In this case, combining the results of micro-Raman spectroscopy with micro-transmission measurements, we were able to ascertain that uniform monolayer ribbons were grown and found also Bernal stacked and misoriented bilayer ribbons. On the Si-face, the situation is completely different. A full graphene coverage of the SiC surface is achieved but anisotropic growth still occurs, because of the step-bunched SiC surface reconstruction. While in the middle of reconstructed terraces thin graphene stacks (up to 5 layers) are grown, thicker graphene stripes appear at step edges. In both the cases, the strong interaction between the graphene layers and the underlying SiC substrate induces a high compressive thermal strain and n-type doping.Micro-Raman and micro-transmission imaging experiments have been done on epitaxial graphene grown on the C- and Si-faces of on-axis 6H-SiC substrates. On the C-face it is shown that the SiC sublimation process results in the growth of long and isolated graphene ribbons (up to 600  Since the first report by C.V. Raman in 1928 -5.μRS) has become an attractive characterization tool in industrial clean-room facilities.Today numerous applications exist that cover the whole development of modern electronic and optoelectronic devices. They run from basic inspection of as-grown semiconductors to advanced device inspection tools. For instance, Raman mapping enables to check the crystalline quality ,7, the cμRS that have been mentioned before. Raman spectroscopy on few layers graphene (FLG) not only can evaluate the crystalline quality but also, the thickness, the stacking order of graphene sheets, the doping level, and finally the residual strain.In this field, graphene is a new comer. Because of its outstanding electronic, thermal, optical, and mechanical properties -24 it caIn this study, we review some recent Raman imaging results collected on epitaxial graphene grown on the C- and Si-faces of 6H-SiC substrates -29. In t2 pieces of on-axis 6H-SiC substrates cut from, either, the Si-terminated (0001) face or the C-terminated ® morphology [All samples were grown using the processes described in -29. We urphology . Then, a-6 Torr. Before sublimation, the samples were heated at 1150°C for 10 min to remove any trace of native oxide. During the growth, the samples were covered by a graphite cap to increase the C and Si partial pressures over the SiC surface. Such graphite coverage lowers the Si out-diffusion process during the growth and enables to perform FLG growth at higher temperature. This promotes better SiC surface reconstruction.For sublimation, we used a high temperature furnace from Jipelec  previousμm wide and 150-μm long, but a longer 1700°C annealing results in longer ribbons (up to 600-μm) with the same width. The width does not depend on the annealing time because the ribbons fully occupy a single terrace of the heavily reconstructed (step-bunched) SiC surface [In this way, on the C-face of 6H-SiC SiC substrates after 15 min annealing at 1700°C in a secondary vacuum, the growth of long (self-organized) graphene ribbons can be reached . These r surface .μm and the average step height 10 nm. Optical microscopy showed that the terraces had a remarkable homogeneity of width and orientation over a scale of 1 cm2.On the Si-face, to increase the FLG anisotropy, a modified growth process was used. The growth was done at 1750°C for 20 min under argon with a graphite cap covering the sample . In thisμm with, typically 1-mW power focussed on the sample. To combine micro-Raman spectroscopy with micro-transmission experiments, a low noise photodiode was inserted between the SiC substrate and the XYZ piezoelectric stage. For details, see [Raman spectra were collected at room temperature, using a Jobin-Yvon Horiba T64000 spectrometer operated in the confocal mode. The 514-nm line of an Ar-Kr ion laser was used for excitation. With a × 100 microscope objective, the spot diameter was about approximately 1 ils, see . In thisThe graphene extinction was deduced using the following equation:T0 is the bare SiC transmittance value and T the modified one with epitaxial FLGs on top. From the work of Ref. [in which  of Ref.  it can bn = 2.68 is the SiC refractive index, N is the number of graphene layers, and σ is the optical conductivity of a single (isolated) graphene sheet that was confirmed experimentally [In this equation, mentally . The relμm2) map collected on two neighboring graphene ribbons. The step size was 0.5 μm for the X direction and 2 μm for the Y direction. Six individual maps are shown. The first one corresponds to the extinction values, the second one to the integrated intensity of the G band normalized to the HOPG peak. The third one gives the normalized integrated intensity of the 2D band, while the fourth and fifth ones give the shift of the G and 2D bands, respectively. Finally, the last one corresponds to the absolute value of the Fermi level computed from the previous results. Of course, because of the limited range of the XY piezostage (100 × 100 μm2) the two ribbons could not be completely probed. But a first point to be noticed is that, on both ribbons, no D band map could be given. This is shown in greater details in Figures In Figure η ranging from 1.2 to 1.4%. This shows that we deal with a true epitaxial monolayer graphene (MLG) ribbon. To ascertain this result, a Raman spectrum collected at the center of ribbon is shown in Figure Let us now consider everything more in detail. The first (extinction) map shows that both ribbons have an excellent thickness uniformity. However, and because the relative extinction is different, it shows also that the left one is thicker than the right one. Concerning the absolute values, from these extinction maps complemented by additional point by point measurements, we find that the thinner (right) ribbon corresponds with relative extinction values -1, with a FWHM of the order of 13 cm-1, while the 2D band ranges from 2682 to 2688 cm-1 with a FWHM around 25 cm-1. This means that the graphene ribbons are strain free, unlike epitaxial graphene grown on the Si-face of SiC. This is confirmed by the presence of wrinkles evidenced by AFM [IG/I2D between the integrated intensities of the G and 2D banda according to [Typically, the G band falls between 1583 and 1587 cmd by AFM . For sucrding to :(3)(4)ep γ= 21 meV is proportional to the electron-phonon scattering rate [v0 = 106 ms-1 is the electron velocity. The 0.06 factor is deduced from the function f(Sr) [εeff = 5.33 for our graphene layer comprised between air and 6H SiC   by usingC = 9.66 ). The ablar ones  gave a h work of  such conConcerning the second ribbon (on the left side), as already said we found a twice larger relative extinction. Ranging from 2.6 to 2.8%, this indicates a bilayer system. The relative extinction and the G band intensity both indicate that this ribbon is a bilayer with an excellent thickness uniformity. On the contrary, the 2D band intensity map in Figure μRS with micro-transmission measurements appears then as a most necessary tool to discriminate (without any ambiguity) between true MLG and misoriented multilayers. Of course, to perform such reproducible intensity measurements, any laser power fluctuation has to be carefully corrected. In this work, this was done using an additional low noise photodiode that measured continuously the laser power during the Raman map acquisitions.Bernal stacking is not that usual for epitaxial graphene grown on the C-face of SiC substrates. Most of the time the graphene planes are slightly misoriented, corresponding to turbostratic stacking. In this work, we also found misoriented ribbons (not shown). The presence of rotational stacking faults between the two (or more) successive graphene planes results in Raman spectra similar to the monolayer one as shown in Figure Coming back to the maps in Figure μm2 micro-Raman map collected on an epitaxial graphene stack grown on the Si-face of a 6H-SiC substrate. The normalized integrated intensity of the G band is compared to its Raman shift and to an optical microscopy image (OM) of the same area. On the OM image, dark areas correspond to central part of the terraces, while the bright areas correspond to the edges of the step bunched SiC reconstructed surface. As already said, the terraces are 5 μm wide and 10 nm high and, from the G band intensity, we find that graphene covers all the SiC surface. It is then impossible to measure directly the relative extinction and (consequently) the FLG thickness. Of course, a thickness estimate can still be done from the G band intensity. We found about 5 layers in the center of terraces  of the K point corresponding to a n-type doping. This interaction causes also deviations from the linear band dispersion leading to a parabolic dispersion with an apparent gap of approximately 0.25 eV. This strong interaction is also responsible for the thermal stress experienced by these graphene layers. This is no longer true on the C-face, on which it has been shown that graphene interacts very weakly with the underlying substrate [From these results, we confirm that there is a strong difference between graphene grown on the C-face and graphene grown on the Si-face of SiC substrates. On the Si-face graphene strongly interacts with the underlying SiC lattice. This interaction leads to the formation of the so-called buffer layer, which is covalently bound to the SiC lattice ,46. Thisubstrate ,48. It i-1 blue shifted compared to bulk silicon. This high blue shift is again due to a strong compressive stress induced by the SiC substrate (-2 GPa or a strain of -1.3%). First-order Raman scattering of the SiC substrate-1 and the corresponds to the two TO modes of E2 symmetry at 764 and 786 cm-1 and the A1(LO) phonon at 964 cm-1. Its second overtone with its characteristic fingerprint [-1 under the sharp G band of FLG around 1590 cm-1. The 2D band is around 2780 cm-1. No D band can be seen on these points. Moreover, we can see that no significant variations of the G band intensity and Raman shift can be observed close to these Si clusters. This means that these Si clusters do not alter the graphene growth. These clusters are located close to the step edges, like the graphitic pits. There might be a link between the presence of different defects at the step edges like these clusters, the higher growth rate and the clear electrical anisotropy that has been evidenced by magnetoresistance experiments performed on several Hall bars with different orientations [Finally, on the G band intensity map we can see several points marked in blue. These blue points correspond to area where crystalline silicon clusters were found. One of the corresponding uncorrected Raman spectrum is shown in Figure gerprint  falls bentations .μm long and 5-μm wide. They are mainly monolayers and Bernal stacked bilayers but turbostratic bi and multilayer areas have also been found. Finally for monolayers graphene, we also illustrated how the absolute value of the Fermi level can be found, in good agreement with electrical results.Reviewing recent Raman imaging experiments performed on epitaxial graphene grown on the C and Si face of 6H SiC substrates, we have shown the benefits of combining Raman spectroscopy with micro-transmissions measurements. Provided the relative extinction of FLGs can be obtained, this enables to determine (without any ambiguity) the thickness, homogeneity, and stacking order  of FLGs. On the C-face of SiC substrates we have shown that long, self-ordered, graphene ribbons can be grown. These ribbons have excellent crystalline quality and are strain relaxed. They are up to 600-On the Si face, on the opposite, a full graphene coverage of the SiC surface has been found. The surface is still heavily step-bunched but a high compressive thermal strain and n-type doping has been observed. It confirms that on the Si face a strong interaction exists between the graphene layers and the underlying SiC substrate. FLG on the Si-face exhibits Bernal stacking with thickness inhomogeneity. Thin (5 layers) FLGs were grown in the middle of terraces, while thicker graphene stripes grew close to the step edges. In the vicinity of these steps disordered graphite pits and crystalline Si clusters were found. There might be a link between the presence of these defects, the thickness inhomogeneity and the clear electrical anisotropy that has been recently evidenced by magnetoresistance experiments.The authors declare that they have no competing interests.NC and PG prepared the samples. AT carried out the micro Raman spectroscopy and microtransmission experiments. AT and JC performed the analysis of the Raman and transmission data. AT wrote the manuscript. All authors read and approved the final manuscript.aThe ratio between the integrated intensities of the 2D and G band depends on the experimental setup. In our case I2D = IG = 1.02 for our HOPG reference sample. For the lack of a better knowledge, we assume that our setup is similar to the ones of Refs. [of Refs. ,35.


Arabidopsis thaliana MHX gene. Moreover, the ability of this element to enhance translation was diminished by a minor downstream shift in the position of introns containing it from the 5′ UTR into the coding sequence. These data suggest that some of the extra length of 5′ UTR introns results from the presence of elements that enhance translation, and, moreover, from the ability of 5′ UTR introns to provide preferable platforms for such elements over downstream introns. The impact of the identified intronic element on translational efficiency was augmented upon removal of neighbouring intronic elements. Interference between different intronic elements had not been reported thus far. This interference may support the bioinformatics-based idea that some of the extra sequence of 5′ UTR introns is also necessary for separating different functional intronic elements.The mechanisms that underlie the ability of some introns to increase gene expression, a phenomenon called intron-mediated enhancement (IME), are not fully understood. It is also not known why introns localized in the 5′-untranslated region (5′ UTR) are considerably longer than downstream eukaryotic introns. It was hypothesized that this extra length results from the presence of some functional intronic elements. However, deletion analyses studies carried out thus far were unable to identify specific intronic regions necessary for IME. Using deletion analysis and a gain-of-function approach, an internal element that considerably increases translational efficiency, without affecting splicing, was identified in the 5′ UTR intron of the  The ability of introns to boost gene expression was identified in many organisms, including plants. In most cases, the presence of introns increases the steady-state levels of mature mRNA in the cell . The latper se is not sufficient for enhancing expression, as evident from the fact that different introns have different abilities to boost expression, and some introns cannot stimulate expression at all. Yet, deletion analysis studies were unable to identify specific intronic regions necessary for IME , 20–24 nucleotides upstream of exon–exon junctions ] of the MHX gene b was prArabidopsis thaliana (L.) (ecotype Col-0) plants were transformed using Agrobacterium by the floral dip technique  constructs of the DEL series has been described , without altering the deduced amino acid sequence. A DNA fragment carrying the downstream region of the AtMHX promoter, the 5′ UTR of this gene, and an intron from the Ricinus communis (L.) CAT1 gene (GenBank accession no. D21161.1) replacing the LI in the 5′ UTR (at its precise location) was synthesized by GenScript . The modifications introduced into this sequence  are detailed in Supplementary Fig. S1 available at JXB online. In particular, a potential cryptic splice site located seven nucleotides before the 3′ end of the CAT1 intron was eliminated here . It was shown later on that elimination of this potential cryptic splice site resulted in full and accurate splicing of the CAT1 intron (http://www.cbs.dtu.dk/services/NetPGene/). The synthesized DNA fragment was used to replace the 5′ UTR and LI between the BglII site of the promoter and the Mva1269I site of GUS. Different elements of the modified LI of AtMHX , which was also synthesized by GenScript, were amplified by PCR and cloned into the middle of the HpaI site of the CAT1 intron, thereby creating the different UTR series constructs. To create the –I construct of the UTR and CDS series, the 5′ UTR of AtMHX with the necessary flanking sequences (the downstream region of the AtMHX promoter and the coding sequence of GUS up to the Mva1269I site) was cloned between the BglII site of the promoter and the Mva1269I site of GUS.To create the UTR series constructs, a AtMHX promoter, the 5′ UTR of AtMHX, and the coding sequence of GUS up to the Mva1269I site was synthesized by GenScript. The GUS coding sequence included the CAT1 intron 12bp downstream of the AUG codon . This DNA fragment was cloned between the BglII site of the promoter and the Mva1269I site of GUS. The different elements of the LI of AtMHX were digested from the corresponding UTR series constructs by NheI and AflII, and introduced into the same sites of the CAT1 intron, thereby creating the different CDS series constructs. All chimeric genes were verified by sequencing, cloned into the binary vector pGA492  according to the manufacturer’s instructions. Quantification of band densities on gels was performed with the ImageJ program (NIH).Total RNA was extracted with TRI-Reagent (Sigma) according to the manufacturer’s instructions. RNA samples were denatured with glyoxal (Sigma) and fractionated on 1% agarose gels as described . Gel preGUS). PCR conditions were as follow: an initial cycle of 94 °C for 3min, followed by 30 cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 2min, and a final cycle of 72 °C for 10min.RNA was treated with DNase I, followed by DNase I removal, using Ambion’s AM1906 kit according to the manufacturer’s instructions. A preliminary PCR with the same primers that were subsequently used for RT–PCR was conducted to verify that no DNA remained. Preparation of cDNA was carried out using an oligo(dT) primer and M-MuLV reverse transcriptase  according to the manufacturer’s instructions. A 1.5 μl aliquot of each cDNA was utilized as template for RT–PCR analysis with the following primers: forward primer 5′-GCAGGATCCACGCTTGACCGATTC-3′ (corresponding to the first exon of the 5′ UTR) and reverse primer 5′-TTCGCGATCCAGACTG-3′  Triton X-100. Following centrifugation , the supernatant was collected and the concentration of proteins was determined using the Bradford reagent (Sigma). Samples including equal amounts of protein were suspended in 250 μl of extraction buffer including 1mM  of the fluorescent GUS substrate 4-methylumbelliferyl-β-d-glucuronide (MUG) (Duchefa Biochemie BV). GUS activity was assayed on a 96-well fluorescent plate-reader  with the excitation wavelength set at 350nm and the emission wavelength at 460nm. GUS activity (milli units mg protein–1) was calculated from the slope of the line generated from measurements taken at 3min intervals during 2h, with respect to the slope of commercial pure GUS enzyme (Roche Diagnostics GmbH).Quantitative measurement of GUS activity was carried out using the fluorometric assay described by AtMHX for its ability to enhance expression was first investigated by deletion analysis. The internal LI sequence  was divided into five regions (Supplementary Table S1 at JXB online). A series of constructs named D1–D7, in which single or multiple internal LI regions were deleted, was created from a basic construct called WT  did not include the LI. This series of constructs is referred to here as the DEL series.The significance of various internal regions of the LI of  regions . The two regions , considealled WT . The WT Arabidopsis. About 45 independent T1 plants were collected for each construct used in this study to compensate the position effect and ensure reliable results. Since splicing is important for efficient IME, RT–PCR analysis was carried out to determine if the deleted introns were correctly spliced. The expected size of the PCR product of a spliced transcript . Unspliced products were particularly noticeable in constructs that lacked the intronic E1 region . Interestingly, plants expressing constructs WT, D6, and D7, which were the only constructs that included the intronic region E3, showed significantly higher GUS activity levels compared with the other constructs. Deletion of E3 alone from the WT construct resulted in a 7-fold reduction in GUS activity (compare constructs WT and D5). This suggests that E3 makes a major contribution to the enhancement capacity of the LI. As shown in GUS activity of plants expressing the different constructs, relative to that of plants expressing the –I construct, is shown in Ricinus communis CAT1 gene, and was chosen because of its relatively low IMEter score and also because it was used as a foreign intron  in the pCAMBIA series of plant vectors. All AUG triplets were eliminated (by keeping the same nucleotides in a different order) from both the CAT1 intron and the LI of AtMHX, in order to eliminate the possibility of nonsense-mediated mRNA decay (NMD) or translational inhibition in the case of incomplete splicing. In brief, upstream AUG (uAUG) codons that are recognized by the ribosome (depending on their sequence context) may lead to transcript degradation by the NMD pathway, and/or translational inhibition  construct of the gain-of-function series of constructs. The modifications almost did not alter the IMEter scores of the introns (Supplementary Table S2 at JXB online).To explore further the role of the various LI regions, a gain-of-function approach was used. Individual or several LI elements were introduced into the middle of a non-related intron predicted to have a relatively low ability to enhance expression. The intron used was derived from the CAT1 intron alone (construct U0) or including various LI elements, was used to replace the LI in the 5′ UTR of AtMHX  and minor (Mp) products of the plasmid carrying the construct that lacked any intron [–I(p)], and did not show a product whose size was similar to that of their unspliced plasmid. This indicated that all introns were spliced with high efficiency, apparently because the CAT1 intron provided all the necessary signals and probably due to the a priori elimination of potential cryptic splice sites . GUS activity of the transformed plants, relative to that of the –I plants, is shown in CAT1 intron resulted in the highest enhancement of GUS activity—670- or 53-fold—compared with the –I or U0 constructs, respectively. This indicated that E3 has a special ability to enhance expression, in agreement with the results of the DEL series. For all constructs used in this study in which the expression level was above the limit of histochemical detection, the sites of GUS expression were similar to those previously reported for the WT construct , or to the CAT1 intron that included either E2 (U12 to U2 ratio of 0.25) or E2+E3 (U123 to U23 ratio of 0.43). The strong inhibition seen when E1 was inserted into the CAT1 intron alone indicated that the negative impact of E1 in the UTR series was not restricted to its interaction with other regions of the LI of AtMHX. The function of the E2 region was less clear—its inclusion almost did not affect the CAT1 intron alone (compare U2 and U0), had a positive effect when added to the inhibitory El element (U12 versus U1), and had a strong negative effect when added to the enhancing E3 region (U23 versus U3). It therefore seems that the positive or negative impact of certain intronic regions on IME can be compensated for by the presence of other intronic elements.The results of the UTR series also supported the conclusion obtained from the DEL series regarding the inhibitory impact of the E1 region. E1 lowered GUS activity when added to the GUS transcript were determined in plants expressing the DEL and UTR series of constructs . In order to understand how the LI elements exert their impact, the levels of nstructs . Each coGUS mRNA or GUS activity correlated with the IMEter scores of the different introns. The levels of GUS mRNA showed high correlation with the IMEter scores . These data indicate that the investigated LI elements differ in their ability to enhance translation.It was then examined to what extent the levels of r scores , F. Thesr scores  or weak r scores  correlatlculated . This molculated . Figure CAT1 intron alone resulted in a 19-fold increase in translational efficiency (CAT1 intron (U12 versus U0). Altogether, these data show that the ability of E3 to enhance translation was abrogated by the presence of elements E2 or E1+E2 at its 5′ end, but not by the presence of elements E4+E5 at its 3′ end.The E3 element made a large contribution to the translational efficiency in both series of constructs. In the DEL series, its omission in D5 resulted in a 2.7-fold reduction in translational efficiency compared with the WT construct . In the CAT1 intron . It thus seems that the indicated inhibitory impact results from interaction between element E3 and elements E2 or E1+E2 (or between putative proteins or RNAs that bind these elements).To determine whether the strong (5- or 6-fold) inhibitory impact of E2 or E1+E2 on E3-mediated translational efficiency is related to a specific interaction between these elements and E3, it is necessary to evaluate the effect of the indicated elements in constructs that lack E3. CAT1 intron alone (CAT1 intron that included E2 (U12 versus U2) or E2+E3 (U123 versus U23).The results presented before showed that element E1 inhibited expression in both the DEL and UTR series. The present data show that E1 decreases the ability of introns that include it to enhance translation. There was a 2-fold increase in translational efficiency in construct D7, in which E1 was removed, compared with the WT construct . At the GUS initiation codon; that is, 82bp downstream of their position in the UTR series . The resulting series of constructs, called CDS, is illustrated in CAT1 intron was inserted into the GUS coding sequence at essentially the same position used in the pCAMBIA vectors. The CDS and UTR series constructs were identical with respect to all sequence elements, and differed only in the position of their introns. Following transformation into Arabidopsis, RT–PCR showed that all constructs were spliced at high efficiency (The significance of intron position for the function of the LI elements was investigated by moving the same introns used in the UTR series into the coding sequence. The introns were inserted only 12bp downstream to the ficiency .CAT1 intron resulted in the highest enhancement of expression—64- and 9-fold—compared with the constructs that lacked the intron (–I) or included the CAT1 intron alone (C0), respectively , and had a minor positive impact (or at least did not have a negative impact) when included together with E2 (C12 versus C2) or E2+E3 (C123 versus C23). The fact that a positive, or at least lack of negative, effect of E1 was observed here for three construct pairs that differed only in this element strengthened the conclusion that the impact of E1 differed between the CDS series and the two 5′ UTR-localized series of constructs.However, in contrast to the findings in both the DEL and UTR series, in which E1 inhibited expression, in the CDS series this element enhanced expression when positioned in the GUS mRNA in plants expressing CDS series constructs is shown in GUS mRNA and GUS activity showed relatively good correlation with the IMEter scores of the different introns (GUS mRNA and GUS activity of pairs of constructs having the same intron in different positions—either the 5′ UTR (black columns) or the coding sequence (grey columns) (the constructs were presented according to the increasing GUS activity of the UTR series). All data were calculated relative to the same construct (–I) and, consequently, the difference in column lengths between each pair of constructs is proportional to the difference in the absolute levels of GUS mRNA or activity between the two constructs.The levels of  introns , D. To bGUS mRNA were, in general, comparable in constructs having the same intron at the two different locations. Still, for most construct pairs, mRNA levels were 10–40% lower in the CDS series. It was shown that the ability to enhance mRNA accumulation is proportional to the intron’s proximity to the 5′ end of the transcript . This indicates that the reduction in the ability to enhance translation due to intron displacement in constructs C3, C23, and C2 primarily resulted from the shift in E3 and E2 location.30% only . It is t30% only , indicat30% only . Specifi30% only . The samElement E1 alone had a much higher ability to enhance translation in the CDS than in the UTR series  was shown to correlate with the extent of increase in mRNA accumulation in  introns , F, 6C.  introns . This inhttp://mfold.rna.albany.edu/?q=mfold/RNA-Folding-Form)   did not CAT1 intron alone) was, however, much lower compared with the 19-fold increase mediated by E3 in the UTR series. It is possible that in the CDS series introns, in which the ability of E3 or other intronic elements to enhance translation was diminished (due to the change in their position), the inhibitory impact of E1 on the function of these elements became less significant, and a positive impact of E1 on translation became unmasked. A positive impact of U-rich intronic motifs on IME, without an impact on splicing, had been reported , it enhanced translation in the CDS series, in which the introns were localized in the coding sequence. The extent of this enhancement  identified only short (3–6 nucleotides) motifs previously shown to affect splicing efficiency or alternative splicing in mammalian cells (data not shown). Since the identified short motifs were implicated in splicing, they are not related to the impact of E3  on translational efficiency. It was shown that the enhancing effect of introns on translation is mediated by the EJC  35S promoter by ~3-fold (AtMHX promoter (AtMHX expression. However, it should be noted that the 35S promoter is much stronger than the AtMHX promoter (The LI of AtMHX transcript could be easily visualized, expression of the corresponding protein is apparently low (AtMHX (It was previously reported that while w (AtMHX . This mew (AtMHX . InteresJXB online.Supplementary data are available at Figure S1. The sequences of the 5′ UTR of AtMHX and the CAT1 intron.Figure S2. The modifications introduced into the LI elements in the UTR and CDS series compared with the DEL series.Figure S3. The coding sequence of GUS.Figure S4. The northern blot shown in Table S1. IMEter scores of individual LI elements.Table S2. IMEter scores of the modified LI elements inside CAT1 compared with putative similar constructs containing the comparable non-modified LI elements.


Amyotrophic Lateral Sclerosis (ALS) is an adult onset neurodegenerative disease, which is universally fatal. While the causes of this devastating disease are poorly understood, recent advances have implicated RNA-binding proteins (RBPs) that contain predicted prion domains as a major culprit. Specifically, mutations in the RBPs TDP-43 and FUS can cause ALS. Cytoplasmic mislocalization and inclusion formation are common pathological features of TDP-43 and FUS proteinopathies. Though these RBPs share striking pathological and structural similarities, considerable evidence suggests that the ALS-linked mutations in TDP-43 and FUS can cause disease by disparate mechanisms. In a recent study, Couthouis et al. screened for protein candidates that were also involved in RNA processing, contained a predicted prion domain, shared other phenotypic similarities with TDP-43 and FUS, and identified TAF15 as a putative ALS gene. Subsequent sequencing of ALS patients successfully identified ALS-linked mutations in TAF15 that were largely absent in control populations. This study underscores the important role that perturbations in RNA metabolism might play in neurodegeneration, and it raises the possibility that future studies will identify other RBPs with critical roles in neurodegenerative disease. Amyotrophic Lateral Sclerosis (ALS) is an adult-onset neurodegenerative disease that is typically fatal within 2–5 years of disease onset. Loss of upper and lower motor neurons leads to paralysis and is usually the cause of death. Most cases are sporadic with no family history, but ~10% of cases are familial [SOD1 underlie ~20% of familial ALS (fALS) cases and ~1% of sporadic ALS (sALS) cases [Efforts to understand the causes of ALS and methods to mitigate its pathogenesis have been severely limited by an inability to identify ALS genes. For the past two decades, most ALS research has focused on the ubiquitously expressed copper/zinc superoxide dismutase 1 (SOD1). Mutations in S) cases –7. TheseTDP43 were found in sALS and fALS cases, but not in control cases [ The identification of a novel ALS disease protein in 2006 dramatically altered our understanding of this disease and brought to light the potential role RNA-binding proteins (RBPs) play in neurodegeneration. TDP-43 was found in cytoplasmic inclusions in diseased brain [ol cases . This fiTDP-43 is a highly conserved, ubiquitously expressed protein that has various roles in RNA metabolism , 9. TDP- The identification of the RBP, TDP-43, as a causative agent of ALS-directed research towards the possibility that other RBPs might also play a role in ALS. This led to the contemporaneous discovery by two groups of the ALS disease protein Fused in Sarcoma, or FUS . Like TDFUS contains an N-terminal QGSY-rich domain, followed by a Glycine-rich domain, an RRM, and two RGG-rich domains , 10. Lik Prions are infectious proteins that are prone to misfold and aggregate and can spread from organism to organism in a DNA/RNA-independent manner –17. TheyTDP-43 and FUS both contain a predicted prion domain similar to those found in yeast prion proteins like Sup35 and Ure2 , 18–20. To test the prediction that these RBPs are intrinsically aggregation-prone, a cell-free system was established to monitor the propensity of pure protein for spontaneous aggregation , 22, 25.Saccharomyces cerevisiae has been extensively used to gain insight into many neurodegenerative processes [ The baker's yeast rocesses . Furtherrocesses . Importarocesses , 27–34.  Though TDP-43 and FUS share many striking similarities , severalMost of the TDP-43 ALS-linked mutations tested to date accelerate aggregation in vitro, whereas the FUS mutations have no effect , 22. Q33 Given the largely overlapping functions and protein domains of TDP-43 and FUS, it is likely that many other RBPs play a crucial role not only in ALS but also in neurodegeneration more generally , 18, 25.First, the entire human genome was surveyed for proteins that contained RRMs . Of the Interestingly, TAF15 shares many similarities with TDP-43 and FUS . Its dom The identification of TDP-43 as an ALS gene altered the genetic landscape of neurodegenerative disease. The importance of altered RNA processing in disease pathogenesis is now much more widely appreciated. Furthermore, the identification of other RBPs that all contain RRMs and prion domains is unlikely to be mere coincidence , 18. IndThough much progress has been made in the identification and characterization of the roles that RBPs play in ALS, much is still beyond our current understanding. For instance, it is still not known whether these proteins cause disease by a loss of function or gain of function or both , 16–18.


IL10) gene polymorphisms are associated with the development of sarcoidosis in Japanese patients.To investigate whether interleukin 10 (IL10 and assessed the allelic diversity between cases and controls.Two hundred and eighty-eight Japanese sarcoidosis patients and 310 Japanese healthy controls were recruited. We genotyped 9 single-nucleotide polymorphisms in IL10 alleles, genotypes, and haplotypes in the sarcoidosis cases compared to the controls were detected.No significant differences in the frequency of IL10 polymorphisms are not significantly related to the pathogenesis of sarcoidosis in the Japanese population.Our results suggest that  Sarcoidosis is a systemic inflammatory disorder characterized by non-caseating granuloma formation in many organs, such as: lung, skin, eye, lymph nodes, central and peripheral nervous system, and heart -3.In Japan, the reported incidence rate of the disease is 1.01 per 100,000 inhabitants . On a glMycobacterium tuberculosis and Propionibacterium acnes DNA have been detected in sarcoid lesions [It is also thought that environmental factors associate with the disease progression. By using polymerase-chain-reaction (PCR) techniques,  lesions -14. Rece lesions .These stIL10 polymorphisms were associated with several inflammatory diseases [IL10 in Japanese sarcoidosis patients.The inflammatory response in sarcoidosis is characterized by the increased production of several inflammatory cytokines produced by type 1 helper T (Th1) cells and macrophages, such as interleukin-2 (IL-2), interferon-gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α) -18. Thesdiseases -22. In tTwo hundred and eighty-eight unrelated patients with a diagnosis of sarcoidosis and 310 healthy controls were recruited from Yokohama City University, Hokkaido University, Fujita Health University, Tokyo University, Keio University, Kumamoto City hospital and Yuasa eye clinic. All patients and control participants were of Japanese ethnicity. Sarcoidosis patients were diagnosed according to the diagnostic criteria developed by the Japanese Society of Sarcoidosis and Other Granulomatous Disorders (JSSOG) previously described . UveitisIL10 SNPs which previously showed a significant association with Japanese Behcet’s disease [rs1878672, rs1554286, rs1518111, rs3021094, rs3790622, rs3024490, rs1800872, rs1800871, and rs1800896 . We selected  disease : rs18786s1800896 , Table 12 test. The Haploview 4.1 program was used to compute pair-wise linkage disequilibrium (LD) statistics [2 value were plotted, and LD blocks were defined according to the criteria [Hardy–Weinberg equilibrium was tested for each SNP among the controls. Differences in allele and genotype frequencies between cases and controls were assessed by the χatistics . Standarcriteria . Haplotycriteria . P valueIL10: rs1878672, rs1554286, rs1518111, rs3021094, rs3790622, rs3024490, rs1800872, rs1800871, and rs1800896. All SNPs were in Hardy–Weinberg equilibrium in the controls (data not shown). All 9 SNPs were located in 1 haplotype block, and the magnitude of LD between each SNP was extremely high, with pair-wise D’≥0.83 (We genotyped nine common SNPs in  D’≥0.83 . The all D’≥0.83 . We analIL10 polymorphisms affect the development of Japanese patients with sarcoidosis. Our results showed that all the IL10 polymorphisms were not significantly associated with any clinical subtype of sarcoidosis including ocular involvement in Japanese patients. Here we report a lack of association between IL10 variants and Japanese sarcoidosis patients, suggesting that the possibility of attributing the pathogenesis of sarcoidosis to IL10 genetic variations is low.The aim of the current study was to investigate whether IL-10 poroduced by Th2 cells suppresses inflammatory cytokines produced by Th1 cells. Although the mechanism of IL-10 in sarcoidosis is unclear, it is thought to be associated with granuloma resolution . Some stIL10 gene polymorphisms were associated with several inflammatory diseases. Wang et al. [IL10 polymorphisms were associated with increased patient serum IL-10 levels. Hudson et al. [IL10 genotypes were associated with systemic sclerosis-related autoantibodies and contribute to the etiology of systemic sclerosis. Recently, Mizuki et al. [IL10 as a disease susceptibility gene. Muraközy et al. [IL10 polymorphisms with sarcoidosis, however they could not find any significant differences. As with the previous report, we could not find any association between IL10 gene polymorphisms and sarcoidosis. On the other hand, Vasakova et al. [IL10 polymorphisms between sarcoidosis and healthy controls in the Czech Caucasian population, whereas they suggested that their findings cannot be generalized since the sample size in the study was small.Recent studies have also reported that g et al.  reportedn et al.  reportedi et al.  performey et al.  investiga et al.  have recIL10 polymorphisms do not appear to be significantly relevant to Japanese sarcoidosis patients. However, further genetic studies in other ethnic populations are required to elucidate the association between IL10 polymorphisms and sarcoidosis.In summary, the


The competition between intermolecular interactions and long-range lateral variations in the substrate–adsorbate interaction was studied by scanning tunnelling microscopy (STM) and force field based calculations, by comparing the phase formation of (sub-) monolayers of the organic molecules (i) 2-phenyl-4,6-bis(6-(pyridin-3-yl)-4-(pyridin-3-yl)pyridin-2-yl)pyrimidine  and (ii) 3,4,9,10-perylene tetracarboxylic-dianhydride (PTCDA) on graphene/Ru(0001). For PTCDA adsorption, a 2D adlayer phase was formed, which extended over large areas, while for 3,3'-BTP adsorption linear or ring like structures were formed, which exclusively populated the areas between the maxima of the moiré structure of the buckled graphene layer. The consequences for the competing intermolecular interactions and corrugation in the adsorption potential are discussed and compared with the theoretical results. It is well known that the formation of highly ordered 2D supramolecular networks on smooth surfaces, such as metal substrates or highly oriented pyrolytic graphite (HOPG), is mainly governed by the intermolecular interactions between adjacent molecules due to hydrogen bonding –4, covalOne example of this effect is in metal supported graphene films. In the case of Ru as a support, it is known from LEED structure analysis and theory that the lattice mismatch between the graphene layer and the underlying metal substrate results in a buckling of the graphene layer and therefore in a distinct height corrugation of 1.5 Å –16. The Recently, we demonstrated, for the adsorption of 2-phenyl-4,6-bis(6-(pyridin-2-yl)-4-(pyridin-4-yl)pyridin-2-yl)pyrimidine  molecules on a Ru(0001) supported graphene film, that such surfaces indeed exhibit not only a height corrugation but also a distinct corrugation in the adsorption potential . This wa…N-type hydrogen bonds are formed which are typically in the range of 100 meV . At. At24]. The influence of thermal activation on the structure formation is illustrated in a series of snapshots recorded at 2 frames per second  at room temperature with a home built video-STM . Upon imTo determine the corrugation of the adsorption potential, we summed up the different contributions to the adsorption energy (molecule–graphene and molecule–Ru interaction) and subtracted the adsorption energy of a “hill” site from that of a “valley” position. The corrugation of the adsorption energy can be compared with the intermolecular interactions. For the adsorption of 3,3'-BTP molecules, the STM images shown above reveal that very similar hexagonal local units are formed upon adsorption on HOPG and on graphene/Ru(0001). On both surfaces, these units consist of six molecules in a hexagonal arrangement. The main difference lies in the spacing between the different units. In the case of graphene/Ru(0001), they are separated by the hills of the graphene. On HOPG, in contrast, they are interconnected and therefore additionally stabilized by hydrogen bonds between adjacent molecules of neighboring units. The additional double hydrogen bonds are in the range of 0.14 eV per double bond . TherefoE ranges from −0.435 to −0.690 eV. To rationalize the different behavior of the PTCDA molecules, we again compare the corrugation of the adsorption energy with the intermolecular interaction between adjacent molecules in the herringbone configuration of PTCDA. Recent DFT calculations by Mura et al. [The same procedure was applied for PTCDA molecules adsorbed on graphene/Ru(0001). a et al.  have shoa et al. . CompariThese results illustrate the detail of microscopic understanding that can be extracted from combined STM experiments and state-of-the-art calculations. They also support the validity of structural concepts for the self assembly of supramolecular networks for cases where the adsorption potential on the substrate is highly corrugated, instead of the normal ‘smooth’ substrates.We have shown by STM imaging that (i) there are distinct differences in the adlayer structures of 3,3'-BTP on HOPG compared to 3,3'-BTP on graphene/Ru(0001), while (ii) for PTCDA, similar structures are formed on both substrates. In the first case, the adlayer forms 1D chains around the hills of the graphene/Ru(0001), but a 2D interconnected network on HOPG, while in the second case 2D networks are formed on both substrates. Qualitatively, these differences can be explained by the competition between intermolecular interactions and the lateral variation of the adsorption potential, i.e., of the molecule–substrate interactions. In the case of the 3,3'-BTP molecules, the intermolecular interactions are significantly weaker than the potential energy corrugation of the surface, rendering the occupation of hill sites, and hence the formation of 2D networks, energetically unfavorable. In the case of PTCDA molecules, the lateral corrugation of the adsorption potential must be overcompensated by stronger intermolecular interactions. These ideas are fully supported on a quantitative scale by a combination of force field and density functional theory based calculations, which reveal much stronger intermolecular interactions for PTCDA than for 3,3'-BTP, while the difference in binding energy on valley sites (favorable) and on hill sites (unfavorable), and hence the lateral corrugation of the adsorption potential on the graphene/Ru(0001) substrate, is of similar magnitude for both molecules.−10 mbar), equipped with a commercial variable temperature scanning tunneling microscope (STM)  and facilities for sample preparation, such as an Ar+ ion sputter gun and evaporation sources for the deposition of organic molecules.The experiments were performed in a standard ultrahigh vacuum (UHV) system , followed by flash annealing to 1650 K. Remaining carbon impurities were removed by oxidation, involving oxygen adsorption (10 L O2: 1 L = 1.33 × 10−6 mbar s−1) and subsequent repeated flash annealing to 1650 K. The freshly prepared surface exhibited 50–200 nm wide, atomically flat terraces, separated by monolayer steps. The graphene layer was prepared by exposing the Ru(0001) surface to ethylene for prolonged time at elevated temperatures . The structural quality of the graphene layer was checked by STM in constant current mode at temperatures of around 100 K.A Ru(0001) crystal (Mateck) was cleaned by standard procedures, including 3–4 cycles of Ar3,3'-BTP molecules  were deposited from a home-built, resistively heated, Knudsen cell at 583 K. The PTCDA molecules  were evaporated from a commercial evaporation source  at 628 K. Prior to evaporation, the PTCDA was cleaned in UHV by a temperature gradient sublimation technique using a resistively heated quartz tube. After deposition the sample was annealed to 583 K to improve the structural quality of the molecular film.To complement and rationalize the experimental findings above, force field calculations were performed to determine the site specific adsorption energies for 3,3'-BTP and PTCDA on graphene/Ru(0001). Due to the large size of the system, quantum chemical calculations were too computationally expensive. The surface was modelled with three layers of Ru and one layer of graphene on top, using a commensurate lattice with a (12 × 12) graphene unit cell on a (11 × 11) Ru(0001) cell with a lattice constant of 29.96 Å, in agreement with experiment, using the coordinates obtained in recent density functional theory calculations from a combined experimental and theoretical study . These s


Sarcoidosis is a granulomatous multisystem disorder of unclear etiology that involves any organ, most commonly the lung and the lymph nodes. It is hypothesized that the disease derives from the interaction between single or multiple environmental factors and genetically determined host factors. Multiple potential etiologic agents for sarcoidosis have been proposed without any definitive demonstration of causality.We report the case of two patients, husband (57 years old) and wife (55 years old), both suffering from sarcoidosis. They underwent a lymph node biopsy by mediastinoscopy which showed a “granulomatous epithelioid giant cell non-necrotising chronic lymphadenitis”. They had lived up to 3 years ago in the country in a farm, in contact with organic dusts, animals such as dogs, chickens, rabbits, pigeons; now they have lived since about 3 years in an urban area where there are numerous chemical industries and stone quarries. The aim of this case report was to focus on environmental factors that might be related to the pathogenesis of the sarcoidosis. Sarcoidosis is a granulomatous multisystem disorder of unclear etiology that involves any organ, most commonly the lung and the intrathoracic lymph nodes. Also heart, skin, and central nervous system are frequently affected. The diagnosis of sarcoidosis is further hindered by the lack of any reliable and specific diagnostic test, as there are no laboratory or imaging findings that enable the definitive diagnosis. Even the histological data are not conclusive because the non-necrotising granuloma, a hallmark of morphologic appearance of the disease, is not unique for sarcoidosis. Granulomas typically consist of a compact central area of macrophages that differentiate into epithelioid cells and then fuse to form multinucleated giant cells surrounded by lymphocytes.Mycobacterium spp. being the most commonly implicated organism. The role of various non-infectious organic and inorganic environmental agents has been studied in the pathogenesis of sarcoidosis.A genetic predisposition to sarcoidosis is epidemiologically evidenced by the demonstration of familial aggregation, differences in disease susceptibility and severity between racial groups, and the significantly increased incidence in monozygotic twins of affected individuals compared to other siblings ,2. It isWe report the case of two patients, husband and wife who had lived up to 3 years ago in the open country, in contact with organic dusts (hay and molds), and animals such as dogs, chickens, rabbits, pigeons; now they live in an urban area where there are numerous chemicals industries and stone quarries.The woman, 55 years old, is a housewife and a former smoker of 1 pack/years. She underwent a hysterectomy for uterine fibromatosis, and a surgical operation for right humerus osteochondroma. She has got a herpes simplex virus type I infection. At the time of our first observation she referred in the last 2 years dry cough with little sputum, dyspnea grade II according to the American Thoracic Society (ATS) scale and mild fever. At the beginning of the symptoms she performed a chest x-ray which showed “interstitial thickening” and a microbiological sputum examination which was negative for tuberculosis and fungal infection. The general practitioner gave her a non-specific antibiotic therapy with low benefit and then a steroid therapy with improvement in symptoms. At the time of our observation, she underwent a total-body CT scan which showed “a series of lung nodules distributed in all lobes; a solid slab in the mantle of the apical segment of the left lower lobe; several small adenomegalies in the anterior mediastinum, around the trachea and below aorta artery; no evidence of effusion in the pleural cavity” in the thorax   was normal.The 6-min walking test was normal and the pulmonary function tests showed a reduction in the diffusing capacity for carbon monoxide (DLO) Table . The artCO  in severe obesity. As his wife, he has got a herpes simplex virus type I infection. He takes regular therapy with aspirin, doxazosin, allopurinol, irbesartan, erythropoietin and insulin. He referred dyspnea ATS grade II-III in the absence of cough. He also reported to have taken steroids in the past at the starting dose of 50 mg/day and gradually tapered. This therapy was recommended in the absence of a definitive diagnosis and on the basis of symptoms and radiological abnormalities of “interstitial thickening” present in the chest x-ray. At the time of our first observation, the pulmonary function tests revealed a mild restrictive pattern with reduction of DLThe environmental exposition of the two patients concerns chemical agents because of the industries near where they lived in the last years and because of the presence of a sand quarry. The industries in the area where they live include those involved in the processing of tires. Thus, factors such as the inorganic powder used in the rubber industry can play an active role in the pathogenesis of their disease. Other factors potentially involved could be silica, silicone and the fibers of the glass. Three recent longitudinal studies regarding the World Trade Center disaster have emphasized the role of airborne inorganic particulate exposure in sarcoidosis -6. TheseGeographic clustering of disease in the southern United States and in other parts of the world has promoted further speculations concerning weather, soil, and foliage in the pathogenesis of sarcoidosis . Past stInterestingly, other studies suggest the hypothesis of either a person-to-person transmission or a shared exposure to an environmental agent. In a well documented clustering that has occurred in the relatively isolated population of the Isle of Man, individuals with sarcoidosis were more likely to have had prior contact with an individual who had sarcoidosis, compared to controls (39.6% versus 1.1%). These contacts included neighbors, unrelated cohabitants, and co-workers . SimilarRhodococcussp[Finally, several observations suggest that sarcoidosis may be caused by an infectious agent. There are many known infectious agents that can cause granulomas that resemble those of sarcoidosis, including mycobacteria, herpes viruses, histoplasmosis, treponematosis, sporotrichosis, coccidioidomycosis, schistosomiasis, listeria, the agent of Whipple’s disease and ococcussp. BecauseBoth our patients have got labial herpes simplex virus and this could suggest a potential role of the infection in the development of the disease. Moreover, before moving to the urban area, the two patients lived in the countryside, reporting exposure to molds and hay. Finally, they were exposed to chemical agents derived from the industry of the tires. Among those, silica is a mix of modern tires and it is known that silica could be potentially involved in the pathogenesis of the disease. However, all the environmental factors that could be considered potential triggers of the disease are not sufficient by themselves to cause sarcoidosis and must interact with a particular immune response of the host. Therefore, the genetic predisposition of the patients plays a key role in the development and progression of the disease.In conclusion, this case report wants to focus on environmental factors that may be potential causative agents for sarcoidosis. Important tool in investigating patients with sarcoidosis is a complete medical history that includes occupational and environmental factors, lifestyle habits and any previous infection. It could be also useful to study and monitor the areas with the highest incidence of this disease.Written informed consent was obtained from the patient for publication of this Case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.IL and IS made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; LF and FV were involved in drafting the manuscript or revising it critically for important intellectual content; SV gave final approval of the version to be published.


After 3 months, she presented with raised nodules on her knees and elbows. After 8 months, she developed painful subcutaneous nodules on her forearms. We diagnosed sarcoidosis with both plaques and subcutaneous nodules associated with combination therapy with peginterferon α-2a and ribavirin. Sarcoidosis with both plaques and subcutaneous nodules is very rare. The patient had a sustained Th2 response in the liver. And a sustained Th1 response occurred only in the skin. It is likely that, for this reason, sarcoidosis was localized to the skin, and the patient developed sarcoidosis with both plaques and subcutaneous nodules.A 64-year-old woman with hepatitis C was treated using combination therapy with peginterferon  Combination therapy with interferon (IFN) and ribavirin is the standard treatment for hepatitis C. Both IFN and ribavirin induce a T helper cell (Th) response. Sarcoidosis is a systemic granulomatous disease that is triggered by a Th1 response. IFN-induced sarcoidosis is well documented, and some cases of cutaneous sarcoidosis have been reported . In the α-2a 180 mg/week and ribavirin 600 mg/day. After 2 months, the peginterferon α-2a dose was reduced by 50 percent due to a decrease in neutrophilic leukocytes. The HCV RNA level decreased, and liver enzymes remained normal. Three months after combination therapy was initiated, the patient presented with several asymptomatic, raised, brawny red nodules on the knees and elbows.A 64-year-old woman with hepatitis C was started on a treatment regimen of ursodeoxycholic acid. Liver enzymes could be normalized, but a negative hepatitis C virus (HCV) ribonucleic acid (RNA) value could not be achieved. Therefore, the patient began combination therapy with peginterferonγ-glutamyltransferase (γ-GTP) 16 IU/mL. The angiotensin-converting enzyme (ACE) level was slightly elevated at 22.7 U/L, while the lysozyme level was normal at 4.8 μL/mL. A chest X-ray showed no active disease, and the cardiac electrogram showed no abnormality. The patient's eye examination was unremarkable. Based on these findings, a diagnosis of sarcoidosis with both plaques and subcutaneous nodules was made. Since we suspected that sarcoidosis was caused by peginterferon α-2a and ribavirin therapy, these drugs were discontinued, and ursodeoxycholic acid therapy was restarted at the original dose. Observation of the superficial and subcutaneous nodules continued, with no additional therapy. Gradually, these nodules disappeared, and the patient was pain free. Four months after stopping the combination therapy, no subcutaneous nodules were found on palpation, and no superficial nodules could be detected with the unaided eye. An MRI scan of the lesions of the knees and elbows showed no abnormality. In addition, the ACE level normalized at 19.7 U/L, and the lysozyme level decreased to 4.2 μL/mL. After several years, the patient has had no reactivation of sarcoidosis, and liver enzymes are maintained within normal limits by the administration of ursodeoxycholic acid. At first, the nodules were characterized by disappearances and reappearances. However, 7 months after starting combination therapy, the patient complained of pain in her knees and elbows. Nodules in these regions were clearly visible Figures  and 2. Xα is known to stimulate the Th1 cells, and those produce leukotrienes that seem to be responsible for the development of the granulomas. Ribavirin also enhances the Th1 response. Therefore, combination therapy with IFN-α and ribavirin is a trigger for sarcoidosis. Many cases of cutaneous sarcoidosis have been reported as a result of combination therapy for hepatitis C [Sarcoidosis is an autoimmune systemic disease characterized by the Th1 immune response. IFN-atitis C . HoweverThe recognition of sarcoidosis with both plaques and subcutaneous nodules was based on several factors. In the current case, the Th1 response was not enhanced in the liver, but the Th2 response improved. It has been reported that patients with chronic hepatitis demonstrate an enhanced Th2 response . In the α-2a has a long duration of action, with maximum plasma concentration (Tmax) occurring from 72 to 96 hours. Therapeutic blood levels of the drug are sustained for 168 hours. In addition, peginterferon α-2a is administered by subcutaneous injection. Thus, the drug was confined to the skin. It is likely that sarcoidosis was localized to the skin for this reason.Peginterferon This paper describes a rare case of sarcoidosis with both plaques and subcutaneous nodules. Th2 cells reached other organs through the bloodstream because of the sustained Th2 response in the liver. However, the skin is far removed from the liver. Thus, only the patient's skin showed an enhanced Th1 response.


Caenorhabditis elegans by combining computational predictions and experimentally derived TF–DNA interaction data. We found that first introns of C. elegans genes, particularly those for families enriched in long first introns, are more conserved in length, have more conserved predicted TF interactions and are bound by more TFs than other introns. We detected a significant positive correlation between first intron size and the number of TF interactions obtained from chromatin immunoprecipitation assays or determined by yeast one-hybrid assays. TFs that bind first introns are largely different from those binding promoters, suggesting that the different interactions are complementary rather than redundant. By combining first intron and promoter interactions, we found that genes that share a large fraction of TF interactions are more likely to be co-expressed than when only TF interactions with promoters are considered. Altogether, our data suggest that C. elegans gene regulation may be additive through the combined effects of multiple regulatory regions.Gene expression is controlled through the binding of transcription factors (TFs) to regulatory genomic regions. First introns are longer than other introns in multiple eukaryotic species and are under selective constraint. Here we explore the importance of first introns in TF binding in the nematode  Saccharomyces cerevisiae, where TFs bind promoters immediately upstream of the transcription start site (TSS)  to regulatory genomic regions such as promoters and enhancers. The regulation of transcription is relatively simple in single cell eukaryotes such as the yeast te (TSS) . However introns ,3. Enhan introns ,5. HowevCaenorhabditis elegans is a primary model system for the study of development, as well as physiological processes such as fat storage, dietary response and aging  or gene-centered approaches (DNA-to-protein) ,11. TF-cal stage . These sal stage ,13, theral stage . Furthert intron . HoweverC. elegans TFs  assays can be used to identify the repertoire of TFs that can bind to a DNA sequence of interest ,19. Collgans TFs . Y1H assC. elegans. However, with the exception of 43 TFs that have been profiled genome-wide by ChIP , the propensity of first introns to interact with TFs has been largely unexplored. Here, we use a combined experimental and computational approach to investigate TF binding to C. elegans (first) introns. We find that the first intron is generally longer than other introns, which is in agreement with previous observations , alternative start sites (i.e. two or more distinct first coding exons) and presence downstream in an operon . Genes that were negative for all three criteria were defined as ‘conventional genes’ (15 621 genes). Promoter regions were defined from the start codon to up to 2-kb upstream unless there was a neighboring gene closer, in which case the region was extended until the start codon or the 3′-end of the neighboring gene.Genomic sequences and annotations were downloaded from C. elegans conventional gene were compared to all coding exons of the C. briggsae ortholog to determine the homologous segment using tBlastx. Matches were tabulated and genes where contiguous one-to-one alignments could be accurately detected were used for downstream analyses. The analysis of length conservation was performed for genes that have an identical exon–intron structure  in C. elegans and C. briggsae (3404 genes) to be able to determine and compare length conservation for all introns. For length conservation analysis, only genes that have a first intron longer than 500 bp in at least one of the species were considered (942 genes).Gene orthology was based on InParanoid data . Individhttp://intermine.modencode.org/ on 29 April 2012. The midpoint of the ChIPped region was defined as the binding site. Gene fragments were analyzed individually to determine whether the TF ChIP peak midpoints were located between the start and end of the fragment.ChIP interaction data from the modENCODE Project  were dowC. elegans TFs that belong to a broad range of TF families. We used a threshold of 0.09 as previously determined   to scoretermined . A prediCaenorhabditis elegans conventional genes with first introns longer than 250 bp were analyzed for gene family enrichment (gene families defined according to InterPro) using DAVID Bioinformatics Functional Annotation tool  and 40–50 genes were randomly selected from each bin. The first introns of the selected genes were cloned as previously described for promoter fragments (C. elegans (N2) genomic DNA by polymerase chain reaction, cloned into a gateway entry vector and subsequently transferred into the HIS3 and LacZ Y1H reporter Destination vectors. The reporter constructs were integrated into the genome of S. cerevisiae (Y1H-aS2) to create Y1H bait strains as described (Supplementary Table S1. Yeast bait strains corresponding to gene promoters were generated using the promoterome resource (Supplementary Table S2). Interactions that were detected in two of four replicates were considered positive. Auto-active and low-confidence DNA baits (i.e. baits with non-uniform background reporter expression) (10 for first intron baits) were removed from further analysis.We selected the first introns of 227 resource . eY1H asSupplementary Table S1). Promoter interactions for the same homeodomain genes were obtained from Reece-Hoyes  database . For eacf is the probability density distribution, x is the number of interactions and 1/λ is the average number of interactions.The promoter and first intron in-degree (number of TF interactions per regulatory element) distributions were modeled using the exponential distribution as previously shown for promoters in several species ,21,32,33The promoter + first intron in-degree distribution was modeled with a hypo-exponential distribution . This diP = 0.145) and the first intron (λI = 0.3) in-degree distributions and used to determine the expected hypo-exponential distribution for the promoter + first intron in-degree.The λ values were estimated for the promoter . We named the remaining set of genes ‘conventional genes’ (76% of C. elegans protein-coding genes). As shown in C. elegans conventional genes are longer than other introns , confirming previous observations  and whose first intron is ≥500-bp long in at least one species (942 genes). Genes with different exon–intron structure were not included in the analysis to be able to compare intron length conservation between introns at different positions. Additionally, introns that are shorter than 500 bp in both species were excluded from the analysis, as length conservation in this case may only reflect a general tendency for worm introns to be short. Briefly, we divided the length of the longer of the two introns by that of the shorter one. When intron length is conserved, the intron length ratio is close to 1, whereas larger discrepancies in intron length result in values >1 (P < 0.01). To confirm that this observation is not specific to the C. elegans and C. briggsae comparison, we compared 100 randomly selected C. elegans genes with orthologs in C. remanei and observed a 28% increase in conservation of first intron length relative to the second intron (data not shown). These results suggest that there is evolutionary pressure to maintain the length of first introns, which points to a functionally important role such as the regulation of gene expression.If first introns are important for gene regulation, one would expect that the length of long first introns is more conserved than the length of long non-first introns, as they could potentially contain more TFBSs and would thus be more sensitive to deletions. To test this prediction, we compared the intron length ratio between conventional genes of two related nematode species, alues >1 B. By usiTo test the potential importance of the first intron in gene regulation, we used TF binding as a proxy. We first evaluated the frequency of TF interactions with different gene regions using ChIP data for 43 TFs obtained from the modENCODE Project . We founC. elegans TFs , with first introns longer than 500 bp having on average 30-fold more TF interactions than first introns shorter than 100 bp.First introns bind on average only 3.4% of the number of TFs compared with promoters as determined by ChIP assays A. HoweveC. elegans TFs and, in most cases, only in a single developmental stage and laboratory condition  in a high-throughput manner . Although this number is lower than the average number of TFs that bind gene promoters   , our resGiven that some first introns, in particular longer ones, bind a large number of TFs, we hypothesized that these introns may compensate for low TF binding to the cognate promoter. To test this hypothesis, we compared the number of ChIP interactions between promoters and first introns on a gene-by-gene basis C. We didWe reasoned that TF interactions with first introns could contribute to gene regulation in two ways. First, TFs that bind the first intron could be overlapping with those that bind promoters, resulting in redundancy and, as a consequence, a robust regulation of gene expression ,41. SecoP = 0.286). The same was true when only first intron interactions were considered (P = 0.466). However, when promoter and first intron interactions were combined, gene-pairs with a high PDI similarity were enriched for gene-pairs with medium and high co-expression (P = 0.0016). This observation provides further support for the notion that promoter and first intron interactions are not redundant, but rather may independently contribute to gene expression.Gene regulation is achieved by the combinatorial action of TFs. We hypothesized that if two genes share a large fraction of TFs binding to their regulatory regions, they are more likely to be co-expressed than two genes that have different interacting TFs. To test this hypothesis, we focused on 24 homeodomain genes, a gene family enriched in long first introns . We firstab-1 and ceh-27 have a relatively high co-expression correlation (0.43), although they have a low PDI similarity (0.17) if only promoter interactions are considered (ceh-83 and ceh-89 have a relatively low co-expression correlation (0.26), although they have a high PDI similarity (0.5) when considering only promoter interactions, but have a low PDI similarity (0.17) if first intron interactions are included  per promoter best follows an exponential distribution, with most promoters binding very few TFs and a few promoters binding a disproportionately large number of TFs  often occur in specific gene families. For instance, GPCR, the endoglin/CD105 protein family, homeodomain TFs and neurotransmitter-gated ion channels on average have long first introns . ImportaC. elegans and C. briggsae than the rest of the conventional genes . Interestingly, in mice, neuronal and developmental genes have been associated with the greatest enrichment of putatively functional noncoding sequences in first introns as defined by sequence constraint, suggesting a more complex regulation for this set of genes  grants  and a postdoctoral fellowship from the Pew Latin American Fellows Program (to J.I.F.B. in part). Funding for open access charge: NIH.Conflict of interest statement. None declared.


Increasing genome data show that introns, a hallmark of eukaryotes, already existed at a high density in the last common ancestor of extant eukaryotes. However, intron content is highly variable among species. The tempo of intron gains and losses has been irregular and several factors may explain why some genomes are intron-poor whereas other are intron-rich.We studied the dynamics of intron gains and losses in an α-amylase gene, whose product breaks down starch and other polysaccharides. It was transferred from an Actinobacterium to an ancestor of Agaricomycotina. This gene underwent further duplications in several species. The results indicate a high rate of intron insertions soon after the gene settled in the fungal genome. A number of these oldest introns, regularly scattered along the gene, remained conserved. Subsequent gains and losses were lineage dependent, with a majority of losses. Moreover, a few species exhibited a high number of both specific intron gains and losses in recent periods. There was little sequence conservation around insertion sites, then probably little information for splicing, whereas splicing sites, inside introns, showed typical and conserved patterns. There was little variation of intron size.Since most Basidiomycetes have intron-rich genomes and this richness was ancestral in Fungi, long before the transfer event, we suggest that the new gene was shaped to comply with requirements of the splicing machinery, such as short exon and intron sizes, in order to be correctly processed. Saccharomyces cerevisiae, Guillardia theta, Encephalitozoon cuniculi. Intron-rich species are often multicellular, for example vertebrates, the worm Caenorhabditis elegans, the fungus Phanerochaete chrysosporium, the sea squirt Ciona intestinalis. Intron-rich unicellular organisms also exist, like the green alga Chlamydomonas)us genes . In S. htronless  and in mtronless .The HGT α-amylase gene could be a suitable model for studying the evolution of information content around intron sites. However, we found a low level of information at positions −2 to +2 surrounding intron sites, contrasting with our results in animals , where tIn contrast, information at both 5' and 3' splicing sites was strong and typical of fungal introns , suggestAltogether, our data suggest that several features were important to confer to the transferred gene suitable characteristics regarding splicing efficiency: short introns; shortening the exons to a small size through multiple intron gains, although exon sizes were more variable than intron sizes; and rather regular intron spacing along the entire gene, perhaps for efficient nonsense mediated decay . It is nOur results need now to be generalized by investigating other genes recently transferred from bacteria, in both intron-rich and intron-poor genomes, in order to confirm whether introns colonized intronless genes rapidly, with a density related to the genome average.HGT: Horizontal gene transfer; CBM: Carbohydrate binding module; GH: Glycosyl hydrolase.The authors declare no competing interests.JLDL designed the study, acquired experimental and database data, analyzed data, and drafted the manuscript. MB brought expertise in Fungi, contributed material and phylogenetic data and analyzed and discussed data. SJ performed GH13 phylogeny, AHV contributed to discussion and manuscript writing, DC performed the datation study and contributed to discussion and manuscript writing. All authors read and approved the final manuscript.List of primers designed for detection of α-amylase genes orthologous to Phchr1|7087|. PCR conditions were: initial denaturation 94°C, 6 mn; denaturation 94°C, 25 s; annealing 58°C, 50s; elongation 72°C, 1 mn, 45 cycles using the Taq Gold polymerase (Applied Biosystems). Various combinations of forward and reverse primers were tried.Click here for fileGenBank or JGI accession numbers of sequences EF1α, RNA polymerase II LSU 1 and LSU2, used for datation estimates.Click here for fileAbbreviations used in Figure Click here for fileAlignment of the α-amylase protein sequences studied, built with MAFFT, showing the intron positions. Pink: phase 0 introns; green: phase 1 introns; blue: phase 2 introns. This alignment was used, without the N-terminal variable region , for gene tree reconstruction . The tree was rooted with two bacterial sequences. Abbreviations are given in Additional file Click here for fileReconciliation tree made from the gene tree (Additional file Click here for fileFungal species tree and divergence times estimated with BEAST using EF1α+LSU1+LSU2, with dates of divergence at nodes. Horizontal bars show the 95% highest posterior intervals of the divergence times.Click here for fileAnalysis of exon size distribution. For each gene, the effective number of exon was computed according to ref. 16. The statistical significance was estimated by 10,000 simulations. Ne: effective number of exons. ns: not significant; *: p<0.05. 5%: value of Ne below which are the smallest 5% simulated Ne values; 95%: value of Ne below which are the smallest 5% simulated Ne values.Click here for fileConsensus sequences at positions -2 and -1, and +1 and +2 around intron positions with different phases, drawn with Weblogo 3.2 . n is thClick here for fileConsensus splicing sites of introns drawn with Weblogo 3.2 . Left toClick here for file


The secretion of Interleukin-4 (IL4) is the characteristic of T-helper 2 responses. IL4 is a cytokine produced by CD4+ T cells in response to helminthes and other extracellular parasites. It has a critical role in guiding antibody class switching, hematopoiesis and inflammation, and the development of appropriate effector T-cell responses. In this study, it is the first time an attempt has been made to understand whether it is possible to predict IL4 inducing peptides. The data set used in this study comprises 904 experimentally validated IL4 inducing and 742 noninducing MHC class II binders. Our analysis revealed that certain types of residues are preferred at certain positions in IL4 inducing peptides. It was also observed that IL4 inducing and noninducing epitopes differ in compositional and motif pattern. Based on our analysis we developed classification models where the hybrid method of amino acid pairs and motif information performed the best with maximum accuracy of 75.76% and MCC of 0.51. These results indicate that it is possible to predict IL4 inducing peptides with reasonable precession. These models would be useful in designing the peptides that may induce desired Th2 response. Cellular immune response to a pathogen is mediated through the processing and presentation of antigen on the surface via major histocompatibility complex (MHC). The exogenous antigens are processed through lysosome and presented by MHC class II. The loaded peptide on MHC class II interacts with CD4+ T cells and a pattern of cytokine is synthesized and secreted. Depending upon the cytokines secreted, the T-helper cells polarize into diverse T-cell populations like Th1, Th2, Th17, or iTregs , cancer Although this identification demands experimentally validating the immune response generated by each antigen, this investigation is time-consuming, cumbersome, and expensive task since the possible antigens and corresponding fragments range in millions –11. ThusIn the past, several methods have been developed for identification of MHC class II binders that may activate T-helper cells. These T-helper cells induce different types of cytokines like IL-4 and IFN-gamma. Presently, available methods provide no information about the type of cytokine a MHC II binding peptide will induce. In order to address this issue, an attempt has been made to develop a method for predicting IL-4 inducing MHC II binding peptides.It is important to select the right dataset for developing a prediction method. The performance of the method is largely dependent on the datasets used for training a model. In this study datasets were generated from publicly available immune epitope database (IEDB) . We extrSince the main dataset includes only MHC class-II binders, the prediction algorithm (based on the above dataset) can only predict IL4 inducing peptides from MHC class-II binders. We are also interested in discriminating the IL4 inducing peptide from the random peptides. Thus, we created an alternate dataset for building prediction models that can be used for mapping IL4-inducing peptides in antigens. Our alternate dataset has random peptides as negative set instead of non-IL4-inducing peptides. We generated IL4 noninducing (negative examples), from SwissProt proteins.We first analyzed the IL4 inducing positive and noninducing negative sequences, to comprehend the preferred peptide length for both positive and negative peptides, by using R-package for creating boxplot . We alsoThe recognition of functional motifs in peptide or proteins constitutes an important element in functional annotation of sequences . In the We further explored 100 degenerate motifs using three kinds of classification: (i) none, (ii) Koolman-Rohm , and  using the following formula and a vector of dimension 20 is used to represent amino acid composition of a peptide:Likewise, the algorithm calculates dipeptide composition (DPC) and a vector of dimension 400, representing a peptide, using the following formula:Amino acids pairs (AAP) based method represents the input epitope by a vector of fixed vector length (400) by incorporating the information from each amino acid pair and their propensity in the given dataset. This approach had shown its potential in past for predicting B-cell epitope .Here, we converted positive and negative examples into binary codes, where each amino acid is represented by a vector of dimension 20 . Using these binary vectors, different peptides were represented, such as a 15-amino acid long peptide which is represented by a vector of dimension 300 (15 × 20).In this study, classification models have been developed using machine learning technique support vector machine (SVM). In order to implement or develop SVM models, we used software SVMlight . SVM modWe further employed a hybrid approach, where we combined the predictions from both motif and model based methods. In a hybrid approach, the weight of +1 was given to the peptide having IL4 motif (exclusively found in IL4 inducing peptides) and −1 was given to peptide having a non-IL4-motif (exclusively found in non-IL4-inducing peptides). We developed several hybrid models depending on the type of vector inputs used for SVM based prediction.In order to develop reliable prediction models, we trained and tested our models using fivefold cross-validation technique. In this analysis, the whole dataset is divided randomly into five equal parts, and each time four sets are used for training our models and remaining set is used for testing. This procedure is repeated five times so that each set is tested once and four times it is used for training. The final performance of the model is evaluated by averaging the performance of models on each set. The performance of models was measured using the following standard parameters, that is, sensitivity, specificity, accuracy, and Matthew's correlation coefficient (MCC).http://dx.doi.org/10.1155/2013/263952).In order to understand the properties of the IL4 inducers, we analyzed both IL4 inducer (IL4+) and noninducer (IL4−) MHC class II binding epitopes extracted from IEDB. There are several studies where authors have exploited physiochemical properties (PCPs) of peptides to discriminate one class of peptide from other classes . We examWe first compared the length of the two types of peptides and observed that average length of Il4 inducing and noninducing peptides is not significantly different. We could not find any significant relation between length of sequence and its potential to induce IL4 production .We also compared amino acid composition of IL4+ and IL4− peptides and found compositional biasness between two types of peptides. It was observed that residues E, F, K, and I are more abundant in IL4 inducing peptides, while IL4 noninducing sequences majorly include G, D, and L .We have analyzed the role of MHC alleles to skew the immune response to induce IL4 cytokine. The dataset that comprises 1759 epitopes was derived from 2845 IL4 assays and MHC alleles were undetermined in 1901 (66.8%) assays. The rest of assays (33.2%) were restricted to 91 MHC alleles. Out of these 91 alleles, 34 alleles were observed in IL4 positive as well as IL4 negative assays . We have also found 10 alleles for which IL4 assays were returned as exclusively negative and 47 alleles for which all the IL4 assays were resulted as exclusively positive . The mosThe amino acid compositional analysis described the overall dominant residues in IL4 inducing and noninducing peptides. However, this information does not specify the positional preference of specific amino acid residues at specific positions. In order to know the preference of a particular amino acid at different positions or at N- or C-terminals, we created the two-sample logo for our positive and negative IL4 peptides. Two-sample logo as depicted in In order to look at position specific proclivity of different PCPs as mentioned in the method section, we calculated the average of every PCP, at every position of N′ and C′ terminal as mentioned in the method section. For every PCP, we found various positions showing discriminating values in plot ; for example, 1, 2, 3, 5, and 12 positions of N′ terminal show high hydrophilicity in IL4+ peptides. Based on these observations we selected discriminating residues for PCP as mentioned in Table 1S. We selected some of the discriminating PCP and looked at the average of the sum of PCP of IL4+, IL4−, IL4 + Nt, IL4 − Nt, IL4 + Ct, and IL − Ct amino acid sequences  (see the Method section). We found hydrophilicity, pI, amphipathicity, steric, and charge properties, discriminating between IL4+ and IL4− data. On the other hand, the sum of PCP of selected residue positions (see the Method section) showed a significant difference in all the PCPs .We next tried to determine exclusive motifs or patterns in IL4 inducing peptides by using MERCI software. We used three types of classification, that is, none, Koolman-Rohm, and Betts-Russell, to determine 100 motifs in peptides. It was observed that Betts-Russell classification significantly discriminated 205 IL4 inducers from noninducers and Koolman-Rohm was significantly distinguished 150 non-IL4-inducers from IL4 inducers .Collectively, motifs generated from all types of classification discriminated 333 positive and 237 negative peptides. The best motifs generated from each classification are listed in We developed prediction models using SVM that is widely used in the past for classification models –30. In tWe further developed another SVM model based on binary profile of amino acids of the peptides, where a vector of dimension 20 represents each residue. The compositional variation plot for each residue in IL4 inducing and noninducing peptides and the performance of SVM model based on binary profile of N-/C-terminal residues were also analyzed (depicted in Table 3S).We adopted a hybrid approach for prediction of IL4 peptides by combining the prediction based on SVM model and motif search. The datasets were first sorted based on the exclusive motifs searched in positive and negative peptides using MERCI. The initial search identified 333 IL4 inducing and 237 IL4 noninducing MHC class II binding peptides, and these sequences were given weightage by adding +1 and −1, respectively, in SVM score in the hybrid method. Additionally, in this hybrid approach, we developed four different models using different input features each time . This teAll the models described above has been developed on main dataset that contain experimentally validated IL4 inducing and noninducing MHC class II binders. These models only can be used for predicting IL4 inducing peptides if users know that their query peptide is MHC class II binders. In order to provide service to the community we developed models on alternate dataset that can be used to discover IL4 peptides in proteins/antigens. As described in Materials and the Methods section our alternate dataset contains negative set/examples random peptide. We developed models on alternate dataset and achieved maximum accuracy of 70% .Performance on independent dataset is one of the best ways to validate a prediction model. As the IEDB database is continuously updated, we extracted the 71-peptide novel entries (deposited after extraction of our dataset) from IEDB for MHC class II binders for which IL4 assay was positive. Out of 71 peptides our best model correctly predicted 49 epitopes at default threshold.With the advent of the next generation sequencing techniques, the designing of rational vaccines based on the immunogenic features of peptides has become a need of the modern era. Identification of peptides or antigenic that can activate all arms of the immune system is important for designing effective immunotherapy or epitope/subunit vaccine. It means vaccine candidates (peptide/antigen) should have antigenic regions that can activate both B-cell and T-cell epitopes (MHC Class I or II peptides). The Th2 response is very important in vaccine or immunotherapy design against extracellular pathogen. IL4 is the principal cytokine that directs commitment of T cells to Th2 phenotype . TherefoWe extracted experimentally validated MHC class II binding peptides from IEDB with and without IL4 inducing potential. We initially analyzed both these datasets to select important features that could lay the basis for the IL4 inducing capability of the peptide. It is well documented that the binding of peptides to MHC complex is largely dependent on the length of the peptides ; thus weMHC alleles are well documented in literature for thier capability to skew the immune response –35. Our Distinction of different immune epitopes has already been reported with different PCPs in the past . In our Next, we tried to discriminate IL4 inducers and noninducers by use of machine learning technique. For analysis of positional feature of a sequence by SVM, binary patterns of the sequences were used as input. Since binary patterns of peptides could only be applied at a fixed length, we generated different binary inputs by varying the length of amino acids from 9 to 15 through both N and C terminals of a peptide. The performance of SVM model based on these inputs showed a MCC of 0.18. Further, we analyzed the residue and dipeptide compositional vector of IL4 inducer and noninducer sequences. It was observed that the models based on compositional profile performed better than models trained on binary patterns, possibly because this attribute of the sequence does not depend upon the length of the peptide as it has a fixed feature input of 20 and 400 for residue composition and dipeptide composition vector, respectively.We have also developed hybrid model, by employing the information from motif and machine learning. In the hybrid approach, the weightage has been given to sequences that could be predicted with exclusive motifs searched using MERCI. We observed that the performance was improved up to a value of MCC 0.51 using hybrid of MERCI and amino acid pair. This could be attributed to the role of propensity and exclusive motifs in prediction of IL4 inducing epitopes as it was also publicized for B-cell epitopes . The AAPin silico prediction method that can aid in understanding the IL4 inducing potential of the antigens in computer aided rational vaccine design for better control of diseases.Performance of our model on independent dataset was that 69% (49 out of 71) is satisfactory. This performance is comparable with 78.76% sensitivity at fivefold cross-validation on training dataset. In summary, we have developed the The tendency of an epitope to induce IL4 and skew the immune response towards Th2 makes it of great significance in immunotherapy and vaccine designing. Although the induction of IL4 response is a very complex issue that depends on a number of factors like cytokine milieu, MHC haplotype, the costimulatory molecules, and peptide itself, a peptide is an important factor that could be controlled easily while designing a vaccine or immunotherapy. However, Th2 response includes other cytokines like IL5 and IL10; we only focused on IL4 cytokine. Although the experimental evidence for Th2 peptides is limited, our computational analysis appears to support their existence.http://crdd.osdd.net/raghava/il4pred/).Keeping this limitation in mind, we have made an attempt to predict the peptides that may induce IL4 response. In this study we evaluate performance of our models using fivefold cross-validation as well as evaluating performance of our method on an independent dataset. It was observed that our model predicts IL4 inducing peptides with reasonable accuracy. In order to facilitate the scientific community working in the area of subunit vaccine, we have used the above models for developing a webserver IL4pred (We are providing three tables and four figures as supplementary document. In these three tables, we are providing the performance of SVM on our main dataset using features like physicochemical properties, compositional feature combined with length of peptide and binary profile of different window length. The figures in supplementary document represent the analysis of physiochemical properties and MHC alleles in our main dataset.Click here for additional data file.