Idiopathic epilepsy (IE) is a naturally occurring and significant seizure disorder affecting all dog breeds. Because dog breeds are genetically isolated populations, it is possible that IE is attributable to common founders and is genetically homogenous within breeds. In humans, a number of mutations, the majority of which are genes encoding ion channels, neurotransmitters, or their regulatory subunits, have been discovered to cause rare, specific types of IE. It was hypothesized that there are simple genetic bases for IE in some purebred dog breeds, specifically in Vizslas, English Springer Spaniels (ESS), Greater Swiss Mountain Dogs (GSMD), and Beagles, and that the gene(s) responsible may, in some cases, be the same as those already discovered in humans.Candidate genes known to be involved in human epilepsy, along with selected additional genes in the same gene families that are involved in murine epilepsy or are expressed in neural tissue, were examined in populations of affected and unaffected dogs. Microsatellite markers in close proximity to each candidate gene were genotyped and subjected to two-point linkage in Vizslas, and association analysis in ESS, GSMD and Beagles.Most of these candidate genes were not significantly associated with IE in these four dog breeds, while a few genes remained inconclusive. Other genes not included in this study may still be causing monogenic IE in these breeds or, like many cases of human IE, the disease in dogs may be likewise polygenic. The prevalence of epilepsy in humans is reported to be in the range of 4 - 10/1000 in most study settings . It is nEPM2B  gene.Canine epilepsy is a naturally occurring, spontaneous condition. Canine seizures exhibit a remarkable resemblance to human seizures  and the Modes of inheritance predicted from examination of pedigrees of dogs with IE show evidence for a gene of major effect or autosomal recessive inheritance, both of which could occur due to a founder effect. Examples include the English Springer Spaniel  , the VizLinebreeding and inbreeding have become commonplace to meet the very specific dog breed standards of the hundreds of dog breeds in existence today. These practices, while creating more uniform offspring, can also result in deleterious diseases due to the concentration of recessive mutations passed from the founders. Almost half of the nearly four hundred genetic disorders that have been described in dogs are recognized exclusively in one or a few breeds , presumaThe aim of the present study was to take advantage of the likely founder effect occurring in purebred dogs with IE in order to investigate an underlying genetic basis for IE in four dog breeds: the Vizsla, the English Springer Spaniel (ESS), the Greater Swiss Mountain Dog (GSMD) and the Beagle. A candidate gene approach was utilized; genes known to be involved in inherited human epilepsy, along with two genes involved in mouse epilepsy models and additional related gene family members, were selected. Most candidate genes tested were ion channels. Microsatellite markers within or very close to these genes were used to generate genotypes which were then subjected to either two-point linkage analysis or association chi-square studies. Using four different breeds, which may each have different forms of IE, increased the chances of potentially finding an associated gene in at least one of the breeds.GABRD remains inconclusive in the ESS, despite testing three different microsatellites.Call rates in each sample set for the microsatellite markers that were ultimately used in each analysis were approximately 90% or higher. Heterozygosity was recorded for each marker as a measure of that marker's extent of utility for this study. Average heterozygosity ± standard error of all markers analyzed for Beagles was 0.574 ± 0.024, for Vizslas was 0.569 ± 0.026, for ESS was 0.425 ± 0.030, and for GSMD was 0.341 ± 0.030. As expected, the average heterozygosity varied by breed and markers that were informative in one breed were not necessarily so in another breed. Out of all tested microsatellites, there were greater numbers of low heterozygosity (cut-off of 0.3) markers in the GSMD (27) and ESS (20) compared to the Vizslas (9) and Beagles (6), resulting in the lower average heterozygosities for the GSMD and ESS. For some genes, despite testing several microsatellites, results remain uninformative. For example, CHRNA2 in the Vizslas. However, not all of the other 30 genes were examined in every breed.From the list of 52 candidate genes, 20 are known to be associated with inherited human epilepsy (indicated by a bolded entry with * in the tables) and two are associated with inherited mouse epilepsy models (indicated by a bolded entry with # in the tables). All 20 of the inherited human epilepsy-associated genes and both mouse model genes were analyzed at least once in all four of the test breeds, except for CHRNA2, and both of the mouse model genes were excluded. Four genes  had low heterozygosity or monomorphism for the markers tested, therefore, these genes cannot be excluded. In addition, two genes (CACNB1 and CACNB2) had LOD values falling between -2.0 and 3.2, a grey zone of being neither linked nor unlinked; thus, conclusions cannot be drawn concerning possible linkage of these genes to IE in Vizslas. The most interesting of these was CACNB1, which had a LOD of 0.820 and a heterozygosity of 0.548.In the two-point linkage study, the two large Vizsla families were divided into ten smaller families to decrease complexity and inbreeding loops. The power of this ten family pedigree to detect significant linkage was demonstrated with simulated linkage analysis. The highest average LOD score for simulated linkage was 3.63, obtained by using a simulated marker with four alleles, no heterogeneity, and a recombination frequency of 0.001. The maximum attainable LOD score in these simulations was 8.46, obtained by using a simulated marker with three alleles, 5% heterogeneity, and a recombination frequency of 0.001. All of the simulation combinations obtained average LOD scores above 2.0 (suggestive linkage), demonstrating that the pedigrees are sufficient to detect linkage for a monogenic mutation. Results for the Vizslas are shown in Table GABRD and CHRNA2), had markers with heterozygosities falling below the 0.3 cut-off, prohibiting the drawing of conclusions about these genes. One gene, GABRA1, which is an inherited human epilepsy gene, initially resulted in marginally significant p-values for two different microsatellites: 0.074 (first set) and 0.033 (second set), however, a third microsatellite's alleles resulted in an insignificant p-value of 0.890. Additionally, when the Bonferroni correction was applied, these results were not significant; therefore, no further follow-up was initiated.Results for the English Springer Spaniels are shown in Table GABRD, KCNA1, and ME2), could not be determined due to low heterozygosity. The first marker analyzed for KCND2 resulted in a p-value of 0.036, prompting the examination of two additional markers. One of these was uninformative, but the other had a heterozygosity of 0.729 and a p-value of 0.155, rendering this gene not significant and unlikely to be associated to IE in GSMD. Additionally, CHRNB2, a gene that is associated with human epilepsy, resulted in a p-value of 0.055. While this p-value is potentially suggestive of association, when correcting for multiple testing with the Bonferroni correction, it does not approach significance.Results for the Greater Swiss Mountain Dogs are shown in Table CACNA1D, CACNG3, and KCNQ5) were inconclusive due to low heterozygosity in this breed. CHRNA1 gave a p-value of 0.05 on its initial marker, leading to the development of primers for additional microsatellites. Two of these, CHRNA1's third set and fourth set of primers, gave p-values of 0.072 and 0.056, respectively, but CHRNA1's second set of primers resulted in a highly insignificant p-value of 0.600, making it unlikely that this locus is associated with IE in this breed. Two other loci, both human epilepsy-associated genes, though insignificant, had lower p-values: KCNQ3 at 0.077 and LGI1 at 0.070. Again, applying correction for multiple testing indicates that these p-values are not significant.Results for the Beagles are shown in Table This study was designed to use LD to exploit a possible founder effect suspected to be responsible for IE in these breeds by testing as many candidate genes as possible using microsatellite markers. Microsatellites have been shown to be a practical and useful tool in candidate gene studies . Marker CACNA1A, which was polymorphic enough to be confidently insignificant after analyzing one or two microsatellites in ESS, GSMD, and Beagles, but remained inconclusive through three markers in the Vizslas and ultimately required a fourth microsatellite to achieve a suitable heterozygosity in this breed. The accepted marker heterozygosity of 0.3 is perhaps low, especially when analyzing only a single microsatellite for each candidate gene, however, for many of the insignificant microsatellite results in all four breeds the heterozygosity was > 0.5.Overall marker informativeness was highly influenced by sample population. The Vizslas and Beagles had excellent average heterozygosities , while the ESS and GSMD were much lower . This may be due, in part, to the sample cohorts: the ESS and GSMD were set up as discordant full- or half-sibling pairs, whereas the Vizslas were constructed as large pedigrees, and the Beagles were case/control matched pairs that did not share common ancestors to at least the grandparent level. Reduced marker heterozygosity across breeds could also be influenced by a higher degree of inbreeding within the ESS and GSMD sample cohorts in this study, and the lower average heterozygosities of these two breeds may suggest a founder effect that could eventually help uncover associations. However, marker informativeness is not entirely dependent on sample population, as evidenced by ARX, CACNA1A, CACNA1H, CACNB4, CHRNA4, CHRNB2, CLCN2, GABRA1, GABRG2, KCNQ2, KCNQ3, LGI1, NHLRC1, SCN1A, SCN1B, and SCN2A) and both of the mouse model genes (CACNA2D2 and CACNG2) were either excluded (with linkage) or demonstrated insignificant association to IE in all four of the breed cohorts. Another two of them (KCNA1 and ME2) were insignificant in three out of four breed cohorts, remaining inconclusive only in the GSMD. One gene, GABRD, was inconclusive in two breeds (ESS and GSMD), and CHRNA2 was inconclusive in ESS and was not tested in Vizslas. Insignificant association to IE was demonstrated in most of the additional candidate genes tested. For every breed, however, there were a handful of markers that were inconclusive, due to low heterozygosity.Ultimately, 16 of the 20 human epilepsy-associated candidate genes  that were not excluded by follow-up markers, including It is possible that the populations tested were underpowered to detect association if it existed. Utilizing a sibling-pair case-control design could decrease power because the control dog may also carry the risk allele. However, this situation is difficult to avoid in highly inbred dog populations, and the sibling-pair case-control design should aid in avoiding population stratification, which can create false positive results. Although sample size requirements for canine association studies have not been precisely defined, based on average linkage disequilibrium estimates in dog breeds, a starting point of approximately 25 cases and 25 controls appears to be adequate to find statistical significance for a completely penetrant recessive trait . The numIn the linkage analysis of Vizsla pedigrees, it is possible to conclude that these candidate gene loci are truly excluded, due to convincingly negative LOD scores at zero centiMorgans. However, if the inheritance and age dependent penetrance assumptions for the Vizsla model are incorrect, then the present linkage study is flawed. These input assumptions were based on previous study of IE in this breed  and on tIt is possible that canine IE, like the majority of human IE, is a genetically complex disease in most breeds and that multiple loci contribute to susceptibility in any given breed. The present study would very likely have detected a major contributing locus, and it is unlikely that a truly causative locus is being excluded as a false negative. In humans, the vast majority of IE remains genetically unexplained and is considered to be polygenic -39. A reUltimately, canine IE found to be significantly associated with DNA markers or with a mutation in a specific gene would allow genetic tests to be developed to assist dog breeders with decreasing the incidence of this disease. This would be most effective for monogenic, highly penetrant forms of IE, but if the disease proves to be genetically complex, with multiple genes contributing and less than 100% penetrance, then such a test could still possibly be used to provide a relative risk for IE development within a breed. In any event, discovery of IE associated gene loci in dogs may not only improve the understanding of canine health, but could advance the study of neurobiology and human health as well.The present study did not identify any obvious association or linkage between IE and the microsatellite markers tested for the various candidate genes in any of the four dog breeds. It is possible that IE may in fact be a polygenic disorder even in highly inbred dog breeds, which underscores the challenging nature of investigating inherited epilepsy. Eventually identifying the genetic causes for IE in dogs would have a significant impact on canine health, as well as providing a useful model for IE in humans.Since seizure activity can result from numerous discernable abnormalities, and only dogs very likely to be affected with IE were to be included in the study, the case definition was designed to eliminate, as much as possible, all other likely causes of seizures. Therefore, in order to qualify for "case" status for IE, each affected dog must have had two or more seizures with clinical normalcy between episodes, a veterinarian-administered normal physical examination and neurological examination, as well as normal bloodwork, including a complete blood count and blood chemistry profile, and, when possible, a bile acids assay. Since IE has been shown to be statistically much more probable when the dog is aged 1 to 5 years at the time of its first seizure , only doDogs in these studies were privately owned and voluntarily enrolled by their owners with informed consent. Cases were solicited through breed publications, presentations at local and national breed club meetings, and word of mouth. For each enrolled dog, a general health survey, a signed owner consent form, and up to ten mLs of whole blood in an EDTA tube were required. Blood samples were obtained via venipuncture either at the dog's regular veterinarian or at specially-organized blood-draws at breed shows across the USA. This study was approved by the University of Minnesota IACUC.Owners of dogs affected with seizures were asked to fill out an additional questionnaire which asked for information about the dog's seizure characteristics, preictal and postictal signs, previous medical history, and any anti-epileptic drug therapy; these were submitted by phone, fax, postal mail or e-mail. Any additional follow-up questions were conducted via telephone interview. Finally, medical records for each affected dog were obtained from the dog's regular veterinarian and any specialists (if applicable); these were inspected for bloodwork results and the veterinarian's physical and neurological examination findings. Dogs were assigned case, control, or unknown status after careful evaluation of this information. "Unknown" status was reserved for those dogs displaying suspicious seizure or seizure-like activity, but couldn't be classified as a case for various reasons; for example, if the dog had only one recorded seizure or had suffered head trauma prior to onset of the first seizure.DNA was isolated from whole blood following standard protocols, utilizing the Puregene DNA Isolation Kit from Qiagen Inc. .Cohorts of affected and unaffected dogs for genotyping were assembled for each of the four test breeds - the Vizsla, the GSMD, the ESS, and the Beagle. Ultimately, the available samples determined what type of genetic analysis was conducted for each breed.For Vizslas, the depth of the available DNA samples from multiple siblings, parents, and many grandparents allowed the development of multi-generation pedigrees Figure  suitableFor the GSMD, discordant full-sibling or half-sibling pairs of affected (n = 24) and unaffected (n = 24) dogs were assembled and used in association studies. Siblings were used to help avoid spurious association from population stratification. In a few instances, two different pairs of siblings were related to each other, such as sharing one parent.Similar to the GSMD, the ESS were assembled into discordant full- or half-sibling pairs of affected (n = 24) and unaffected (n = 24) dogs and were used in association studies. As in the GSMD, occasionally the pairs of siblings were also related to one another; this was impossible to avoid due to the inbred nature of the breed.Association studies were also conducted for the Beagles. Due to lack of available sibling pairs, pedigree analyses were used to ensure that only dogs with no common grandparents were utilized. This strategy has previously shown to be adequate for canine genetic mutation detection . Twenty-KCNA1, KCNQ2, and KCNQ3; calcium channels CACNA1A, CACNA1H, and CACNB4; sodium channels SCN1A, SCN2A, and SCN1B; GABA receptors GABRA1, GABRD, and GABRG2; acetylcholine receptors CHRNA2, CHRNA4, and CHRNB2; and chloride channel CLCN2; as well as novel  genes malic enzyme 2 (ME2), leucine-rich glioma inactivated 1 (LGI1), NHL repeat containing 1 (NHLRC1), and aristaless related homeobox (ARX), for a total of 20 human epilepsy-associated genes [CACNA2D2, (the ducky mouse) [CACNG2 (the stargazer mouse) [A non-exhaustive list of candidate genes known to be involved with inherited human epilepsy was assembled, including potassium channels ed genes ,48-54. Ty mouse)  and CACNr mouse) .CACNA1B, CACNA1D, CACNA1E, CACNA1F, CACNA1G, CACNA1I, CACNB1, CACNB2, CACNB3, CACNG3, CACNG4, and CACNG6; sodium channels SCN3A, SCN3B, SCN8A, and SCN11A; GABA receptors GABRA2, and GABRA6; acetylcholine receptors CHRNA1, CHRNA3, CHRNA5, CHRNA9, CHRNA10, CHRNB1, and CHRND as well as CHRNA7, which has had suggested linkage with human IE [KCND2, KCNQ1, and KCNQ5. Finally, the dynamin-1 gene (DNM1) was also examined. This gene was recently found to be mutated in the canine disease termed exercise induced collapse [In addition to this core set of genes, further genes were selected from the same gene families based on their presence in neural tissue, including: calcium channels human IE ; and potcollapse  and a mucollapse . TherefoCHRNA4 and KCNQ2 were close enough to one another in the canine genome that one marker was used for both genes.Human mRNA sequence was obtained for each gene from the National Center for Biotechnology Information . This seTaq DNA polymerase , with dH2O to final volume. PCR cycling conditions were 95°C for 20 minutes, then 40 cycles of the following: 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 30 to 60 seconds (depending on product size), and a final extension of 72°C for 15 minutes. PCR products were prepared for capillary gel electrophoresis according to manufacturer's recommendations and size separated using the Beckman CEQ 8000 automated DNA-fragment analyzer with fluorescence detection . Genotype data were analyzed with instrument software, and alleles were manually verified.Each microsatellite was PCR amplified in 15-μL reactions including 12.5 ng canine DNA, 10X PCR Buffer, 2.5 pmol of the forward primer and 0.75 pmol of the reverse-tailed primer, 0.78 pmol of a fluorescent dye-labeled primer complementary to the tail sequence, 20 μM dNTP mix, and 0.5 units of H = 1 - Σip2, where i pis the population frequency of the ith allele, and H is the probability that a random individual is heterozygous for any two alleles at a gene locus with allele frequencies, i p[Marker heterozygosity was calculated using the following formula: cies, pi . A cut-ocies, pi .In order to determine the power of the ten Vizsla family pedigrees to detect linkage, simulated linkage was performed with the FASTSLINK program -67. MultParametric two-point linkage analysis was utilized for Vizsla marker data, with significant evidence for linkage a LOD (logarithm of odds) score of 3.2 or greater . A LOD sFor the GSMD, ESS, and Beagles, the allele frequencies were subjected to a nonparametric chi-square test for independence  . AllelesKJE participated in conceiving the study, carried out the methods including choosing microsatellites, designing primers, genotyping, statistical analyses, and drafted the manuscript. EEP conceived the study and study design, and coordinated sample accession. KMM participated in carrying out methods including constructing pedigrees and genotyping, as well as sample accession. JRM conceived the study and study design and contributed significant manuscript review. All authors read and approved the final manuscript.Primer sequences for candidate gene microsatellites. Sequence information and product sizes are presented for primers designed for microsatellites examined for each candidate gene. Bolded genes marked with a * indicate those associated with human epilepsy, and bolded genes marked with a # indicate those associated with mouse models of epilepsy. Two of the DNM1 marker primer pairs, DNM1 Third Set and DNM1 Fifth Set, are taken from the University of California-Davis Canine Genetic Linkage Map [kage Map , and areClick here for fileCandidate gene locations and primer locations. All pertinent canine chromosomal location information is presented for each gene and all microsatellites examined for that gene. Bolded genes marked with a * indicate those associated with human epilepsy, and bolded genes marked with a # indicate those associated with mouse models of epilepsy. Two of the DNM1 marker primer pairs, DNM1 Third Set and DNM1 Fifth Set, are taken from the University of California-Davis Canine Genetic Linkage Map [kage Map , and arekage Map  and the kage Map . CFA = cClick here for file


In this study, we examine the interactive effects of elevated atmospheric CO2 concentrations , temperature  and invertebrate biodiversity on MPB biomass in experimental systems. We assembled communities of three common grazing invertebrates  in monoculture and in all possible multispecies combinations. This experimental design specifically addresses interactions between the selected climate change variables and any ecological consequences caused by changes in species composition or richness.Understanding the effects of anthropogenically-driven changes in global temperature, atmospheric carbon dioxide and biodiversity on the functionality of marine ecosystems is crucial for predicting and managing the associated impacts. Coastal ecosystems are important sources of carbon (primary production) to shelf waters and play a vital role in global nutrient cycling. These systems are especially vulnerable to the effects of human activities and will be the first areas impacted by rising sea levels. Within these coastal ecosystems, microalgal assemblages (microphytobenthos: MPB) are vital for autochthonous carbon fixation. The level of 2 concentration, temperature and invertebrate diversity were not additive, rather they interacted to determine MPB biomass, and overall this effect was negative. Diversity effects were underpinned by strong species composition effects, illustrating the importance of individual species identity.The effects of elevated COOverall, our findings suggest that in natural systems, the complex interactions between changing environmental conditions and any associated changes in invertebrate assemblage structure are likely to reduce MPB biomass. Furthermore, these effects would be sufficient to affect the net metabolic balance of the coastal ecosystem, with important implications for system ecology and sustainable exploitation. The infosystems  is of palevels 27 ppmv to pmv yr-1  and the pmv yr-1 . Studiespmv yr-1 ,10. Thispmv yr-1  across mpmv yr-1  and effeEmpirical research to date has concentrated on the responses of a variety of ecosystems to individual anthropogenic drivers of change . This distinction has been widely applied in the study of lotic systems  within the context of a range of biodiversity levels  on the biomass of MPB using a model multi-trophic level [In the face of growing concern about climate change, the net carbon status of many coastal and estuarine systems has received increasing attention . Net all concept ) and has concept ,31. Rela concept  having p concept , and enh concept ,35. Our  concept -38 and hic level  experime3).Surface sediment (< 2 cm depth) was collected from tidal mud flats on the Ythan Estuary, Aberdeenshire, Scotland, UK  and sieved (500 μm) in a seawater bath  to remove macrofauna. The sediment was left to settle for 48 h before the supernatant was removed, the sediment homogenised and placed in the mesocosms to a depth of 10 cm  between mesocosms prior to the addition of seawater.To standardize the biomass of the MPB, MPB-rich surface sediment was collected from the Ythan estuary, spread onto a shallow tray (< 1 cm depth) and left under constant light for 48 h. This material was then homogenised and distributed (125 cmHediste (Nereis) diversicolor (HD), the gastropod Hydrobia ulvae (HU) and the amphipod Corophium volutator (CV) were collected from the study site. These species represent a range of functional types in the way that they bioturbate sediments, and hence drive nutrient flux [The polychaete ent flux ,41, and ent flux .Replicate (n = 3) macrofaunal communities were assembled in single and multispecies treatments . These unique species permutations eliminate pseudoreplication  and allo3 of MPB- rich sediment and 2.35 l of seawater  were added to give an overlying depth of 20 cm. This initial fill of water was replaced after 24 h to remove the nutrient pulse associated with assembly [2 atmospheric level within the chamber. A total of 216 mesocosms were required.Mesocosms were Perspex cores 33 cm high with an internal diameter of 10 cm. Following the addition of sediment (10 cm deep) to each mesocosm, 125 cmassembly  and macr-2 s-1). Nine environmental regimes were employed, using three constant temperatures  with a digital controller . An Infra-Red Gas Analyser  was used to calibrate and validate the CO2 regulation (± 30 ppm). Mesocosms within an environmental chamber were randomly assigned positions to factor out any effects of spatial heterogenity. Each experiment was run for 7 days.Mesocosms were placed in two environmental chambers  with temperature control (± 0.1°C). The experiments were run with a 12 h light - 12 h dark (L/D) cycle using high intensity discharge sodium lamps  of MPB [0-15 . This indicates that the minimum fluorescence was determined after 15 minutes of dark adaptation [The biomass of the MPB was measured at the end of the experiment using a PAM fluorometer . This is a widely accepted proxy method for measuring the surfice biomass (chlorophyll ) of MPB ,46. Fluoaptation -49. Thisaptation  by cell 2 concentration and temperature as nominal explanatory variables. Initially, a linear regression model was fitted and assessed for normality (Q-Q plots), homogeneity of variance, and outlying values (Cook's distance) [MPB biomass was treated as a response variable, with macrofaunal species richness (or species combination), COistance) ,51. As oistance) , and thiistance) -52 mixedistance) ,42,53. Tistance) . The infistance)  in the 'istance) .2 × temperature; L-ratio = 18.23, d.f. = 12, p = 0.0011). Of the two climatic variables, temperature  was more influential than CO2 . There was an apparent decline in MPB biomass with increasing temperature , with MPB biomass as the dependent variable, included a two-way interaction . Species richness was the most influential variable , followed by temperature , and CO2 concentration . The effect of species richness on MPB biomass was most apparent at 6°C . Species composition was the most influential variable , followed by temperature , and CO2 . Increased species diversity had a detrimental effect on MPB biomass, but the magnitude of these effects was dependent on the composition of the invertebrate assemblage within each treatment  available [C. volutator) illustrates how the extinction of influential (rare or common) species may have profound effects on the ecosystem - and that these effects may be direct or indirect [Individual species responses to climate change are often highly uncertain , and envvailable . Therefovailable ,11,20. Iindirect ,68.Many studies focusing on the ecological consequences of altered biodiversity within the context of specific drivers of environmental change have concentrated on single variables and few species ,64,65,69The interactive effects between climate drivers and other anthropogenic factors, such as pollution, habitat destruction and overfishing ,69,71 adNH carried out the experimental procedures, coordinated the study, performed the statistical analysis and drafted the manuscript. MTB assisted in all experimental procedures, provided statistical support and helped draft the manuscript. MS and DMP participated in the design and coordination of the study, and assisted interpretation of statistical analysis. DR and PCLW conceived initial ideas for the study. All authors read, edited and approved the final manuscript.Supporting Information. •Additional graphs of raw data, summary of study site temperatures, final models in full, figures referred to in the text as Figures S, schematics of experimental set up and design. •Supporting material for the data presented in the main body of the paper, including annual temperature graph of study site, supplementary figures referred to in the text, box plots of raw data, bootstrapping of models, and full final models.Click here for file


Bryophyte distribution is primarily associated with the depth of litter, the air temperature and the precipitation. Further studies are necessary to include other epiphytes types and vascular vegetation in a larger altitudinal range.An investigation of terrestrial bryophyte species diversity and community structure along an altitudinal gradient from 2,001 to 4,221 m a.s.l. in Gongga Mountain in Sichuan, China was carried out in June 2010. Factors which might affect bryophyte species composition and diversity, including climate, elevation, slope, depth of litter, vegetation type, soil pH and soil Eh, were examined to understand the altitudinal feature of bryophyte distribution. A total of 14 representative elevations were chosen along an altitudinal gradient, with study sites at each elevation chosen according to habitat type  and accessibility. At each elevation, three 100 m × 2 m transects that are 50 m apart were set along the contour line, and three 50 cm × 50 cm quadrats were set along each transect at an interval of 30 m. Species diversity, cover, biomass, and thickness of terrestrial bryophytes were examined. A total of 165 species, including 42 liverworts and 123 mosses, are recorded in Gongga mountain. Ground bryophyte species richness does not show any clear elevation trend. The terrestrial bryophyte cover increases with elevation. The terrestrial bryophyte biomass and thickness display a clear humped relationship with the elevation, with the maximum around 3,758 m. At this altitude, biomass is 700.3 g m Bryophytes are an important component of ecosystem biodiversity and make up a significant part of species richness Several altitudinal patterns of bryophyte richness and distribution have been reported, such as decreasing Much research has been conducted to find out the main factors influencing bryophyte richness and distribution pattern. Pharo and Beattie Views about the factors mainly influencing bryophyte growth and distribution are still debated. The ecological mechanisms of bryophyte richness and distribution pattern along altitudinal gradients still need to be further investigated. In addition, the biomass, thickness and cover of bryophytes are key indices reflecting bryophyte growth status. Such data are useful for modeling whole ecosystem response to climate change, modeling ecosystem carbon and nutrient cycling, and improving our understanding of the ecological roles of bryophytes in forest ecosystem. However, most of the previous research did not consider the biomass, thickness and cover of bryophytes.The objectives of the present study were (1) to describe the distribution pattern of terrestrial bryophytes along an altitudinal gradient in Gongga Mountain, Sichuan, China, and (2) to find out the major factors which influence bryophyte diversity and distribution. This investigation will be helpful in identifying strategies and opportunities for the conservation of bryophyte species, and would also be the basis of climate change research in this area. We hypothesized that climate is the main factor that influences bryophyte distribution.The study was conducted in Gongga Mountain , which is located in Sichuan, southwestern China, at the eastern edge of the Tibetan Plateau. The peak of Gongga Mountain is 7556 m a.s.l., the summit in the Hengduan Mountain Range. Within a horizontal distance of 28 km the relative height drops 6,400 m to the eastern bottom of Gongga Mountain. On the eastern slope of Gongga Mountain, the annual precipitation varies between 1,068 and 3,210 mm , the mean annual temperature is between −2.6 and 14.5°C depending on the altitude. The mountain has an intact vertical zonality from subtropical vegetation to alpine cold vegetation In June 2010, bryophytes were collected along an altitudinal transect between 2,001 and 4,221 m a.s.l. All permits required to carry out the field studies were obtained from the Natural Park authorities. A total of 14 sites at representative elevations were chosen along the altitudinal gradient, with each site chosen according to habitat type  and accessibility . At eachA screen with 400 grids (2.5 cm × 2.5 cm) was then placed on each quadrat. The percentage cover of the whole ground floor bryophytes was recorded based on the number and space of grids occupied with bryophytes 2R = 0.9677, P<0.01), RH , precipitation , and soil temperature  were calculated using the yearly climatic data of the seven meteorological stations.Climate indices, including air temperature, relative humidity (RH), precipitation, soil temperature, and soil moisture in each sampling elevation, were obtained from meteorological stations set up by the Alpine Ecosystem Observation and Experiment Station of Gongga Mountain. There are seven meteorological stations along the altitudinal gradient. For sites without a meteorological station, the climatic conditions were estimated after adjusting the meteorological data from the closest meteorological station to consider the effect of altitude. To this end, air temperature gradient . The environmental and climatic factors along the altitudinal gradient were presented in Vegetation type, canopy height and closure, aspect, slope, and the geographic position were investigated in each transect. Canopy height was determined by measuring the height of one representative tree in each transect using a ruler and a tape measure For analyses of species richness we used the total species number per transect (n = 42). Coverage and biomass values were averaged in each transect and elevation.S-plus 8.0 statistical software  All environmental variables measured on the quadrat scale were averaged in each transect. The generalized additive models (GAMs), a widely used biological nonparametric generalization of multiple linear regression, were used to describe responses of species richness, coverage, biomass and thickness of bryophyte to elevation change. In the GAMs, a link function is related to predictor variables by scatterplot smoothers instead of least-squares fits, and is subject to less restrictive distributional assumptions than multiple linear regression 695 specimens were collected across all transects and identified to 165 species level, including 42 liverworts and 123 mosses, representing 64 genera within 30 bryophyte families . The groR  = 0.605, P  = 0.01). Elevation trend of species richness is highly curvature as the elevation varies from 2,001 m to 4,221 m a.s.l.   . The bry−2 around 3,758 m a.s.l. (−2) between 2,001 and 2,784 m a.s.l., where the ecotones are evergreen broad-leaved forest, mixed evergreen and deciduous broad-leaved forest, and mixed broadleaf-conifer forest Click here for additional data file.


The aim of this study was to evaluate the discriminant capability of the patient acceptable symptom state (PASS) according to disease activity, in a cohort of Italian patients affected by systemic Lupus erythematosus (SLE).Consecutive SLE patients were enrolled. At each visit, the patients underwent a complete physical examination and the clinical/laboratory data were collected in a standardized, computerized, and electronically-filled form. The evaluation of serum complement C3 and C4 levels and determination of autoantibodies was obtained. Disease activity was assessed with the SLEDAI-2K and ECLAM, while chronic damage was measured with the SLICC. Finally, PASS was assessed in all patients by asking to answer yes or no to a single question.One hundred sixty-five patients were enrolled . No patients refused to answer, suggesting the acceptability of PASS. A total of 80% of patients rated their state as acceptable. The patients with an acceptable status had significantly lower mean SLEDAI-2K and ECLAM scores than the others . No significant differences were observed when considering chronic damage, evaluated with SLICC.In the clinical practice, SLE patients assessment performed by using complex disease activity indices such as SLEDAI-2K and ECLAM, could be time consuming. In our study, for the first time, we used PASS, a quick and easily comprehensible tool, to evaluate the patients’ status, this single question seems to be able to discriminate patients with different disease activity, especially when this is determined by musculoskeletal involvement. Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease, involving genetic and environmental factors, characterized by a wide range of autoantibodies and clinical manifestations –10. MoniThrough the years, many indices have been developed and validated to measure disease activity in SLE patients, such as the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) and the European Consensus Lupus Activity Measurement (ECLAM) ,13. FlarFurthermore, in daily clinical practice, evaluation of disease activity is not always feasible due to time consuming and lacking data. Thus, it could be of interest the development of a feasible and time-sparing tool to assess patients’ status.As recently pointed-out, the Outcome Measures in Rheumatology Clinical Trials (OMERACT) recommended the measurement of patient well-being, identified by a dichotomous conditions: satisfactory versus unsatisfactory status ,20.The patient acceptable symptom state (PASS) is a single-question outcome tool to evaluate the level of symptoms at which patients consider themselves well . Data puHowever, no data are available concerning a possible application of PASS in patients affected by SLE. Thus, the aim of the present study was to evaluate the discriminant capability of PASS according with disease activity in a cohort of Italian SLE patients.Consecutive SLE patients were enrolled between January 2010 and June 2012 at the Lupus Clinic of the Rheumatology Unit, Sapienza University of Rome (“Sapienza Lupus Cohort”).SLE diagnosis was performed according to the revised 1997 American College of Rheumatology (ACR) criteria . PatientAt each visit, the patients underwent a complete physical examination, the clinical and laboratory data were collected in a standardized, computerized, and electronically-filled form, which includes demographics, education level, past medical history with date of diagnosis, co-morbidities, previous and concomitant treatments.Crithidia Luciliae (titer ≥1:10), ENA  by ELISA assay considering titers above the cut-off of the reference laboratory, anti-CL of IgG or IgM isotype, by ELISA, in serum or plasma at medium or high titers , anti-β2Glycoprotein-I (GPI) of IgG or IgM isotype, by ELISA, in serum or plasma (above the 99th percentile), and finally Lupus anticoagulant (LA) was assessed according to the guidelines of the International Society on Thrombosis and Hemostasis (Scientific Subcommittee on lupus anticoagulant/phospholipid-dependent antibodies) [The evaluation of serum complement C3 and C4 levels and determination of autoantibodies was obtained. ANA were determined by means of indirect immunofluorescence (IIF) on HEp-2 ; anti-dsDNA with ELISA assays (considering levels two folds higher than the cut-off of the reference laboratory) or IIF on ibodies) . For allDisease activity was assessed at each visit with the SLEDAI-2K and ECLAM, while chronic damage was measured by the System Lupus International Collaborating Clinics (SLICC) Damage Index (SDI) ,13,29.> 4, excluding serology alone [In patients with more than 2 consecutive visits with a minimum interval of 2 months between them, the assessment of PAD could be performed and PAD was defined as a SLEDAI-2K score gy alone .In a subgroup of the enrolled patients the evaluation of quality of life by using the Medical Outcomes Study-Short Form 36 (SF-36) was performed .PASS was assessed in all patients by asking to answer yes or no to a single question  that wasThe original question was "Considering all the different ways your disease is affecting you, if you would stay in this state for the next months, do you consider that your current state is satisfactory?” . For thiversus no) with the disease activity indices SLEDAI-2K, ECLAM and PAD, recorded during the same visit. Moreover, the association with the chronic damage, assessed by SDI, was evaluated.The discriminant ability of PASS for use in SLE patients was assessed by comparing the relationship of PASS responses  were evaluated.No patients refused to answer, suggesting the acceptability of PASS. In our cohort, 136 patients (82.4%) showed a high educational level (more than 8 years of education), while 29 patients showed a low level of education (less than 8 years of education). No patients asked the examiner to repeat the question due to non-comprehension, and there was no difference in the percentage of patients answering yes or no according to the educational level , suggesting that the question was easy understood.According with the answer given to PASS, the patients were dichotomized in group 1 (patients answering yes) and group 2 (patients answering no).In versus 21.1%, P=0.2).Interestingly, group 1 patients had significantly lower mean SLEDAI-2K and ECLAM scores than group 2 patients [1.8±2.7 versus 3.4±2.3(P=0.004); 0.7±0.9 versus 1.4±1.1(P=0.0027), respectively; No significant differences were observed when considering chronic damage, evaluated with SDI .Aiming at evaluating the impact of the quality of life on PASS status, 30 patients  completed the SF-36 questionnaire. Among these, 21 patients (70%) answered yes to PASS question. In In the present study, we demonstrated for the first time the ability of the Patient Acceptable Symptom State (PASS) in discriminating patients affected by SLE with different level of the disease activity.As emphasized during the OMERACT 6 meeting, the patients’ perspective evaluation is a very important outcome to perform a complete assessment, and it could influence clinical decision making . In thisSo far, PASS was applied in patients affected by different rheumatic conditions, such as OA, AS, and RA, showing a significant correlation with disease activity –26. At fIn patients affected by AS, a significant association between the presence of an acceptable symptom state and a reduced disease activity, assessed with BASDAI, BASFI and/or ASDAS, was found, underlining the external validity of PASS –34. RodrMore recently, PASS was administered to patients affected by RA. It has been shown that a positive response to PASS is associated with a range of moderate disease activity assessed with several composite indices, such as DAS28, CDAI and SDAI ,37.Following these previous experiences, we evaluated the discriminant capability of PASS in a cohort of Italian SLE patients. A significant association with disease activity, assessed by SLEDAI-2K and ECLAM was identified. In particular, patients with a positive response to PASS showed significant lower levels of SLEDAI-2K and ECLAM. In this same group, a lower percentage of patients with persistently active disease, defined by PAD, was observed.When analyzing the clinical manifestations, a significant lower frequency of musculoskeletal involvement was found in patients with an acceptable symptom state. Joint involvement in SLE is very common, affecting up to 90% of patients at any stage of disease. The clinical presentation of joint involvement can widely vary, ranging from arthralgia, without erosions or deformity, to an erosive arthritis with severe functional disability . The resIn our cohort, a significant higher percentage of autoantibodies in patients with a negative response to PASS was found. We could hypothesize that the presence of autoantibodies might be associated with a greater disease activity, influencing the acceptability of the patient’s status. Indeed, it’s widely known the association between anti-SSA and anti-RNP antibodies and mucocutaneous involvement in SLE patients . Since bThe absence of an association between PASS and SDI in this cohort is not surprising. Indeed, the response to PASS freezes the actual disease status, reflected by the disease activity but not by chronic damage indices. Moreover, in our population the low mean values of SDI probably don’t influence the patient’s status.Even though SF-36 was administered only to a subgroup of SLE patients, overall a worse quality of life was identified in the patients answering no to PASS question.PASS is a simple, reliable, and valid patient-reported outcome to assess patients’ well-being. It could provide a highly feasible tool for clinicians to focalize the disease activity status in SLE patients. In daily clinical practice PASS should not be applied as replacement but administered together with the commonly used activity indices. Larger studies are needed to confirm these results and verify its actual application in a disease characterized by a great clinical heterogeneity, such as SLE.Table S1Translation to Italian language of original question of patient acceptable symptom state (PASS).(DOC)Click here for additional data file.


To investigate small-nucleolar RNAs (snoRNAs) as reference genes when measuring miRNA expression in tumour samples, given emerging evidence for their role in cancer.Four snoRNAs, commonly used for normalisation, RNU44, RNU48, RNU43 and RNU6B, and miRNA known to be associated with pathological factors, were measured by real-time polymerase chain reaction in two patient series: 219 breast cancer and 46 head and neck squamous cell carcinoma (HNSCC). SnoRNA and miRNA were then correlated with clinicopathological features and prognosis.Small-nucleolar RNA expression was as variable as miRNA expression . Normalising miRNA PCR expression data to these recommended snoRNAs introduced bias in associations between miRNA and pathology or outcome. Low snoRNA expression correlated with markers of aggressive pathology. Low levels of RNU44 were associated with a poor prognosis. RNU44 is an intronic gene in a cluster of highly conserved snoRNAs in the growth arrest specific 5 (GAS5) transcript, which is normally upregulated to arrest cell growth under stress. Low-tumour GAS5 expression was associated with a poor prognosis. RNU48 and RNU43 were also identified as intronic snoRNAs within genes that are dysregulated in cancer.Small-nucleolar RNAs are important in cancer prognosis, and their use as reference genes can introduce bias when determining miRNA expression. There is increasing interest in measuring the levels of microRNAs (miRNAs) in tumours using real-time polymerase chain reaction (RT–PCR) methods as markers of pathology and prognosis  . The snoogenesis . RNU6B (GAS5), which encodes multiple snoRNAs, induces growth arrest and apoptosis in breast cancer cell lines, and is significantly downregulated in breast cancer .Ethical approval was obtained from the local research Ethics Committees (Oxford and South Manchester). The breast cancer series consisted of 219 patients with early-first primary breast cancer, treated in Oxford between 1989 and 1992. Patients received surgery followed by adjuvant treatment. The data set was complete for age, nodal status, definitive surgery, relapse and survival. The patient demographics and details of treatments given are provided in (Patient tumour samples taken at operation were placed in RNAlater (Applied Biosystems) for up to 12 h before cryopreservation in liquid nitrogen. Subsequently, the samples were divided and half paraffin embedded for histological analysis. The RNA was extracted from the remaining tissue using Tri Reagent . Quality and quantity of RNA were confirmed using the NanoDrop ND-1000 spectrophotometer and the Agilent 2100 Bioanalyzer . Cases were excluded in which tumour was present in <10% of a representative hematoxylin and eosin section.t method. In which discussed, results were also normalised using RNU43, RNU44, RNU48 and U6 (for the subset of breast cases), the average of two snoRNAs or the median of three snoRNAs.Expression of miR-210, miR-21, miR-10b, miR-342 and miR-30a-3p, and three snoRNAs, RNU43, RNU44 and RNU48 were measured by RT–PCR according to the TaqMan MicroRNA Assay protocol (Applied Biosystems) in the entire breast cancer series. U6 was also measured on a subset of 48 of the 219 breast cancer cases, chosen as they were the most representative of the various molecular subtypes of breast cancer . The expression of miR-210, miR-21, miR-10b, RNU43, RNU44 and RNU48 was measured in the HNSCC series. Complementary DNA was synthesised from 5 ng of total RNA using TaqMan miRNA primers and the TaqMan MicroRNA Reverse Transcription Kit (both from Applied Biosystems). Real-time polymerase chain reaction was carried out in triplicate as described previously .A total of 72 of the 219 samples in the breast cancer series and the 46 HNSCC samples were also expression profiled using Affymetrix GeneChips , with standard pre-processing and normalisation n=152, . Publishhttp://www.oncomine.org).Small-nucleolar RNAs were mapped to genomic regions using the gene accession numbers and the UCSC browser. Publically available data sets were accessed using Oncomine (M), mean pairwise variation for a gene compared with all other tested reference genes, was derived from the GeNorm algorithm (http://www.bioconductor.org). Unless otherwise stated, correlations of snoRNAs and miRNAs, with clinical and pathological variables, were assessed using the appropriate non-parametric methods . Correlation between snoRNA and probe sets was performed using Pearson correlations. Tumour recurrence and death were calculated from the time of surgery, for the breast series according to STEEP criteria (http://www.r-project.org) were used for statistical analyses.The gene stability measure . We found in the breast series that RNU43 RNU44 and RNU48 had M value as 1.32, 1.22 and 1.27, respectively. The values for the 48 breast cancer subset were 1.59, 1.42 and 1.76, respectively. The corresponding values for the 46 HNSCC were 0.70, 0.72 and 0.62, respectively.We first noted that the expression of snoRNAs was highly variable in cancer, with similar ranges of values to some of the most varying miRNAs . In the ρ) was 0.94 when comparing raw and normalised microarray breast cancer data, whereas it was 0.89 for raw and normalised RT–PCR data (P<0.001 for both)  . ConcernP-values in P-values 0.027 and 0.716, for raw and normalised data, respectively). The opposite effect  was observed for miR-210 in the breast cancer cases, which goes up in association with a poor prognosis. The P-values for miR-210 and RFS were 0.01 and 0.003 for raw and normalised data, respectively, data not shown.For the five miRNA measured in the 219 breast cancer cases and the three miRNA measured in the 46 HNSCC cases, contrasting results were obtained for normalisation. Normalisation could either unmask or mask associations between miRNA expression and clinicopathological factors . The forDicer and Argonaute 2, henceforth called AGO2). The uncorrected snoRNAs were directly associated with multiple factors, including oestrogen receptor (ER) status, grade, microarray-based markers of proliferation and invasion, and miRNA processing genes , with a hazard ratio of 0.5 and 95% confidence intervals of 0.26–0.99. This was not significant in multivariate analysis . The GAS5 transcript, which contains a cluster of highly conserved snoRNAs, can be interrogated with multiple probes available on Affymetrix arrays  in a microarray of 22 glioblastoma multiforme compared with normal brain samples . Results for the other three probe sets are shown in In our series of HNSCC, when expression values for the probes to Next, we examined the subset of the series of 219 patients with breast cancer for which Affymetrix gene expression data were available (the overlapping series of 152 cases). A similar trend was observed with lower levels of GAS5 being associated with a poor prognosis but this was not statistically significant .H. sapiens ribosomal protein L3 . Alternate transcriptional splice variants, encoding different isoforms, have been characterised. RPL3 belongs to the L3P family of ribosomal proteins and encodes a ribosomal protein that is a component of the 60S subunit. In publically available data sets, interrogated using Oncomine, RPL3 was downregulated: two-fold in ovarian adenocarcinoma compared with normal ovary   , mapped to an intronic region of RPL3 was not associated with prognosis. In the breast series, there was a poor correlation between RPL3 and RNU43, but low levels of RPL3 were associated with a poor prognosis in this series.When mapped to Affymetrix probe sets  in our sc6orf48 gene are associated with susceptibility to infection , suggesting that these probe sets are not a helpful way of interrogating RNU48 values.RNU48 is also an intronic snoRNA, within chromosome 6 open reading frame 48 (c6orf48 or G8). C6orf48 is part of the major histocompatability complex III tissue-protective factor produced in response to chronic inflammation, and polymorphisms in the U6 was not directly associated with clinicopathological factors, with the exception of invasion gene expression signature score. It was associated with several factors when normalised to the average of other snoRNAs, suggesting that these were introducing bias into the results . We wereNormalisation of miRNA data measured by RT–PCR is critical to interpreting clinical significance and developing miRNA as tumour markers. Our work highlights several key problems in the way this has been carried out, and may explain many opposing or non-confirmatory studies in this area. The first issue we noted was that snoRNAs had highly variable expression and introduced noise when used as controls. For microarray data, there was a good internal correlation between quantile normalised and non-normalised data for a given miRNA, however, the internal correlation between the relative expression by RT–PCR of miR-210, and miR-210, normalised to three control genes, was noisier. This is likely to be because of the robustness of standardisation of gene array data, with thousands of probes contributing to normalisation of arrays and well-validated methodology. miR-210 is among the miRNAs most reproducibly associated with outcome, is highly expressed and has a wide range of expression levels, but the above issue will be even more important in the case of less varying and lower expressed miRNAs.M values for the snoRNAs were higher than expected: for example, in a study of multiple mRNA housekeeping genes, M values were less than 0.5 . This may be because of biological differences, such as the existence of multiple subtypes of breast cancer, with different intrinsic snoRNA levels, and variation among samples induced by ER and HER2 status, the heterogeneity in HNSCC site subtypes and differences in relative amounts of tumour and stromal tissue between samples. In addition, it may relate to sample collection methods. The sample collection for HNSCC was carried out by clinical fellows in the operating theatre to a highly standardised protocol, while for the breast samples it was performed according to standard surgical procedure. Although this may have contributed to reduced variability in the HNSCC, it did not eliminate the problems of normalisation, and illustrates the pitfalls of attempting to introduce miRNA-based signatures to routine clinical practice.Indeed, the noise introduced by normalising to snoRNAs may explain why RT–PCR has been questioned as the ‘gold standard’ for validating miRNA results by Raw data are expected to be noisy but normalisation to genes, which also have variable expression, introduces more noise and possibly bias in the results. Normalisation of miR-10b to the three control genes  abrogated the association of miR-10b with a good prognosis, whereas for miR-210, which is a marker of poor prognosis, the opposite effect was observed , although the results were significant whether normalised or not. This suggested that in some cases, use of snoRNAs as reference genes introduces bias as well as noise. For miRNA, with a large expression range and strong signal, such as miR-210, normalisation problems may be less important than for miRNA with a small range or subtle, although important, biological effect .Dicer and AGO2. This implies that regulation of ribosomal RNA cannot be considered independently from, and may interact both with, these biologically important factors, and with the processing of other noncoding RNA such as miRNA by their interaction with Dicer and AGO2. Of note, the snoRNAs varied significantly across molecular subtype classification, implying the alteration of multiple species of RNA, both ‘coding’ and ‘non-coding’, across the groups. Furthermore, the finding that RNU44 levels were highest in Luminal A type breast tumours may have been expected from the observation that RNU44 is linked to ER status – however, it is interesting that RNU43, which is not linked to ER status, is even more highly correlated with Luminal A tumours. Recently, poor concordance between molecular classification when using different algorithms has been described  , these microarray data can be mined for prognostic association, however, where there is a poor correlation , data mining was not possible . Indeed,This was reinforced by the complex relationship between RNU43 and RPL3. In many cases, introns containing snoRNAs occur within protein-coding transcripts, encoding ribosomal proteins, and others involved in ribosomal biogenesis, and RNU43 would initially appear to be an example of this type of parallel genetic output. However, the mapping of RNU43 to RPL3-linked Affymetrix probe sets has not shown a good link between RPL3 and RNU43, so further work is required to elucidate the reason for the difference .U6, highly conserved across evolution, has multiple copies in the genome, and appears to be upregulated in cervical cancer and correlate with progression .Taken together, these data suggest that RNU44, commonly used for normalisation of miRNA , is a poor choice of normalisation gene. U6 appears the most stable of the snoRNAs tested in breast cancer. Caution should be exercised in using RNU43 and RNU48. RNU43, RNU44 and RNU48 were recommended by manufacturers, because of their stability across multiple normal tissue types  samples.


Therefore, an effective strategy for controlling ozone pollution may be achieved by the reduction of vehicle emissions in Guangzhou. In recent decades, high ambient ozone concentrations have become one of the major regional air quality issues in the Pearl River Delta (PRD) region. Non-methane hydrocarbons (NMHCs), as key precursors of ozone, were found to be the limiting factor in photochemical ozone formation for large areas in the PRD. For source apportioning of NMHCs as well as ozone pollution control strategies, it is necessary to obtain typical seasonal and diurnal patterns of NMHCs with a large pool of field data. To date, few studies have focused on seasonal and diurnal variations of NMHCs in urban areas of Guangzhou. This study explored the seasonal variations of most hydrocarbons concentrations with autumn maximum and spring minimum in Guangzhou. The diurnal variations of most anthropogenic NMHCs typically showed two-peak pattern with one at 8:00 in the morning and another at 20:00 in the evening, both corresponding to traffic rush hours in Guangzhou, whereas isoprene displayed a different bimodal diurnal curve. Propene, ethene,  NMHCs, as key precursors of ozone, are found to be the limiting factor in photochemical ozone formation for large areas of the PRD [3 pollution in urban areas can be obtained by controlling the emission of NMHCs. For source apportionment of NMHCs as well as ozone control strategies, it is necessary to obtain typical seasonal and diurnal patterns of NMHCs in the PRD.In recent decades, high ambient ozone O concentret al. [et al. [Several studies ,5,6,7 haet al.  reported [et al.  found th3 pollution in Guangzhou and even the larger areas in the PRD.Although the two studies mentioned above have investigated the diurnal variations of NMHCs in Guangzhou, there is still a lack of studies on the seasonal and diurnal variations of NMHCs. Moreover, Guangzhou is considered to be the major source area of volatile organic compounds (VOCs) in the PRD. Therefore, there is an urgent need to better understand the characteristics of temporal variation of NMHCs in Guangzhou for source apportionment of NMHCs and effective control of photochemical OIn this study, NMHC measurements were carried out at an urban site in Guangzhou from March to December 2005. The ambient concentrations of NMHCs in Guangzhou were examined and compared with other cities. The seasonal and diurnal variations of NMHCs concentrations, as well as the photochemical reactivity of individual NMHCs were characterized. To our knowledge, our research is the first to systemically investigate the comprehensive characteristic of NMHCs in the urban atmosphere of Guangzhou. Therefore, this study is to be a very significant work to formulate ozone pollution control strategies and help the local government implement air quality standards.The sampling site is located at the Guangzhou Institute of Geochemistry, Chinese Academy of Sciences in the Tianhe District of Guangzhou, South China. The site is surrounded with scattered schools, commercial shops and residential buildings. In addition, the east, south and west of the site have separately several large factories such as petrochemical plants and Guangzhou Honda Co., Ltd, the Guangyuan Expressway and Huanan Expressway Trunk. Therefore, the site is expected to be adequate for representing the mixture of various VOCs emission sources. Air samples were collected on the roof of a 4-floor building (about 12 m above ground). Pre-vacuated 2 L stainless steel electro polished canisters provided by University of California, Irvine (UCI) were used to collect air samples. Prior to sampling, all canisters were cleaned to –50 millitorr using a vacuum cleaner (Entech 3100A), and then each canister was joined to a flow controlling device to obtain one-hour integrated air sample (2 L). The field sampling was carried out on 6–7 March, 2–4 August, 1, 14 and 28 September, 12 October, 9 November and 9–12 December 2005, respectively. An air sample was collected at one or two-hour intervals from 6 a.m. till 22 p.m. during sampling days to explore the diurnal variations of the hydrocarbons. A total of 145 air samples were collected during the measurement periods. The analysis of the canister samples was performed in VOCs laboratory of the State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry. A cryogenic pre-concentrator (Entech Instrument 7100A) together with a gas chromatograph  equipped with a nonpolar capillary column  and a quadrupole mass spectrometer detector  were used to detect VOCs. 2O. Secondly, the second cryotrap was cooled to −40 °C, allowing trapping of VOCs and leaving CO2. Subsequently, VOCs were backflushed from Module 2 at 180 °C and focused in the Module 3 trap at −150 °C again. Finally, after concentrated by a 3-stage cryotraps, VOCs along with helium entered into a gas chromatograph column. Column was initially held at −50 °C for 3 min, then was raised up to 10 °C at the rate of 15 °C·min–1, later to 250 °C at 5 °C·min−1 and was remained for 10 min. Thus, VOCs were separated on capillary column, and subsequently identified by MSD in Selected Ion Mode.First, an ambient air sample was pumped into the pre-concentrator with a 3-stage cryotraps (Module 1~3), initially trapped cryogenically by liquid nitrogen at −150 °C on glass beads of Module 1, and then desorbed at 10 °C to remove most of the liquid H2-C11 NMHCs were quantified by multipoint calibration. The detection limit for C2-C11 NMHCs was 3 pptv. The measurement accuracy was 5–10% for NMHCs. The measurement precision ranged 0.5–15% for NMHCs. The response of the instrument to NMHCs was calibrated after every eight samples using standard runs of a calibration gas with ambient concentrations.At least three samples were withdrawn from each canister for the reduction of analytical error and the quality check of the analysis. Standard gas containing 108 components (supplied by UCI) was used for the identification 79 different hydrocarbons and the target 59 CSeasons in Guangzhou are divided into spring , summer , autumn  and winter . The 59 NMHCs in urban atmosphere of Guangzhou were quantified. These 59 species including 27 alkanes, 12 alkenes, one alkynes(ethyne), 16 aromatics and three biogenic hydrocarbons  added up to 47.3 ppbv, in which alkanes, alkenes, alkynes, aromatics and biogenic hydrocarbons accounted for 49, 14, 12, 23 and 2% of the total NMHC concentration, respectively. The average mixing ratios and standard deviations of selected hydrocarbons in the ambient air samples are presented in In order to better understand the general characteristics of ambient level and various source emissions of NMHCs in Guangzhou, we compare the NMHC mixing ratios of Guangzhou with other cities. A comparison of some NMHC mixing ratios in various cities is illustrated in Toluene is both the most abundant aromatic hydrocarbon in all cities compared and the top most abundant hydrocarbon in Hong Kong, Dongguan, Shanghai and Kaohsiung as well. However, toluene is the third most abundant hydrocarbon in Guangzhou, and the ambient level of toluene in Guangzhou is lower than that of Kaohsiung, Dongguan and Shanghai, respectively . TolueneIsoprene was primarily emitted from biogenic sources , but isoi/n-butane, i-pentane, ethene, propene, ethylbenzene and m,p-xylene show autumn maximum concentrations while propane and toluene exhibit winter maxima. Besides, ethyne and isoprene have summer maximum concentrations. On the other hand, all selected NMHC concentrations exhibit spring minima. The seasonal variations of NMHCs in the troposphere were generally affected by various factors such as source emission strength variations, meteorological conditions and seasonal variability of OH radicals. Since OH radicals exhibited the highest concentration in summer and the lowest in winter, and the reaction with OH radicals was the major sink of NMHCs [The seasonal variations of selected NMHCs in Guangzhou are illustrated in of NMHCs , the NMHof NMHCs ,23,24. Hof NMHCs . et al. [Guangzhou enjoys a typical subtropical climate under a strong influence of the Asian monsoon. Northerly and northeasterly winds from Mainland China prevail during the autumn-winter season while southerly and southwesterly winds from the South China Sea are predominant in the summer-spring season. The continental outflows in autumn-winter enrich much more air pollutants than the marine air masses in spring-summer, as a result, the enhanced pollutants concentrations appear in autumn—winter, while the lowest concentrations occur in spring-summer. In addition, a hazy-weather phenomenon was very common in the PRD region in autumn and the air was relatively dirty under such weather conditions . Consequet al. .in situ prevailing winds in summer. The predominant source of ethyne was traffic emissions in urban areas, and traffic emissions combined with weather conditions remarkably contributed to the ethyne concentration. As mentioned in Ethyne concentration in the four seasons shows a summer maximum. This can be explained by the strong source emissions and Similar to the seasonal variation of ethyne concentration, isoprene also has a summer maximum concentration among the four seasons . Isoprenn-butane, i-butane, i-pentane, propene, benzene and ethyne exhibit a generally similar shape with the first concentration peak in the morning (8:00 a.m.) and the second one in the afternoon (20:00 p.m.), both corresponding to traffic rush hours in Guangzhou. The two peak patterns indicate that the predominant sources of these hydrocarbons are traffic emissions.The average diurnal variations of selected NMHCs in Guangzhou are shown in The toluene time series shows higher early morning and nighttime concentrations and lower levels from 12:00–16:00 . In detaThe diurnal fluctuation of isoprene concentration shows a bimodal pattern as well. That is, isoprene has one peak concentration at 10:00 a.m. and another at 16:00 p.m. in a diurnal cycle . The diux and sunlight. However, different hydrocarbons react at different rates and exhibit differences in reactivity with respect to ozone formation. To assess individual hydrocarbons’ activity related to photochemical ozone formation, a propylene-equivalent concentration equation (1) proposed by Chameides [et al. [As mentioned in the previous sections, the photochemical reactions with OH radicals are an important factor controlling the seasonal and diurnal variations of NMHCs. Moreover, photochemical oxidation of NMHCs results in ozone formation in the presence of NOhameides  was used [et al.  and hereOH(i) is the rate constant between species i and OH radical cited from Atkinson and Arey [OH(C3H6) is the rate constant between C3H6 and OH radical. In Equation (2), MIR coefficients are cited from Carter [In Equation (1), Propy-Equiv(i) is a measure of species i on an OH reactivity-based scale, normalized to the reactivity of propylene, Kand Arey , and KOHm Carter .m,p-xylene, propene and toluene are the top four hydrocarbons based on propylene-equivalent concentrations, accounting for 16%, 12%, 9% and 8% of the total concentration, respectively. The rests mostly are alkenes. This result was consistent with the estimates in the PRD [These NMHCs accounted for about 86% of the total propylene-equivalent concentration and about 87% of the total OFP at the urban site in Guangzhou. Isoprene, m,p-xylene and toluene are the top four contributors to the OFP at the Guangzhou urban site, which separately accounted for 17%, 17%, 10% and 9% of the total OFP. The high contributions of propene, ethene, m,p-xylene and toluene to the total OFP had also been reported by other studies [Propene, ethene,  studies ,18. etc.) and the aromatics were mainly emitted from vehicular source although toluene and m,p-xylene were also from gasoline and solvent evaporation. Therefore, vehicles emissions can dramatically contribute to the ozone formation in Guangzhou. The alkenes  were measured at an urban site in Guangzhou during March–December 2005. Alkanes provided the largest contribution to the total NMHCs, accounting for over 49%. Alkenes, alkynes, aromatics and biogenic hydrocarbons separately accounted for 14, 12, 23 and 2% of the total NMHC concentrations. Ethyne, propane and toluene were the three top most abundant individual hydrocarbons in the urban atmosphere of Guangzhou. The seasonal variation trends of most anthropogenic emissions of NMHCs generally showed high concentrations in autumn and low concentrations in spring. Such seasonal behaviors were mainly attributed to the seasonal change of monsoon winds in the PRD. Isoprene revealed its highest summer concentration among the four seasons, suggesting its well-known biogenic emissions. The diurnal profiles for anthropogenic NMHC concentrations generally exhibited a first peak in the morning and a second one in the afternoon, both corresponding to traffic rush hours in Guangzhou, indicating that the predominant sources of these hydrocarbons are traffic emissions. Isoprene concentrations display a bimodal diurnal curve different from those of anthropogenic NMHCs, which was significantly affected by meteorological conditions and source emissions as well as photochemical removal.m,p-xylene and toluene were the four largest contributors to the OFP, indicating that reduction of vehicles emissions could probably effectively control ozone pollution. In addition, biogenic emissions can also contribute to the ozone formation since isoprene is the fifth largest contributor to the OFP in Guangzhou.Propene, ethene,


Previous studies in patients with hip and knee osteoarthritis (OA) have advocated the relevance of assessing the number of painful joint sites, other than the primary affected joint, in both research and clinical practice. However, it is unclear whether joint-pain comorbidities can simply be summed up.A total of 401 patients with hip or knee OA completed questionnaires on demographic variables and joint-pain comorbidities. Rasch analysis was performed to evaluate whether a sum score of joint-pain comorbidities can be calculated.Self-reported joint-pain comorbidities showed a good fit to the Rasch model and were not biased by gender, age, disease duration, BMI, or patient group. As a group, joint-pain comorbidities covered a reasonable range of severity levels, although the sum score had rather low reliability levels suggesting it cannot discriminate well among patients.Joint-pain comorbidities, in other than the primary affected joints, can be summed into a joint pain comorbidity score. Nevertheless, its use is discouraged for individual decision making purposes since its lacks discriminative power in patients with minimal or extreme joint pain. Previous studies in patients with hip and knee osteoarthritis (OA) advocated the relevance of assessing the number of painful joints, other than the primary hip or knee joint, in both research and clinical practice -3. JointTo date, the use of joint counts to indicate the level of joint-pain comorbidity in people with knee or hip OA is common -5. By siTherefore, the aim of this study was to examine the validity of summing painful joint sites in people with hip or knee OA.Data were used from a previous study, which examined the prevalence of joint-pain comorbidities in patients with a physician-based diagnosis of hip or knee OA . The patAt baseline, information was gathered on gender, age, body-mass index (BMI), and disease duration. In addition, the patients received a questionnaire within 14 days after their visit, assessing their joint pain at 19 joint sites, including the hands, wrists, elbows, shoulders, cervical spine, thoracic spine, lumbar spine, hips, knees, ankles, and feet. Patients had to indicate whether they experienced symptoms from particular joints and whether these symptoms were present for more than half of the time during the last month. If both questions were positive, the joint was counted as being painful.The study was approved by the Institutional Review Board of the University Medical Centre, Nijmegen and all patients enrolled in the study gave informed consent.Whether the joint-pain comorbidities can simply be summed up was evaluated using Rasch analysis. A feature of the Rasch model is that it assumes all items to be equally discriminating . As a reIn order to perform Rasch analysis, the data must conform to a number of underlying assumptions of the Rasch model -10 incluThe first assumption, unidimensionality, was tested with a principal component analysis of the tetrachoric correlation matrix in SPSS Statistics 18.0, using oblimin rotation. The scale was assumed to be sufficiently unidimensional (i.e. there is one dominant underlying factor) if the ratio of the first and second eigenvalue was >3 : 1 .The second assumption concerns the model’s ability to reflect the true relationship among the underlying construct and the item responses -10. ThisThe final assumption assumes that the items are not further associated with each other once the Rasch factor is taken into account. Violation of this assumption might point to response dependency  or to multidimensionality of the scale ,12. ItemIn case the Rasch assumptions were satisfied, joints were checked for differential item functioning (DIF). DIF is present when subgroups of patients with similar levels of the measured underlying construct (i.e. the degree of joint-pain comorbidity as measured by summing the number of painful joints) give different responses (i.e. painful or not painful) to a specific joint ,10. DIF Finally, the performance of the sum score was examined by evaluating its test information function with associated reliability levels; showing whether precise and reliable joint-pain comorbidity scores can be obtained across the range of joint pain comorbidity severity. Reliabilities >0.7 were deemed acceptable for group use . In addiRasch analyses were performed using Winsteps, version 3.65 .2. The majority of the participants had a K/L grade of ≥ 2 and a disease duration of >5 years, respectively 76% and 61%. Mean (SD) pain levels were 45 (21) points for the hip group and 50 (20) points for the knee group, as measured with the Hip disability and Osteoarthritis Outcomes Score and Knee Injury and Osteoarthritis Outcome Score respectively [Data were collected from 401 patients (58% n=231] women and 42% n=170] men), of which 71% were referred for knee OA and 29% for hip OA. The average (SD) age of the study sample was 58 (13) years, with an average (SD) body mass index of 27 (5) kg/mectively ,17. More0 men), o31 women ectively .The test of unidimensionality showed that the ratio between the first and second eigenvalue was 9.701 : 2.117, which is >3 : 1. Thus, the 19 items formed a sufficiently unidimensional scale for Rasch analysis.The results of the Rasch analysis showed an acceptable fit of the comorbid joints (i.e. other than the primary affected joints) to the Rasch model Table . Infit MAlthough some Infit and Outfit values fell outside the specified range for a number of joints, these analyses were based on very strict sample size corrected Infit and Outfit statistics. A more commonly applied, less strict, rule of thumb is that both the Infit and the Outfit MNSQ are allowed to be somewhere between the 0.7 and 1.3. When these values were held against the data, fit was even better with none of the joints showing Infit MNSQ values <0.70 or >1.30 and only 5 joints showing Outfit MNSQ values <0.70 or >1.30.r > 0.70, within a range of -1.04 to +1.18, corresponding to the presence of 5 to 14 painful joints. Although this indicates that the instrument can discriminate well between patients within these wide range of joint scores, it only covered 11.2% of the patient sample since most patients (86.8%) had less than 5 painful joints. The instrument’s discriminative power for this large majority of patients with few complaints is much lower, diminishing the instrument’s usefulness.The test information function Figure  shows thThe past decades show a marked increase in item response theory applications within the rheumatic field . Item reComorbidity is very common in people with OA ,22 and iThe results of this study showed that it is valid to sum the number of painful joint sites to assess joint-pain comorbidity in hip or knee osteoarthritis. However, prudence is in order when using this instrument for treatment decisions on an individual level.OA: Osteoarthritis; BMI: Body mass index; MNSQ: Mean square; DIF: Differential item functioning.None of the authors have financial, commercial, or other associations that might pose a conflict of interest in connection with the work.LS was responsible for the conceptualization of the manuscript. EVDE and TH were responsible for data collection. PTK, TH, EVDE, and MVDL supervised the whole study and the interpretation of the results. All authors critically evaluated the manuscript, contributed to its content, and approved the final version. LS and TH take responsibility for the integrity of the work as a whole, from inception to finished article .The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/14/234/prepub


Biomphalaria glabrata, B. straminea, B. tenagophila. In the Biomphalaria genus, the myoglobin gene has three introns; these were amplified by PCR, analyzed by PCR-RFLP. Results showed that the size (number or nucleotides), the nucleotide sequence of the coding gene of the myoglobin are variable in the three species. We observed the presence of size polymorphisms in intron 2, 3; this characterizes a homozygous/heterozygous profile, it indicates the existence of two alleles which are different in size in each species of Biomphalaria. This polymorphism could be explored for specific identification of Biomphalaria individuals.Introns are common among all eukaryotes, while only a limited number of introns are found in prokaryotes. Globin, globin-like proteins are widely distributed in nature, being found even in prokaryotes, a wide range of patterns of intron-exon have been reported in several eukaryotic globin genes. Globin genes in invertebrates show considerable variation in the positions of introns; globins can be found without introns, with only one intron or with three introns in different positions. In this work we analyzed the introns in the myoglobin gene from  In 1977, several research groups published their findings about DNA coding sequences (exons) broken by non-coding sequences (introns) in eukaryotes ,3. IntroCavalier-Smith  suggesteIndependently in 1978, Doolittle , DarnellIn the evolutionary history of introns, the selective stretch which modulates their evolution, the relative contribution of its eukaryotic gene loss or insertion is not understood . In inteMany authors have attempted to interpret the distribution of intron in terms of a mixture of movement, removal of introns, but the difficulty of phase-shifting movement causes serious doubts about these interpretations. In contrast, some gene families for which phylogenies can be traced show patterns that clearly indicate intron insertions .The protein coding sequences are interrupted by introns at specific points, which could be used to investigate, for instance, the evolution of protein families. The gene encoding the ancestral globin chain is assumed to be interrupted by three introns inserted in the B, E, G helices. However, the conservation of this intron pattern, the exact insertion positions of the introns during evolution is a matter of ongoing discussion .Intron location, sequence may also shed light on the origin of some protein genes, for instance the polymeric globin genes occurring in mollusks, arthropods . In thisIdentifying individuals from the Biomphalaria genus is a difficult task because they are very similar in morphology. Classic methodology of identification is based on comparative morphology of shells, anatomy of the reproductive organs, however there is much intra-specific variation . ClassicIn this work, we analyze the introns of the myoglobin gene of mollusks from the Biomphalaria genus in order to evaluate their potential use for precise identification in systematic studies. The size (in base pairs) of the amplicons of intron 1 was analyzed by polyacrylamide gel electrophoresis. All individuals of the three Biomphalaria species showed a DNA fragment of about 1,300 bp .Amplification of intron 2 of B. straminea generated an amplicon larger than 1,300 bp, of about 1,500 bp. In B. glabrata, B. tenagophila, some individuals were observed to have a large amplicon of about 1,400 bp,, some individuals showed a smaller fragment , whereas some individuals showed the two fragments .The amplification of the intron 3 presented a similar homozygous/heterozygous pattern in B. straminea. Some individuals showed an amplicon of about 1,400 bp, other individuals showed an amplicon of about 1,200 bp,, some ones showed the two amplicons. In B. glabrata, was observed one amplicon only of about 750–850 bp,, B. tenagophila showed an amplicon of about 1,300 bp . Clear, satisfactory amplification using genomic DNA as a template is not a simple task, since nonspecific amplifications, artifacts could contaminate the true results. After obtaining the best protocol for amplification, this was repeated at least five times. The monitoring of all amplifications showed the results on the size of introns from Biomphalaria, which are described in the text; the gels presented as figures have some artifacts that do not refer to amplification of introns.The models for putative alleles from the three Biomphalaria species are shown in Figure 4. They were built based on the nucleotide sequence of the myoglobin gene from B. glabrata deposited in data bank, sequence of the cDNA myoglobins of B. tenagophila, B. straminea that were deposited in Genbank , which are very similar to B. glabrata myoglobin (>96% identity). PCR-RFLP was used to obtain rapid information about the nucleotide sequence of the myoglobin introns from Biomphalaria; for individuals who had introns of different sizes, the largest fragment was digested.In no individual from the Biomphalaria species was fragmentation of intron 1 observed by restriction enzymes. The intron 2 from B. straminea, B. tenagophila was digested by Hind III only, however it was digested in different places, generating fragments of different sizes.Intron 3 from B. straminea was cutted by Xba I, Xho I, Bgl II, Hind III,, the same intron from B. tenagophila was cutted by Xba I, Hind III. Intron 3 from B. glabrata was not cut by any enzyme .In 2004, Sokolova, Boulding  describeThe first intron of B. glabrata has 1,116 bp, the second intron has 1,008 bp, third has 582 bp . The difThe first elucidation of globin gene structure was that of the human hemoglobin α, β chains. These genes have two introns conserved exactly at positions B12.2 (α-helix B in the protein), G6.3 . AccordiThe typical gene structure of three exons/two introns are found in all vertebrate globins, in most annelids . The resThe globin genes of vertebrates, plants contain, respectively, two, three introns, their insertion positions are conserved. The three intron/four exon pattern of plants is proposed to be ancestral, all other globin gene structures would be derived mainly by intron loss . SeveralB. straminea presents a size polymorphism in the third intron of the myoglobin gene. B. tenagophila, B. glabrata contain size polymorphism in the second intron,, any of the three species showed size polymorphism in the first intron. This kind of polymorphism, the homozygous/heterozygous pattern was not shown for the myoglobin gene of B. glabrata which had been described.Analysis of unique individuals indicates that there are two alleles of different sizes which code for myoglobins in the Biomphalaria genus. The difference is about 200 bp, each Biomphalaria specie presents polymorphism in one intron only. The smaller alleles were observed in B. glabrata, the bigger ones were observed in B. straminea. By analyzing the size it is believed that the nucleotide sequence of the myoglobin gene of the B. glabrata deposited in data bank could be the minor allele of this specie.No differences were observed in size of the primary sequences (amino acids) of the myoglobins from the three Biomphalaria species such that the contribution of the exon to the size of the amplicons is the same for all three Biomphalaria species. The difference among the myoglobin introns from B. glabrata, B. straminea e B. tenagophila suggests a potential for using these as molecular markers to carry out the specific identification of these mollusks. Analysis of the distribution of the possible alleles of the gene encoding myoglobin among individuals of three species of Biomphalaria do not indicated a relation to the geographical origin of the mollusks, but this relationship cannot be ruled out, since it would be necessary to analyze a larger number of individuals.The sequencing of heterozygous genotypes with different alleles in size is a difficult task,, RFLP analysis is a way to get quick information about the nucleotide sequence . AnalysiThe Biomphalaria genus covers organisms morphologically very similar, making their identification difficult. The classical identification is based on comparative morphology of the shells, anatomy of reproductive organs, but the enormous intraspecific variation of morphological, anatomical features commits to a specific classification. Identification may also be hampered by the small size of the specimens, whose distinctive morphological characters are not very evident .Monis  highlighIn the same context, Knight et al. , using tB. glabrata, B. tenagophila, B. straminea from several Brazilian provinces  belonginGenomic DNA was purified from foot tissue of each individual of Biomphalaria using a DNA purification kit  according to recommendations from the manufacturer. The DNA quantification, purity were analyzed by spectrophotometry at 260, 280 nm using a UV-160A UV-Visible recording spectrophotometer (Shimadzu). The DNA quality was determined by 1% agarose gel electrophoresis. The DNA was not pooled at any step.Amplification of the introns were performed by PCR using three sets of primers which were drafted based on nucleotide sequence from the B. glabrata myoglobin gene available in GenBank Accession Code U89283. The primers were BIO 1F, BIO 2 , the other primers annealing in exon regions or in the UTR (untranslated region) to provide the complete sequence of the three introns of the myoglobin gene . All amplifications were reproduced at least five times.Primer pair BIO 1F (forward) (5’ TACTGTCACACAACCAGCCC 3’), BIO 1R (reverse) (5’ GCGTTCTTTTTGCCGTCAGC 3’) were used to amplify the first intron (intron 1), primers BIO 4F (5’ GGCAAAAAGAACGCTGGAAT 3’), BIO 4R (5’ GAGTCCGATTTTCAGGTTG 3’) were used to amplify the second intron (intron 2), primers BIO 2, BIO 3 (5’ GAGTTGGTCAGGATCCGTGG 3’) were used to amplify the third intron (intron 3).The PCR for intron 1 was performed for 30 cycles of 94 °C for 30 s, 65 °C for 60 s, 72 °C for 2 min, a final elongation step at 72 °C for 10 min. The amplification of the introns 2, 3 used the same reaction, except for an annealing temperature of 60 °C. The amplicons were analyzed by 6% polyacrylamide gel electrophoresis with silver stain; the base pair markers φX174/Hae III digested (Amersham Biosciences), 1 kb Plus DNA ladder (Invitrogen) were applied.PCR-RFLP was used to analyze the similarity of the nucleotide sequence from myoglobin introns. The three introns of the myoglobin gene from three Biomphalaria species were amplified as previously described, amplicons were submitted to digestion with the restriction endonucleases Xba I, Bgl II, Xho I, Hind III, Bam HI (Invitrogen). Only intronic fragments were digested, because the aim was to observe if the nucleotide sequence of the introns differed among species of Biomphalaria. The use of the full gene (whole gene amplification) in RFLP would not allow identification of the differences (cuts) belonging to the introns, exons,, in which intron. Reactions were performed according to the manufacturer, were evaluated by 6% polyacrylamide gel electrophoresis with silver staining; the base pair marker φX174/Hae III digested (Amersham Biosciences) was applied. The gene encoding myoglobin from B. glabrata, B. straminea, B. tenagophila displays size polymorphisms (base pairs) in the intronic regions, featuring a profile of homozygosity/heterozygosity in this gene. Considering the difference among the introns of myoglobin from B. glabrata, B. straminea, B. tenagophila, there is the possibility of using these introns as molecular markers for specific identification of these mollusks.