Since several of the cognitive functions typically deficient in children with Attention Deficit Hyperactivity Disorder (ADHD) are mediated by prefrontal dopamine (DA) mechanisms, we investigated the relationship between a functional polymorphism of the COMT gene and neuropsychological task performance in these children.An association has been observed between the Val108/158 Met polymorphism of the COMT gene was genotyped in 118 children with ADHD (DSM-IV). The Wisconsin Card Sorting Test (WCST), Tower of London (TOL), and Self-Ordered Pointing Task (SOPT) were employed to evaluate executive functions. Neuropsychological task performance was compared across genotype groups using analysis of variance.The Val/Val, Val/Met and Met/Met genotypes were similar with regard to demographic and clinical characteristics. No genotype effects were observed for WCST standardized perseverative error scores , TOL standardized scores , and SOPT error scores .ADHD children with the Val108/158 Met polymorphism of the COMT gene in both healthy and schizophrenic adults, this polymorphism does not appear to modulate executive functions in children with ADHD.Contrary to the observed association between WCST performance and the  SLC6A3 gene . Conversely, parents transmitted the Met allele to their affected children in 29 occurrences, whereas this same allele was not transmitted in 28 occurrences . In addition, results from the QTDT revealed no evidence of linkage or within-family association between the three quantitative phenotypes and the COMT gene.The genotype distribution conformed to a Hardy-Weinberg equilibrium  on WCST standardized perseverative error scores , WCST number of categories completed , WCST number of trials to first category , TOL standardized total item scores , TOL raw item scores , TOL time to complete each trial , TOL number of problems solved  and SOPT raw error scores .A one-way ANOVA performed on these data revealed no significant difference between the LL, HL, and HH genotypes according to WCST standardized perseverative error scores [FCOMT polymorphism and a variety of indices reflecting executive control both in healthy [COMT appears to be important to the regulation of dopamine metabolism within the PFC [COMT Val108/158Met polymorphism, which is known to be associated with a significant change in the catabolic capacity of this enzyme, modulates the risk for ADHD or various indices of executive control. Contrary to our expectations and findings in both healthy [Val108/158 Met functional polymorphism of the COMT gene and neuropsychological task performance reflecting executive control was not observed in children with ADHD. This result is consistent with the findings of a recent case-control study conducted by Mills et al. (2004), which, to our knowledge, is the only other study to investigate the relationship between the COMT Val108/158Met polymorphism and neuropsychological task performance in children with ADHD [Previous studies have identified an association between the  healthy ,37 and s healthy -41. The  the PFC -35. Sinc the PFC -11,60,61 healthy ,37 and s healthy -41, an aith ADHD . HoweverCOMT Val108/158Met polymorphism and behavioral indices of executive function in children with ADHD may be explained by the young age of the population of patients included in the present study. Indeed it is possible that, due to age-related changes in the functional importance of the COMT within the prefrontal cortex, this association is observable only in adults. This possibility is supported by data in both rats [COMT activity [COMT in the catabolism of dopamine is developmentally regulated, with children relying less on this catabolic pathway than adults. Conversely, it has been reported that DAT density is inversely correlated with age [COMT activity on the other hand, may suggest that dopamine metabolism relies more on the DAT than on COMT activity in children compared to adults. This hypothesis is compatible with the fact that several studies have identified an association between the DAT [COMT, gene and ADHD.The absence of an association between the oth rats -65 and hoth rats ,67 suggeactivity -70. Thiswith age . Taken t the DAT ,60,72-74It is also possible that the negative result observed in the present study is due to a type II error  secondary to the lack of power of our sample to detect an association. However, using results from the WCST, the variable for which relevant genetic data already exists, we conducted a power analysis and determined that our sample size has sufficient power (80% at α = .05) to detect a mean difference of 11.2 on this measure. Furthermore, it is possible that some of the tests used in our assessment are mediated by the PFC but insensitive to PFC DA levels .An additional limitation of the present study is that some genotype groups included few subjects. Increasing the sample size to achieve larger genotype groups would be necessary to reach firmer conclusions. This is particularly true for female subjects who were significantly underrepresented in the study . In order to generalize these negative results to females, a more comparable gender representation is required, particularly in view of some previous research indicating that the allelic distribution of the COMT may be gender dependent .Val108/158 Met polymorphism of the COMT gene in increasing the risk for ADHD or in modulating several indices of executive functions in children with ADHD. This result is contrary to previous findings in both healthy and schizophrenic adults and may be related to developmental specificities.This study does not support the involvement of the The author(s) declare that they have no competing interests.ET performed the data analysis and drafted the manuscript. NG was involved in the conception of the study and provided clinical support. LBA provided clinical support and aided in data collection. PL provided clinical support. VM aided in neuropsychological testing and data collection. RD and ATZ performed the genotyping for the study and aided in data management. MTS coordinated the clinical aspects of the study and was involved in data management. CB provided clinical support. RJ was responsible for the conception of the study, drafting of the manuscript, and supervision of the research project.The pre-publication history for this paper can be accessed here:


Hairless animals in the lab, on the other hand, can be very instructive. Understanding how hair develops sheds light on the fundamental processes that generate a wide range of tissues and organs, including the lungs, cornea, and mammary glands.If you're a cat fancier, you're well aware that hair follicles are expendable. The product of a spontaneous mutation that caught a cat breeder's eye, How complex, three-dimensional structures emerge from single sheets of cells is a fundamental question in developmental biology. The dispensability of hair follicles makes them the perfect model system for studying this question—specifically, how structures and organs develop from buds. In a new study, Elaine Fuchs and colleagues use a three-pronged approach—involving gene expression analysis, transgenic mice, and cell cultures—to study how epithelial buds, the precursors of hair follicles, form. Their experiments point to two key actors in a signaling pathway that molds a targeted cluster of cells into a hair bud.During the budding process, overlapping signaling pathways from two adjacent embryonic cell layers—the epithelium and the mesenchyme—direct morphogenesis. The mesenchymal cells initiate the cell-to-cell “crosstalk” that controls bud formation by first directing a small cluster of epithelial cells to form a placode, the pouch that forms hair plugs. The placode in turn directs underlying mesenchymal cells to form the base of the hair follicle, called the dermal papilla, and both structures contribute to the mature hair follicle. During development, cells are constantly bombarded with external signals. The trick is figuring out which signals trigger the transcriptional and behavioral properties in cells that spur bud formation.In previous experiments, Fuchs and colleagues showed that reducing expression of E-cadherin—a membrane protein that forms the adhesive junctions between epidermal cells—is essential for allowing the cell remodeling required for bud formation. Here, the authors analyze the timing of external signals against the response of targeted cells to determine how targeted cells translate signals into changes in cell adhesion and remodeling, proliferation, and differentiation—the agents of most types of organogenesis.Since Snail, a protein that impedes the transcription of a subset of genes, functions in many developmental processes requiring epithelial remodeling, the authors reasoned it might do the same in hair bud formation. Working with developing mouse embryos, they saw a spike in Snail expression on embryonic day 17.5, coinciding with hair bud formation, enhanced cell proliferation, and the down-regulation of E-cadherin. Artificially sustaining Snail expression in the skin of transgenic mice caused abnormal levels of cell proliferation in the epidermis and reduced cell adhesion.Working with skin keratinocytes, precursors of hair fibers, Fuchs and colleagues explored several signaling proteins known to be involved in bud formation as possible activators of Snail expression. When the authors treated keratinocytes with small amounts of one stimulator, TGF-β2, they saw “rapid and transient induction of Snail.” Snail proteins were absent from 17.5-day-old knockout mice lacking TGF-β2 but not from their nonmutant littermates. Conversely, transgenic mice with elevated TGF-β2 signaling activity displayed ectopic expression of Snail. Knockout mice lacking TGF-β2 also showed higher levels of E-cadherin—normally down-regulated by Snail—than their nonmutant littermates.Altogether, these findings suggest that TGF-β2 signaling transiently induces Snail, which in turn down-regulates E-cadherin and activates a proliferation pathway in the developing bud. Reduced E-cadherin, the authors conclude, appears to contribute to Snail-mediated enhanced proliferation by allowing proteins normally sequestered at the membrane to operate in a proliferation pathway after the number of cellular junctions diminishes. By identifying which molecules are active in specific cell types at specific developmental stages, this study lays the foundation for dissecting the mechanisms that connect two key processes—intercellular remodeling and proliferation—in epithelial development. And since the consequences of TGF-β2 activity seen here in the hair bud more closely resemble certain types of skin cancer progression than skin development, a mechanistic understanding of hair follicle development promises to shed light on how skin cancer develops as well.


Tortula ruralis. To establish a sound basis for characterizing the Tortula genome in regards to desiccation tolerance, we analyzed 10,368 expressed sequence tags (ESTs) from rehydrated rapid-dried Tortula gametophytes, a stage previously determined to exhibit the maximum stress induced change in gene expression.The cellular response of plants to water-deficits has both economic and evolutionary importance directly affecting plant productivity in agriculture and plant survival in the natural environment. Genes induced by water-deficit stress have been successfully enumerated in plants that are relatively sensitive to cellular dehydration, however we have little knowledge as to the adaptive role of these genes in establishing tolerance to water loss at the cellular level. Our approach to address this problem has been to investigate the genetic responses of plants that are capable of tolerating extremes of dehydration, in particular the desiccation-tolerant bryophyte, Tortula clusters with an identical analysis of 9,981 clusters from the desiccation sensitive bryophyte species Physcomitrella patens, revealed, and accentuated, the differences between stressed and unstressed transcriptomes. Cross species sequence comparisons indicated that on the whole the Tortula clusters were more closely related to those from Physcomitrella than Arabidopsis (complete genome BLASTx comparison) although because of the differences in the databases there were more high scoring matches to the Arabidopsis sequences. The most abundant transcripts contained within the Tortula ESTs encode Late Embryogenesis Abundant (LEA) proteins that are normally associated with drying plant tissues. This suggests that LEAs may also play a role in recovery from desiccation when water is reintroduced into a dried tissue.The 10, 368 ESTs formed 5,563 EST clusters  of which 3,321 (59.7%) exhibited similarity to genes present in the public databases and 2,242 were categorized as unknowns based on protein homology scores. The 3,321 clusters were classified by function using the Gene Ontology (GO) hierarchy and the KEGG database. The results indicate that the transcriptome contains a diverse population of transcripts that reflects, as expected, a period of metabolic upheaval in the gametophyte cells. Much of the emphasis within the transcriptome is centered on the protein synthetic machinery, ion and metabolite transport, and membrane biosynthesis and repair. Rehydrating gametophytes also have an abundance of transcripts that code for enzymes involved in oxidative stress metabolism and phosphorylating activities. The functional classifications reflect a remarkable consistency with what we have previously established with regards to the metabolic activities that are important in the recovery of the gametophytes from desiccation. A comparison of the GO distribution of Tortula ruralis, an important plant model for plant stress responses and vegetative desiccation tolerance, is an important step in understanding the genome level response to cellular dehydration. The type of transcript analysis performed here has laid the foundation for more detailed functional and genome level analyses of the genes involved in desiccation tolerance in plants.The establishment of a rehydration EST collection for  Understanding how plant cells tolerate water loss is a vital prerequisite for developing strategies for improving tolerance of, and biomass/seed production under drought conditions.The cellular response of plants to water deficits has both economic and evolutionary importance directly affecting plant productivity in agriculture and plant survival in the natural environment. Ramanathan  has arguArabidopsis thaliana [Arabidopsis microarrays [Tortula ruralis [Hedw.] Gaertn. Meyer & Scherb., that is representative of the primitive genetic strategy for the acquisition of desiccation-tolerance ..ArabidopDesiccation tolerance, the ability to recover from the almost complete loss (90%) of protoplasmic water, is a phenomenon common in the reproductive structures of green plants: pollen, spores and seeds. However, the ability to survive vegetative desiccation is a demonstrable but uncommon occurrence in the plant kingdom -18. WithRecent physiological phylogenetic analyses indicate that vegetative desiccation tolerance was primitively present in the bryophytes , but was lost in the evolution of tracheophytes. Desiccation-tolerant bryophytes are found worldwide and occupy a variety of ecological niches, most of which could, during some period of the year, be considered as extreme either on a macro or microhabitat level. In most cases the extremes that these plants experience are both in water availability and temperature -23.Craterostigma plantagineum, Sporobolus stapfianus, and other desiccation-tolerant angiosperms, desiccation-tolerant bryophytes appear to express this trait constitutively [Desiccation-tolerant bryophytes, because of their simple architecture, have few, if any, morphological  characteristics or adaptations that can limit water loss or regulate plant temperature. As a result of this, the internal water content of their photosynthetic tissues rapidly equilibrates to the water potential of the environment once free water is lost from the surface of the plant. This in turn means that these plants experience drying rates that are much faster than those experienced by their more complex pteridophyte or angiosperm counterparts. In fact, the drying rates that desiccation-tolerant bryophytes experience are generally lethal to desiccation-tolerant ferns and flowering plants . The raptutively ,24. Thistutively . In thistutively ,24. The T. ruralis and other mosses to rapidly recover synthetic metabolism when rehydrated. The speed of this recovery was inversely dependent upon the rate of prior desiccation: the faster the rate of desiccation, the slower the recovery. In addition, although the pattern of protein synthesis in the first two hours of rehydration of T. ruralis is distinctly different from that of hydrated controls, novel transcripts were not made in response to desiccation [T. ruralis responds to desiccation by an alteration in protein synthesis upon rehydration that is in large measure the result of a change in translational control. Changes in transcriptional activity were observed for nearly all transcripts studied [T. ruralis relies more upon the activation of pre-existing repair mechanisms for desiccation tolerance than it does on either pre-established or activated protection systems.The repair aspect of the mechanism of desiccation tolerance in these plants, although demonstrated to be a major component of tolerance, is difficult to detail and characterize. Most work has focused on the proteins whose synthesis is induced immediately upon rehydration of desiccated gametophytic tissue. Early work  establisiccation . Hence i studied  but did T. ruralis, Oliver [T. ruralis does not synthesize specific proteins in response to applied ABA. The accumulation of these transcripts was postulated to be the result of an increase in mRNA stability brought about by the removal of water from the cells [In a detailed study of the changes in protein synthesis initiated by rehydration in , Oliver  demonstr, Oliver ,29 at a , Oliver , M. J. Ohe cells . Recent per se. We have some limited knowledge of the functions (or postulated functions) of a few of the rehydrins from classical molecular analyses, [Tortula gametophytes from the desiccated state. The first step in this process was to establish an EST collection that is representative of the transcripts available to the moss during the first few hours following rehydration. In this report we present a bioinformatic analysis of 10, 368 ESTs from the early phases of recovery following rehydration of rapidly-dried Tortula ruralis gametophytes; rapid dried gametophytes were chosen in order to maximize the recovery and repair response upon rehydration. The bioinformatics approach we have taken is based on that used by McCarter et al., [Meloidogyne incognita L2 ESTs, and includes cluster analyses, transcript abundancy estimations, and functional classifications based on InterPror domains, Gene Ontology hierarchy, and KEGG biochemical classifications. The overall goal was to gain an appreciation of the Tortula transcriptome during the time period following rehydration when the desiccation driven alteration in gene expression is at its peak. During this period we hypothesize the processes of cellular repair and recovery are the main focus of the metabolism of the gametophytic cells.The major question arising from these studies concerns the identity of rehydrins and what possible functions and roles they may play in regards to the response to desiccation and rehydration and to desiccation tolerance nalyses, ,32 and fnalyses, . However et al.,  to conduTortula ruralis rehydration library and subjected to single-pass 5' directional sequencing to generate 10,368 primary ESTs of which 9,159 (88%) passed through quality control, vector trimming, E. coli contamination, and cloning artifact removal. The 9,159 ESTs averaged 648 nucleotides in length and totaled 5.93 million nucleotides submitted to Genbank. These submitted ESTs form the basis of the subsequent transcriptome analysis utilizing the High Throughput-Gene Ontology-Genome Annotation Toolkit (HT-GO-GAT), a software developed by S.E. Dowd (unpublished).Ten thousand three hundred and sixty eight individual cDNA clones were selected from a Tortula ESTs, which derive from a population of individual gametophytes, alleles of the same gene. The 9,159 ESTs formed 7,272 contigs and 5,563 clusters, both of which exhibit an average size of 669 nucleotides. However, the longest sequence increased from 1,575 nucleotides for contigs to 2,229 nucleotides for clusters. Clusters varied in content from a single EST (singletons) in 4,362 cases (78%) to 48 ESTs for a single cluster , the 9,159 ESTs were grouped into contigs and clusters by establishing assembly stringencies that generated groupings defined by the analysis protocols of McCarter et al., 2003 . Contigs-7 were assessed manually for all possible alignments taking into account the alignment length, number of identical matches, gaps, and positive replacements. Utilizing these criteria we were able to annotate 3,321 (59.7 %) of the 5,563 clusters by their similarity to known genes within the database. This also meant that 2,242 of the clusters, or 40.3%, represent sequences that have no known counterpart in the public databases and that we categorize as unknowns.The consensus Cluster sequences were subjected to a BLASTx style search of a custom curated non-redundant database derived from UNIPROT and annotated according to the degree of similarity of the cluster to the highest scoring match in the database of known gene sequences. The degree of similarity was based on the quality of the BLASTx statistical outputs as well as a visual inspection of the aligned sequences between the query and the target. Clusters that generated an HSP-bit score below 70 and or E values higher than 10Tortula rehydration EST collection. These transcripts only account for 8.4% of the generated ESTs, however. Ten of the most abundantly represented transcripts encode proteins that do not match any of the sequences in the databases searched in this study and are designated as unknowns. Seven abundant transcripts appear to encode proteins that belong to a class of proteins known as Late Embryogenesis Abundant (LEA) proteins, although the relatively low HSP bit scores and high E-values for most of these BLASTx matches has to be considered as a caveat in this assessment. LEA proteins have long been ascribed a protective role for cells that are experiencing dehydration [Tortula ruralis, which is consistent with some of our earlier findings [Tortula and what has been reported for angiosperms may also be a reflection of what we believe to be a more primitive mechanism of dehydration tolerance, and perhaps a more primitive role for and control of LEA gene expression. Other abundant transcripts appear to fall into the membrane transport  and proteins that can be associated with plant stress events . The Early Light Inducible Protein A (ELIP-A) transcript is the only member of the abundant transcripts that we have previously reported [Tortula chloroplast and may play a protective or repair function for the photosynthetic apparatus [Table ydration ,35. Thisfindings , then itfindings , or are reported  as belonreported . These ppparatus .Tortula rehydration transcriptome does represent the first major step for such a pursuit.Although transcript abundance may reflect the metabolic or physiological needs of the moss during the rehydration phase of a wet/dry/wet cycle it would be more desirable to know how these transcripts are recruited and utilized by the translational machinery to make the proteins that actually contribute to the recovery of the gametophytes following rehydration. Such information is beyond the scope of this analysis but the identification and isolation of the Clusters that represent the Tortula transcripts present during the initial phases following rehydration using the Gene Ontology (GO) classification system paints a broader view of the possible metabolic activity of the gametophytic cells at this time. This does, of course, come with the understanding that these are transcript based analyses and do not directly reflect protein levels which would offer a more definitive assessment of the metabolic capability of the cells during rehydration.Physiological, biochemical, and molecular data all point towards an active period of cellular activity, presumably for repair and recovery from desiccation induced damage, during the first two hours following rehydration of dried gametophytes ,15. The Tortula rehydration transcripts was achieved by matching the Tortula clusters to characterized protein domains in a combined protein database, using HT-GO-GAT , which allowed us to assign GO terms to each cluster. The assignment of GO terms made it possible to place the clusters into the GO hierarchy which can be viewed by use of an AmiGo browser. Of the 3, 321 clusters that exhibited significant similarity to known genes in the public databases, 2,203  represent genes that contain conserved protein domains that have known biochemical and physiological functions in other organisms and map to the GO hierarchy. The GO representations for the Tortula rehydration clusters are presented in Tables .Functional classification of the Physcomitrella patens following various hormonal treatments designed to illicit developmental perturbations and metabolic switching  map into the Biological Processes classification, 1641 (98%) of these fall into the Physiological Processes category and 409 24.5%) cross-map into the Cellular Processes category . The importance of the protein synthetic machinery and its re-establishment following rehydration is also highlighted by the preponderance of clusters associated with the Ribosomal subcategory of the Cellular Component Classification; 40% of the Cytoplasm category .The subcategory distributions within the Physiological and Cellular processes also seem to reflect the nature of the cellular disturbances that result from a desiccation-rehydration event. Processes involved in metabolite and ion transport within and between cells are represented by 17% of the clusters that map to Biological Processes, and almost 86% of those that map to the Cell Growth and Maintenance subcategory. Within the Metabolism subcategory of Physiological processes 40%  of the clusters map to protein metabolism (synthesis) and 37%  map to biosynthetic processes. The considerable representation within the aforementioned three subcategories is consistent with much of our biochemical and physiological evidence concerning the metabolic activity and emphasis in gametophytic cells during rehydration ,24,25. ITortula leaf cells [Only 982 clusters  map within the Cellular Component Classification and are presumably associated with structural functions  and translational level (54 clusters). These clusters, along with those that represent biochemical control mechanisms for signaling and gene expression at the protein level, such as kinases (90 clusters) and phosphate-transfer activities (154 clusters), may represent critical elements in the activation and execution of the cellular recovery processes necessary for the mechanism of desiccation tolerance exhibited by Tortula rehydration clusters throughout the GO mapping system is indicative of the emphases on, but not expression levels of, particular cellular activities represented in the moss gametophytes during this period of a wet and dry cycle. In an attempt to assess if the accents on particular cellular activities indicated for rehydrated gametophytes are characteristic of the rehydration induced metabolic state or are simply indicative of processes associated with normally active bryophyte cells, we compared the representation of the Tortula rehydration clusters within the GO categories with similar "clusters" from Physcomitrella patens, the only other bryophyte that has similar genomic level information. The majority of the Physcomitrella "clusters" are derived from a large EST collection representing transcripts from both untreated and hormone induced  cells of protonemal cultures. The Physcomitrella ESTs are described by Nishiyama et al., [ as assembled contigs . In total 22,885 Physcomitrella contigs, derived from 102553 ESTs obtained from Physcobase and Genbank, were subjected to a BLASTx search, as described for the Tortula clusters using HT-GO-GAT. Of the 22,885 contigs 9,981 (43.6%) represent genes that contain conserved protein domains that have known biochemical and physiological functions in other organisms and map to the GO hierarchy.The representation of the  et al.,  and wereTortula and Physcomitrella, or 2) differences in representation may reflect differences in the emphasis on individual classes of cellular activities between mature gametophytes (Tortula) and protonema (Physcomitrella). Until a comparison of GO mapping distributions can be made directly between clusters derived from an EST collection from hydrated control gametophytes from Tortula with those from the rehydration collection these caveats remain important. Nevertheless, even with these difficulties and limitations the comparison is still useful for developing new hypotheses as to what cellular processes might be important in the recovery of moss cells from desiccation.There are two caveats for this comparison; 1) differences in representation may simply reflect species differences in the emphasis on individual classes of cellular activities between Tortula clusters within the GO mappings are similar if not identical to the distribution of the Phycomitrella contigs gleaned from Physcobase. This can be seen especially at the GO map 2 and 3 relationship-level categories (Table ). However, there are several notable differences and in general the differences are consistent with what has been determined, in earlier studies, to be important in the recovery of the moss from desiccation following rehydration [Tortula to representation for Physcomitrella (T:P). In the Biological category the most striking differences in GO representation of Tortula clusters occurs in categories where the percentage representations are relatively low but the numbers of clusters are substantial. In the level 4 relationship, Photosynthesis, the representation for Tortula is 3.31% compared to 1.38% for Physcomitrella, a 2.4 fold difference, the majority of which is accounted for by the difference in the representation levels for the light reaction category. The rapid recovery of photosynthesis is critical for the recovery of bryophyte cells, particularly in regards to the production of energy and reducing power for the metabolic activity associated with repair and reconstitution of the gametophytic cells. Chloroplast structure in Tortula is severely disrupted, especially if desiccation occurred rapidly, in the first few hours following rehydration [Tortula than in Physcomitrella cells that have not experienced a disruption in the photosynthetic apparatus. Similar inferences can be made concerning the differences in representation observed for carbon utilization (T:P of 2.0) and oxidative phosphorylation (T:P of 2.1) for Tortula in that mitochondrial activity and integrity are also compromised during rehydration [Tortula and Physcomitrella mappings relate to a higher representation for Tortula in the categories that relate to responses to external stimuli and responses to stress both of which would seem consistent with the emphasis that cellular activity for Tortula would have in comparison to unstressed Physcomitrella cells. In particular the increased representation within the response to oxidative stress is of note, as an elevated protection of cellular integrity from the damaging reactive oxygen species (ROS) typically associated with a desiccation rehydration event is a distinctive component of desiccation tolerant bryophytes when compared to their desiccation sensitive relatives such as Physcomitrella [In general the distribution of the ydration ,23 and wydration ,13. The ydration ,40 but rydration ,36. Othemitrella .Tortula are mirrored in the Cellular Component Level 2 classification mappings. In the Cellular Component classification Tortula exhibits an almost two fold difference in representation within categories associated with either the chloroplast or mitochondria, such as Extrachromsomal DNA (T:P of 1.8), Thylakoid (T:P of 1.8), Mitochondrial membrane (T:P of 1.9), and both Inner and Outer membranes (T:P of 1.8). In this case the categories are generally related to genes representing membrane components and since it is the organelle membranes that exhibit the majority of the damage during desiccation and rehydration it is consistent that these categories would be better represented in the Tortula cluster mappings than those for Physcomitrella. In addition to the organellar related classifications the Tortula clusters also exhibit a higher representation within the Ribonucleoprotein complex category (T:P of 1.8) which in all likelihood reflects an emphasis on ribosomal components since a similar difference in representation is seen in the Structural Constituent of the Ribosome category (T:P of 2.0) within the Molecular Function Level 2 classification. Again such differences in representation in the comparison between Tortula and Physcomitrella GO mappings are consistent with our previous studies on the responses of Tortula gametophytes to desiccation and rehydration and comparisons to non-stressed bryophyte tissues. Protein synthesis is critical to the recovery of Tortula cells following a desiccation event [Tortula clusters are derived from ESTs of rehydrated moss that was dried rapidly, the greater representation in ribosome related GO mappings for this collection compared to the Physcomitrella clusters, that represent transcripts from cells where presumably normal ribosomal turnover and synthetic rates are prevalent, is consistent with the biological state of the Tortula cells. The emphasis on protein synthesis in rehydrated Tortula cells compared to those of Physcomitrella is also evident in the comparison of representations within the Translation regulator, Translation factor, and Translation elongation factor activity GO mappings.The above observations concerning the more extensive representation of clusters within the GO mappings related to organelle function in on event ,13,15 noon event ,29. Earlon event ,43, rapiTortula collection of clusters involved in Two-component Sensor or Channel/pore class Transporter activity designated mappings, offer novel possibilities for investigation into rehydration metabolism that have not been indicated as important until now. The individual identity of the clusters that map to these categories should offer possible hypotheses that can be tested in our future research.Other differences seen in the Molecular function classification, such as the greater representation within the ) metabolic pathways. Of the 2203 clusters that map within the GO hierarchy only 642 clusters had assigned EC numbers generating 325 unique mappings (see Tortula clusters. The KEGG metabolic pathways that are well represented by Tortula clusters are Carbohydrate Metabolism (83 enzymes represented), Amino Acid Metabolism (72 enzymes), Energy Metabolism (40 enzymes), Lipid Metabolism (27 enzymes), and Metabolism of Cofactors (24 enzymes). All of these pathways have been previously associated with the cellular recovery processes associated with rehydrated moss gametophytes [Tortula clusters only the lack of ascorbate metabolizing enzymes appears unusual as ascorbate has been well documented as an important metabolite in the protection of moss gametophytes from oxidative damage during stress. This however appears to be the result of a limitation in the assignment of EC numbers since several Tortula clusters show significant similarities to enzymes involved in ascorbate metabolism in the original BLASTx search used to generate the GO mappings. The limitation of the KEGG based classification can also been seen in the poor representation of Tortula clusters in the other KEGG pathways  which is surprising given the number of clusters that were identified in the BLASTx search and in the GO databases as being contained within these classifications. As an example, none of the 239 clusters that map in the GO hierarchy as structural components of the ribosome  40.3% failed to exhibit sufficient similarity  with known sequences to allow for an accurate annotation of the cluster. It is possible that these contigs, rather than containing novel amino-acid coding regions, contain mainly 3' or 5' untranslated regions (UTRs) or coding regions that are so short as to render them incapable of generating a significant similarity score. In order to investigate this possibility we examined the three classes of contigs, those with significant similarity scores; "good hits", those that generated poor similarity scores; "false hits", and those that failed to generate any scored similarity; "no hits", to determine the longest open reading frame (ORF). We limited the ORF determination to those clusters that contain an AUG codon in the 5' to 3' direction of the clone in any one of the three possible reading frames . The results of this analysis are shown in Figure Physcomitrella and Arabidopsis databases were independently used in a BLASTx search using the 5,563 Tortula clusters as query sequences. Even though we used the entire 5,563 cluster sequences as individual queries only 3,321 actually represent sequences that could be annotated using the criteria for similarity discussed previously. When the Tortula clusters were used as queries against the Physcomitrella database, 5,554 generated HSP Bit scores however only 282 exhibited a level of conservation of sequence that passed the criteria established for annotation of our Tortula clusters . Against the Arabidopsis database only 927 Tortula clusters generated HSP Bit scores but 612 had a level of conservation sufficient to be considered reliable matches to Arabidopsis genes. These observations are difficult to rationalize and may simply reflect the inequality of the target databases. The Physcomitrella database, which is generated from a relatively small EST collection, is much smaller than that for Arabidopsis, which is gleaned from the full genomic sequence. This may explain why although most Tortula clusters generated HSP Bit scores only a relative few were capable of producing scores sufficiently high enough, and with low enough error probabilities, for confident assignment of co-identity with a Physcomitrella contig. Obviously Tortula ruralis is more closely related to Physcomitrella patens, as they are both bryophytes, than to Arabidopsis thaliana but the phylogenetic distance between the two bryophytes is still substantial, which may also help explain the paucity of high value matches and the large number of low value hits. The Arabidopsis database is much more comprehensive and would be expected to generate more significant matches as observed and perhaps because of the evolutionary distance between the two plants more queries that do not generate an HSP.In this analysis the Physcomitrella  and Arabidopsis  are presented in Table Tortula and Physcomitrella did not register as highly conserved between Tortula and Arabidopsis, the most conserved protein between Tortula and Physcomitrella, the rRNA intron-encoded homing endonuclease, did not generate a match at all with an Arabidopsis counterpart. Interestingly, the most conserved photosynthesis related protein between Tortula and Physcomitrella is a chlorophyll a/b-binding protein which generates a HSP Bit score of 205 and E value of 3.0E-53, the Arabidopsis counterpart generates a HSP Bit score of 82 and E value of 8.9E-15. In this case the level of conservation of this protein within the comparisons appears to reflect the phylogenetic relationships that exist between the three plants. However, the most conserved photosynthesis related protein between Tortula and Arabidopsis is a photosystem I P700 apoprotein A2 which generates a HSP Bit score of 1120 and E value of 0, the Physcomitrella counterpart generates a HSP Bit score of 34 and E value of 1.25E-1, which appears contrary to what is expected at what is seen for the chlorophyll a/b-binding protein. This type of relationship is also seen for the Ribulose 1 protein (Cluster 2170) that is conserved between Tortula and Arabidopsis but only to a low degree with the Physcomitrella counterpart. If such relationships can be substantiated with further study and complete sequences (both the Tortula and Physcomitrella clusters are based on ESTs) then some interesting evolutionary questions can be raised concerning both gene function and selection pressures on the individual plants and metabolic processes.The twenty gene products that exhibit the highest level of conservation for both comparisons, Physcomitrella patens as a model plant for molecular genetic studies, fired by its particular ability to perform efficient and homologous recombination in vitro, that bryophyte genomics has become a topic of some interest. Physcomitrella is rapidly becoming the model of choice for developmental and transgenic studies [Tortula ruralis on the other hand has long been established as an attractive model for the analysis of environmental stress tolerance, in particular desiccation tolerance, It has been a very useful model in assessing structural, physiological, biochemical and genetic (gene expression) aspects of severe dehydration of plant cells and mechanisms by which primitive plants respond to and survive protoplasmic water loss [Tortula model into genomics is a critical aspect in the development, along with a transformation system and assessment of its ability for efficient homologous recombination, into a more useful and manipulable model for understanding desiccation tolerance and the nature of extremophiles. In addition to the importance of Tortula for stress biology, the establishment of a second and contrasting bryophyte model, especially with regards to genomics, is essential for the validation and usefulness of the information gained from the analysis of the Physcomitrella genome and the general principles gleaned from its use as a plant model. It is to these ends that we have initiated this study into the transcriptome of Tortula gametophytes as they respond to a major stress event, in this case a combination of desiccation and rehydration.Bryophytes have an important and underestimated place in the study of plant responses to water deficits, in particular desiccation tolerance. Bryophytes occupy what we believe to be one of the most primitive states, along with algae, in the evolution of desiccation tolerance and represent, in all probability, the stage in the emergence of plants from a fresh water environment to occupy the various niches available on dry land . Unfortu studies  because  studies . Tortulater loss ,25,46. TTortula, as defined by the clusters presented herein, is remarkably consistent with what we know about the desiccation response for this bryophyte and its metabolic activity during the first two hours following rehydration. The GO mapping of the Tortula clusters enabled a broad look at what cellular activities appear to be emphasized in the rehydrated gametophytes and in agreement with our previous biochemical analyses highlighted the prominence of the protein synthetic machinery, both in structure and control, membrane structure and metabolism, and the need to reestablish plastid integrity. These observations were bolstered by the comparative GO analysis using the extensive EST collection generated for the desiccation sensitive moss Physcomitrella patens. The GO analysis has also provided fuel for new investigations and hypotheses into the role of other cellular processes, such as membrane transport, phosphorylation and signal transduction, in the mechanisms that enable desiccation tolerance in plants. Signal transduction is especially intriguing with regards to desiccation tolerance in this bryophyte as it appears to rely on alterations in translational control to effect a response to desiccation in contrast to the well characterized transcriptional responses, and associated signaling pathways, associated with abiotic stress in the Angiosperms [Tortula ruralis and what can be inferred from the analyses of the Tortula rehydration EST collection gives a measure of confidence not only in the value of a bioinformatics approach to gain a view of a particular transcriptome but also in the basis for new hypotheses and research directions that are generated from them.The rehydration transcriptome of iosperms ,7. In adTortula gametophyte microarray designed for the profiling of both the extant transcriptome and the associated translatome and their response to desiccation and rehydration under various conditions. The array and the experimental design of the expression profiling will allow us to generate a more accurate assessment of both transcript levels and transcript recruitment into the protein synthetic machinery during, and recovery from, desiccation in Tortula ruralis. In combination with the bioinformatics analyses presented here, the expression profiling will allow us to generate a more complete picture of the cellular response of a tolerant plant species, an extremophile, to an extreme abiotic stress event.Although the type of analyses presented here are extremely useful in assessing the types of transcripts present in a particular tissue at a particular time and in response to some perturbation, either external or internal in origin, and generating hypotheses concerning the functional aspects of the transcriptome they are intrinsically correlative in nature. In order to gain a more direct picture of the transcriptome and more importantly, with regards to functional assessments, the "translatome", the bioinformatics must be linked to a detailed expression profile of the transcripts represented within the ESTs generated to investigate the particular biological response or mechanism of interest. To this end the clusters described in this report form the basis of a Tortula ruralis , also classified as Syntrichia ruralis, gemetophytes were collected, harvested, and stored as described previously [eviously . For expTotal RNA was isolated from the rehydrated gametophytes by a series of phenol extractions as described by Lane and Tumiatis Kennedy . PolyA RHigh throughput sequencing of the inserts contained in the 10,368 individual clones was performed using "rolling circle amplification" of the individual plasmids to generate suitable sequencing templates at the Joint Genome Institute, Walnut Creek, CA, U.S.A. Clones were sequenced using primers specific for vector sequence upstream of the multiple cloning site and at the 5' end of the cDNA insert. The sequences were delivered as primary binary files (raw trace files), which were then processed through our sequencing, pipeline as described below.Trace files were entered into SeqMan II and quality screening performed with medium stringency corresponding to a phred threshold value of 12. Vector searching was performed using the pSPORT™ vector both in forward and reverse orientations with minimum match length of 7, connect distance of 3, Maximum register shift of 10, Minimum NW percent match of 90, gap weight of 0 and length weight of 2. Contaminant screening was performed with minimum match of 25. Following the quality screening the sequences were exported as a single FASTA file and polyA tails were removed using a custom PERL script. Several hundred sequences that contained internal polyA stretches were manually identified. These sequences were manually edited to remove polyA and trailing sequences.Edited sequences were then entered into SeqMan for assembly into contigs. Assembly for contigs was performed with match size of 50, minimum match percentage of 97, minimum sequence length of 100.Consensus contig sequences were exported from SeqMan as individual files and entered into a separate SeqMan project and reassembled using the same parameters used for contigs but with a minimum match percentage of 90. These were considered clusters.. A total of 9159 EST sequences were submitted to GenBank and assigned accession numbers CN200321-CN209479EST submission to GenBank was performed using the USDA-ARS Livestock Issues Research Unit's (LIRU) High Throughput-Gene Ontology-Genome Annotation Toolkit (HT-GO-GAT). HT-GO-GAT can be obtained from the LIRU website at .Functional annotations, Enzyme commission numbers, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway assignments were assigned to cluster sequences using HT-GO-GAT. Consensus sequences derived from Clusters were entered into the software that utilizes custom BLASTx, RPS-BLAST, and relational mySQL databases to identify potential functional assignments based upon sequence and functional domain similarity matching. The software was set to identify high stringency matches and the resulting data manually curated using the high throughput results viewer interface of HT-GO-GAT. HT-GO-GAT produces various reports related to given datasets including ORF analysis, KEGG pathway reports and visualization, and also exports gene ontology association files. These association files were imported into a mySQL database and visualized using the USDA-ARS-LIRU's DrZooView2.0 GO database browser MJO conceived of the study, generated the libraries and ESTs, annotated the EST database, performed much of the analyses, interpreted the results, and drafted the manuscript. SED designed the bioinformatics software and database, performed the sequence alignments, performed the ORF analysis, and generated the Bryobase website. JZ coded the HT-GO-GAT software and created the mySQL database. SAM participated in the design of the bioinformatics and design of the sequence analysis. PRP participated in the design of the database and sequence analysis, and participated in the annotation of the individual EST sequences.Tortula rehydration clustersKEGG biochemical pathway mappings for . An alternative functional classification of clusters based on established biochemical activities of gene products. The classification is achieved by the use of HT-GO-GAT to assign clusters to individual Kyoto Encyclopedia of Genes and Genomes (KEGG ) metabolic pathways. This file contains the tabulated results of this classification.Click here for file


The sperm penetration assay (SPA) is used to predict the fertilizing capacity of sperm. Thus, some programs rely on SPA scores to formulate insemination plans in conjunction with in-vitro fertilization (IVF) cycles. The purpose of this study was to evaluate if a relationship exists between SPA scores and polyspermy rates during conventional IVF cycles.A total of 1350 consecutive IVF patients using conventional IVF insemination were evaluated in the study. Oocytes were inseminated three hours post-retrieval by the addition of 150,000 to 300,000 progressively motile sperm. Approximately 18 hours after insemination, the oocytes were evaluated for fertilization by the visualization of pronuclei. The presence of three or more pronuclei was indicative of polyspermy. Polyspermy rates, fertilization success, embryo quality, and pregnancy rates were analyzed retrospectively to evaluate their relationship with SPA score, count, motility, number of progressively motile sperm inseminated, oocyte pre-insemination incubation time, patient age, and diagnosis.s = 0.10, p < 0.05). Patients with a normal SPA score had significantly higher polyspermy rates than those with abnormal SPA scores . Fertilization percentage was significantly lower in the group with severely abnormal SPA scores versus all other SPA groups . Although embryo quality was not affected, both clinical pregnancy and implantation rates improved slightly as SPA score increased. In addition, there was a decrease in the rate of spontaneous abortion as SPA score increased.A significant positive relationship was observed between SPA score and polyspermy rate (rThese data indicate SPA score is positively correlated with polyspermy rates and IVF fertilization percentage. Additionally, there is a slight increase in clinical pregnancy rates, and embryo implantation rates with increased SPA. Furthermore, there is a slight decrease in spontaneous abortions rates related to increased SPA. The sperm penetration assay (SPA) is used to evaluate the fertilizing capacity of human spermatozoa . Some inOver the past decades success rates have steadily improved for human IVF embryo transfer programs. Recent advances in ovulation induction, micromanipulation, culturing conditions, and media formulations have fostered a technological revolution for IVF leading into the 21'st century . However, many common problems still remain.One common problem associated with conventional IVF insemination is polyspermic fertilization when more than one sperm successfully penetrates and fertilizes the oocyte. These pre-embryos are considered abnormal and typically discarded. Thus, increased rates of polyspermy ultimately result in a reduced number of embryos.Studies have been conducted that evaluate the factors associated with human IVF polyspermy -5. HowevThe purpose of this study was to evaluate if a relationship exists between SPA scores and polyspermy rates during IVF cycles utilizing conventional insemination. We also evaluated the relationship between SPA score and fertilization rate, embryo quality, and pregnancy rate.After Institutional Review Board approval, we conducted a retrospective study of 1350 consecutive IVF patients using conventional IVF insemination. Intracytoplasmic sperm injection (ICSI) patients were excluded from the study. The fertilizing capacity of each patient was assessed with the SPA. The SPA was performed on patients undergoing IVF using techniques previously described . The SPADuring the IVF cycle ovarian stimulation was performed using standard techniques of gonadotrophin-releasing hormone (GnRH) agonist down-regulation combined with controlled ovarian stimulation using a combination of recombinant follicle stimulating hormone (rFSH) and urinary-derived gonadotropin stimulation. Ovarian follicles were aspirated using a trans-vaginal ultrasound-guided needle.Oocytes were inseminated three-hours post-retrieval by the addition of 150,000 to 300,000 progressively motile sperm. Approximately 18-hours post-insemination, the oocytes were evaluated for fertilization by visualization of pronuclei. The presence of three or more pronuclei was indicative of polyspermy in which case the fertilized oocyte was discarded. Embryos were cultured in HTF medium and transferred 72-hours post-retrieval. Embryo quality was assessed using a previously reported embryo scoring system that took into account the number of cells present and the level of cellular fragmentation [Polyspermy rates, fertilization success, embryo quality, and pregnancy rates were analyzed retrospectively with respect to their relationship with SPA score. The correlation between SPA score and polyspermy was conducted using Spearman's correlation coefficient. Additionally, polyspermy rates, fertilization percentage, and embryo quality for the different SPA groups were analyzed for statistical difference using Kruskal-Wallis analysis. Lastly, pregnancy rates in the different groups were evaluated with a Chi-square analysis.s = 0.10, p < 0.05). Patients with normal SPA scores (Group 3) had significantly elevated polyspermy rates over those with severely abnormal SPA scores  compared with the studies of van der Ven 06 sperm . Our resPoly-pronuclear formation in in-vitro fertilized eggs usually arises from polyspermic fertilization but may also be a product of second polar body retention . Thus, oConsistent with other reports, the SPA was correlated with fertilization percentage ,6. FurthDone correctly, the SPA provides a reliable assessment of the fertilizing ability of human spermatozoa with very low false negative rates (< 0.03%) and serves a valuable tool for clinicians to treat infertility patients with the appropriate modality . The SPAVWA designed the study, carried out statistical evaluation, and was the primary author of the manuscript. CMP, KPJ, HHH, MG, and IH were involved in management of the IVF cases and collection of the clinical data. DTC was responsible for the implementation of the study, administrative requirements including IRB approval, statistical evaluation of the data, and preparation of the manuscript.Table 1. Relationship between SPA and IVF outcome measuresClick here for file


Epidemiological studies suggest a non-monotonic effect of alcohol consumption on cardiovascular risk, while there is strong evidence concerning the involvement of homocysteine levels on thrombosis. The aim of this work was to evaluate the association between usual ethanol consumption and homocysteine levels, in cardiovascular disease free adults.From May 2001 to December 2002 we randomly enrolled 1514 adult men and 1528 women, without any evidence of cardiovascular disease, stratified by age – gender (census 2001), from the greater area of Athens, Greece. Among the variables ascertained we measured the daily ethanol consumption and plasma homocysteine concentrations.Data analysis revealed a J-shape association between ethanol intake  and total homocysteine levels (mean ± standard deviation) among males  and females , after controlling for several potential confounders. The lowest homocysteine concentrations were observed with ethanol intake of < 12 gr/day (Bonferroni α* < 0.05). No differences were observed when we stratified our analysis by type of alcoholic beverage consumed.We observed a J-shape relationship between homocysteine concentrations and the amount of ethanol usually consumed. Alcoholic beverages are widely consumed throughout the world and it has long been known that heavy alcohol consumption is hazardous to various body organs. In several countries alcohol is considered as one of the leading causes of preventable deaths, after smoking . HoweverSome investigators consider increased homocysteine levels as an independent risk factor of cardiovascular disease, and its involvement in mechanisms of thrombosis has well been documented ,7. MoreoWe therefore studied the relation between amount of ethanol consumption and homocysteine levels, in 3042 adults enrolled in the ATTICA Study.The "ATTICA" study  is a heaThe selected sample was population-based and reflecting the underlying population with respect to sex, age and residence. The number of the participants was determined by power analysis and chosen to evaluate greater than 0.5 standardised differences between ethanol groups and homocysteine levels, with statistical power > 0.80 at < 0.05 probability level .The questionnaire included demographic characteristics , detailed medical history and lifestyle habits, such as food items consumed, smoking habits and physical activity status.Dietary intake during the year before enrolment was assessed through a semi-quantitative food frequency questionnaire provided by the EPIC-Greece Study. The questionnaire was administrated in person by specially trained dieticians and has been validated . The dai2.Regarding the rest of the investigated parameters the educational level of the participants  was measured in years of school. Information about smoking habits was collected using a standardized questionnaire developed for the Study. Current smokers were defined as those who smoked at least one cigarette per day. Former smokers were defined as those who had stopped smoking more than one year previously. The rest of the participants were defined as non smokers. For the multivariate statistical analyses cigarette smoking was quantified in pack-years (cigarette packs per day × years of smoking), adjusted for a nicotine content of 0.8 mg / cigarette. All participants were classified at entry according to their habitual physical activity. Class 1 were sedentary, engaging in little exercise; class 2 were moderately active during a substantial part of the day; and class 3 performed hard physical work much of the time. Classification was based on the responses to questions about the occupation and usual activities, including part-time jobs and notable non-occupational exercise . Body maBlood samples were collected from the antecubital vein between 8 to 10 a.m., in a sitting position after 12 hours of fasting and avoiding of ethanol. For the determination of plasma fibrinogen blood was anticoagulated with 3.8% trisodium citrate (9:1 vol/vol) and cooled on ice until centrifugation. For determination of homocysteine, blood was collected in a cool vacutainer containing EDTA, which was stored on ice for a maximum of 2 hours till the centrifugation at 3000 g for 5 minutes at 4°C. Plasma homocysteine levels were measured with an automatic Abott Axsym analyzer, which is based on the technology of polarized immunofluorescence. The intra and inter-assay coefficients of variation of homocysteine did not exceed 5%.Arterial blood pressure was automatically measured at the end of the physical examination with subject in sitting position. Hypertension was defined as a systolic blood pressure >/= 140 mmHg, a diastolic blood pressure >/= 90 mmHg, or the use of any antihypertensive medication; hypercholesterolemia was defined as total cholesterol levels greater than 220 mg/dl or the use of lipid lowering agents and diabetes mellitus as a fasting blood glucose > 125 mg/dl or the use of antidiabetic medication.rd order interpolating polynomials.Continuous variables are presented as mean values ± standard deviation, while qualitative variables are presented as absolute and relative frequencies. Associations between categorical variables were tested by the use of contingency tables and the calculation of chi-squared test. Comparisons between normally distributed continuous variables and categorical variables were performed by the calculation of Student's t-test and multi way Analysis of co-Variance (multi-ANCOVA), after controlling for homoscedacity and various potential confounders. In the case of asymmetric continuous variables the tested hypotheses were based on the calculations of non-parametric tests, such as Mann – Whitney and Kruskal – Wallis. Kolmogorov-Smirnov criterion assessed normality of continuous variables. Finally, correlations between continuous variables were tested through multiple regression analysis after the adjustment for the potential confounders and interactions. The J- shape association between the exposure variable (ethanol intake) and homocysteine levels was illustrated by connecting the mean values of the investigated parameters using 3P-values are based on two-sided tests and compared to a significance level of 5%. However, due to multiple significance tests we used the Bonferroni correction (since the number of comparisons was less than ten) in order to account for the increase in Type I error. SPSS 11.0 software  was used for all the statistical calculations.All reported Thirty four percent of males and 62% percent of females reported ethanol abstinence within the recorded 7-day period p < 0.001). In addition, 37% of males and 33% of females consumed < 12 gr of ethanol per day, 16% of males and 4% of females consumed 12 – 24 gr of ethanol per day, and 13% of males and 1% of females consumed > 24 gr of ethanol per day , during the preceding week. Furthermore, middle-aged male participants (45 – 65 years old) consumed higher quantities of ethanol compared to younger (< 45 years) or older individuals , while no statistically significant differences were observed between ethanol consumption and age, in females . Ethanol intake comes from wine in 65% of men and 77% of women, from beer in 22% of men and 11% of women and from spirits or other drinks 13% of men and 12% of women. Further, descriptive characteristics of the studied population by ethanol consumption level are presented in Table . In addith percentile for men was 8.6 μmol/L and for women 6.8 μmol/L, while the 90th percentiles were 18 μmol/L and 14 μmol/L, for men and women, respectively. Due to the significant differences observed between genders in homocysteine levels, all the following analyses will be gender-specific.Homocysteine values were higher in males as compared to females . The 10Unadjusted analysis revealed a J-shape association between ethanol quantities consumed during the past week  and homocysteine levels in both males  and females . Post hoc analysis revealed that the lowest values of homocysteine levels were observed in people who reported moderate daily ethanol intake of <12 gr . No differences were observed when we stratified our analysis by alcoholic beverages primarily consumed. Figure However, since several potential confounders may influence the relationship between ethanol intake and homocysteine concentration we repeated our analysis after taking into account age, gender, pack-years of smoking, presence of hypertension, hypercholesterolemia, and diabetes, body mass index, fruits and vegetables consumption, especially leafy green vegetables, legumes, citrus fruits and juices that are reached in folic acid, as well as years of school. Multivariate regression analysis showed that <12 gr/d ethanol intake was inversely associated with homocysteine levels  as compared to no consumption. On the other hand, increased ethanol intakes, i.e. 12 – 24 gr/d, 24 – 48 gr/d or > 48 gr/d were positively associated with homocysteine concentration . No differences were observed when we stratified our analysis by gender.The results of the present study revealed a J-shape association between ethanol consumption and homocysteine levels, of a large, random and population representative sample, free of cardiovascular disease. The lowest values of homocysteine were observed in daily ethanol intake of less than 12 gr, both in men and women and remained significant after adjustment for several potential confounders.Our results are in line with that of some other studies. For example, De Bree et al.  observedOn the contrary, there are several studies that have shown a linear relationship between ethanol intake and homocysteine levels. For example, Folsom et al.  in a stuOur data were analyzed according to total ethanol intake and did not distinguish between different types of ethanol. Rimm et al. reviewed 12 and pyridoxal phosphate, as well as to ethanol intake per se [12, B6, betaine) remains unclear and more intervention and experimental studies are necessary.Chronic alcoholism has been found to be associated with hyper-homocysteinaemia, which could attribute to disturbed folate metabolism and to changes in circulating concentrations of vitamin Be per se . Finallye per se . Neverth6 and B12 intake (through food groups consumed) on homocysteine concentrations. In addition, kidney function is a strong determinant of homocysteine; however, we have not measured serum creatinine. The later may be another limitation of our study. Additionally, misreporting of ethanol consumption, due to social class can be a potential confounder.This study as a cross-sectional one cannot establish causal relations but only generate hypothesis for associations. The population studied in this work is homogeneous and may reflect lifestyle habits in similar cultures, like Western Europe, Mediterranean etc. However, our findings could not extrapolate into other populations without further investigation and consideration. Also, the numbers of participants in categories of high intake (>48 gr of ethanol /d) were rather small, and the impression of the effects in homocysteine levels in even higher ethanol consumption may be misleading. Although this analysis has been adjusted for several known confounders, we have indirectly investigated the impact of serum folate and vitamins BThe present study supports the existence of a J-shape association between ethanol consumption and homocysteine levels in both males and females, of a large, random and population representative sample, free of cardiovascular disease. Therefore our results indicate that daily consumption of 1–2 units of ethanol is associated with lower homocysteine concentrations and provide further evidence for a variant association between ethanol intake and coronary heart disease risk in both genders.