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 (number of slices = 49, thickness = 3 mm, voxel size = 3 × 3 × 3 mm3) 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 (D1 = thumb, D5 = little finger) 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 [0, 0, 0] 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 [0, 0, 0].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 (10 U/μl, Boehringer) 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 (Life Technologies, Inc.). 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 (150 mM NaCI, 50 mM Tris-HCI pH 8.0, 0.5% deoxycholate, 0.1% SDS, 0.5% Nonidet P-40, protease inhibitors, 80 U/ml endonuclease) 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 (BA, Schleicher & Schuell). 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 (Immunotech, France) or anti-mouse (Dako) Immunoglobulin G-horse radish peroxidase-conjugated antibody. Blots were then developed using an enhanced chemiluminescence detection system (Renaissance, NEN, Life Science Products). 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 (5 × 10For immunostaining, C91PL cells were centrifuged on cytoslides using a cytospin (Thermo Shandon, Pittsburgh, PA), 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 (SD 13, range 18 to 78) 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 (95% CI 1.1, 2.30).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 (SD 13, range 18 to 78) 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 [Puf3p associated almost exclusively with transcripts of nuclear genes that encode mitochondrial proteins  of annotated Puf1p targets and 24 out of 104  annotated Puf2p targets  (YPD, May 2003). This group includes transporters for spermine (Tpo1, Tpo2, Tpo3), proteins (Nce101, Nce102, Ast1, Vps72, Mas6, Sfk1, Mup3), vesicles (Sso2, Snc2, Yip1, Aps3, Ypr157w), and lipids (Pdr16, Ykl091c, Fps1 [glycerol]). .p < 3 × 10−6]). 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 (Kar1, Ccd31, Spc19, Spc42, Bbp1, Cnm67, and Nuf2) (Among the Puf5p targets see , we founnd Nuf2) . 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  (puf3Δ mutant, 11 (70%) 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 (Heidelberg, Germany) . 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 (buffer A plus 0.5 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonylfluoride, 0.5 μg/ml leupeptin, 0.8 μg/ml pepstatin, 20 U/ml DNase I, 100 U/ml RNasin [Promega, Madison, Wisconsin, United States], and 0.2 mg/ml heparin). Cells were broken mechanically with glass beads, and extracts were incubated with 400-μl slurry (50% [v/v]) IgG–agarose beads (Sigma, St. Louis, Missouri, United States) for 2 h at 4°C. The beads were washed four times for 15 min at 4°C with buffer C (20 mM Tris–HCl [pH 8.0], 140 mM KCl, 1.8 mM MgCl2, 0.5 mM DTT, 0.01% NP-40, 10 U/ml RNasin). 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) (2 median ratios were retrieved from SMD and exported into Microsoft (Redmond, Washington, United States) 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) (S. cerevisiae genome was obtained by searching “Pattern Match” in the SGD (As the exact 5′- and 3′-UTR lengths are unknown for most of the Puf target mRNAs, we used the estimated average lengths from yeast . Hence, ro.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 (Oberkochen, Germany) 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 (Cbf5, Gar1, and Nhp2 [(B) 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 (92 and 87%, respectively, while that of the medium control was 92%), 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 (Mayo Clinic, Rochester, MN). These cells were maintained in RPMI 1640 media (GibcoBRL, Rockville, MD), 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 (Tel-Test, Inc., Friendswood, Texas) [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 (Orem, UT). 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 (3 × 10, Texas) . 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 (Promega, Madison, WI), and 35 fmol of double stranded NF-κB consensus oligonucleotide (5' AGT TGA GGG GAC TTT CCC AGG C 3') (Promega, Madison, WI) end labeled with γ-P32 ATP (Amersham Biosciences, Piscataway, NJ). 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 (Bio-Rad Laboratories, Hercules CA) 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 (50 mM Tris HCL, 150 mM NaCl, 1 mM EDTA, 1% Triton × 100, and 0.1% SDS) to be analyzed for p38 MAPK activation by Western blot . Briefly1 or β2 adrenergic receptor antibodies (Santa Cruz, Santa Cruz, CA) 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 (-233 to + 50, relative to the transcription initiation site) of the IL-13 gene was fused to the luciferase coding sequence. Plasmid DNA was obtained with double-cesium chloride purification (BioServe Biotechnologies, Laurel, MD), 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 (10t-test using Statistica software (StatSoft, Inc., Tulsa, OK). 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 (TriPath Care Technologies, Burlington, NC) and processed routinely using the PrepStain Slide Processor (TriPath Care Technologies, Burlington, NC). 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 (Cary, NC). 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 (Richmond, IL) 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:


We developed a method to make a various high quality random peptide libraries for evolutionary protein engineering based on a combinatorial DNA synthesis.16 diversity, and the construction of a library with random sequence coding 120 amino acids containing few stop codons.A split synthesis in codon units was performed with mixtures of bases optimally designed by using a Genetic Algorithm program. It required only standard DNA synthetic reagents and standard DNA synthesizers in three lines. This multi-line split DNA synthesis (MLSDS) is simply realized by adding a mix-and-split process to normal DNA synthesis protocol. Superiority of MLSDS method over other methods was shown. We demonstrated the synthesis of oligonucleotide libraries with 10Owing to the flexibility of the MLSDS method, it will be able to design various "rational" libraries by using bioinformatics databases. This library leads to premature short peptides and a particular bias of the amino acid composition, which makes the effective searchable sequence space biased. A slightly improved library NNK or NNS (K / S = equimolar mixture of T and C / G and C) has been conventionally used. Several methods have been developed for a more improved library. Various "rational" libraries in which the nucleotide mixtures were optimized for a target amino acid composition by using a computer calculation have been developed [The combinatorial synthesis method has been demonstrating its effectiveness in discovering novel functional molecules. Examples of this method in the field of evolutionary protein engineering are selections of a novel functional peptide from a random library on solid support , phage ddisplay) -5. The eeveloped -8.Removal of stop codons to obtain long ORFs is important for the evolutionary design of a novel protein starting from a random library. Several methods based on random block-ligation were reported ,10. Two We report in this article on a convenient method for the construction of a high quality library based on combinatorial DNA synthesis. This library has few stop codon and has an optimized amino acid composition for various purposes. A random library based on the split synthesis  is made Scheme of the MLSDS method is shown in Fig. Designed biased libraries are useful for creating various novel proteins such as a functional peptide without Cys  or an enF value (0.96 on three lines splits) and gave no stop codon even if the effect of a point deletion was included in the GA calculation. A Met-less random library may be the best starting library for global search of the protein sequence space. This speculation is supported by the report [Among these wide spectra of requirements, we designed DNA libraries that code peptide libraries having various characteristics and have no stop codons. Examples are: a library with the average amino acid composition of natural proteins , which ie report  stating Internal deletion problem in the oligonucleotides synthesis process is important. It destroys the codon-based design, leading to stop-codon generation and undesirable amino acid composition. Our program incorporated deletion effects into the GA calculation and succeeded to minimize the deletion problem. Moreover it was reported that contamination of deletion products could be decreased on a denaturing PAGE for DNA of this length .F value but suitable for designing actual synthesizing operations. These results showed MLSDS method gave a high quality library even with three DNA synthesizers.We also investigated the practical number of DNA synthesizers. For this purpose, we calculated the final correlation coefficient between the designed and the various target compositions with up to 6-line DNA synthesizers. As shown in Fig. F = 0.99 (on three lines) in the GA calculations. This is reasonable, because the average amino acid composition among natural proteins highly correlates to the number of synonymous codons in the standard genetic code table [When we took the natural abundance as the target amino acid composition, we got a highest fitness value de table .F = 0.85 and 0.66, respectively) without stop codons in full-length DNAs. The deletion rate was about 0.3% per coupling. For the total DNAs including deletants, F = 0.90 and 0.60, respectively.We synthesized a "Natural" library and a "Uniform except [Cys] = 0" library mentioned in the previous section. In Table F = 1.00)  Table . So far,In order to make long ORFs, we assembled 8 units of the oligomers. Stem sequences of them did not have any stop codons. A DNA library encoding 120 amino acids plus nine 5'- and 3'-flanking semi-random di-peptides  was constructed  and purity (data of PAGE) of synthesized DNA. With an in vivo selection, there is a diversity limit by the transformation step. But with an in vitro selection, there is no such limitation. Thus exploration of huge sequence space by in vitro virus [The diversity of the synthesized library is about 10ro virus -5 or relro virus ,29 will So far, a really random library has been generated by four methods. Other methods do not give a really random library, because they can not provide a library in which all the 20 amino acids are encoded at all sites. A comparison of library quality for three methods is shown in Table Triticum aestivum. It was reported that the reaction efficiency of 3NPs was far from uniform. The sequence data of synthesized DNA using an equimolar mixture of 19 kinds of 3NPs (without Cys) showed 12-fold (maximum) difference in composition [An application of 3NPs method to mutagenesis of antibodies  or coileposition  or more position . Thus itposition  (Table 3position  method iposition .A pre-selecting method using an mRNA display  was fruiThe Y-Ligation Block Shuffling (YLBS) method  has highMLSDS produced libraries with high quality as shown in Table in vitro.We applied the split synthesis to oligodeoxyribonucleotide synthesis and developed a new procedure, Multi-Line Split DNA Synthesis (MLSDS), based on the synthesis of designed codon mixtures using three-line DNA synthesizers. MLSDS can make various "rational" libraries of huge diversity with few stop codons by using bioinformatics databases. Combination of an MLSDS library with a screening method for huge diversity will accelerate the protein evolution A random MLSDS library was synthesized as follows. A standard DNA synthesis method was used in three lines of DNA synthesizer running in parallel. The randomized regions were combinatorialy synthesized in codon units. Triplet codons were synthesized separately in the three synthesizers as an elongation reaction of oligonucleotides on beads made of controlled pore glass (CPG). CPG beads were mixed together manually, and then splitted again into three reaction tubes manually and the next triplet codons were synthesized Fig. .AC TGS N(123/456/789)15 AC TGS NGAG GCT GGC TGC CAC-3', where N and S denote A/T/G/C and G/C, respectively. The A:T:G:C mixing ratio in each letter of three codon groups 123, 456, and 789 was shown in Table BbsI and BbvI, respectively. In order to make longer sequences, we ligated 2 to 8 units of oligomers at the cohesive ends (the underlined sequences shown above) generated by the restriction enzyme treatment. The assembly method was as described in Ref. [The sequence of a 87 mer library was 5'-GAT GAG GCG AAG ACG  in Ref. . The itaThe synthesized DNA libraries were amplified by PCR using KOD Dash polymerase (TOYOBO), inserted into pCR2.1TOPO vector (Invitrogen) and cloned, avoiding cloning bias. The clones were sequenced.pT = (pT1, pT2, pT3,..., pT21), was established, where normally pT21 = 0 for stop codons. Secondly, we calculated an expected amino acid (plus stop codons) composition, p = (p1, p2, p3,.., p21), from the molar mixing ratio of the bases, x = (x1, x2, x3,....., xL12), where 12L is equal to 4(number of bases) × 3(number of codon letters) × L(number of synthesizer-line). For example, the mixtures for the first letter and the second letter of the first DNA synthesizer have the molar mixing ratio [A]: [T]: [G]: [C] = x1 : x2 : x3 : x4 and x5 : x6 : x7 : x8, respectively. And for example, when L = 3, the expected alanine composition p1 is given by:Computer calculations to determine the optimum molar mixing ratio of four bases in the codon synthesis step were performed by using Mathematica (Wolfram Research). We made a GA program for this purpose. Firstly, the target amino acid composition, for the full-length sequence without deletion.L-dimensional optimization problem) having the solution xi as integer (0,1,2,3,4,5). The reason for 6-digits "integer" was to simplify the DNA synthesizer handling and also to simplify the calculation. As the fitness F of x in the GA, we took a correlation coefficient between the expected (or designed) amino acid composition and the target amino acid composition:We solved an integer-programming problem  considering the single nucleotide deletion.In the additional WORD file , the detail of calculation method is described for the the expected amino acids composition Click here for file


Governments around the world have turned their attention to the issue of taxpayer access to primary research literature stemming from publicly funded investigations The global debate over access to primary research literature heated up this summer, fueled by a slew of congressional and parliamentary recommendations, claims of political victory by critics and proponents of open access, and redoubled lobbying efforts on every side of the issue. After months of often dizzying rhetoric from virtually all camps, one concrete development has indisputably emerged from the fray: governments around the world have begun to take an interest in the question of who can and can't read the results of the scientific research they fund. “We are convinced,” concluded a recent report from the Science and Technology Committee of the United Kingdom's House of Commons, “that the amount of public money invested in scientific research and its outputs is sufficient to merit Government involvement in the publishing process” . United PLoS Biology and PLoS Medicine, that currently make their content immediately free online in centralized repositories. A US House of Representatives Committee on Appropriations, for example, recently passed language that would allow many, though not all, publishers six months between the date of publication of NIH-funded research articles and the date of their deposition in a free-to-use archive. (At the time of this writing, the bill is awaiting further discussion in the House and Senate.)While such pronouncements may sow fear in the hearts of some scientists and publishers, concerns that governments are poised to tell researchers where or how to publish seem largely unfounded. Both the UK report and rumblings from the US government suggest that any legislative dictates on access to scientific literature are likely to be structured to minimize potentially deleterious implications for established, subscription-based journals, for-profit and not-for-profit alike. Mandates for open access to articles summarizing the results of publicly funded research would not be mandates for scientists to submit work only to the handful of journals, like included in publicly funded electronic archives like PubMed Central, as the US Congress might, would be less a paradigm shift or a radically interventionist mandate than a sensible extension of existing policy for most governments and their funding agencies.In any case, it is a perfectly reasonable premise that governments should attach conditions to grants mandating public access to resulting peer-reviewed, published articles. Making funding for research contingent on the results of the work being disseminated as widely as possible is hardly a revolutionary proposition. All funders expect, of course, that scientists won't simply stash their findings in a desk drawer. Most, like NIH, include in their mission statements clauses about “fostering the communication of medical and health sciences information” . The US Increasingly, it seems, this is the view being adopted by policy makers—that it is the status quo, rather than prospective policy revision, that is anomalous or hard to justify. “We would be very surprised,” the Science and Technology Committee notes, “if Government did not itself feel the need to account for its investment [in research] in the publishing process. We… hope that this report will be a catalyst for change” .Washington Post last year  report was the product of a seven-month investigation, featuring some 127 submissions of written evidence and four days of oral testimony from the likes of Nature Publishing Group, Reed Elsevier, and indeed, the Public Library of Science. NIH has promised a period of public comment on its plan for implementing the Appropriations Committee's requirement before moving forward, in addition to the information-gathering meeting of publishers in July and subsequent meetings hosted by Dr. Zerhouni. All told, the current spate of government attention to the issue of public access to research results seems methodical, inclusive, and likely to prove productive for scientific communities and the public.As a matter of sheer principle, it strikes many people as odd that “anyone can download medical nonsense from the Web for free, but citizens must pay to see the results of carefully conducted biomedical research that was financed by their taxes,” as Rick Weiss noted on the front page of the ast year . While n


As key regulators of mitotic chromosome segregation, the Aurora family of serine/threonine kinases play an important role in cell division. Abnormalities in Aurora kinases have been strongly linked with cancer, which has lead to the recent development of new classes of anti-cancer drugs that specifically target the ATP-binding domain of these kinases. From an evolutionary perspective, the species distribution of the Aurora kinase family is complex. Mammals uniquely have three Aurora kinases, Aurora-A, Aurora-B, and Aurora-C, while for other metazoans, including the frog, fruitfly and nematode, only Aurora-A and Aurora-B kinases are known. The fungi have a single Aurora-like homolog. Based on the tacit assumption of orthology to human counterparts, model organism studies have been central to the functional characterization of Aurora kinases. However, the ortholog and paralog relationships of these kinases across various species have not been rigorously examined. Here, we present comprehensive evolutionary analyses of the Aurora kinase family.Phylogenetic trees suggest that all three vertebrate Auroras evolved from a single urochordate ancestor. Specifically, Aurora-A is an orthologous lineage in cold-blooded vertebrates and mammals, while structurally similar Aurora-B and Aurora-C evolved more recently in mammals from a duplication of an ancestral Aurora-B/C gene found in cold-blooded vertebrates. All so-called Aurora-A and Aurora-B kinases of non-chordates are ancestral to the clade of chordate Auroras and, therefore, are not strictly orthologous to vertebrate counterparts. Comparisons of human Aurora-B and Aurora-C sequences to the resolved 3D structure of human Aurora-A lends further support to the evolutionary scenario that vertebrate Aurora-B and Aurora-C are closely related paralogs. Of the 26 residues lining the ATP-binding active site, only three were variant and all were specific to Aurora-A.In this study, we found that invertebrate Aurora-A and Aurora-B kinases are highly divergent protein families from their chordate counterparts. Furthermore, while the Aurora-A family is ubiquitous among all vertebrates, the Aurora-B and Aurora-C families in humans arose from a gene duplication event in mammals. These findings show the importance of understanding evolutionary relationships in the interpretation and transference of knowledge from studies of model organism systems to human cellular biology. In addition, given the important role of Aurora kinases in cancer, evolutionary analysis and comparisons of ATP-binding domains suggest a rationale for designing dual action anti-tumor drugs that inhibit both Aurora-B and Aurora-C kinases. The Auroras are a conserved family of serine/threonine kinases which have essential functions in cell division ,2. In miDrosophila melanogaster (fruitfly) that caused defective spindle-pole formation [Drosophila was found to have a second Aurora homolog [Caenorhabditis elegans, similarly has two Aurora-like genes [Saccharomyces cerevisiae and Schizosaccharomyces pombe, have a single Aurora, known as increase-in-ploidy 1 (Ipl1) [Xenopus laevis, which has two kinases; Aurora-A and Aurora-B [Model organism studies have played a pivotal role in functional characterization of Aurora kinases. Aurora kinases were first identified as mutant alleles in ormation . Subsequ homolog , and theke genes ,6. The f1 (Ipl1)  and Auro1 (Ipl1) , respectAurora-B ,10. MoreAurora-B .The Aurora kinases are mitotic kinases that generally associate with chromosomes, often in complexes with other proteins, and interact with cytoskeletal components in cell division. The three mammalian Aurora kinases appear at specific locations during mitosis. Aurora-A, the "polar kinase", primarily associates with the separating centrosomes while Aurora-B, the "equatorial kinase", is a chromosomal passenger protein . The leaRecent studies indicate that all three Aurora kinases have strong associations with cancer. Aurora-A has been mapped to a region in the human chromosome 20q13.2-13.3) that is amplified in cancer cell lines and primary tumors .2-13.3 t. TransfeDespite the importance of model organisms in understanding Aurora kinase function, the evolutionary relationships among these variants are unclear. Two previous phylogenetic analyses of Aurora kinases were incomplete because the contemporary complement of Aurora kinases was unavailable  or certaTakifugu rubripes [Danio rerio [Ciona intestinalis [In order to construct a comprehensive phylogenetic tree, GenBank was searched for all possible Aurora kinases. In addition to previously published Aurora kinase sequences, further chordate and urochordate Aurora homologs were found by using mammalian Aurora-A, Aurora-B and Aurora-C protein sequences as queries in BLASTP or TBLASTN  searchesrubripes , the zebio rerio , and thestinalis .Multiple sequence alignments show that the Aurora kinase family is highly conserved among species Fig. . PairwisC. intestinalis. The use of alternative kinase families, other than PLK4, to root the tree did not alter the internal topology of the Aurora clade. Although its genome sequence is incomplete, C. intestinalis likely has only a single Aurora homolog since other probable kinase open reading frames associated with the next top five BLASTP [Phylogenetic trees constructed using four methodologies, all rooted using polo-like kinases type 4 (PLK4), show that all vertebrate Auroras form a clade distinct from those of invertebrates Fig. . The phye BLASTP  hits didXenopus laevis, rodents and humans as well as a new putative ortholog in T. rubripes.Among true vertebrates, our phylogenetic tree shows that the Aurora kinases underwent two major gene duplication events. The first split in cold-blooded vertebrates lead to the formation of two Aurora subfamilies. One branch encompasses all known vertebrate Aurora-A sequences in a single orthologous lineage that includes fishes, amphibians and mammals. This family includes previously identified Aurora-A kinases in X. laevis) and fish (D. rerio and T. rubripes). Searches of T. rubripes and D. rerio protein and DNA sequence databases detected several other putative serine/threonine kinase homologs but none were Auroras according to phylogenetic analyses. Thus, cold-blooded vertebrates appear to have only a single Aurora-A ortholog, a single Aurora-B-like homolog, and lack an Aurora-C ortholog. Conversely, Aurora-A, Aurora-B and Aurora-C appear to be ubiquitous to mammals  where they are encoded by separate chromosomal loci. It would appear that mammalian Aurora-B and Aurora-C evolved from a duplication event involving the ancestral Aurora-B found in cold-blooded vertebrates. This depiction of the evolutionary relationships of vertebrate Auroras was consistently determined by four different phylogenetic methods with high bootstrap or Bayesian posterior probability values  and S. pombe (Ark1). Other basal branches are the amitochondrial fungi, Encephalitozoon cuniculi, and the kinetoplast protist, Leishmania major [The Aurora kinases of plants and invertebrates are all outgroup lineages to chordates / urochordates Fig. . Althougia major .C. elegans and D. melanogaster, occupy separate early branches and are not, as their current names suggest, orthologs to either Aurora-A or Aurora-B of vertebrates. An unrooted phylogenetic tree with only model organism species shows the same topology of vertebrate Auroras as the more species-rich tree rooted by PLK4 kinases  and ZM4Evolutionary analysis shows that cell division Aurora kinases, while consistent in theme throughout eukaryotes, have undergone lineage-specific expansions and specialization in metazoans. Aurora-C is the least known of the Aurora kinases. Yet as an evolutionary innovation in mammals, further studies are very much warranted from the perspectives of better understanding its potential roles in both cell replication and tumor progression. A better functional understanding of Aurora-C would help clarify the evolutionary relationships of Aurora-B and Aurora-C in mammals relative to the ancestral Aurora-BC in cold-blooded vertebrates. Additionally, the close evolutionary and structural relationships between mammalian Aurora-B and Aurora-C offers the tantalizing opportunity to design dual kinase inhibitors that might circumvent potential tumor cell resistance to mono-target chemotherapeutics.Takifugu rubripes, Danio rerios and Ciona intestinalis homologs were obtained by BLASTP and TBLASTN [T. rubripes and D. rerios had orthologs to X. laevis Aurora-A and Aurora-B but not mammalian Aurora-C. C. intestinalis had a single Aurora-like kinase.All Aurora kinase orthologs and paralogs were initially collected from GenBank nonredundant protein database by performing separate searches using BLASTP  with hum TBLASTN  of speciPLK4 kinases were selected as the outgroup for phylogenetic analyses because they were the most similar non-Aurora kinases to either human Aurora-A, Aurora-B or Aurora-C in multiple BLASTP  searches6 generations, burn-in values were set for 104 generations, and trees sampled every 100 generations. All trees were visualized using the program TREEVIEW v1.6.6 [We constructed phylogenetic trees using distance neighbor-joining (NJ), maximum parsimony (MP), maximum likelihood quartet puzzling (QP), and Bayesian posterior probabilities (BP). NJ trees were based on pair wise distances between amino acid sequences using the programs NEIGHBOR and PROTDIST (Dayhoff option) of the PHYLIP 3.6 package . The proW v1.6.6 .For the Aurora kinase phylogeny rooted with PLK4 kinases shown in Fig. For the unrooted phylogeny of Aurora kinases of model organisms shown in Fig. The SwissPDBviewer program  was usedJRB conceived the study, performed the phylogenetic analysis and drafted the manuscript. KKK performed the 3D structural analyses and contributed to the draft manuscript. MLB participated in the preliminary phylogenetic analysis and contributed to the draft manuscript. PS and DRP both contributed to the draft of the manuscript and added key references.Multiple sequence alignment of edited Aurora and Plk4 kinases used to produce the phylogeny shown in Fig. Click here for fileAccession numbers (gi), Locus link Ids (LID) or genome predicted protein Ids for Aurora and PLK kinases as labelled in the Supplementary Data multiple sequence alignment file S1.Click here for file