TDP-43 accumulation and ALS-linked mutations within degradation pathways implicate failed TDP-43 clearance as a primary disease mechanism. Here, we report the differing roles of the ubiquitin proteasome system (UPS) and autophagy in the clearance of TDP-43. We have investigated the effects of inhibitors of the UPS and autophagy on the degradation, localisation and mobility of soluble and insoluble TDP-43. We find that soluble TDP-43 is degraded primarily by the UPS, whereas the clearance of aggregated TDP-43 requires autophagy. Cellular macroaggregates, which recapitulate many of the pathological features of the aggregates in patients, are reversible when both the UPS and autophagy are functional. Their clearance involves the autophagic removal of oligomeric TDP-43. We speculate that, in addition to an age-related decline in pathway activity, a second hit in either the UPS or the autophagy pathway drives the accumulation of TDP-43 in ALS and FTD. Therapies for clearing excess TDP-43 should therefore target a combination of these pathways.TAR DNA-binding protein  is the major pathological protein in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Large TDP-43 aggregates that are decorated with degradation adaptor proteins are seen in the cytoplasm of remaining neurons in ALS and FTD patients  Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that are characterised by the deposition of pathological protein aggregates composed of TAR DNA-binding protein 43 (TDP-43 or TARDBP) . These ‘Evidence that the dysregulation of TDP-43 can cause disease comes from many animal models. A neurodegenerative ALS-like phenotype can be generated by either overexpression or knockdown of TDP-43 in flies, fish and mice . Indeed,Two of the major pathways for cellular protein degradation are the ubiquitin proteasome system (UPS) and macroautophagy, herein referred to as ‘autophagy’. Autophagy has been shown to degrade soluble and aggregated protein substrates that are too large to enter the UPS pore . UbiquitDetermining the relative importance of these two protein degradation pathways in maintaining TDP-43 proteostasis is challenging owing to the complex interplay between them, yet is crucial to understanding the mechanisms underpinning TDP-43 accumulation in disease. Although the role of TDP-43 macroaggregation remains controversial, the cytoplasmic accumulation of TDP-43 is definitely linked to toxicity, and therapeutic strategies designed to enhance TDP-43 clearance are of great appeal. We therefore sought to investigate how TDP-43 protein degradation is regulated and the potential for targeting degradative pathways as a treatment for ALS and FTD.50; HEK293, 0.97 ng/ml; SH-SY5Y, 0.35 ng/ml).Stable cellular models of ALS and FTD based on doxycycline (DOX)-inducible expression of various TDP-43 constructs  were genNext, we sought to investigate the relative degradation rates of the TDP-43 constructs. Because of its ability to autoregulate , quantifIn HEK293 lines assayed over 72 h , wild-tyt1/2; HA–TDP-43 WT, 29.2 h; HA–TDP-43 ΔNLS, 16.6 h; HA–TDP-43 CTF, 10.2 h).In order to verify that the assay was not simply measuring the dilution of HA–TDP-43 protein by cell division or the slow decay of DOX-induced transcripts, we performed a modified experiment over a shorter timecourse in SH-SY5Y lines , and useHaving determined that TDP-43 degradation rates were consistent between cell lines and timecourses, we next analysed the pathway(s) by which TDP-43 was being degraded, using HEK293 lines. Inhibitors of the UPS or autophagy were included during the final 48 h of the DOX-washout period when protein degradation occurs (for schematic see P≤0.05) and, to a lesser extent, the degradation of wild-type and ΔNLS TDP-43. 3MA showed minimal effect on the autophagy reporters p62 and LC3 (microtubule-associated protein 1 light chain 3 β) at the timepoint chosen for assay of TDP-43 levels (48 h 3MA); however, shorter timecourses of 3MA treatment revealed autophagy inhibition had occurred . The related UPS inhibitor epoxomicin also caused significant accumulation of wild-type, ΔNLS and CTF TDP-43 . The inhibition of both degradation pathways (by combined treatment with 3MA and MG132) did not inhibit the degradation of any of the protein constructs any more than MG132 alone. Activators of the autophagy pathway validated these findings because the addition of trehalose and LiCl during DOX washout substantially accelerated the degradation of CTF TDP-43  and p62 , and polyubiquitin chains with different internal lysine linkages (K48 and K63) . These aWe next sought to specifically investigate the handling of insoluble TDP-43. We used 1 µg/ml DOX plus MG132, which acts reversibly, to induce the formation of insoluble TDP-43 WT or ΔNLS cellular aggregates and then we followed the fate of those aggregates after washout of MG132. DOX was maintained in the medium at a concentration of 1 µg/ml during washout to ensure that our findings reflected TDP-43 aggregate handling in the face of continued expression of TDP-43. End-point imaging showed that for both WT  and ΔNLSt1/2; EGFP–�TDP WT, 29.3 h; HA�–TDP WT, 29.2 h). We also verified that EGFP-tagged TDP-43 was functional, as evidenced by its ability to downregulate endogenous TDP-43 . Owing to the low fluorescence intensity of EGFP–TDP WT aggregates, their clearance could not readily be measured in living cells.We therefore examined the clearance of aggregates in live cells, using stable SH-SY5Y cells transfected with EGFP-tagged TDP-43. We verified that the replacement of the N-terminal HA-tag with EGFP did not alter the timecourse of TDP-43 degradation by performing doxycycline pulse-chase experiments  are mobile, readily exchange with the diffuse protein pool and can be cleared by the UPS . To deteP≤0.01; MG132 with aggregates, 48%±1.6, P≤0.001; ±s.e.m.). Furthermore, we found that the TDP-43 within the aggregates was almost completely immobile, with the bleached portions of the aggregates remaining dark for the duration of FRAP imaging and not visibly exchanging with the diffuse pool (P≤0.001). At 8–10 h after the washout of MG132, there was a significant increase in the proportion of diffuse TDP-43 that was mobile in cells still containing large aggregates, suggesting there was mobilisation or clearance of previously immobile species . However, direct bleaching of macroaggregated TDP-43 showed that this species was not mobilised, as it showed no increase in the mobile fraction after washout.We examined cells that were induced to express EGFP–TDP-43 using 1 µg/ml DOX alone, cells induced to form aggregates with 1 µg/ml DOX plus MG132, and cells induced to form aggregates then washed out and left in fresh medium containing 1 µg/ml DOX without MG132 for 8–10 h. Firstly, by bleaching diffuse cytosolic EGFP–TDP-43 ΔNLS we found that the proportion of diffuse TDP-43 protein that was mobile was reduced by inhibition of the UPS, both in cells devoid of aggregates and, more strikingly, in cells that contained large aggregates (t1/2 = 3.4 s). Diffuse TDP-43 in cells exposed to UPS inhibition but without aggregates showed a similar rate of recovery (t1/2 = 3.6 s), whereas TDP-43 in cells with aggregates showed significantly slower recovery (t1/2 = 6.9 s). A decrease in the fluorescence recovery rate indicates increased binding or increased size of the TDP-43 particle, consistent with oligomerisation of TDP-43. At 8–10 h after the washout of MG132, there was an increase in the rate of recovery of diffuse TDP-43 in cells with large aggregates (t1/2 = 6.9 s before versus 4.0 s after washout), suggesting that there had been removal or dissociation of slow-moving species.Next, we examined the rate of fluorescence recovery (the speed of molecular movement) for mobile species of TDP-43 protein . DiffuseP≤0.01; ΔNLS, 31±9 MG132 versus 6±2 DOX, P≤0.05; ±s.e.m.).Our findings suggested that during washout, slow-moving and immobile species were either resolved into mobile or faster-moving species, or were removed directly. Oligomeric or microaggregated species cannot be cleared directly by the UPS, and their clearance might require aggregate autophagy or ‘aggrephagy’ . TherefoP≤0.01, Student’s t-test with Welch’s correction; ±s.e.m.). For cells that survived the period of imaging, we plotted the relationship between the load of aggregated protein and the rate of clearance from the cell . However, these aggregates then persisted in the cells. The slope of the line relating initial aggregate load with clearance time therefore flattened in the presence of autophagy inhibitors  but not by knockout of a key molecule in autophagy induction, Atg7  then subcloned into Gateway. EGFP-tagged TDP-43 ΔNLS was generated by site-directed mutagenesis of the WT construct in pDONR . Primer sequences are available on request.Constructs are shown schematically in ® Terminator v1.1  on an ABI3130 genetic analyser . Sequence chromatograms were analysed using Geneious v5.4.6 . All primers were obtained from Sigma-Aldrich . mCherry–GFP–LC3 plasmid (All constructs were verified in-house by DNA sequencing using Big-Dye plasmid  was kind plasmid  was a ki3MA (used at 10 mM), bafilomycin (400 nM) and lithium chloride (10 mM) were purchased from Acros Organics . MG132 (0.5 µM), epoxomicin (100 nM) and rapamycin (0.2 µg/ml) were purchased from Cayman Chemicals . Trehalose (100 mM) was purchased from Sigma-Aldrich.Stable tetracycline-inducible (Tet-ON) clonal cell lines were generated in human neuroblastoma SH-SY5Y cells  and human embryonic kidney HEK293 , using the T-REx system. Briefly, stable clonal lines transfected with pcDNA6/TR (encoding a constitutively expressed Tet-repressor protein) were transfected with pDEST30-TDP-43 constructs using Lipofectamine 2000, selected using 600 µg/ml geneticin and clonally isolated.2.SH-SY5Y cells were maintained in DMEM/F12 plus Glutamax, supplemented with 10% fetal bovine serum (FBS) , 100 U/ml penicillin and 100 µg/ml streptomycin. HEK293 cells (stable Tet-ON) were maintained in DMEM plus Glutamax, supplemented with 10% FBS (Tet-free), 100 U/ml penicillin and 100 µg/ml streptomycin. All cells were maintained at 37°C, under 5% CO2, HEK293 lines were plated at 25,000 cells/cm2 and TDP-43 expression was induced using 1 µg/ml DOX, unless otherwise stated. All reagents were purchased from Invitrogen Life Technologies unless otherwise stated.Transient transfection of mCherry–GFP–LC3 into stable SH-SY5Y lines was performed using Lipofectamine 2000, as per the manufacturer’s instructions. Transient transfection of FLAG–Ub into stable HEK293 lines was performed using Fugene HD  as per the manufacturer’s instructions. For experimental analyses, SH-SY5Y lines were plated at 75,000 cells/cmFor immunofluorescence analyses, SH-SY5Y lines were plated onto untreated 13-mm coverslips (1.5 thickness). HEK293 lines were plated onto poly-D-lysine-treated coverslips. After treatment, cells were fixed in 4% paraformaldehyde  for 10 min and rinsed with phosphate-buffered saline (PBS). The cells were incubated with primary antibody overnight at 4°C, then with fluorescent secondary antibodies for 6 h at room temperature, each diluted in PBS with 0.2% Triton X-100 and 1% serum (goat or donkey as appropriate). For thioflavin T staining, cells were stained for 10 min with freshly prepared and filtered 0.1% thioflavin T (Acros Organics) in PBS and rinsed. After immunodetection, the coverslips were mounted onto slides using fluorescence mounting medium  and left to harden overnight.The antibodies used for immunofluorescence were against TDP-43 , HA.11 , p62 , ubiquitin , K48-linked ubiquitin , K63-linked ubiquitin , ubiquilin-2 . Invitrogen Life Technologies goat secondary antibodies  or Jackson ImmunoResearch donkey secondary antibody  were used at 1∶500.Wide-field imaging was performed using a Zeiss Axiovert S100  with a ×63/NA 1.25 Plan Neofluar oil immersion objective, fitted with a CoolSnap EZ digital camera . Confocal images were acquired using a Leica TCS SP5 confocal laser scanning microscope  with a ×63/NA 1.4 Plan Apo oil immersion objective and a pinhole of 95.6 µm (Airy 1). Images (1024×1024 pixels) were acquired at 400 Hz with a line averaging of 8, using Leica LAS-AF software and 405 nm, 488 nm and 633 nm laser lines.http://rsb.info.nih.gov/ij/) on merged RGB images for a representative experiment. GFP–LC3 autophagosomes were quantified using the Granularity tool in Metamorph  and the granules were normalised to the number of transfected cells by manual counting of GFP-positive cells. 113–622 granules were counted in 20–50 transfected cells in a representative experiment.Line intensity profiles were quantified using the colour profiler plug-in (Dimiter Prodanov) in ImageJ  pulse–chase as described previously . Briefly® III First-Strand Synthesis SuperMix (Invitrogen Life Technologies). Quantitative PCR was performed using FASTStart SYBR Green Mastermix on a 7900HT Fast Real-Time PCR System (Applied Biosystems) running SDS v.2.3 software. The primers that were used to detect HA–TDP WT and ΔNLS transcripts were 5′-TACCCATACGATGTTCCAGATTAC-3′ and 5′-GCATGCAGAATTCCTTCTACC-3′. HA–TDP CTF transcripts were quantified using the same HA-specific forward primer with the reverse primer 5′-CCCGTACTGAGAGAAGAACT-3′. GAPDH acted as a reference transcript, using the primers 5′-CAGCCTCAAGATCATCAGCA-3′ and 5′-GGCATGGACTGTGGTCATGAG-3′. PCR cycling conditions were: activation, 10 min at 95°C; cycling, 15 s at 95°C, 30 s at 60°C and 30 s at 72°C for 40 cycles; followed by the thermal denaturation protocol. The expression levels of HA–TDP transcripts relative to GAPDH transcripts (reference), with normalisation to no DOX (control), were determined using the Pfaffl method  and cDNA was reverse transcribed using oligo(dT) primers with SuperScriptl method .The sequential biochemical fractionation of cellular proteins was performed as described previously for ALS and FTD brain homogenates , with seRIPA/urea solubility fractionation was performed as described by Winton and colleagues , using cProtein concentrations were quantified using the BioRad DC Protein Assay  and equivalent protein was loaded for each sample . For solubility experiments, whole-lysate protein was quantified and the equivalent liquid volume of the soluble and insoluble fractions was loaded. Gels were transferred onto nitrocellulose using the iBlot (Invitrogen Life Technologies), stained with Ponceau S and then blocked in TBS with 0.05% Tween-20 (TBS-T) and 5% non-fat dried milk  for 30 min. The blots were probed overnight at 4°C with primary antibody, then for 3 h at room temperature with secondary antibodies . The blots were scanned on the Li-Cor Odyssey gel scanner  and were then reprobed (without stripping) for loading-control proteins using the same antibody conditions and scanning protocol.Antibodies used for blotting were against TDP-43 , HA.11 , Histone H3 , p62 , GAPDH , GFP , LC3B  and Dylight fluorescent secondary antibodies .The blot images in TIF format were quantified using the gel analyser tool in ImageJ. Integrated band intensities were normalised to the band intensities of loading controls and also to relative input for the solubility assays.2 and were left to recover overnight. TDP-43 expression was induced with DOX for 24 h and then aggregate formation was induced using DOX with 0.5 µM MG132 for a further 48 h. For washout experiments, the cells were then washed and left in fresh medium containing DOX without MG132, in the presence or absence of inhibitors for the stated time periods.The clearance of TDP-43 aggregates was assessed by RIPA/urea solubility fractionation and immunofluorescence (HA-tagged constructs) and live-cell imaging and fluorescence recovery after photobleaching (FRAP) (EGFP-tagged constructs). For all aggregate clearance assays, stable SH-SY5Y TDP-43 WT or ΔNLS cells were plated at 100,000 cells/cmLive imaging was used to assess whether EGFP–TDP-43 ΔNLS aggregates were cleared. Stable SH-SY5Y cells were plated onto Hi-Q4 dishes . Immediately before imaging, the cells were washed and left in fresh medium without MG132, containing DOX and either vehicle (0.05% DMSO), 3MA (10 mM) or bafilomycin (400 nM).2. The image sets were analysed using ImageJ. The aggregate load was defined as the total integrated density (area×intensity) of all aggregates in a given cell, with aggregate boundaries determined by intensity thresholding. The clearance time was defined as the time at which aggregates were indistinguishable from diffuse TDP-43.Epifluorescent and phase images (1280×960 pixels) of selected cells with aggregates were acquired twice per hour for 15 h using a BioStation IM-Q  fitted with a ×20/NA 0.5 Plan Fluor objective and maintained at 37°C, under 5% COStable SH-SY5Y EGFP–TDP-43 WT or ΔNLS cells were plated onto 18-mm coverslips (1.5 thickness). To investigate protein mobility after MG132 washout, the cells were washed and left in fresh medium containing DOX, but without MG132, for 8–10 h before FRAP.2. Fluorescence recovery was followed for 1 min, with a laser power of 1–3% to minimise acquisition bleaching.FRAP was performed using a Nikon A1 plus laser scanning confocal microscope fitted with an environmental chamber maintained at 37°C . Confocal images were acquired using a ×60/NA 1.4 Apo oil immersion objective, a confocal pinhole of 34.8 µm (Airy 1.2) and a 488-nm laser line. FRAP was performed using NIS-Elements AR software (v. 4.00.04). Images of 512×512 pixels were acquired. Five pre-bleach frames were acquired with a laser power of 1–3%, before bleaching EGFP–TDP-43  using a square region of interest (ROI) of 2 µmtI is the fluorescence intensity of the ROI at a given time, I0 is the fluorescence intensity of the ROI immediately after bleaching and Ii is the average fluorescence intensity of the ROI before bleaching. The data were fitted to a one-phase exponential decay and plotted using GraphPad Prism 6.02.Data analysis was performed using ImageJ with the Bio-Formats plugin . The meat-tests were performed after F-testing for equal variance and Welch's correction was applied where stated. The Pearson correlation test was performed after D'Agostino and Pearson normality testing. Statistical significance was set at P≤0.05 . All figures were prepared using Adobe Photoshop CS3.Data handling and graphical representations were performed using Microsoft Excel unless otherwise stated. Statistical analyses as described in the figure legends were performed using GraphPad Prism 6.02. One-way ANOVA was performed after Bartlett's testing for equal variance. For both one- and two-way ANOVA, Bonferroni post-tests were used to compare the selected data sets. Two-tailed Student’s


Sarcoidosis is a multisystem granulomatous disease characterized by hilar lymphadenopathy, involvement of internal organs, and diverse skin lesions. Systemic sclerosis is an autoimmune disease characterized by skin hardening and different internal organ fibrosis, including vascular abnormality. Immune response associated with Th-2 has been shown in the early and active stage of the disease. In this paper, we report coexistence of systemic sclerosis with sarcoidosis in a female patient presenting with granulomatous dermatitis, interstitial lung disease, and Raynaud's phenomenon complaints. Sarcoidosis is a systemic disease characterized by the involvement of multiple tissues and organs and a noncaseating granulomatous reaction, which is not well understood . AlthougWe present 52-year-old woman, who did not seek medical care for complaints that continued for six years and suggested Raynaud's phenomenon in the finger joints of the hands. One year ago, brown-red skin lesions developed at the pretibial part of the right foot and the patient consulted a dermatologist. However, the patient did not receive any results from the dermatological examination and visited our rheumatology polyclinic after arthralgia, morning stiffness, and effort dyspnoea started together with the Raynaud's complaints. Upon a physical examination, telangiectasia on the face, lessening of the mouth opening, sclerodactilia and Raynaud's phenomenon pallor phase of the fingers, and a brown-red skin lesion of approximately 15 cm on the right foot pretibial site not protruding from the skin were found Sarcoidosis is a systemic, Th1-mediated granulomatous disease, with unknown etiology, involving more than one organ. Sarcoidosis may mimic a number of rheumatic diseases or may coexist side by side. It can commonly present with connective tissue diseases, including primary Sjögren's syndrome, SLE, and RA, including clinical findings that are indicative of vasculitis and/or spondyloarthritis. Enzenauer and West have shown that 6 out of 569 patients followed with connective tissue disease developed concomitant sarcoidosis within 10 years . SakamotCoexistence SS with sarcoidosis is an entity that should be rarely seen, when the incidence of both diseases are considered  . MoreoveAs a result, we are reporting the coexistence of sarcoidosis with SS. The overlapping of SS with sarcoidosis is a very rare entity. The Th1/Th2 paradigm is one of its most important causes. Since they both can exhibit similar clinical manifestations, it is a crucial difference to the overlapping syndromes for prognosis and therapy. Although the causes for this coexistence are not clear, further research is warranted to determine whether there is a common etiopathogenesis and/or coincidental coexistence.


We previously have studied the insertion and deletion polymorphism by sequencing no more than one hundred introns in a mixed human population and found that the minimal introns tended to maintain length at an optimal size. Here we analyzed re-sequenced 179 individual genomes  from the data released by the 1000 Genome Project to study the size dynamics of minimal introns.We not only confirmed that minimal introns in human populations are selected but also found two major effects in minimal intron evolution: (i) Size-effect: minimal introns longer than an optimal size (87 nt) tend to have a higher ratio of deletion to insertion than those that are shorter than the optimal size; (ii) GC-effect: minimal introns with lower GC content tend to be more frequently deleted than those with higher GC content. The GC-effect results in a higher GC content in minimal introns than their flanking exons as opposed to larger introns (≥125 nt) that always have a lower GC content than that of their flanking exons. We also observed that the two effects are distinguishable but not completely separable within and between populations.We validated the unique mutation dynamics of minimal introns in keeping their near-optimal size and GC content, and our observations suggest potentially important functions of human minimal introns in transcript processing and gene regulation. Intron is an important and necessary subassembly of eukaryotic genes and plays precise and complicated roles especially in the case of mRNA processing and alternative splicing There have been several comparative studies within and among species, revealing situations where short introns  are under selection, and most of them have focused on model organisms, such as Drosophila. There have been several interesting observations. First, shorter introns tend to have higher recombination rates As data from the 1000 Genome Project are being released To obtain reliable sequence and allele information, we limited our dataset to (1) ancestor alleles were determined based on sequences from all three primates and (2) intron sequences must be mapped precisely in genomic sequences. This filter yielded 202,009 YRI, 146,842 CEU, and 136,175 JPTCHB indels. When plotted the derived allele frequency (DAF) of the indel data, we found that all curves showed a uni-modal distribution, slightly skewed toward left . The moroptimal value based on the intron length distribution corresponding to the peak value (mode) of minimal introns ratio of deletion to insertion or RDI when we investigated strength variations around an optimum: 87 nt . The results suggested that shorter introns are subjected to stronger selective constraints than longer introns when we defined the length of 87 nt as an optimum. We believe that the insignificance of the test is largely due to insufficient data. The stronger negative selection acting on the nucleotide content of protein coding sequences provides additional evidence to support the proposal that minimal introns in a size range of 50–86 nt are more likely to subject to purifying selection resisting to the overall deletion force acting in all introns.In the current study, our samples are still limited to the three populations and limited number of indels in minimal introns (50–124 nt). Although the statistics based on the current data is not as striking as the previous result, they are more robust and conclusive. In the previous study, we chose a set of introns that cover a full range of intron GC content (16.4–78.7%) in order to avoid biases in sampling. As a matter of fact, the GC content of minimal introns in the human genome tends to be higher (median: 60.4%), and in a rather random sampling scheme, our analysis is biased toward minimal introns with higher GC content whereas in a more selective case we actually sampled more AT-rich minimal introns. In either case, size-effect is indisputable. In the previous study, by using low-frequency alleles (indels with DAF<0.06), we found the size-effect but failed when look at higher frequencies, such DAF>0.35. Similarly, in this study we also tried to examine various DAF cut-offs  and found that the size-effect became weaker when DAF cut-off is above 0.1 (data not shown). Our data collectively confirmed the proposal that minimal introns have an optimal size or a narrow range and the distribution of minimal introns has a mode of 87 nt. If introns drift off this optimal size, there should be a tendency to be drawn back. To look for evidence supporting our hypothesis, we chose to focus on minor alleles that are sensitive to selection but not yet fixed in populations as we have preciously pointed out that rare polymorphisms (as a threshold of 0.06) are subjected to selections (e.g. adaptive selection on protein-coding sequences) In the current study, we also addressed GC-effect in addition to size-effect. In the previous study, we noticed a GC-effect where almost all insertions happened in GC-rich minimal introns whereas all deletions are among GC-poor minimal introns. Therefore, our previous data were not informative when we tried to separate size effect from GC effect. Although the current data did not give us a clear-cut answer but we believe that GC-effect should be resolved with statistical significance based on adequate data from a broad GC-content range.Finally, we would like to emphasize that sequence variations represent a rich data resource for interrogating biological questions, especially in a context of structure-function relationship such as the size of minimal introns and exons. In a long enough generation period, indels (or SNPs) with higher derived allele frequencies are more likely to be related to positive selections (adaptive selections), as those with lower derived allele frequencies tend to be under negative selections (purifying selections) for deleterious variants, in particular, the extremely rare variations. In contrast, what we found is that the DAF<0.1 variations appear largely adaptive in human populations because the maintenance of an optimal minimal intron length results a better functionality (efficiently processed and exported to the cytosol) for minimal intron-containing genes. We considered the indels of minimal introns as a dynamic parameter for reaching the size optima under various selective forces. Both positive and negative selections can work on these indels; positive selection leads to the fixation of the minimal introns with optimal sizes and purifying selection tends to remove the minimal intron-containing genes  that possessing over- or under-sized minimal introns.Drosophila melanogasterD. simulansin silico.Parsch et al. observed the insertion bias in short introns when examining indel divergence between or within ftp://ftp-trace.ncbi.nih.gov/1000genomes/). We retrieved the three blastz alignment files from UCSC databases ftp://hgdownload.cse.ucsc.edu/) between human and another species: chimpanzee (hg18vsPanTro2), orangutan (hg18vsPonAbe2), and rhesus monkeys (hg18vsRheMac2). We compared the indel variations with the corresponding position of these three primate references to identify ancestral alleles. We calculated the nonsynonymous and synonymous substitution rates between human and mouse from γ-MYN method We defined intron locations based on human Refseq data (NCBI build 36) described previously


A 2 μW power dissipation, voltage-output, humidity sensor accurate to 5% relative humidity was developed using the LFoundry 0.15 μm CMOS technology without post-processing. The sensor consists of a woven lateral array of electrodes implemented in CMOS top metal, a Intervia Photodielectric 8023–10 humidity-sensitive layer, and a CMOS capacitance to voltage converter. After nIn the current work, we investigated the possibility of employing the standard passivation layer of the CMOS chip as the humidity sensing element with electrodes shaped similar to those described in ,6, but u2.Sensing of humidity with hygroscopic polymer films is the most suitable approach for integrating humidity sensors in CMOS, but this approach has problems to solve. First, most hygroscopic films produce humidity-dependent capacitance, so a capacitance-to-voltage on-chip converter is necessary for simplified processing of the sensor output. Second, implementation of the sensing element is not straightforward. To obtain a large relative capacitance change, the electrical flux must be confined to the hygroscopic film. The simplest way to achieve this objective is if electrodes enclose the hygroscopic film from all sides, but for the moist air to gain access to the sensing film, a large area of the polymer must be exposed to the air. In the implemented humidity sensor, the electrical flux reaching the CMOS substrate was confined by the woven electrode mesh below the main sensing electrodes see . The wovFurthermore, previously proposed comb-type electrodes have a 50%metal density if sensitivity is maximized . For theA C/V (capacitance-to-voltage) converter derived from  was deve3.Experimental results from the manufactured humidity sensors are briefly summarized in Humidity range was limited by dryer capability of the humidity chamber and surface condensation. Performance measurements were done only in the 5–100 °C range. At 100–120 °C, only reliability testing was done. The accuracy mentioned in


The transcription factor p53 regulates cellular integrity in response to stress. p53 is mutated in more than half of cancerous cells, with a majority of the mutations localized to the DNA binding domain (DBD). In order to map the structural and dynamical features of the DBD, we carried out multiple copy molecular dynamics simulations . Simulations show the loop 1 to be the most dynamic element among the DNA-contacting loops (loops 1-3). Loop 1 occupies two major conformational states: extended and recessed; the former but not the latter displays correlations in atomic fluctuations with those of loop 2 (~24 Å apart). Since loop 1 binds to the major groove whereas loop 2 binds to the minor groove of DNA, our results begin to provide some insight into the possible mechanism underpinning the cooperative nature of DBD binding to DNA. We propose (1) a novel mechanism underlying the dynamics of loop 1 and the possible tread-milling of p53 on DNA and (2) possible mutations on loop 1 residues to restore the transcriptional activity of an oncogenic mutation at a distant site. Although p53 activates transcription most efficiently as a tetramer , both moin vivo ,16. Morein vivo –19. Indiin vivo . The DBD is an approximately 25 kDa chain consisting of an immunoglobulin-like β-sandwich  that provides the scaffold for the DNA binding surfaces . The secBoth L2 and L3 are stabilized by a zinc ion that is tetrahedrally held by the side chains of a histidine (H179) and three cysteine residues  . The zinProteins exist as inter-converting conformational states under ambient conditions and their conformational dynamics are known to be intricately linked to their functions  and chanWe begin by exploring the conformational space that is spanned by the available experimental structures of DBD. As of July 2011, the RCSB Protein Data Bank (PDB) contains 57 PDB entries of monomeric and multimeric p53 DBD as well as p53 DBD in complex with DNA. From these entries, we excluded structures that have unresolved residues, particularly in the mobile loop regions. The final set consists of 141 distinct conformers . The lowest sequence identity among these conformers is 84.4%. This difference of more than 15% in sequence identity arises from species differences (human versus mouse p53 DBD), but they are highly similar in structures with Cα RMSDs of 0.32- 0.59 Å. p53 DBD from species other than human and mouse were excluded because of the higher sequence differences. We did not see any separation between human and mouse p53 DBD in the first three dominant principal components (PC) space; the details of our principal component analysis (PCA) results are presented below. The final set also includes DBD with multiple mutations such as those introduced to stabilize the DBD.To perform PCA, we first determine, through iterated rounds of structural superposition , 35 struThe NMR and crystal conformers are well separated along PC 1. To identify residues that are responsible for the conformational differences retained by a given PC, the relative displacement of each residue along that PC is examined as atomic displacements from the mean structure . PC 1 deRadius of gyration can be used to understand the degree of compactness of folded protein conformers. We calculated the radius of gyration, surface area and volumes of the experimentally available conformers of each DBD and plot them against PC 1 . PC 1 clPC 2 separates conformers with major differences in L3 conformations; L3 is thought to be crucial for interactions of the DBD with the minor groove of DNA . There aAlong PC2, one of the two major clusters is made up of the crystal conformers (PDB code: 2H1L) that were crystallized with the large T-antigen (LTag) oncoprotein of simian virus 40 (SV40) . The DBDIn the only NMR study reported on isolated p53 DBD, M246 seems to occupy two conformations based on the detection of line broadening . This daBesides separating conformers with distinct methionine switch conformations in L3, PC2 also provides clues on the zinc ion. The zinc ion is coordinated by C176 of L2, H179 of H1, and C238 and C242 of L3. The crystal conformer (PDB code: 2P52), which was crystallized in the absence of zinc ion , lies inSimilar to PC 1 in the context of L1 diversity, PC 3 captures the diversity of conformers especially at L1. Two major clusters emerge along PC 3, in which one cluster (PC 3 > 15) has recessed L1 conformations and the other has extended L1 conformations . Only 6.To further probe the conformational space of monomeric p53 DBD, we performed two copies of MD simulations, each starting from four different structures, resulting in eight independent copies of 100 ns each. The RMSD profiles of the structural changes along the MD conformers suggest that our simulations are stable especially for the core residues . hitherto. The extended L1, particularly its K120 , seems to be stabilized by contacts formed with symmetry related molecules  approximation. The energetic distribution of GB free energy and solvation energy of free p53 DBD conformers with extended L1 is lower than those of recessed L1 , i.e. inNuclear Overhauser effect (NOE) measurements by Canadillas et al. suggested that there are compatible alternative conformations of L1 . In FiguWe further analyzed p53 DBD with extended and recessed loop 1 states using a simplified elastic network model that computes the normal modes of motion . This teIn order to understand the dynamical behavior of wild-type DBD as a whole, we further concatenate the eight trajectories of wild-type p53 DBD. From the RMSF profiles we see that as expected the N-terminal residues are the most flexible . L1 is tIt is known that enhanced dynamics are important for molecular recognition . Since LThe fluctuations of L2 are higher than those of L3 . The N-tnd copy of a simulation started from the crystal structure with PDB code 2OCJ) can sample the recessed L1 conformational space  in L1 in six NMR conformers . We also observed the transient helix in L1 in 4.9 % of the wild type MD conformers as determined using DSSP , L1 is disordered . To comping DSSP . In the We discover that isolated (DNA-free) monomeric p53 DBD can intrinsically sample both extended and recessed L1 conformations in MD simulations. In simulations initiated from the crystal and NMR structures with extended L1, MD conformers can adopt L1 conformations that are recessed, as evidenced by the smaller RMSD with respect to recessed L1 than the RMSD with respect to extended L1 . Again, To address these questions, we classified available MD conformers (of the cumulative 0.8 μs simulation time) into either extended L1 or recessed L1 states based on the stringent criteria that the L1 RMSD ≤ 2 Å with respect to crystal structures with extended L1  or recessed L1 (PDB code 3Q05) respectively. The extended L1 dataset has 3113 MD conformers, whereas the recessed L1 dataset has 357 MD conformers. The flexibility of L1, L2, the S5-S6 loop and the S6-S7 loop is higher in MD conformers with extended L1 than in those with recessed L1 . In termTo further understand the distinct dynamics between extended and recessed L1 conformers, we analyzed their correlated motions by calculating the dynamic cross-correlation matrix (DCCM) . DCCM haIn both extended and recessed L1 conformers, the following regions have strong correlated motions: between S1 and S3, S2’ and the S10-H2 loop, S3 and S8, H1 and L3, S5 and S8 regions . The corThe absence of the anti-correlated motion between L1 and L2 in the recessed L1 states may arise from the increasing separation between L1 and H2 , where HNext, we compare hydrogen bonds present in p53 DBD MD conformers with extended versus recessed L1. In the recessed state, L1 forms an average of 8 hydrogen bonds, whereas extended L1 forms an average of 5 hydrogen bonds. The total number of hydrogen bonds appears to be similar for both p53 DBD with extended and recessed L1 , with a 2) is larger than with recessed L1 (490.6 Å2) as calculated using NACCESS [Given these differences, we ask why in a single complex of DNA and p53 DBD tetramer such as in PDB code 3Q05  and 3TS8 NACCESS . The benL1 is important for binding to DNA, turning on trans-activation and apoptosis based on results from yeast-based assays ,78, cellin vitro [L1 (residues F113-T123) is a mutational cold spot , as oppoin vitro . S121F min vitro . T123A min vitro . It seemin vitro . In wildin vitro . The subin vitro , and thein vitro . Such a in vitro , dihydroin vitro , and actin vitro . Why is it that the wild-type p53 DBD has flexible L1 that binds DNA well but not as well as the S121F and T123A mutants do? A recent work based on the transcription factor Rap1  suggestsThere exists a complex relationship between protein flexibility and stability ,57,86,87per se, (ii) analyzes the differences in dynamical behaviors of recessed and extended L1 conformational states, and proposes their distinct involvements in binding to DNA, and (iii) highlights how the dynamics of L1 provides a promising avenue for therapeutic interventions through long-range  modulations. Such efforts have been given an optimistic boost recently by the computer aided discovery of a small molecule that re-stabilizes a mutant form of p53 [In conclusion, through multiple copies of MD simulations of the p53 DBD, this study (i) shows that the structure of p53 DBD is substantially more dynamic than what has been apparent from crystal structures m of p53 . We retrieved homologous structures to p53 DBD (PDB code 2OCJ) from the RCSB PDB and performed bioinformatics-based principal component analysis (PCA) using the Bio3D package of R . To identify core positions, iterated rounds of structural superposition were used to identify the most structurally invariant region of the p53 DBD structure. This procedure, implemented in the Bio3D package, entailed excluding those residues with the largest positional differences, before each round of superposition, until only the invariant core residues remained. We then used the structurally invariant core as the reference frame for structural alignment of crystal, NMR and simulation conformers (structures). The simulation conformers were obtained from performing molecular dynamics (MD) simulations.Next, we used the Cartesian coordinates of aligned Cα atoms to define the elements of a covariance matrix. The covariance matrix was then diagonalized to derive principal components (PCs) with their associated variances. PCA enables the projection of crystal, NMR and MD conformers on the subspace defined by PCs with the largest variances. Such an approach has been successfully employed to study the conformational diversity in the Ras ,90 and HSystems for molecular dynamics (MD) simulations were prepared from high-resolution crystal structures and a model of NMR structure of wild-type p53 DBD. In the original crystal structures, the p53 DBD structure is DNA-bound in the PDB code 2AHI  and 3Q052+ ion, from each entry to perform dual copies MD simulations for each starting structure, by varying the initial velocities. Therefore, we have eight independently simulated systems (and hence trajectories of 100 ns each) to enhance the sampled conformational space. The total simulation time for the combined trajectories is 0.8 microseconds.We extracted the monomeric p53 DBD structure, together with the Zn2+ ion, neutralizing the systems with charge neutralizing counter ions at pH 7, and adding TIP3P water molecules [Each system was simulated using the ff99SB force field  in the Aolecules  with theolecules . The sysolecules . Energy minimization with decreasing constraints on the positions of heavy atoms was initially performed for 1000 steps. Constant volume heating (to 300 K) was carried out for 50 ps. Next, equilibration at constant temperature (300 K) and constant pressure (1 atm) was performed for 2 ns.-1 and the pressure was maintained using weak-coupling with a pressure relaxation time of 1 ps. The SHAKE algorithm [The NPT  ensemble at 300 K, 1 atm, and long-range non-bonded interactions with a 10 Å atom-based cutoff were used for the production simulations. The temperature was maintained using Langevin dynamics with a clgorithm  was usedptraj module of AMBER, and the Bio3D package of R [solv,np = 0.00542*SASA +0.92 [Trajectory analyses were performed using the age of R . The timage of R ,101 apprage of R  with ΔGsSA +0.92 .We performed coarse-grained AD-ENM normal mode analysis , that usFigure S1The residual contribution of PC 1 to PC 3 reflects the dynamics of individual residues. (Left panel) Secondary structures of alpha helices and beta sheets are represented as black and grey boxes on horizontal axes. (Right panel) For each PC, equidistant atomic displacements from the mean structure are mapped onto the structure of p53 DNA binding domain. The N- and C-terminal of p53 DBD are labeled in cyan and magenta, respectively. The important loops in the p53 DBD: loop 1, loop 2 and loop 3 are colored blue, green and red, respectively.(TIFF)Click here for additional data file.Figure S2A) surface area (B) volume, and (C) radius of gyration.The relationships between PC 1 and Click here for additional data file.Figure S3The Cα RMSD with respect to the first conformer as a function of simulation time of wild-type p53 DBD started from the crystal structures of 2OCJ, 2AHI, 3Q05, 2FEJ. Different copies indicate different initial velocities in the beginning of simulation in order to allow enhanced conformational sampling. Black lines indicate all Cα atoms, grey lines indicate the Cα atoms of core residues. (TIFF)Click here for additional data file.Figures S4Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figures S5Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figures S6Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figures S7Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figures S8Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figures S9Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figures S10Projection of MD conformers (diamonds) onto the first three PCs defined by the experimentally-determined crystal (yellow circles) and NMR (magenta circles) structures see . The MD conformers are color mapped based on simulation time from 0 to 100 ns. The PDB code of starting structure used for each MD simulation is indicated on upper left.(TIFF)Click here for additional data file.Figure S11The contacts between K120 of DNA-free DBD crystal structure and residues of 3 different crystal symmetry mates (in each panel).(TIFF)Click here for additional data file.Figure S12Comparison of energetic profiles of p53 DNA binding domain with extended and recessed loop 1. The energetic distribution of (A) solvation energy (B) total molecular mechanics energy of p53 DBDs with extended and recessed L1. (C) The average and standard deviations of the energetic components. ELE is the non-bonded electrostatic energy. VDW is the non-bonded van der Waals energy. INT is the sum of bond, angle and dihedral energies. GAS is the sum of ELE, VDW and INT, i.e. the molecular mechanical energy in vacuum. GBSUR is the hydrophobic contribution to solvation free energy for generalized Born (GB) calculation.(TIFF)Click here for additional data file.Figure S13None of NMR conformers (PDB code 2FEJ) adopts recessed L1 conformation, their L1 is colored in cyan. For clarity, only L1 of NMR conformers is shown. Recessed L1 of PDB code 3Q05 chain A is colored blue. L2 and L3 are colored green and red, respectively.(TIFF)Click here for additional data file.Figure S14The time-dependent secondary structure profiles of MD conformers, with α helices and β sheets shown in black and grey respectively, except that the transient helix present in L1 is shown in red. The PDB code of starting structure used for each MD simulation is indicated on the top of each panel.(TIFF)Click here for additional data file.Figure S15The RMSDs of L1 residues in MD conformers with respect to the L1 residues in the crystal structures with extended L1  and recessed L1 (PDB code 3Q05 in black). The L1 RMSDs with respect to the L1 residues in the NMR structure (PDB code 2FEJ) are plotted in red. TS: Transitioning state between extended and recessed L1.(TIFF)Click here for additional data file.Figure S16Hydrogen bonds in p53 DNA binding domain. (A) The total number of hydrogen bonds in the p53 DBD MD conformers with extended L1 (magenta +) and recessed L1 (cyan x). (B) Stable hydrogen bonds that are present in MD conformers with extended L1 but are lost in MD conformers with recessed L1. (C) The mapping of residues involved in stable hydrogen bonds onto the crystal structure with extended L1 (PDB code 2OCJ). The secondary structure coloring is the same as in Figure 1C.(TIFF)Click here for additional data file.Figure S17Visualization of dominant motions (gray) obtained from NMA of p53 DBD with extended (green) and recessed (cyan) loop 1.(TIF)Click here for additional data file.File S1(DOC)Click here for additional data file.Table S1(XLS)Click here for additional data file.


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