = 1. We choose to work with the PK and PU families as the PK families were used by Janssen et al. [m children in all PK families and m + 1 children in all PU families. As these two types of families have different distributions [T = 2) under the classification scheme according to distributions.In our contrived dataset, the linked group consists of two types of families: Phase Known (PK) double backcross families in proportion n et al.  to evalum children is:Now, let us consider the ELOD of each family. The ELOD of a PK family with θ is the parameter for recombination fraction while r is the true recombination fraction. Similarly, one can find the ELOD of a PU family with m + 1 children:where, as before, r close to 0, it can be seen that ELODPK(θ) and ELODPU(θ) are both approximately m log10 2, when they are evaluated at θ = r [α parameters.For at θ = r  LUFED com/0, m - 1/1,..., 1/m - 1, 0/m) offspring.It is worth noting that this dataset may serve as an example for understanding classification based on distributions and family realizations that make up a family type. The phase information as well as the number of offspring are part of the family structure, which is the basis of classification according to distributions. However, phenotypes and genotypes do not configure into this classification scheme. For example, the PK data type consists of families with several phenotypes, i.e., various combinations of recombinant/non-recombinant , q = ; and (b) p = , q = . For each of these two settings, we take m = 3 children in PK families and m + 1 = 4 children in PU families. In both settings, the overall proportion of linked families, a, is 0.5, and the true  recombination fraction, r, is 0.02. So, as discussed above, the distribution of informativeness among the linked families, under the ELOD measure, is the same as that among the unlinked families. The two pictures in Figure α, θ) where the maximum expected log-likelihood occurs are not the same as their true values. So the estimates are inconsistent. The extent of this inconsistency depends on several factors, including how close p is to q. As seen in Figure p differs greatly from q, the parameter estimates are far away from the true values. On the other hand, in Figure p is close to q, the estimates are not too far from the truth. In fact, the estimates are expected to converge to the true parameter values when the difference between p and q approaches 0.To illustrate our results, we consider two specific settings for the distributions of families in the linked and unlinked groups under the classification scheme according to distributions: (a) = 0.9, 0., q =  in the two groups. Although the problem considered in Vieland and Logue [The first question of interest was addressed in Whittemore and Halpern , where tnd Logue  is diffeno under the ELOD measure for informativeness. The difference between the estimates and the true values of the parameters, even with infinite amount of data, can be large, if one would carry out the analysis under the admixture approach. This should serve as a warning against complacency when the LUFED condition is met. We do realize that, with phase known and phase unknown data, these examples can be quite extreme in human genetic studies, although such data arise frequently in experimental crosses. In situations where data from more general family structures are available, the effect of violation of the admixture approach assumptions may be much smaller.The second question stems from our curiosity on an argument made by Janssen et al.  for the The criteria for classification of families deserve further clarification because it is the heart of the problem. We discuss two criteria for classification, one according to the distributions of the data, and the other according to a measure of informativeness of the data for linkage studies (leading to the LUFED condition). The classification scheme based on the distribution criterion leads to a necessary and sufficient condition for validating the admixture approach. This result is a by-product of our theoretical development for finding an answer to the first question. The classification scheme based on the informativeness criterion, on the other hand, leads to the conclusion that LUFED is only a necessary, but not a sufficient, condition for the admixture approach, contrary to Janssen et al.'s  contentiFinally, we note that although the general formulation of likelihood based on forming groups of families with the same distribution gives consistent estimators, its practical utility seems to be limited. This is mainly due to the difficulty of classifying families according to distributions, in most applications, except for simple situations such as those involving only the PK and PU families. The general likelihood formulation is used here as a vehicle to further the understanding of potential problems in using the two-parameter admixture approach. A practical solution that embeds the correct likelihood formulation in a Bayesian framework is being pursued in a separate study.Both authors were involved in all aspects of this research project. Both authors read and approved the final manuscript.


The rate of total edentulism is said to be increasing in developing countries and this had been attributed mainly to the high prevalence of periodontal diseases and caries. Several reports have shown that non-disease factors such as attitude, behavior, dental attendance, characteristics of health care systems and socio-demographic factors play important roles in the aetiopathogenesis of edentulism. The aim of this study was to assess the relationship between socio-demographic factors and edentulism.A total of 152 patients made up of 80 (52.6%) males and 72 (47.4%) females who presented in two prosthetic clinics located in an urban and a rural area were included in the study. The relationship between gender, age, socio-economic status and edentulism in this study population was established.rd decade and then started to decline. A significant relationship was found between denture demand and the level of education with a higher demand in lower educational groups (p < 0.001). In addition, the lower socio-economic group had a higher demand more for prostheses than the higher group.No significant relationship between gender and denture demand was noted in the study. The demand for complete dentures increased with age while the demand for removable partial dentures also increased with age until the 3The findings in this study revealed a significant relationship between socio-demographic variables and edentulism with age, educational level and socio-economic status playing vital roles in edentulism and denture demand. Edentulism  is an indicator of the oral health of a population . It may While the rate of total edentulism is decreasing in developed countries, the reverse is the case with developing countries and this had been attributed mainly to the high prevalence of periodontal diseases and caries -7.Previous studies have also shown that several non-disease factors such as attitude, behavior, dental attendance, characteristics of health care system and socio-demographic factors play important roles in the aetiopathogenesis of edentulism .Some studies reported that the incidence of edentulism correlated with educational levels and income status, with those in the lower levels exhibiting higher risks of becoming totally edentulous ,9. In adAlthough, Hoover and McDermount  reportedStudies among Nigerians have linked some of these socio-demographic factors with the prevalence, pattern and rate of dental diseases ,14 but tThe aim of this study therefore was to assess the relationship between socio-demographic variables with types of edentulism.All patients that attended and were treated in the removable prosthetic units of Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife  between the months of March and May year 2002 and Lagos University Teaching Hospitals (LUTH), Lagos  between December 2002 and March 2003 were included in the study. Information such as age, gender, occupation and level of education attained were documented. The types of partial denture received following treatment at the clinics were also documentedThere has not been a consensus on various socio-economic classifications in Nigeria because of the unstructured nature of the society. Therefore, for the purpose of this study, a standard occupational classification system designed by Office of population Census and Surveys, London (OPCS 1991)  modifiedClass 1 = Skilled worker e.g professionals and managerial officers and retirees of this cadre.Class 2 = Unskilled workers e.g. Artisans and tradersClass 3 = Dependants. e.g. Retirees of class 2, those not on pensions, house wives of class 2 cadre, students whose parents are unskilled workersData was analysed using SPSS for Windows version 10.0, . Analysis included frequency, cross tabulations, calculation of means. Association between discrete variables was tested by Chi-Square and the level of significance was set at 5%.One hundred and fifty two patients attended the prosthetic clinics during the study period. Eighty (52.6%) were males while 72 (47.4%) were females Table . Their aThere were no statistically significant age p = 0.312) and gender (p = 0.113) differences between the populations from the two centers.  .  patients belonged to class I; 43(28.29%) patients belonged to class II while 81(53.29%) belonged to class III. There was no statistically significant difference in the socio-economic status of patients from the two centers  received removable partial dentures, 13 patients (8.6%) received complete dentures while 5 patients (3.3%) received either upper or lower complete dentures. There was no significant difference in the demand for different types of dentures between the study locations. (P = 0.315). . Table It was observed that as the age increased, the proportions demanding for complete dentures also increased. In addition, those in age group 21–40 years demanded more for removable partial denture than any other age groups. While those above 61 years asked more for removable complete dentures than removable partial dentures. (Table No significant relationship between gender and pattern of denture demand (p = 0.812) was noted and no statistically significant difference was noted in the pattern of denture demand between the two centers (p = 0.277). Table .The lower educational groups demanded more for complete dentures among those asking for complete denture, while those with higher level of education asked more for removable partial denture. (P < 0.001). Table . More ovAmong the patients that were completely edentulous, there was no significant difference in the demand for complete dentures between those with lower educational status and those with higher educational status. P = 0.276 , hence the likelihood of retaining teeth in the mouth becomes higher as the educational level increases. Although the educational status of the patients from the two centers differ, independent analysis of these centers still showed the same significant effect of educational status on the pattern of denture demand. This is in agreement with the findings of Brodeur et al, where the proportion of completely edentulous adults decreased from 26% in 1980 to 20 % in 1993 due to improved income and educational status [, hence tThe association between edentulism and educational status may be as a result of improved dental health awareness, increased utilization of oral health facilities, proper oral hygiene habits acquired during learning process and peer group influence.Interestingly, about 23.3 % of those in class II who need dentures asked for complete dentures as opposed to 3.6% in class I and 8.6% in class III. The reason for this may be as a result of the fact that they may not be able to afford the exorbitant cost of restorative procedures hence they wait until they have lost their set of teeth to have a complete removable denture which is cheaper.The present study showed that people with low socio-economic status demanded for more dentures than the high socio-economic group. Studies have long established a gradient relationship between socio-economic status and health . In so mThe importance of socio-economic status is further reflected in the urban-rural variance noted in this study population's demand for denture. The Ile-Ife study group, a rural population with a lower socio-economic status, demanded for more dentures than the Lagos study group.However, this study population was an all-inclusive hospital based sample; the result may not be representative of the population at large. Hence, its use can only be limited to the study population. A randomized population based survey may be able to present a better picture among Nigerians.This study observed that edentulism is due to a combination of various factors. Poor education, a risk factor for poverty, has been identified as a major factor in edentulism. So also is the socio-economic status of the patient. These two factors, which are non-disease factors, affect the mortality of teeth arising from disease factors. There is therefore a need for oral health policy formulators to focus on improving the educational and socio-economic status of its citizens (a down stream approach) rather than the present emphasis on disease control (an up stream approach) in oral health care delivery.On the other hand, with increasing level of literacy, and positive social changes in Nigeria, Prosthodontists should brace up to face the challenges that may arise from increased removable partial denture demand and decreased demand for complete dentures. This is because, with increase in level of education and socio-economic status of patients, the demand for removable partial dentures is likely to increase while dentists may be confronted with a significant increase in the number of difficult edentulous mouths requiring treatment.In addition to addressing the non-disease factors, dental education should be targeted at the uneducated populace, the rural dwellers and low-income groups to reduce the rate of total edentulism.No gender relationship with denture demand was noted this study. In addition, the demand for complete dentures increased with age. There was a statistically significant inverse relationship between educational levels and demand for dentures. There was more demand for prostheses among the lower socio economic groups.The pre-publication history for this paper can be accessed here:


To contribute further to the classification of three CFTR amino acid changes  either as CF or CBAVD-causing mutations or as neutral variations.The CFTR genes from individuals who carried at least one of these changes were extensively scanned by a well established DGGE assay followed by direct sequencing and familial segregation analysis of mutations and polymorphisms.Four CF patients (out of 1238) originally identified as carrying the p.I148T mutation in trans with a CF mutation had a second mutation (c.3199del6 or a novel mutation c.3395insA) on the p.I148T allele. We demonstrate here that the deletion c.3199del6 can also be associated with CF without p.I148T. Three CBAVD patients originally identified with the complex allele p.R74W-p.D1270N were also carrying p.V201M on this allele, by contrast with non CF or asymptomatic individuals including the mother of a CF child, who were carrying p.R74W-p.D1270N alone.These findings question p.I148T or p.R74W-p.D1270N as causing by themselves CF or CBAVD and emphazises the necessity to perform a complete scanning of CFTR genes and to assign the parental alleles when novel missense mutations are identified. Frameshift, splice-site, nonsense, and in-frame but nonfunctional deletions (such as p.F508del) are disease-causing mutations. By contrast, the status of some missense mutations is extremely difficult to assess and functional studies are not available to diagnostic laboratories. Missense mutations may represent, depending on the populations, up to 45% of mutations responsible for CF or CBAVD . Moreover, due to improved scanning strategies, a growing number of complex alleles  are thought to affect the expression of the disease phenotype by modulating the effect of a mutation  in patients with a CBAVD phenotype in this series.With the start of population screening for CF carriers, new data on the prevalence of some missense mutations have been provided, questioning their involvement as disease-causing mutations. In North American populations, missense mutations p.I148T and p.D1270N were found >100 times and >200 times, respectively, more frequently in carrier screening than in CF patients . Moreovetrans.From 1990 to 2003, we have analysed for CFTR mutations genomic DNA from 437 families with CF and 170 with isolated azoospermia caused by CBAVD, using a combination of mutation screening for known and scanning for unknown mutations. The first step was the search of 31 CF mutations detected by the ABI oligonucleotide ligation assay and 3 common intronic mutations by using restriction analysis. The second step was the scanning of coding/flanking sequences by DGGE (Denaturing Gradient Gel Electrophoresis) using 32 GC-clamped amplimers (which in our experience detected 98% of CFTR mutants), followed by sequencing to resolve abnormal PCR products (BigDye terminator cycle sequencing on ABI 310 automate sequencer). Whenever possible, family members were assayed for the mutations and associated polymorphisms. We detected 160 different mutations in the CF group accounting for 97 % of CF alleles, and 64 different mutations in the CBAVD group accounting for 85% of CBAVD alleles, which represents one of the highest allelic heterogeneity reported so far. Usually, mutation scanning is stopped when two mutations are found to be in trans of c.3195del6  and assayed their relatives for the additional sequence changes identified. We also re-analyzed the CFTR gene of a CF patient who had been originally described with c.394delTT in .Gene variants and mutants are described using DNA and protein designation: intronic changes, deletions, insertions and frameshifts are reported at the cDNA level (c.) and amino acid changes at the protein level (p.), as recommended in the Human Genome Variation Society web page Two out of 437 CF patients analyzed in Montpellier and two out of 801 CF patients analyzed in Lyon were found to carry p.I148T, which was initially thought to be one of the two mutations responsible for CF in these patients. However, thorough re-analysis of the entire CFTR sequence determined that a CF mutation (c.3395insA or c.3199del6) was present on the same gene in both cases  on the same chromosome in three unrelated individuals with CBAVD ; in addition, a completely asymptomatic adult compound heterozygote for p.D1270N and p.I148T has been identified . Althougtrans that are in fact complex alleles.This report further corroborates the recent hypothesis  that p.INone declared.JPA, CG, CT and FC carried out the molecular genetic studies. MDG coordonated the molecular analysis. MC conceived the study and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Retinopathy of Prematurity (ROP) is a common retinal neovascular disorder of premature infants. It is of variable severity, usually heals with mild or no sequelae, but may progress to blindness from retinal detachments or severe retinal scar formation. This is a preliminary report of the effectiveness and safety of a new and original use of topical ketorolac in preterm newborn to prevent the progression of ROP to the more severe forms of this disease.From January 2001 to December 2002, all fifty nine preterm newborns with birthweight less than 1250 grams or gestational age less than 30 weeks of gestational age admitted to neonatal intensive care were eligible for treatment with topical ketorolac (0.25 milligrams every 8 hours in each eye). The historical comparison group included all 53 preterm newborns, with the same inclusion criteria, admitted between January 1999 and December 2000.Groups were comparable in terms of weight distribution, Apgar score at 5 minutes, incidence of sepsis, intraventricular hemorrhage and necrotizing enterocolitis. The duration of oxygen therapy was significantly longer in the control group. In the ketorolac group, among 43 children that were alive at discharge, one (2.3%) developed threshold ROP and cryotherapy was necessary. In the comparison group 35 children survived, and six child (17%) needed cryotherapy . Adjusting by duration of oxygen therapy did not significantly change these results. Adverse effects attributable to ketorolac were not detected.This preliminary report suggests that ketorolac in the form of an ophthalmic solution can reduce the risk of developing severe ROP in very preterm newborns, without producing significant adverse side effects. These results, although promising, should be interpreted with caution because of the weakness of the study design. This is an inexpensive and simple intervention that might ameliorate the progression of a disease with devastating consequences for children and their families. We believe that next logical step would be to assess the effectiveness of this intervention in a randomized controlled trial of adequate sample size. Retinopathy of Prematurity (ROP) is a common retinal neovascular disorder of premature infants. It is of variable severity, usually heals with mild or no sequelae, but may progress in some infants to partial vision loss or blindness from retinal detachments or severe retinal scar formation . ROP remThe active disease appears in the premature about 4 to 8 weeks after birth. In this period, the levels of vascular endothelial growth factor (VEGF) increase in the retina, as well as other chemical mediators of inflammation such as platelets activator factor (PAF), prostaglandins (PGs), and eicosanoids which would put again under way the process of vascularization that had stopped in the period of oxidative injury. This vascularization is now degenerated and invasive -17.In models of animal experimentation it was possible to diminish the degree of retinal neovascularization with use of indometacin , dexametKetorolac is a non-steroid anti-inflammatory drug (NSAID) derived from indometacin. Its mechanism of action is developed through the interruption of the synthesis of prostanoids, inhibiting the way of COX 1 and 2 in arachidonic acid metabolism; in this way the tissue levels of prostaglandin F2alfa and thromboxane B2 decrease. Its adverse effects are linked predominantly to their inhibitory action of platelet aggregation. It does not alter either the platelet count, or factors of clotting. High digestive hemorrhage is the principal adverse reaction. Nervous and cardiovascular systems are not generally affected by the use of ketorolac to habitual doses -24.Ketorolac administered as conjunctival topical diminishes prostaglandin E2 concentration in aqueous humor, without modifying the intraocular pressure . In addiKetorolac ophthalmic solution is usually used in older adults with retinal disorders. It is used to diminish the Cystoid Macular Edema that complicates the surgery of cataracts. In this pathology ketorolac has proved to be effective in diminishing the macular edema and improving the visual acuity, providing evidence that their conjunctival instillation produce effects on the most internal layers of the eye. The ophthalmic use of ketorolac only reports occasional episodes of discomfort and ocular burning -28.The use of ketorolac ophthalmic solution in pediatrics is frequent as an analgesic in corneal abrasions, and in allergic and post surgical conjunctivitis. The FDA recognizes its indication for allergic conjunctivitis, ocular pain, post surgical ocular inflammation, ocular pruritus and photophobia ,29.On the basis of experimental evidence and physio-pathogenic rationality, we treated preterm newborns admitted to neonatal intensive care unit (NICU) of our hospital with ketorolac in ophthalmic drops with the aim of decreasing the progression and severity of ROP. This study compares this group of children treated with ketorolac with historic controls to assess the impact of this treatment on the incidence of severe ROP and adverse effects.This is a preliminary report, that compares a cohort of children treated with ketorolac with historic controls that did not received such treatment. From January 2001 to December 2002 all preterm newborns with birthweight less than 1250 g or gestational age less than 30 weeks of gestational age, admitted in the NICU of the University Hospital of Maternity and Neonatology of the city of Córdoba, Argentina, were eligible for treatment with topical ketorolac. The comparison group included all preterm newborns, with the same inclusion criteria, admitted between January 1999 and December 2000. None of these newborns received treatment with ketorolac.There were no differences either in treatment guidelines, equipment or in the number of physicians and nurses that took care of the patients in the two periods. All the children were examined by the same group of ophthalmologists, which have many years of experience in treating patients with ROP. The international classification for ROP  was usedDuring the period in which ketorolac tromethamine was used, when risk signs for ROP were identified   treatmenThe variables considered to assess the comparability among both groups were: birthweight, Apgar score at birth less than 6 at 5 minutes, duration of oxygen therapy, peri-intraventricular hemorrhage (PIVH) equal or greater than 3 degrees, necrotizing enterocolitis (NEC) equal of greater than II degrees, and late sepsis. The analyses included only those children that were alive at discharge, but the number of deaths was reported for both study periods. The presence of undesirable effects of ketorolac such as hemorrhages, oliguresis, local manifestations of intolerance, and conjunctival infection were analyzed.Fischer exact test was used to obtain two tailed p-values. Relative risks (RR) with 95% confidence intervals where also calculated. Adjusted analyses were performed by using Poisson regression with robust estimates . StatistDuring the analyzed period 112 eligible preterm newborns were admitted, 53 between 1999 and 2000 (historic controls) and 59 between 2001 and 2002 (ketorolac group). The number of newborns that died before discharge was similar between groups  than in the control group  is large and in our opinion unlikely to be explained completely by confounding factors, although this possibility cannot be completely ruled out. To our knowledge, no significant change in the standard of care took place between the study periods, but mortality was lower during the period were ketorolac was used, although the difference was not statistically significant. The groups were comparable in terms of their birthweight distribution, Apgar score, and indicators of severe morbidity. A significant difference was found in duration of oxygen therapy, but in our data there was no association between duration of oxygen therapy and severe ROP, and adjusting for oxygen did not change the results. The principal factor involved in the genesis and severity of ROP is the injury due to O2 radicals; there were no substantial changes in the management of O2 supply to the children of our unit in both periods, the equipment used to measured O2 saturation and to administer the oxygen.The prevalence of severe ROP in our maternity was relatively high, considering that for 1998 the Vermont-Oxford Network reported an incidence of 9.5% for severe ROP and of 57.2% for ROP of any degree , but simKetorolac was apparently safe. No patients in the group that received ketorolac presented oliguric, or with biochemical signs of renal failure during treatment. There were no hemorrhagic manifestations that could be attributed to ketorolac; hemorrhages observed in vitreous evolved favorably with collyrium. Neither local intolerance episodes to the drops nor purulent conjunctivitis among the treated cases were observed.The effect on ROP we have observed is compatible with an impact of Ketorolac in the active phase of neovascularization, a fact that is also observed in numerous reports in animal models of ROP with NSAIDs and steroid drugs administered systemically. The hypothesis of an inflammatory component in active ROP has been quite recently recognized by experts, extensive leukocyte adhesion was observed at the leading edge of pathological neovascularization . Recent ROP is a problem that modern medicine has generated due to the survival of very small preterm children and which has been unable to solve so far. About 2% of the children with birthweight less than 1500 grams are blind because of this pathology. Current preventive interventions can prevent blindness in nearly 70% of the cases. Earlier treatment using ablation of the avascular retina in pre-threshold ROP has been proposed , but a pThis preliminary report provides evidence suggesting that ketorolac in the form of an ophthalmic solution can be used to reduce the risk of developing severe ROP in very preterm newborns, without producing significant adverse side effects. These results, although promising, should be interpreted with caution because of the weakness of the study design. We cannot exclude the possibility of bias been responsible of the observed effect. On the other hand, if confirmed, these results can have important public health implications. This is an inexpensive and simple intervention that might ameliorate the progression of a disease with devastating consequences for children and their families. We believe that next logical step would be to assess the effectiveness of this intervention in a randomized controlled trial of adequate sample size to provide a definite answer to this research question.None declared.MAV and RM conceived the study. MAV, MLC, EB and RM design the study. MF carried out the revision of the clinical histories. MS carries out the ophthalmologic exams of all the cases. RM verified the ophthalmologic finds. EB carried out the statistical analysis. MAV and EB drafted the article. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: