Solanum lycopersicum cv. Micro-Tom) was used as a model for this study using LC-FTICR-MS. Collected mass spectral features, together with predicted molecular formulae and putative structures, were provided as metabolite annotations for 869 metabolites. Comparison with public databases suggests that 494 metabolites are novel. A grading system was introduced to describe the evidence supporting the annotations. Based on the comprehensive characterization of tomato fruit metabolites, we identified chemical building blocks that are frequently found in tomato fruit tissues, and predicted novel metabolic pathways for flavonoids and glycoalkaloids. These results demonstrate that metabolite annotation facilitates the systematic analysis of unknown metabolites and biological interpretation of their relationships, which provide a basis for integrating metabolite information into the system-level study of plant biology.A large number of metabolites are found in each plant, most of which have not yet been identified. Development of a methodology is required to deal systematically with unknown metabolites, and to elucidate their biological roles in an integrated ‘omics’ framework. Here we report the development of a ‘metabolite annotation’ procedure. The metabolite annotation is a process by which structures and functions are inferred for metabolites. Tomato ( Large-scale biology studies supported by high-throughput data acquisition technologies require a method to bridge the gap between the data obtained and their biological interpretation. In genomics, without an analytical method to define genes, the nucleotide sequence of a whole genome is merely a series of letters . Using tOne approach to overcoming this obstacle is to quantify all detected peaks and compile them as un-annotated variables  fruit was analyzed as a model plant for two reasons. First, tomato contains a number of secondary metabolites that are not present in other model plants such as Arabidopsis and rice. Second, a tomato genome sequencing project is currently underway . Based on comprehensive characterization of tomato fruit metabolites, we identified chemical building blocks that appear frequently in the tomato fruit tissues. We also assigned several unknown flavonoids and glycoalkaloids to novel metabolic pathways based on the annotations of putative structures. These results demonstrate that metabolite annotation allows us to systematically analyze unknown metabolites and facilitates biological interpretation of their roles in metabolic processes.Tomato metabolite data were collected in a non-targeted manner. We then compiled a dataset comprised of mass spectral features including retention time, UV/visible absorption spectrum, m/z values of mass signals were calibrated with those of internal standards detected in the same scan . Fourth, we searched for pairs of peak groups that had m/z intervals (Δ) of 1.0033 and 1.9958 to identify 12C/13C1 isotopic peak pairs and 32S/34S1 isotopic peak pairs, respectively  and database search results were attached to each metabolite as annotations . Biologically relevant metabolite pairs were screened according to two criteria. First, the Δ[m/z ] value observed between the metabolites must be observed in more than one pair of MS/MS fragments. Second, metabolite pairs must have more than one identical MS/MS fragment. The relative intensity of the MS/MS fragment ions was not taken into account. For example, m/z values of 1372.5 , caffeic acid (162.032), hexose (162.053 and 162.054), malonic acid (86.001) and the amino group (17.027) . The analysis indicated that addition of chemical building blocks such as an amino group, caffeic acid, a C3H7NO2S moiety or hexose occurred frequently during ripening. According to the annotations of putative structure and database hits, these chemical building blocks are frequently associated with secondary metabolism.We then checked whether Δ−) were frequently produced together with [M-H]− ions, and were assigned using the same criteria. Metabolite annotations were provided for the adduct ion species with the highest intensity, i.e. [M+H]+ and [M-H]− in positive- and negative-ionization ESI modes, respectively, for the majority of the metabolites detected in the present study (A program written in Microsoft VCcriteria : (i) thent study . After tm/z values, predicted molecular formula, retention time, MS/MS data and λmax of the UV/visible absorption spectra was compiled. As MS/MS data, the m/z value, raw intensity and relative intensity of the 20 highest-intensity MS/MS fragment ions were retrieved. References for each annotated metabolite were searched for in the public databases PubChem (http://pubchem.ncbi.nlm.nih.gov/), the Dictionary of Natural Product (http://www.chemnetbase.com/scripts/dnpweb.exe?welcome-main), KNApSAcK (http://kanaya.naist.jp/KNApSAcK/), KEGG (http://www.genome.jp/kegg/kegg2.html) and MotoDB (http://appliedbioinformatics.wur.nl/moto/). To browse and search the annotation information, a web-based database (http://webs2.kazusa.or.jp/komics/) was constructed using MySQL and PHP.For database construction, a dataset comprised of accurate max and reference information. In grade C, multiple molecular formulae were assigned to each metabolite. Annotations in grade C were classified into eight sub-grades according to the availability of MS/MS and λmax information.To each metabolite, an annotation grade was added to describe the evidence supporting the annotations for that metabolite . First, m/z ]) were calculated for pairwise combinations of m/z values shown in m/z ] values were calculated separately for m/z datasets of tomato tissue samples and for m/z datasets obtained in positive- and negative-ionization ESI modes. Δ[m/z ] values were calculated in the 500 Da range. To identify Δ[m/z ] values that occurred more frequently than others, a threshold probability was determined based on the standard deviation of the probability distribution within each sample. Probabilities of 10-, 20-, 30-, 40-, 50-, 60- and 70-fold standard deviation levels were tested, and the 40-fold standard deviation level was used as the threshold  the probability was above the 40-fold standard deviation level, (ii) the frequency of the Δ[m/z ] value increased in the later stages, and (iii) the probability of the Δ[m/z ] value increased in the later stages. To obtain the chemical information of KEGG compounds, compound files were first retrieved from the KEGG ftp site , and then compounds containing C, H and O in the molecular formula were selected. Finally, compounds with a non-redundant compound ID were chosen. The theoretical molecular weight of KEGG compounds were calculated using accurate masses of the elements C, H, N, O, P and S. Programs for calculating Δ[m/z ] and elemental composition difference were written in Perl. The program for the selection of KEGG compounds was written in Java.Mass difference values (Δ[hreshold . MS/MS d


Rhipicephalus (Boophilus) microplus, economically impact cattle industry in tropical and subtropical regions of the world. The morphological and genetic differences among R. microplus strains have been documented in the literature, suggesting that biogeographical and ecological separation may have resulted in boophilid ticks from America/Africa and those from Australia being different species. To test the hypothesis of the presence of different boophilid species, herein we performed a series of experiments to characterize the reproductive performance of crosses between R. microplus from Australia, Africa and America and the genetic diversity of strains from Australia, Asia, Africa and America.The cattle tick, The results showed that the crosses between Australian and Argentinean or Mozambican strains of boophilid ticks are infertile while crosses between Argentinean and Mozambican strains are fertile. These results showed that tick strains from Africa (Mozambique) and America (Argentina) are the same species, while ticks from Australia may actually represent a separate species. The genetic analysis of mitochondrial 12S and 16S rDNA and microsatellite loci were not conclusive when taken separately, but provided evidence that Australian tick strains were genetically different from Asian, African and American strains.R. microplus. These species could be redefined as R. microplus  (for American and African strains) and probably the old R. australis Fuller, 1899 , which needs to be redescribed. However, experiments with a larger number of tick strains from different geographic locations are needed to corroborate these results.The results reported herein support the hypothesis that at least two different species share the name  Rhipicephalus (Boophilus) microplus, is distributed in tropical and subtropical regions of the world [R. microplus economically impact cattle industry by reducing weight gain and milk production, and by transmitting pathogens that cause babesiosis (Babesia bovis and B. bigemina) and anaplasmosis  [R. microplus strains have been documented in the literature [R. (B.) microplus strains from South Africa and Australia were sterile. Thus, R. microplus from South Africa and Australia might be regarded as different species [The cattle tick, he world . Infestarginale) ,3. The mterature ,5. Althoterature  argued tterature . These a species .R. microplus strains collected at different locations. It has been [R. microplus strains and among tick strains from Australia, Brazil, Kenya and South Africa. However, a clear divergence in the acetylcholinesterase sequence between Australian and African/Mexican strains of R. microplus has been detected [R. microplus strains is lower than the divergence between these strains and the R. microplus from Australia and Nepal [Contradictory results have been reported after the analysis of different gene loci in has been  found thdetected . The mitnd Nepal .R. microplus, but being actually separate species [R. microplus ticks, herein we performed a series of experiments to characterize the the reproductive performance of crosses between ticks from Australia, Africa and America and the genetic diversity of R. microplus strains from Australia, Asia, Africa and America.These results suggested that biogeographical and ecological separation between boophilid lineages may have resulted in different species of ticks in America/Africa and those from Australia, collectively known as  species . To testR. microplus from Argentina (ARG), Mozambique (MOZ) and Australia   Table . In expen) Table . Each crAll engorged females recovered from each sleeve (each separate cross) were individually weighed and left in an incubator at 25°C and 85% RH. The total egg mass produced by each female was weighed and its hatchability was determined as described . The eggIn experiment II, tick-naïve New Zealand rabbits were infested with larvae from ARG, AUS, and MOZ strains inside a cotton sleeve (20 cm diameter) glued to the shaved dorsum skin of the rabbit. At 12–15 days post-infestation, sleeves were opened and engorged nymphs attached to the skin were manually removed from each sleeve and held in an incubator at 34°C and 95% RH for molting. Emerged adults, less than 24 hours from their ecdysis, were sexed to form the infestation male × female crosses in sleeves glued to the dorsum of a calf. A total of 12 sleeves were mounted on the calf to encompass the nine possible crosses between the three strains, as well as three additional sleeves for virgin control females for each strain (Table 1 larvae) were used to infest a calf for experiment I-F1 ticks, and another calf for experiment II-F1 ticks (one sleeve used for infestation with larvae generated from each separate cross). The resulting engorged females were processed as previously described.In order to verify successful reproductive compatibility between strains, unfed larvae (20–30 days old) obtained from the crosses  were compared by the Student R. microplus from America, Africa, Asia and Oceania and one strain of each R. annulatus and R. decoloratus from Africa were used for DNA extraction and sequencing of mitochondrial 12S and 16S rDNA , R. annulatus, USA (L34311), R. microplus, USA (L34310), R. microplus, strain 9 from Taiwan (AY974232), Dermacentor andersoni, USA (L34299), D. marginatus, Spain (Z97879), D. variabilis, USA (L34300), Haemaphysalis juxtakochi, Uruguay (AY762323), H. leporispalustris, USA (L34309), H. punctata, Spain (Z97880), Hyalomma dromedarii, UK (L34306), H. lusitanicum, Spain (Z97881), Rhipicephalus appendiculatus, UK (L34301), R. bursa, Spain (Z97878), R. pusillus, Spain (Z97883) and R. sanguineus, Spain (Z97884).R. annulatus (U95866), R. annulatus, Italy (AM410573), R. annulatus, Israel (AF133058), R. microplus, Nepal (AF150042), R. microplus, Australia (AF031847), R. decoloratus, Zimbabwe (AF150044), R. decoloratus, Kenya (AF031846), R. kohlsi, Jordan (AF150043), R. kohlsi, origin unknown (AY008686), Dermacentor albipictus, USA (AF150041), D. andersoni, USA (AF150040), D. reticulatus, France (AF150038), Haemaphysalis leachi, Zimbabwe (AF150035), H. punctata, Switzerland (AF150032), Hyalomma dromedarii, Morocco (AF150036), H. truncatum, Zimbabwe (AF150031), Rhipicephalus appendiculatus, Uganda (AF150028), R. bursa, Spain (AF150053), R. evertsi evertsi, Zimbabwe (AF150052), R. pusillus, France (AF150022), R. sanguineus, France (AF150020), and R. turanicus, France (AF150018).Haemaphysalis were treated as out-group.Multiple sequence alignments were done using Clustal W . Phylogestrain 1 × malestrain 2 and femalestrain 2 × malestrain 1 homologous and heterologous crosses were used. For each cross, egg masses from two separate crosses were used for genotyping. Tick DNA was obtained from egg batches , pooled larvae  or pooled whole ticks (India and Indonesia strains). Tick tissues were homogenized in liquid N or with a 1 ml tuberculin syringe with a 25-G needle to extract DNA with Tri Reagent  following manufacturer's recommendations. The purified DNA was dissolved in distilled water and the concentration determined using the NanoDrop 1000 .Microsatellite polymorphism was analyzed in tick strains from Argentina, Australia, Mozambique, India and New Caledonia Table . For ArgDQ001904), BmA06 (DQ001905), BmB12 (DQ001906), BmC03 (DQ001907), BmC07 (DQ001909) and BmD12 (DQ001911) using oligonucleotide primers and PCR cycling conditions described [4, 1X avian myeloblastosis virus (AMV) RT/Thermus flavus (Tfl) reaction buffer, 0.2 mM each deoxynucleoside triphosphate (dNTP), 5 u Tfl DNA polymerase, 0.2 μM of each oligonucleotide primer) employing the Access RT-PCR system . Reactions were performed in an automated DNA thermal cycler . The reaction was terminated after a final extension at 68°C for 5 min. Control reactions were done using the same procedures, but without DNA added to check contamination of the PCR reaction. PCR products were electrophoresed on 1% agarose gels to check the size of amplified fragments by comparison to a DNA molecular weight marker . Fragments were separated using an ABI 3730 automated DNA sequencer  and sized relative to a ROX-labeled internal size standard . The data were analyzed using program Peak Scanner (Applied Biosystems).Polymorphism was analyzed at microsatellite loci BmA12  and absence (0) of the PCR bands for each cross. Based on the binary matrix, similarity matrixes were calculated using Jaccard's and Dice's coefficients ,24. The R. microplus strains  between American and African strains  sequence (98% bootstrap support) whereas the strains from Australia, New Caledonia and Indonesia grouped together in a different cluster  . The strains from Australia, New Caledonia and Indonesia formed another cluster with a relatively high bootstrap support (78%). Finally, a strong support (99%) was found for a third clade containing R. microplus from India and Nepal (AF150043). In this analysis, the R. annulatus 12S rDNA sequences grouped in a separate cluster with a low bootstrap support (65%).The phylogenetic analysis of 16S sequences showed that sequences of an AY97422 sequencan AY97422 sequencR. microplus crosses showed that homologous and heterologous crosses involving AUS had lower similarity coefficients when compared to other homologous and heterologous crosses between MOZ and ARG strains  and America (Argentina) have no biological barrier for reproduction, being therefore the same species. All the reproductive parameters were significantly reduced when the Australian strain was involved in heterologous crosses suggesting the existence of a separate species. The genetic analysis of mitochondrial 12S and 16S rDNA as well as nuclear microsatellite loci sequences further suggested that strains from Africa and South America are conspecific. According to these analyses, ticks from Australia, Indonesia and New Caledonia would be also conspecific, different from the American-African strains. While clustered in a different clade, support for a separate species in India and Nepal was inconclusive. Interestingly, the results of the microsatellite analysis suggested the absence of transmission ratio distortion between paternal and maternal alleles even in the offspring of the AUS crosses with either ARG and/or MOZ strains, reducing the possibility of parental genetic effects to explain strain incompatibility.To test this hypothesis, we conducted experiments to characterize the reproductive performance and genetic diversity of vice versa. Attractiveness and cross-mating between R. microplus and R. annulatus has been reported, resulting in sterile hybrids [It is well known that in the absence of males, Metastriata females increase their feeding period, have smaller repletion weights and oviposit none or only few fertile eggs when compared to females mated with conspecific males . In the  hybrids , similarOne possible drawback of this study is the use of the Australian Yeerongpilly strain, which has been kept in the laboratory for over 60 years . This stR. microplus in general. However, despite the limitations of the study, the results reported herein support the hypothesis that at least two species exist under the name R. microplus. These species could be redefined as R. microplus  (for American and African strains) and probably R. australis Fuller, 1899 . After its description, R. australis was reported in Africa. However, these records were synonymized under R. annulatus. Interestingly, R. annulatus appeared within the monophyletic clade of R. microplus in the 16S mtDNA tree or as a paraphyletic branche in the 12S mtDNA tree suggesting that the denomination of R. annulatus may needs revision. Currently, R. australis is regarded as a synonym of R. microplus. Biological features reported here, including the lack of fertile F1 progeny in heterologous crosses and smaller, significantly different weight of engorged Australian (Yeerongpilly strain) females as well as molecular findings involving different Australian strains are clearly supportive of the hypothesis of a reproductive isolation between Australian and American/African ticks. We can not ascribe the Australian-Indonesian-Caledonian specimens to that species until the finding of its types and further morphological comparisons are conducted. It is also possible that other boophilid tick strains may inhabit small areas of the South Pacific. This issue warrants further studies by conducting detailed morphologic, genetic and reproductive studies with multiple tick strains collected at different geographic locations and cured from their endosymbionts which may affect tick reproductive performance.Although mitochondrial 12S and 16S rDNA phylogenies were constructed with sequences from multiple Australian strains, the studies of the reproductive performance and microsatellite polymorphism were conducted with a single Australian (Yeerongpilly) strain, a fact that should be taken with caution when generalizing these results to Australian R. microplus into Africa took place with animals from India, after the epidemic of Rinderpest in the 19th century. Although it is hypothesized that R. microplus entered into South America along with Indian cattle, information is currently not available to support this hypothesis. However, the introduction of R. microplus in Australia took place with animals from Indonesia (most probably cattle from Java around 1870) [R. microplus, but adequate biological evidence is lacking and molecular evidence presented herein are not conclusive and/or may be the result of allopatric evolution. Detailed morphological, genetic and reproductive studies of tick strains from Africa, America, Australia, Indonesia, New Caledonia, India and Nepal would be necessary before confirming the redescription and possible resurrection of other boophilid tick species. Nevertheless, until further studies are conducted, caution should be taken in extrapolating results from studies on Australian ticks determined as R. microplus. These data have important implications in the field of animal health, because many studies involving ecology and control of cattle ticks performed on Australian tick strains considered as R. microplus may not be directly applicable to tick strains in America or Africa.It is important to note that the introduction of nd 1870)  and thenR. microplus and suggested a reproductive isolation for Australian-Indonesian-Caledonian strains resulting in a different species. These results support the hypothesis that at least two different species have evolved under the name R. microplus. These species must be redefined after careful inspection of type specimens and further analysis of local tick fauna, including studies with several tick strains from each geographic location.The results reported herein provided biological and genetic evidences of allopatric speciation in MBL conducted the tick crosses. MBL and AE-P analyzed the results of tick crosses. VN and AJM obtained new tick sequences. AJM and AAG conducted phylogenetic analyses. VN, CT and JF obtained and analyzed tick microsatellites. MBL, AE-P, AAG, FJ and JF conceived and participated in study design. MBL, AE-P, AAG, AJM, FJ and JF helped to draft the manuscript. All authors read and approved the final manuscript.Microsatellite genotypes of individual crosses. The data provided represent the microsatellite alleles identified in all tick crosses.Click here for file


Caenorhabditis elegans. Two species of parasitic nematodes also utilize the ASH neuron to avoid certain stimuli. We wanted to extend our knowledge of avoidance behaviors by comparing multiple stimuli in a set of free-living nematode species.Avoidance of noxious stimuli is essential for the survival of an animal in its natural habitat. Some avoidance responses require polymodal sensory neurons, which sense a range of diverse stimuli, whereas other stimuli require a unimodal sensory neuron, which senses a single stimulus. Polymodality might have evolved to help animals quickly detect and respond to diverse noxious stimuli. Nematodes inhabit diverse habitats and most nematode nervous systems are composed of a small number of neurons, despite a wide assortment in nematode sizes. Given this observation, we speculated that cellular contribution to stereotyped avoidance behaviors would also be conserved between nematode species. The ASH neuron mediates avoidance of three classes of noxious stimuli in C. elegans, the bilaterally symmetric polymodal ASH neurons detect all three classes of repellant. We identified the putative ASH neurons in different nematode species by their anatomical positions and showed that in all six species ablation of the ASH neurons resulted in an inability to avoid noxious stimuli. However, in the nematode Pristionchus pacificus, the ADL neuron in addition to the ASH neuron contributed to osmosensation. In the species Caenorhabditis sp. 3, only the ASH neuron was required to mediate nose touch avoidance instead of three neurons in C. elegans. These data suggest that different species can increase or decrease the contribution of additional, non-ASH sensory neurons mediating osmosensation and mechanosensation.We used comparative behavioral analysis and laser microsurgery to examine three avoidance behaviors in six diverse species of free-living nematodes. We found that all species tested exhibit avoidance of chemo-, mechano- and osmosensory stimuli. In The overall conservation of ASH mediated polymodal nociception suggests that it is an ancestral evolutionarily stable feature of sensation. However, the finding that contribution from non-ASH sensory neurons mediates polymodal nociception in some nematode species suggests that even in conserved sensory behaviors, the cellular response network is dynamic over evolutionary time, perhaps shaped by adaptation of each species to its environment. Caenorhabditis elegans, which is 1 mm long, has 302 neurons [Ascaris lumbricoides, which grows to be 20 cm long, has 298 neurons [Despite a more than 1000-fold range in the sizes of different nematode species, nematode nervous systems are composed of a small number of neurons: C. elegans, the functions of several of the amphidial neurons and their roles in various behaviors have been characterized in detail [Strongyloides stercoralis) [Haemonchus contortus) [C. elegans and several parasitic nematodes such as H. contortus and Ancylostoma caninum, have shown that the sensory neuroanatomy is remarkably similar between the different species [H. contortus, a passively ingested parasite of sheep, shows the presence of 12 sensory neurons just like in C. elegans [H. contortus and C. elegans is in perfect correlation with the current phylogeny wherein this parasitic nematode is in the same order as C. elegans [S. stercoralis, there is remarkable similarity of the amphid structure with C. elegans [Free-living nematodes use amphids and phasmids as sensory structures to seek food and avoid harmful stimuli ,4. In C.n detail -5. Parascoralis) ,7 or pasntortus) ,9. Elect species ,7,10,11. elegans ,9,11. Th elegans . However elegans ,7,10. GiH. contortus and the dog hookworm A. caninum act as thermosensors just as in C. elegans [S. stercoralis, ablation of the ASE neuron and ASH neuron resulted in loss of attraction to sodium chloride at lower concentrations and avoidance of high salt concentration respectively [C. elegans can serve as a useful model in understanding the role of different sensory neurons in diverse nematode species.Studies by Schad and colleagues on the role of homologous neurons mediating chemotaxis, thermotaxis and development in parasitic nematodes have shown that positionally homologous neurons perform similar functions in these species, suggesting that neuronal function is conserved between free-living and parasitic nematodes ,14,15. I elegans ,18. Evenectively ,20. ThesA. caninum suggested that the sensory repertoire could change in nematodes [C. elegans, the bilaterally symmetric pair of 'polymodal nociceptive' ASH neurons mediates stereotyped avoidance to high osmotic strength, certain odorants, nose touch, acidic pH, heavy metals, and alkaloids [C. elegans [S. stercoralis [Involvement of other amphidial neurons such as the ADL neuron in chemorepulsion to SDS in ematodes . We havelkaloids ,22-25. A elegans ,27 , Ceanorhabditis briggsae (AF16), and the less closely related Caenorhabditis sp. 3 (PS1010) [C. elegans and C. briggsae were isolated from compost and Caenorhabditis sp. 3 (PS1010) was isolated from galleries of palm and sugarcane weevils [Cruznema tripartitum (SB202) is also a member of Eurhabditis but belongs to a different branch than Caenorhabditis [Pristionchus pacificus (PS312) [Panagrellus redivivus (PS2298) was chosen as a representative of the outgroup and has been isolated from sugar-rich environments such as sap of rubber trees and brewery mash [For our comparative analysis, we chose six representative free-living species from different groups of rhabditids with a range of phylogenetic distances to s Figure . Based ois clade . From th(PS1010) ,13. C. e weevils . Cruznemhabditis . From th PS312) ,31, whic2 ,31, wh (PS312) . Panagreery mash   arranged in the shape of a 'V'  [C. elegans, in the presence of food, response to 100% octanol is primarily mediated by the ASH neuron, and in the absence of food ASH, ADL, and AWB neurons are responsible for sensing 100% octanol, suggesting that feeding status of the animal affects response to 1-octanol in C. elegans [P. redivivus responded after ~2–3 seconds of octanol exposure, indicating that these species are similar in sensitivity to C. elegans [P. redivivus showed significantly delayed sensitivity to 1-octanol, responding in an average of 5.9 seconds. The delayed response could be due to the requirement for a higher concentration of the chemical to generate a response. To confirm the identity of the putative ASH neuron identified by position and morphology  ,41 Addit elegans . All the elegans . P. redi Figures , we usedC. tripartitum, after the first few positive trials, the animals failed to respond to nose touch, showing an overall avoidance of < 10%, suggesting that C. tripartitum adapts to the nose touch stimulus faster than C. elegans and the other species tested  did not significantly differ from ASH-ablated animals  . Hence, we believe that the ASH neuron is the primary sensory neuron for mediating osmotic avoidance in all nematode species, but additional sensory neurons, such as ADL neuron in P. pacificus, may contribute to mediating osmotic avoidance.Ablation of the ASH neuron almost completely abolishes osmotic avoidance response in  elegans . In the H neuron . Ablatiopellents . The rese Figure . Ablatios Figure . In P. ps Figure . Ablatio) Figure . AblatioC. elegans, the ADL neuron, along with ASH and ASE neurons, mediates avoidance response to heavy metal ions, but does not play a role on its own in mediating this avoidance [A. caninum, both ASH and ADL neurons are required to mediate avoidance of the detergent sodium dodecyl sulphate [P. pacificus, ablation of ADL neuron resulted in a significant reduction of avoidance response, indicating that this neuron is necessary for mediating avoidance to high osmotic conditions. The role of the ADL neuron during avoidance behaviors in different species thus remains unclear. Perhaps analysis of the genes expressed in the ADL neuron would provide insight into the role of this neuron in the different species.In voidance . In the sulphate . Ablatiosulphate . In P. pWe cannot rule out the possibility that ablation of specific neurons results in functional replacements by other neurons. However, if this occurs, then this aspect of nematode neurobiology varies among cells, behaviors, and species.We hypothesized that nematodes would display habitat-specific sensitivity to the same stimuli rather than phylogeny based sensitivity. Therefore, we tested several different concentrations of the different noxious stimuli (chemosensation and osmosensation), and used a cluster algorithm to generate a behavioral dendrogram for the different avoidance behaviors (see Methods).P. redivivus displayed the least sensitivity to octanol compared with the other species at the different concentrations . s Figure . C. elegl Figure . Also, i' Figure .P. redivivus was the least sensitive to glycerol compared with the other species . On the other hand, we see an increase in the set of sensory neurons mediating osmotic stress with the ADL neuron partially mediating osmotic avoidance in P. pacificus along with the ASH neuron  [C. elegans eat-4(ky5) MT6308 [C. elegans glr-1(n2461) KP4 [C. briggsae (AF16)[Caenorhabditis sp. 3 (PS1010), P. pacificus (PS312) [C. tripartitum (SB202), and P. redivivus (PS2298/MT8872) [All strains were raised at 20°C unless indicated otherwise, using standard methods . Strainsisolate) , C. eleg) MT6308 , C. eleg461) KP4 , C. brigae (AF16), Caenorh (PS312) , C. trip/MT8872) .A stock solution of DiI (2 mg/ml) was prepared in dimethyl formamide and stored at -20°C . Worms fFor all species tested, we used the L1 larva stage for our ablations as described previously . All behFor octanol avoidance sensitivity, we made different dilutions of 100% octanol (0.1%–100%) and each species was tested with each concentration at least on three different days. Data was represented as average avoidance time for each concentration.Similarly, for sensitivity to osmotic avoidance, we tested concentrations ranging from 0.1 M to 4 M glycerol. Data was represented as mean a.i. . The x-axis of the dendrogram indicates relative distance between the different species.The statistical tests employed are described in Additional file JS and PWS designed the experiments, JS and OD performed the experiments, and JS wrote the paper with input from PWS.Supplementary Materials and Methods. Supplemental data including detailed Materials and Methods and Additional figure legends.Click here for fileAdditional Figures S1–4. Additional figures.Click here for file


Geobacter species in a phylogenetic cluster known as subsurface clade 1 are often the predominant microorganisms in subsurface environments in which Fe(III) reduction is the primary electron-accepting process. Geobacter bemidjiensis, a member of this clade, was isolated from hydrocarbon-contaminated subsurface sediments in Bemidji, Minnesota, and is closely related to Geobacter species found to be abundant at other subsurface sites. This study examines whether there are significant differences in the metabolism and physiology of G. bemidjiensis compared to non-subsurface Geobacter species.G. bemidjiensis indicates several differences in metabolism compared to previously sequenced non-subsurface Geobacteraceae, which will be useful for in silico metabolic modeling of subsurface bioremediation processes involving Geobacter species. Pathways can now be predicted for the use of various carbon sources such as propionate by G. bemidjiensis. Additional metabolic capabilities such as carbon dioxide fixation and growth on glucose were predicted from the genome annotation. The presence of different dicarboxylic acid transporters and two oxaloacetate decarboxylases in G. bemidjiensis may explain its ability to grow by disproportionation of fumarate. Although benzoate is the only aromatic compound that G. bemidjiensis is known or predicted to utilize as an electron donor and carbon source, the genome suggests that this species may be able to detoxify other aromatic pollutants without degrading them. Furthermore, G. bemidjiensis is auxotrophic for 4-aminobenzoate, which makes it the first Geobacter species identified as having a vitamin requirement. Several features of the genome indicated that G. bemidjiensis has enhanced abilities to respire, detoxify and avoid oxygen.Annotation of the genome sequence of G. bemidjiensis offers surprising insights into the metabolism and physiology of Geobacteraceae in subsurface environments, compared to non-subsurface Geobacter species, such as the ability to disproportionate fumarate, more efficient oxidation of propionate, enhanced responses to oxygen stress, and dependence on the environment for a vitamin requirement. Therefore, an understanding of the activity of Geobacter species in the subsurface is more likely to benefit from studies of subsurface isolates such as G. bemidjiensis than from the non-subsurface model species studied so far.Overall, the genome sequence of  Geobacter bemidjiensis is a member of the Geobacteraceae, a family of Fe(III)-respiring Deltaproteobacteria that are of interest for their role in cycling of carbon and metals in aquatic sediments and subsurface environments as well as the bioremediation of organic- and metal-contaminated groundwater and the harvesting of electricity from complex organic matter .-42.GeobaG. bemidjiensis , which is 52% identical to the catalytic subunit of sodium-translocating glutaconyl-CoA decarboxylase of Acidaminococcus fermentans [G. bemidjiensis genome, indicating that decarboxylation of glutaconyl-CoA is not coupled to a sodium pump. Although the GcdA protein of A. fermentans on its own is capable of decarboxylating glutaconyl-CoA with free biotin as a cofactor [gcdA in G. bemidjiensis (oadA Gbem_1454) contains two biotin attachment domains, whereas its sodium pump-associated homologs contain only one. The possibility that the two decarboxylases cooperate as a complex deserves investigation.Degradation of 3-hydroxypimelyl-CoA in s Figure  is predireducens . Subsequrmentans . No homoG. bemidjiensis corroborates the observation that it cannot degrade as many aromatic compounds as G. metallireducens [G. bemidjiensis can detoxify some aromatic pollutants without degrading them. A homolog of the broad-specificity aldo-keto reductase YvgN of B. subtilis [G. bemidjiensis may convert chloro- and nitro- derivatives of benzaldehyde to the corresponding benzol derivatives. Although YvgN was previously described as a methylglyoxal reductase [G. bemidjiensis, as a methylglyoxal synthase was not found in G. bemidjiensis, but only in G. lovleyi (Glov_0611).Although the genome of reducens ,42,46, isubtilis  is preseeductase , detoxifG. bemidjiensis is auxotrophic for PABA  30% identical to that of E. coli [G. metallireducens. Furthermore, an arylamine N-acetyltransferase (Gbem_0306) not found in other Geobacteraceae may act in detoxification of aromatic compounds by G. bemidjiensis, and a putative amidohydrolase in the benzoate degradation gene cluster (Gbem_1458 = Gmet_2056) may also be involved in metabolism of aromatic compounds. Further studies of subsurface Geobacter species such as G. bemidjiensis are necessary to characterize their abilities to transform aromatic compounds.The fact that A Figure  indicate E. coli , which iS. lividans [G. bemidjiensis possesses broad-spectrum resistance to mercury in various organic forms. A homolog of this enzyme was not found in any other Geobacter species, but genes that may encode mercuric reductases  exhibit vertical inheritance in the family, indicating that Geobacteraceae may generally have the ability to detoxify inorganic Hg(II) ions, whereas subsurface Geobacter species such as G. bemidjiensis may have acquired additional resistance to organomercuric compounds.The presence of an alkylmercury lyase (Gbem_0319) 36% identical to that of lividans ) suggestG. sulfurreducens and G. metallireducens, the G. bemidjiensis genome contains duplicate transporters, triplicate regulators, and quadruplicate phosphate-binding proteins  transport system for phosphate consists of a periplasmic phosphate-binding protein (PstS), two membrane proteins (PstC and PstA), an ATP-binding protein (PstB) and a regulatory protein (PhoU). Whereas these are encoded by single-copy genes in s Figure . This exmitation .G. bemidjiensis  in contrast with its location on the 5' side of modABC in G. sulfurreducens. The possibility that these expansions and rearrangements are a response by subsurface Geobacter species to molybdate limitation deserves to be investigated.The ABC transport system for molybdate, consisting of a molybdate-binding protein (ModA), membrane protein (ModB) and ATP-binding protein (ModC), has also expanded in s Figure : the modG. sulfurreducens and G. metallireducens are capable of oxygen respiration using a cytochrome 3 caaoxidase complex, which is also found in G. bemidjiensis (Gbem_0042-Gbem_0047). Near this operon in G. bemidjiensis only . The G. bemidjiensis genome contains two pairs of genes encoding cytochrome bd quinol oxidases: one (Gbem_1269-Gbem_1270) is closely related to its counterparts in G. sulfurreducens and G. metallireducens, while the other (Gbem_2016-Gbem_2017) is not. Thus, G. bemidjiensis may have enhanced capabilities for oxygen respiration compared to G. sulfurreducens and G. metallireducens, due to oxygen stress in its subsurface niche, and may have the ability to respond to oxygen anaerotactically through chemotaxis-type signalling.Genome sequencing led to the surprising discovery that y Figure  is a genvulgaris . In addiG. bemidjiensis, suggesting that it may have expanded its ability to sequester molecular oxygen and deliver it to respiratory or detoxifying enzymes, in contrast to G. metallireducens with two hemerythrin homologs; G. sulfurreducens has six hemerythrin homologs. To detoxify reactive oxygen species, G. bemidjiensis possesses a desulfoferrodoxin (Gbem_3292) 60% identical to that of Desulfoarculus baarsii [D. gigas [G. sulfurreducens and G. metallireducens. Phylogenetic analysis (not shown) indicates that although the characterized cytochrome c peroxidase of G. sulfurreducens [G. bemidjiensis (Gbem_0020), this is actually an ortholog of MacA, implicated in Fe(III) reduction [G. metallireducens, there is no catalase in G. bemidjiensis, meaning that no oxygen is produced from detoxification of hydrogen peroxide; detoxification by rubrerythrins produces only water. All Geobacteraceae encode at least one iron-sulfur-oxygen hybrid cluster protein, thought to detoxify an unidentified reactive compound in response to nitric oxide stress [G. bemidjiensis and Geobacter sp. M21 alone have three hybrid cluster protein genes , evidently derived by expansion of a single ancestral gene. G. bemidjiensis also has a quinol-oxidizing nitric oxide reductase (norZ Gbem_3901) 40% identical to that of Cupriavidus necator [G. metallireducens. Overall, the genome annotation indicates that G. bemidjiensis has evolved to cope with many kinds of reactive oxygen species, a finding that should improve models of Geobacter metabolism in the subsurface.Eight hemerythrin family proteins  were predicted from the genome of  baarsii  and a ruD. gigas , in addieduction . As in Ge stress , as welle stress ; G. bemi necator , with a Geobacter metabolism and physiology must include the c-type cytochromes that mediate electron transfer to extracellular electron acceptors such as insoluble Fe(III) oxides. The number of genes encoding c-type cytochromes in the G. bemidjiensis genome is 84, fewer than G. metallireducens (90 genes) and G. sulfurreducens (104 genes), despite the G. bemidjiensis genome being the largest of the three. The distribution of genes with a given number of heme-binding motifs , and the most complex c-type cytochrome in each genome contains 34-37 hemes. However, G. sulfurreducens and G. metallireducens encode more monoheme c-type cytochromes than G. bemidjiensis, and while the mode number of hemes in multiheme c-type cytochromes of G. bemidjiensis is 5 hemes, the other two genomes have two modes with 4 and 8 hemes. Of the c-type cytochrome genes implicated in Fe(III) and U(VI) reduction in G. sulfurreducens, G. bemidjiensis possesses macA (Gbem_0020) [7 cfamily  [omcS family  [omcB family (Gbem_3379) [omcF-related genes  [omcE [G. bemidjiensis was reported not to grow with a graphite electrode as electron acceptor [omcZ (Gbem_3056), which is required for G. sulfurreducens to grow by transferring electrons to an electrode [c-type cytochromes of G. bemidjiensis (49%) have full-length homologs in G. sulfurreducens and/or G. metallireducens  of G. bemidjiensis are absent from G. sulfurreducens and G. metallireducens with one exception . Of the c-type cytochromes of G. bemidjiensis that have one, two or five heme-binding motifs, approximately two-thirds in each category have no match in the other two genomes, whereas of those with three, four or six to twelve heme-binding motifs, at least half in each category are shared. Thus, extensive similarities as well as notable differences in the complement of c-type cytochromes exist between the subsurface species G. bemidjiensis and its non-subsurface relatives.An understanding of subsurface reducens 0 genes aem_0020) ,60,61, tem_3455) , five meem_2680) , one memem_3379) , and twoem_2183) . There i3) [omcE . Althouglectrode . OverallG. bemidjiensis genome . The ancestral flagellin gene has also expanded into triplicates , as has an ancestral GEMM riboswitch-regulated gene encoding a fibronectin type III domain protein  that may be localized to the outer surface of the cell. Interestingly, phylogenetic analysis (not shown) indicates that G. bemidjiensis has also recently triplicated  an ancestral gene related to omcS of G. sulfurreducens, which encodes an outer surface c-type cytochrome that is important for reduction of insoluble Fe(III) [G. bemidjiensis, although the significance of this expansion to life in subsurface environments is unknown.Several genes of pilus biogenesis are present in triplicate in the e Figure , all app Fe(III) . Thus, mG. bemidjiensis genome was examined for features of gene regulation conserved between it and its non-subsurface relatives. Of the six sigma factors of RNA polymerase in G. sulfurreducens, G. bemidjiensis has orthologs of five: RpoD (Gbem_3694), RpoS (Gbem_2683), RpoN (Gbem_0869), RpoH (Gbem_0573), and FliA (Gbem_3764). No homolog of the putative stress response sigma factor RpoE was found. There are also two additional sigma factors (Gbem_1696 and Gbem_3169) unrelated to the unique sigma factor of G. metallireducens.The G. bemidjiensis genome encodes 127 putative sensor histidine kinases containing HATPase_c domains , of which 47 genes (37%) have full-length homologs in G. sulfurreducens and/or G. metallireducens. There are 163 proteins with response receiver (REC) domains  are found in 12% of regions between protein-coding genes in G. bemidjiensis, and multiple sequences are present in 31% of such intergenic regions, indicating that insertion is not random. One nucleotide sequence family was found inserted into protein-coding genes  as well as between genes, as previously observed for a different family in G. metallireducens [Geobacter species than in those of its non-subsurface relatives remain to be elucidated.Riboswitches that have been identified in the non-subsurface species reducens ,67 were reducens . The impG. bemidjiensis reveals many differences from the previously published genomes of non-subsurface Geobacter species. Enzymes that account for the metabolic versatility of G. bemidjiensis were identified, and further metabolic, physiological and genomic peculiarities were discovered, including a more efficient pathway for oxidation of propionate, a pathway of fumarate disproportionation without excretion of acetate, a reductive TCA cycle, utilization of glucose, a defective folate biosynthesis pathway, and enhanced abilities to respond to oxygen stress. This information is of utmost value for an understanding of the activity of Geobacteraceae in subsurface environments undergoing bioremediation accompanied by reduction of Fe(III).The complete genome sequence of G. bemidjiensis Bem(T) [http://www.phrap.com was used for sequence assembly and quality assessment [G. bemidjiensis Bem(T) contains 67990 reads, achieving an average of 11-fold sequence coverage per base with an error rate less than 1 in 100,000.The genome of s Bem(T)  was sequsessment -70. Aftevs. Genbank's non-redundant database (NR of Nov. 2007) at 1e-05 evalue was conducted. Matches to the Geobacter genus were excluded and the alignment of the N-terminus of each gene model vs. the best NR match was used to pick a preferred gene model. The gene/protein set was searched using BLASTP, hmmer, RPS-BLAST and Interpro. BLASTP searches were done vs. Swiss-Prot/TrEMBL, NR, and KEGG databases with a cutoff evalue of 1e-05. Hmmer searches were done vs. Pfam and TIGRfam databases using the trusted cutoff. RPS-BLAST searches against PRIAM used its 1e-30 high-confidence cutoff and searches against COGS used a 1e-10 cutoff. Interpro was run using its default cutoffs. Automated product assignment was made using the following hierarchy of data sources: PRIAM, TIGRFam, Pfam, Interpro profiles, Swiss-Prot/TrEMBL, KEGG, and COG group. tRNAs were annotated using tRNAscan-SE (v1.23). rRNAs were annotated using RNAmmer v 1.1 [rnpB and ssrA genes were located using the Rfam database and Infernal.The protein-coding genes were predicted using Prodigal V1.0 . A BLASTer v 1.1 . The srpG. bemidjiensis and the manually curated genome annotations of G. sulfurreducens and G. metallireducens [G. bemidjiensis genes were emended for consistency with their counterparts in G. sulfurreducens and G. metallireducens. The coordinates of numerous genes were adjusted according to the criteria of full-length alignment, plausible ribosome-binding sites, and minimal overlap between genes on opposite DNA strands. The annotations of G. bemidjiensis genes that were not matched to genes in G. sulfurreducens or G. metallireducens were checked by BLAST searches of NR and the Swiss-Prot database. Functional annotations in all three genomes were updated to match the experimental characterization of highly similar full-length homologs, with extensive reference to the EcoSal online textbook http://www.ecosal.org and the MetaCyc database [The automated genome annotation of reducens  were quereducens  as implereducens  using thdatabase . Genes tPhylogenetic analysis of selected proteins was performed. In each case, the protein sequence of interest was included, along with its relatives, as identified by BLAST , and theG. bemidjiensis was cultured under strictly anaerobic conditions at 30°C in an atmosphere of N2 and CO2 (80%:20%), as previously described for G. sulfurreducens [G. bemidjiensis was tested in rubber-stoppered 26 ml glass tubes containing 10 ml of NBAF medium [To monitor the disproportionation of fumarate,  and CO2 %:20%, asreducens  from whireducens . The 4-aF medium  from whiABC: ATP-binding cassette; ATP: adenosine 5'-triphosphate; CoA: coenzyme A; DNA: deoxyribonucleic acid; FAD: flavin adenine dinucleotide; GTP: guanosine 5'-triphosphate; NAD(H): nicotinamide adenine dinucleotide (reduced); NADP(H): nicotinamide adenine dinucleotide 2'-phosphate (reduced); PABA: 4-aminobenzoate; rRNA: ribosomal RNA; RNA: ribonucleic acid; TCA: tricarboxylic acid; tRNA: transfer RNA; UDP: uridine 5'-diphosphate.CH supervised the genome sequencing, HK performed genome sequence finishing, and ML oversaw the automated annotation process. MA performed manual curation of the genome annotation (assisted by MC and NY) and wrote the manuscript. NY did the phylogenetic analyses. DH, CR and MA conducted physiological experiments. DL conceived of the study and offered guidance with the writing. All authors read, assisted with editing, and approved the final manuscript.Table S1. Genes of G. bemidjiensis in which frameshifts occur in homopolymeric regions.Click here for fileTable S2. Enzymes of acyl-CoA metabolism in G. bemidjiensis.Click here for fileTable S3. Selected sugar interconversion genes of G. bemidjiensis.Click here for fileTable S4. Predicted c-type cytochromes of G. bemidjiensis.Click here for fileTable S5. Signalling proteins of G. bemidjiensis.Click here for fileTable S6. Multicopy nucleotide sequences in G. bemidjiensis.Click here for fileFigure S1. Multicopy nucleotide sequences of the G. bemidjiensis genome: base coordinates and alignments. .Click here for fileFigure S2. Multicopy nucleotide sequences of the G. bemidjiensis genome: base coordinates and alignments. .Click here for fileFigure S3. Multicopy nucleotide sequences of the G. bemidjiensis genome: base coordinates and alignments. .Click here for fileFigure S4. Multicopy nucleotide sequences of the G. bemidjiensis genome: base coordinates and alignments. .Click here for fileFigure S5. Multicopy nucleotide sequences of the G. bemidjiensis genome: base coordinates and alignments. .Click here for fileFigure S6. Multicopy nucleotide sequences of the G. bemidjiensis genome: base coordinates and alignments. .Click here for fileFigure S7. Multicopy nucleotide sequences of the G. bemidjiensis genome: base coordinates and alignments. .Click here for fileFigure S8. Multicopy nucleotide sequences of the G. bemidjiensis genome: base coordinates and alignments. .Click here for fileFigure S9. Multicopy nucleotide sequences of the G. bemidjiensis genome: base coordinates and alignments. .Click here for file


Helicobacter pylori (H. pylori) plays an important role in chronic gastritis, peptic ulcer disease and gastric malignancies, and its eradication has been advocated. The association between H. pylori infection and liver cirrhosis in patients with hepatitis C virus has been documented in different parts of the world; nevertheless, no conclusive data is available in Egypt.Chronic hepatitis C virus (HCV) infection is a leading cause of end-stage liver disease worldwide. It has been shown that H. pylori infection was sought in 90 patients with chronic HCV infection and in 66 HCV-free healthy controls.In the present study, the status of H. pylori positivity was increased significantly (P = 0.03) in the HCV-infected patients when compared to that in healthy controls, where H. pylori infection was found in 50 (55.6%) out of 90 of the HCV-infected patients versus 26 (39.4%) out of 66 of the healthy controls. In HCV-infected patients, the prevalence of H. pylori infection was increased significantly (P = 0.04) from chronic active hepatitis to cirrhosis. H. pylori infection was present in 6/18 (33.3%), 10/21 (47.6%), 16/27 (59.3%), 18/24 (75.0%) patients with chronic active hepatitis, Child-Pugh score A, Child-Pugh score B and Child-Pugh score C, respectively. More importantly, the prevalence of H. pylori infection in HCV-infected patients was increased very significantly (P = 0.003) with increasing Meld (model for end-stage liver disease) score. The prevalence of H. pylori was documented in 9/28 (32.1%) patients with Meld score >10 and in 41/62 (66.1%) patients with Meld score >10.The study showed that the H. pylori prevalence with advancing hepatic lesions, and the eradication treatment may prove beneficial in those patients with chronic hepatitis C.It may be stated that our results collectively reflect a remarkable increase in  Hepatitis C virus (HCV) is the leading cause of chronic liver disease globally and is eH. pylori is recognized as a pathogen of upper gastrointestinal diseases, such as acute and chronic gastritis, duodenal and gastric ulcers[H. pylori has also been closely associated with development of gastric adenocarcinoma.[H. pylori has been reported to induce hepatotoxicity in vitro.[H. pylori and other Helicobacter species. Furthermore, several investigators have reported a high prevalence of H. pylori infection in patients with chronic liver diseases.[H. pylori is generally believed to be sensitive to bile,[H. pylori is detectable in the liver and biliary tract[H. pylori can survive in bile-rich environment.[H. pylori may survive in the liver and biliary tract.ic ulcers5 and mucic ulcers H. pylorarcinoma. H. pylorin vitro. A solubldiseases.10 Althou to bile,–13 severary tract–17 and tironment.–20 TheseH. pylori infection and cirrhosis in patients with hepatitis C virus has been documented in different parts of the world; nevertheless, no conclusive data is available in Egypt. Therefore, the rationale of the present study was to investigate the status of H. pylori infection in HCV-infected patients with and without liver cirrhosis.The association between The present study was conducted on 90 patients from among the out-clinic patients of National Liver Institute (NLI), Minufiya University, Minufiya, Egypt. Sixty-six healthy controls were recruited from among the blood donor clientele of the NLI for comparison purposes. All patients and controls were subjected to thorough history-taking; complete clinical examination; abdominal ultrasound; and laboratory investigations, including total bilirubin (TB), direct bilirubin (DB), total protein (TP), serum albumin (S. Alb), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP) and serum creatinine. Hepatitis B surface antigen (HBsAg), anti-HCV antibodies were detected by ELISA  and RT-PCR for HCV RNA . All patients were strictly positive for anti-HCV and HCV RNA and negative for HBV. On the other hand, subjects of the control group were free of both HCV and HBV.et al.[Liver biopsy was performed for the patient groups only. Two pathologists did the histopathological assessment separately, and then a consensus between them was made on discordant assessments. The two pathologists were not aware of the clinical data, at the time of assessment. Histological grading of hepatic cirrhosis was done as defined by Ishak et al. AccordinThe study protocol respected the most recent Declaration of Helsinki, and all Assessment of severity of liver disease was performed using both Child-Pugh scoring system and the http://depts.washington.edu/uwhep/calculations/meldscore.htm. The Meld score stratified the patients into two categories — the first group with Meld score ≤10; and the second group, >10.Meld score = 0.957 × loge (creatinine mg/dL) + 0.378 × loge (bilirubin mg/dL) + 1.120 × loge (INR) + 0.643. The Meld score can be easily calculated using a web site. The web site that we have used for our calculations is H. pylori antibodies . A positive result in either of the tests was considered as indicative of active H. pylori infection as proved by gastric biopsy positivity. Methodology described by us earlier was followed.[Gastric mucosal biopsies were used for routine rapid urease testing. The patients were tested for serum followed.U test, while Kurskal-Wallis test was performed to compare more than two groups. Nominal data were analyzed using chi-square test, and P values <0.05 were considered statistically significant. Data were tabulated and analyzed using the SPSS 11 statistical package .Quantitative data were expressed as mean ± standard deviation. Comparing of two groups was analyzed by Mann-Whitney H. pylori positivity was increased significantly (P = 0.03) in the HCV-infected patients when compared to that of healthy controls, where H. pylori infection was found in 50 (55.6%) out of 90 of the HCV-infected patients versus 26 (39.4%) out of 66 of the healthy controls. In HCV-infected patients, the prevalence of H. pylori infection was increased significantly (P = 0.04) from chronic active hepatitis to cirrhosis. H. pylori was present in 6/18 (33.3%), 10/21 (47.6%), 16/27 (59.3%), 18/24 (75.0%) patients with chronic active hepatitis, Child-Pugh score A, Child-Pugh score B and Child-Pugh score C, respectively. More importantly, the prevalence of H. pylori infection in HCV-infected patients was increased very significantly (P = 0.003) with increasing Meld score. The prevalence of H. pylori was documented in 9/28 (32.1%) patients with Meld score ≤10 and in 41/62 (66.1%) patients with Meld score >10 [core >10 .P = 0.0001 for each) in the mean values of ALT, AST, prothrombin concentration, serum total bilirubin, serum albumin and platelet count between chronic hepatitis C patients and controls. In HCV patients, the number of patients with elevated ALT, AST and total bilirubin was increased in the H. pylori–positive group of patients when compared with the H. pylori–negative group despite no significance being observed. In the HCV-infected patients, the H. pylori–positive patients were likely to be older in age, but there was no significant difference (P = 0.11). Also, there were more H. pylori–positive male patients than female patients (P = 0.029). In the HCV-infected patients, the concurrent schistosoma infection was documented largely in 38/50 (76%) H. pylori–positive patients versus 12/50 (24%) patients that were free of schistosoma infection (P = 0.010) [There were significant differences (= 0.010) .H. pylori–positive patients [It seems clear that sex being male, age being above 40 years and positive schistosoma infection are risk factors for increase in the severity of liver disease, as revealed by Meld score in patients .H. pylori.[H. pylori infection in our HCV patients was found to increase non significantly with age and significantly with sex being male. These results are comparable with the data reported by Pellicano et al.[H. pylori infection increases with age but is quite different among the various populations. In United States, the prevalence is less than 20% at 20 years of age, and then increases to approximately 50% at 50 years of age.[Approximately 50% of the humanity is infected with . pylori. H. pylorno et al. The prevs of age. In Japans of age. In Koreas of age.H. pylori among cirrhotic subjects.[1030The discovery of the presence of helicobacter species DNA in liver material from patients with liver disease has led to the challenging hypothesis that these bacteria may play a role in the evolution of hepatic lesions, from chronic viral hepatitis to cirrhosis and hepatocellular carcinoma (HCC). From an subjects.103031 DeH. pylori infection was investigated among patients with HCV with chronic active hepatitis and cirrhosis with different staging. The results revealed that this bacterial infection was significantly higher in the patient groups compared with the healthy control group. Moreover, the H. pylori infection was found to increase with the increase in both Child and Meld scores. These results reflect high prevalence of H. pylori infection, and this may explain the frequent occurrence of gastro-duodenal ulcer in cirrhotic patients.[et al.[H. pylori is implicated in the pathogenesis and progression of cirrhosis, particularly in HCV-infected individuals, and the involvement of Helicobacter spp in HCC seems highly possible.In the present study, the patients. Furtherms.[et al. proposedHelicobacter spp could also secrete a liver-specific toxin that causes hepatocyte necrosis in cell culture and might therefore be involved in damaging liver parenchyma in vivo , tumor necrosis factor (TNF) and by the presence of lympho-mono cellular infiltrate and lymphoid follicle formation. Viruses hn et al.)Schistosoma mansoni were repeatedly shown to augment pathogenesis induced by HBV and HCV hepatitis.[H. pylori, and in the absence of treatment, infection would persist throughout life. However, based on seroepidemiological studies in adults and children from both developing and developed countries, it appears that the spontaneous elimination of H. pylori infection may occur.[H. pylori is not definitively known; however, epidemiological studies suggest person-to-person transmission, by either fecal-oral or oral-oral route, to be the major mechanism.[H. pylori.[H. pylori infection,[Hepatitis C virus (HCV) is the major agent in non-A non-B hepatitis with serious complications ranging from chronic inflammatory disease to hepatic cirrhosis and end-stage liver failure or hepatocellular carcinoma. Egypt has unusually high prevalence of hepatitis C, resulting in high morbidity and mortality from liver disease. Approximately 20% of blood donors are seropositive for HCV antibodies. Schistos39epatitis. Coinfectepatitis. It was gay occur. The modeechanism. In devel. pylori. The casenfection, and thesH. pylori–positive patients versus those who were free of schistosoma infection (P = 0.01). Concurrent S. mansoni infection with H. pylori is associated with reduced prevalence of gastric atrophy, a precancerous condition. It was demonstrated that concurrent S. mansoni infection may modify the inflammatory response to gastric H. pylori infection, by the reduction of oxyradical-induced DNA damage, apoptosis, cellular proliferation activity and increase in antioxidant production.[Schistosoma mansoni secretes a chemokine-binding protein with prominent anti-inflammatory activity,[H. pylori infection. The Th2-like response stimulated by helminth infections might modulate the Th1-like immune response induced by H. pylori infection despite chronic inflammation leading to a substantial reduction in mRNA for cytokines associated with a gastric inflammatory response of Th1 cells.[In our HCV patients, the concurrent schistosoma infection was documented largely in oduction. Recentlyactivity, and thish1 cells.H. pylori–positive patients [et al.[H. pylori clearly augment the pathogenesis induced by HCV.[42It seems clear that sex being male, age above 40 years and positive schistosoma infection are risk factors for increase in the severity of liver disease as revealed by Meld score in patients . A compas [et al. Coinfectd by HCV.4250H. pylori is the most interesting result of the current study. However, the major drawback is the failure to address the relation between liver cirrhosis and the presence of other enterohepatic Helicobacter spp., such as H. hepaticus, H. bilis and H. pullorum. Further studies are recommended to explore that relation and to detect the antigenic cross-reactivity between H. pylori and other species as reported earlier.[Revealing a relation between liver cirrhosis in HCV-infected patients and the presence of  earlier. These fuH. pylori prevalence with advancing hepatic lesions, and the eradication treatment may prove beneficial in those patients with chronic hepatitis C. Future studies should be prospectively carried out to investigate the effect of H. pylori treatment on the cirrhotic status of those patients.In conclusion, it may be stated that our results collectively reflect a remarkable increase in the


Cryptosporidium is a protozoan parasite that causes diarrheal illness in a wide range of hosts including humans. Two species, C. parvum and C. hominis are of primary public health relevance. Genome sequences of these two species are available and show only 3-5% sequence divergence. We investigated this sequence variability, which could correspond either to sequence gaps in the published genome sequences or to the presence of species-specific genes. Comparative genomic tools were used to identify putative species-specific genes and a subset of these genes was tested by PCR in a collection of Cryptosporidium clinical isolates and reference strains.C. parvum and C. hominis. PCR product sequence analysis revealed interesting SNPs, the majority of which were species-specific. These genetic loci allowed us to construct a robust and multi-locus analysis. The Neighbour-Joining phylogenetic tree constructed clearly discriminated the previously described lineages of Cryptosporidium species and subtypes.The majority of the putative species-specific genes examined were in fact common to Cryptosporidium sp. are in fact present in multiple species and only appear species specific because of gaps in published genome sequences. Nevertheless SNPs may offer a promising approach to studying the taxonomy of closely related species of Cryptosporidia.Most of the genes identified as being species specific during bioinformatics in  Cryptosporidium species infect humans [C. hominis and C. parvum [Cryptosporidium parvum is zoonotic and infects a wide range of animal hosts including humans, whereas C. hominis is generally restricted to humans [C. hominis and C. parvum is the host range [Cryptosporidium species differ in geographical and temporal distribution and pathogenicity [Cryptosporidium cuniculus  [At least eight t humans ; however. parvum -5. Crypto humans . Therefost range -3. In adgenicity ,8. Diffegenicity ,9. Howevgenicity . These agenicity ,12. Subsi, 1979) .C. parvum and C. hominis has driven a bias in Cryptosporidium research towards these two species. Indeed, the genomes of C. parvum and C. hominis  have been sequenced [C. muris, a less relevant Cryptosporidium species from a public health perspective, is underway [http://CryptoDB.org. The genome sizes for C. parvum and C. hominis are 9.11 and 9.16 Mb, respectively. The GC content is ~ 30% and the coding region is of about 6 Mb [C. hominis than in C. parvum: 3,994 genes versus 3,952 genes. The significance of these 42 missing genes is not clear. The average gene length is comparable between the 2 species: 1.57 kb and 1.72 kb, for C. hominis and C. parvum, respectively. Genome comparison showed that C. hominis and C. parvum are very similar. This high level of sequence similarity limited the ability of comparative genomics to improve annotation, identify conserved non-coding sequence elements and study gene and protein evolution [C. hominis and C. parvum genomes exhibit only 3-5% sequence divergence, with no large insertions, deletions or rearrangements [C. parvum genes not found in C. hominis are proximal to known sequence gaps. However, uncertainty about the amount of sequence variation between C. parvum and C. hominis persists due to the incomplete status of the C. hominis genome. Nevertheless, it has been concluded that the phenotypic differences between C. hominis and C. parvum are caused by polymorphisms in coding regions and differences in gene regulation [C. hominis and C. parvum in the phenotypic differences is now much more accessible for investigation. In fact, these genes may include hitherto valuable epidemiological markers and previously unnoticed genetic determinants of host specificity and virulence. In addition, such markers would also serve as typing targets.The public health relevance of equenced ,15. The underway . The genvolution . In factgulation . The rolC. parvum and C. hominis genomes for incomplete regions and missing genes in order to identify novel genotyping markers. These genes are likely to contribute to the phenotypic differences between C. parvum and C. hominis and therefore might be potential genetic determinants of host tropism.The aim of this study was to survey the published C. hominis and C. parvum, respectively. The majority of C. parvum putative specific genes were annotated, while C. hominis putative specific genes corresponded mainly to hypothetical proteins. Subsequently, the secondary screen decreased the number of the predicted genes to 93 and 211 genes for C. hominis and C. parvum, respectively.Initial screening by Reciprocal Blast and retention of coding sequences showing a level of similarity below 10%  identified 117 and 272 putative species-specific genes for Cryptosporidium clinical isolates and reference strains , only, Cgd2_2430 and Chro.20156 PCR reactions were negative  of primers tested. C. baileyi DNA was not amplified by any of the primers tested .Sequence analysis of these novel genetic loci showed interesting genetic polymorphisms and 78 Single nucleotide polymorphisms (SNP) were detected. These SNPs were detected from a total number of 4150 nucleotides, corresponding to an average of 1 SNP every 53 bp. The number of SNPs was variable for each gene, ranging from 1 SNP every 30 bp for Cryptosporidium. It is noticeable that the genetic differences of C. hominis and C. parvum to C. meleagridis were comparable . This analysis showed a minimal genetic variability between C. hominis and C. parvum (1.72%) .SNP analysis was performed in a pair-wise manner between isolate groups and subtypes using the logical function "IF" of the Microsoft Excel software to discriminate between variables. When the SNPs were identical between the 2 groups, the value "0" was attributed, while if the 2 SNPs were different, the value "1" was assigned and the values summed for each group. The number of base pair differences between the groups is shown in Table COWP  gene were used for Multi-locus Analysis (MLA). All the retrieved sequences allowed comparison of a total 4469 bp. A Neighbour-Joining Tree was generated based on these sequences using MEGA software. The tree showed clear discrimination between C. parvum and C. hominis isolates  phylogenetic method was also tested to construct phylogenetic trees and gave the same topology with similar bootstrap values (data not shown). There was no discrimination between the different isolates belonging to the main species groups, despite distinct gp60 subtypes. However, TU502 strain showed some sequence divergence and was grouped separately within the C. hominis cluster. This is due to the presence of a unique SNP at position 132 on Cgd8_2370 gene, which was confirmed by 3 independent rounds of sequencing. Cryptosporidium meleagridis sequences were included in the MLA and used as an out group. Cryptosporidium meleagridis DNA did amplify 8/10 loci tested, however, for 2 loci (Cgd8_2370 and Chro.50330 genes) the generated sequences were not of high quality and were not used for analysis. Therefore, the differences between this strain and the other isolates were based only on 2853 bp comparisons for 7 genetic loci. The phylogenetic tree with C. meleagridis as the out group also allowed discrimination of Cryptosporidium species and subtypes in a similar manner than the tree presented in Figure Sequences of the ten genetic loci and of the s Figure . Within C. hominis and C. parvum based on published genome sequences. The initial bioinformatics primary and secondary screening allowed the identification of 93 and 211 genes for C. hominis and C. parvum, respectively. This finding is somewhat lower than the number of orthologous gene clusters for C. parvum and C. hominis reported previously in a study of the Apicomplexa [Cryptosporidium clinical isolates and reference strains. PCR screening of the predicted putative species-specific genes showed that the majority of the genes were not as predicted. In fact, 90% of the genes tested were present in both C. hominis and C. parvum isolates. This would suggest caution when using lineage-specific genes for taxonomic analysis at least until published genomes are known to be complete [In this study, comparative genomic tools were used to identify putative species-specific genes for complexa . Initialcomplete .C. hominis TU502 genome is neither completed nor fully assembled, which is consistent with the smaller number of putative C. hominis specific genes as compared to those specific to C. parvum. However, this seems to be in disagreement with the finding that the C. hominis genome has 42 genes more than the C. parvum genome. Nevertheless, it is plausible that the status of the C. hominis genome had hindered the accuracy of the initial comparative genomic analysis because the selected genes may correspond to sequence gaps reported by the authors [C. muris genome. At the time of writing, 8.9 Mb from the C. muris genome have been made available for download from CryptoDB, of which 7.2 Mb corresponding to coding sequences. Based on these newly added genomic sequences, 7/10 (70%) of the selected putative species-specific genes appear to have orthologs in C. muris. This information, if known previously, would have decreased dramatically the number of putative species-specific genes predicted by comparative genomics. Despite this limitation, only one C. parvum and one C. hominis gene were shown experimentally by PCR to be putatively specific, the characterisation of these genes is ongoing.The discrepancy between bioinformatics and PCR is likely to be caused, at least in part, by the fact that the  authors . FurtherC. hominis and C. parvum or whether these primers would also amplify orthologous genes from other Cryptosporidium species by testing DNA from C. andersoni, C. felis, cervine genotype, C. meleagridis and C. baileyi. Cryptosporidium meleagridis DNA amplified using 80% of the primers tested, while, C. andersoni, cervine genotype and C. felis DNA amplified with only 10% of primers. This result is in accordance with the taxonomy and evolution of Cryptosporidium species [C. meleagridis is the closest species to the cluster formed by C. hominis, C. parvum and C. cuniculus based on partial SSU rRNA gene [Cryptosporidium meleagridis DNA did not amplify with primers of Cgd2_2430 and Chro.20156. This could be explained by either nucleotide mismatch in the primer region or that the genes were missing.We considered whether the observed ubiquity of the predicted specific genes represented the closeness between  species . In factRNA gene . CryptosCryptosporidium genomes and was used to examine polymorphism in these regions. PCR product sequence analysis revealed interesting genetic variation as SNPs. In this study, 78 SNPs were detected, 78.3% (61) of which were species-specific. The presence of species-specific SNPs was reported previously from several genetic markers and has been exploited for Cryptosporidium genotyping and subtyping [SSU rRNA [COWP [dihydrofolate reductase (DHFR) gene [thrombospondin related adhesive protein of Cryptosporidium-1 (TRAP-C1) [TRAP-C2 [polythreonine (Poly-T) repeats [and heat shock protein 70 (HSP70) [Cryptosporidium species from various sources. In a similar manner, the newly identified SNPs could be also used for Cryptosporidium genotyping, especially by PCR-RFLP and/or sequencing. The majority of the SNPs detected (64.2%) were synonymous. It has long been assumed that synonymous SNPs are inconsequential as the primary sequence of the protein is preserved. However, it has been demonstrated that synonymous mutations can alter the structure, function and expression level of the protein by affecting messenger RNA splicing, stability, protein folding and structure [C. parvum and C. hominis orthologous protein coding genes. The authors also reported a high number of non-synonymous SNPs in genes involved in host-parasite interactions, mainly genes with transmembrane domains or signal peptides [PCR screening and sequencing of genes found experimentally to be common to both species provided de novo sequence information at incomplete regions of the ubtyping . PCR-RFLSSU rRNA , COWP [2NA [COWP , dihydroFR) gene , thromboTRAP-C1)  and TRAP[TRAP-C2 , polythr repeats and heat  (HSP70)  genes altructure . In additructure  used a gpeptides .C. meleagridis PCR products allowed data enrichment as this species is distant from C. hominis and C. parvum. In fact, among the genes assessed here, C. meleagridis species had 108 additional SNPs, 20 of which are in the Chro.30149 gene. For Chro.30149 gene, C. meleagridis has in average 1 SNP every 15 nucleotide. Surprisingly, all C. meleagridis SNPs are synonymous. Interestingly, no SNP was detected in this gene from C. hominis and C. parvum DNA. Chro.30149 has a predicted function as Ubiquitin ligase. This gene is a housekeeping gene and shows a low level of sequence divergence between species and isolates when compared to contingency genes consistently under environmental pressure and characterized by higher spontaneous mutation rates [The sequence analysis of on rates .Cryptosporidium species and subtypes tested. This analysis showed that the genetic difference between C. hominis and C. parvum was only 1.72%. Within C. parvum group, the anthroponotic subtype isolates showed only 0.12% from the main zoonotic C. parvum isolates. The C. cuniculus isolates exhibited 0.27% genetic differences to C. hominis isolates. In addition, extremely low sequence variability between C. hominis and C. cuniculus was observed using the common genotyping loci [C. cuniculus [The newly identified SNPs were used to determine genetic differences between the main ing loci . Based ouniculus .Cryptosporidium subtypes. Therefore, these genetic loci represent potential powerful targets for Cryptosporidium genotyping and subtyping purposes. Especially since these genes are stable and slow mutating, unlike the currently used Cryptosporidium typing targets .In addition, sequence analysis allowed us to perform a robust and novel MLA. The Neighbour-Joining phylogenetic tree clearly grouped and discriminated with high bootstrap values the previously described lineages of gp60 is hypervariable and under selective pressure as it mediates parasite attachment to host cells [Cryptosporidium typing have been reported. Hunter and colleagues [gp60 might not be a reliable marker of C. parvum and C. hominis population structure. The ten novel loci, described in this study, showed excellent discriminatory power and consistency to assess phylogenetic relationships at the species and infra-species levels. These findings suggest that these loci could be alternative valuable genotyping and subtyping targets for Cryptosporidium. However, their stability should be assessed in an extensive collection of isolates from different subtype families and geographical locations to validate their discriminatory power.Mini and Microsatellites are repetitive versatile DNA repeats known to influence the structure and expression of protein-coding genes and to be responsive to environmental signals ,33. The st cells . In factlleagues  describelleagues  reportedC. parvum and C. hominis species-specific genes. Despite the fact that the majority of the predicted genes were common to both species and some to C. meleagridis, experimental evidence was found for one specific gene for each species. The ten novel genetic loci studied showed an interesting polymorphism. In fact, sequence analysis of PCR products revealed multiple SNPs, the majority of which were species-specific. These SNPs were stable and consistent across Cryptosporidium species and subtypes. These results showed that the ten novel genetic loci can potentially be used to assess the phylogenetic distance and relationships at the species and infra-species level of human infective Cryptosporidium isolates. In addition, the paired SNP analysis was found to be a good strategy to assess the genetic divergence of the isolates tested.In this study, comparative genomics were used to identify putative C. parvum and C. hominis. This is a variant of Blast , originally described by Altschul and colleagues [http://www.ncbi.nlm.nih.gov/blast/blast_overview.shtml. Subsequently, each of the ~ 3900 genes of C. parvum and C. hominis was assigned a similarity score. Only sequences which returned genes with less than 10% sequence similarity from the other genome were considered. These coding sequences are putatively species-specific genes. A secondary screen was performed as follows: each gene was individually tested using Blastn algorithm http://blast.ncbi.nlm.nih.gov/Blast.cgi to confirm specificity and reveal any sequence similarity to genes from other Cryptosporidium species. Furthermore, orthology queries were performed using CryptoDB database. Whenever a gene showed sequence similarity, it was eliminated from the selection. This secondary screen increased the prediction stringency.Reciprocal Blast was used to identify genes with high sequence variability between lleagues  and is ahttp://www.invitrogen.com and supplied by Operon/Eurofins MWG . Table COWP gene [Cryptosporidium Reference Unit (CRU) [Cryptosporidium oocyst wall protein (COWP) gene [C. parvum and C. hominis [SSU rRNA and gp60 genes [Cryptosporidium clinical isolates was tested , 3 C. parvum isolates  and 4 C. parvum anthroponotic subtype isolates . The anthroponotic C. parvum group isolates were previously identified as gp60 subtype family IIc (CRU unpublished data). This subtype family was reported to infect only humans, and was never reported in an animal species [C. cuniculus) isolate from the Northamptonshire outbreak [C. parvum Iowa , C. parvum Moredun  and C. hominis TU502  were tested. Table Cryptosporidium species, therefore, DNA from other Cryptosporidium species and genotypes was kindly donated by CRU and tested; this includes C. andersoni (W13086), C. felis (W14508), cervine genotype (W15916), C. meleagridis (W10509) and C. baileyi (W14184).Amongst the putative species-specific genes, initially 10 genes were selected with preference to annotated genes and tested experimentally by PCR. For each gene, a pair of primers was designed using OligoPerfect™ Designer software OWP gene  were useOWP gene . PCR scrOWP gene  and archit (CRU) . Each isWP) gene  and by r hominis  prior to60 genes ,45. A toed Table . This in species -48. In aoutbreak  and thre® PCR purification Kit . Purified PCR products were sequenced in both directions using PCR primers. We used 2 independent sequencing facilities: the genome lab, John Innes Centre http://www.jicgenomelab.co.uk and the sequencing service at the University of Dundee http://www.dnaseq.co.uk, both using Dye-terminator chemistry technology and Applied Biosystems automated capillary DNA sequencer . Sequences were assembled using CAP3 software http://pbil.univ-lyon1.fr/cap3.php[® application of Vector NTI Advance™ 10 software http://www.Invitrogen.com. Phylogenetic analysis was performed using MEGA  software http://www.megasoftware.net[Positive PCR products were purified using QIAquick/cap3.php and aligtware.net.MB carried out the experimental testing of the predicted putative species-specific genes, sequence alignment and data analysis and drafted the manuscript. KMT conceived the study, provided technical guidance, coordinated the study and helped to draft the manuscript. RC performed the comparative genomic analysis. RMC participated in the design of the study and helped to draft the manuscript. KE carried out DNA extraction from clinical samples and genotyping and subtyping of the isolates at CRU and helped to draft the manuscript. PRH coordinated the study and carried out data analysis and MLA. All authors read and approved the final manuscript.Cryptosporidium clinical isolates and reference strainsAlignment of PCR product sequences of . This file shows the PCR product sequences for the ten novel genetic loci and the COWP gene. The sequences are available online (see result section). The alignment shows the position of each SNP detected. The totality of the SNPs was used for MLA and calculation of genetic differences between Cryptosporidium species and isotypes tested.Click here for file


Subtilisin-like proteases (SLPs) form a superfamily of enzymes that act to degrade protein substrates. In fungi, SLPs can play either a general nutritive role, or may play specific roles in cell metabolism, or as pathogenicity or virulence factors.Epichloë festucae. Phylogenetic analysis indicated that these SLPs belong to four different subtilisin families: proteinase K, kexin, pyrolysin and subtilisin. The pattern of intron loss and gain is consistent with this phylogeny. E. festucae is exceptional in that it contains two kexin-like genes. Phylogenetic analysis in Hypocreales fungi revealed an extensive history of gene loss and duplication.Fifteen different genes encoding SLPs were identified in the genome of the grass endophytic fungus This study provides new insights into the evolution of the SLP superfamily in filamentous fungi. Proteases catalyse the cleavage of polypeptides to oligopeptides or amino acids. In fungi, aspartic, cysteine, metallo-, serine and threonine proteases, as well as uncharacterised classes of proteases, have been identified, which have been classified according to the amino acid residues required for catalytic activity . The serTritirachium album proteinase K -dCTP  labeled probes , 50 μM of each dNTP, 200 nM of each primer, 0.5 U of Taq DNA polymerase (Roche) and 5 ng of genomic DNA. The thermocycler conditions used were: 94°C for 2 min; 30 cycles of 94°C for 30 s, 60°C for 30 s and 72°C for 1 min per kb, followed by a final step at 72°C for 5 min.Standard PCR amplifications of genomic DNA templates were carried out in 25 μL reactions containing 10 mM Tris-HCl, 1.5 mM MgClN. lolii or E. festucae. Products were separated on either an ABI Prism 377 sequencer  or an ABI 3730 analyzer  at the Allan Wilson Centre Genome Service, Massey University, Palmerston North, New Zealand.DNA fragments were sequenced by the dideoxynucleotide chain-termination method  using Bi using the Brookhaven (PDB), SWISSPROT, GenBank (CDS translation), PIR and PRF databases employing algorithms for both local (BLASTX and BLASTP) and global (FASTA) alignments [Fusarium graminearum parameters . There were some instances where SLPs were not annotated in genome sequences. TBlastN analysis, using a conserved region of the peptidase S8 domain as the query sequence, was used to identify all putative SLPs in genome sequences. Where additional SLP genes were identified they were included in the analysis. The presence of signal peptides was analyzed using SignalP3.0 [Sequence data were assembled into contigs with SEQUENCHER  version 4.1 and analyzed and annotated using MacVector 7.2 . Sequence comparisons were performed at the National Center of Biotechnology Information (NCBI) site ignments . PotentignalP3.0 . PolypepgnalP3.0  in MEGA4gnalP3.0 ).. The Newick files were imported into MEGA 4.0 [Phylogenetic analyses were conducted in MEGA4 . The evoMEGA 4.0  to view MEGA 4.0 . The booMEGA 4.0 . BrancheMEGA 4.0  and are Assignment of subtilisin-like proteases to different families was done on the basis of domain structure, similarity to other proteases and grouping in phylogenetic trees. Proteinase K type enzymes have propeptide and peptidase S8 domains. Subfamilies sf1, sf2 and sf3 were previously described . SubfamiN. lolii and E. festucae sequences have been submitted to DDBJ/EMBL/Genbank databases: prtA and prtE (nucleotide accession EU515143/protein accessions ACB30133 and ACB30132), prtB (EF015481/ABK27194), prtC (FJ648718/ACN30265), prtD (EU515141/ACB30128), prtF (EU515139/ACB30123), prtG (FJ648719/ACN30268), prtH (EU515135/ACB30121), prtI (EU515134/ACB30118), prtJ (FJ648720/ACN30270), prtK (EU515134/ACB30119), prtL (EU515136/ACB30120), prtM (FJ648721/ACN30271), kexA (EU515138/ACB30122) and kexB (EU515140/ACB30127).The following E. festucae strain 2368 genome sequence data are available at .The E. festucae . prtI, prtJ, prtK, prtL, prtM and kexA were identified from the genome sequence of this E. festucae strain  genes (prtA and prtB) were identified in a genomic library from N. lolii [Epichloë typhina At1 sequence [prtE, was identified directly upstream of the prtA gene , or degenerate primers designed to conserved SLP sequences   Figure . The kexc25 gene , was alsy Figure .E. festucae SLPs grouped into four of the six different subtilisin families [prtA, B, C, D, E, F, I and J) encoded predicted proteins belonging to the proteinase K family  are predicted to encode proteases from the previously described class I or subtilisin family [E. festucae pyrolysin-like enzymes have this domain structure  domains [A. nidulans, A. niger and mammalian furins, but not S. cerevisiae Kex2p [S. cerevisiae, the propeptide of the KEX2 gene product is removed by autocatalysis, with cleavage on the carboxyl-terminal side of a Lys-Arg site [The predicted y Figure . The E.  domains   [prtL gene product contained many insertions in the peptidase S8 catalytic domain relative to subtilisin Carlsberg, as well as a carboxyl-terminal extension of unknown function that may be required for structural integrity of these enzymes.The predicted s (OSPs)   . Like otprtA, prtB, prtE and kexB gene products validated the FGENESH predictions for these genes. In the proteinase K family, all of the genes except prtJ had a first intron at a conserved position .Intron gains and losses are important in the evolution of gene families ,49. Intrn intron . HoweverprtH, K and M have a first intron at a conserved position  and prtD (vacuolar SLP) genes have highly specialized functions within the cell, which may suggest that conservation of the region around these genes is linked to their conserved function, a hypothesis supported by an analysis of regions of conserved microsynteny between the genomes of Magnaporthe grisea and Neurospora crassa [E. festucae genome [prtG and prtK genes may have.Sequence analysis revealed that four a Figure . These ga crassa  and othee genome ,52. HoweE. festucae compares with those in other fungal species, a comprehensive survey of these genes in fungal genomes was performed. Numbers of predicted SLP-encoding genes varied from a minimum of three (Aspergillus nidulans and Aspergillus oryzae), to a maximum of 32 (Trichoderma virens) . The prtE homologues appear to have been lost from the Trichoderma spp. lineages.In the proteinase K family, a complicated pattern of gene duplication and loss has occurred. Analyses suggested that the common ancestor of the Hypocreales must have contained at least six proteinase K type genes, two of which must have belonged to subfamily 1. In prtB/prtC/prtI group was represented in the Trichoderma spp. as a single gene, but was lost in the Fusarium spp. and N. haematococca. The gene ancestral to prtB/prtC/prtI appeared to have undergone extensive gene duplication in the Clavicipitaceae lineages (E. festucae and M. anisopliae) to produce prtC (Fl1)/pr1G (M. anisopliae), prtB/pr1I, and prtI/pr1A, along with a further duplication to give the M. anisopliae pr1B gene. Pr1A and related enzymes in M. anisopliae are thought to act as virulence factors [prtB, prtC, prtE and prtI genes all sharing two common introns, with a third intron shared by prtB, prtC and prtI.The  factors , which aprtF, prtA and FGSG_03315 (F. graminearum) genes. E. festucae appears to have lost genes from the FGSG_03315 group, which is present in one copy in the T. reesei and T. virens genomes, but in two copies in the N. haematococca and Fusarium spp. genomes, suggesting that gene duplication has taken place in the common ancestor of these species. In the prtF homologues, a single gene is present in all the lineages except the Trichoderma spp., where gene loss appears to have occurred, and F. graminearum, where a gene duplication event appears to have taken place. The prtA homologues may have arisen from duplication of one of the other subfamily 2 genes, as it only contains genes from Fusarium spp., N. haematococca and the Clavicipitaceae fungi, E. festucae and M. anisopliae. While the E. festucae and M. anisopliae genomes contain only a single copy of this gene, two subsequent gene duplications have taken place in the Fusarium spp. and N. haematococca species to give three prtA-like genes for this group.In proteinase K subfamily 2, there also appears to be three ancestral genes, represented by the M. anisopliae genome is the pr1E and pr1F genes, which are located in tandem. Bagga et al [M. anisopliae contained pr1D and pr1J genes, duplication and divergence of a pr1J-like (prtF group) gene giving rise to a pr1F-likegene, which subsequently reduplicated to give the pr1E and pr1F genes within the Metarhizium genus. The pr1E gene appears to have arisen by tandem duplication of the pr1F gene within M. anisopliae, after divergence of the f. spp. anisopliae and acridum [An unusual case in the ga et al  suggest  acridum .prtJ gene is representative of the new subfamily 4, which is present in a single copy in all of the Hypocreales strains except M. anisopliae, where the genome is unsequenced. This gene may be present in the M. anisopliae genome, but was undetected during expression studies used to identify SLPs in this organism. The presence of this gene in the Hypocreales genomes , whereas one of the F. oxysporum genes, FOXG_02695, also appears to have undergone a similar frameshift.The newly proposed subfamily 5, characterized by the CHGG_10086 gene from s Figure . This geM. anisopliae (possibly due to not having the complete genome sequence). This subfamily was previously shown to have undergone extensive gene duplication in the more distantly related M. grisea [prtK-like gene, with multiple copies in the Fusarium spp. and E. festucae.Gene duplication and gene loss was studied in the fungal pyrolysin family. For subfamily 2, a single representative was found in each of the Hypocreales genomes, except . grisea  (AdditioM. anisopliae) have at least one kexin gene . The role of these proteins within the cell is unknown, but potentially the ankyrin repeats, which are involved in protein-protein interactions [A complicating factor in assessing the evolution of the Hypocreales SLP superfamily is the presence of many sequences with lower SLP similarity in p Figure . These sractions , could tE. festucae. Fifteen predicted SLP genes were present in the E. festucae genome, representing four different SLP families. New subfamilies within the proteinase K family were identified, as well as a new family, the oxidatively stable proteases previously thought to be present only in bacteria. Phylogenetic studies showed that many gene duplications and loss events have occurred during evolution of the SLP gene family within the Hypocreales.In this study, we aimed to study the evolution of the SLP gene family in E. festucae Fl1 DNA sequencing, bioinformatics and drafted the manuscript. BS participated in the design and coordination of the study and helped to draft and revise the manuscript. CLS assisted with the phylogenetic analysis and provided the E. festucae E2368 sequence. All authors read and approved the final manuscript.MKB carried out the Table describing list of probes. Probes used for Southern hybridization and E. festucae genomic library screening.Click here for fileTable of primer sequences. Sequences of primers used to amplify prt genes.Click here for fileE. festucae subtilisin-like protease genesTable showing Bioinformatic analysis of . Bioinformatic analysis of E. festucae strain Fl1 subtilisin-like protease genes.Click here for fileEvolutionary relationships of fungal proteinase K family genes based on PhyML analysis. The phylogram  is rooted using the Bacillus subtilis subtilisin Carlsberg protein (accession P00780) as an outgroup. Numbers at branches indicate the percentage of 1000 bootstrap replicates that supported each branch. E. festucae sequences are marked by black circles.Click here for fileEvolutionary relationships of fungal pyrolysin genes based on PhyML analysis. The phylogram  is rooted using the Bacillus subtilis subtilisin Carlsberg protein (accession P00780) as an outgroup. Numbers at branches indicate the percentage of 1000 bootstrap replicates that supported each branch. E. festucae sequences are marked by black circles.Click here for fileEvolutionary relationships of fungal kexin genes based on PhyML analysis. The phylogram  is rooted using the Bacillus subtilis subtilisin Carlsberg protein (accession P00780) as an outgroup. Numbers at branches indicate the percentage of 1000 bootstrap replicates that supported each branch. E. festucae sequences are marked by black circles.Click here for fileEvolutionary relationships of fungal OSP genes based on PhyML analysis. The phylogram  is rooted using the Bacillus subtilis subtilisin Carlsberg protein (accession P00780) as an outgroup. Numbers at branches indicate the percentage of 1000 bootstrap replicates that supported each branch. E. festucae sequences are marked by black circles.Click here for fileTable showing taxonomic distribution of subtilisin like proteases. Taxonomic distribution of subtilisin-like proteases in fungal genomes.Click here for fileTable on distribution of subtilisin like proteases. Distribution of Hypocreales subtilisin-like proteases in known families and subfamilies.Click here for file