Arabidopsis splicing related genes includes classification of genes encoding snRNAs and other splicing related proteins, together with information on gene structure, alternative splicing, gene duplications and phylogenetic relationships.The database of  Arabidopsis genome by sequence comparison and motif searches, including the previously elusive U4atac snRNA gene. Most of the genes have not been studied experimentally. Classification of these genes and detailed information on gene structure, alternative splicing, gene duplications and phylogenetic relationships are made accessible as a comprehensive database of Arabidopsis Splicing Related Genes (ASRG) on our website.A total of 74 small nuclear RNA (snRNA) genes and 395 genes encoding splicing-related proteins were identified in the  Although the splicing mechanisms in plants and animals appear to be similar overall, incorrect splicing of plant pre-mRNAs in mammalian systems (and vice versa) suggests that there are plant-specific characteristics, resulting from coevolution of splicing factors with the signals they recognize or from the requirement for additional splicing factors (reviewed in [Most eukaryotic genes contain introns that are spliced from the precursor mRNA (pre-mRNA). The correct interpretation of splicing signals is essential to generate authentic mature mRNAs that yield correct translation products. As an important post-transcriptional mechanism, gene function can be controlled at the level of splicing through the production of different mRNAs from a single pre-mRNA (reviewed in ). The geiewed in ,3).Drosophila and fission yeast genomes in an effort to identify potential homologs [Drosophila and fission yeast. The availability of the near-complete genome of Arabidopsis thaliana [et al. [et al. [et al. [Arabidopsis protein kinases and computationally compared derived protein clusters with established gene families in budding yeast. Previous surveys of Arabidopsis gene families that contain some splicing-related genes include the DEAD box RNA helicase family [Arabidopsis Information Resource (TAIR) links to more than 850 such expert-maintained collections of gene families [Genome projects are accelerating research on splicing. For example, with the majority of splicing-related genes already known in human and budding yeast, these gene sequences were used to query the homologs ,5. Most thaliana  provides [et al.  identifi [et al.  catalogu [et al.  surveyede family  and RNA-e family . At presfamilies .Arabidopsis genes involved in pre-mRNA splicing. Recent mass spectrometry analyses revealed more than 200 proteins associated with human spliceosomes , which should facilitate genome-wide studies of pre-mRNA splicing in plants.Here we present the results of computational identification of potentially all or nearly all eosomes  and TATA box -27. All 3-10AGTNNAA in U snRNAs  transcribed by RNA polymerase II (Pol II) [Nicotiana plumbaginifolia protoplasts [Arabidopsis.Other conserved transcription signals were also identified in most active snRNAs, including the sequence element CAANTC  in U2 snRNAs (located at -6 to -1) , and the(Pol II) ,24,32. T(Pol II)  is also toplasts , the extArabidopsis [U6atac gene (atU6atac-2) by sequence mapping. This gene has a USE and a TATA box in the promoter region. The atU6atac-2 gene is more than 90% similar to atU6atac in both its 5' and 3' ends, with a 10-nucletotide deletion in the central region. The putative U4atac-U6atac interaction region in atU6atac-2 is 100% conserved with the interaction region previously identified in atU6atac [The minor snRNAs are functional in the splicing of U12-type (AT-AC) introns. Four types of minor snRNAs, which correspond to four types of major snRNAs, exist in mammals. U11 is the analog of U1, U12 is the analog of U2, U4atac is the analog of U4, and U6atac is the analog of U6. The U5 snRNA seems to function in both the major and minor spliceosome . Two minbidopsis . Both haatU6atac ,35.Arabidopsis. BLAST searches using the human U11 and U4atac homologs as queries against the Arabidopsis genome failed to find any significant hits, indicating divergence of the minor snRNAs in plants and mammals. Using the strategy described below, we successfully identified a putative Arabidopsis U4atac gene. It is a single-copy gene containing all conserved functional domains. We also found a single candidate U11 snRNA gene  that has the USE and TATA box in the promoter region. This gene also contains a putative binding site fr Sm protein and a region that could pair with the 5' splice site of the U12-type intron.U11 and U4atac have not been experimentally identified in Arabidopsis homologs. Therefore, we deduced the tentative AtU4atac stem II sequence (CCCGTCTCTGTCAGAGGAG) from AtU6atac snRNA and searched for matching sequences in the Arabidopsis genome. Hit regions together with flanking regions 500 base-pairs (bp) upstream and 500 bp downstream were retrieved and screened for transcription signals (USE and TATA box). One sequence was identified that contains both the USE and TATA box in appropriate positions, as shown in Figure Like U4 snRNA and U6 snRNA, human U4atac and U6atac snRNAs interact with each other through base pairing . The samU4atac snRNA gene contains not only the stem II sequence, but also the stem I sequence that presumably base-pairs with U6atac snRNA stem I. Furthermore, a highly conserved Sm-protein-binding region exists at the 3' end. The predicted secondary structure is nearly identical to hsU4atac, with a relative longer single-stranded region (data not shown). With the highly conserved transcriptional signals, functional domains and secondary structure, this candidate gene is likely to be a real U4atac snRNA homolog. We named it AtU4atac and assigned At4g16065 as its tentative gene model because it is located between gene models At4g16060 and At4g16070 on chromosome 4.The tentative Arabidopsis chromosomes [Arabidopsis U1-U4 clusters have the U1 snRNA gene located upstream of the U4 snRNA gene, with a 180-300-nucleotide intergenic region. Five of the U1-U4 arrays are located on chromosome 5 , and the remaining two on chromosome 1 (U1-10/U4-6 and U1-14p/U4-10). The U2-17 and U5-10 occur in tandem array on chromosome 5, separated by fewer than 200 nucleotides.Some snRNAs genes exist as small groups on the omosomes . We idenDrosophila are known [Arabidopsis. Sequence-similarity scores and comparison of gene structure and protein domain structure were used to assign the genes to families. Each gene was assigned a tentative name based on the name of its respective animal homolog. Different homologs within a gene family were labeled by adding an Arabic number  to the name. Close family members with similar gene structure were indicated by adding -a, -b, and -c to the name. The 395 genes were classified into five different categories according to the presumed function of their products. Ninety-one encode small nuclear ribonucleoprotein particle (snRNP) proteins, 109 encode splicing factors, and 60 encode potential splicing regulators. Details of EST evidence, alternative splicing patterns, duplication sources and domain structure of these genes are listed in Table Arabidopsis proteins corresponding to 54 human spliceosome-associated proteins. The remaining 51 genes encode proteins with domains or sequences similar to known splicing factors, but without enough similarity to allow unambiguous classification. These two categories are not discussed in detail here, but information about these genes is available at our ASRG site [Most of the proteins involved in splicing in mammals and re known ,37,38. Ire known ). Using Arabidopsis. All U snRNPs except U6 snRNP contain seven common core proteins bound to snRNAs. These core proteins all have an Sm domain and have been called Sm proteins. The U6 snRNP contains seven LSM proteins ('like Sm' proteins). Another LSM protein (LSM1) is not involved in binding snRNA  involved in the formation of the major spliceosome, corresponding to five snRNAs. Five snRNPs  have been isolated experimentally in yeast or human -45. Eachiewed in ).Arabidopsis, and eight of them are duplicated. It is likely that these genes existed as single copies in the ancestor of animals and plants, but duplicated within the plant lineage. Only one of the 24 genes  has been characterized experimentally in Arabidopsis. The LSM5 gene was cloned from a mutant supersensitive to ABA (abscisic acid) and drought . LSM5 it stress . cDNA anArabidopsis proteins corresponding to the 35 snRNP-specific proteins used as queries in our genome mapping. Very few of them have been characterized experimentally, including U1-70K, U1A and a tandem duplication pair of SAP130 [U1-70K was reported as a single-copy essential gene. Expression of U1-70K antisense transcript under the APETALA3 promoter suppressed the development of sepals and petals [U1-70K (At2g43370) and named it U1-70K2. The U1-70K2 proteins showed 48% similarity to the U1-70K protein according to Blast2 results. Both genes retain the sixth intron in some transcripts, a situation which would produce truncated proteins [Arabidopsis U1 snRNP proteins, including the U1-70K-coding genes, may undergo alternative splicing.There are 63 f SAP130 -50. U1-7d petals . We idenArabidopsis genome. The atSAP114 family has five members, including two that occur in tandem (atSAP114-1a and atSAP114-1b). Three members have EST/cDNA evidence  subunit, which are clustered on chromosome 4. Both the U4/U6 90 kDa protein and the U4/U6 15.5 kDa protein also have three gene copies, and the 116 kDa and 200 kDa subunits in U5 snRNP have four copies apiece.Several genes in U2, U5, U4/U6 and U4.U6/U5 snRNPs, but none in U1 snRNP, occur in more than three copies in the Arabidopsis also lacks homologs of Prp42, a component of U1 snRNP in yeast [Arabidopsis has two copies of the gene for Prp39, which are similar to Prp42. Furthermore, atPrp39a can produce a shorter protein isoform with a novel amino-terminal sequence by exon skipping. It is possible that the duplicates and alternative isoforms of plant U1 snRNP proteins are functional homologs of the yeast-specific proteins.The yeast U1 snRNP contains several specific proteins that are not present in mammalian U1 snRNPs . As in min yeast . HoweverArabidopsis. The human 18S U11/U12 snRNP contains several proteins found in U2 snRNP as well as seven novel proteins [Arabidopsis, while the remaining three  have no clear homologs. Interestingly, all four Arabidopsis genes are single copy in the genome, and three of them are apparently alternatively spliced  is not lethal and is non-pleiotropic. The abh1 plants show ABA-hypersensitive closure of stomata and reduced wilting during drought [We divided the splicing factors into eight subgroups according to recent human spliceosome studies ,14,16,18on yeast ) and capon yeast ), deriveon yeast . These s drought .Arabidopsis, including two U2AF65, two U2AF35, and 18 SR proteins [Arabidopsis genome. Although the Urp gene is expressed ubiquitously in human tissues, no ESTs from this gene were found in Arabidopsis. Three copies of PTB/hnRNP-I genes were identified in Arabidopsis. The PTB protein competes for the poly-pyrimidine tract with the U2AF large subunit, thus negatively regulating splicing [Many splicing factors have been identified previously in proteins -67. The proteins , is alsosplicing .65 (At2g33440) and an additional SR protein (At2g46610). The U2AF65-related protein  has 247 amino acids and shares over 40% similarity with the carboxy terminal region of the two atU2AF65 homologs. Only one RRM can be identified in atULrp, in contrast to three RRMs and one amino-terminal RS domain in atU2AF65 proteins, and there is no apparent RS domain in atULrp. No animal homolog of atULrp could be identified. The function of this one-RRM U2AF65-related protein is not clear. As it lacks other functional motifs, it might act as a competitor of U2AF65. A two-RRM U2AF65 protein can be produced through alternative splicing. The 11th intron of atU2AF65a can be retained  to produce a truncated protein with only the first two RRMs. Interestingly, the last RRM in atU2AF65a contains several amino-acid variations from the consensus pattern such that it could not be detected by InterPro and NCBI-CDD searches using default values, also suggesting that perhaps only the first two RRMs are essential.We also identified a homolog related to atU2AFatRSp32 is similar to that of atRSp31. Two other genes (atRSp40 and atRSp41) are in the same family and also have similar exon and intron sizes . It shares 70% identity and 78% similarity with atRSp31. The protein is 250 amino acids in length and contains two RRMs and some RS dipeptides in the carboxy-terminal region. The gene structure of 0. It shaArabidopsis has a single copy of the SC35 ortholog and four SC35-like proteins , which appear to have diverged significantly from SC35. It seems that this divergence predates the split of plants and animals because a similar SC35-like gene family exists in the human genome (SRrp35 and SRrp40). The SRrp35 and SRrp40 were found to antagonize other SR proteins in vitro and function in 5' splice-site selection [Arabidopsis, with three  containing one CCHC-type zinc finger and two  containing two CCHC-type zinc fingers in addition to an RRM and an RS domain. Interestingly, several SR proteins  were found to interact with atU1-70K, and some SR proteins can interact with each other, thus forming a complicated interaction network to facilitate splice-site selection and spliceosome assembly [RNPS1 gene, which encodes an EJC protein. Other human SR proteins  lack clear orthologs in Arabidopsis. We conclude that SR protein families evolved differently in animals and plants from three to four common ancestors, including SC35, SF2/ASF and 9G8/RSZ. The SRrp (SC35-like in plants) family may not be classical SR proteins but they play important roles in splice-site selection.The atRSp31 family  belongs to a novel plant SR family and has no clear animal ortholog. Other families include the SC35 (or SRrp/TASR2) family, SF2/ASF family, and the 9G8 family. election . SF2/ASFassembly -63. atSRArabidopsis. The BΔU1 complex is the spliceosome complex captured immediately before catalytic activation. Most proteins in the 35S U5 snRNP are absent in the BΔU1 complex but present in the active B complex, indicating the important roles of 35S U5 snRNP-associated proteins in spliceosome activation [Arabidopsis revealed the same pathway of spliceosome activation in plants. A subcomplex named Prp19 complex in 35S U5 snRNP has a critical role in spliceosome activation [Arabidopsis, including a chromosomal duplication pair of Prp19 genes and a single copy of the CDC5 gene. For the BΔU1 complex, six human genes have homologs, and five of them are single copy in Arabidopsis. Two genes (NPW38BP/SNP70 and p220(NPAT)) in the human BΔU1 complex have no apparent Arabidopsis homologs.Proteins in other subgroups, such as 17S U2 snRNP-associated proteins, 35S U5 snRNP-associated proteins, and protein specific to the BΔU1 complex, are also conserved in tivation . Conservtivation ,72. All Arabidopsis also lacks an SMN protein complex. In human, the SMN protein  can interact with a series of proteins including Gemin2, Gemin3 (a helicase), Gemin4, Gemin5 and Gemin6 to form an SMN complex, which has important roles in the biogenesis of snRNPs and the assembly of the spliceosome through direct interactions with Sm proteins and snRNA [Arabidopsis genome.nd snRNA . AlthougArabidopsis. A total of eight SR protein kinases were identified in Arabidopsis, including three Lammer/CLK kinases , two SRPK1 homologs, and three SPRK2 homologs. The three Lammer/CLK kinases were identified previously, and AFC2 was shown to phosphorylate SR protein in vitro [Arabidopsis changed the alternative splice patterns of several genes, including atSRp30, atSR1/atSRp34 and U1-70K [Splicing regulators are proteins that can either modify splicing factors or compete with splicing factors for their binding site. Important splicing regulators are hnRNP proteins and SR protein kinases. The exact role of phosphorylation of SR proteins in splicing is not yet clear, but SR protein kinases are well conserved and exist as multiple copies in in vitro ,74. Overd U1-70K , indicatArabidopsis by sequence-similarity searches, including a superfamily of glycine-rich RNA-binding proteins. This family contains 21 members similar to human hnRNP A1 and hnRNP A2/B1. It can be further divided into two subfamilies. One includes eight proteins containing one RRM, and another has 13 members with two RRMs. 12 of these proteins were identified previously, including AtGRP7, AtGRP8, UBA2a, UBA2b, UBA2c and AtRNPA/B1-6 [AtGRP8 transcripts [The heterogeneous nuclear ribonucleoproteins (hnRNPs) bind to splice sites and to binding sites for splicing factors on nascent pre-mRNAs, thus competing with splicing factors to negatively control splicing (reviewed in ). HumansNPA/B1-6 ,77,78. Anscripts . UBA2 prArabidopsis. BLAST searches of the human (CUG)n triplet repeat RNA-binding protein (CUG-BP) against all Arabidopsis proteins revealed three putative homologs, including atFCA. atFCA and CUG-BP share similarity within the RRMs and a region approximately 40 amino acids in length. An additional protein (At2g47310) related to FCA was identified and named FCA2, as it shares about 50% similarity with FCA. The FCA proteins have two RRMs and a WW domain, which interact with the FY protein, a homolog of yeast polyadenylation factor Psf2p [Other human hnRNPs related to splicing also have homologs in or Psf2p ,81. The or Psf2p ,82. FCA or Psf2p . We alsoor Psf2p , whereasor Psf2p ,85.Arabidopsis proteins corresponding to human spliceosome-associated proteins identified in recent proteomic studies [Arabidopsis. We also identified a total of 51 Arabidopsis protein-coding genes similar to known splicing proteins. They have conserved domains and some level of sequence similarity to known splicing factors. We did not include these two categories in Table In addition to the 260 proteins in the above three categories, there are also 84  studies -18. SomeArabidopsis snRNA and splicing-related proteins across the genome is shown in Figure atU2A-atCdc5 and atCUG-BP1-atU1C, include genes from different functional categories. No clear clusters of genes for snRNA-splicing-related proteins were identified. Although about one third of snRNA genes are located near other protein-coding genes, none of their neighboring genes is related to splicing. As a caveat, we should point out that our snRNA gene determination strongly suggests annotation errors in overlapping protein-coding gene models. Thus, atU2-1, atU2.3, atU4.2, atU4-11p, atU5-13 and atU6.26 overlap gene models At1g16820, At3g57770, At3g06895, At1g68390, At5g53740 and At3g13857, respectively, but none of these models is well supported by cDNA or EST evidence (see displays linked at ASRG [The distribution of  at ASRG ).Arabidopsis transcription factors [The 260 proteins in the first three categories could be grouped into 130 families, 66 of which consist of multiple members. The duplication rate is over 50%, which is higher than the 44% duplication rate of  factors . As showU2-14/U2-10, U5-3/U5-5 and (U6.1 U6.26)/(U6-8p U6-9p), may also have been produced by chromosome duplication. The C.D.2-3 region  and four U2 snRNA genes  also are located in the same region on chromosome 3. No U5 and U2 homologs exist in the corresponding region on chromosome 2, suggesting that the snRNA duplication events in that region may have happened after the chromosome duplication event, or that the snRNA duplicates were lost subsequent to chromosome duplication.At least 130 duplication events are required to yield the 260 proteins from 130 families given one single-copy ancestor per family. Thirty-three duplication events  are likely to be the result of chromosome duplications. The chromosomal duplication ratio is 18.9-27.5% among the three groups  has theChromosomal duplication rather than individual gene duplication appears to be the predominant mode of amplification for some types of genes. As shown in Table Arabidopsis, which is about 13% using the same criteria . As shown in Table According to EST/cDNA alignments, 80 of the 260 protein coding genes show 66 alternative splicing events. This rate (30.8%) is much higher than the overall frequency of alternative splicing in Arabidopsis alternative splicing events , the ratio of intron retention in splicing-related genes is similar and the ratio of exon skipping is higher. Interestingly, only one of the 20 splicing regulator genes processed by alternative splicing (about 5%) shows exon skipping, indicating that exon skipping is an important post-transcriptional method for controlling the expression of splicing factor coding genes but not the splicing regulator genes.Among different types of alternative splicing, intron retention is the most abundant of the alternative transcripts identified for the 260 classified splicing-related genes. As shown in Table Arabidopsis , BAD09319 (osSC35b), AAP46199 (osSR33-1), BAC799901 (osSCL30a/osSR33-2), and BAD19168 (osSCL30-1). As shown in the phylogenetic tree displayed in Figure Arabidopsis chromosomal duplication pair atSR33 - atSCL30a forms a clade, while their rice copies (osSR33-1 and osSCL30a) form another clade. Also there are additional duplications for the rice SC35 and SCL30 genes. We are currently working to identify all rice splicing related genes. The complete sets of these genes in two plant species should provide a good foundation for assessing similarities and differences in splicing mechanisms used by monocot and dicot plants.To confirm the above hypothesis, we searched the recently sequenced rice genome using the five Arabidopsis indicates that other organisms also have integrated new proteins or pathways into the splicing mechanism over the course of evolution relative to other eukaryotes. Other evidence supporting the conserved splicing but flexible regulating mechanism include differential conservation among U snRNAs (U1 snRNAs are less conserved than U6 snRNAs) and high alternative splicing frequency in U1 snRNP proteins, SR proteins and hnRNP proteins. The SR proteins and U1 snRNP proteins are involved in early steps of splicing and 5' and 3' splice-site selection; multiple isoforms of these proteins may be functionally significant in the control of splicing.As introns evolve rapidly, the mechanism to recognize and splice them should either evolve correspondingly or be flexible enough to accommodate the changes. It seems that plants deploy the most economic and practical way by keeping a largely conserved splicing mechanism and a very flexible recognition and control mechanism. Direct evidence comes from the presence of plant-specific splicing proteins, such as the novel SR protein family and the superfamily of hnRNP A/B. The absence of SMN complex and some yeast U1 snRNP proteins in Arabidopsis genes. More than half of SR proteins and U1 snRNP proteins show alternative splicing. Alternative splicing might increase protein diversity derived from splicing-related genes, which would further add flexibility to the splicing mechanism. The high frequency of alternative transcripts from splicing related genes raises another interesting question - how is splicing regulated in these splicing-related genes? One possible answer is that some splicing-related genes may be autoregulated. Accumulation of one transcript would feed back to inhibit/promote other isoforms. Several splicing-related genes have been reported to be regulated in this way. For example, AtGRP7 (hnRNP A/B superfamily) is a circadian clock-regulated protein which negatively autoregulates its expression [AtGRP7 and atSRp30 is known to affect the splicing of AtGRP8 and atSR1, respectively [SR1 gene. The ratio of two transcripts from the SR1 gene (SR1B/SR1) increases in a temperature-dependent manner [It is interesting to note that the overall alternative splicing frequency in splicing related genes is much higher than the frequency averaged over all pression . When thpression . atSRp30pression . Anotherectively ,79. A tht manner . GeneralArabidopsis splicing-related genes were computationally identified in this study by means of sequence comparisons and motif searches, including a tentative U4atac snRNA gene containing all conserved motifs, a new SR protein-coding gene (atRSp32) belonging to the atRSp31 family, and several genes related to genes encoding known splicing-related proteins (atULrp and atFCA2). A web-accessible database containing all the Arabidopsis splicing related genes has been constructed and will be expanded to other organisms in the near future. This compilation should provide a good foundation to study the splicing process in more detail and to determine to what extent these genes are conserved across the entire plant kingdom. Our data show that about 50% of the splicing-related genes are duplicated in Arabidopsis. The duplication ratios for splicing regulators are even higher, indicating that the splicing mechanism is generally conserved among plants, but that the regulation of splicing may be more variable and flexible, thus enabling plants to respond to their specific environments.A large number of Arabidopsis genome at the AtGDB BLAST server [Arabidopsis genome . Local BLAST [-5 were regarded as possible homologs. A total of 70 major snRNAs and three minor snRNAs were identified by this method. Each major snRNA type has 10-18 copies in the genome. A tentative gene name and gene model were assigned to each snRNA gene after comparison with the snRNAs identified in MATDB [Sequences of the 15 experimentally identified major snRNAs were downloaded from GenBank. The two minor snRNAs sequences were compiled from the literature . These gT server  and at tT server . Our inial BLAST  was usedin MATDB . SequencArabidopsis splicing related protein-coding genes. First, sequences of splicing-related proteins from human and Drosophila were downloaded from GenBank according to several recent proteomic studies [Arabidopsis annotated proteins . These candidates were regarded as true homologs.A three-round BLAST search strategy was used to identify  studies -18 and t studies . Human h studies  were dow TIGR at ). All hiArabidopsis protein set again. An e-value of 10-20 was used as a cutoff value to find possible paralogs of the true homologs. Sequences identified in both rounds of BLAST hits were regarded as main candidates for splicing related proteins.In the second step, all identified true homologs were used to query the Finally, the main candidates were queried against GenPept and all annotated human proteins (obtained from Ensembl ). All caThe gene structure and chromosomal locations for the genes encoding splicing-related proteins were retrieved from AtGDB . The chrInterProScan 3.3 was downloaded from  and was The gene families with multiple copies were inspected to determine whether they were likely to have derived from chromosome-duplication events. Gene models of the duplicated gene were searched against the gene list of each chromosome redundancy region at MATDB . If the Arabidopsis ESTs and cDNAs were aligned against the genome using the spliced alignment program GeneSeqer as made available through AtGDB [All gh AtGDB . We retrDetails about each splicing-related gene were saved in a MySQL database. PHP scripts were written to interact with the database and generate the interface web pages. Text and BLAST searches were implemented by Perl-cgi scripts.


The primary sensory cortex (S1) in the postcentral gyrus is comprised of four areas that each contain a body map, where the representation of the hand is located with the thumb most laterally, anteriorly and inferiorly and the little finger most medially, posteriorly and superiorly. Previous studies on somatotopy using functional MRI have either used low field strength, have included a small number of subjects or failed to attribute activations to any area within S1. In the present study we included twenty subjects, who were investigated at 3 Tesla (T). We focused specifically on Brodmann area 3b, which neurons have discrete receptive fields with a potentially more clearcut somatotopic organisation. The spatial distribution for all fingers' peak activation was determined and group as well as individual analysis was performed.Activation maps from 18 subjects were of adequate quality; in 17 subjects activations were present for all fingers and these data were further analysed. In the group analysis the thumb was located most laterally, anteriorly and inferiorly with the other fingers sequentially positioned more medially, posteriorly and superiorly. At the individual level this somatotopic relationship was present for the thumb and little finger, with a higher variability for the fingers in between. The Euclidian distance between the first and fifth finger was 17.2 mm, between the first and second finger 10.6 mm and between the remaining fingers on average 6.3 mm.Results from the group analysis, that is both the location of the fingers and the Euclidian distances, are well comparable to results from previous studies using a wide range of modalities. On the subject level the spatial localisation of the fingers showed a less stringent somatotopic order so that the location of a finger in a single subject cannot be predicted from the group result. The first somatotopic maps of the homuncular organisation of the primary somatosensory cortex S1) were established in 1937 by using intra-operative electrical stimulation of the brain surface  were est-6. StudiPrevious studies on somatotopy in the hand area using functional Magnetic Resonance Imaging (fMRI) have yielded varying results. Gelnar et al. failed to show a somatotopy in S1 when applying vibratory stimuli to three of the fingers of the right hand . MaldjiaMethods differ considerably between these three studies with regard to anatomical considerations, number of subjects studied, and the field strength used. Maldjian et al. used the highest field strength, 4 Tesla (T), while both Kurth et al. and Gelnar et al. used 1.5 T -5. HowevIn the present study we readdressed the issue of somatotopy in the hand area as assessed with fMRI. Our aim was to optimise results by including a larger number of subjects, by focussing on area 3 b, where homuncular organisation expectedly is most distinct and by performing fMRI at 3 Tesla (T).Tactile stimulation of the fingers of the dominant hand yielded activation in contra-and ipsilateral S1, contra- or bilateral secondary sensory cortex S2), ipsilateral cerebellum, and in some subjects the contralateral thalamus. Significant activation for all five fingers in area 3b was present in seventeen subjects and these data were further analyzed. In the one volunteer excluded from the analysis, activation was present for four fingers. The spatial distribution of the activations in the contralateral S1 for tactile stimulation versus rest in one subject is shown in Figure , ipsilatA somatotopic organisation with the representation of the thumb located laterally to the little finger was present in 16 out of 17 subjects, with the thumb located anteriorly to the little finger in 14 out of 17 subjects and with the thumb located inferiorly to that of the little finger in 16 out of 17 subjects.Group averages of the distances from D2 to D1 (D2-D1), D3 to D1(D3-D1), D4 to D1 (D4-D1) and D5 to D1 (D5-D1) are presented in the Table and shown as graphs in Figure The Euclidian distance from D1 to D2 was 10.6 mm (SEM ± 1.5). The distance from D2 to D3 was 5.5 mm (± 0.9), from D3 to D4 7.4 mm (± 1.1) and from D4 to D5 6.8 mm (± 1.2), resulting in an average for D2-D3, D3-D4 and D4-D5 of 6.6 mm.The spatial extension of the representation of the hand in area 3b, defined as the Euclidian distance between D1 and D5 was 17.2 mm (± 2.0 mm).In the group average from the present study the strict somatotopic organisation in the primary sensory cortex known from studies using a variety of modalities, was reproduced -6. The fThe average extension of the hand representation in area 3b of 17 mm with a somatotopic arrangement of fingers 1-5 as described above is consistent with results from previous studies using a range of modalities -4,7. Als3, resampled to 1.5 × 1.5 × 1.5 mm3, activation for all five fingers was found in 17 of 18 subjects. For comparison, Kurth et al. used a resolution of 1.7 × 1.7 × 1.7 mm3 and electrical stimulation with ring electrodes and found activation in area 3b for all five fingers in 7 out of 10 subjects (70%) [4]. The present study showed activations for all fingers in 94 % of subjects. This difference might be due to the higher field strength used in this study as the higher magnetisation vector and sensitivity to changes in susceptibility increase the signal-to-noise ratio.With the spatial resolution used in this study, 3 × 3 × 3 mmIn one subject excluded from the analysis, activation was present for four fingers and another was excluded due to general lack of activation. Lack of activation may be due to subjects being 'low-activators' in fMRI experiments, due to inadequate stimulation or to some other, unknown factor.The type of stimulation used is decisive of what area in S1 can be expected to be activated. For example, neurons in area 3a are responsive to deep receptor and proprioceptive stimulation and in one study punctate tactile stimulation did not activate area 3a . Also, rWe found activation in the anterior wall of the postcentral gyrus, defined as area 3b according to our operational definition during tactile stimulation Fig . More prIn the group analysis, a somatotopic organisation for all the fingers in the hand representation of area 3b could be demonstrated using fMRI; the Euclidian distance between the thumb and the little finger was well comparable to that determined in previous studies. On the subject level the cortical somatosensory representation of the thumb was located laterally, anteriorly, and inferiorly to that of the little finger in 14 out of 17 subjects. The spatial localisation of the remaining fingers showed a less stringent somatotopic order when compared individually.Twenty healthy, self-reportedlly right-handed volunteers  were included in the study. The protocol was approved by the local ethics committee and written informed consent was obtained. All volunteers had normal images on a fluid-attenuated inversion recovery (FLAIR) sequence.Tactile stimulation consisted of brushing the glabrous skin of the two distal phalanges of each finger continuously forwards and backwards with a commercially available tooth brush. During the experiment, volunteers were positioned on the MR table with their right arm from the elbow down in a padded cast, that also provided support for the dorsal part of the hand. They were instructed to rest their arm against the magnet bore so that both arm and hand were relaxed. Pieces of soft cloth were placed between the fingers in order to avoid that stimulation also involved a neighbouring finger.The frequency was 1 Hz; no forced pressure was exerted. Consistency was tested on a finger model firmly taped onto a computerized electronic scale : the intra-examiner error was 18% based on a mean pressure of 6.64 g, standard deviation 1.199 g.3 using a magnetisation prepared gradient echo sequence (MPRAGE) were acquired. Functional echo-planar image volumes of the whole brain  sensitised to the Blood Oxygenation Level Dependent (BOLD)-effect (echo time = 30 ms) were acquired. Five scanning sessions were performed. Each session included 92 functional volumes with a temporal resolution of 3 seconds. The first two volumes in each session were discarded from further analysis to allow for initial T1-equilibrium effects.MRI was performed using a 3 T head scanner (Siemens Allegra) with a quadrature birdcage coil. Morphological T1-weighted images with a resolution of 1 × 1 × 1 mmFingers 1 to 5  of the right hand were stimulated sequentially in separate sessions according to a block design that included four periods of stimulation and five of rest for each finger. The epoch length for both stimulation and rest periods was 30 seconds.3 and then realigned to the first image and coregistered to the T1-weighted image volume. All data were spatially filtered using an isotropic 4 mm, full-width, half-maximum Gaussian kernel. A high pass filter (cut off frequency 0.008 Hz) was applied to eliminate low frequency signal fluctuations. In order to preserve each subject's somatotopic arrangement in area 3b no normalization to a common brain atlas was performed.Image processing and analysis were carried out using the SPM99 soft ware package . All funFunctional data from two subjects were excluded due to major motion artefacts (subject no. 7) and global lack of activation (subject no.18). Task specific effects were estimated using the general linear model (GLM) that included a box car function convolved with the canonical hemodynamic response function in SPM99. The effect of sensory stimulation of each finger versus rest was determined using a one-sample t-test of pertinent linear contrasts of parameter estimates in each subject with a significance level of p < 0.001 (uncorrected). Then the spatial coordinates of the peak activation voxel in area 3b were determined. Due to lack of neuroanatomical landmarks, exact delineation of the cytoarchitectonically defined areas within S1 cannot be achieved in MR images. Therefore we used an operational definition based on cytoarchitectonic studies of S1 on 10 post-mortem brains. In >50 % of these brains area 3a was located in the fundus of the central sulcus, area 3b in the rostral bank of the postcentral gyrus and area 1 on its crown reaching down into the postcentral sulcus . Fig 1 iThe spatial coordinates of the peak voxel for the thumb (D1) was defined as being at origo  in a 3D Cartesian coordinate system.When the spatial coordinates for all fingers were known in all subjects, somatotopy was assessed by determining the average distances for the whole group from each finger to the thumb.Euclidian distances between fingers as well as from each finger to the thumb (D1) were calculated as: DI, yDI and zDI representing the coordinates of the finger DI and xDII, yDII and zDII representing the coordinates of DII in the three directions x, y, z in a system where the coordinates of D1 are at origo .with xEach finger's distance to the first finger was compared to that of the directly neighbouring fingers using the Wilcoxon matched pairs test with a significance level of p < 0.05. The Euclidian distances to the first finger were compared for each finger using the Wilcoxon matched pairs test with a significance level of p < 0.05.DvW conceived of the study, performed the image analysis and drafted the manuscript. PF guided the image analysis. JO designed the MR-parameters. EML participated in the design of the study. BR and GL initiated and conducted the stimulation consistency study. All authors read and approved the final manuscript.


In this study, we have examined the role of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in viral gene expression in T lymphocytes transformed by HTLV-1.hnRNPA1 gene using RNA interference could inhibit Rex-mediated export of viral mRNAs in HTLV-1 producing C91PL T-cells. We show that the expression of NLS-A1-HA does not modify the export of Rex-dependent viral mRNAs. Conversely, inhibiting A1 expression in C91PL cells by RNA interference provoked an increase in the Rex-dependent export of unspliced and singly spliced mRNAs. Surprisingly, we also observed a significant increase in proviral transcription and an accumulation of unspliced mRNAs, suggesting that the splicing process was affected. Finally, A1 knockdown in C91PL cells increased viral production by these cells. Thus, hnRNP A1 is implicated in the modulation of the level of HTLV-1 gene expression in T cells transformed by this human retrovirus.We have previously observed that hnRNP A1 (A1) down-modulates the post transcriptional activity of Rex protein of HTLV-1. Here, we tested whether the ectopic expression of a dominant negative mutant (NLS-A1-HA) defective in shuttling activity or knockdown of the These observations provide an insight into a new cellular control of HTLV-1 replication and suggest that hnRNP A1 is likely part of the regulatory mechanisms of the life cycle of this human retrovirus in T cells. We hrex mRNA .The ubiquitously expressed hnRNP A1 is an abundant nuclear protein that participates in RNA processing, alternative splicing and chromosome maintenance as well as in the nucleocytoplasmic transport of mRNAs -18. ThisAs indicated above, we have provided evidence that hnRNP A1 impairs the post-transcriptional regulation of HTLV-1 gene expression, by interfering with the binding of Rex to the XRE . In the luc gene  were then transiently transfected in Jurkat cells in the absence or in the presence of a Rex-expressing plasmid. It was observed that, in presence of Rex, luc expression in cells transfected with the CMV/mutXRE vector was more than 3-fold higher than that in cells transfected with the CMV/XRE vector  and singly-spliced (env) mRNAs. After a few days of culture in presence of puromycin, immunostaining of the resistant population revealed that about 30% of the cells were displaying HA labelling  was observed, when compared to the respective control cells (678 ± 104 pg/ml). Taken together, these results indicate that the impairment of the hnRNP A1 functionality might favour the translation of cytoplasmic viral mRNAs.We next investigated whether overexpression of this defective hnRNP A1 mutant was interfering with the expression of viral mRNAs. Quantification of the nuclear and the cytoplasmic levels of unspliced RNA Fig. . The comNAs Fig. . The ratcted Fig . As the We next evaluated whether HTLV-1 replication is modulated by RNA interference with hnRNP A1 gene expression. To that aim, two oligonucleotides encoding siRNA directed against hnRNP A1, one targeting an RNA sequence located on the 5' end (34-nt after the translation start site), and the other an RNA sequence close to the 3'end (548-nt after translation start site) were each inserted in the pRS retroviral vector , as indiin situ synthesis of siRNAs and to suppress specifically hnRNP A1 gene expression in C91PL cells. Retroviruses produced from pRS-siRNA+548 and from the pRS empty vector were used to transduce these cells with a m.o.i. of 5. Four days after transduction, hnRNP A1 depletion was assessed by quantitative PCR analysis of cytoplasmic mRNAs. In siRNA-transduced C91PL cells, that transcript represented 32% of that in control pRS transduced cells  assessing the amount of total viral mRNAs Fig  revealedred Fig. . A slighgag producing cells in siRNA-transduced C91PL cells and in control cells, the quantification of 19gag in the supernatant medium of siRNA-transduced cells revealed a 1.5-fold increase of the p19gag amount (1017 ± 26 pg/ml), compared to that in control cells (678 ± 104 pg/ml).These results suggest that inhibition of hnRNP A1 in C91PL cells mainly correlates with a defect in the splicing of genomic mRNAs. The NER of the unspliced and singly spliced mRNAs was significantly higher in siRNA-treated cells than in control cells, whereas the cytoplasmic expression of tax/rex mRNAs, which is Rex-independent was not modified Fig. . As the gag/pol transcript and increases the post-transcriptional activity of Rex leading to an increase of viral production.Collectively, these data support that the hnRNP A1 depletion in HTLV-1-producing T cells increases viral transcription, is correlated with a defect in the splicing process at the level of the The ubiquitously expressed hnRNP A1, an RNA-binding protein, is a nucleocytoplasmic shuttling hnRNP that accompanies eukaryotic mRNAs from the active site of transcription to that of translation. As such, hnRNP A1 is involved in a variety of important cellular functions, including RNA splicing, transport, turnover and translation. We have previously shown that hnRNP A1 decreases the post-transcriptional activity of the Rex protein of HTLV-1, by interfering with the binding of the viral protein on its response element, present on the 3' LTR of all viral RNAs. Here we first report that the mutation of a putative binding site of hnRNP A1 in the XRE enhances the functional activity of Rex. This observation obtained through transient transfection experiments, confirms that A1 proteins could antagonize the post-transcriptional activity of Rex, by a competitive mechanism.gag/pol, the singly spliced env and the doubly spliced tax/rex) mRNAs, which encode the structural and regulatory proteins. The gag/pol and env mRNAs are dependent on Rex for their cytoplasmic expression. To determine whether hnRNP A1 interferes with viral replication, we first examined the effect of the ectopic expression of an hnRNP A1 mutant (NLS-A1-HA) defective in nuclear export activity. This mutant was previously used to assess the potential role of hnRNP A1 in nucleocytoplasmic shuttling activity in normal and leukemic myelopoiesis. Interestingly it was found that the ectopic expression of this dominant negative form of hnRNP A1 resulted in the downmodulation of the nucleocytoplasmic trafficking of cellular mRNAs that encode proteins affecting the phenotype of normal and transformed myeloid progenitors [We have next investigated the role of hnRNP A1 in HTLV-1 transformed C91PL cells, which produce HTLV-1 virions. These express the three differentially spliced . This is consistent with other studies showing that si-RNA-mediated reduction in A1 levels did not affect cell division nor provoke cell death in normal cell lines .gag protein. Since hnRNP A1 has been implicated in nuclear export of cellular mature mRNAs [We next performed siRNA depletion of hnRNP A1 in C91PL cells and have observed a significant increase in proviral transcription, as demonstrated by the higher level of viral transcripts than that in control cells Figure . Furtherre mRNAs  as well in vitro HTLV-1-transformed T-cells, that consisting in the depletion of this cellular protein by RNA interference provides evidence for the role of hnRNP A1 in restraining the viral life cycle at both transcriptional and post-transcriptional levels. We conclude from these findings that down-regulation of hnRNP A1 has an important role on the replicative potential of HTLV-1 in T lymphocytes. Consequently, these data allows us to define hnRNP A1 as a cellular protein endowed with an anti-HTLV-1 activity.Of the two experimental approaches used in the present study to apprehend the implication of hnRNP A1 on HTLV-1 replication in The vector pRetro-SUPER (pRS) was used to generate biologically active siRNAs from the Pol III H1-RNA gene promoter . Two annluc gene and the XRE are located within the intron  was derived from the reporter plasmid pDM138 containing the CAT gene and the XRE sequences . It exprsee Fig. . The mut5 cells per well of a 12-well plate were transfected using the calcium phosphate coprecipitation technique [Jurkat lymphoblastoid T-cells were incubated at 37°C in a 5% CO2 atmosphere, in RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (FCS) and 20 IU/ml penicillin, 20 μg/ml streptomycin. The HTLV-1-transformed T-cell line, C91PL  was cultechnique . Jurkat 5 cell/well of a 6-well plate) with 2 μg of pRS or pRS-siRNA together with 1 μg of pgag-pol/MLV and 0,45 μg of env/VSV-G with ExGen 500 reagent (Euromedex). Twelve hours later, the cells were washed once with PBS, and newly produced virions were harvested over 24 h in 1,5 ml of fresh medium. Viral supernatants were clarifed by passage through a 0.45-μm syringe filter and aliquots were stored at -80°C. Titers of virus stocks were determined by infecting rhabdomyosarcoma human TE cells (60% confluent) with serially diluted viral stocks. After infection, cells were split and plated in the presence of puromycin (5 μg/ml); puromycin-resistant colonies were scored after 7 days. Virus titers generally ranged from 3 to 5 × 105 transducing units per ml.Fresh viral stocks were prepared by transfecting 293T cells (seeded at 5 × 106) plated in a 24-well plate were infected at a multiplicity of infection (moi) of 5 with viral stocks in a final volume of 1.0 ml containing 4 μg of polybrene/ml, for 18 h and allowed to recover for 24 hr with fresh medium. When necessary, transduced cells were selected with puromycin 4–5 μg/ml for 4 days and maintained in culture for long time period with 1 μg/ml puromycin.Transduction of Jurkat or of C91 PL T cells with retroviral vectors was carried out as followed: briefly, cells (1 × 106 cells by using an Rneasy RNA-preparation kit (Qiagen) according to the manufacturer's instructions. To reduce the amount of DNA originating from lysis, samples were treated with Rnase-free Dnase  for 30 min at 20°C and then for 15 min at 65°C. 500 ng of RNA sample were reverse transcribed by using oligo(dT)12–18 and Superscript II . Reverse transcription was performed for 50 min at 42°C. The total cDNA volume of 20 μl was frozen until real-time quantitative PCR was performed. After thawing for PCR experiments, the cDNA was diluted in distilled water and 2 μl of diluted cDNA was used for each PCR reaction. The realtime quantitative PCR (RQ-PCR) was performed in special lightcycler capillaries (Roche) with a lightcycler Instrument (Roche), by using the LightCycler-FastStart reaction Mix SYBR-Green kit (Roche). The following specific primers were used to detect: hnRNP A1, sense 5'-AAGCAATTTTGGAGGTGGTG-3' and antisens, 5'-ATAGCCACCTTGGTTTCGTG-3', gag/polHTLV-1sense, 5'-CCCTCCAGTTACGATTTCCA-3' and antisens, 5'-GGCTTGGGTTTGGATGAGTA-3', envHTLV-1sense, 5'-CTGTGGTGCCTCCTGAACT-3' and antisens, 5'-AAAGTGGCGAGAAACTTACCC-3', pXIII sense, 5'-ATCCCGTGGAGACTCCTCAA-3' and antisens, 5'-CCAAACACGTAGACTGGGTATCC-3'. β-actin sense,5'-TGAGCTGCGTGTGGCTCC-3' and antisens: 5'-GGCATGGGGGAGGGCATACC-3'.Nuclear and cytoplasmic RNAs were extracted from 2 × 10The thermal cycling conditions consisted of 40 cycles at 95°C for 10 sec, 61°C for 5 sec, 72°C for 10 sec. The fluorescence signal increase of SYBR-GREEN was automatically detected during the 72°C phase of the PCR. Omission of reverse transcriptase in the RT-PCR protocol led to a failure of target gene amplification in the positive controls. Light cycler PCR data were analyzed using LightCycler Data software (Idaho Technology). The software first normalizes each sample by background subtraction of initial cycles. A fluorescence threshold is then set at 5% full scale, and the software determines the cycle number at which each sample reached this threshold. The fluorescence threshold cycle number correlates inversely with the log of initial template concentration. β-actin transcript levels were used to normalize the amount of cDNA in each sample. Melting curve profiles were used to confirm amplification of specific transcripts.Cells were washed and harvested in ice-cold PBS containing protease inhibitors . Cells were lysed in RIPA buffer  and incubated for 30 min at 4°C. After centrifugation at 12,000 rpm for 10 min at 4°C, the supernatant was assayed for protein content by Bradford assay (Bio-Rad). Equal amounts of proteins were separated by SDS/PAGE.Cells were lysed in Laemmli buffer and equal amounts of proteins were subjected to 12% SDS-PAGE. They were subsequently blotted onto nitrocellulose membrane . The membrane was then blocked overnight at 4°C in blocking buffer (PBS and 0.1% Tween-20) supplemented 10% non-fat powdered milk and probed with the appropriate antibody diluted in blocking buffer plus 10% non-fat powdered milk. The following antibodies were used: rabbit anti-actin (Sigma), mouse anti-ASF/SF2 (gift from Dr. J. Stevenin) mouse monoclonal anti-hnRNP A1 and anti-hnRNP C antibodies , followed with an anti-rabbit  or anti-mouse (Dako) Immunoglobulin G-horse radish peroxidase-conjugated antibody. Blots were then developed using an enhanced chemiluminescence detection system . Bands were visualized by using Hyperfilm (Amersham Pharmacia Biotech).5) were washed twice with PBS, resuspended in 3% (vol/vol) paraformaldehyde/PBS for 45 min at room temperature, and permeabilized with 0.5% Triton X-100/PBS for 5 min. After washing with PBS, the cells were incubated with specific antibodies (4B10) diluted in 1% BSA/PBS for 1 h. Cells were washed twice with PBS and were then incubated with FITC-conjugated goat anti-mouse, PE-conjugated goat anti-rabbit in 1% BSA/PBS for 40 min. Cells were washed three times with PBS and resuspended in a 2% paraformaldehyde/PBS solution. The fluorescence intensity was measured on a FACScan instrument . The integrated fluorescence of the gated population was measured, and data from 10,000 analyzed events were collected.Cells , fixed on slides with 3.7% paraformaldehyde for 15 min at room temperature, and permeabilized with 0.5% Triton X100 for 5 min in 4°C. The samples were saturated with PBS containing 0.5% gelatin and 0.25% bovine serum albumin for 1 h and stained for 1 h with a 1/100 dilution of a rabbit polyclonal serum directed against HA (Y11 from Santa Cruz Biotechnology) (NLS-A1-HA staining) or 1/1000 dilution of mouse monoclonal antibodies (4B10) (hnRNP A1 staining) in the same saturation solution. The samples were then washed three times with PBS containing 0.25% gelatin and incubated for 1 h with a 1/100 dilution of the following secondary antibodies: goat anti-rabbit immunoglobulin G conjugated to fluorescein isothiocyanate (green color for HA) and goat anti-mouse immunoglobulin G conjugated to lissamine rhodamine sulfchloride (red color for hnRNP A1) (Jackson Immunoresearch). The samples were washed three times in PBS with 0.25% gelatin and mounted for analysis on a Zeiss LSM 510 laser scanning confocal microscope.p19gag was measured in culture medium using the RETROTEK HTLV p19 Antigen ELISA kit (Zeptometrix). Medium of the cell culture was centrifuged at low speed to remove the cell debris, and filtrated through a 0,45-μm filter. The amount of Gag protein was quantified in the resultant supernatant according to the manufacturer procedure. Results are expressed as pg/ml of p19 protein and are the mean of two different experiments, each point tested in quadruplicate.The author(s) declare that they have no competing interests.


A systematic review identified a range of methods, which can influence response rates. However, analysis specific to a healthcare setting, and in particular, involving people expected to be poor responders, was missing, We examined the effect of pre-warning letters on response rates to a postal survey of sedentary patients whom we expected a low rate of response.Participants were randomised to receive a pre-warning letter or no pre-warning letter, seven days before sending the main questionnaire. The main questionnaire included a covering letter and pre-paid return envelope. After seven days, non-responders were sent a reminder letter and seven days later, another reminder letter with a further copy of the questionnaire and return envelope.627 adults, with a mean age of 48 years  of whom 69.2% (434/627) were women, were randomised. 49.0% (307/627) of patients were allocated to receive a pre-warning letter and 51.0% (320/627) no pre-warning letter, seven days in advance of posting the main questionnaire. The final response rate to the main questionnaire was 30.0% (92/307) amongst those sent a pre-warning letter and 20.9% (67/320) not sent a pre-warning letter, with an adjusted odds ratio of 1.60 .The relatively low cost method of sending a pre-warning letter had a modest impact on increasing response rates to a postal questionnaire sent to a group of patients for whom a low response rate was anticipated. Investigators should consider incorporating this simple intervention when conducting postal surveys, to reduce the potential for nonresponse bias and to increase the study power. Methods other than postal surveys may be needed however when a low response rate to postal surveys is likely. Postal surveys are routinely used to obtain information from patients and groups within the general population, over a range of topics. Postal surveys are a cost-efficient method compared with intensive methods such as face-to-face interviews and capable of obtaining systematically, information on many thousands of people. A key quality component of postal surveys relates to the number of people sampled, and the proportion returning a completed useable questionnaire . Lower rMany studies conclude that non-responders in surveys and other epidemiological studies can differ to responders with respect to a range of specific health, lifestyle and social variables. Non-responders have been found to differ with respect to their sex, age, race, social class, home circumstances, education, and healthy lifestyle behaviours -7. They A systematic review  found a In the current study, we carried out a randomised controlled trial to examine the effect of a pre-warning letter on response rates to a postal questionnaire. The questionnaire was sent to patients who had previously been referred to a community based exercise referral scheme because of a sedentary lifestyle and sought information on the quality of the service offered. An earlier study  of a simThe sample consisted of patients who had been referred to a community based exercise referral scheme during the past 12 months, identified from the service register. Patients were referred by a primary care practitioner because of concerns about their sedentary behaviour and its impact on their health. The questionnaire formed part of a project examining the relationship between patient service-expectations and service outcomes.We examined the effect of a pre-warning letter, posted to patients seven-days before sending the main questionnaire, compared with no pre-warning letter. The pre-warning letter was printed on one-side of letter-headed paper, and informed the respondent that a survey would be sent to them within the next few days. It informed them about the purpose of the survey and the importance of it being completed and returned.The main questionnaire was sent with a covering letter and a pre-paid business franked addressed envelope for its return . A standThe primary outcome was the final response rate to postal questionnaires after sending all reminder letters. This was calculated at least 6 weeks after sending the initial questionnaire, to allow for late responders. Differences in proportions between groups were examined using Pearson's chi-square test and logistic regression to adjust for age and sex with 95% confidence intervals (95% CI). To observe a difference of at least 10% between trial arms required 752 participants, based on a return rate of 60% in the control groups, with 80% power. Approval for the study was received in advance from the local research ethics committee and the research governance committee.The number of patients referred to the exercise referral scheme in the past year with complete name and address information was 627. Their mean age was 48 years  and 69.2% (434/627) were women. Randomisation allocated 49.0% (307/627) of patients to receive a pre-warning letter and 51.0% (320/627) no pre-warning letter  .The final response rate to the postal survey, after completing two stages of follow-up was 25.4% (159/627). In the pre-warning group, the response rate was 30.0% (92/307) compared with 20.9% (67/320) in the control group (χSending a pre-warning letter seven days in advance of mailing out a postal questionnaire had a modest impact on increasing final response rates by almost 10%, with a relative 43% increase compared with sending no pre-warning letter. Patients in our study were selected on the basis of a previous referral to a community based exercise referral scheme, and the questionnaire sent to them sought information about their perceptions of the service quality. An earlier trial examining the impact of this service on a similar group of patients, in terms of increasing physical activity, had achieved average response rates of 60% . The mucOur findings are consistent with the evidence from a systematic review  that preThe authors declare that they have no competing interests.RAH conceived the study design, drafted the main protocol, carried out randomisation, analysed the results and wrote the first draft of the paper.DC was responsible for completing the study, for data entry, for assisting with data analysis and significant comments on the manuscript.The pre-publication history for this paper can be accessed here:


Saccharomyces cerevisiae and DNA microarrays to comprehensively identify the associated mRNAs. Distinct groups of 40–220 different mRNAs with striking common themes in the functions and subcellular localization of the proteins they encode are associated with each of the five Puf proteins: Puf3p binds nearly exclusively to cytoplasmic mRNAs that encode mitochondrial proteins; Puf1p and Puf2p interact preferentially with mRNAs encoding membrane-associated proteins; Puf4p preferentially binds mRNAs encoding nucleolar ribosomal RNA-processing factors; and Puf5p is associated with mRNAs encoding chromatin modifiers and components of the spindle pole body. We identified distinct sequence motifs in the 3′-untranslated regions of the mRNAs bound by Puf3p, Puf4p, and Puf5p. Three-hybrid assays confirmed the role of these motifs in specific RNA–protein interactions in vivo. The results suggest that combinatorial tagging of transcripts by specific RNA-binding proteins may be a general mechanism for coordinated control of the localization, translation, and decay of mRNAs and thus an integral part of the global gene expression program.Genes encoding RNA-binding proteins are diverse and abundant in eukaryotic genomes. Although some have been shown to have roles in post-transcriptional regulation of the expression of specific genes, few of these proteins have been studied systematically. We have used an affinity tag to isolate each of the five members of the Puf family of RNA-binding proteins in  Messenger RNAs that are associated with each of the five Puf RNA-binding proteins can be grouped according to function and localization, suggesting that tagging of transcripts by specific RNA-binding proteins coordinates the control of their localization, translation, and decay The dynamic structure and physiology of a cell depend on coordinated synthesis, assembly, and localization of its macromolecular components . The timSaccharomyces cerevisiae cell during exponential growth in rich medium , regulate the switch from spermatogenesis to oogenesis by repressing fem-3 translation, and they are implicated in the propagation of germline stem cells through binding and inhibition of gld-1 mRNA expression  (Puf) proteins from karyotes . Puf prokaryotes . The Pufkaryotes . For exapression . Less ispression .S. cerevisiae, five proteins, termed Puf1p to Puf5p, bear six to eight Puf repeats -tagged proteins were purified from whole-cell extracts of revisiae . The TAPrevisiae , a sequerevisiae . This derevisiae . To contrevisiae . Two samnome see . The ratS. cerevisiae Puf proteins for which direct in vitro interaction with an mRNA (COX17) has previously been described, thereby providing an internal positive control   see . To defiale) see . Althougale) see B, the ocale) see .S. cerevisiae genome. Of these, 90 transcripts interact with more than one Puf protein. The largest overlap was observed between the groups of transcripts associated with Puf1p and Puf2p—which also have the greatest overall similarity in amino acid sequence among the Puf proteins ; 36 of the 40 Puf1p targets were also associated with Puf2p. Twenty-eight mRNAs were bound by both Puf4p and Puf5p, and 16 were bound both by Puf2p and Puf5p. Seven transcripts were enriched with three different Puf proteins . The remaining 645 target mRNAs were each associated with only one of the Puf proteins. Thus, each Puf protein associates with a distinct and highly specific subset of mRNAs   annotations for process, function, and compartment from the se (SGD) . (The tase (SGD) . We thense (SGD) .p < 10−88; see S. cerevisiae ). Second, the Puf5p-associated transcripts included a substantial fraction of the mRNAs known to encode components or regulators of the mitotic spindle apparatus in yeast: 14 mRNAs that encode microtubule-based spindle components, including seven of the 25  structural components of the spindle pole body   . MessageS. cerevisiae genome (p < 10−12). Of these 36, 29 are directly involved in ribosomal RNA (rRNA) synthesis, processing, and ribosome maturation (p < 10−15), major functions of the nucleolus (Transcripts encoding nucleolar proteins were highly enriched among the Puf4p-bound mRNAs: 36 of the 133 (27%) annotated genes in this group encode nucleolar proteins, as compared to 3% of all the annotated genes in the ucleolus  (see Tabucleolus  and S8.p < 10−11), among them the four core histone proteins .Twenty-eight transcripts were enriched in both the Puf4p and Puf5p affinity isolations, including six transcripts encoding components of the nucleosome , which synthesizes pseudouridine in rRNAs     were bou NCE102) .  as a motif discovery tool   were among the transcripts identified as Puf3p targets by our co-purification experiments, and all encode mitochondrial proteins. This result could reflect a direct effect of Puf3p on its target mRNAs, for example, by promoting mRNA decay  . Of the NA decay . HoweverS. cerevisiae genome, we found that more than 700 transcripts appeared to be specifically bound by one or more RBPs, with each of the five Puf family proteins “tagging” a distinct set of mRNAs. These sets encode functionally and cytotopically related proteins. For three of the Puf proteins, we identified distinct short sequences in the associated specific set of mRNAs, typically in the 3′-UTR, which were sufficient for specific binding to the cognate Puf protein in vivo. Many sets of mRNAs encoding proteins localized to the same subcellular compartment, protein complex, or functional system were bound by the same Puf protein. Puf3p, which specifically associated with cytoplasmic mRNAs encoding mitochondrial proteins, generally affected the steady-state levels of its mRNA targets as reflected by their increased abundance in puf3 mutant cells.In an analysis of just five of the hundreds of RBPs encoded by the The selective “tagging” by sequence-specific RBPs of mRNAs that share common physiological roles suggests a general and widespread mechanism for coordinated control of their expression. Previous reports have identified coordinated regulation of small sets of functionally related mRNAs by specific RBPs. For example, mammalian stem–loop binding protein (SLBP) associates with all five classes of histone mRNAs and guides proper 3′-end formation . Iron reS. cerevisiae have been subjected to a similar genome-wide survey of their mRNA targets. She2p, which plays a critical role in selective targeting of specific mRNAs to the bud tip  was obtained from Cellzome  . TAP-tagon Davis .PUF3 was amplified by PCR with primers Puf3-F1 and Puf3-R1 from S. cerevisiae genomic DNA and cloned into pCR2.1 using the TOPO TA Cloning Kit . The PUF3 ORF was sequenced and subcloned into pACTII via NcoI and EcoRI restriction sites, resulting in plasmid pACTII-Puf3. A full-length Puf5p construct pGAD-MPT5 was a gift from Dr. Kenji Irie (The ORF of nji Irie .Sequences encoding the Pum-HD domains of Puf3p (amino acids 535–879), Puf4p (amino acids 557–888), and Puf5p (amino acids 202–578) were PCR-amplified from genomic DNA with oligo pairs Puf3-F6nco/Puf3-R1, Puf4-F2nco/Puf4-R1eco, and Puf5-F2nco/Puf5-R1xho, respectively. Products were ligated into pCR2.1-TOPO, sequenced, and further cloned into pACTII via restriction sites present in the oligonucleotides used for amplification.PUF3 RNA consensus sequence spanning nucleotides 24–33 in the 3′-UTR of YBL038w/MRPL16 was constructed with oligonucleotides M16-1 and M16-2. In M16mut the conserved UGU motif was changed to ACA. The PUF4 consensus (nucleotides 24–34 in the 3′-UTR of YOR145c) was constructed with oligonucleotides Yor-1 and Yor-2. The PUF5 consensus (nucleotides 105–114 in the 3′-UTR of YNL278w/CAF120) was constructed with oligonucleotides Caf-1 and Caf-2.The RNA consensus sequences interacting with Puf proteins plus ten nucleotides of flanking sequences were cloned into the SmaI and SphI sites of the vector pIIIA/MS2-2  using an2, 0.1% Nonidet P-40 [NP-40], 0.02 mg/ml heparin) and resuspended in 5 ml of buffer B . Cells were broken mechanically with glass beads, and extracts were incubated with 400-μl slurry (50% [v/v]) IgG–agarose beads  for 2 h at 4°C. The beads were washed four times for 15 min at 4°C with buffer C . Puf proteins were released from the beads by incubation with 80 U of TEV protease (Invitrogen) for 2 h at 15°C. RNA was isolated from the TEV eluates, which corresponds to the purified fraction and from extracts (input) by extraction with phenol/chloroform and isopropanol precipitation.For a detailed protocol, see the Supporting Information on our Web site. In brief, 1 l of cells were cultured in YPAD medium (yeast–peptone–dextrose [YPD] supplemented with 20 mg/ml adenine–sulfate) at 30°C and collected during exponential growth by centrifugation. Cells were washed twice with ice-cold buffer A . Microarrays were scanned with an Axon Instruments  Scanner 4000. Scanning parameters were adjusted to give similar fluorescent intensities for B. subtilis spots in both channels. Data were collected with the GENEPIX 3.0 Program (Axon Instruments), and spots with abnormal morphology were excluded from further analysis. Arrays were computer normalized by the Stanford Microarray Database (SMD)  Excel after filtering for regression correlation of greater than 0.6 (filters for large variations in the ratios of pixels within each spot), CH1I/CH1B of greater than 1.8 , and CH2I/CH2B of greater than 1.0  and for data from at least two independent measurements. Average log2 ratios were calculated for each gene across the four independent experiments performed for each Puf protein (microarrays and raw data can be downloaded from our supporting Web sites [http://microarray-pubs.stanford.edu/yeast_puf/ and http://genome-www5.stanford.MicroArray/SMD/]). Genes for which the enrichment ratios were at least two standard deviations above the median across all genes were selected. A total of 923 genes were selected in this way. To eliminate nonspecifically enriched RNAs from this gene list, the results from the affinity enrichments for each of the Puf proteins and the data obtained from four independent mock affinity enrichments were clustered by the Pearson correlation algorithm  . Log2 melgorithm . Transcrlgorithm , most wepuf3 mutant and wild-type cells were cultured in minimal media supplemented with 3% glycerol and harvested during exponential growth (OD600 = 0.5). Total RNA (8 μg) isolated from wild-type and mutant cells were used to prepare Cy3 and Cy5 fluorescently labeled cDNA as described above, except that only an oligo(dT) primer was used. The two differentially labeled cDNAs were mixed together and hybridized to yeast DNA microarrays. Arrays were scanned and the data were collected, entered into SMD, and computer normalized .rmalized . Log2 mehttp://meme.sdsc.edu/meme/website/intro.html)   (see Tabro.html) . The num the SGD . TherebyThree-hybrid assays were performed as described elsewhere .http://www.med.unc.edu/%7Ehdohlman/IF.html. Fixed and permeabilized cells were treated with 5 μg/ml purified rabbit immunoglobulin (Sigma) for 1 h at room temperature. After washing, cells were incubated with Cy3 goat anti-rabbit antibodies (1:400). Images were obtained on a Zeiss  Axioplan-2 microscope using an Axiocam HRC camera.Immunofluorescence was performed as described at http://microarray-pubs.stanford.edu/yeast_puf/ and at http://genome-www5.stanford.MicroArray/SMD/.Full microarray results and other supporting information can be viewed at Figure S1See (167 KB EPS).Click here for additional data file.Figure S2PMA1 and PMA2 encode plasma membrane proteins that comprise the major ATP-dependent proton transporters and regulate cellular pH levels. Pmp1p, Pmp2p, and Pmp3p are small isoproteolipids, which are present in a physical complex with Pma1p and act as regulators of its activity upon stress conditions ((A) Puf2p-bound mRNAs encode diverse proteins involved in regulation of ATP-dependent proton transport. nditions . Hrk1p inditions . Ast1p inditions .O-ribose methylation of pre-rRNA, a process guided by small nucleolar RNAs (snoRNAs) of the box C/D family. Cbf5p catalyzes pseudouridine formation with box H/ACA snoRNAs, and three of the four components of the H/ACA core complex were Puf4p-associated  Puf4p-bound mRNAs encode the nucleolar GAR proteins (blue), members of the H/ACA core complex (boxed), and Hmt1p, a dimethylase acting on GAR proteins. Nop1p performs 2′-and Nhp2 ; no data(38 KB EPS).Click here for additional data file.Figure S3Serial dilutions (1:10) of cells were spotted on plates supplemented with the indicated media. Plates were incubated for 3 d at 30°C. Abbreviations: YPD, yeast–peptone–dextrose; YPGE, yeast–peptone–3% glycerol–2% ethanol; SC, synthetic complete.(264 KB PDF).Click here for additional data file.Table S1(15 KB XLS).Click here for additional data file.Table S2Cells were grown to mid-log phase in YPAD medium and the number of cells was counted. Whole-cell extracts were prepared as described previously . In brief, cells were resuspended in 1× SDS-PAGE sample buffer, incubated at 100°C for 10 min, and vortexed for 2 min with glass beads. After a short centrifugation, eight dilutions of cell extracts and protein A , which served as a reference standard, were spotted on a nitrocellulose filter. Expression of IgG-binding domains was monitored with rabbit peroxidase–anti-peroxidase soluble complex at 1:5,000 (Sigma). Chemiluminescence was measured with a Typhoon 8600 Imager  and quantified with the ImageQuant 5.2 software. Averaged numbers from two independent measurements were used for calculations. The total number of mRNA copies in the pool associated with each Puf protein was estimated as follows: copy numbers for individual mRNAs were retrieved from two independent genome-wide measurements . For gen(30 KB XLS).Click here for additional data file.Table S32 ratios of enrichment across four independent Puf affinity isolations; standard deviations; association of mRNA with other Puf proteins; mRNA copy numbers.Columns indicate the following (from left to right): ORF; gene name; GO annotations; classification of gene products (soluble/membrane-associated); average log(28 KB XLS).Click here for additional data file.Table S4Notations are as in (52 KB XLS).Click here for additional data file.Table S52 ratios of enrichment across four independent Puf affinity isolations; standard deviations; association of mRNA with other Puf proteins; location of consensus motif identified by MEME; mRNA copy numbers.Columns indicate the following (from left to right): ORF; gene name; GO annotations; classification of gene products (soluble/membrane-associated); average log(70 KB XLS).Click here for additional data file.Table S6Notations are as in (61 KB XLS).Click here for additional data file.Table S7Notations are as in (64 KB XLS).Click here for additional data file.Table S8p values of less than 0.001 are indicated. GO annotations were retrieved from the SGD with GO Finder (http://db.yeastgenome.org/cgi-bin/SGD/GO/goTermFinder) on May 21, 2003. Respective p values are indicated in a column next to the names of the GO term.Only annotations with (30 KB XLS).Click here for additional data file.Table S9(63 KB XLS).Click here for additional data file.Table S10(55 KB XLS).Click here for additional data file.Table S11(34 KB XLS).Click here for additional data file.Drosophila, or C. elegans proteins are from the SwissProt database (http://www.ebi.ac.uk/swissprot/): CPEB (Q18317), GLD1 (Q17339), DAZL (Q92904), FBF-1 (Q9N5M6), FEM3 (P34691), IRP (P21399), NANOS (P25724), Drosophila PUMILIO (P25822), human PUMILIO-1 (Q14671), human PUMILIO-2 (Q9HAN2), and SLBP (P97330).All accession numbers for human, S. cerevisiae genes are from SGD (http://genome-www.stanford.edu/Saccharomyces/) (ORF/SGD identification number): ADA2 (YDR448W/S0002856), AME1 (YBR211C/S0000415), APS3 YJL024C/S0003561), AST1 (YBL069W/S0000165), BBP1 (YPL255W/S0006176), BDF1 (YLR399C/S0004391), BDF2 (YDL070W/S0002228), BOI2 (YER114C/S0000916), BSP1 (YPR171W/S0006375), BUB1 (YGR188C/S0003420), BUD9 (YGR041W/S0003273), CBF5 (YLR175W/S0004165), CDC31 (YOR257W/S0005783), CNM67 (YNL225C/S0005169), COX17 (YLL009C/S0003932), DAD2 (YKR083C/S0001791), DHH1 (YDL160C/S0002319), ELM1 (YKL048C/S0001531), FPS1 (YLL043W/S0003966), GAR1 (YHR089C/S0001131), GIC1 (YHR061C/S0001103), HDA1 (YNL021W/S0004966), HFI1 (YPL254W/S0006175), HMT1 (YBR034C/S0000238), HOS1 (YPR068C/S0006272), HOS3 (YPL116W/S0006037), HST1 (YOL068C/S0005429), HTA1 (YDR225W/S0002633), IFM1 (YOL023W/S0005383), KAR1 (YNL188W/S0005132), KAR9 (YPL269W/S0006190), KHD1 (YBL032W/S0000128), MAS6 (YNR017W/S0005300), MEX67 (YPL169C/S0006090), MUP3 (YHL036W/S0001028), NCE101 (YJL205C/S0003742), NCE102 (YPR149W/S0006353), NHP2 (YDL208W/S0002367), NOP1 (YDL014W/S0002172), NSR1 (YGR159C/S0003391), NUF2 (YOL069W/S0005430), PDR16 (YNL231C/S0005175), PMA1 (YGL008C/S0002976), PUF1 (YJR091C/S0003851), PUF2 (YPR042C/S0006246), PUF3 (YLL013C/S0003936), PUF4 (YGL014W/S0002982), PUF5 (YGL178W/S0003146), RAX2 (YLR084C/S0004074), RSC1 (YGR056W/S0003288), RSC2 (YLR357W/S0004349), RSC4 (YKR008W/S0001716), SBP1 (YHL034C/S0001026), SCP160 (YJL080C/S0003616), SFK1 (YKL051W/S0001534), SFL1 (YOR140W/S0005666), SHE2 (YKL130C/S0001613), SIN3 (YOL004W/S0005364), SNC2 (YOR327C/S0005854), SNT1 (YCR033W/S0000629), SPC19 (YDR201W/S0002609), SPC42 (YKL042W/S0001525), SPT7 (YBR081C/S0000285), SPT8 (YLR055C/S0004045), SSO2 (YMR183C/S0004795), STE7 (YDL159W/S0002318), SUR7 (YML052W/S0004516), TPO1 (YLL028W/S0003951), TPO2 (YGR138C/S0003370), TPO3 (YPR156C/S0006360), VPS72 (YDR485C/S0002893), YIP1 (YGR172C/S0003404), YKL091c (YKL091C/S0001574), YPR157w (YPR157W/S0006361), and YRA1 (YDR381W/S0002789).The accession numbers for


Catecholamines, such as epinephrine, are elaborated in stress responses, and mediate vasoconstriction to cause elevation in systemic vascular resistance and blood pressure. Our previous study has shown that IL-1 can induce mast cells to produce proinflammatory cytokines which are involved in atherogenesis. The aim of this study was to determine the effects of epinephrine on IL-1-induced proatherogenic cytokine production from mast cells.6 cells/ml) were cultured with epinephrine (1 × 10-5 M) in the presence or absence of IL-1β (10 ng/ml) for 24 hrs. HMC-1 cultured alone produced none to trace amounts of IL-6, IL-8, and IL-13. IL-1β significantly induced production of these cytokines in HMC-1, while epinephrine alone did not. However, IL-6, IL-8, and IL-13 production induced by IL-1β were significantly enhanced by addition of epinephrine. The enhancing effect appears to involve NF-κB and p38 MAPK pathways. Flow cytometry showed the presence of β1 and β2 adrenoreceptors on resting mast cells. The enhancing effect of proatherogenic cytokine production by epinephrine was down regulated by the β1 and β2 adrenoceptor antagonist, propranolol, but not by the β1 adrenoceptor antagonist, atenolol, suggesting the effect involved β2 adrenoceptors. The enhancing effect of epinephrine on proatherogenic cytokine production was also down regulated by the immunosuppressive drug, dexamethasone.Two ml of HMC-1 (0.75 × 10These results not only confirm that an acute phase cytokine, IL-1β, regulates mast cell function, but also show that epinephrine up regulates the IL-1β induction of proatherogenic cytokines in mast cells. These data provide a novel role for epinephrine, a stress hormone, in inflammation and atherogenesis. Atherogenesis involves the cellular infiltration of several cell types, including monocytes, T lymphocytes, and mast cells. Cytokine secretion by these cells and endothelial cells are contributing factors in the growth and propagation of atherosclerotic plaques as well as the stability and degradation of fibrous caps. Cytokines implicated in atherogenesis include Interleukin (IL)-1β, IL-6, IL-8, IL-13, and Tumor Necrosis Factor (TNF) .IL-1β is secreted mainly by macrophages and virtually by every cell type in the body. IL-1β is produced in response to various stimulants, such as cytokines, bacteria, and viruses, but most interestingly to epinephrine . IL-1β het al. further supports the role of mast cells in atherogenesis [Mast cells are found preferentially around blood vessels and beneath the epithelium of the skin and mucus membranes ,10-12. Togenesis . They deStress is known to influence immune function -17. An iin vitro [Epinephrine and norepinephrine increase the uptake of low density lipoprotein in atheroscelotic plaques in rabbits and rats  as well in vitro . Most rein vitro . Both epin vitro  and thatin vitro ,29. Thesin vitro  stress i-3 M did not induce production of IL-6 in HMC-1  compared with that induced by IL-1β alone  , nor induced production of IL-6, IL-8 or IL-13   Fig .-7 M) alone did not induce proatherogenic cytokine production  nor SB203580 (1 × 10-5 M) affected the cell viability of the cultures , nor did they induce proatherogenic cytokine production  proteins have been demonstrated as risk factors for atherosclerotic heart disease . Recent In this study, IL-1β, an acute phase cytokine, activated mast cells to produce proatherogenic cytokines, IL-6, IL-8, and IL-13, in a dose-dependent manner Fig , and 3. 2-adrenoreceptor-mediated signaling pathways in inducing proatherogenic cytokine production. Several reports have shown that the effect of catecholamines on immune function is due to β-adrenoceptors [1 and β2 adrenoceptors in small amounts , 10 mM HEPES (GibcoBRL), Gentamycin 50 μg/ml, 5 μg/ml insulin transferrin, 2 mM L-glutamine, and 5% heat inactivated fetal bovine serum , at 37°C and in 5% CO2 mixture [2 flasks. To each well of a 6 well culture plate, two ml of HMC-1 mast cells at 0.75 × 106 cells/ml concentration were cultured with epinephrine at 1 × 10-5 M concentration in the presence and absence of IL-1β (10 ng/ml) for 24 hrs. The cultures were carried out in triplicate. Supernatants were harvested for measuring IL-6, IL-8, and IL-13 by ELISA and cell viability and numbers of the culture were analyzed.HMC-1 cell line, established from a patient with mast cell leukemia, were graciously provided by Dr. Butterfield . These cells were maintained in RPMI 1640 media , supplemented with 5 × 10 mixture . HMC-1 cCytokine ELISA was performed for the following cytokines: IL-6, IL-8, and IL-13. ELISA was carried out on cell-free culture supernatants using commercially available ELISA kits, according to manufacturers instructions as earlier described . Results were analyzed on an ELISA plate reader (Dynatech MR 5000 with supporting software) ,48.At the end of incubation, the cells were subjected to the viability count by trypan blue (TB) dye exclusion technique. Two tenths ml of cell cultures were mixed with 0.05 ml of TB, applied to hemocytometer, and counted under a microscope. The cell viability is calculated by dividing the number of live cells (unstained cells) by the total number of all cells (TB-stained and unstained cells) and expressed as a percent.6 cells) by the addition of 1 ml of RNAzol B  [2 (5 mM), and oligo(dT)16 (2.5 μM) as a primer. The samples were incubated at 42°C for 20 minutes, 99°C for 20 minutes, and 5°C for 5 minutes in a DNA thermocycler  for reverse transcription. PCR of cDNA was done with MgCl2 (1.8 mM), each of the dNTPs (0.2 mM), AmpliTaq polymerase (1 U/50 μl), and paired cytokine-specific primers (0.2 nM of each primer) to a total volume of 50 μl. Cycles consisted of 1 cycle of 95°C for 2 min, 35 cycles of 95°C for 45 sec, 60°C for 45 sec, and 72°C for 1 min 30 sec, and lastly, 1 cycle of 72°C for 10 min. Ten microliters of the sample were electrophoresed on a 2% agarose gel and stained with ethidium bromide for viewing. Primer sequences used are as follows: HPRT: 5' CGA GAT GTG ATG AAG GAG ATG G 3' and 5' GGA TTA TAC TGC CTG ACC AAG G 3'; IL-6: 5' ATG AAC TCC TTC TCC ACA AGC GC 3' and 5' GAA GAG CCC TCA GGC TGG ACT G 3'; IL-8: 5' ATG ACT TCC AAG CTG GCC GTG GCT 3' and 5' TCT CAG CCC TCT TCA AAA ACT TCT C 3'; and IL-13: 5' GGA AGC TTC TCC TCA ATC CTC TCC TGT T 3' and 5' GCG GAT TCG TTG AAC CGT CCC TCG CGA AA 3'. Densitometry was done by normalizing target genes to house keepers using Un-Scan-It Version 5.1 software . The PCR experiment was repeated twice.HMC-1 were treated with the appropriate reagents and allowed to incubate at 37°C before being harvested for RNA. RNA was extracted from HMC-1  . After s3VO4, and 20 mM p-nitrophenyl phosphate. Cells were incubated over ice for 30 minutes and then vortexed after the addition of 6.25 μl of 10% of Nonidet P-40. After 2 minutes of centrifugation at 30,000 × g, supernatants were kept at -80°C while the pellets were collected and vortexed every 20 minutes for 3 hours in 60 ml of a hypertonic salt solution: 20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 12 mM DTT, 1 mM PMSF, 1 μM aprotinin, 1 μM pepstatin, 14 μM leupeptin, 50 mM NaF, 30 mM β-glycerophosphate, 1 mM Na3VO4, and 20 mM p-nitrophenyl phosphate. Nuclear translocation of NF-κB was analyzed by the Electrophoretic Mobility Shift Assay (EMSA) using the nuclear fraction. Seven micrograms of nuclear protein were added to 2 ml of binding buffer , and 35 fmol of double stranded NF-κB consensus oligonucleotide (5' AGT TGA GGG GAC TTT CCC AGG C 3')  end labeled with γ-P32 ATP . The samples were incubated at room temperature for 20 minutes and run on a 5% nondenaturing polyacrylamide gel for 2 hours. The gel was then dried on a Gel-Drier  and exposed to Kodak X-ray film at -80°C.HMC-1 were stimulated with PMA, IL-1β and/or epinephrine and then harvested for EMSA analysis ,50. CellCells were treated and lysed in lysis buffer  to be analyzed for p38 MAPK activation by Western blot . Briefly1 or β2 adrenergic receptor antibodies  using normal rabbit serum as a control. The samples were washed with PBS with 0.01% sodium azide and resuspended in 100 ml PBS. FITC labeled goat anti-rabbit Ig's antibody was added to the samples and allowed to bind for 20 minutes. The samples were once again washed with PBS with 0.01% sodium azide and resuspended in 100 μl of PBS. In addition, HMC-1 were pretreated with normal rabbit serum and incubated with FITC labeled goat anti-rabbit Ig's antibody as a control for nonspecific binding [Resting HMC-1 were centrifuged, washed in PBS at room temperature, and resuspended in 100 μl of PBS. The cells were incubated for 20 minutes with rabbit polyclonal anti β binding . Cell su-5 M), and IL-1β plus epinephrine to investigate IL-13 promotor activity. Untreated cells were used as a control. Transient transfection assays were performed using a reporter gene construct containing the minimal promoter sequence of IL-13. The promoter sequence  of the IL-13 gene was fused to the luciferase coding sequence. Plasmid DNA was obtained with double-cesium chloride purification , while SuperFect reagent (Qiagen) was used for transient transfections of HMC-1 cells. Two micrograms of plasmid DNA and 8 μl SuperFect reagent were used for transfection of 1 × 106 HMC-1 cells. Luciferase expression was monitored by chemiluminescence of cell lysates 24 hrs after transfections using the Enhanced Luciferase Assay Kit .HMC-1 were treated with IL-1β (10 ng/ml), epinephrine . All data were reported as means ± SE. A p-value of less than 0.05 was considered significant.All experiments were done in triplicate. The data were analyzed by Student's two-tailed HMC-1, human mast cell – 1Epi, epinephrinePro, propranololAte, atenololDex, dexamethasoneMAPK, mitogen-activated protein kinaseDSC designed experiments, oversaw research, and wrote paper. SMF designed and conducted experiments and wrote paper. SP helped with experiments. KC conducted experiments. EK conducted experiments. SAL conducted experiments. SKH conducted experiments. GK oversaw research.


Publication fees are not borne purely by authors, but are shared by the many organizations whose missions depend on the broadest possible dissemination and communication of scientific discoveries In the wake of declarations supporting open access to research literature from international bodies including the Organization for Economic Cooperation and Development (OECD) and the United Nations' World Summit on the Information Society (WSIS), advocates and critics of the movement appear to have agreed that the issue warrants a robust, ongoing dialogue—a development undoubtedly in the interest of the scientific community, regardless of its ultimate outcome.PLoS Biology, we plan to explore the more pervasive of these misunderstandings, in an effort to expose the real challenges that need to be overcome and to identify some possible solutions. Here we address the first of these—the perception that the publication-charge model puts an unfair burden on authors. Subsequently, we will address concerns about the long-term economic viability of the open-access model, the integrity and quality of work published in open-access journals, and the effect that open access will have on scholarly societies.To the extent that listserv messages, editorials, and conference presentations are representative of more widespread reactions to the debate, there appear to be a number of common misconceptions about what open access is and what problems it can or cannot solve. Over the next few months in By charging authors a fee to have their work published in lieu of charging readers to access articles, open-access publishers such as the Public Library of Science (PLoS) and BioMed Central (BMC) have transformed the traditional publishing system. This reliance on a seemingly untested revenue stream has generated skepticism that authors will be both willing and able to pay publication charges.EMBO Journal, for example, authors are allowed six pages of text free, but are then charged $200 per page beyond that. A review of recent issues shows that almost all authors exceed six pages, voluntarily paying on average over $800 to publish their articles.Publication fees are not a phenomenon born of the open-access movement. Many authors regularly pay several thousands of dollars in page charges, color charges, correction costs, reprint costs, and other fees to their publisher, even when such costs are entirely voluntary. In the Proceedings of National Academy of Science (PNAS) found that although PNAS already makes its content freely available after six months, nearly 50% of PNAS authors expressed a willingness to pay an “open-access surcharge” of $500 or more to make their papers available for free online immediately upon publication—this above and beyond the $1,700 in page charges that the average PNAS author already pays The concern about authors' ability to pay publication charges will become less pressing as governments, funding organizations, and institutions increasingly support open-access publication on their researchers' behalf. More funding agencies are joining the Howard Hughes Medical Institute, the Wellcome Trust, and others who have already designated funds for open-access publication.  already pays such costs for universities and other organizations in a number of countries in which the foundation is active by way of a PLoS Institutional Membership that grants waived publication charges to authors while providing compensatory revenue for PLoS.Perhaps the real misconception about the unfair burden that open access places on authors resides in the terminology—the term “author charge” is itself misleading. Publication fees are not borne purely by authors, but are shared by the many organizations whose missions depend on the broadest possible dissemination and communication of scientific discoveries. Some of those may provide funding for open-access publication as intermediaries between authors and journals, as OSI does. Others—including many government-financed funding agencies—do so directly through their research grants to scientists. In both cases, funding open access is an effective way to fulfill mandates for public access to and accountability over scientific research and to ensure that all worthy research is published.


We have previously shown that use of an EC brush device in combination with the Rovers Cervex-Brush (SurePath broom) offered no significant improvement in EC recovery. Here we determine if use of additional collection devices enhance the diagnostic utility of the SurePath Pap for gynecologic cytology.After informed consent, 37 women ages 18–56 receiving their routine cervical examinations were randomized into four experimental groups. Each group was first sampled with the SurePath broom then immediately re-sampled with an additional collection device or devices. Group 1: Rover endocervix brush (n = 8). Group 2: Medscand CytoBrush Plus GT (n = 7). Group 3: Rover spatula + endocervix brush (n = 11). Group 4: Medscand spatula + CytoBrush Plus GT (n = 11).Examination of SurePath broom-collected cytology yielded the following abnormal diagnoses: atypia (n = 2), LSIL (n = 5) and HSIL (n = 3). Comparison of these diagnoses to those obtained from paired samples using the additional collection devices showed that use of a second and or third device yielded no additional abnormal diagnoses. Importantly, use of additional devices did not improve upon the abnormal cell recovery of the SurePath broom and in 4/10 cases under-predicted or did not detect the SurePath broom-collected lesion as confirmed by cervical biopsy. Finally, in 36/37 cases, the SurePath broom successfully recovered ECs. Use of additional devices, in Group 3, augmented EC recovery to 37/37.Use of additional collection devices in conjunction with the SurePath broom did not enhance diagnostic utility of the SurePath Pap. A potential but not significant improvement in EC recovery might be seen with the use of three devices. In gynecologic cytology, sampling of both the ecto and endocervix is critical to increasing Pap test sensitivity . ControvAfter study design approval from the Carle Clinic Association Institutional Review Board and informed consent, 37 women ages 18–56 receiving their routine cervical examinations were sampled with the SurePath broom. This device, which is packaged with the SurePath Pap, is the Rovers Cervex-Brush . Its use followed the manufactures recommendations of 5 full clockwise rotations. The same patient was immediately re-sampled with either the Rovers endocervix brush, patient group 1; the Medscand Cytobrush Plus GT , patient group 2: the Rovers endocervix brush + Rovers Spatula, patient group 3; or the Medscand Cytobrush Plus GT + Medscand Pap Perfect Spatula, patient group 4. All devices had "pop-off" heads. The SurePath broom device was collected into CytoRich Preservative vials  and processed routinely using the PrepStain Slide Processor . In samples using multiple collection devices, all devices, including the SurePath broom, were placed in a single collection vial and processed as above.The diagnostic terminology used was derived from the 1991 revision of the Bethesda System (TBS) -9. The dAll cases where the cytology diagnosis was ASC-US or more serious underwent colposcopic-guided cervical biopsy . Cases were excluded from analysis if the: 1) cytology and corresponding tissue diagnosis were separated in time by more than 6 months; 2) cytology and/or corresponding cervical biopsy were not performed and interpreted within the Carle Clinic Association/Hospital system. In cases where multiple cervical biopsies were performed, the cytology closest in temporal relationship to the cervical biopsy was correlated. No cervical biopsies met the exclusion criteria.All analyses were performed using SAS statistical software . Data comparisons were made using the Student's paired t-test, the Sign Test and the Wilcoxon Signed Rank test for analysis of non-parametric data.We have previously shown that use of the Surgipath C-E brush  in combination with the SurePath broom did not increase EC recovery in women undergoing a SurePath Pap . This prWhen group 2 patients were examined by cytology after using the SurePath broom  of SurePath Pap limited by diagnoses are due to lack of an EC component . To overIn this study, we found that 3% of women sampled with just the SurePath broom lacked EC cells. These findings were consistent with but better than our previous report based on 3,994 women in which we found that 6% of SurePath Paps were "satisfactory for evaluation but limited by no EC cells" -16. ThisDebate over cervical sampling devices often focuses on EC sampling. Unfortunately, few studies are available that report both the EC absence rate and the abnormal cell detection rate in studies where multiple devices are compared. The retrospective study by Boon et al  stands oAs with any study, the strength of statistical analysis increases as the sample size is increased. However, even with small sample sizes, compelling results can be obtained if the statistical significance is large. Here the data was analyzed using three different nonparametric statistical tests (for non-continuous or non-numeric data) to ensure stringency. In addition, we chose to include the p-values for each of these statistical tests to show that multiple analyses yield the same result and that no single statistical test was chosen to favor a desired outcome. The hypothesis being tested, in this study, is that the use of additional collection devices in conjunction with the SurePath broom device does not enhance diagnostic utility. Normally, p-values <0.05 indicate that one should reject the hypothesis being tested and conclude enhanced utility. In this study, the large p-values generated from analysis of the data indicate a very high probability that the hypothesis be rejected and that no enhanced diagnostic utility is realized with the use of additional collection devices.Since liquid-based Pap testing is relatively new, little work has been done to examine sampling device effectiveness utilizing this technology. Selvaggi et al compared the ThinPrep broom to the ThinPrep spatula + cytobrush and the ThinPrep broom +cytobrush . These aIn conclusion, the SurePath broom appears to be a very effective cervix sampling device when coupled with the SurePath Pap. In 60 patients examined prospectively  of uncertain significance (ASC-US), endocervical cell (EC), Food and Drug Administration (FDA), high grade SIL (HSIL), low grade SIL (LSIL), Papanicolaou (Pap), quantity not sufficient for diagnosis (QNS), squamous intraepithelial lesion (SIL), SurePath Liquid-Based Pap Test (SurePath Pap), SurePath Liquid-Based Pap Test Broom Device (SurePath broom), The Bethesda System (TBS).SJD coordinated the study and analyzed the study data, DLO collected all samples, JCO performed the statistical analysis. GGF constructed the manuscript.The pre-publication history for this paper can be accessed here: