PLoS Medicine suggests that oats are not safe in all cases.Most patients with celiac disease can eliminate their symptoms—at a price: life-long adherence to a gluten-free diet. This means no wheat, rye, barley, and, until recently, no oats. Then some recent studies suggested that oats did not cause the intestinal inflammation characteristic of the disease, and thus oats are now often included in the celiac disease diet. This is good news for patients coping with severe restrictions on what they can and must not eat, but a study by Ludvig Sollid and colleagues in this issue of Like other chronic inflammatory diseases, celiac disease is caused by a complex interplay between genetic and environmental factors, but it is better understood than most. Long believed to be a relatively rare disorder, it is now thought to affect about one in 250 people worldwide. Clinical symptoms are present in less than half of patients and vary considerably. Genetically, almost all patients have one of two predisposing HLA molecules, which determine the context in which their immune system encounters foreign antigens, including gluten proteins found in wheat and other cereals. In individuals with celiac disease, the immune system mounts an abnormal response to gluten, which is characterized by gluten-reactive intestinal T cells and by inflammation and compromised function of the small intestine.Ludvig Sollid and colleagues applied the current understanding of celiac disease and a range of molecular pathology tools to studying the response to oats of nine patients with celiac disease. The nine patients were not a random sample: all of them had been eating oats, and four of them had shown clinical symptoms after oats ingestion. The goal of the study was to characterize the intestinal T cell response to oats in these patients, and to relate it to clinical symptoms and intestinal biopsy results. All patients were on a gluten-free diet and ate oats that were free of contamination by other cereals.Three of the four patients who had reported problems after eating oats showed intestinal inflammation typical of celiac disease, and Sollid and colleagues studied intestinal T cells from these three patients. Two of the five patients who seemed to tolerate oats also had oats-reactive intestinal T cells. Functional study of these T cells showed that they were restricted to celiac-disease-associated HLA molecules and that they recognized two peptides derived from oat avenin that are very similar to peptides of gluten.Taken together, the findings show that intolerance to oats exists at least in some patients with celiac disease, and that those patients have the same molecular reaction to oats that other patients have to wheat, barley, or rye. However, identical reactions were also seen in two of the patients who were clinically tolerant to oats. The authors suggest that these reactions could develop into symptomatic disease after some time delay, but there is no proof that the presence of oats-reactive T cells is an indicator of future symptoms or even of enhanced susceptibility to clinical oats intolerance.Oats are not safe for all patients with celiac disease, but future studies are needed to determine the frequency of oats intolerance.


It is predominantly localized to various brain regions and retina where it may play a role in protection against ischemia and nitric oxide-induced neural injury. Cerebrospinal fluid was collected from 12 chronic regional or systemic pain and 5 control subjects. Proteins were precipitated by addition of 50% 0.2 N acetic acid, 50% ethanol, 0.02% sodium bisulfite. The pellet was extensively digested with trypsin. Peptides were separated by capillary liquid chromatography using a gradient from 95% water to 95% acetonitrile in 0.2% formic acid, and eluted through a nanoelectrospray ionization interface into a quadrapole – time-of-flight dual mass spectrometer . Peptides were sequenced  and proteins identified using MASCOT Six different neuroglobin peptides were identified in various combinations in 3 of 9 female pain subjects, but none in male pain, or female or male control subjects.This is the first description of neuroglobin in cerebrospinal fluid. The mechanism(s) leading to its release in chronic pain states remain to be defined. The protein constituents (proteome) of cerebrospinal fluid (CSF) are altered in disease states such as meningitis, but may also be more subtly altered in many other neural conditions. CSF has been difficult to investigate because of the need for invasive lumbar punctures and the small volumes of CSF available for analysis. This situation is now rapidly changing as methods requiring microliter volumes and sophisticated analysis tools such as proteomics become available ,2. ProteAphrodite aculeate intracellular globin (30% identify) [One such protein is neuroglobin. Neuroglobin is a recently identified member of the globin family. It binds oxygen with an affinity between that of myoglobin and hemoglobin ,4. Neurodentify)  than to r) for each peptide. The numbers of matching peptides were 5 in sample #1, 2 in sample #2, and 3 in sample #3. The peptides mapped 31.1%, 13.9% and 17.2%, respectively, of the total neuroglobin protein sequence. They matched amino acids 1–10 and 15–30 of the N-terminal and 131–151 of the C-terminal. Trypsin digestion missed cleavage sites at amino acids 18 and 146. Reproducibility was demonstrated by the consistent retention times for the same peptides from different subjects. No low abundance neuroglobin peptides were found in other samples. BLAST sequence analysis of all six peptides identified only one protein: hypothetical 16.9 kDa protein .Five peptides derived from neuroglobin (NCBInr ID accession 10864065) were identified using both the MASCOT software with NCBInr database and ProteinLynx Global Server with SwissProt database  in a new globin subfamily that forms hexacoordinated heme iron complexes ,13. ThesThe source of neuroglobin in the CSF is likely to be brain regions such as the subthalamic nuclei , frontal lobe, thalamus, occipital pole, pituitary gland, and medulla oblongata ,14. ImmuNeuroglobin mRNA was also present in adrenal cells and the β cells of the pancreatic islets of Langerhans . Roles iNeuroglobin is likely to serve as an intracellular oxygen depot to facilitate oxygen diffusion to the mitochondria. A role in oxygen supply was supported by the very high expression of neuroglobin mRNA in retinal neurons but not the supporting ocular epithelium and other structures . RetinalNeuroglobin might act in certain circumstances to limit neural cellular damage during hypoxia. Neuroglobin expression was inversely correlated to the sensitivity of the brain regions to ischemia . For exa2+-E7His) and displace the protein from the globin [Plasmodium and Trypanosoma from the antiparasitic effects of nitric oxide [Neuroglobin was also colocalized with nitric oxide synthase in the lateral tegmental nuclei, stria terminalis, habencule, nucleus of the tractus solitarius, periaqueductal grey matter, amygdala and subfornic organ . The proe globin . This afic oxide . Neuroglic oxide .In this study, neuroglobin was qualitatively identified in CSF from 3 female subjects with chronic pain conditions. Females have greater pain sensitivity to pressure and other stimuli (lower pain thresholds) , but paiIt is tempting to speculate that the source of neuroglobin in our samples was from nuclei involved in pain transmission or regulation such as the thalamus, prefrontal cortex, amygdala, or spinal cord dorsal horn somatic pain synaptic regions (e.g. layers 1 and 2 of Rexed). The fact that neuroglobin was not detected in any of the control females in our study makes it unlikely that the expression was related to gender. Examination of additional normal and chronic pain subjects is underway to determine the factors that may be responsible for neuroglobin expression. It is also possible that the proteomic detection of neuroglobin varies depending upon sample preparation, signal-to-noise ratio for relatively low abundance proteins compared to albumin and immunoglobulins that are present in high abundance, duration of storage, factors related to trypsin digestion, capillary liquid chromatography, mass spectrometry or bioinformatic neuroglobin peptide detection. These technical factors are unlikely to be significant since our samples were treated identically and were stored for approximately equal amounts of time.In contrast to neuroglobin's localization, cytoglobin-immunoreactive material was localized to the cellular nucleus in all tissues examined . MammaliThis is the first description of neuroglobin in cerebrospinal fluid and in humans. Neuroglobin was identified in 3 of 9 female pain subjects. The role(s) for this ancient oxygen and nitric oxide binding protein in humans, and potential links to pain, remain to be fully determined.After obtaining informed consent, lumbar punctures were performed on 17 subjects as part of an evaluation of pain mechanisms. Twelve patients had musculoskeletal pain and five were healthy control subjects. Cerebrospinal fluid samples were aliquoted and frozen at -70°C. Lipids and peptides were extracted from 200 μl of thawed CSF by adding an equal volume of 50% ethanol, 50% 0.2 N acetic acid 0.02% sodium bisulfite ("acid-ethanol") . Centrif and NCBInr protein database [) [The column eluate was pumped through a nanoelectrospray interface into a quadrapole – time of flight  mass spectrometer. MASSLYNX version 3.5 software was used to control the CapLC and Q-ToF-2, data acquisition, processing, and determination of peptide sequences. The protein identification was performed with the MASCOT MS/MS ion search software database ,8, and wabase [) ,10, protSamples were assessed by CapLC-Q-ToF-2 in triplicate. At least 2 separate peptides from neuroglobin had to identify in each individual sample to ensure that this protein, and not a related protein, was present. In an attempt to detect low abundance expression of neuroglobin peptide ions that were not selected by MS-MS , all MS data from the appropriate CapLC retention times were reassessed at high resolution. Positive results (MS data) were checked to see if ions in MS were present but not in MS-MS for other pieces. All putative neuroglobin peptide spectra were sequenced using PepSeq (Waters) and confirmed by visual inspection.The author(s) declare that they have no competing interests.1,4, Sample preparation, chromatography, mass spectrometry and manuscript preparationCasado B 2, Supervision and assistance with chromatography and mass spectrometry and manuscript preparationPannell LK 1, Sample preparationWhalen G 3, Clinical investigation of subjectsClauw DJ 1,*, Organization of study and selection of samples, preparation of manuscriptBaraniuk JN


Cryptosporidium parvum genes of likely endosymbiont or prokaryotic origin supports the hypothesis that C. arvum evolved from a plastid-containing lineage.An analysis of  Cryptosporidium parvum is an emerging pathogen capable of causing illness in humans and other animals and death in immunocompromised individuals. No effective treatment is available and the genome sequence has recently been completed. This parasite differs from other apicomplexans in its lack of a plastid organelle, the apicoplast. Gene transfer, either intracellular from an endosymbiont/donor organelle or horizontal from another organism, can provide evidence of a previous endosymbiotic relationship and/or alter the genetic repertoire of the host organism. Given the importance of gene transfers in eukaryotic evolution and the potential implications for chemotherapy, it is important to identify the complement of transferred genes in Cryptosporidium.The apicomplexan parasite Cryptosporidium evolved from a plastid-containing lineage and subsequently lost its apicoplast during evolution. Expression analyses of candidate genes of algal and eubacterial origin show that these genes are expressed and developmentally regulated during the life cycle of C. parvum.We have identified 31 genes of likely plastid/endosymbiont (n = 7) or prokaryotic (n = 24) origin using a phylogenomic approach. The findings support the hypothesis that Cryptosporidium is the recipient of a large number of transferred genes, many of which are not shared by other apicomplexan parasites. Genes transferred from distant phylogenetic sources, such as eubacteria, may be potential targets for therapeutic drugs owing to their phylogenetic distance or the lack of homologs in the host. The successful integration and expression of the transferred genes in this genome has changed the genetic and metabolic repertoire of the parasite. Cryptosporidium is a member of the Apicomplexa, a eukaryotic phylum that includes several important parasitic pathogens such as Plasmodium, Toxoplasma, Eimeria and Theileria. As an emerging pathogen in humans and other animals, Cryptosporidium often causes fever, diarrhea, anorexia and other complications. Although cryptosporidial infection is often self-limiting, it can be persistent and fatal for immunocompromised individuals. So far, no effective treatment is available [Cryptosporidium continues to be a security concern as a potential water-borne bioterrorism agent [vailable . Furthersm agent .Cryptosporidium is phylogenetically quite distant from the hemosporidian and coccidian apicomplexans [Cryptosporidium contained a plastid, even though many of its descendants  appear to lack a plastid. Although indirect evidence has been noted for the past existence of an apicoplast in C. parvum [mplexans  and, depmplexans ,5. It ismplexans ,7. The amplexans . All thamplexans . The apimplexans ,11. In amplexans . Plastidmplexans -17. Amonmplexans ,18. If t. parvum ,20, no rCryptosporidium is thus of evolutionary and practical importance.Gene transfers, either intracellular (IGT) from an endosymbiont or organelle to the host nucleus or horizontal (HGT) between species, can dramatically alter the biochemical repertoire of host organisms and potentially create structural or functional novelties -23. In pC. parvum ) [C. parvum. The fate of several of these transferred genes in C. parvum is explored by expression analyses. The significance of our findings and their impact on the genetic makeup of the parasite are discussed.In this study, we use a phylogenomic approach to mine the recently sequenced genome of es (Mb))  for evidCryptosporidium, like that of Plasmodium falciparum [Arabidopsis and Oryza than to any other non-apicomplexan organism currently represented in GenBank. The program Glimmer predicted 5,519 protein-coding sequences in the C. parvum genome, 4,320 of which had similarity to other sequences deposited in the GenBank nonredundant protein database. A significant number of these sequences, 936  or 783 , had their most significant, non-apicomplexan, similarity to a sequence isolated from plants, algae, eubacteria (including cyanobacteria) or archaea  were used as input for phylogenetic analyses using the PyPhy program [The Glimmer-predicted protein-coding regions of the  program . In thisC. parvum predicted gene set with the 551 P. falciparum predicted nuclear-encoded apicoplast-targeted proteins (NEAPs) yielded 40 significant hits , 23 of which were also identified in the phylogenomic analyses. A combination of these two approaches identified 410 candidates requiring further detailed analyses. Of these candidates, the majority were eliminated after stringent criteria were applied because of ambiguous tree topologies, insufficient taxonomic sampling, lack of bootstrap support or the presence of clear vertical eukaryotic ancestry . Thirty-one genes survived the screen and were deemed to be either strong or likely candidates for gene transfer . Two experiments were performed. In the first, nearly complete genomic sequences (generated in a different laboratory) from the closely related species C. hominis were screened using BLASTN for the existence of the predicted genes. Twenty out of 21 C. parvum sequences were identified in C. hominis. The remaining sequence was represented by two independently isolated expressed sequence tag (EST) sequences in the GenBank and CryptoDB databases (data not shown). In the second experiment, genomic Southern analyses of the IOWA isolate were carried out , as confirmed by similarity searches against ApiDB . In P. falciparum, leucine aminopeptidase is a predicted NEAP and possesses an amino-terminal extension with a putative transit peptide. Consistent with the lack of an apicoplast, this gene in Cryptosporidium contains no evidence of a signal peptide and the amino-terminal extension is reduced. Similarity searches of the GenBank nonredundant protein database revealed top hits to Plasmodium, followed by Arabidopsis thaliana, and several cyanobacteria including Prochlorococcus, Nostoc and Trichodesmium, and plant chloroplast precursors in Lycopersicon esculentum and Solanum tuberosum (data not shown). A multiple sequence alignment of the predicted protein sequences of leucine aminopeptidase reveals overall similarity and a shared indel among apicomplexan, plant and cyanobacterial sequences  , plants , cyanobacteria , a ciliate (Tetrahymena) and the kinetoplastids (Leishmania and Trypanosoma). Arabidopsis thaliana apparently contains at least two copies of this gene; the protein of one (accession number NP_565734) is predicted by ChloroP [Another 1) Table . Similar ChloroP  to be chP. falciparum, T. gondii [Giardia intestinalis and Spironucleus and the parabasalid Trichomonas vaginalis [Giardia, Spironucleus and Trichomonas group with the cyanobacterium Nostoc, as previously published. In the midsection of the tree, the G6PI of apicomplexans and ciliates forms a well-supported monophyletic group with the plants and the heterokont Phytophthora. The multiple protein sequence alignment of G6PI identifies several conserved positions shared exclusively by apicomplexans, Tetrahymena, plants and Phytophthora. This gene does not contain a signal or transit peptide and is not predicted to be targeted to the apicoplast in P. falciparum. The remainder of the tree shows a weakly supported branch including eubacteria, fungi and several eukaryotes. The eukaryotes are interrupted by the inclusion of G6PI from the eubacterial organisms Escherichia coli and Cytophaga. This relationship of E. coli G6PI and eukaryotic G6PI has been observed before and may represent yet another gene transfer [Only one gene of algal nuclear origin, glucose-6-phosphate isomerase (G6PI), was identified by the screen described here. Several other algal-like genes are probable, but their support was weaker Table . A 'plan. gondii ) and a 'aginalis . Figure transfer .Our study identified HGTs from several distinct sources, involving a variety of biochemical activities and metabolic pathways Table . NotablyC. parvum is tryptophan synthetase β subunit (trpB). This gene has been identified in both C. parvum and C. hominis, but not in other apicomplexans. The relationship of C. parvum trpB to proteobacterial sequences is well-supported as a monophyletic group by two of the three methods used in our analyses  monophyletic grouping with eubacterial homologs (data not shown). The eubacterial origin of 1,4-α-glucan branching enzyme is shown in Figure T. gondii. It is possible that these genes are of plastidic origin and were transferred to the nuclear genome before the divergence of C. parvum and T. gondii; the phylogenetic analysis provides little direct support for this interpretation, however.Other HGTs of eubacterial origin include the genes encoding α-amylase and glutamine synthetase and two copies of 1,4-α-glucan branching enzyme, all of which are overwhelmingly similar to eubacterial sequences. α-amylase shows no significant hit to any other apicomplexan or eukaryotic sequence, suggesting a unique HGT from eubacteria to trpB gene and the gene for aspartate ammonia ligase are located 4,881 base-pairs (bp) apart on the same strand of a contig for chromosome V; there is no annotated gene between these two genes. Both genes are of eubacterial origin and are not found in other apicomplexan organisms. While it is possible that they have been acquired independently with this positioning, or later came to have this positioning via genome rearrangements, it is interesting to speculate that these genes were acquired together. The origin of trpB is proteobacterial. The origin of aspartate ammonia ligase is eubacterial, but not definitively of any particular lineage. In the absence of genome sequences for all organisms, throughout all of time, exact donors are extremely difficult to assess and inferences must be drawn from sequences that appear to be closely related to the actual donor.We examined the transferred genes for evidence of non-independent acquisition, for example, blocks of transferred genes or evidence that genes were acquired together from the same source. Examination of the chromosomal location of the genes listed in Table C. parvum encodes two genes for 1,4-α-glucan branching enzymes. Both are eubacterial in origin and both are located on chromosome VI, although not close together. They are approximately 110 kb apart and many intervening genes are present. The evidence that these genes were acquired together comes from the phylogenetic analysis presented in Figure C. parvum and T. gondii represent one copy each of this ancient duplication. This suggests that the ancestor of C. parvum and T. gondii acquired the genes after they had duplicated and diverged in eubacteria.In the second case, C. parvum-infected HCT-8 cells was shown to be free of contaminating C. parvum genomic DNA by the lack of amplification product from a reverse transcriptase reaction sham control. RT-PCR detected no signals in cDNA samples from mock-infected HCT-8 cells. On the other hand, RT-PCR product signals were detected in the C. parvum-infected cells of six independent time-course experiments for each of the genes examined ). The expression profiles of the acquired genes show that they are regulated and differentially expressed throughout the life cycle of C. parvum in patterns characteristic of other non-transferred genes , gene trees obtained from automated PyPhy analyses indicate a strong monophyletic grouping of the C. parvum gene with plant or eubacterial homologs, but this topology disappears when sequences from other unicellular eukaryotes, such as Dictyostelium, Entamoeba and Trichomonas are included in the analysis (data not shown).The limitations of similarity searches and incomplete taxonomic sampling are well evidenced in our phylogenomic analyses. From similarity searches, lciparum , is moreia Table . In manyC. parvum instead of a definitive list. As genomic data are obtained from a greater diversity of unicellular eukaryotes and eubacteria, phylogenetic analyses of nearest neighbors are likely to change.The list of genes in Table C. parvum sequences of cyanobacterial and algal origin reported here had to enter the genome at some point during its evolution. Formal possibilities include vertical inheritance from a plastid-containing chromalveolate ancestor, HGT from the cyanobacterial and algal sources (or from a secondary source such as a plastid-containing apicomplexan), or IGT from an endosymbiont/plastid organelle during evolution, followed by loss of the source. Cryptosporidium does not harbor an apicoplast organelle or any trace of a plastid genome [Cryptosporidium or the lineage giving rise to it. The exact position of C. parvum on the tree of life has been debated, with developmental and morphological considerations placing it within the Apicomplexa, and molecular analyses locating it in various positions, both within and outside the Apicomplexa [C. parvum is an apicomplexan, and if the secondary endosymbiosis which is believed to have given rise to the apicoplast occurred before the formation of the Apicomplexa, as has been suggested [C. parvum would have evolved from a plastid-containing lineage and would be expected to harbor traces of this relationship in its nuclear genome. Genes of likely cyanobacterial and algal/plant origin are detected in the nuclear genome of C. parvum  provides the best evidence against this scenario as multiple independent transfers would be required and so far there is no evidence for intra-alveolate gene transfer. Vertical inheritance is more difficult to address as it involves distinguishing between genes acquired via IGT from a primary endosymbiotic event versus a secondary endosymbioic event. Our data, especially the analysis of G6PI and BT-1 are consistent with both primary and secondary endosymbioses, provided that the secondary endosymbiosis is pre-alveolate in origin. As more genome data become available and flanking genes can be examined for each gene in a larger context, positional information will be informative in distinguishing among the alternatives.What about other interpretations? While it is formally possible that these genes were acquired independently via HGT in C. parvum leucine aminopeptidase does contain an amino-terminal extension of approximately 85-65 amino acids  relative to bacterial homologs, but this extension does not contain a signal sequence. The extension in P. falciparum is 85 amino acids and the protein is believed to be targeted to the apicoplast [C. parvum and P. falciparum amino-terminal extensions (data not shown).The plastidic nature of some genes is particularly apparent. There is a shared indel among leucine aminopeptidase protein sequences in apicomplexans, cyanobacteria and plant chloroplast precursors Figure . The C. icoplast ,37. No sP. falciparum [Trypanosoma [C. parvum and P. falciparum, with an identity of 60%. In our phylogenetic analyses, C. parvum clearly forms a monophyletic group with Plasmodium, Toxoplasma and Eimeria. This branch groups with Dictyostelium, Kinetoplastida and cyanobacterial lineages, but bootstrap support is not significant. The sister group to the above organisms are the plants and additional cyanobacteria, but again with no bootstrap support . Another gene, enolase, contains two indels shared between land plants and apicomplexans (including C. parvum) and was suggested to be a plant-like gene [Other genes were less informative in this analysis. Among these, aldolase was reported in both lciparum  and the panosoma  as a plaike gene , but altike gene .C. parvum [Cryptosporidium genome sequences deposited in CryptoDB using plant , cyanobacterial  and other bacterial  homologs.The biochemical activity of the polyamine biosynthetic enzyme arginine decarboxylase (ADC), which is typically found in plants and bacteria, was previously reported in . parvum . HoweverC. parvum [C. parvum genome sequence , trpB and the gene for aspartate ammonia ligase).Several HGTs from bacteria have been reported previously in . parvum ,32,40. WC. parvum which does not contain a typical mitochondrion [The endosymbiotic event that gave rise to the mitochondrion occurred very early in eukaryotic evolution and is associated with significant IGT. However, most of these transfer events happened long before the evolutionary time window we explored in this study . Many IGhondrion ,42-44) aC. parvum are known to be expressed. IMPDH has been shown to be essential in C. parvum purine metabolism [C. parvum no longer possesses the ability to synthesize nucleotides; instead it relies entirely on salvage.Gene transfer is an important evolutionary force ,22,45,46tabolism  and TK htabolism . It is nC. parvum, acquired plastidic proteins are theoretically destined to go elsewhere. In the absence of an apicoplast, it is tempting to suspect that plastid-targeted proteins would have been lost, or would be detected as pseudogenes. No identifiable pseudogenes were detected and at least one gene is still viable. The C. parvum leucine aminopeptidase, which still contains an amino-terminal extension , is intact and is expressed, as shown in Figure C. parvum genome. One copy has a long amino-terminal extension (97 amino acids) beginning with a signal peptide. The extension does not contain characteristics of a transit peptide. Expression of a fluorescent reporter construct containing this extension in a related parasite, T. gondii, did not reveal apicoplast targeting but instead secretion via dense granules . Exactly how and where intracellularly transferred genes  have become incorporated into other metabolic processes remains a fertile area for exploration.Many apicoplast and algal nuclear genes have been transferred to the host nuclear genome, where they were subsequently translated in the cytosol and their proteins targeted to the apicoplast organelle. However, as there is no apicoplast in Cryptosporidium is the recipient of a large number (31) of transferred genes, many of which are not shared by other apicomplexan parasites. The genes have been acquired from several different sources including α-, β-, and ε-proteobacteria, cyanobacteria, algae/plants and possibly the Archaea. We have described two cases of two genes that appear to have been acquired together from a eubacterial source: trpB and the aspartate ammonia ligase gene are located within 5 kb of each other, while the two copies of 1,4-α-glucan branching enzyme represent copies of an ancient gene duplication also observed in cyanobacteria.Arabidopsis genome [Giardia [Trypanosoma [Entamoeba [Euglena [Cryptosporidium [Once thought to be a relatively rare event, reports of gene transfers in eukaryotes are increasingly common. The abundance of available eukaryotic genome sequence is providing the material for analyses that were not possible only a few years ago. Analysis of the s genome  has reve[Giardia ,49, Tryppanosoma , Entamoentamoeba ,49, Eugl[Euglena , Cryptosporidium ,32,40 anporidium .C. parvum genome, and in two cases (IMPDH and TK), the transferred genes have been shown to be functional [Cryptosporidium genome has changed the genetic and metabolic repertoire of the parasite.As discussed earlier, genes transferred from distant phylogenetic sources such as eubacteria could be potential therapeutic targets. In apicomplexans, transferred genes are already some of the most promising targets of anti-parasitic drugs and vaccines ,25,52. Wnctional . The sucC. parvum and C. hominis were downloaded from CryptoDB [C. parvum (IOWA) sequence as previously described using the Glimmer program [Cryptosporidium coding sequences [C. parvum annotation did not consider ORFs of less than 100 amino acids that did not have significant BLAST hits and thus may be a slight underestimate [Genomic sequences for CryptoDB . Genes w program  trained equences . A few pequences ,20; thusestimate .C. parvum, ORFs of more than 120 amino acids from Theileria annulata, and more than 75 amino acids from consensus ESTs for several apicomplexan organisms. Genomic sequences for T. gondii (8x coverage) and clustered ESTs were downloaded from ToxoDB [Entamoeba histolytica [D. discoideum [Leishmania major [T. brucei [T. cruzi [Tetrahymena thermophila [T. annulata, E. histolytica, D. discoideum, L. major and T. brucei were produced by the Pathogen Sequencing Unit of the Sanger Institute and can be obtained from [T. thermophila was obtained from TIGR and can be accessed at [An internal database (ApiDB) containing all available apicomplexan sequence data was created . A seconm ToxoDB ,56. Genom ToxoDB  and extetolytica , D. discscoideum , the kinia major , T. bruc. brucei , T. cruzT. cruzi , and a crmophila . Sequencned from . Prelimiessed at .The source code of the phylogenomic software PyPhy  was kindC. parvum gene does not form a monophyletic group with prokaryotic or plant-related taxa regardless of rooting, the subject gene was eliminated from further consideration. If the topology of the gene tree is consistent with a phylogenetic anomaly caused by gene transfer, but may also be interpreted differently if the tree is rooted otherwise, it was removed from consideration at this time. If the top hits of both nr and dbEST database searches are predominantly non-plant eukaryotes, and the topology of the tree was poor, the subject gene was considered an unlikely candidate. Finally, all 551 protein sequences predicted to be NEAPs in the malarial parasite P. falciparum [C. parvum genome and the results were screened using a BLAST cutoff E-value of 10-5 and a length coverage of 50%. Sequences identified by these searches were added to the candidate list  for manual phylogenetic analyses to verify their likely origins. It should be noted that all trees were screened for the existence of a particular phylogenetic relationship. In some cases the proteins utilized to generate a particular tree are capable of resolving relationships among many branches of the tree of life, and in others they are not. Despite these differences in resolving power, the proteins which survive our phylogenetic screen and subsequent detailed analyses described below exhibit significant support for the branches of the tree in which we are interested. Similar procedures were used to characterize the complement of nuclear-encoded genes of plastid origin in the Arabidopsis genome [Output gene trees with phylogenetic connections   to prokalciparum  were uses genome . BLAST ss genome .Detailed phylogenetic analyses of candidate genes identified by phylogenomic screening: candidate genes surviving the PyPhy phylogenomic screen were reanalyzed with careful attention to taxonomic completeness, including representative species from major prokaryotic and eukaryotic lineages when necessary and possible. New multiple sequence alignments were created with ClustalX , followeC. parvum (IOWA) oocysts (108) were obtained from the Sterling Parasitology Laboratory at the University of Arizona and were lysed using a freeze/thaw method. Genomic DNA was purified using the DNeasy Tissue Kit (Qiagen). Genomic DNA (5 μg) was restricted with BamH1 and EcoR1 respectively and electrophoresed on a 0.8% gel in 1x TAE buffer, transferred to a positively charged nylon membrane (Bio-Rad), and fixed using a UVP crosslinker set at 125 mJ as described in [C. parvum genomic DNA for the probes  was amplified by PCR .ribed in . C. parvC. parvum (IOWA isolate) oocysts were used to infect confluent human adenocarcinoma cell monolayers at a concentration of one oocyst per cell as previously described [C. parvum-infected HCT-8 cultures at 2, 6, 12, 24, 36, 48 and 72 h post-inoculation by directly lysing the cells with 4 ml TRIzol reagent (GIBCO-BRL/Life Technologies). Purified RNA was resuspended in RNAse-free water and the integrity of the samples was confirmed by gel electrophoresis.Sterilized escribed . Total RC. parvum 18S rRNA were used to normalize the amount of cDNA product of the candidate gene to that of C. parvum rRNA in the same sample. PCR products were separated on a 4% non-denaturing polyacrylamide gel and signals from specific products were captured and quantified using a phosphorimaging system (Molecular Dynamics). The expression level of each gene at each time point was calculated as the ratio of its RT-PCR product signal to that of the C. parvum 18S rRNA. Six independent time-course experiments were used in the analysis.Primers specific for several transferred genes identified in the study were designed  and a semi-quantitative RT-PCR analysis was carried out as previously described . PrimersC. parvum phosphoglucomutase in T. gondii; table of primers used for RT-PCR experiments; phylogenetic tree of aldolase; alignment files for phylogenetic analyses in Figure Additional data is provided with the online version of this paper, consisting of a PDF file (Additional data file C. parvum phosphoglucomutase in T. gondii; table of primers used for RT-PCR experiments; phylogenetic tree of aldolase; alignment files for phylogenetic analyses in Figure 4; and the alignment of 1,4-α-glucan branching enzyme sequences used in Figure 5This file contains the materials and methods for genomic Southern analysis; the amino-acid sequences of genes listed in Table 2; accession numbers for sequences used in Figure 4; accession numbers for sequences used in Figure 5; expression of Click here for additional data file


Quantitative studies are becoming more recognized as important to understanding health care with all of its richness and complexities. The purpose of this descriptive survey was to provide a quantitative evaluation of the qualitative studies published in 170 core clinical journals for 2000.All identified studies that used qualitative methods were reviewed to ascertain which clinical journals publish qualitative studies and to extract research methods, content , and whether mixed methods  were used.60 330 articles were reviewed. 355 reports of original qualitative studies and 12 systematic review articles were identified in 48 journals. Most of the journals were in the discipline of nursing. Only 4 of the most highly cited health care journals, based on ISI Science Citation Index (SCI) Impact Factors, published qualitative studies. 37 of the 355 original reports used both qualitative and quantitative (mixed) methods. Patients and non-health care settings were the most common groups of people studied. Diseases and conditions were cancer, mental health, pregnancy and childbirth, and cerebrovascular disease with many other diseases and conditions represented. Phenomenology and grounded theory were commonly used; substantial ethnography was also present. No substantial differences were noted for content or methods when articles published in all disciplines were compared with articles published in nursing titles or when studies with mixed methods were compared with studies that included only qualitative methods.The clinical literature includes many qualitative studies although they are often published in nursing journals or journals with low SCI Impact Factor journals. Many qualitative studies incorporate both qualitative and quantitative methods. Quantitative studies provide answers or insights for many important questions or issues in health care and clinical research. Other important questions dealing with why, how, contexts, and experiences of individuals or groups, can be best addressed using qualitative methods. Other issues benefit from interleaving or integration of both research traditions. Miller and Crabtree , describMost studies in the major clinical journals have been quantitative studies. Very few qualitative studies and even fewer that combine both qualitative and quantitative approaches are published. An example of the breadth of qualitative studies and how findings and results can be combined across paradigms is a study by Jolly and Wiles ,4 who usAnother example of the use of mixed methods was research done by Willms and Wilson and their colleagues -7 on smoAnother effective example of integrated qualitative (ethnography) and quantitative (epidemiology) methods was a study done by Borkan and colleagues  to deterDonovan and colleagues  used mixWe postulate that qualitative studies, either stand-alone reports or studies with mixed methods, are occurring more frequently in health care. This paper was done to describe the publishing of qualitative studies in 1 year of clinical literature, document and present the range of content and techniques in these studies, and establish a baseline for subsequent studies. We defined our sample to include all articles published in a set of major general medical, mental health, or nursing journals during 2000. We determined how many qualitative studies were published and in which journals, and extracted design methods and healthcare content, and how often studies used mixed methods and analyses. Because the nursing literature published a higher proportion of qualitative studies in our sample we also compared studies published in nursing journals with other journals to ascertain if quantitative differences exist across disciplines in the use of qualitative methods. Our analysis is a quantitative review of qualitative studies in health care in 2000.ACP Journal Club , Evidence-Based Medicine , Evidence-Based Nursing , and Evidence-Based Mental Health . Their purpose is to provide enhanced abstracts and commentaries on important high-quality original studies and review articles for their respective clinical audiences. To identify these studies and review articles, 6 research staff read major clinical journals to ascertain if articles were in 1 or more categories of therapy, diagnosis, prognosis, etiology, economics, clinical prediction guides, differential diagnosis, and qualitative studies and if so, did each meet predefined methodology criteria for study quality[The Health Information Research Unit of the Department of Clinical Epidemiology and Biostatistics, Faculty of Health Sciences at McMaster University in Hamilton, Ontario, Canada was the editorial office for four evidence-based summary journals in 2000: y quality. For 200y quality. One hunThe staff of the Health Information Research Unit has established quality criteria for the 8 categories of clinical literature that must be met before articles are judged appropriate for clinical application and publication in an abstract journal. Qualitative studies have 3 criteria:• content relates to how people feel or experience certain situations, specifically those that relate to health care• data collection methods and analyses are appropriate • units of collection and analysis are ideas, thoughts, concepts, phrases, incidents, or stories that become categories or themes.The reading methods have been developed during the past 13 years and inter-rater reliability kappa (chance adjusted agreement) for identifying categories and applying criteria is consistently > 80%.For this paper, KAM, one of the readers, analyzed the qualitative studies. Qualitative systematic reviews were excluded leaving only reports of original studies. These were assessed to extract journal title, qualitative study type, data collection methods, research question, persons studied, setting, and disease or health condition considered. In addition, studies with mixed methods were further analyzed although we did not use stringent criteria for assessing the quality  of the cIn addition, Giacomini and Cook ,15, as pnd edition by Denzin and Lincoln [Data were taken from article abstracts and if needed, the full text was reviewed. Methodologies assessed were phenomenology, grounded theory, ethnography, case studies, narrative analysis, participant action, critical incident techniques, and discourse analysis. Author descriptions were used and if an additional methodology was found it was added to the list of types using definitions and descriptions from the Handbook of Qualitative Analysis, 2 Lincoln . Data coThe 170 journals included 60 330 articles of which 31 496 (52%) contained original data or were review articles. 3830 of these (6%) passed criteria for being high-quality and clinically relevant in 1 of the 8 categories. 367 articles met quality criteria for original studies or reviews of qualitative studies. Table The reproducibility of the categorization was measured by kappa (chance adjusted agreement): 0.92 for disease/condition, 0.83 for groups studied, 0.81 for setting, 0.73 for data collection, and 0.63 for data analysis type. The agreement for data analysis type was disappointing but not surprising in that 20% of the studies did not label their analyses necessitating assignment of analysis type by the data extractors. Agreement was low for participant selection methods (kappa 0.5) and therefore data on participant selection methods are not reported.The 355 qualitative studies appeared in 48 journals . These 48 journals were only 28% of the 170 clinical journals being read. Most of the qualitative studies were published in nursing journals: The 17 nursing titles included 214 qualitative studies .BMJ publishing 12. The highest-ranking journal with qualitative studies was Annals of Internal Medicine, ranked number 6. JAMA, ranked number 2, published articles about qualitative studies in 2000 [Few qualitative studies were published in the high-circulation, general healthcare journals. Using SCI Impact Factor ranking for 2000, only 4 of the top 20 journals  included qualitative and quantitative methods and analyses. These were published in 17 journals with only 1 article in 1% includContent of the studies is shown in Table Disease/condition breakdowns represented common health care situations: cancer (11%), mental health (10%), pregnancy and childbirth (9%), cerebrovascular disease (10%), general issues such as vaccinations or Internet use, and nonspecific spectrum of diseases  (12%). Many uncommon issues were also assessed. For example, Tongprateep  reports Analysis of the 37 articles with mixed methods showed similar patterns for settings, persons studied, and disease/condition evaluated except that more physicians were studied (P < 0.025) and more situations dealing with injury (P < 0.001) were evaluated. For the 211 articles in Nursing journals, very little difference was also seen except that fewer physicians were studied (P < 0.001) and more studies were done outside clinical settings (P < 0.001).Phenomenology (37%), grounded theory (35%), and ethnography (18%) were used most often with some case studies (7%), narrative analysis (6%), participant action (3%) research, critical incident techniques (1%), and discourse analysis (1%) Table . More thSemi-structured interviews were used (77%) with some focus groups (18%) and observation (14%). These methods are major data gathering techniques in qualitative studies. Questionnaires (7%), document analysis (6%), and structured (4%) and unstructured interviews (1%) were used less often. For mixed methods studies, patterns were similar although questionnaires were used more frequently . Nursing studies did not differ for data gathering techniques.Sampling is important in all studies–often no single right way exists for a study question. Purposive, snowball, and theoretical sampling are often used in qualitative studies and random and consecutive sampling for quantitative studies. All methods were represented in this analysis but the breakdowns are not reported because of low inter-rater agreements for categorization and missing author information.BMJ and Annals of Internal Medicine also published several. Three of the high circulation medical journals  and 16 of the top 20 clinical journals, based on SCI Impact Factors, did not publish any qualitative studies. This is likely a reflection on the emphasis on a positivist, numerical approach that many of these journals embrace. The difference in proportion of qualitative studiers published in nursing journals is probably because of two historical, but linked factors. Qualitative studies have roots in women's studies and the nursing profession has always dealt with the patient as much more of a whole person rather than basic sciences facts and numbers. Both of these factors lead to more emphasis on understanding and embracing qualitative methods for research and practice. This view is substantiated by the fact that MEDLINE indexes most of the qualitative studies under the term Nursing Methodology Research until 2003.In 2000 the major clinical journals published many qualitative studies–approximately 9% of all high-quality, clinically relevant articles. Most of the qualitative studies were reports of original research although 12 (3%) were systematic reviews. Most of the qualitative studies were in nursing journals although some medical journals such as A substantial proportion of the qualitative studies 11%) included both qualitative and quantitative (mixed) data. In general, these mixed methods studies were similar to the single methodology studies except they did more assessments of physicians and relied more on questionnaires to gather data for analysis. The presence of these mixed methods or multipardigmatic studies as described by Miller and Crabtree 1% includ is encouIt is also interesting comparing qualitative studies in Nursing and non-Nursing journals. Regardless of the differences in proportion of qualitative studies published, from a content point of view few differences exist between the Nursing and non-Nursing journals except that more physicians were studied in the non-Nursing journals and fewer studies were done in clinical settings–not unsurprising findings. This indicates that the content and methods of qualitative studies seem to be similar across disciplines or if the methods are combined with quantitative methods.This review of the publication of qualitative studies is limited in several ways. The proportion of journals studied was very low in relation to the total number of journals published. MEDLINE indexes over 4000 journals and this number is still a relatively small proportion of all journals that deal with health care. In addition, all of the journals searched were published in English so we do not know about qualitative studies in other languages. Although our criteria were relatively strict for including qualitative studies, our criteria for mixed methods studies could certainly have been stronger. We did not count the number of high-quality quantitative studies that could have included some qualitative analyses. We studied only 1 year of publishing; much could have changed since 2002.Qualitative studies provide insight into social, emotional, and experiential aspects of health and health care and as such, they have an important place in understanding health and health care. Hopefully more studies will be published and more will be published in the high impact (high circulation) journals. This paper provides a basis for measuring increases.Qualitative studies are being done and are published in a wide range of healthcare journals. These journals however are not the highest impact journals. It is encouraging to see that the number of qualitative studies that were published in 2000 and also the number of studies that combined qualitative and quantitative methods. More can be done however to complete and publish qualitative studies, and where appropriate, integrate the best of both methodologies. Both qualitative and quantitative researchers and clinicians need to work together to make this happen. Journal editors can also encourage submission of qualitative and mixed methods studies and facilitate publication of those they do receive.SCI ISI Science Citation IndexNone declared.This work was done in partial fulfillment of PhD requirements for KAM. Both authors have supplied intellectual input in designing and implanting the survey. KAM has collected and analyzed the data and both authors have contributed to writing the paper and agree on its content.The pre-publication history for this paper can be accessed here:


Late-stage cancers are notoriously unresponsive to treatment, making certain hard-to-detect cancers particularly insidious. Ovarian cancer, for example, most often escapes diagnosis until the tumor has already metastasized. At this stage, ovarian cancer is classified as peritoneal carcinomatosis, a terminal condition characterized by widespread tumor growth throughout the peritoneum, the large serous membrane that lines the abdominal cavity, pelvis, and associated organs. Advanced cases of peritoneal carcinomatosis are largely resistant to chemotherapy and account for the bleak 15%–20% survival rate of ovarian cancer.Biologists often view cancer as an evolutionary process in which cells that would normally cooperate with their neighbors begin to compete with them. Selective advantage for cancer cells often begins with mutations that inhibit tumor suppressor pathways. p53, like other tumor suppressor genes, arrests cell growth and induces apoptosis (programmed cell death) in response to cellular stress, such as chromosomal damage. Cells with p53 mutations often escape these constraints, leading to the uncontrolled growth characteristic of “immortal” cancer cells. Nearly all types of tumors have mutations in the p53 pathway. Treatments focused on restoring p53 function—which is likely to be defective only in cancer cells—should prove more effective than chemotherapies, which indiscriminately kill all dividing cells, healthy or cancerous. With the goal of developing targeted therapeutic strategies, Steven Dowdy and colleagues show that restoring p53 protein function in tumor cells not only dramatically increases lifespan in mice but also eliminates disease.While past efforts to restore tumor suppressor function in cancer cells have focused on gene therapy, Dowdy and colleagues introduced modified p53 peptides, or protein fragments, into cancer cells. p53 works as a “transcriptional” activator that binds to specific sequences of DNA and triggers apoptosis in response to DNA damage. Its biological function flows from this binding ability. One region of this large protein, called the C-terminal domain, facilitates effective binding. In cancer cells, synthesized peptides  derived from this region can induce apoptosis by activating p53—which is normally present in low levels in a biologically inactive form—and restoring function to p53 proteins with DNA-binding mutations.To get p53C′ peptides into cancer cells, Eric Snyder et al. used a technique pioneered by Dowdy that delivers large proteins into the cell interior. Since the cell membrane normally limits passage to only small molecules , this is no small feat. The technique exploits the ability of a small peptide region from the HIV TAT protein to smuggle macromolecules through cell membranes that normally prohibit entry to such large molecules. After synthesizing a structurally modified form of p53C′ less prone to degradation, the researchers first confirmed that the peptide was functional and then that it activates p53-specific genes in tumor cells, but not in normal cells. Testing the effectiveness of the peptide therapy on mouse strains used to model human metastatic disease, they found that mice treated with the TATp53C′ peptide showed a significant reduction in tumor growth and lived six times longer than both mice treated with a control peptide and untreated mice, with some mice remaining disease-free more than 200 days after treatment.This macromolecular delivery approach, Snyder et al. argue, works with greater specificity and avoids the tumor-generated neutralizing effects observed in small molecule strategies. Because a mutation in the p53 gene is one of the most common events in the development of cancer, these results could have implications for a wide variety of cancers. And by working with mouse models that approximate the physiological burdens metastatic cancer imposes on humans, Dowdy's team demonstrates the promise of developing targeted “intracellular biologic” therapeutics that treat the systemic pathology of cancer—inhibiting tumor growth as well as alleviating the lethal complications associated with the disease.


The genes encoding proteins in the thrombomodulin-protein C pathway are promising candidate genes for stroke susceptibility because of their importance in thrombosis regulation and inflammatory response. Several published studies have shown that the Ala455Val thrombomodulin polymorphism is associated with ischemic heart disease, but none has examined the association with stroke. Using data from the Stroke Prevention in Young Women Study, we sought to determine the association between the Ala455Val thrombomodulin polymorphism and the occurrence of ischemic stroke in young women.All 59 hospitals in the greater Baltimore-Washington area participated in a population-based case-control study of stroke in young women. We compared 141 cases of first ischemic stroke (44% black) among women 15 to 44 years of age with 210 control subjects (35% black) who were identified by random digit dialing and frequency matched to the cases by age and geographical region of residence. Data on historical risk factors were collected by standardized interview. Genotyping of the thrombomodulin Ala455Val polymorphism was performed by pyrosequencing.The A allele (frequency = 0.85) was associated with stroke under the recessive model. After adjustment for age, race, cigarette smoking, hypertension, and diabetes, the AA genotype, compared with the AV and VV genotypes combined, was significantly associated with stroke . The AA genotype was more common among black than white control subjects (81% versus 68%) but there was no significant interaction between the risk genotype and race (adjusted odds ratio 2.7 for blacks and 1.6 for whites). A secondary analysis removing all probable (n = 16) and possible (n = 15) cardioembolic strokes demonstrated an increased association .Among women aged 15 to 44 years, the AA genotype is more prevalent among blacks than whites and is associated with increased risk of early onset ischemic stroke. Removing strokes potentially related to cardioembolic phenomena increased this association. Further studies are needed to determine whether this polymorphism is functionally related to thrombomodulin expression or whether the association is due to population stratification or linkage to a nearby functional polymorphism. Thrombosis is a dynamic balance between factors that promote clot formation, antithrombotic mechanisms, and fibrinolysis. Central to this balance is the thrombomodulin-protein C antithrombotic mechanism. Thrombomodulin forms a 1:1 complex with thrombin on the vascular endothelium, thereby inhibiting the procoagulant actions of thrombin and converting protein C to activated protein C -5. TheseTHBD) maps to chromosome 20p11.2, contains a single exon and no introns, and spans 4 kb . The thrombomodulin protein is expressed primarily on the luminal surface of vascular endothelial cells and consists of 557 amino acids (aa) : an N-terminal lectin-like module (aa 1–154), a hydrophobic region (aa 155–222), six epidermal growth factor (EGF)-like modules (aa 223–462), a serine and threonine rich region (aa 463–497), a single transmembrane segment (aa 498–521), and a short cytoplasmic tail (aa 522–557) [The thrombomodulin gene  is a population-based case-control study that was initiated to examine risk factors for ischemic stroke in young women. In that study the term "population-based" means that cases and their comparison group were identified from the same defined population. The study area included all of Maryland (except the far Western panhandle), Washington DC, and the southern portions of both Pennsylvania and Delaware. Cases were female patients 15 to 44 years of age with a first cerebral infarction as identified by discharge surveillance at 59 regional hospitals and through direct referral by regional neurologists. The methods for discharge surveillance, chart abstraction, and case adjudication have been described previously ,13,14. TTHBD genotyping at the Ala455Val polymorphism for 141 cases and 210 control subjects. This included all case and control samples that were available at that time. Genotyping was performed blinded to case-control status. Genomic DNA was extracted from stored peripheral blood lymphocytes by using standard protocols . The THBD Ala455Val polymorphism was determined by pyrosequencing. The single-nucleotide polymorphism region of the gene was amplified by polymerase chain reaction (PCR) with the use of published primers [We performed  primers ,16. The THBD Ala455Val polymorphism and stroke: age, race, current cigarette smoking, hypertension, diabetes mellitus, history of angina or myocardial infarction (angina/MI), use of oral contraceptive pills (OCP) or hormone replacement therapy (HRT), sickle cell disease, and sickle cell trait. Age, race, current cigarette smoking status, use of OCP or HRT was determined by subject reports . Hypertension and diabetes mellitus, sickle cell disease or sickle cell trait were determined by asking study participants (or a proxy) if a physician had ever told them that they had the condition.We assessed the following potential confounders of the association between the alleles of the We compared means by t tests and proportions by χ2 tests. The probability values presented are based on two-sided tests. Because of the low frequency of the V455 allele, we compared the frequency of the combined AV/VV genotype between cases and controls. Adjusted odds ratios derived from logistic regression were used to determine whether the presence of the Ala455Val test allele was associated with an increased risk for stroke after differences in age, race, current cigarette smoking, hypertension, and diabetes mellitus were controlled for. Additional analyses included: 1). adding ischemic heart disease (angina/MI) into the logistic regression model; 2). evaluation for interactions between genotype and OCP/HRT 3). an analysis excluding sickle cell trait, and 4). an analysis excluding cardioembolic strokes.Characteristics by case-control status are described in Table The distribution of genotypes was in Hardy Weinberg equilibrium for the pooled set of cases and controls, both in total and by race.Among control subjects, the prevalence of the AA genotype was 81% (59/73) for blacks and 68% (93/137) for whites. The relationship between the Ala455Val genotypes and selected stroke risk factors in control subjects is summarized in Table Table The strength of association between the AA genotype and stroke remained unchanged including history of angina or myocardial infarction in the logistic regression model . Neither OCP/HRT use, nor sickle cell trait demonstrated an interaction with genotype and additional adjustment for these factors did not alter the association between the AA genotype and stroke.Among the 141 stroke patients, 70 (50%) had a least 1 probable cause, 30 (21%) had no probable cause but a least one possible cause, and 41 (29%) were indeterminate. Table A secondary analysis removing all probable (n = 16) or possible (n = 15) cardioembolic strokes was performed using the same adjusted model including age, race, smoking, hypertension, and diabetes. An increased association between non-cardioembolic stroke and the AA genotype was demonstrated .THBD Ala455Val polymorphism, the prevalence of the AA genotype among our control population was similar to that previously reported for the Atherosclerosis Risk in Communities (ARIC) Study population [In our study of the pulation . Our respulation . VasculaTHBD Ala455Val polymorphism and coronary artery disease (CAD) have yielded conflicting results. A Swedish case-control study found the alanine allele was associated with CAD [THBD Ala455Val polymorphism and CAD [THBD Ala455Val polymorphism, ours included, is due to population-stratification bias, a functionally neutral polymorphism that serves as a marker for a nearby functional mutation (linkage disequilibrium), or the true existence of different associations in the different study populations.Several recent studies evaluating the with CAD . In contwith CAD . A Briti and CAD . Consist and CAD , we obsePopulation-stratification bias is due to confounding by population admixture . An unidTHBD Ala455Val locus may be in linkage disequilibrium with an unobserved "high-risk" susceptibility locus. Linkage disequilibrium is a function of the history of the population, and thus true associations can occur in one population and not another.The THBD Ala455Val polymorphism, thereby defining a susceptibility locus for the disease. An important criterion for a true susceptibility locus is that the polymorphism is associated with a change in protein expression or function. The THBD Ala455Val polymorphism has not been associated with variation in soluble thrombomodulin concentrations [Our results are also consistent with a causal association between stroke and the trations , but soltrations ,8,20,21.trations . Furthertrations .Thrombomodulin has not previously been examined as a candidate gene for stroke susceptibility. We found that among women aged 15 to 44 years, the AA genotype is more prevalent among blacks than whites and is associated with increased risk of early-onset ischemic stroke. Removing strokes potentially related to cardioembolic phenomena increased this association. Further studies are needed to determine whether this association is due to population stratification, linkage to a nearby functional polymorphism, or variation in thrombomodulin expression or function.The author(s) declare that they have no competing interests.All authors certify that they participated in the conceptual design of this work, the analysis of the data, and the writing of the manuscript to take public responsibility for it. All authors reviewed the final version of the manuscript and approve it for publication. J.W.C., S.J.K., B.D.M., M.A.W. and R.F.M. participated in the writing of the initial draft. M.G., K.K.S., W.H.G. and S.C.R. participated in the genotyping. J.W.C., S.J.K., B.D.M., and L.J.R. participated in the data analysis. All authors provided critiques of the final manuscript.Dr. Cole's effort on this project was supported in part by an American Academy of Neurology Clinical Research Training Fellowship, by the National Institutes of Health Research Training in the Epidemiology of Aging (Grant T32-AG00262-04), and by the Department of Veterans Affairs, Baltimore, Office of Research and Development, Medical Research Service, and Stroke Research Enhancement Award Program. Dr. Kittner was supported in part by the Department of Veterans Affairs, Baltimore, Office of Research and Development, Medical Research Service, Geriatrics Research, Education and Clinical Center, and Stroke Research Enhancement Award Program; a Cooperative Agreement with the Division of Adult and Community Health, Centers for Disease Control and Prevention; the National Institute of Neurological Disorders and Stroke and the NIH Office of Research on Women's Health; the National Institute on Aging Pepper Center Grant P60 12583; and the University of Maryland General Clinical Research Center (Grant M01 RR 165001), General Clinical Research Centers Program, National Center for Research Resources, NIH.The pre-publication history for this paper can be accessed here:


Micro computed tomography (micro CT) has been shown to provide exceptionally high quality imaging of the fine structural detail within urinary calculi. We tested the idea that micro CT might also be used to identify the mineral composition of urinary stones non-destructively.Micro CT x-ray attenuation values were measured for mineral that was positively identified by infrared microspectroscopy (FT-IR). To do this, human urinary stones were sectioned with a diamond wire saw. The cut surface was explored by FT-IR and regions of pure mineral were evaluated by micro CT to correlate x-ray attenuation values with mineral content. Additionally, intact stones were imaged with micro CT to visualize internal morphology and map the distribution of specific mineral components in 3-D.Micro CT images taken just beneath the cut surface of urinary stones showed excellent resolution of structural detail that could be correlated with structure visible in the optical image mode of FT-IR. Regions of pure mineral were not difficult to find by FT-IR for most stones and such regions could be localized on micro CT images of the cut surface. This was not true, however, for two brushite stones tested; in these, brushite was closely intermixed with calcium oxalate. Micro CT x-ray attenuation values were collected for six minerals that could be found in regions that appeared to be pure, including uric acid , struvite (7242 – 7969 AU), cystine (8619 – 9921 AU), calcium oxalate dihydrate (13815 – 15797 AU), calcium oxalate monohydrate (16297 – 18449 AU), and hydroxyapatite (21144 – 23121 AU). These AU values did not overlap. Analysis of intact stones showed excellent resolution of structural detail and could discriminate multiple mineral types within heterogeneous stones.Micro CT gives excellent structural detail of urinary stones, and these results demonstrate the feasibility of identifying and localizing most of the common mineral types found in urinary calculi using laboratory CT. Clinical laboratory assessment of urinary stones is typically conducted using destructive methods of analysis -5 and isKnowing the mineral composition of a patient's stones has obvious value in determining a treatment plan, but stone structure may be important as well. It has long been appreciated that there is variability in stone fragility to shock waves in lithotripsy, that stones of a given mineral type do not all break the same . Stone fThe purpose of this report is to present micro computed tomography (micro CT) as a potential method for the analysis of urinary stone composition and morphology in a nondestructive manner at very high resolution. Micro CT, which has seen considerable use as a research tool in bone biology , has theHuman urinary stones were obtained following percutaneous nephrolithotomy procedures or from a stone analysis laboratory . A representative collection of urinary stones of pure and heterogeneous mineral content was selected for use in this study.Stones for this work had already been analyzed using conventional methods , with portion used here typically the half of the stone remaining intact after that analysis. For some stones, pre-CT analysis was completed on a cohort stone taken during the same surgical procedure. Compositions of the 11 stones used for combined micro CT and IR microspectroscopy included pure COM, pure calcium oxalate dihydrate (COD), 91% COD/9% COM, 99% COM/1% hydroxyapatite, 52% COM/48% uricite, pure uricite, pure struvite, two pure cystine stones, and two pure brushite stones.-1. Spectra for COM, COD, apatite, uric acid, struvite, cystine, and brushite were all easily identified and distinguishable from one another. Uricite and uric acid dihydrate could not be distinguished above the background noise of the spectra, and so regions are reported as uric acid.Stones were embedded within methyl methacrylate and sliced into 1–2 mm slabs using a diamond wire saw . A Perkin-Elmer AutoImage infrared microscope interfaced to a Perkin-Elmer Spectrum 2000 fourier transform infrared (FT-IR) spectrometer  was used to collect infrared spectra on the flat surface of the stone slices. The spectrometer directs infrared light onto the specimen through a microscope lens, and collects reflected light onto a cooled mercury-cadmium-telluride detector. Spectra were collected in grids of 200 × 200 μm intervals, averaging 16 individual scans on each spot, yielding spectral resolution of 4 cmThe same stone slices analyzed by FT-IR microspectroscopy were also scanned by micro CT, using a Scanco MicroCT 20 instrument  Figure . The sysThe FT-IR analysis yielded a 2-D map of mineral composition for a defined region of each stone slice. Regions of the 2-D map showing pure mineral were compared to the identical region on the micro CT image slice taken just below the cut surface  affords much greater spatial resolution than conventional single-detector helical CT. Williams et al. used four-row MDCT to show that some degree of internal structure can already be seen in urinary stones . Zarse aere used . Eight-rere used . Sixteenere used . Moreovein vitro imaging of urinary calculi for nondestructive stone analysis. Fine resolution coupled with the 2-D and 3-D reconstruction capabilities of micro CT yields an imaging diagnostic that offers excellent images of surface and internal stone structure. Mineral deposition pattern and regions of potential structural weakness, such as voids, were easily visible. Six common stone minerals were found to occupy non-overlapping ranges of attenuation value, which allows the identification of mineral types using micro CT alone. This technology carries the potential for immediate application to non-destructive analysis of stone structure and composition in clinical stone analysis laboratories. The demonstration that stone composition can be determined by micro CT is proof of concept and an important step toward the use of helical CT to provide similar analysis in patients.Micro CT provides high resolution The author(s) declare that they have no competing interests.CZ did the experimental work for calibrating the attenuation values, put together several of the figures, including the image stack movie, and drafted the manuscript. AJ performed the FT-IR analyses. EH did many of the micro CT scans and managed the library from which stones were chosen. SK and JL provided some of the stones from patients, and SK performed the 3-D imaging of cystine stone. JM, JL, and AE initiated much of the intellectual direction for the study. JM and AE also played important roles in the FT-IR analysis part of the study. JW performed the 3-D image construction in MacVol, drafted several of the figures, and performed the primary editing of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Micro CT image stack through a urinary stone.MOV 6.61 MBClick here for file


These tablets consist of dimethylfumarate (DMF) and salts of monoethylfumarate (MEF) and its main bioactive metabolite is monomethylfumarate (MMF). Little is known about the pharmacokinetics of these FAE. The aim of the present study was to investigate the hydrolysis of DMF to MMF and the stability of MMF, DMF and MEF at DMF is hydrolyzed to MMF in an alkaline environment (pH 8), but not in an acidic environment (pH 1). In these conditions MMF and MEF remained intact during the period of analysis (6 h). Interestingly, DMF was hardly hydrolyzed to MMF in a buffer of pH 7.4, but was rapidly hydrolyzed in human serum having the same pH. Moreover, in whole blood the half-life of DMF was dramatically reduced as compared to serum. The concentrations of MMF and MEF in serum and whole blood decreased with increasing time. These data indicate that the majority of the FAE in the circulation are metabolized by one or more types of blood cells. Additional experiments with purified blood cell fractions resuspended in phosphate buffered saline (pH 7.4) revealed that at concentrations present in whole blood monocytes/lymphocytes, but not granulocytes and erythrocytes, effectively hydrolyzed DMF to MMF. Furthermore, in agreement with the data obtained with the pure components of the tablet, the enteric-coated tablet remained intact at pH 1, but rapidly dissolved at pH 8.in vitro data indicate that hydrolysis of DMF to MMF rapidly occurs at pH 8, resembling that within the small intestines, but not at pH 1 resembling the pH in the stomach. At both pHs MMF and MEF remained intact. These data explain the observation that after oral FAE intake MMF and MEF, but not DMF, can be readily detected in the circulation of human healthy volunteers and psoriasis patients.Together, these  In the present study, we investigated the hydrolysis of DMF to its most bioactive metabolite monomethylfumarate (MMF) and the stability of MMF, DMF and MEF in different environments representing various body compartments using this methodology.Psoriasis is a chronic inflammatory skin disease characterized by epidermal hyperplasia and infiltration of inflammatory cells into skin lesions. Anti-psoriatic therapies are mainly anti-inflammatory. Long-term use of many of these anti-psoriatic therapies is often hampered by serious adverse effects -5. In thDMF, MMF and MEF remained completely intact in a buffer of pH 1 mimicking the pH in the stomach Figure  and 1F. To further examine the pH-dependency of the hydrolysis of DMF to MMF, we measured concentrations of DMF and MMF in phosphate buffers (with pH ranging from 6.5–8) supplemented with DMF or the combination of DMF and MEF. The results revealed that the half-life of DMF dramatically decreased with increasing pH values and the maximal hydrolysis of DMF to MMF was seen at pH 8 . For example, at pH 7.4 the half-life of DMF amounted to 12.7 ± 1.0 hr (n = 3) Figure . In agreSince FAE must enter the circulation to exert their anti-psoriatic effects at the affected skin site , we deteTo find out which blood cell type(s) is (are) responsible for the hydrolysis of FAE in whole blood, hydrolysis of DMF in a buffer of pH 7.4 by purified blood cell fractions was analyzed. The results revealed that monocytes/lymphocytes Figure , but notin vitro pharmacokinetics of FAE. For example, no enzymes, e.g. esterases, are present in these buffers whereas they are in these body compartments. In this connection, Werdenberg and collegues . Our observation that the MMF is more rapidly removed from whole blood than from serum could indicate that MMF (and MEF) is taken up by blood cells and perhaps further metabolized into FA, which subsequently fuels the citric acid cycle, as suggested earlier by Joshi . The different interactions between FAE and blood cells may affect their functional activities, as has been reported earlier [It should be realized that MMF (and MEF) most likely enter the circulation of psoriasis patients in order to exert their antipsoriatic effects in the skin lesions. In agreement we detected MMF and MEF, but not DMF, in the circulation of healthy volunteers and psoriasis patients after oral intake of Fumaraat 120tablets [, Litjens earlier ,17-19, tin vitro data indicate that DMF is almost completely hydrolyzed to MMF at an alkaline pH, but not at an acidic pH, suggesting that this hydrolysis occurs mainly within the small intestines and not in the stomach. Most likely, MMF and MEF are then absorbed in the circulation where they interact with blood cells and perhaps cells in the psoriatic lesions. The different interactions between these FAE and the various cell types may explain the beneficial effects of FAE in psoriasis. Finally, these in vitro experimental data will be key to the pharmacokinetic analysis of oral FAE in human healthy volunteers and psoriasis patients.Together, these ®; TioFarma), containing 120 mg of DMF and 95 mg of calcium-MEF was investigated in this study.The following FAE were used: dimethylfumarate , calcium-monoethylfumarate , monomethylfumarate . In addition, the enteric-coated, magisterial manufactured tablet  and pH 8 (0.1 M of sodium phosphate buffer), the tablet was placed in these buffers and at various intervals samples were taken and prepared for measurement of the concentrations of DMF, MMF and MEF by high-performance liquid chromatography (HPLC) as described below.To investigate the stability of DMF, MMF and MEF and the hydrolysis of DMF to MMF in several environments representing various aspects of different body compartments, 0.1 N HCl with pH 1 and 0.1 M sodium phosphate buffer with pH 8 were spiked with 2 mg/L of DMF, MMF, MEF or the combination of 2 mg/L of DMF and 1.4 mg/L MEF, to resemble the ratio of these two components in the Fumaraat 120To further investigate the effect of the pH on the hydrolysis of DMF to MMF, 0.1 M sodium phosphate buffers with pH values ranging from 6.5–8 were spiked with DMF and the combination of DMF and MEF. At several intervals thereafter, samples were taken, and then prepared for measurement of the various FAE by HPLC.As the current buffers lack proteins, no extraction procedure was necessary and the concentrations of the various FAE in the samples could be directly quantified by HPLC (see below).As described above, serum and whole blood from 3 volunteers was spiked with 2 mg/L of DMF, MMF, MEF or the combination of 2 mg/L of DMF and 1.4 mg/L MEF. All volunteers were healthy as assessed by a full medical screening.At several intervals, samples were taken, and then prepared for measurement of the various FAE by HPLC. In short, serum and whole blood contained proteins known to interfere with the measurement of FAE. To overcome this problem, the various FAE were extracted from serum and whole blood samples and subsequently the concentrations were measured by HPLC (see below).6 monocytes and lymphocytes/mL PBS, 4 × 106 granulocytes/mL PBS, and 5 × 109 erythrocytes/mL PBS were spiked with 2 mg/L DMF. Again, at several intervals samples were taken and concentrations of the various FAE were measured as described below.The various blood cell fractions were obtained from blood of healthy volunteers using centrifugational techniques as described earlier ,19. In set al., submitted for publication). Briefly, after precipitation of serum proteins with acetonitrile, DMF in the samples was quantitated by HPLC. The sample preparation for MMF and MEF required a protein precipitation step with metaphosphoric acid followed by extraction with diethylether and additional pH-lowering to pH 0.5. Next, sodium chloride was added before centrifugation at 12,000 g. Thereafter, the ether layer was transferred to a glass vial and after evaporation the residue reconstituted in methanol: 0.1 M potassium phosphate buffer  supplemented with 5 mM tetrabutylammonium dihydrogen phosphate 1:1 (v/v).The concentrations of the various fumarates in serum samples were determined as described  equipped with an Alltima C18  column and an Alltima Guard C18 precolumn  using methanol:water 30:70 (v/v) as an eluent for DMF and methanol: potassium phosphate buffer supplemented with 5 mM tetrabutylammonium dihydrogen phosphate 20:80 (v/v) as eluent for MMF and MEF. The limit of detection for all three compounds amounted to 0.01 mg/L, the coefficient of variation for MMF, DMF and MEF was 7%, 8% and 9% at 0.5 mg/L, respectively (n = 4), and the recovery of MMF, DMF and MEF amounted to 75 ± 7%, 98 ± 3%, 67 ± 7% (n = 6). Standard curves constructed with purified FAE in buffers or human serum were used to quantify the concentrations of FAE in the various samples of these buffers and human serum or whole blood, respectively.NL participated in the design of the study, analysis of the data and drafted the manuscript. ES carried out the optimisation of the HPLC method and performed all HPLC analyses. CvG was responsible for the optimisation of the HPLC method and participated in the design of the study. HM participated in the design of the study and analysis of the data. JvD, HT and PN conceived of the study and participated in its design and coordination. All authors read and approved the final manuscript.


How did language develop and evolve? Here, linguists, cognitive scientists, behavioural ecologists, and theoretical biologists all offer their disparate views on this emerging field Language Evolution, a compilation of essays by a diverse group of respected researchers, is amongst the first books that try to tackle what is arguably one of the hardest scientific problems. The editors set themselves the ambitious target of creating an up-to-date book about this emerging field, and they have to be congratulated for their efforts. Linguists, cognitive scientists, behavioural ecologists, and theoretical biologists all offer their view on the origin of human language and, refreshingly, do not shy from pointing out the real or assumed weaknesses of the other approaches.A ban in the 1866s by the French Academy of Sciences on publications about the origin of human language must have been one of the strangest bans in the history of sciences. Yet it was highly effective. After the ban, scientists and interested laymen had to wait for more than a century to hold a textbook on language evolution in their hands. One of the main themes of the book is the evolutionary approach and the importance of biological structures and properties that were co-opted in the development of language (pre-adaptations). In one essay, Michael Studdert-Kenedy and Louis Goldstein propose that speech, as a motor function, draws on phylogenetically ancient mammalian oral capacities for sucking, licking, swallowing, and chewing. Thus, our hominid ancestors adopted an apparatus already divided neuroanatomically into discrete components. Complementing this evidence, Marc Hauser and Tecumseh Fitch compare human speech production and perception with that of nonhuman species. They conclude that many traits that were formerly thought to have evolved specifically for speech  are also present in other species.But perhaps the most interesting idea about pre-adaptation comes from the work of neuroscientist Michael Arbib on ‘mirror’ neurons in monkeys. These neurons are a subset of the grasp-related premotor neurons that discharge not only, as other premotor neurons do, when the monkey executes a certain class of actions, but also when the monkey observes more or less similarly meaningful hand movements made by the experimenter (or by another monkey). The area in which these grasp-related neurons are found is analogous with the Broca's area in human brains, which is involved in assessing the syntax of words. This observation serves as the basis for the mirror-system hypothesis, which postulates that Broca's area in humans evolved from a basic mechanism not originally related to communication but rather from the mirror system for grasping in the common ancestor of monkey and human. As a result, the mirror system provides a possible ‘neural link’ in the evolution of human language.There is still much debate about the selection pressures that led to the evolution of language. Observing the overabundance of potential selective scenarios for why language evolved, the linguist Derek Bickerton voices his scepticism: ‘The fact that these and similar explanations flourish side by side tells one immediately not enough constraints are being used to limit possible explanations.’ One frequent source of confusion, he notes, is equating language with speech by not distinguishing between modality, lexicon, and structure. Hauser and Fitch share Bickerton's scepticism and urge scientists to rely more on the traditional comparative approach, which was always the strength of Darwinian evolutionary theory.Primatologist Robin Dunbar, who originally proposed that grooming (group bonding) could have provided the stimulus for language, dismisses two other possible scenarios—hunting and tool-making—as potential ecological contexts for the evolution of human language. Gestural origins are also dismissed in his theory, because gestural languages do not seem to develop spontaneously and also require a line-of-sight contact making them useless at night.Interestingly, Steven Pinker rules out both Dunbar's theory of grooming and Geoffrey Miller's theory of sexual selection, whereas Bickerton rules out grooming, gossip, mating contract, and Machiavellian intelligence as likely contexts for the origin of human language.Also under fire in the book is the idea that the human brain is somehow equipped at birth with a ‘universal grammar’ out of which all human languages later develop. Several authors try to provide alternatives to innate predispositions, such as the importance of function to categorization (Michael Tomasello) and the importance of cultural transmission to the structure of language (Simon Kirby and Morton Christiansen). Arbib explicitly questions the traditional Chomskyan theory of innate linguistic predispositions and argues that what humans have and had in the past is ‘language readiness’ rather than a fixed universal grammar.Neuroscientist Terrence Deacon also puts an alternative theory forward. According to Deacon, many of the language universals reflect semiotic constraints inherent in the requirements for producing symbolic reference rather than innate predispositions. Thus, neither evolved innate predispositions nor culturally evolved and transmitted regularities can be considered as the ultimate source of language universals. He draws a parallel with mathematical operations  and with prime numbers. Symbolic reference, he argues, is constrained by the structure it refers to.The editors claim, in the light of this diversity, that ‘this book is intended to bring together, for the first time, all the major perspectives on language evolution’. We have two concerns with this aim. First, two books of the same organization and scope have been published in the past six years based on the material from language evolution conferences . AlthougOne of these missing themes is the selective advantage of early language. As discussed, many of the contributors express their scepticism towards the selective scenarios found in the literature—and indeed towards such constructions in general—but there is no review and no balanced evaluation of these selective scenarios. Since one of the key questions of language evolution is the selective advantage of early language, the lack of such a review is a major weakness. A balanced account could have been presented even if the editors and most of the contributors are frustrated by the plethora of selective scenarios.Related to the possible selective advantage of language is the issue of genetic background. Although there is mention of the so-called FOX genes—some mutations of which are associated with language disorders—there is no detailed discussion of our current knowledge of genetics related to language.Another lightly treated theme is the neural basis of language and language evolution. Understandably it is one of the most difficult issues concerning human language, and no one expects the editors or any of the contributors to come up with an answer to all the questions. What is missing again is a good survey outlining the problems and the current findings of the field.The weaknesses of the book come from its structure and organization. The editors, instead of outlining a structure and asking specialists to contribute to that structure, appear to have let every contributor write freely about their current ideas and current research without regard to the bigger picture. This definitely shows the interests of the contributors and outlines the current state of the art; it leaves gaps, however, in the coverage of crucial topics related to the evolution of human language.