Allergic rhinitis (AR) is a common airway disease. In order to study the prevalence of AR in high school students in Kerman, the Score for Allergic Rhinitis (SFAR) was used and the quality of life in the students affected by rhinitis was evaluated using the SF-36 questionnaire.This was a cross-sectional, analytical, descriptive study, based on the SFAR scale. Quality of life in students with AR was evaluated using the SF-36 questionnaire.From 1511 students who completed the SFAR questionnaire, 291  had AR. Domestic dust was the most common cause of the disease. The most common symptoms of AR were rhinorrhea (76.6%), epiphora (76.3%), nasal congestion (64.3%), and itching (54.3%). According to the ARYA scale, (Allergic Rhinitis and its Impact on Asthma), 41.9% of students had moderate-to-severe rhinitis and 58.1% had mild rhinitis. A total of 43.1% of patients with moderate-to-severe rhinitis had a persistent condition and 56.9% had an intermediate condition. Results of the SF-36 questionnaire among students with AR showed a significant difference in physical functioning and bodily pain in comparison with healthy students.The results of this study show that the prevalence of AR among Kerman high school students is 19.3%. Because of the effect of this disease on the life quality of high school students in terms of both physical functioning and bodily pain, efforts should be made to reduce allergen levels as far as possible. Allergic rhinitis (AR) is an upper respiratory disease caused by immunoglobulin E (IgE) interference following contact with allergens . AllergiSymptoms of AR usually appear before the age of 20 . To date, a number of studies have been undertaken in Iran investigating the prevalence of AR. One such study performed in Hamadan reported an AR prevalence among high school students of 34.3% , while oAs the Kerman geographical zone is surrounded by a dry climate as well as agricultural land, and because a large number of allergens have been recorded in the area, we decided to investigate the prevalence of AR in this city.This was a descriptive, analytical, cross-sectional study. The population of this study was high school students in the city of Kerman.The survey was carried out in two phases. In the first phase, the prevalence of AR was investigated; while in the second phase, the quality of life of students with AR was compared with that of healthy students.In the first phase, 1840 students selected through cluster sampling were investigated. Sixteen schools were chosen at random among Kerman high schools, and all students from one class at each level of study were chosen at random and questioned. All students in the study were given the project questionnaire. The questionnaire consisted of three parts. In the first part, the demographic characteristics of the students were registered, while the second part included the score for AR (SFAR) scale. This scale runs from 0 to 16, with a sensitivity of 74% and a specificity of 83% for SFAR≥7, satisfactorily discrimi- nating between outpatients with AR and those without AR. The advantage of this scale is that it requires only a short time for completion, and is easy to understand .In the second phase of the research, and in order to study the quality of life of students with AR, the SF-36 questionnaire was used. This questionnaire investigates quality of life in eight domains, including physical functioning, role limitation due to physical problems, general health, vitality, social functioning, mental health, role limitation due to mental problems, and bodily pain. The quality of life score in each domain ranges between 0 and 100. A score close to zero indicates a poorer quality of life, while a score close to 100 indicates better quality of life. The Farsi equivalent of this questionnaire was available and its validity and reliability were approved . StudentFor statistical analysis, t-test and Chi-Square tests were performed, using SPSS 11.5 software. In the first phase of this study, 1840 questionnaires were distributed, of which 1511 (82%) were completed and returned. Respondents included 825 (54.6%) girls and 686 (45.4%) boys, all of whom were high school students. Based on the SFAR scale, 291 students (19.3%) had AR, of whom 153 (52.6%) were girls and 138 (47.4%) were boys. Symptoms during the preceding 12 months among students with AR included nose itching (54.3%), congestion (64.3%), and rhinorrhea (76.6%). The rate of eye itching and epiphora was 76.3%. There was a significant difference in symptoms compared with healthy students (P< 0.001). Of the students with AR, 176 (61.6%) were aware of their allergy, with 38 (13.1%) already having had an allergic test and 33 (87%) having received positive results. Asthma had already been diagnosed in 18.6% of cases, compared with eczema in 4.1% of cases and AR in 12% . Compared with healthy families, these diseases were more common among individuals with a family history of allergy (P<0.001). However, students with an allergy background had greater contact with tobacco smoke and opium compared with healthy controls (P<0.001). A comparison of AR symptoms between students with and without AR is shown .In students with AR, 122 (41.9%) had moderate-to-severe disease and 169 (58.1%) had mild disease. However, 43.1% of those with moderate-to-severe AR had a persistent condition and 56.9% had intermediate symptoms. It was also calculated that among patients with mild rhinitis, 28.6% had a persistent condition and 71.4% had intermediate symptoms, showing significant differences among those with AR . In the second phase of the study, the quality of life of the 126 students with AR as well as 133 healthy students was investigated using the SF-36 questionnaire. After calculating the scores, quality of life was studied across the eight domains, and it was determined that the lowest scores were related to general health then social functioning, while the highest score was related to physical functioning. A comparison of quality of life between students with and without AR is shown in The main objective of this research was to study the prevalence of allergic rhinitis among high school students (15–18 years of age) in the city of Kerman (south east of Iran) using the SFAR scale. Based on this study, the prevalence of AR is estimated at 19.3%. This compares with other studies carried out in Iran using the International Study of Asthma and Allergic in Childhood protocol (ISSAC), which have previously show a prevalence of 17.7% in Hamadan (west of Iran) , and 29.In this study, there were no significant differences between the two sexes, while there was a significant difference between the two sexes in the study undertaken in Rasht . In the The SAFARI study conducted in Hamadan  showed tIt has previously been shown that the quality of life at school of students with AR is lower than that of peers without AR, and it has also been estimated that AR is responsible for the loss of 3.5 million working days and 2 million school days each year . One stuIn our study the lowest score was related to general health and the highest score was related to physical functioning, and the only significant difference between groups related to physical functioning and bodily pain. These findings were in contrast with some other studies reported. Hellgren studied quality of life in noninfectious rhinitis (NIR) and demonstrated that NIR patients scored lower in vitality, physical function, and social function than healthy persons . Bunny eAs the scales used in this study were not the same as the scales used in similar studies in other cities, the prevalence of rhinitis in Kerman cannot be compared directly with previous reports from other cities. However, considering the value of the scale used in this study and the prevalence calculated in this study, as well as our knowledge about the effect of this disease, we can state with some confidence that AR reduces physical function and increases bodily pain among young people.As observed in this study, AR is highly dependent on smoking and opium habits. Therefore, reducing contact with these factors might reduce the allergic attacks which have such a negative impact on individuals’ performance. This finding highlights the negative consequences and high price associated with drug usage.


Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1) that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA) accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response.The Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica, Arabidopsis and Carica papaya. The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors ). To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants.A putative TcNPR1 is a functional ortholog of Arabidopsis NPR1, and is likely to play a major role in defense response in cacao. This fundamental knowledge can contribute to breeding of disease resistant cacao varieties through the application of molecular markers or the use of transgenic strategies.Our data indicate that the  PR) genes [Plants have evolved a complex network of defense responses, often associated with a response local to the site of infection -4. In adR) genes ,15-17.Arabidopsis [Non-Expressor of PR1 (NPR1). Studies that followed further documented that npr1 mutants displayed reduced expression of PR genes upon SA treatment and were more susceptible to pathogens [NPR1 was overexpressed, the resulting transgenic plants displayed increased resistance to pathogens, and were able to induce increased levels of PR genes in a dose-dependent fashion [NPR1 was originally identified by screening for mutants that were insensitive to SA  or benzothiadiazole (BTH)) in bidopsis ,18-20. Tathogens ,18,20,21 fashion .NPR1 encodes a protein containing ankyrin repeats and a BTB/POZ domain, both of which mediate protein-protein interactions in animals [NPR1 is constitutively expressed, and NPR1 protein is present as inactive oligomers in the cytoplasm of the cell. Upon SAR induction, the redox state of the cell is altered, resulting in the reduction of NPR1 to its active monomeric form. Monomeric NPR1 moves into the nucleus where it can affect the induction of PR genes. Although NPR1 itself has no DNA binding domains, it participates in the regulation of defense gene transcription via interactions with TGA transcription factors [Arabidopsis, two conserved cysteine residues (C82 and C216) have been shown to be essential to the oligomerization and cytoplasmic localization of AtNPR1 [ animals . NPR1 sh animals ,24. Rece animals ,17,25-27 factors ,28-33. IIt is believed that NPR1 also plays a role in the jasmonic acid (JA) signaling pathway and mediates the crosstalk between SA-JA defense pathways to fine-tune defense responses ,30,34-36NPR1-like genes have been characterized from Vitis vinifera (grapevine) [Nicotiana benthamiana leaves, the proteins were localized predominantly to the nucleus and triggered the accumulation of pathogenesis-related proteins PR1 and PR2. In addition, the silencing of a tomato NPR1-like gene leads to increased bacterial growth upon Ralstonia solanacearum infection in tomato [NPR1-like gene leads to increased susceptibility to tobacco mosaic virus [MpNPR1 gene in transgenic apple plants resulted in the up-regulation of PR genes and enhanced resistance to bacterial and fungal pathogens [Arabidopsis NPR1 confers resistance to Fusarium head blight in susceptible cultivar Bobwhite [nahG) are unable to accumulate salicylic acid and display increased susceptibility to rice blast [Arabidopsis NPR1 gene in rice leads to enhanced resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae [NPR1 has been isolated from rice (OsNPR1/NH1), and the overexpression of OsNPR1 in rice leads to enhanced resistance to both bacterial and oomycete pathogens [OsNPR1 is able to complement the Arabidopsis npr1-1 mutant [Arabidopsis NPR1, mutation of the corresponding cysteines (C82 and C216) in OsNPR1 also resulted in constitutive nuclear localization [A growing body of evidence has revealed that the salicylic acid dependent, NPR1-mediated defense pathway is also conserved in other plant species across wide phylogenetic distances. Two apevine) . When trn tomato . In tobaic virus . Similarathogens . In wheaBobwhite . Major eBobwhite . In addice blast . Overexp. oryzae . An orthathogens . Moreove1 mutant . Like Atlization . Thus, iTheobroma cacao L, (cacao) is a small tropical tree species endemic to the Amazon rainforest of South America. Cacao seeds are harvested and processed into cocoa beans and chocolate, providing an income for millions of small-holder farmers in West Africa, Central and South America, the Caribbean, Malaysia, Indonesia and other tropical areas. Pathogens are a major problem for cacao production, causing annual crop losses estimated at 30-40% [Moniliophthora perniciosa, the causal agent of witches' broom disease, Moniliophthora roreri, the causal agent of frosty pod rot [Phytophthora spp., the causal agent of black pod disease [t 30-40% . In its  pod rot -45 and s disease ,47. Outs disease -50.Theobroma cacao have been identified through gene expression analyses after hormone treatments [Colletotrichum gloeosporioides [PR genes have been isolated from cacao [Several defense-related genes in eatments ,47,51. Aorioides . Moreoveom cacao -55. Seveom cacao . All togNPR1 homologue from the tropical tree, Theobroma cacao. We show that Theobroma cacao NPR1 (TcNPR1) shares similar functions as Arabidopsis NPR1. It is able to partially complement the Arabidopsis npr1-2 mutation in transgenic Arabidopsis plants in a leaf infection assay and translocate into nucleus upon SA induction in the same manner as the endogenous Arabidopsis NPR1 protein.In this paper, we report the isolation and characterization of an Theobroma cacao NPR1 (TcNPR1). The degenerate primers were designed based on the alignment of NPR1 homologs from Arabidopsis, Brassica and Carica papaya and cDNA from cacao genotype Scavina6 (SCA6) leaf was used as template. A fragment of 1776 bp was isolated, cloned into pGEM sequencing vector and sequenced to reveal an intact coding sequence of the expected length and with high homology to the Arabidopsis NPR1 gene.Degenerate PCR was utilized to clone the full length cDNA of TcNPR1 gene was obtained by screening Clemson University Genomics Institute (CUGI) cacao BAC library using the putative cacao TcNPR1 cDNA clone as probe. Two BAC clones were found to contain the TcNPR1 gene: 2K13 and 11K17. The genomic sequence of TcNPR1 was isolated by primer walking sequencing from known sequence using clone 2K13. A similar strategy was performed to sequence a region of 1.1 kb containing the promoter sequence upstream of ATG start codon. The full sequence consisted of a 4.5 kb genomic region of TcNPR1 containing 1.1 kb promoter, four exons and three introns . Both the s Figure . Each hahttp://www.dna.affrc.go.jp/PLACE/[http://bioinformatics.psb.ugent.be/webtools/plantcare/html/[PR1 [AtNPR1. W-box was shown to be the binding site for SA-induced WRKY DNA binding proteins [Nicotiana tabacum) class I chitinase gene [TcNPR1 gene might be regulated by SA in a manner similar to AtNPR1. Interestingly, several cis-elements involved in light responsiveness and circadian control are also presented in the TcNPR1 promoter, suggesting that TcNPR1 might be also regulated by light.We analyzed the 1.1 kb promoter region of the TcNPR1 gene Figure  using pljp/PLACE/ and Planare/html/,62. Althare/html/,64. A vatml/[PR1 . In addiproteins , and wasase gene . All of TcNPR1 in various cacao tissues of Scavina6, including leaves from stage A (young/expanding), C (expanded/soft), E (mature/hardened), open flowers, unopened flowers, roots, seeds and fruit exocarps. TcNPR1 transcript was detected in all tissues tested  and lower in the later stages of development (stage E). The lowest expression of TcNPR1 in all tested tissue was observed in seeds whereas the expression was relatively high in fruit exocarps. In flowers, expression of TcNPR1 was higher in open flowers than in unopened ones. The expression of TcNPR1 in roots was at a moderate level, comparable to that in flowers and younger leaves.Semi-quantitative RT-PCR was performed to illustrate the basal expression level of d Figure , an exprNPR1 transcript accumulation can be increased by SA treatment of Arabidopsis leaves, we tested if TcNPR1 can respond to exogenous SA in the same manner. We applied various concentrations of SA to stage C leaves of two genotypes, Scavina6 and ICS1, which differ in their resistance to witches' broom disease (Scavina6 is more resistant) [Arabidopsis, the NPR1 gene is induced approximately 2-3 fold 24 hrs after treatment of leaves with 1 mM SA [TcNPR1 24 hours after SA application  . In Arab 1 mM SA . Semi-qun Figure . To quanTcNPR1 gene under the control of the E-12 omega promoter and introduced it into the Arabidopsis npr1-2 mutant to test if it can restore the mutant phenotype. One of the well characterized phenotypes of this mutant is the lack of SA-dependent activation of the PR1 gene [PR1 gene is thought to encode a protein active in defense response and has been used as a marker of SA pathway activation in many studies and in different plant species.To assess the function of TcNPR1, we placed the cacao PR1 gene ,21. The TcNPR1 transgenic lines, wild type Arabidopsis Col-0 along with the npr1-2 mutant were sprayed with 1 mM SA, and the expression of TcNPR1 and AtPR1 was determined by semi-quantitative RT-PCR 24 hr after induction. Five transgenic lines all showed heterologous TcNPR1 expression with varied expression levels , though the level of induction was not as high as in wild type Col-0. No PR1 gene induction was observed for transgenic lines 1 and 5. These results suggest that the TcNPR1 gene can at least partially complement the Arabidopsis npr1mutant and act to mediate SA dependent PR1 gene expression in Arabidopsis leaves but it may not act as efficiently as the endogenous NPR1 itself.Five independent s Figure . As expe0 Figure , but thes report . There wArabidopsis npr1 mutation is increased pathogen growth after bacterial infection of leaves [TcNPR1 overexpression in npr1-2 mutant can complement the mutant disease susceptible phenotype, we infected leaves from 5 transgenic lines with Pseudomonas syringae pv. tomato DC3000 (P.s.t.) by syringe infiltration. The results indicated that the npr1-2 mutant was more susceptible than Col-0 , which exhibited significant up-regulation of the PR1 after SA treatment, showed a 30 to 100 fold reduction of bacterial growth compared to the npr1-2 mutant. There was no significant change in bacterial growth rates in leaf disks of the other two transgenic lines tested (Line 1 and 5). To assess the relationship between the level of SA-dependent induction of PR1 and the degree of bacterial growth in the transgenic lines, we plotted the values as depicted in Figure 2 = 0.88), suggesting that the resistance conferred by TcNPR1 is via the SA dependent resistance pathway and further supports our hypothesis that TcNPR1 plays a similar function to Arabidopsis NPR1 in plant defense response.To quantify the disease symptom, bacterial assays were carried out to measure the titer of bacterial on infected leaves. The levels of bacterial in infected s Figure . The thrArabidopsis NPR1, we created transgenic Arabidopsis plants containing a TcNPR1-EGFP translational fusion and observed the subcellular localization of the fusion protein using confocal microscopy  was stably transformed into the npr1-2 mutant and we observed the localization of EGFP fusion protein before and 24 hrs after SA treatment in both leaf and root cells of four independent transgenic lines. We observed no EGFP fluorescence in negative control plants transformed with the identical vector lacking the TcNPR1-EGFP fusion gene . Consistent with the findings of others [Another hallmark of AtNPR1 function is its nuclear localization in response to treatment with SA ,59,70,71e Figure  and 5B. f others ,59, we oArabidopsis NPR1-EGFP protein based on reduced nuclear fluorescence observed in TcNPR1-EGFP transgenic plants, our results taken together indicate that TcNPR1, like Arabidopsis NPR1, can translocate into nucleus after SA induction and participate in the induction of defense related gene expression.The TcNPR1-EGFP fusion protein appeared to be evenly distributed in cytoplasm of leaf guard cells from water-treated 4-week-old soil grown plants, however, the protein accumulated moderately in guard cell nucleus 24 hours after SA application Figure . AlthougArabidopsis NPR1 can mediate the antagonism between SA and jasmonic acid (JA) by suppressing JA-responsive genes [npr1-2 mutant and five independent 35S:TcNPR1 transgenic Arabidopsis lines. Semi-quantitative RT-PCR showed that all five lines carrying the cacao transgene expressed TcNPR1 at moderate levels, and these did not change much during hormone treatments  in 0.015% Silwet L-77, two well established MeJA inducible genes (VSP2 and PDF1.2) were up-regulated in wild-type plant and in npr1-2 mutant, consistent with previous reports [PDF1.2 PCR products, and we determined that the smaller molecular weight band resulted from cDNA amplification and the large fragment resulted from amplification of genomic DNA (data not shown). As predicted, all five 35S:TcNPR1 transgenic lines exhibited levels of increased VSP2 and PDF1.2 that were similar to those seen in Col-0 plants. Upon treatment with a combination of 1 mM SA and 0.1 mM MeJA in 0.015% Silwet L-77, PR1 was expressed at a level similar to seen when plants were treated with SA alone, indicating that MeJA had no effects on SA-responsive PR1 expression. Both VSP2 and PDF1.2 expressed at significantly lower levels in Col-0 compared to that in npr1-2 mutant after SA and MeJA combined treatment, demonstrating the function of AtNPR1 in antagonistic repression of JA-responsive genes. All five transgenic lines containing TcNPR1 gene displayed reduced expression levels of JA-responsive gene expression upon SA and JA combined treatment compared to npr1-2 mutant, suggesting that TcNPR1 can also mediate SA-JA cross-talk in a manner similar to AtNPR1.It has been previously demonstrated that ve genes ,34,35, ss Figure . Exogeno reports . Two DNAVSP2 and PDF1.2 after the treatment of the combination of SA and MeJA, we measured the band intensity as above , a pattern similar to wild-type Col-0, suggesting that TcNPR1 restored the npr1 phenotype. These data support our hypothesis that TcNPR1 may play a role in mediating SA-JA cross talk as does Arabidopsis NPR1.To quantify the expression of e Figure . The datNPR1 homologous gene from the tropical tree, Theobroma cacao, and have generated transgenic Arabidopsis npr1-2 mutant lines overexpressing TcNPR1. All of our results are consistent with the hypothesis that TcNPR1 is a functional orthologue of the well characterized Arabidopsis gene. TcNPR1 complemented each of the major Arabidopsis npr1-2 mutant phenotypes that were tested. Over-expression of TcNPR1 in the npr1-2 mutant conferred PR1 up-regulation after SA treatment and increased resistance to Pseudomonas syringae pv. tomato DC3000  for witches' broom disease resistance is tightly linked to the TcNPR1 gene [TcNPR1 gene serves as a key candidate gene for generation of molecular markers that can be used for marker assisted selection of new disease resistant varieties. In addition, TcNPR1 expression levels could be modified in transgenic cacao varieties to develop broad-spectrum disease resistance. This approach has already been successful in several species but to our knowledge, has not yet been deployed in commercial production for any species. However, consumer and industry reluctance to accept transgenic plant technology remains a formidable barrier to development of any transgenic cacao varieties for commercialization.Plant diseases, especially pathogenic fungi, are estimated to cause about 30-40% yield loss on cacao annually ,81, and PR1 gene , thus thTcNPR1 gene and its heterologous complementation in Arabidopsis allowed us to rapidly characterize the function of this defense-related gene. The up-regulation of PR1 and increased bacterial resistance in transgenic Arabidopsis npr1-2 mutants strongly supported that TcNPR1 is a functional ortholog of Arabidopsis NPR1, and vital component in SA-dependent signaling pathway in Theobroma cacao. Our results provide potential opportunities to enhance disease resistance in this crop species through conventional breeding or biotechnological approaches. Further investigation is needed to identify the TcNPR1 interacting transcription factors and their downstream targets in cacao and to reveal further details of the molecular mechanisms of the role TcNPR1 plays as a central mediator of the plant defense response.The isolation of the NPR1 cDNA sequences from Arabidopsis (U76707), Brassica napus (AF527176), and Carica papaya (AY550242) were aligned using the ClustalW program v1.8 [ram v1.8 . Degenerhttp://www.clontech.com/) according to the manufacturer's instructions.Total RNA was isolated from cacao leaves from variety Scavina6 as previously described . Cacao lPCR reactions were performed using cacao leaf 2.5 μl cDNA from first strand synthesis from SMART RACE cDNA Amplification Kit, 10 μl Redi-prime PCR mix  and 5 μM of the above degenerate primers. Following denaturation (94° for 5 min.), PCR was performed for 32 cycles using the following condition , followed by a 5 min. extension at 72°. PCR products were resolved on 1% agarose gels, purified with the GENECLEAN II Kit  and cloned into the pGEM-T-Easy vector  according to the manufacturer's instructions. DNA sequencing was performed at the Penn State Genomics Core Facility using an ABI Hitachi 3730XL DNA Analyzer. The resulting clone was designated as pGEM-TcNPR1.Theobroma cacao BAC filter arrays constructed using genomic DNA from genotype LCT-EEN 37 were purchased from the Clemson University Genomic Institute http://www.genome.clemson.edu/. Filter arrays were blocked for 4 hours at 60°C in a solution containing 1% BSA, 1 mM EDTA, 7% SDS, and 0.25 M sodium phosphate. PCR generated TcNPR1 cDNA fragment labeled with 32P dCTP using the MegaPrimer DNA Labeling System  according to the manufacturer's instructions was added and hybridized overnight at 60°C. The next day, the filter arrays were washed twice in 2× sodium chloride/sodium citrate (SSC), 0.5% sodium dodecyl sulfate (SDS) for 20 minutes at 60°C. Radiographic imaging was performed via storage phosphor imaging . After filter alignment and clone number identification, a BAC clone (2K13) containing a putative TcNPR1 fragment was obtained from a frozen stock. The sequence of TcNPR1 genomic fragment was acquired by series sequencing of the BAC clone from ATG start codon. Sequencing primer was designed based on the TcNPR1 cDNA at the first round and following series primers were designed based on the known sequence resulting from previous sequencing. Introns were identified by aligning the genomic sequence and full length cDNA using SPIDEY software http://www.ncbi.nlm.nih.gov/spidey/. The same strategy was applied to clone the 1.1 kb promoter region upstream of the ATG. Forward and reverse sequencing was also performed to validate the sequence.Arabidopsis NPR1 protein sequence (At1g64280) and putative cacao NPR1 protein sequences (genbank accession HM117159) were aligned using the ClustalW program v1.83 [http://smart.embl-heidelberg.de/.For sequence verification the am v1.83 . The TcNTotal RNA was isolated from Scavina6 leaves stages A, C and E , open fTcNPR1  and TcActin . RT-PCR reactions were set up using 1 μl of 1/2 diluted cDNA and 5 μM of the TcNPR1 or TcActin primers. Titration of cycles was carried out and it was determined that the PCR amplification of TcNPR1 was within its linear range at 27 cycles using the following condition: 94°C for 30 sec., 56°C for 30 sec, 72°C for 1 min. Similarly, PCR of TcActin was performed under non-saturation conditions within the linear range . TcActin served as a cDNA loading control.Semi-quantitative RT-PCR was performed using intron-spanning primers for TcNPR1 expression as described above. The PCR products were analyzed on 1% agarose gel, stained with ethidium bromide. The expression values of TcNPR1 and TcActin were quantified using ImageQuant software  as described in [TcNPR1 was calculated by comparing with the expression of TcActin.The leaves of two to three-month old cacao plants generated by rooted cuttings from two different genotypes (ICS1 and Scavina6) were sprayed with salicylic acid (SA) dissolved in water at three different concentrations, 1 mM, 2 mM and 4 mM. Control plants were treated with water. Plants were grown in a greenhouse under conditions previously described  and leafribed in  and relaAll binary plant transformation vectors were constructed by incorporating the genes of interest into pCAMBIA-1300 binary transformation vector containing plant selectable marker for hygromycin resistance .TcNPR1 coding sequence fragment was generated by PCR using pGEM-TcNPR1 as described above and included XmaI and NotI restriction sites at the 5'- and 3'-ends respectively . The fragment was cloned into pGEM T-Easy  according to the manufacturer's instructions and sequenced for integrity. The TcNPR1 coding sequence was then cloned into the XmaI and NotI sites of an intermediate cloning vector (pE2113) between E12-Ω promoter [TcNPR1 gene cassette was excised from pE2113 using PvuII and ligated into the SmaI site of pCAMBIA-1300.Binary Vector p35S:TcNPR1 - The promoter  and 35SCLigations were performed overnight at 16° with 3Units of T4 DNA ligase .AtNPR1 coding sequence fragment was generated by PCR using the AtNPR1 cDNA clone U13446 from Arabidopsis Biological Resource Center http://www.biosci.ohio-state.edu/~plantbio/Facilities/abrc/abrchome.htm and included NcoI sites at the 5'- and 3'-ends . The PCR fragment was cloned into pGEM T-Easy and sequenced. The resulting intermediate plasmid was designated pGEM-AtNPR1. The AtNPR1 coding sequence was then excised by NcoI from pGEM-AtNPR1, and blunt-end cloned into pE2113 between E12-Ω promoter [XmaI and NotI fragment. Contently 3.1 kb fragment containing the AtNPR1 gene cassette was obtained by digestion with PvuII, and blunt-end ligated into the SmaI site of pCAMBIA-1300.Binary vector p35S:AtNPR1 - The promoter  and 35SCEcoRI and HindIII sites of pCambia1300. The resulting binary vector was designated pXCGH. PCR generated TcNPR1fragment, including SmaI and KpnI sites at the 5'- and 3'-ends  was first cloned into pGEM T-Easy (pGEM-TcNPR1-EGFP). The sequence was verified, the DNA fragment was excised with SmaI and KpnI and blunt ends ligated into the blunt-ended NcoI site of pXCGH positioned between the E12-Ω promoter and at the 5'end of the EGFP coding sequence to generate the binary vector p35S:TcNPR1:EGFP.Binary Vector p35S:TcNPR1:EGFP - The cassette of E12-Ω promoter and EGFP on the intermediate cloning vector pE2113 was cloned into AtNPR1 coding sequence was digested with NcoI and the fragment was ligated into the NcoI site of pE2113 as described above. The 3.6 kb fragment containing the AtNPR1- EGFP fusion gene cassette was digested with SalI and EcoRI and cloned into the SalI and EcoRI sites of pCAMBIA-1300.Binary vector p35S:AtNPR1:EGFP - The pGEM-AtNPR1 containing Agrobacterium tumefaciens strain AGL1 by electroporation, as previously described in [Arabidopsis Col-0 plants were grown in a Conviron growth chamber (Model No. MTPS144) maintained at 22°C, under a 16:8::L:D cycle. Light intensity was maintained at 200 μM/m2·s with Octron 4100K Ecologic bulbs . To increase the number of inflorescences, plants were cut back after bolting, and allowed to re-grow. The floral dip method was used to transform Arabidopsis as described previously [600 = 1.2. Cells were centrifuged at 1,500 × g for 6 minutes and re-suspended in 300 mls of a solution containing 2.15 g L-1 MS salts, 5% sucrose, 0.02% Silwet-77. The flowers were dipped in the solution for three seconds, domed to remain humidity and covered with black cloth. The cloth was removed the next day and plants were regularly watered until seed maturation.The binary vectors described above were introduced into ribed in . Arabidoeviously . Briefly-1 hygromycin B. Plates were place in a Conviron growth chamber under the same light and temperature conditions as above. After 10 days, germinated seedlings were examined for leaf development and root elongation. Those seedlings that showed root elongation were transferred to soil and allowed to grow. Transformations were performed with the following vectors: p35S:TcNPR1, p35S:AtNPR1, p35S:TcNPR1:EGFP, and p35S:AtNPR1:EGFP constructed as described above, and control vectors p35S: EGFP  supplemented with 25 μg ml00.0126, )and pCamArabidopsis Col-0 and npr1-2 mutants and five independent transgenic lines growing in soil were sprayed with 1 mM SA, along with water-treated control plants. Three biological replicates, each containing leaves from 5 individual plants were collected 24 hrs after treatment. Total RNA was isolated from treated and control samples using RNeasy plant mini kit . cDNA was generated as described above. Semi-quantitative RT-PCR was performed as described above to measure the expression of TcNPR1 and AtPR1. Arabidopsis Ubiquitin served as cDNA loading and normalization control. The following primer sets and conditions were employed:Four week-old wild type TcNPR1-5': ATGGATTCCCGTCTGGAACTTGGT; TcNPR1-3': TCTGGAGTGTCATTTCCTCCGCAT . AtPR1-5': CTCGAAAGCTCAAGATAGCCCACA; AtPR1-3': CTTCTCGTTCACATAATTCCCACG . Ubiquitin-5': ACCGGCAAGACCATCACTCT; Ubiquitin-3': AGGCCTCAACTGGTTGCTGT . The conditions of PCR were determined by cycle titration to avoid saturating conditions. The relative expression levels were determined as described above.Pseudomonas syringae pv. tomato DC3000 (P.s.t.) was grown on Difco Pseudomonas agar (PA)  supplemented with rifampicin (100 μg ml-1) and kanamycin (25 μg ml-1) at 25°C for 48 hrs. Cells were scraped from plates using a bacterial inoculating loop and re-suspended in water. Plant infection assays and bacterial growth assays were carried out as described previously in [npr1-2 mutant overexpressing TcNPR1 coding sequence were infected with P.s.t. at OD600 = 0.002. Briefly, three days after inoculation leaf disks from treated leaves of 2 independent replicate plants were pooled for a single sample. Data represents means ± SE (cfu/mg FW) of three biological replicates per treatment and statistical differences were determined by Single factor ANOVA analysis.ously in . Five inFor observations of green leaves, four week-old soil-grown transgenic plants containing one of transgenes 35S:AtNPR1:EGFP, 35S:TcNPR1:EGFP, 35S:EGFP and plants transformed with empty binary vector pCambia 1300 were sprayed with either a 1 mM solution of SA in water or water. For root observations, control and transgenic seed were germinated on MS agar or MS agar supplemented with 0.5 mM SA  and seedArabidopsis Col-0, npr1-2 mutants and five independent transgenic lines containing p35S:TcNPR1 were sprayed with a combination of 1 mM SA and 0.1 mM MeJA in 0.015% Silwet L-77. Plants treated with 1 mM SA alone in water, 0.1 mM MeJA alone in 0.015% Silwet L-77 and water with 0.015% Silwet L-77 served as control treatment. Three biological replicates each consisting of leaves from 5 individual plants were collected at 48 hrs after treatment, total RNA was isolated, cDNA was synthesized and semi-quantitative RT-PCR was performed as described above to determine the transcripts level of TcNPR1 and AtPR1. For expression analysis of VSP2 and PDF1.2, following primer sets and conditions were used to maintain the reaction in its linear amplification range. VSP2 Forward: TACGGTCTCGGCATCCGTTC; VSP2 Reverse: CCTCAAGTTCGAACCATTAGGCT . PDF1.2 Forward: TCATCATGGCTAAGTTTGCTTCCATC; PDF1.2 Reverse: TGTCATAAAGTTACTCATAGAGTGAC . The PCR products were analyzed on 1% agarose gel, stained with ethidium bromide. The expression values of AtVSP2 and AtPDF1.2 were quantified using ImageQuant software  as described in [AtUbiquitin.Four weeks old soil-grown wild type ribed in  and relahttp://esttik.cirad.fr/ under the following accession numbers: At1g64280 (NPR1), At2g14610 (PR1), At5g24770 (VSP2), At5g44420 (PDF1.2), At3g52590 (ubiquitin), HM117159 (TcNPR1) and CL33contig2 (TcActin).Sequence data from this article can be found in the Arabidopsis Genome Initiative, GenBank/EMBL databases or Esttik database NPR1: non expressor of PR genes 1; SA: salicylic acid; INA: 2,6-dichloroisonicotic acid; BTH: benzothiadiazole; BTB/POZ: broad complex, tramtrack and bric a brac/pox virus and zinc finger; JA: jasmonic acid; PR: pathogenesis related; SAR: systemic acquired resistance; NLS: nuclear localization signal; MEJA: methyl jasmonate; VSP2: vegetative storage protein 2; PDF1.2: plant defensin 1.2; QTL: quantitative trait locus.ie, sequence analysis, gene expression studies, phenotypic analysis of transgenic Arabidopsis plants, confocal microscopy observations and drafted the manuscript. SNM participated in the design of the study, directed the transformation vector construction and transgenic lines generation, and participated in drafting of the manuscript. YL participated in transgenic Arabidopsis plants analysis and helped to analyze the sequence. JV cloned the TcNPR1 gene. MJG conceived the study, drafted the manuscript and gave advice on experimental design, data analysis and execution. All authors read and approved the final manuscript.ZS performed most of the experiments,


Vicia faba L.) is an important food legume crop, grown for human consumption globally including in China, Turkey, Egypt and Ethiopia. Although genetic gain has been made through conventional selection and breeding efforts, this could be substantially improved through the application of molecular methods. For this, a set of reliable molecular markers representative of the entire genome is required.Faba bean (A library with 125,559 putative SSR sequences was constructed and characterized for repeat type and length from a mixed genome of 247 spring and winter sown faba bean genotypes using 454 sequencing. A suit of 28,503 primer pair sequences were designed and 150 were randomly selected for validation. Of these, 94 produced reproducible amplicons that were polymorphic among 32 faba bean genotypes selected from diverse geographical locations. The number of alleles per locus ranged from 2 to 8, the expected heterozygocities ranged from 0.0000 to 1.0000, and the observed heterozygosities ranged from 0.0908 to 0.8410. The validation by UPGMA cluster analysis of 32 genotypes based on Nei's genetic distance, showed high quality and effectiveness of those novel SSR markers developed via next generation sequencing technology.V. faba genome. These novel markers are valuable for constructing genetic linkage maps, future QTL mapping, and marker-assisted trait selection in faba bean breeding efforts.Large scale SSR marker development was successfully achieved using next generation sequencing of the  Vicia faba L.) is an important temperate legume, grown for human consumption and animal feed due to its high protein and fibre content [Faba bean  technologies enable the generation of large volumes of sequence efficiently and cost-effectively ,6. This et al. developed primers to 25 SSR loci detected in chromosome 1 DNA libraries [et al. developed primers to 54 SSR loci [et al. developed 11 EST-SSR loci primers [A limited number of characterized SSR loci <120) which have been validated over relatively few genetic backgrounds are available for faba bean. Initially, Pozarkova 0 which hSSR loci  and Gong primers . Most re primers .in silico analysis, this approach has the potential to substantially broaden the field of comparative studies to species where limited or no sequence information is available.Besides providing a cost-effective valuable source for molecular marker generation, the identification of SSR within ESTs is an effective approach for gene discovery and transcript pattern characterization, particularly if through mapping an EST-SSR or EST marker is significantly associated with a QTL -18. ThisThe present study identified high-quality putative SSR loci and flanking primer sequences cheaply and efficiently using the Roche 454 GS FLX Titanium platform. The resultant SSR sequences were characterized and validated through successful amplification of randomly selected target loci across a selection of faba bean genotypes from diverse geographic origin.A total of 247 faba bean accessions were selected from the National Genebank of China held at the Institute of Crop Science (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing. Of these, 100 originated from China, 54 were from other Asian countries, 39 were from Europe, 30 were from Africa, 14 were from the America, 9 breeding lines were sourced from the ICARDA  faba bean breeding program and one was from Oceania  short read archive and given the accession number SRP006387.Subsequently, entire libraries were equally pooled and subjected to 454 sequencing with GS-FLX Titanium reagents at Beijing Autolab Biotechnology Co., Ltd (China). All processing and analyses of the sequencing data was performed with GS-FLX Software v2.0.01 . Using a series of normalization, correction and quality-filtering algorithms, the 454 sequencing data were processed to screen and filter for weak signals and low-quality reads, and to trim the read ends for 454 adaptor sequences using the EMBOSS  softwareMicrosatellite identification) tool (http://pgrc.ipk-gatersleben.de/misa/) was configured to locate a minimum of 10 bp: monomers (×10), 2-mers (×6), 3-mers (×5), 4-mers (×5), 5-mers (×5) and 6-mers (×5). This tool allowed the identification and localization of perfect microsatellites as well as compound microsatellites. The maximum size of interruption allowed between two different SSR in a compound sequence was 100 bp. Subsequently, Primer 3.0 (http://www-genome.wi.mit.edu/genome_software/other/primer3.html.) was used to design primer pairs to the flanking sequences of each unique SSR.The software MISA  and SSR position was searched and analyzed for using a bespoke program written in MISA files  and plotTaq DNA polymerase , 1 × PCR BufferII, 1.5 mM MgCl2, 25 μM of dNTP, 0.4 μM primer, and 50 ng of genomic DNA. Microsatellite loci were amplified on a Heijingang Thermal Cycler  with the following cycle: 5 min initial denaturation at 95°C; 35 cycles of 30s at 95°C, 30s at the optimized annealing temperature (Table Polymerase chain reactions (PCR) were performed in 20 μl reaction volumes containing 0.5 U of V. faba accessions , expected (He) heterozygosities and observed (Ho) heterozygosities were calculated using POPGEN1.32 [The genotyping data was subsequently used to determine genetic relationships among 32 PGEN1.32 . The cluPGEN1.32  by usingPGEN1.32 .V. faba library was 73.9%. Among the 276 clones sequenced, 31.9% contained SSR sequences within an insert that ranged from 0.2 to 1.0 kb in size.The recombination rate within the constructed SSR-enriched A total of 578,251 reads were generated from the pooled library, and 532,599 read sequences were used for further analysis after adaptor removal. Adenine was the most abundant nucleotide (30%), followed by thymine (27%), guanine (22%) and cytosine (21%). The mean GC content was 43%. The average length of read sequence was 305 bp, with a maximum length of 635 bp  was located within compound repeats. The majority of identified SSR motifs (83%) were located between the 5’-terminus and mid regions of the cloned sequences, and within 200 bp of the 5’-terminus n, particularly at the 11–12 bp length. The dinucleotide repeats (AC/GT)n and (AG/CT)n were predominant, representing 99.2% of all of the dinucleotides characterised. Triucleotide (AAC/GTT)n repeats were the most abundant (96.5%). Twenty two unique tetranucleotide repeat motifs were identified, with the most common being AGAT/ATCT (66.4%), ACAG/CTGT (19.3%) and ACAT/ATGT (9.1%). Pentanucleotide and hexanucleotide motifs were far less frequent, together comprising only 0.1% of the total SSR detected. The dominant pentanucelotide motif was AGAGT/ATCTC (23.8%) and the most common hexanucelotide motif was ACACGC/CGTGTG (49.5%) 12(ACG)37 and noted as C* type) and those with an interruption between two motifs ( ie (AAC)7gtcaat(AAC)5 and noted as C type). In total, 1,893 C* type and 59,369 C type compound SSR loci were detected among those sequenced, reflecting the complexity of the faba bean genome.Of the 150 primer pairs selected for validation of SSR locus amplification, 102 produced a reproducible and clear amplicon of the expected size. Of these, 94 63%) were polymorphic among thirty-two genotypes assessed (Table % were poThe dendrogram showed that the 32 faba bean genotypes fell into four distinct clusters Figure . ClusterVicia faba. Given the huge number of marker loci identified in this study, future SSR marker optimisation may be best focussed on those comprising trinucleotide repeats. These repeats are generally more robust since they are reported to give fewer “stutter bands” than those based on dinucleotide repeats [This study demonstrated that massively parallel sequencing technology offers opportunity to quickly identify large numbers of high quality SSR with diverse motifs from a genetically orphaned species such as  repeats ,32. Also repeats -35. Whil repeats .V. faba[The conversion of SSR-containing sequences into single locus markers may have a low success rate due to complex and/or insufficient flanking sequence. For example, just 20% of the identified dinucleotide repeats from spruce were converted to clear, discrete markers . SimilarV. faba. AnotherPreviously, a total of 304,680 reads were generated and 802 EST-SSR primer pairs were designed from transcriptome sequencing of faba bean . From thV. faba by application of 454 GS FLX Titanium sequencing technology.This work represents a major advance in the identification of large numbers of informative SSR loci in Na: Number of alleles; He: Expected heterozygosities; Ho: Observed heterozygosities.SSR: Simple sequence repeat; QTL: Quantative Trait Locus; MAS: Marker-assisted selection; NGS: Next generation sequencing; EST: Express sequence tag; NCBI: National Center for Biotechnology Information; CTAB: Cetyltrimethylammonium bromid; MISA: Microsatellite identification; The authors declare that they have no competing interests.V. faba. XLS and JYJ took charge of quality inspection of the DNA library. JJH and XYZ participated in conceiving the study and the manuscript drafting. XXZ designed and coordinated the study, and assisted in preparing the manuscript. All authors read and approved the final manuscript.TY performed bioinformatic analysis, primer design and drafted the manuscript. SYB created the SSR sequences rich DNA library, and participated in 454 sequencing. RF assisted in designing experiment and preparing the manuscript. TJJ tested SSR markers. JPG and YHH prepared all the seeds of  Table S1. The information of Vicia faba L. germplasm used in this study.Click here for file Table S2. The primer pairs were successfully designed by Primer3.Click here for file Figure S1. Frequences of different SSR repeat motif types in mononuceotide.Click here for file Figure S2. Frequences of different SSR repeat motif types in dinuceotide.Click here for fileFrequences of different SSR repeat motif types in trinuceotide.Click here for file Figure S4. Frequences of different SSR repeat motif types in tetranuceotide.Click here for file Figure S5. Figure S3. Frequences of different SSR repeat motif types in pentanuceotide.Click here for file Figure S6. Frequences of different SSR repeat motif types in heaxanuceotide.Click here for file


Pinus sylvestris var. mongolica (Mongolia pine) and Larix principis-rupprechtii (larch) in a forest-steppe ecotone, northern China (June 2006 to October 2009). We analyzed the relationship between seasonal dynamics of SR, HR, AR and soil temperature (ST), soil water content (SWC) and normalized difference vegetation index . Our results showed that ST and SWC were driving factors for the seasonal dynamics of SR rather than plant greenness, irrespective of stand age and forest type. For ∼15-year-old stands, the seasonal dynamics of both AR and HR were dependent on ST. Higher Q10 of HR compared with AR occurred in larch. However, in Mongolia pine a similar Q10 occurred between HR and AR. With stand age, Q10 of both HR and AR increased in larch. For Mongolia pine, Q10 of HR increased with stand age, but AR showed no significant relationship with ST. As stand age increased, HR was correlated with SWC in Mongolia pine, but for larch AR correlated with SWC. The dependence of AR on NDVI occurred in ∼35-year-old Mongolia pine. Our study demonstrated the importance of separating autotrophic and heterotrophic respiration components of SR when stimulating the response of soil carbon efflux to environmental changes. When estimating the response of autotrophic and heterotrophic respiration to environmental changes, the effect of forest type on age-related trends is required.We examined the effects of forest stand age on soil respiration (SR) including the heterotrophic respiration (HR) and autotrophic respiration (AR) of two forest types. We measured soil respiration and partitioned the HR and AR components across three age classes ∼15, ∼25, and ∼35-year-old  We analyzed the relationships between SR, HR, AR and ST, SWC and NDVI . Our objective was to determine if forest type modulated the seasonal dynamics of SR and the AR and HR components and their responses to ST, SWC and NDVI across different aged stands. We tested the following hypotheses: (1) ST, SWC and NDVI will have significant effects on the seasonal dynamics of SR across varied aged stands, irrespective of forest type. This was based on the findings that SR depended on ST, SWC Previous studies on the effect of stand age on forest carbon efflux focused on the measurements of total SR in a single forest type. Researchers have observed that total SR increased The administration of the Saihanba Forestry Center gave permission for this research at each study site. We confirm that the field studies did not involve endangered or protected species.−3 with litter and fine root biomass increasing with stand age  and L. principis-rupprechtii (larch) the most dominant species.The study was conducted at Saihanba Forestry Center, Hebei Province, northern China  adjacent to the Beijing-Tianjin region. Our study site lay within a typical forest-steppe ecotone in a temperate region. The climate is semi-arid, semi-humid, with long cold winters (November to March) and a short spring and summer. The annual mean air temperature and precipitation from 1964 to 2004 were −1.4°C and 450.1 mm, respectively. The soils are predominantly sandy, accompanied by meadow and marsh-type. The soil has low nutrient content with the organic carbon levels at 0.71–1.88% and total nitrogen at 0.08–0.19%. The bulk density ranged from 0.74 to 1.06 g cmtand age . PrimaryLeymus chinensis. In combination, these stands made up a chronosequence from 15 to 35 years old, with the oldest stand ready for intermediate cutting. Neither fertilization nor drainage works had been carried out since tree establishment in any of the stands. Moreover, the topography at all six stands is predominantly flat with a similar slope, position and elevation (To assess the effects of forest age on soil respiration (SR), autotrophic respiration (AR) and heterotrophic respiration (HR) across different forest types, we selected six stands with the same planting density (4995 stem/ha), including three age classes of Mongolia pine and larch. These were pure plantations established on former meadow grassland dominated by levation . The sel2 flux system . During the growing season, five polyvinyl chloride (PVC) collars  were inserted 3 cm into the soil in each plot and left in situ throughout the study. The five PVC collars were placed in each plot, one in each of the four corners and the fifth in the middle. Live plants inside the collars were clipped at the soil surface 1 day before each measurement. SR was measured every 15–20 days. To minimize the daily variation in SR and represent the daily mean, measurements were made between 0800 and 1100 h 2 flux system was kept in an isolated/heated container to maintain the temperature above freezing. Winter SR was measured monthly except in February.SR was measured from June 2006 to October 2009 using a Li-8100 soil CODuring respiration measurements, ST was recorded in each collar at 5 cm soil depth with the Li-8100 temperature probe. Continuous measurements of ST were recorded at 30-min intervals with StowAway loggers  inserted in the soil at each site. SWC at 0–10 cm was measured inside the collars using time domain reflectometry . No data for SWC were obtained during the winter because the probe could not be fully inserted into the frozen soil.https://wist.echo.nasa.gov/api.NDVI is derived from the red: near-infrared reflectance ratio:Harmonic (Fourier) analysis was used to remove the excess noise remaining in the NDVI time-series from satellite-borne data sources and to obtain reasonably smooth continuous daily data 2 and 70 cm deep) in October 2006. The 70-cm-long PVC collars isolated old plant roots, preventing new roots from growing inside the collars. This method has been successfully applied in various ecosystems to separate HR from the total SR To detect the response of HR and AR to environmental changes, five additional soil collars per plot were placed with deep PVC collars  with the soil CO2 efflux measured at the stands believed to represent HR. AR was calculated as the difference between SR and HR.To examine the transient response of dead root decomposition, we commenced the COR is observed SR , t is the concurrent ST (5 cm depth), with 0R being the basal respiration at temperature of 0°C and β the fitted parameters obtained using least squares nonlinear regression with SigmaPlot V. 8.02. The β values were used to calculate apparent Q10 values, which describes the change in respiration rate over a 10°C increase in t using:We examined the relationships between SR, HR, AR and ST by fitting exponential functions to the data from each stand using the following equation:15, the respiration values at a reference temperature of 15°C (without stimulation introduced by photosynthetic activity and without water limitations) 10 coefficient calculated from 15 and SWC and NDVI. 15R is the respiration flux at a constant temperature of 15°C, R the measured respiration rate, Q10 from T the ST at a 5 cm depth.In addition, we calculated RTo evaluate if SR, HR and AR significantly differed among different aged stands, forest type and measurement time, we used a three-way ANOVA. The relationships between SR, HR, AR and ST, SWC and NDVI were examined using regression analysis. All statistical analyses were performed with a significance level of 0.05 using SPSS .10 of SR across three stand ages ranged from 3.49–4.31 and 3.10–3.67 for larch and Mongolia pine, respectively  was similar across three stand ages of both forest types . HR showed no significant differences between the two stands . In contrast, Mongolia pine showed a similar Q10 for HR compared with AR (2.61 vs. 2.76). As the stand age increased, the Q10 of both HR  and AR  increased in larch , but AR showed no significant relationship with ST in ∼35-year-old stand  that ST, SWC and NDVI will impose significant effects on the seasonal dynamics of SR across different aged stands, irrespective of forest type. However, we found that the seasonal dynamics of SR were principally driven by ST and SWC, not by NDVI, across different aged stands, irrespective of forest type. Forest SR is closely related to gross primary productivity (GPP) and leaf area index (LAI) d stands . These rd stands . Our res10 for HR compared with AR in larch and a similar Q10 for AR with HR in Mongolia pine , who observed maximal contribution of AR to total SR in summer for evergreen forest and in autumn for deciduous forest In our second hypothesis, we expected greater sensitivity in HR to variation in ST compared with AR. However, we observed a mixed result. In ∼15-year-old stand, we found a higher Qlia pine , 7a. TheAccording to our second hypothesis, we expected that the seasonal dynamics of AR were more dependent on NDVI rather than HR. However, we found that AR was significantly affected by NDVI only in ∼35-year-old Mongolia pine . We did et al. (2010), who found a significant correlation between AR and SWC in Quercus-dominated deciduous stand, not evergreen stands of Abies holophylla. One possible reason for this difference between Mongolia pine and larch in the dependence of AR and HR on SWC may be associated with reduced root longevity of deciduous tree species (<1 year) compared with evergreen species (<1–12 years) We expected a higher dependence of HR on SWC compared with AR because AR was reported as less responsive to water variability compared with HR In summary, we tested the effects of forest type on SR, AR and HR across different aged stands in the same location to prevent confounding from climatic and edaphic conditions. We observed that ST and SWC were significant factors controlling the seasonal dynamics of total SR, irrespective of forest age and type. However, the response of AR and HR to ST, SWC and NDVI differed across various aged stands, with the effects of stand age dependent on the forest type. These results suggest that in stimulating the response of forest carbon efflux to environmental changes, we should consider the effects of stand age and the influence of forest type on this age-related trend. Furthermore, our study emphasized considering the response of HR and AR to environmental changes separately when predicting the response of soil carbon efflux in different aged forests to global climate changes. Future research should attempt an in-depth understanding of the effects of more functional types on carbon efflux across different aged stands.We thank two anonymous reviewers for their constructive suggestions on an early version of this manuscript.Figure S115) and NDVI across different aged stands. The relationships are shown for ∼15 (a), ∼25 (b), ∼35-year-old stands (c) of Larix prinicipis, ∼15 (d), ∼25 (e), and ∼35 year-old stands (f) of Pinus sylvestris.Relationships between soil respiration at 15°C (SR(TIF)Click here for additional data file.Figure S215) and soil water content (SWC) across different aged stands. The relationships are shown for ∼15 (a), ∼25 (b) and ∼35-year-old stands (c) of Larix prinicipis, ∼15 (d), ∼25 (e) and ∼35- year-old stands (f) of Pinus sylvestris.Relationships between soil respiration at 15°C (SR(TIF)Click here for additional data file.Figure S3Larix prinicipis, ∼15 (d), ∼25 (e) and ∼35-year-old stands (f) of Pinus sylvestris.Relationships between soil respiration (SR) and NDVI across different aged stands. The relationships are shown for ∼15 (a), ∼25 (b) and ∼35- year-old stands (c) of (TIF)Click here for additional data file.Figure S4Larix prinicipis, ∼15 (c), ∼25 (d), and ∼35-year-old stands (e) of Pinus sylvestris. The data of ∼35-year-old stands L. prinicipis were absent because of the damage of StowAway loggers inserted in the soil.Relationships between ST (soil temperature at 5 cm depth) and NDVI for non-growing season (dotted line) and growing season (solid line). The relationships are shown for ∼15 (a) and ∼25-year-old stands (b) of (TIF)Click here for additional data file.


Purpose. This study examines the effects of fundus fluorescein angiography (FFA) procedure on ocular pulse amplitude (OPA) and intraocular pressure (IOP). Materials and Methods. Sixty eyes of 30 nonproliferative diabetic retinopathy patients  were included in this cross-sectional case series. IOP and OPA were measured with the Pascal dynamic contour tonometer before and after 5 minutes of intravenous fluorescein dye injection. Results. Pre-FFA mean OPA value was 3.05 ± 1.36 mmHg and post-FFA mean OPA value was 2.93 ± 1.28 mmHg (P = 0.071). Pre-FFA mean IOP value was 17.97 ± 1.99 mmHg and post-FFA mean IOP value was 17.81 ± 2.22 mmHg (P = 0.407). Conclusion. Although both mean OPA and IOP values were decreased after FFA procedure, the difference was not statistically significant. This clinical trial is registered with Australian New Zealand Clinical Trials Registry number ACTRN12613000433707. Fundus fluorescein angiography (FFA) is a procedure for the examination of chorioretinal circulation. It can be used to confirm the diagnosis, monitor the progress of posterior segment diseases, and assess the efficacy of certain treatments like laser photocoagulation and intravitreal injections. Although FFA is a safe procedure, some serious complications like allergic skin rash and anaphylaxis could be seen rarely . The effThe dynamic contour tonometry (DCT) is a noninvasive and direct intraocular pressure (IOP) measuring device and is considered to accurately measure the IOP independent of the corneal thickness or corneal elasticity . It alsoThe OPA is generally considered as the difference between systolic and diastolic values of the pulsatile IOP. It gives us an opinion about the choroidal blood flow corresponding with the heart pulse as a function of time . ChoroidFFA is an imaging procedure using fluorescence ability, and it has been thought to have little or no effect on ocular physiology. The rationale of this study was that fluorescein sodium passes freely through the choroidal vessels into the extravasal space and this would have an influence on choroidal blood flows, either by an osmotic effect or a hemorheological effect. Also, influence of fluorescein sodium on the erythrocyte aggregation was shown in the past literature, which might be related with alterations in retina-choroidal microcirculation . AlthougThis study was a prospective cross-sectional case series and involved 60 eyes of 30 patients. The study was conducted in accordance with the ethical standards of Declaration of Helsinki and was approved by the Institutional Ethical Committee.All of the eyes had nonproliferative diabetic retinopathy (NPDRP). The diagnosis of NPDRP was based on indirect retinoscopy and fundus fluorescein angiography (FFA). All of the subjects underwent an ophthalmic examination including visual acuity assessment, biomicroscopy, air-puff tonometer, indirect retinoscopy, pachymeter, and macular optical coherence tomography before their participation in the study. Exclusion criteria were any ocular surgery other than phacoemulsification, corneal irregularities, history of glaucoma, uveitis, and any other retinal disorders except NPDRP. Also, the patients who had nausea and vomiting after FFA were excluded, since these situations could affect the OPA and IOP. Patients had refractive errors between −1.50 and +1.50 diopters spherical equivalent.FFA measurements were done with Topcon TRC 501X angiography . FFA procedure was done as follows. 5 mL 10% fluorescein sodium was injected rapidly from median antecubital vein or dorsal hand veins. After injection, serial retinal photographs were taken for 5 minutes. Between postinjection 5 and 10 minutes, IOP and OPA were measured. In order to separate the effect of rapid photography from fluorescein sodium injection, we had taken immediate postcolored retinal photography OPA and IOP measurements of 10 eyes before the dye injection.Intraocular pressure (IOP) and ocular pulse amplitude (OPA) measurements were done with the Pascal dynamic contour tonometer . This is a slit-lamp biomicroscopy mounted, self-calibrating, 7 mm tip diameter, and 1.2 mm pressure sensor diameter device. One drop of Alcaine  was instilled just prior to IOP and OPA measurements. Measurements were performed within a 5-minute period. Three DCT measurements were taken in order to have one good-quality measurement. Only quality 1 and 2 measurements were taken into consideration. All OPA and IOP measurements were performed by the same investigator (GP) before and after 5 minutes from fluorescein dye injection.Optical coherence tomography (OCT) measurements were done with spectral-domain OCT . Only central macular thickness was assessed by OCT. Corneal pachymetry measurements were done with Nidek UP-1000 ultrasonic pachymeter and central corneal thickness was recorded. The pachymeter probe was placed on the center of the cornea and the mean of five readings was calculated.t-test between pre-FFA and post-FFA OPAs and IOPs were performed to evaluate the effect of FFA on OPA and IOP. The Wilcoxon signed-rank test was used to analyze pre- and postretinal photography OPA and IOP values of 10 eyes. Age and macular thickness were assessed for a relationship with pre-FFA and post-FFA OPA and IOP differences using univariate linear regression analysis. The effects of lens status and gender on pre-FFA and post-FFA OPA and IOP differences were analyzed with independent samples t-test.Statistical analysis was performed with SPSS for Windows statistical software version 17.0 . All data are written as mean ± standard deviation (SD). Paired The study evaluated 30 nonproliferative diabetic retinopathy patients . Bilateral eyes were recruited for all the patients. The distribution of eyes according to NPDRP severity was as follows: mild NPDRP in 10 eyes, moderate NPDRP in 38 eyes, and severe NPDRP in 12 eyes.P = 0.071). Pre-FFA mean IOP value was 17.97 ± 1.99  mmHg and post-FFA mean IOP value was 17.81 ± 2.22  mmHg (P = 0.407). Best corrected visual acuity values evaluated with Snellen chart ranged between 0.1 and 0.9 in decimals (mean 0.54 ± 0.28).Pre-FFA mean OPA value was 3.05 ± 1.36  mmHg and post-FFA mean OPA value was 2.93 ± 1.28  mmHg (P = 0.45). Pre-retinal photography mean IOP value was 18.36 ± 1.49 mmHg and postretinal photography mean IOP value was 18.28 ± 1.99 mmHg (P = 0.78).In order to evaluate the individual effect of successive retinal photography, we recorded post-photography OPA and IOP values of only ten eyes. Pre-retinal photography mean OPA value was 2.70 ± 0.66 mmHg and postretinal photography mean OPA value was 2.63 ± 0.61 mmHg  showed statistically significant association with pre-FFA and post-FFA OPA difference, while macular thickness  did not show significant association with OPA change.When considered alone, age  showed statistically significant association with pre-FFA and post-FFA IOP difference, while macular thickness  did not show significant association with IOP difference.Age (P = 0.001). In males, mean IOP value decreased by 0.027 mmHg and in females mean IOP value decreased by 0.317 mmHg (P = 0.468).In males, mean OPA value increased 0.093 mmHg; whereas in females, mean OPA value decreased 0.330 mmHg after FFA (P = 0.101). In phakic eyes, mean IOP value decreased by 0.10 mmHg and in pseudophakic eyes, mean IOP value decreased by 0.38 mmHg (P = 0.548).In phakic eyes, mean OPA value decreased by 0.058 mmHg and in pseudophakic eyes, mean OPA value decreased by 0.300 mmHg , advanced retinitis pigmentosa, and exudative age-related macular degeneration –21. SomeCentofanti et al. investigated influence of sex and hormonal status on choroidal blood flow . They usSchmidt et al. examined OPA in diabetes mellitus and reported that choroidal circulation remained unaffected as diabetic retinopathy advanced . Their sThere are some restrictions related to this study. For example, ideal timing of OPA and IOP measurements after FFA is difficult to determine. Since taking photographs is very important in the first five minutes, it was not appropriate to disturb the examination. But we quickly measured OPA and IOP after 5 minutes from fluorescein injection. We did not perform FFA on normal subjects, but in order to maintain standardization of the patients, only NPDRP patients were included in the study. One may say if there were an issue, 50 years and millions of fluorescein angiograms later, we would have learned of it. But, according to us, it is important to document these data about FFA for the first time in the literature.In conclusion, FFA procedure does not affect OPA and IOP values. But, age showed significant association with pre-FFA and post-FFA OPA and IOP differences. Interestingly, mean OPA value of males increased, whereas mean OPA value of females decreased after FFA. More comprehensive studies including various ocular and systemic diseases and measurements at different FFA stages are needed to reveal more effects of FFA on OPA.


Androgens induce male characters by activating androgen receptors (AR). Previous quantitative studies on AR in fishes have been limited to few tissues and/or a single season/reproductive state. The aim of this investigation was to study the possible role of AR-beta expression levels in the control of male traits in the three-spined stickleback. To that end, AR-beta expression levels in major tissues in breeding and post-breeding male and female sticklebacks were examined.AR-beta mRNA levels were quantified in ten tissues; eye, liver, axial muscle, heart, brain, intestine, ovary, testis, kidney and pectoral muscle in six breeding and post-breeding males and females using reverse transcription quantitative PCR.Breeding in contrast to post-breeding males built nests and showed secondary sexual characters (e.g. kidney hypertrophy) and elevated androgen levels. Post-breeding females had lower ovarian weights and testosterone levels than breeding females. AR-beta was expressed in all studied tissues in both sexes and reproductive states with the highest expression in the gonads and in the kidneys. The kidney is an androgen target organ in sticklebacks, from which breeding males produce the protein spiggin, which is used in nest-building. There was also high AR-beta expression in the intestine, an organ that appears to take over hyperosmo-regulation in fresh water when the kidney hypertrophies in mature males and largely loses this function. The only tissue that showed effects of sex or reproductive state on AR-beta mRNA levels was the kidneys, where post-breeding males displayed higher AR-beta mRNA levels than breeding males.The results indicate that changes in AR-beta mRNA levels play no or little role in changes in androgen dependent traits in the male stickleback. Anguilla japonica [Oncorhynchus mykiss [Pagrus major [Androgens stimulate masculine traits such as development of male secondary sexual characters, male reproductive behaviour and spermatogenesis via interactions with nuclear androgen receptors (AR). Among teleost fishes, complete AR cDNAs have been cloned in e.g. Japanese eel, japonica , rainbows mykiss  and red us major .Astatotilapia burtoni [Gambusia affinis [In some teleost fishes, two different AR genes have been cloned e.g. Japanese eel , Burton' burtoni  and West affinis . The nom affinis . We base affinis ,7, to naGasterosteus aculeatus [d in the nM range) for androgens and low number of binding sites (Bmax in the pmol/g tissue range), which is in consistent with nuclear receptors [Recently, an AR-beta with two splicing variants was cloned from the kidney of the three-spined stickleback culeatus . In thiseceptors . FurtherRecently, a second stickleback AR has been reported . Based uSpinibarbus denticulatus [Expression of AR transcripts has been studied in several fishes with different techniques e.g. Northern blot, semi-quantitative reverse transcriptase PCR and real-time PCR. However, most studies have been limited to a few tissues, the male sex (sometimes with the ovary added), and/or qualitative or semi-quantitative techniques. AR-beta mRNA was measured in eye, liver, brain, gonads, kidneys, skin and muscle in adult male and female zebrafish  and in liculatus  at diffeIn order to obtain a more complete picture of the roles of AR in fishes, expression profiles need to be obtained from more organs and under different reproductive conditions. To that end, 15 tissues/organs were sampled from breeding and post-breeding male and female sticklebacks and AR-beta mRNA levels were measured using reverse transcription quantitative PCR (rt-qPCR).Handling of the fish and experiment design were approved by the Northern Stockholm Animal Research Ethical Committee.Gasterosteus aculeatus were caught in the Öresund, in the south of Sweden. The fish were kept in aquaria with filtered and aerated brackish water , under short photoperiod (8 h light: 16 h darkness), at 5-8°C to maintain them in non-breeding "winter" condition. The bottom was covered with sand and there were tubes of ceramic material that provided hiding places. The fish were fed daily with frozen red midge larvae or mysids.Adult non-breeding three-spined sticklebacks, In order to induce maturation, groups of fish were transferred to long photoperiod (LD 16:8) and 20°C, otherwise as above. Only fish that matured, i.e. females that developed roe-swollen bellies and males that developed red breeding colors were used. The breeding fish were transferred to individual 50-liter aquaria containing sand, filamentous algae and filtered water. All males in this group built nests and all females had ovulated at least once. The breeding fish were sampled after one and a half months in LD 16:8 and 20°C. The post-breeding fish were sampled after three months in LD 16:8 and 20°C, when the fish had entered the refractory phase, i.e. the males had lost their breeding coloration and the females showed no swollen bellies.The fish were anaesthetized with 0.1% 2-phenoxyethanol  and weighed. Blood samples for steroid measurement were collected from the severed caudal peduncle and stored at - 70°C until processed.Part or whole organs/tissues; i.e. eye, brain (pituitary included), spleen, gills (filaments separated from the gill arches), heart, liver, a c. 1 cm long posterior segment of the intestine, trunk kidneys (referred to as "kidney"), head kidney, right pectoral muscle (both inner and outer musculature), axial muscle (sampled from the back beneath the third back-spine), pectoral and caudal fins, testes and ovary were snap frozen and stored at -70°C. Intestines were rinsed with sterile 0.9% NaCl before freezing to reduce numbers of bacteria. Kidneys and gonads were weighed prior to freezing in order to calculate kidney somatic index  and gonad somatic index .3 dpm/50 μl and 200 μl of steroid antiserum . All samples were run in duplicate. Vials were vortexed and incubated over night at 4°C. Free, unbound steroid was separated from bound steroid with dextran charcoal suspension . Following 5 min centrifugation at 3900 rpm, the supernatant was poured into scillation vials containing 4 ml scintillation fluid  and run in the counter . The detection limit for the assay was ca. 2 ng/ml, and intra- and interassay coefficients of variance were 5.4 and 7.0%, respectively.Testosterone (T) and 11KT levels were assessed by radioimmunoassay according to [Tissue samples from six breeding and post-breeding males and females were homogenized and total RNA was extracted using TRI reagent (Sigma-Aldrich). Total RNA was treated with DNase Turbo  and quantified using RiboGreen RNA reagent  and the GENios Pro (TECAN)., 200 units of MMLV-RT (Invitrogen) and 40 units of RNase Inhibitor (Invitrogen) incubated at 25°C for 10 min, followed by 37°C for 50 min and 70°C for 15 min.First-strand cDNA synthesis was performed on 100 ng of total RNA using 50 μM random decamer primers A standard for AR was prepared from sense AR cRNA generated from the three-spined stickleback AR-beta cDNA clone (Gene Bank Accession no AY24706) using MEGAscript T7 kit . A serial dilution of AR cRNA was reverse transcribed to cDNA as described above and used as a standard in the quantitative PCR (qPCR) assay.Gene-specific primers for AR (Forward)  5'-CGTGCCCTGCGTTCAC-3') and probe (5'-CACCTCGGGTTCAATG-3') based on the stickleback AR-beta sequence (Gene Bank Accession no AY24706) were designed by TaqMan . Sequence analysis of the primer and probe sequences against the newly reported stickleback AR-alpha cDNA (ENSEMBL Transcript ID ENSGACT00000024538) [Reverse transcription qPCR was performed on ABI Prism 7000 Sequence Detection System . All samples were run in duplicate in a final volume of 50 μl, containing 25 μl TaqMan Universal PCR mastermix, 2.5 μl primer (18 μM), probe (5 μM), 1 μl cDNA and 21.5 μl nuclease free water . Following amplification conditions were applied: at 50°C for 2 min., 95°C for 10 min. followed by 40 cycles of 95°C for 15 sec, 60°C for 1 min.T). The amount of AR and 18S rRNA in each sample was determined according to Standard Curve Method  by comparing mean sample CT values to the standards. PCR efficiency analysis was performed on each reaction.AR and 18S rRNA standards and negative controls  were included in each PCR run. Calibrator samples for AR and 18S rRNA were included in each PCR run, to calculate the inter-assay variation. The inter-assay variation was included in the calculation of sample cycle threshold  except for PCR runs involving head kidneys, pectoral and caudal fins, where standards showed higher intercepts. Therefore, head kidney, pectoral and caudal fins are not comparable with the other organs. The intra- and interassay coefficient of variation for ar mRNA levels were 9.9% and 17.8%, respectively.Serial dilutions of AR and 18S rRNA standards produced linear plots of input cDNA vs CStatistical analyses were performed using Prism version 3.0 . All data were tested for normality and homogeneity of variance using Kolmogorov-Smirnov and Bartlett's test, respectively. KSI and GSI values were arcsine square root transformed, plasma androgen levels were log transformed and AR/18S rRNA values were (-1 * log (y)) transformed, prior to analysis to meet assumptions of homogeneity of variance. Statistical differences in KSI and 11KT and T plasma levels and 18S rRNA expression between experimental groups were tested using one-way ANOVA followed by the Tukey's post-hoc test. Differences in GSI between breeding and post-breeding females and males respectively were assessed by ANCOVA. Differences in slope and intercept for AR and 18S rRNA standards, respectively, in different PCR runs were tested in Linear regression analysis. Two-way ANOVA followed by Bonferroni post-hoc test was used to examine differences in tissue AR-beta mRNA levels with sex and reproductive state as factors. One-way ANOVA followed by the Tukey's post-hoc test was used to determine relative AR-beta mRNA levels in different tissues. In this comparison all samples from each tissue regardless of sex and state of maturity were pooled. This was considered justified since there was only one case with significant effects of sex and maturity within organs (see below). Results were considered statistically significant at p < 0.05. All data are presented as mean ± S.E.Breeding females displayed higher body weight compared to the other groups Table . All breThere was a difference in the 18S rRNA levels per ng cDNA in different tissues , for this reason the AR-beta levels in the former (which appeared to be similar to the other intestine samples) is not shown.AR-beta was expressed in all tested tissues with the highest AR-beta mRNA levels in the kidneys and gonads while the lowest levels were observed in the eyes Figure . The AR-The highest AR-beta mRNA levels were found in kidneys and gonads, which were significantly higher than those in all other tissues . The level in the intestine was significantly higher than in all other tissues except kidneys and gonads (p < 0.001).There was no effect of sex or reproductive state on the AR-beta mRNA levels in brain, axial musculature, eye, pectoral muscle, heart, gonads and liver Figure  and 3B. AR-beta was expressed in all tissues in both sexes and reproductive states. The ubiquitous AR-beta expression in adult stickleback tissues is largely consistent with a number of earlier studies in teleosts  [Monopterus albus) [Similar to sticklebacks, the AR expression was higher in the kidneys than in many other studied tissues in zebrafish , male hailaevis)  and rices albus) . For theSpinibarbus denticulatus (AR-beta) [Halichoeres trimaculatus) (AR-beta) [High AR mRNA levels in the testes have also been found in several previous studies on fishes (e.g. zebrafish (AR-beta) , SpinibaAR-beta) , sea basAR-beta) , three sAR-beta) ). This iAR-beta) ,28. In cAR-beta) ,30. MaleAR-beta) . SpermatAR-beta) . AdminisAR-beta) ) at the AR-beta) ,32.Spinibarbus denticulatus [Pimephales promelas [In the stickleback, AR-beta mRNA levels were as high in the ovaries as in the testes. Similar findings were reported in the three spot wrasse . High mRiculatus . It is niculatus , displayiculatus . Howevericulatus , includiiculatus . Testostpromelas . High dopromelas . The lowAR-beta mRNA levels were also high in the stickleback intestine. The intestine has been proposed to take over the kidneys' role as a freshwater osmoregulatory organ in mature stickleback males . The andAR-beta was present in the stickleback brain, but the levels were low. Androgens stimulate reproductive behavior in the male stickleback , but theSpinibarbus denticulatus [The AR-beta mRNA level in the stickleback liver was low. This is in general agreement with findings in the zebrafish . Howevericulatus , Mozambiiculatus  and riceiculatus  liver ARiculatus . In thisiculatus .In the stickleback, both pectoral and axial muscle contained low levels of AR-beta mRNA. AR(s) in musculature have also been found in some other fishes ,9,10. MaSpinibarbus denticulatus [There was no significant effect of sex on AR-beta mRNA levels in any studied organ in the present study. There are very few other studies where AR mRNA has been studied quantitatively in both sexes of fishes. In zebrafish there were about twice as much AR-beta mRNA in testes as in ovaries and in male than in female muscle, whereas there was no significant effect of sex in brain, kidney, liver, skin and eye . AR-betaiculatus . The levWe found no significant differences in AR-beta mRNA levels in the kidneys between breeding male and female sticklebacks whereas in another study a 1.4-fold higher kidney AR-beta expression in mature males than in mature female sticklebacks was reported .Although breeding and post-breeding fish, particularly in males, showed large differences in 11-KT and T plasma levels, there were no differences in AR-beta mRNA levels in most studied tissues, except for the kidneys. Unexpectedly, we found higher AR-beta mRNA levels in the kidneys of post-breeding compared to breeding males. Breeding males in the present study displayed well-developed hypertrophied kidneys and high T and 11KT levels. Conversely, none of the post-breeding males showed hypertrophied kidneys and their androgen levels were very low, indicating that they had left the reproductive state. High AR-beta mRNA levels in the kidneys of breeding males are likely necessary for the high spiggin synthesis. What function(s), if any, males may have of continuous high AR-beta expression in the kidneys shortly after the reproductive period is unknown.These results suggest that androgens have a little, if any, effect on AR-beta expression in most organs and at least no positive effect in the kidney. No support for auto-regulation of AR-beta mRNA or protein levels (binding capacity) in stickleback kidneys was found when intact females and castrated males were treated with androgens i.e. T, 11KA or 5-alpha dihydrotestosterone . FurtherHere we demonstrated that AR-beta is expressed in all major tissues in adult three-spined sticklebacks. Most tissues exhibited low AR-beta mRNA levels but in the known androgen target organs i.e. the testes and kidneys, and in the ovary and the intestine, the AR-beta mRNA levels were considerably higher, up to 100-fold. There were no clear effects of sex and reproductive states in AR-beta mRNA levels except for the kidneys, where post-breeding males displayed higher AR-beta mRNA levels than breeding males.Despite large differences in androgen levels between breeding and post-breeding fish, most investigated tissues exhibited similar AR-beta mRNA levels. These results suggest that changes in androgen target tissues during the reproductive cycle are controlled by changes in androgen levels and not by changes in AR-beta mRNA levels.T: Testosterone; 11KT: 11-ketotestosterone; AR: Androgen receptor.The authors declare that they have no competing interests.EH, BB, JK and P-EO designed the experiments and prepared the manuscript. EH and BB participated in the preparation of the animals and tissues. EH and AW performed RNA extraction, cDNA synthesis. EH performed the RIA, the real time quantitative PCR and the analysis of data. All authors have read and approved the final manuscript.


Sepsis-associated delirium (SAD) increases morbidity in septic patients and, therefore, factors contributing to SAD should be further characterized. One possible mechanism might be the impairment of cerebrovascular autoregulation (AR) by sepsis, leading to cerebral hypo- or hyperperfusion in these haemodynamically unstable patients. Therefore, the present study investigates the relationship between the incidence of SAD and the status of AR during sepsis.Cerebral blood flow velocity was measured using transcranial Doppler sonography and was correlated with the invasive arterial blood pressure curve to calculate the index of AR Mx (Mx>0.3 indicates impaired AR). Mx was measured daily during the first 4 days of sepsis. Diagnosis of a SAD was performed using the confusion assessment method for ICU (CAM-ICU) and, furthermore the predominant brain electrical activity in electroencephalogram (EEG) both at day 4 after reduction of sedation to RASS >-2.30 critically ill adult patients with severe sepsis or septic shock (APACHE II 32 ± 6) were included. AR was impaired at day 1 in 60%, day 2 in 59%, day 3 in 41% and day 4 in 46% of patients; SAD detected by CAM-ICU was present in 76 % of patients. Impaired AR at day 1 was associated with the incidence of SAD at day 4 (p = 0.035).AR is impaired in the great majority of patients with severe sepsis during the first two days. Impaired AR is associated with SAD, suggesting that dysfunction of AR is one of the trigger mechanisms contributing to the development of SAD.NCT01029080clinicalTrials.gov ID  Intensive care unit (ICU) patients with sepsis commonly develop neurological deficits as part of multi organ failure. A frequent form of central nervous system dysfunction is sepsis-associated delirium (SAD) that can be observed in septic patients . AccordiBeside the inflammatory pathway many other mechanisms have been discussed that might cause SAD, such as microvascular dysfunction -6. LaborFollowing ethical care committee approval of Rhineland-Palatinate  and written informed consent from legal surrogates, 30 adult patients were included in the study. All patients had clinical diagnoses of severe sepsis or septic shock as defined by the international sepsis definition conference . Exclusi2) between 4.7 and 6.0 kPa (35 and 50 mmHg). Inspiratory oxygen fraction and positive end expiratory pressure were individually adjusted to achieve an arterial partial pressure of oxygen (paO2) > 9.0 kPa (70 mmHg). After stabilisation of the cardiopulmonary condition sedation was reduced and ventilation was switched to an assisted spontaneous breathing mode aiming for the same ventilatory settings. Invasive arterial blood pressure and the haemodynamic monitoring were measured by pulse contour analysis using a PiCCO®-Catheter . According to the local sepsis treatment protocol, saline solution was used to reach a global end-diastolic volume index of 650 to 800 ml/m2, norepinephrine was used to reach a mean arterial blood pressure > 65 mmHg, dobutamine was used to reach a target cardiac index > 3.5 l/min/m2 and a central venous oxygen saturation > 70 %. Serum lactate level was measured to monitor adequate sepsis treatment.Patient management was performed according to the international guidelines for management of severe sepsis and septic shock . In all ®, DWL, Sipplingen, Germany). The probes were positioned over the temporal bone window above the zygomatic arch and were fixed with a special frame . This procedure ensured that the angle and the individual depth of insonation were constant during the daily investigation.Measurement of the dynamic AR was performed daily for the first four days using transcranial Doppler (TCD) ultrasound. Cerebral blood flow velocities (CBFV) in both middle cerebral arteries were measured using 2-MHz TCD-probes . Spontaneous variations in CBFV and arterial blood pressure were measured over a 60-minute period, digitized and then processed calculating the time-averaged values using waveform time integration for 6-s intervals. The index of cerebrovascular autoregulation (Mx) was calculated as a Pearson's correlation coefficient between 30 samples of mean CBFV and mean arterial pressure (MAP) . PositivDelirium as a clinical diagnosis was assessed using the confusion assessment method for the ICU (CAM-ICU) ,4. The CFor assessment of the severity of inflammation, serum concentrations of C-reactive protein (CRP) and procalcitonin (PCT) were measured daily. Furthermore, serum concentrations of neuron-specific enolase (NSE) and S100 were determined daily.2 by day . A P-value < 0.05 was considered statistically significant.Physiological data were expressed as the mean ± SD or the median  for nonparametric data. The primary endpoint of the present study was to investigate the association between the status of AR at day 1 and SAD at day 4. A power analysis calculated a need for 20 patients, which was increased to 30 because of the expected inhomogeneous distribution of the incidence of SAD. Data on AR and SAD were dichotomized in intact/impaired AR and SAD/non-SAD and then analysed using the Fisher exact test for the primary target (association between AR and SAD), and the Kendall-Tau-c test to compare the diagnosis of SAD using either CAM-ICU or EEG. To investigate whether other factors correlate with SAD, analysis of variance (ANOVA) correlations between AR, APACHE II scores, age and serum markers were performed and furthermore, spearman-Rho-correlations between Mx and serum markers and paCOThirty patients, five with severe sepsis and twenty-five with septic shock, APACHE II score of 32 ± 6, with a mean age of 64 ± 17 years were included in the study  showed an impaired AR with an Mx > 0.3 during the first four days, 18/30 (60 %) at day 1, 17/29 (59%) at day 2, 12/29 (41%) at day 3 and 11/24 (46%) at day 4. The highest level of Mx was on the first day with a median of 0.42  followed by a decrease with a median of 0.30  between day 2 and day 3 . The status of AR at day 1 and the incidence of SAD using the CAM-ICU at day 4 showed an association (P = 0.035)  patients. EEG could be performed in 18/30 (60%) patients. Of these, 17 patients showed an EEG pattern which is typical for delirium . There was no association between the clinical diagnosis of delirium based on CAM-ICU and the EEG (P = 0.028). The serum level of NSE, the severity of illness assessed by the APACHE II and paCO2 did not correlate with Mx or SAD  was detected in 22/29 patients at day 4, which was associated with an impaired AR at day 1. Further minor findings were: the index of AR Mx correlated weakly at day 2 with inflammatory parameters but not with markers of brain damage . Nevertheless, the marker S100 was elevated in the patients with SAD. There was no correlation between the diagnosis of delirium using CAM-ICU compared to the EEG. Results from the present study therefore, suggest that impairment of AR and SAD is common in ICU patients, and impaired AR might be causative to trigger SAD.In the present study the TCD-derived index of AR Mx was used for the assessment of the status of AR; this index has been already calculated for many different clinical conditions: in traumatic brain injury, in carotid artery disease, in vasospasm, in volunteers, and in sepsis ,17. Mx wSAD occurs in many septic patients and may be a risk factor for morbidity . This is2 reactivity [2-dependent autoregulation of CBF is much more resistant towards influences compared to the blood pressure-dependent CBF autoregulation, which was tested in the present study. This might explain why the CO2 reactivity study showed no impaired vasomotor reactivity, while in the present study the MAP-dependent AR was impaired in patients with SAD. On the other hand, in 16 septic patients the index Mx was measured once during the first 48 hours after stabilisation and disturbance of AR was correlated with the diagnosis of SAD (CAM-ICU) [Disturbed AR may lead to global brain ischaemia due to hypotension in the absence of adequate regulatory compensation. This ischaemia may be a trigger for brain dysfunction like SAD. In the present study an association between impaired AR at day 1 and SAD at day 4 was shown. An investigation including 10 patients with pathological EEG results and sepsis showed intact COactivity  and therCAM-ICU) . This asLevels of CRP and PCT, validated markers of inflammation, correlated only at day 2 with the index of AR Mx. Possibly, disturbance of AR is fostered by the underlying inflammation caused by endothelial dysfunction due to endotoxins from bacteria, or cytokines like ICAM-1 from the immune response ,23. The The sedation management with propofol in combination with sufentanil did not influence AR in patients under general anaesthesia and after head injury -26. It iTo minimize any bias the same technical equipment was always used. TCD is an operator-dependent tool because of the dependency of the angle of insonation. However, for the assessment of AR only the relation of changes in CBFV in correspondence to changes in MAP was used rather than the absolute values of CBFV.2 in the present study were in the upper normal range and increased from 43 ± 9 mmHg at day 1 to 48 ± 9 mmHg at day 4, and might have influenced the AR [2 on the present results should be acceptable as high paCO2 further impairs AR. Therefore, the observed recovery of AR until day 4 should be even more pronounced if paCO2 was held constant during the investigation.The levels of paCOd the AR . This waAnother possible influencing factor is hyperthermia, because an increase of core temperature above 40°C leads to impairment of AR . HoweverThe present study showed that AR is impaired in the majority of patients with severe sepsis and septic shock during the first 2 days. Furthermore, SAD was frequently diagnosed in these patients . Impairment of AR was associated with the occurrence of SAD, suggesting that AR is one of the factors contributing to SAD. This should be taken into account for the treatment of these patients.• Cerebrovascular AR is impaired in 83% of patients with severe sepsis and septic shock• Among patients with severe sepsis and septic shock, 76% have sepsis-associated delirium• Impaired cerebrovascular AR possibly triggers sepsis-associated delirium• Possibly, mean arterial blood pressure should be tightly controlled during the first 2 days in septic patients2: arterial partial pressure of carbon dioxide; paO2: arterial partial pressure of oxygen; PCT: procalcitonin; PiCCO: pulse contour cardiac output; RASS: Richmond Agitation and Sedation Scale; SAD: sepsis-associated delirium; SOFA: Sequential Organ Failure Assessment; TCD: transcranial Doppler.ANOVA: analysis of variance; APACHE: Acute Physiology and Chronic Health Evaluation; AR: cerebrovascular autoregulation; CAM-ICU: confusion assessment method for the intensive care unit; CBF: cerebral blood flow; CBFV: cerebral blood flow velocity; CI: cardiac index; CRP: C-reactive protein; EEG: electroencephalography; HR: heard rate; ICU; intensive care unit; MAP: mean arterial pressure; Mx: Index of cerebrovascular autoregulation; NSE: neuron specific enolase; paCOThe authors declare that they have no competing interests.PS, KUK, LF, DC performed the TCD and CAM-ICU, PS and KJW performed the EEG and KJW interpreted the EEG; PS, DC, KE drafted the document; PS, MD, CW and KE participated in planning the design of the study; MB, LF and PS performed statistical analysis. All authors read and approved the final manuscript.KJW is a board-certified neurologist with special experience in EEG; MB is statistician with experience in clinical trials; LF is medical student and intensive care nurse; PS is an anaesthesiologist and intensive care physician with experience in neurology and neurophysiologic monitoring; KUK, DC, KE, MD and CW are anaesthesiologists with experience in intensive care and neurophysiologic monitoring.