Transcriptional interference has been recently recognized as an unexpectedly complex and mostly negative regulation of genes. Despite a relatively few studies that emerged in recent years, it has been demonstrated that a readthrough transcription derived from one gene can influence the transcription of another overlapping or nested gene. However, the molecular effects resulting from this interaction are largely unknown.in silico chromosome walking, we searched for prematurely terminated transcripts bearing signatures of intron retention or exonization of intronic sequence at their 3′ ends upstream to human L1 retrotransposons, protein-coding and noncoding nested genes. We demonstrate that transcriptional interference induced by intronic L1s (or other repeated DNAs) and nested genes could be characterized by intron retention, forced exonization and cryptic polyadenylation. These molecular effects were revealed from the analysis of endogenous transcripts derived from different cell lines and tissues and confirmed by the expression of three minigenes in cell culture. While intron retention and exonization were comparably observed in introns upstream to L1s, forced exonization was preferentially detected in nested genes. Transcriptional interference induced by L1 or nested genes was dependent on the presence or absence of cryptic splice sites, affected the inclusion or exclusion of the upstream exon and the use of cryptic polyadenylation signals.Using Our results suggest that transcriptional interference induced by intronic L1s and nested genes could influence the transcription of the large number of genes in normal as well as in tumor tissues. Therefore, this type of interference could have a major impact on the regulation of the host gene expression. Genes in mammalian chromosomes are distributed between gene-poor and gene-rich territories. While individual genes in gene-poor regions can be regulated independently without interference from others, genes in gene-rich regions can be regulated via complex network of positive and negative interactions. Gene-gene interactions, occurring at transcriptional level, have been recently recognized as a widespread and unexpectedly complex process, termed transcriptional interference (TI) One interesting example, a gene within a gene, or so called nested gene, has been in the focus in several recent studies  insertion into an intron of the host gene. Human genome contains 80–100 retrotranspositionally competent L1s Transcriptional activity of human L1 is determined by convergently arranged sense (SP) and antisense (ASP) promoters, located in its 5′ untranslated region (5′ UTR) in positions +1 to +101 and +400 to +600, respectively Here we studied the effect of L1 SP- and ASP-induced TI on human genes. We also extended our studies to protein-coding and noncoding (nc) nested genes, i.e. a category of genes, for which only a limited information about transcriptional regulation is available. Our results show that intron retention, forced exonization of intronic sequence and cryptic polyadenylation at the 3′ ends of prematurely terminated transcripts are the major effects induced by intronic L1s or nested genes. These effects were deduced from aberrant endogenous transcripts expressed in different human cell lines and tissues and confirmed in minigene transfection experiments.in silico chromosome walking with UCSC Genome Browser  A and B. The term intron retention, used here, refers to the unspliced intron, which is retained in the 3′ end of spliced exons most likely due to the premature termination of Pol II possibly induced by L1. Similarly, a different molecular effect, forced exonization of the intronic region upstream to L1, describes TI effect, which may be caused by either transcriptional activity of L1 or TFs bound to L1. Exonization depends on the presence of upstream cryptic acceptor splice site.Using Table S1). Of the L1s analyzed, intron retention was observed in 22 genes (45 %) and forced exonization in 19 genes (39 %). Both, intron retention and forced exonization were observed in 8 genes (16 %). Cryptic polyadenylation was detected in 13 genes (27 %). Of these genes, 5 showed intron retention, 3 exonization and 5 had both effects.Our genome-wide search revealed 50 intronic L1s in 49 genes which had a tandem arrangement with respect to the host gene transcription and possibly caused intron retention, forced exonization and cryptic polyadenylation in the upstream region (Table S2). None of this type of ESTs were found in 17 genes (35 %). However, for the remaining 31 genes (63 %) we detected additional potential TI effects in a single intron of 15 genes and in two or more introns in 16 genes. For these 31 genes intron retention was observed in 10 genes (32.5 %), exonization in 11 genes (35 %) and both effects in 10 genes (32.5 %). The occurrence of aberrant ESTs in these cases may be explained by the presence of either repeated DNA  or a putative nested gene, which could influence the transcription across the intron . Indeed, their direct influence, i. e. intron retention, exonization, termination or polyadenylation within the repeated DNA or nested gene was detected in 18 genes (58 %). Of 49 genes, only in one case (KIAA0586) we were unable to explain the intron retention in mRNA AK302718 (marked with ? in Table S2). From these data we conclude that in addition to L1, other repeated DNAs as well as nested genes, located in introns of host genes, could possibly cause TI effects in these genes.In order to determine if the L1s analyzed are the only potential inducers of TI, we searched for additional aberrant expressed sequence tags (ESTs) in our dataset of 49 genes  and selected putative single exon nested genes, for which at least one TI-predicting EST was found. Representative examples of one protein-coding and nc nested genes are shown in  C and D. Our search revealed 104 nested genes, of which 94 (90 %) were potential nc genes and 10 (10 %) were protein-coding (Table S3). Of these protein-coding genes, 4 were detected earlier (Table S1) also revealed that in the case of nested genes exonization was more prevalent (about 2-fold) than in the case of intronic L1s (see above).Despite the fact that biological function of the vast majority of nc RNA transcripts, an abundant class of RNAs (over 55 00)  earlier Table 1.Table S4). Of the 74 host genes analyzed, 26 (35 %) showed no additional aberrant ESTs demonstrating that in these cases the nested gene was the only potential TI inducer. The next large group of 43 host genes (58 %) showed variable number of aberrant ESTs in different introns; intron retention in 15 genes, exonization in 14 and both effects in 16 host genes. Their presence in these introns may be explained by the TI effect induced by repeated DNA  similar to that described above for L1-containing genes. Consistent with this explanation, their direct influence, i. e. intron retention, exonization, premature termination or polyadenylation within the repeated DNA was detected in 17 cases (40 %). Finally, we also detected 5 host genes , which expression gave rise to aberrant ESTs (exonization in 1 case and intron retention in 4 cases) in other introns (marked with ? in Table S4). Their presence in these few cases remained unknown. Taken together, these data show that in addition to intronic nested genes, pseudogenes or repeated DNAs can potentially induce the major TI effects compared to minor effects (43 vs 5), caused by other unknown factors .To test whether the potential TI effect in host gene expression is specific only to the intron, wherein the nested gene is located, we searched for aberrant ESTs over the entire length of each host gene (Table S3). For these genes, we tested the presence of normal transcripts for host and nested and aberrant transcripts for host genes in various human tissues. Widespread transcription of host and nested genes was detected in all tissues analyzed, which in most cases matched the potential TI effect induced by nested gene  were randomly chosen  is located in chromosome 17q24.2, consists of 39 exons and encodes a transporter protein which transports different molecules through extra- and intracellular membranes ABCA9, a full-length L1 PA3 is located in intron 25 and it was previously shown that this L1 has a functional ASP ABCA9 exon 23  is located in chromosome 11q23.1, consists of 19 exons and encodes a protein involved in neural cell adhesion NCAM1 (Table S1) and may interfere with its transcription by causing intron retention and cryptic polyadenylation about 2 kb upstream to L1 (TXNDC12 (thioredoxin domain containing 12) is located in chromosome 1p32.3, consists of 7 exons and encodes a thioredoxin domain-containing protein involved in redox regulation and defense against oxidative stress TXNDC12 contains a tandemly oriented single exon gene (KTI12), which encodes a chromatin associated protein of unknown function  , cf. lanes 1 and 5–7). Consistent with this result, a small (ΔASP292) or a large (ΔASP613) deletion encompassing L1 ASP region , lanes 1 and 3). This transcript was absent in negative control  and in ΔSV40 experiments (lanes 5–6), suggesting that SP activity was dependent from the SV40 promoter activity. Finally, SV40 spliced transcripts of the expected size (319 bp) with retained intron 23 were detected for all constructs, except for ΔSV40 . Since this method is based on solution hybridization and allows quantitative and simultaneous detection of different (un)spliced transcripts, contributions of each promoter or transcription unit can be determined.  cf. lanes 3 and 6–8, and 13 and 16–18). We also tested potential TI effect of L1 PA3 derived from the ABCA9 intron 25 and compared it to the #11AS in ABCA Fl and retrotranspositionally active L1 RP  cf lanes 3, 9 and 10 or 13, 19 and 20). Full-length L1 PA3 and RP reduced significantly SV40 transcription, suggesting that these elements are rather difficult to transcribe ( lanes 2–4 and 9–11). Full-length L1 RP and ORFs (to a lesser extent) in sense orientation had much greater effect in TI than antisense orientation (lanes 2–3 and 5–6), consistent with previous studies Previous studies have suggested that SOX family TFs can modulate L1 SP activity  and et al. et al. The next series of results were obtained from probing transcripts, derived from the expression of transfected ABCA minigenes, with ASP and SP probes  and 5A. ThiBecause some of the SV40 transcripts contained cryptic exon  increased exon 9 skipping about 5-fold , indicating that these regions are required for efficient SV40 transcription.Using the third minigene, we investigated how a single exon nested gene KTI12 were determined. These transcripts and less abundant endogenous transcripts of the expected size (505 nt), encompassing KTI12 positions 595–1099, were detected for all four constructs . However, deletion in a putative promoter region of KTI12 showed transcription level comparable to that of TE, suggesting endogenous transcription (cf. lanes 8 and 10). In addition, KTI12 transcription was detected for ΔSV40, indicating that KTI12 activity was independent from SV40 promoter activity (data not shown). Consistent with our bioinformatic study, a 554 nt-long SV40 transcript containing exons 1, 2 and a cryptic exon (Ex) generated by using an acceptor splice site 251 nt upstream to the most 5′ transcription start site of KTI12 was detected (lane 14). Two additional transcripts, a 389 nt transcript contained alternative acceptor splice site (86 nt upstream to KTI12) and a 909 nt transcript, derived from the readthrough of the intron between exon 2 and KTI12 were also detected. Therefore, these results suggest that KTI12 decreases the efficiency of SV40 transcription by forcing exonization using alternative cryptic acceptor splice sites located upstream to KTI12.In the following series of RT-PCR experiments transcripts derived from KTI12  increased SV40 transcription about 5-fold, suggesting that KTI12 interferes strongly with the elongating Pol II (1 (cf. lanes 3 and 4) suggesting that KTI12 interferes with intron retention and forces exonization in its upstream region. Deletion of KTI12  eliminated these effects, thus further supporting this conclusion. Consistent with RT-PCR results described above  ranging from low level or endogenous transcription for ΔP to high level for Δ395. Deletions made in the initiation region (Δ115 and Δ395) did not abolish the KTI12 transcription (lanes 14 and 15) suggesting the redundancy in transcriptional initiation. Heterogeneous transcription initiation from about 300 nt interval was also supported by ESTs (data not shown).To further investigate the TI effects observed in RT-PCR experiments, we next used RPA. The following results were obtained from probing transcripts expressed from transfected minigenes using Fl and KTI riboprobes. Deletion of KTI12 promoter region (ΔP) would increase SV40 transcription. However, no such increase was observed. Instead, about 2-fold decrease in transcription was detected, while the amount of intron-retained and exonized transcripts remained unchanged . The ratio of Fl transcripts to intron-containing transcripts was comparable in Fl, Δ115 and Δ395 , but it was significantly increased in ΔKTI2, (cf. lanes 9 and 12) suggesting that KTI12 contributed to the intron retention. The high level of intron retention in ΔKTI1 (lane 10) most likely reflected transcriptional readthrough.To further analyze the essence of non-Fl transcripts shown in KTI12 promoter and initiation region to TI was difficult to interpret, our data support the conclusion that KTI12 interferes strongly with SV40 transcription by forcing exonization and causing intron retention in its upstream region. All these data suggest that, similarly to L1, a tandemly located nested gene interferes with the transcription and splicing of the host gene.In summary, despite the fact that in some deletion experiments the contribution of In this paper, we show that TI induced by L1s and nested genes may be characterized by three different effects: intron retention, forced exonization and cryptic polyadenylation Figure 8et al. In ABCA minigene, the presence of cryptic acceptor splice site upstream to L1 5′ UTR determined the efficiency of L1-induced TI, because its deletion increased intron retention about 5-fold. Therefore, the occurrence of cryptic acceptor splice sites is somehow beneficial for SV40 Pol II to guarantee processive transcription coupled with splicing across exons 23, ExSP and 3 . However, this number is clearly an underestimate and does not consider inter-individual variation and expression differences in different cell types or tissues  and nc genes was exonization (Table S3). And 16 (16 %) nested genes showed TI ESTs in both types of tissues. Similar preferential expression in normal tissues was observed for L1 (Table S1). We speculate that about 3-fold preferential expression in normal tissues indicates that TI between nested genes may be part of their normal regulation, although the possibility of misregulation in some cases cannot be excluded. Our results also suggest that TI between nested genes may be widespread and frequently restricted to different human tissues, although we cannot exclude that for some genes tissue-specific patterns (depending on the TF binding or promoter activity) may be observed.Differently from the intronic L1s, which showed comparable intron retention and exonization effects, the main effect determined for nested protein-coding . Interestingly, Zhang et al.In this study, several lines of evidence suggest that L1 or nested gene is able to cause TI in normal gene transcription. Not only ESTs, showing unequal distribution in introns of host genes, but also the orientation bias (tandem orientation being twice less frequent than convergent orientation) Although, in our experiments, we were unable to distinguish between the DNA binding of TFs and the act of transcription as a cause of TI effect in nested genes, it seems likely that DNA-bound TFs may play a role in reducing the speed of transcription elongation. In support of this kinetic model L1 retrotransposons have been shown to influence the expression of host genes by variety of effects, including insertional mutagenesis, mobilization and transcriptional regulation of cellular genes We have characterized TI between host genes and intronic L1 retrotransposons as well as between the nested genes. Based on our bioinformatic study, we first predicted the existence of TI between tandemly arranged host genes and intronic L1 retrotransposons and nested genes, and thereafter proved it experimentally using the expression of three different minigenes in cell culture. Analysis of endogenous transcripts derived from different human cell lines and tissues also supported TI between protein-coding and nc nested genes. Our results suggest that TI induced by intronic L1s and nested genes may be widespread and could influence the expression of many genes. Further analysis of TI effects between nested genes should reveal their role in the expression of a large number of host genes in normal as well as in tumor tissues. Also, it would be interesting to determine whether other retroelements  could exert similar TI effects as L1 retrotransposons and whether their effects may be due to DNA-bound TFs not just transcriptional activity or the mere presence of splicing and polyadenylation signals.In silico chromosome walking with 100 kb steps was carried out on the human genome with UCSC Genome Browser ABCA9 fragment containing exon 23 (108 bp) and its 5′ and 3′ flanking regions  was amplified from genomic DNA derived from NTera2D1 cell line using PCR with primers 22 int Dir and 23 int Rev. Similarly, NCAM1 genomic region was amplified in three overlapping fragments. First, a 1733 bp fragment A containing exon 9 (30 bp) and its flanking regions was amplified with primers 8 int Dir and 9 int Rev 2. Second, a 1908 bp fragment B containing intron 9 was amplified with primers 9 int Dir 2 and 9 int Rev 1. Third, a 848 bp fragment C containing intron 9 and L1 5′ UTR was amplified with primers L1 Dir and L1 Rev. Fragments A, B and C were used to restore a 3637 bp NCAM1 genomic region containing L1 5′ UTR (see Minigene constructs). TXNDC12-KTI12 genomic region was amplified in two fragments. A 226 bp TXNDC12 fragment containing exon 2 (61 bp) and its 5′ and 3′ flanking intronic sequences  was amplified with primers TX ex Dir and TX ex Rev. A 1896 bp KTI12 and its 5′ and 3′ flanking sequences  was amplified with primers KTI Dir 2 and KTI Rev. PCR was carried out with recombinant Taq DNA polymerase (Fermentas) according to manufacturer's protocol. For cloning, genomic fragments were treated with Klenow polymerase (Fermentas) and purified by TAE agarose gel electrophoresis using InvisorbSpin kit (Invitek). All primers used in this study were purchased from TAG Copenhagen A/S and their sequences are listed in Table S5.A 1036 bp ABCA9, NCAM1  and TXNDC12 gel-purified genomic fragments were first cloned into pBlueScript (pBS) SK+ vector (Stratagene) digested with EcoRV. KTI12 genomic fragment was cloned into pBS KS+ vector (Stratagene) digested with SmaI. Positive recombinant plasmids were isolated with alkaline lysis minipreparation method Ecl136II and HindIII, treated with Klenow polymerase and cloned downstream to the ABCA9 insert in pBS SK+ vector blunted at ClaI site. From this plasmid, ABCA9-L1 5′ UTR fragment was separated after digestion with SalI, then treated with Klenow polymerase and digested with EcoRI. The obtained fragment was further cloned into exon trapping vector pSPL3 (Invitrogen), from which a 1 kb intron fragment containing cryptic splice site at position 1134 bp was removed with NheI, after blunting the ends with Klenow polymerase and digestion with EcoRI, thus leaving appropriate sites (blunt and EcoRI) for its insertion. The final plasmid was termed ABCA Fl (7.2 kb) and contained SV40 promoter-ABCA9-L1 5′ UTR-SV40 polyA signal , named ΔASP292 and ΔASP613, were made with BspT1 and NheI, respectively. ΔYY1 (deletion +13 to +21 in L1 5′ UTR) was generated by cloning a 132 bp fragment, starting with YY1 primer sequence and ending with PauI site (obtained from PCR with primers YY1 and L1 ASP10) into ΔASP292 or ABCA Fl construct between blunted XhoI and PauI site. A 96 bp deletion (positions +1 to +96) in L1 SP was made with XhoI and KpnI and the construct was named ΔSP. Double deletions were made consecutively. A 2 bp deletion (ag) in the acceptor splice site (ΔSA) in intron 23 in ctttccagATGGTGCTAAA was made in the ABCA Fl with PCR mutagenesis. First, PCR was carried out with two primer pairs: 23 ex Dir – SA mut2, and SA mut1 – L1 SF1. The obtained two fragments were combined and amplified with primers 23 ex Dir and L1 SF1. Then, the PCR product was digested with Acc65I and inserted into ABCA Fl from which the same, but nonmutated fragment (740 bp) was removed. Sox mutations in the ABCA Fl  was removed. A 423 bp deletion in SV40 promoter (positions 68–491 nt) in ABCA Fl was made with PaeI and NcoI generating ΔSV40. Similarly, partial SV40 promoter deletion (positions 68–140 nt) was made with PaeI generating ΔSV40part.L1 5′ UTR-containing #11AS fragment (990 bp) was obtained from a recombinant pGL3 vector ABCA9 locus was amplified from genomic DNA derived from HeLa cell line using PCR with primers L1 PA3 5′ UTR Dir and L1 5′ UTR Rev. RP 5′ UTR (903 bp), RP ORFs (5121 bp) and RP 5′ UTR+ORFs fragments (6006 bp) were amplified from L1 RP clone  with L1 RP 5′ UTR Dir – L1 5′ UTR Rev, L1 RP ORF1 Dir – L1 3′ UTR Rev and L1 RP 5′ UTR Dir – L1 3′ UTR Rev primers, respectively. We were unsuccessful in amplification of the full-length L1 PA3 from the ABCA9 locus, because of high content of repeated DNAs in the 3′ flanking region. Therefore, we fused L1 5′ UTR with RP ORFs which were 98% identical to L1 PA3 ORFs. Obtained fragments were treated with Klenow polymerase and cloned into pBS SK+ vector blunted at EcoRV site with different orientation. Thereafter these fragments were digested from pBS SK+ with SalI and SmaI and subcloned downstream to the ABCA9 insert into ABCA Fl, from which #11AS 5′ UTR was removed with NheI, after blunting the ends wiht Klenow polymerase and digestion with XhoI. These constructs were named PA3 5′ UTR, RP 5′ UTR, RP ORFs +, RP ORFs –, RP 5′ UTR+ORFs +, RP 5′ UTR+ORFs – and PA3 5′ UTR+ORFs.A 960 bp L1 PA3 5′ UTR fragment from NCAM1 genomic fragments A, B and C were ligated together as follows. Fragments A and B obtained from the appropriate recombinant pBS SK+ digested with BglII and SmaI were ligated and inserted into pBS SK+ digested with EcoRV. Fragment C, obtained after digestion with HindIII, blunted with Klenow polymerase and digested with SpeI, was inserted downstream to the fragments A–B using SpeI and NotI, blunted with Klenow polymerase. Finally, a 3637 bp NCAM1 genomic fragment cloned in pBS SK+ was digested with SacII, blunted with Klenow polymerase and digested with SalI. This fragment was further cloned into exon trapping vector pSPL3 from which a 1 kb intron fragment was removed with NheI as described above. The final plasmid was termed NCAM Fl (8.7 kb) and contained SV40 promoter-NCAM1-L1 5′ UTR-SV40 polyA signal (SpeI and Acc65I. This deletion removed L1 SP (L1 5′ UTR positions +1 to +97) generating ΔSP. ΔL1 was made by removing a 3249 bp fragment (A–B) from NCAM Fl construct with SpeI and SalI. After blunting the ends, this fragment was inserted into pSPL3 vector digested with NheI and XhoI. A 611 bp deletion of SV40 promoter (positions 74–685 nt in pSPL3) was made in NCAM Fl with SalI and Mph1103I generating ΔSV40.TXNDC12 genomic fragment, containing exon 2 obtained from recombinant pBS SK+ after digestion with XhoI and SmaI, was cloned upstream to KTI12 in pBS KS+ digested with XhoI and EcoRV site in the same transcriptional orientation. From this cloning a 2674 bp TXNDC12-KTI12 fragment obtained after digestion with XhoI and XbaI was inserted into exon trapping vector pSPL3 digested with the same restriction enzymes. As described above, from this vector a 1 kb intronic fragment was removed before cloning. The final plasmid was termed TX-KTI Fl (7.7 kb) and contained: SV40 promoter-TXNDC12-KTI12-SV40 polyA signal  generating ΔP. The entire KTI12 coding sequence (1910 bp) was removed with MluI and BamHI generating ΔKTI2. A 115 bp (Δ115) and 395 bp (Δ395) deletions were made within the initiation region of KTI12 with PCR deletion mutagenesis, similarly as described above (ABCA ΔSA) using TX-KTI pBS construct and two primer pairs: T3 – KTI Δ1 as2 and KTI Δ1 s2 – KTI Rev RT in the case of Δ115 and T3 – KTI Δ2 as and KTI Δ2 s – KTI Rev RT in the case of Δ395. PCR fragments were purified from agarose gel and amplified with fusion PCR with T3 and KTI Rev RT primers. After digestion with XhoI and KpnI these fragments were gel-purified and cloned into pSPL3 vector digested with the same restriction enzymes. Deletion of SV40 promoter (positions 72–685 in pSPL3) in TX-KTI Fl was made with PaeI and SalI generating ΔSV40.A 273 bp In cloning experiments, where deletions were made with heterologous restriction enzymes, the ends were blunted with Klenow polymerase and ligated using T4 DNA ligase (Fermentas). Restriction fragments were separated and purified from TAE gel with InvisorbSpin (Invitek) kit. Structures of the final plasmid DNAs were confirmed by restriction mapping and sequencing using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's protocol. Sequences of all Fl minigene constructs are available upon request.2) in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine calf serum, penicillin (100 u/ml) and streptomycin (100 µg/ml) (Invitrogen). Cells were plated in 6-well tissue culture dishes with appropriate density 24 hour before transfection. Transient transfection was carried out with calcium phosphate 2) was used as a negative control. All transfections were made in three parallels. After 4 hours, transfection mix was replaced by fresh medium and incubation continued 24–40 hours.Human teratocarcinoma , adenocarcinoma , and mouse neuroblastoma  were purchased from LGC Standards. Human neuroblastoma (Kelly) was a gift from A. Veske. Cells were grown at 37°C in an atmosphere containing 5% carbon dioxide (CO2) and incubated with proteinase K (Fermentas) (0.2 µg/µl) at 37°C for 2 hours. After incubation, RNA was treated with phenol and precipitated with ethanol. DNA was removed from RNA samples by DNase treatment  at 37°C for 2 hours. Finally, RNA was treated with phenol, precipitated with ethanol, dissolved and kept in TE at −20°C. The amount and quality of RNA was checked by electrophoresis of one tenth of RNA sample.Total RNA was isolated from transfected or nontransfected cells using TRIzol reagent (Invitrogen), extraction with chlorophorm and precipitation with isopropanol as described by the manufacturer's protocol. The RNA pellet was washed with 70% ethanol, dissolved in PK buffer  and kept at −20°C.Cytoplasmic and nuclear RNA fractions were isolated from Kelly and NTera2D1 cell lines using the following protocol. Cells were washed with PBS, placed on ice and lysed directly on dish with 1 ml of NP-40 lysis buffer containing 0.5% Nonidet P-40, 3 mM MgClAGC primer for SV40 exonization and polyadenylated transcripts in ABCA Fl and NCAM Fl; 9 int Rev B primer for NCAM 1–9 transcripts; KTI Rev RT primer for KTI12 transcript; EST1 Rev for SV40 exonization transcripts in TX-KTI Fl, incubated at 65°C for 5 min and chilled on ice. RT reactions were carried out with SuperScript III RTase (Invitrogen)  in a final volume of 5 µl according to manufacturer's protocol. For specific primers and oligo dTAGC incubation temperatures were 50°C and 40°C, respectively.Reverse transcription (RT) was performed essentially as follows, 0.2–1.0 µg of total RNA was hybridized with appropriate primers: pSPL3 Rev primer for SV40 and L1 SP transcripts; 22 int Dir primer for ABCA L1 ASP transcripts; 23 int Rev primer for ABCA SV40 intronic transcripts; oligo dTTable S5) and recombinant Taq DNA polymerase (Fermentas) using 30–40 amplification cycles and a 10 µl final volume. PCR products were treated with Klenow polymerase, identified by restriction mapping, cloned into pBS SK+ vector EcoRV site and sequenced. PCR amplifications of human cDNAs derived from the multiple tissue cDNA panel provided by P. Pruunsild Figure S2). DNA-free total RNAs from human tissues were purchased from BioChain and used in experiment shown in TXNDC12 and KTI12 transcripts were detected with the following primer pairs: 2 ex Dir - 7 ex Rev, KTI Dir 1 - KTI Rev RT, 2 Ex Dir - EST1 Rev and 2 Ex Dir - EST2 Rev for nested PCR (30 cycles). The primers used for the detection of host gene, nested gene, potential TI-specific transcripts induced by nested genes and PPIA (peptidylprolyl isomerase A) are shown in Table S5.PCR was conducted using different combinations of primers (listed in 32P-labeled riboprobes as described in + plasmids linearized with different restriction enzymes. Hybridization was carried out in a 8–10 µl volume. Single-stranded RNAs were removed with RNase A (40 µg/ml) and T1 (100 u/ml) (Fermentas) at 30°C (or 40°C for polyA probes) for 1 hour. RNases were removed with proteinase K (Fermentas) (200 µg/ml) in the presence of 0.5% sodium dodecyl sulfate (SDS) at 37°C for 1 hour. After phenol treatment dsRNAs were precipitated with ethanol in the presence of carrier tRNA (3 µg) and were dissolved in gel loading buffer containing 95% formamide. Protected 32P-labelled RNA fragments were analyzed on a 5% polyacrylamide gel and were detected by Personal FX phosphoimager (BioRad). Quantitation of the bands corresponding to transcripts was carried out by comparing their intensities to the reference, i.e. two-fold serial dilutions of the probe, and if necessary, size normalization was made. All RPA experiments were reproduced using total RNAs obtained from at least two separate transfections.Equal amounts of total RNAs isolated after transfection were dissolved in FA hybridization cocktail and hybridized with different Figure S1TI effects induced by L1 5′ UTR and ORFs. Quantitative detection of various minigene transcripts by RPA. Riborobes Fl1-23-3 and Int1-23 (lanes 1 and 11) are schematically shown above each panel. (A) Different types of 5′ UTR , deletion (Δ) and mutation (M) constructs and (B) RP 5′ UTR+ORFs +/−, PA3 5′ UTR+ORFs and ORFs +/− used in transfection experiments are shown on top of each lane. Their structures are mapped to the schemes above panels. Protected transcripts (marked with arrows) are schematically shown by boxes and their sizes are given in nucleotides. Dashed lines/boxes show the remaining exon(s) not protected by the riboprobe used. In the case of RP 5′ UTR + ORFs (+/−) very faint signals were detected in the original autoradiogram. TE – transfection simulated with buffer.(TIF)Click here for additional data file.Figure S2L1-induced TI determined from NCAM1 endogenous transcripts. (A) Detection of alternatively spliced NCAM1 transcripts in different human cell lines using RT-PCR. (B) Detection of endogenous NCAM1 transcripts in neuroblastoma cell line (Kelly) by RPA. Various transcripts (structures shown on the right) were detected with three different riboprobes shown above panels. Their abundance was determined from the comparison to 2-fold serial dilution of riboprobe (bottom right) after size normalization. (C) Genomic structures of the human and mouse NCAM1 containing exons 7–1. Hatched boxes show intronic regions observed in RT-PCR. (D) Detection of NCAM1 transcripts in mouse and human neuroblastoma cell lines and brain cells. Separation of the RT-PCR products with and without exon 9 are shown at the bottom of panels. Various alternatively spliced transcripts in all panels  were detected with primers specific to exons 7 and 11 and introns 8 and 9. (E) Analysis of the NCAM1 transcripts containing exons 7–11. Endogenous transcripts (Ex-exon and Int-intron) shown on the right of panels were detected by RT-PCR. Intron-containing transcripts were further amplified by nested PCR. Note that NCAM1 primary PCR product  is also visible in some lanes.(DOC)Click here for additional data file.Figure S3TI dependence on the location of L1 (A) or nested gene (B) relative to the intron retention and exonization effects in their upstream region. Data analysis from (TIF)Click here for additional data file.Figure S4TI determined from endogenous NCAM1 transcripts derived from different human cell lines and tissues.(DOC)Click here for additional data file.Table S1Prediction of TI between human genes and intronic L1 retrotransposons.(DOC)Click here for additional data file.Table S2Distribution of additional aberrant transcripts in human genes containing intronic L1 retrotransposons.(DOC)Click here for additional data file.Table S3TI between host and nested genes.(DOC)Click here for additional data file.Table S4Search for additional aberrant transcripts in selected host genes shown in (DOC)Click here for additional data file.Table S5Primers used in RT-PCR and construction of minigenes.(XLS)Click here for additional data file.


Twintrons represent a special intronic arrangement in which introns of two different types occupy the same gene position. Consequently, alternative splicing of these introns requires two different spliceosomes competing for the same RNA molecule. So far, only two twintrons have been described in insects. Surprisingly, we discovered several such arrangements in vertebrate genomes, which are quite conserved throughout the lineages.This article was reviewed by Fyodor Kondrashow and Eugene Koonin. The length of the introns also differs ranging from 209 nt for the shortest intron to 4,226 nt for the longest one. Interestingly, the U12-type intron belongs to the AT-AC type with an unusual AA terminus at the 3′ SS. Upon splicing, it produces a splice variant, which results in the Premature Termination Codon (PTC) and consequently is subjected to nonsense-mediated mRNA decay (NMD) in both the human and mouse genomes. Although the conserved motifs of minor intron are present in several vertebrate genomes, including opossum and Xenopus, we found solid evidence of the U12-type intron splicing only in humans and mice. Interestingly, in the platypus genome splicing signals for U12-type spliceosome has been muted and cannot be recognized by a minor spliceosome any longer. This may suggest that a twintron arrangement was a mediator of U12-type intron elimination from the host gene, in agreement to our original hypothesis [One of the interesting genes harboring a twintron is c for H4 . There apothesis .HNRPLL and NCBP2. Although the protein product of HNRPLL shows slight variations in the RNA recognition motif (RRM) for major and minor intron splice variants, the changes in the protein sequence and structure are insignificant , which has RRM from the 42nd to the 112th amino acid of the U12-type splice variant (PDB ID – 3FEX) [To elucidate the role of alternative SSs in protein architecture, we scanned protein splice variants with InterProScan. Most of the protein isoforms of twintrons did not show any changes in the conserved motifs and structures, except for  – 3FEX) . When thNCBP2 is expressed mainly in brain, thymus, uterus, lungs, testis, and several other tissues, whereas the U2-type splice variant is expressed mostly in tumors and cancerous tissues  definition a twintron is any set of nested introns, belonging to the same splicing mechanism or not. This is at odds with the definition used by the authors and perhaps should be resolved. Perhaps a figure that demonstrates what a twintron looks like is called for: it would have been clearer to me what the authors mean.Our definition of twintrons differs slightly from the original one and includes both nested and shifted arrangements. Although we provide a short twintron definition in the abstract, the full one is now provided in the body of the paper and accompanied by a figure.Authors’ response: Second, the authors suggest that having such nested introns that are excised by two different spliceosomes can be an evolutionary mechanism of switching between the two intron types. However, perhaps this is at odds with the apparent conservation of such a setup - if this is a “safe pathway for intron switching” then certainly it does not appear to be a neutral one. Additionally, the mechanism that could turn an internal exon into an external one in a nested situation is not immediately clear to me.We think that either of two introns can be switched off and this might be random process. We agree that presented examples don’t provide direct evidence that such a switch occurs. However, the fact that twintron arrangement is more common phenomena than anticipated provides indirect evidence that such a mechanism could be used during gene structure evolution.Authors’ response: Is there a preference for U-2 or U-12 introns to be the external ones in the nested setup?No, there’s no bias in the two types introns arrangement. In six cases U12-type intron is the internal one, while in four cases the arrangement is reversed. The rest of twintrons display shifted arrangement.Authors’ response: This is quite an interesting short paper that reports a number of previously unnoticed twintrons and most importantly demonstrates their evolutionary conservation at considerable phylogenetic depths, with the implication of functional importance of the twintron structure itself. This is the major finding of the work, and it is certainly valuable. I am less enthusiastic of the two hypotheses proposed in the article, namely that twintrons could be an important intermediate along the path of elimination of U12 introns and that multiple protein isoforms produced by expression of twintron-containing genes might play a role in carcinogenesis. The first hypothesis, which is one of the main themes in the article, is of interest but I find the evidence quite limited. The authors might wish to expand the discussion. The idea about carcinogenesis, to me, is sheer, unwarranted speculation. The presence of additional splice variants in cancer samples might be caused by a variety of factors, above all the general deterioration of regulatory processes in tumors, and have nothing to do with carcinogenesis. I am not sure this is even worth a mention.We agree that two hypotheses are highly speculative. As suggested, we have expanded the discussion of the first hypothesis and removed the second one from the manuscript.Authors’ response: The authors declare that they have no competing interests.WM conceived and designed the project. JJ and MJ performed genomic analysis and JJ drafted the manuscript. All the authors contributed to the final version of the manuscript. All authors read and approved the final manuscript.Conservation of major and minor splice sites in vertebrates. Click here for fileHNRPLL. The structure is colored based on the secondary structure: red color for alpha-helices and yellow for beta-sheets. A black arrow indicates the variable amino acids. Superimposed 3D structures of U12 and U2-type splice variants of the gene Click here for fileNCBP2. The 3D structure is colored based on the secondary structure: red color for alpha-helices and yellow for beta-sheets. RRM domain missing in the U2 splice variant is shown in blue. Superimposed 3D structures of U12 and U2-type splice variants of the gene Click here for fileExpression data of splice variants of twintrons. Click here for file


The development of transparent radio-frequency electronics has been limited, until recently, by the lack of suitable materials. Naturally thin and transparent graphene may lead to disruptive innovations in such applications. Here, we realize optically transparent broadband absorbers operating in the millimetre wave regime achieved by stacking graphene bearing quartz substrates on a ground plate. Broadband absorption is a result of mutually coupled Fabry-Perot resonators represented by each graphene-quartz substrate. An analytical model has been developed to predict the absorption performance and the angular dependence of the absorber. Using a repeated transfer-and-etch process, multilayer graphene was processed to control its surface resistivity. Millimetre wave reflectometer measurements of the stacked graphene-quartz absorbers demonstrated excellent broadband absorption of 90% with a 28% fractional bandwidth from 125–165 GHz. Our data suggests that the absorbers' operation can also be extended to microwave and low-terahertz bands with negligible loss in performance. Graphene has attracted much attention in recent years due to its extraordinary electronic and optical properties such as high electron mobility, truly atomic thickness, mechanical flexibility and saturable absorption12349101112111416192022232526272830Radar absorbing materials (RAMs) significantly reduce an object's observable radar cross-section within specific bandwidths3332In this letter, we report for the first time on the fabrication and characterization of transparent broadband absorbers consisting of several stacked multilayer graphene sheets on quartz substrates backed with a ground plate. The large-area CVD multilayer graphene films have been fabricated through a repeated etch-and-transfer process to minimise PMMA residue between the layers, thereby yielding high-quality and optically transparent multilayer graphene films. Herein, analytical expressions are developed for the reflection and absorption coefficients consisting of an arbitrary number of stacked graphene layers. These expressions serve as a practical design guide to estimate the influence of the sheet resistance and substrate parameters, as well as the impact of oblique incidence. To validate the broadband absorption, millimetre wave reflectometer experiments have been carried out with single and stacked absorbers from 110 GHz to 170 GHz. For 5-unit stacked structures, 90% absorbance can be achieved with 28% fractional bandwidth from 125 GHz to 165 GHz. The broadband absorption can be further improved by using thinner substrates with lower relative permittivity or stacking more graphene-quartz substrates at the expense of reduced optical transparency.2/Si wafers and were found, by optical and electron microscopy to be free of pin-holes. Samples were of high uniformity with >90% monolayer coverage, as confirmed by Raman spectroscopic mapping and optical microscopy2/Si substrates and etching them in an aqueous ammonium persulfate solution before finally transferring the released PMMA/graphene onto the quartz substrates. This method avoids significant PMMA residue build-up between the stacks of graphene layers yielding reduced mean sheet resistance of ~0.9 kΩ/sq for 2 L and ~0.6 kΩ/sq for 3 L. The number of graphene layers was confirmed via UV-Vis spectro-photometery. Optical transmittances of 85%–91% at 700 nm for quartz-supported 1–4 L graphene was noted  plane wave is assumed to arrive at the absorber surface with an oblique incidence angle of θ. Since the thickness of quartz is a few millimetres, high-order modes become evanescent and can be ignored in the millimetre wave regime. The graphene sheets can be modelled as thin, two-sided surfaces characterized by surface conductivity, which is governed by the intraband contributions at low-terahertz frequencies and can be expressed asω is the radian frequency, cμ is the chemical potential, Γ is the phenomenological scattering rate, T is temperature, e is the charge of an electron, Bk is the Boltzmann's constant. Although sσ is frequency dependent in sσ is dominated by the surface conductance and can be regarded as frequency independent.To predict the absorber performance, we have derived an analytical expression based on a circuit model equivalent, as shown in dβ and characteristic admittance dY, with the resistive graphene sheets represented as a shunt admittance, sY = sσ ≈ 1/sR, where sR is the sheet resistance  graphene-quartz absorber, the input admittance is given by NYin, ≈ Y0 or S11 ≈ 0; the absorption peaks correspond to reflection zeros. In this case the incident wave will go through multiple reflections and be fully absorbed by the lossy resistive graphene sheets.The highest absorption is achieved when In order to investigate the nanostructured absorber, reflection spectra are measured by a well-established free space millimetre wave reflectometery technique and then transformed to absorption spectra according to Firstly, single (N = 1) absorbers with 1–4 L graphene were measured and then two graphene-quartz samples were stacked together to construct a 2-unit (N = 2) absorber . Measurecμ = 0 eV and Γ = 7 meV, this corresponds to a sheet resistance of 5044 Ω/sq which makes the input impedance challenging to match with free space. The peak absorption is lower than 40% indicating a poor absorption. As the chemical potential increases, in steps of cμ = 0.1 eV, the sheet resistance of graphene is reduced and the peak absorption improves. When cμ = 0.3 eV and Γ = 5 meV, the corresponding sheet resistance is 430 Ω/sq, which tends toward the free space impedance. Good impedance matching leads to 100% peak absorption around 148 GHz.The measured reflection and absorption spectra of single graphene-quartz absorbers with 1–4 L graphene on quartz are compared with analytical calculations in Graphene surface conductivity is mainly dominated by the intraband contribution from microwaves to far infrared waves. The real part of the intraband conductivity, which contributes to energy absorption or dissipation3718cμ = 0.0 eV and Γ = 7 meV  and the air gap (~0.1 mm) between quartz and ground plate. The absorption peak increase marginally (+1.2%) for the 3 L case and fall slightly (−1%) for the 4 L case. Multilayer graphene can thusly be used to derive a turbostratic, stacked, artificial graphite-like material of sufficiently reduced sheet resistance capable of near matching the free space impedance; however, it is challenging to improve the sheet resistance further for samples consisting of more than 3 layers, which could be due to water residue between the layers that prevents good contact between the interfacing layers in the present samples.First, the bare quartz substrate on the ground plate was tested in the reflectometer as a background reference. A total reflection with no absorption is observed across the whole frequency range, except for the intrinsic systematic noise at high frequencies (>160 GHz). The single unit graphene-quartz absorbers with 1–4 L graphene were then measured. A 1 L absorber has a small absorption peak around 30% at 148 GHz, which is similar to the case of  = 7 meV . In contif = (2i − 1)f0  have opposite phase difference which leads to periodic reflection zeros and absorption peaksThe single graphene-quartz absorber is based on a Salisbury screen absorber see , which epfi, = (2i − 1)f0/N and z,if = 2if0/N , respectively. As shown in Although a Salisbury screen absorber has multiple absorption bands, maximum absorption is limited to a narrow bandwidth around the resonant frequency. In order to achieve wideband absorption we instead consider a Jaumann absorber, which utilizes a series of a quarter wavelength thick dielectric layers that separate parallel resistive sheets, introducing a mutual coupling of Fabry-Perot resonators over a metallic ground. Here we use stacked graphene-quartz samples to realize a transparent millimetre wave Jaumann absorber. The zero-degree reflection phase condition can be used to predict the locations of absorption peaks and zeros . For thecμ = 0.15 eV) which corresponds to a sheet resistance of 859 Ω/sq. The calculated reflection spectra in The calculation results for stacked graphene-quartz absorbers are depicted in For a dielectric substrate, the resonance frequency will be decreased by increasing either the permittivity or thickness of the slabs. Moreover, the relative permittivity will affect the characteristic admittance of the dielectric according to 3O4 are added to graphene to result in high complex permeability values over the GHz rangeThere are two contributions to microwave absorption: dielectric and magnetic lossIn this work, in order to achieve optical transparency, we have not used multilayer graphene as a dielectric filler but as an ultra thin, lossy resistive component separated by the transparent quartz dielectric filler, backed by a ground plate, forming a Jaumann absorber. We aimed to match the impedance to free space by engineering the surface resistance characteristics of the multilayer graphene combined with the impedance transformation of the transparent dielectric filler. We are able to achieve absorption of 90% of the incident millimetre wave without necessarily taking permeability into consideration.θ < 30° do not have a significant impact on the absorption spectra. However, when θ > 60° absorption is reduced and the magnitude variations become significant for TE-polarized waves, whilst they improve for TM-polarized cases. We also observe that absorption bandwidth increases significantly when the relative permittivity is reduced from rε = 3.8 to rε = 1.1; suggesting that a dielectric with low relative permittivity is required to achieve both wide bandwidth and high absorption.Although data on oblique incidence is not presented, an analytical prediction can be obtained which we have validated in the normal incidence case . We obseBy stacking nanometre-thick multilayer graphene films and millimetre-thick quartz slabs, we have developed a facile and optically transparent broadband millimetre wave absorber. This absorber has been modelled using transmission line theory and an analytical expression has been derived to predict its performance.Each graphene-quartz structure forms a Fabry-Perot resonator. When backed with a ground plate, absorption peaks or reflection zeros occur at odd numbers of the fundamental resonant frequency. We have determined that the sheet resistance of the graphene is an important factor for determining the input impedance and free space impedance matching; here the multilayer CVD graphene films were used to achieve the required resistivity through a repeated transfer process developed to minimise PMMA residues.When multiple graphene-quartz samples are stacked together, the mutual coupling of the Fabry-Perot resonators introduces multiple absorption peaks in each band leading to broadband absorption. A 5-unit stacked graphene-quartz absorber seems adequate to counterbalance the absorption performance and the manufacturing complexity. In addition, the permittivity and thickness of the dielectric slabs provide additional degrees of freedom to adjust the bandwidth and in-band performance. Furthermore, for the case of oblique incidence, the absorber is found to be suitable for angles <30° for TE-polarized waves while good for all angles for TM-polarized waves. In order to achieve excellent absorption for wide incidence angles under TE-polarization, pyramidal or inhomogeneous structures may be used for future work.The measurements of transparent graphene-quartz absorbers have been carried out using a millimetre wave reflectometer across the 110–170 GHz band. Our measured results validate that multilayer graphene achieves reduced sheet resistance assessed by tuning the chemical potential in the calculation, and 5-unit stacked absorber achieves 90% absorbance with a 28% fractional bandwidth. Moreover, the derived analytical formulae predicted the periodic absorption spectra over a wide frequency region, and the proposed absorber can be designed to meet different requirements with the added benefit of optical transparency.2:Ar atmosphere at 4 mbar. Graphene growth was initiated by introducing 7 sccm CH4 (99.9%) under maintained H2 (99.98%) and Ar (99.998%) dilution. All samples were cooled to <300°C under ultra-high purity N2 (99.999%) before venting to atmospheric pressure. Temperatures were monitored using two type K bimetallic thermocouples and a surface infrared interferometer. Pressures and temperatures were accurate to within ±0.1 mbar and ±1°C, respectively.Graphene was grown by thermal chemical vapour deposition in a cold-walled, commercially available reactor (Aixtron Black Magic). 500 nm-thick Cu was magnetron sputtered onto 200 nm thermally oxidised Si-100 four inch wafers which were subsequently annealed at 850°C for 30 min in a 20:1500 standard cubic centimetres per minute H2/Si samples were first diced to 17 mm × 8.5 mm and then cast with 200 nm poly (methyl methacrylate)-(PMMA 950 A4) and were annealed at 180°C for 1 minute. Substrates were then immersed in an aqueous ammonium persulfate ((NH4)2S2O8, 2.2 g/100 mL DI) for 15 hours to etch the Cu catalyst. The floating PMMA-graphene films were then transferred into DI water baths using microscope glass slides and rinsed several times to remove the etchant. For monolayer graphene, the PMMA-graphene film was transferred to Saint-Gobain Spectrosil® 2200 Optical Fused Quartz substrates, and dried at atmospheric pressure for 24 hours. For monolayer samples the films were transferred to occupy only half side of the quartz substrate, leaving the other half exposed for UV-VIS spectro-photometry baseline calibration. For multilayer samples the films were transferred onto slightly larger diced wafer substrate pieces, in order to make PMMA/bilayer graphene stacks. The bilayer stacked substrates were etched again in (NH4)2S2O8 and were rinsed and transferred onto diced wafer quartz substrates, followed by acetone washes to remove the PMMA. The remaining PMMA/bilayer graphene films were transferred onto larger substrates and the process was repeated until stacked samples of up to 5 layers were processed.The graphene on Cu/SiOA HP N5244A vector network analyser and its millimetre wave extension heads were used to drive the reflectometer . The verY.H. proposed and supervised the project. W.I.M. advised on the project. B.W. carried out the absorber design and data analysis, M.T.C. synthesised the CVD graphene. H.M.T. fabricated the multilayer graphene samples and carried out sheet resistance and UV-Vis optical transmission measurements. B.Y. set up the millimetre wave measurement system. B.W., B.Y. and M.N. performed the measurements. Finally, the manuscript was prepared by B.W. and H.M.T. and revised by Y.H., M.T.C. and W.I.M.Supplementary


Yarrowia lipolytica. On the other hand, Basidiomycota and Perizomycotina have introns in their histone genes. We compared the distributions of 81, 47, 79, and 98 introns in the fungal histone H2A, H2B, H3, and H4 genes, respectively. Based on the multiple alignments of the amino acid sequences of histones, we identified 19, 13, 31, and 22 intron insertion sites in the histone H2A, H2B, H3, and H4 genes, respectively. Surprisingly only one hot spot of introns in the histone H2A gene is shared between Basidiomycota and Perizomycotina, suggesting that most of introns of Basidiomycota and Perizomycotina were acquired independently. Our findings suggest that the common ancestor of Ascomycota and Basidiomycota maybe had a few introns in the histone genes. In the course of fungal evolution, Saccharomycotina and Taphrinomycotina lost the histone introns; Basidiomycota and Perizomycotina acquired other introns independently. In addition, most of the introns have sequence similarity among introns of phylogenetically close species, strongly suggesting that horizontal intron transfer events between phylogenetically distant species have not occurred recently in the fungal histone genes.Saccharomycotina and Taphrinomycotina lack intron in their histone genes, except for an intron in one of histone H4 genes of  Eukaryotic genomic DNA is packaged with histone proteins to form chromatin Aspergillus nidulans and Neurospora crassa contain introns Aspergillus nidulans contain a poly(A) signal Interestingly, histone genes of the filamentous ascomycetes Phylogenetic tree based on histone H2B proteins shows that fungi are more closely related to animals than plants, which is consistent with the phylogeneny of organisms Schizosaccharomyces of Taphrinomycotina and Saccharomycotina) lack intron in their histone genes, except for an intron in one of the histone H4 genes of Yarrowia lipolytica  was shared with one of 2 hot spots in the histone H4 genes of Perizomycotina , and between Botryotinia fuckeliana and Sclerotinia sclerotiorum   . Those rAspergillus fumigatus lacks intron at the position 17 (hot spot)  of histone H4_2 of A. fumigatus has a sequence similarity with the 243-nt intron of N. fischeri (The histone H2A_2 gene of ot spot) . Howeverfischeri . The hision site . Howeverfischeri . In ordeLaccaria bicolor histone genes   . It sugghttp://www.ncbi.nlm.nih.gov/projects/genome/guide/fungi/) and performed the sequence-similarity search for the 102 fungal genome sequences. As the result, the distribution of most of the introns (except for some introns) is so limited among the phylogenetically closely related species (Aspergillus nidulans H4_2 intron (88-nt) has a unique insertion site ; Ashbya gossypii, Candida albicans, Debaryomyces hansenii, Kluyveromyces lactis, Pichia stipitis, Saccharomyces cerevisiae, Vanderwaltozyma polyspora, and Yarrowia lipolytica. Perizomycotina of Ascomycota (11 species); Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Botryotinia fuckeliana, Fusarium graminearum, Magnaporthe oryzae, Neosartorya fischeri, Neurospora crassa, Podospora anserine, and Sclerotinia sclerotiorum. Basidiomycota (4 species); Cryptococcus neoformans, Laccaria bicolor, Malassezia globosa, and Ustilago maydis. We extracted all core histones H2A, H2B, H3, and H4 genes from each fungal genome. The locations and the DNA lengths of introns were identified using the sequence viewer on the NCBI web site (http://www.ncbi.nlm.nih.gov/).We compared the following 24 fungal genome sequences. Taphrinomycotina of Ascomycota (1 species); Amino acid sequences of the histones were multiple-aligned using the CLUSTAL W of MEGA software http://fasta.bioch.virginia.edu/fasta_www2/fasta_list2.shtml) using 200 PRSS iterations E-value <0.02 as introns with nucleotide sequence similarity.We selected introns with nucleotide sequence similarity using the SSEARCH with Smith-Waterman similarity scores Click here for additional data file.Figure S2Alignment of fungal histone H2B proteins and the intron insertion site. The character “I” on the first row indicates the intron insertion site.(TXT)Click here for additional data file.Figure S3Alignment of fungal histone H3 proteins and the intron insertion site. The character “I” on the first row indicates the intron insertion site.(TXT)Click here for additional data file.Figure S4Alignment of fungal histone H4 proteins and the intron insertion site. The character “I” on the first row indicates the intron insertion site.(TXT)Click here for additional data file.Table S1Distribution of introns in fungal histone H2A genes.(DOCX)Click here for additional data file.Table S2Distribution of introns in fungal histone H2B genes.(DOCX)Click here for additional data file.Table S3Distribution of introns in fungal histone H3 genes.(DOCX)Click here for additional data file.Table S4Distribution of introns in fungal histone H4 genes.(DOCX)Click here for additional data file.Table S5Introns with sequence similarity. Yellow indicates the intron pair with different insertion sites in the same histone gene. Orange indicates the intron pair of different histone genes. The other indicates the intron pair with the same insertion site in the same histone gene.(DOCX)Click here for additional data file.Table S6E-value <0.02) of the Fungi Genome BLAST in the Fungal Genomes Central on the NCBI web site for each intron used in this study. The hit sequence ID with the score (bits) and E-value are shown.Results ((TXT)Click here for additional data file.


Idiopathic Pulmonary Fibrosis is a lethal fibrotic disease characterized by the unrelenting proliferation and persistence of fibroblasts in a type I collagen-rich matrix that result in an expanding reticular network of fibrotic tissue. However, the underlying mechanism responsible for the persistence of myofibroblasts in IPF remains unclear. During normal tissue repair, unwanted fibroblasts are eliminated during collagen-matrix contraction by a mechanism whereby high PTEN activity suppresses Akt. We have previously found that FoxO3a, a transcriptional activator of apoptosis-inducing proteins, is inactivated in IPF fibroblasts resulting from aberrantly high PI3K/Akt activity due to inappropriately low PTEN activity. Here we demonstrate that this low FoxO3a activity confers IPF fibroblasts with resistance to collagen-mediated apoptosis. We show that the mechanism by which low FoxO3a activity confers IPF fibroblasts with an apoptotic resistant phenotype involves suppression of Fas expression as a result of down regulation of cav-1 expression via a PTEN/Akt-dependent pathway. We demonstrate that PTEN over-expression or Akt inhibition increases FoxO3a expression in IPF fibroblasts, resulting in up-regulation of caveolin-1. We show that FoxO3a binds to the cav-1 promoter region and ectopic expression of FoxO3a transcriptionally increases cav-1 mRNA and protein expression. In turn, we show that overexpression of caveolin-1 increases Fas levels and caspase-3/7 activity and promotes IPF fibroblast apoptosis on polymerized type I collagen. We have found that the expression of caveolin-1, Fas and cleaved caspase-3 proteins in fibroblasts within the fibroblastic foci of IPF patient specimens is low. Our data indicate that the pathologically altered PTEN/Akt axis inactivates FoxO3a down-regulating cav-1 and Fas expression. This confers IPF fibroblasts with an apoptosis-resistant phenotype and may be responsible for IPF progression. Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disorder of unknown etiology The FoxO3a transcription factor controls the expression of proteins regulating both the cell cycle and cell viability. Active FoxO3a functions as a powerful inhibitor of the cell cycle and also promotes apoptosis During normal tissue repair, excess fibroblasts are eliminated by apoptosis. The mechanism involves collagen contraction-mediated activation of PTEN thereby suppressing phosphorylated Akt levels This study involves the analysis of human IPF patient specimens. Primary fibroblast lines were obtained from unused, existing pathological human tissue samples, and therefore is exempt (exemption 4). Tissue samples were stripped of all identifiers and designated as waste. All patients underwent procedures for diagnostic or therapeutic procedures. Written informed consent was obtained on all patients prior to the procedure being performed. Use of human tissues was approved by the Institutional Review Board (IRB) at the University of Minnesota.Cell lines were derived from lungs removed at the time of transplantation or death. The diagnosis of IPF was supported by history, physical examination, pulmonary function tests, and typical high resolution chest computed tomography findings of IPF. In all cases, the diagnosis of IPF was confirmed by microscopic analysis of lung tissue and demonstrated the characteristic morphological findings of usual interstitial pneumonia. All patients fulfilled the criteria for the diagnosis of IPF as established by the American Thoracic Society (ATS) and the European Respiratory Society (ERS). For this study, new primary IPF fibroblast lines were generated as tissue became available. To reduce technical variability, we routinely utilize cells between passages 5 and 7 because of concern that the phenotype of the cells is altered at higher passage. To address concerns of biological variability, we studied 8 IPF cell lines and 6 control cells lines.8 IPF primary human lung fibroblast lines were established and analyzed for this study. Six control primary adult human lung fibroblast lines were established from histologically normal lung tissue distant from carcinoid tumor or radiation-induced fibrotic lung tissue or anatomically normal lung tissue not used at the time of transplantation. Primary lung fibroblast lines were generated by explant culture and cultured in high glucose DMEM containing 10% FCS. Fibroblasts were used between passages five and seven. Cells were characterized as fibroblasts as previously described 4 of control or IPF fibroblasts were cultured on type I polymerized collagen coated 96 well plates in 100 µl of serum free medium as a function of time. 20 µl of Cell Titer Blue reagent was then added to each well and further incubated for 1 to 2 h. Cell viability was measured by a 96 well plate reader with a fluorescence filter set 560(Ex)/590(Em). For caspase-3/7 activity assay, Apo-ONE Homogeneous Caspase-3/7 assay kit was used . 3×104 of control or IPF fibroblasts in 100 µl of serum free DMEM buffer cultured on polymerized collagen on 96 well plates were incubated with 100 µl of the mixture of caspase substrate and Apo-one caspase-3/7 buffer for 12 h. The fluorescence of each well with a filter set 485(Ex)/527(Em) was used to measure caspase-3/7 activity in samples.To precisely measure viable cells on collagen matrix, Cell-Titer-Blue Cell Viability Assay  was used. Briefly, 3×10CAG CCA CCC GAG ATTT GAG CA 3′), human reverse 18S rRNA (5′-TAG TAG CGA CGG GCG GTG TG 3′), mouse forward 18S rRNA (5′-AGG GGA GAG CGG GTA AGA GA-3′), mouse reverse 18S rRNA (GGA CAG GAC TAG GCG GAA CA-3′), human forward cav-1 (5′- TGA CTG AGA AGC AAG TGT ATG ACG-3′), human reverse cav-1 (5′- GCA GAA GGT ATG GAC GTA GAT-3′), mouse forward cav-1 (5′-TGA CTG AGA AGC AAG TGT ATG ACG-3′), mouse reverse cav-1 (5′-GCA GAA GGT ATG GAC GTA GAT-3′) were prepared from the Biomedical Genomic Center at the University of Minnesota. Quantitative PCR was performed using a Light Cycler FastStart DNA Masterplus SYBR Green I Kit. Samples were quantified at the log-linear portion of the curve using LightCycler analysis software and compared to an external calibration standard carve. Cav-1 levels were normalized to 18S rRNA. Quantitative PCR parameters were as follows. Mouse 18SrRNA, 60°C annealing, 10 second extension for 40 cycles; mouse cav-1, 56°C annealing, 15 second extension for 40 cycles; human 18SrRNA, 62°C annealing, 11 second extension for 40 cycles; human cav-1, 56°C annealing, 15 second extension for 40 cycles. For the analysis of putative FoxO3a binding site on the cav-1 promoter region, MATCH program (version 1.0) was used. This software is designed to search potential binding sites for transcription factor nucleotide sequences using the library of mononucleotide weight matrices from TRANSFACT 6.0. The search is performed using 0.75 and 0.70 as cutoffs for core and matrix similarity, respectively using MATCH program. For the confirmation of FoxO3a binding to the cav-1 promoter, 2×105 IPF fibroblasts were infected with 1×106 PFU of adenovirus expressing wild type FoxO3a and agarose ChiP kit from Pierce was used according to manufacturer’s protocol. After crosslinking and micrococcal nuclease digestion steps, FoxO3a bound complex was immunoprecipitated with or without FoxO3a antibody (2 µg). Rabbit IgG was also used as a negative control. After DNA was recovered, C1 forward primer containing putative FoxO3a binding sites , 5′-CTC CAC CCC TGC TGA GAT GAT-3′ and C1 reverse primer 5′-AGT GAG AAC GTT GTC CCG CGC TGG-3′ were used for ChIP-PCR. PCR was carried out with 2 µl of recovered DNA using 0.25 µM of C1 primers as follows. 94°C for 7 min for 1 cycle, then 94°C for 30s, 55°C for 40 s and 72°C for 40 s for 35 cycles and 72°C for 1 cycle for 7 min. Amplified products were analyzed using 2% agarose gel. For the analysis of FoxO3a dependent transcriptional regulation, a luciferase reporter construct that contains Forkhead consensus binding sites (FHRE-Luc) were obtained from Addgene, MA. 2×105 IPF and control fibroblasts expressing wild type FoxO3a, dominant negative FoxO3a or empty vector were transfected with 2 µg of pGL3-FHRE-Luc plasmid using FuGene HD transfection reagent . Cells were then cultured on polymerized collagen in serum free medium for 24 h and lysed in 250 µl of lysis buffer. The luciferase activity of one fifth of the samples were assayed using Bright-Glo™ Luciferase Assay system  according to manufacturer’s protocol using Lumat LB 9507 luminometer.For Western analysis, PTEN, p-Akt (s473), cav-1, Fas antibodies were obtained from Cell Signaling Technologies, MA. GAPDH and actin antibodies and FoxO3a antibody for immunoprecipitation for ChIP assay were purchased from Santa Cruz biotechnologies. FoxO3a and inactive FoxO3a (S253 phosphorylated FoxO3a) antibodies for Western analysis were obtained from Millipore, MA and Cell Signaling Technologies, MA. Anti-human Fas activating clone CH11 was also obtained from Millipore. Immunohistochemical analysis of myofibroblasts within the fibroblastic foci of IPF patient specimens was performed using an α smooth muscle actin antibody . Cav-1, Fas siRNA and non-coding scrambled control siRNA were purchased from Life Technologies and Santa Cruz Biotechnologies, respectively using X-treme GENE siRNA transfection reagent (Roche Applied Science) according to manufacturer’s protocol. At 24 h post transfection, fibroblasts were cultured on polymerized collagen in serum free medium and assay was performed as described. For quantitative RT-PCR assay, total RNA was extracted from cells using Trizol  according to manufacturer’s protocol. cDNA was prepared using the Taqman Reverse Trascription kit (Life Technologies) with random hexamer primers and 1 µg of total RNA. Human forward 18S rRNA  and adenovirus titer was measured by plaque method in 0.5% soft agar. The cells were infected with adenoviral vectors at a multiplicity of infection of 1∶20. For the transient transfection assay, plasmids expressing wild type (WT), mutant (S253A) or triple mutant FoxO3a (TM) which three phosphorylation residues recognized by Akt were mutated to alanine were obtained from Addgene. Adenovirus expressing HA-tagged Akt with c-src myristolyation sequence fused in frame to the N terminus (Hyper active Akt), HA-tagged Akt dominant mutant  and wild type FoxO3a were purchased from Vector Biolabs . Adenovirus expressing GFP tagged wild type FoxO3a, dominant negative FoxO3a with the deletion of the transactivation domain from the c-terminus and empty vector were also purchased from Vector BioLabs. Control or IPF fibroblasts were infected with adenovirus expressing PTEN, Akt or empty vector in 2 ml of tissue culture dishes. 1 to 2×105 control or IPF fibroblasts per ml of DMEM medium were infected with 1×106 PFU of each adenovirus for assay.For PTEN adenovirus construction, wild type (WT) PTEN cDNA was generated from normal human lung fibroblasts (HLF-210) by RT-PCR. The primers used for wild type PTEN, which span the entire coding region of PTEN are Patient specimens were fixed overnight with paraformaldehyde (4%) at RT. The specimens were rinsed in 6% sucrose/0.1 M Dulbeccos PBS (DPBS) at RT, infiltrated with increasing concentrations of Sucrose/DPBS with rotation at 4°C over a 72 h period, and cryoprotected first with 20% sucrose/DPBS overnight, then in a 1∶1 solution of 20% sucrose in DPBS/O.C.T , and rotated overnight at 4°C. After cryoembedding in O.C.T., 4 µ sections were cut and placed on silane-coated slides using a Leica 1950 Cryostat. Cryosections were fixed in 100% methanol at −20°C for 10 minutes, air dried and then quenched with 0.3% Hydrogen peroxide in methanol for 15 minutes at RT, and again air dried. They were then rinsed in PBS, placed in a steamer for 10 min with Citrate Buffer for antigen retrieval, and blocked for 30 min with normal serum determined by the host species of the antibody, to block nonspecific binding of secondary antibodies. Endogenous Avidin and Biotin binding sites were blocked by sequential incubation with Avidin/Biotin Blocking Kit . The sections were then incubated overnight at 4°C in their respected concentrations for each antibody, α- smooth muscle actin (1∶100) (Vector Labs), p-FoxO3A (1∶150) , cleaved caspase-3 (1∶100) and cav-1(1∶1000)  and Fas (1∶100) . For detection of the primary antibody, the LSAB Method was used. The sections were first rinsed with PBS, then placed in a species specific biotinylated secondary antibody for 30 min (1∶500), and transferred to the HRP-Streptavidin  conjugate solution for 30 min. The enzyme was then visualized with the application of the chromagen substrate 3, 3′ Diaminobenzene and counterstained with Hematoxylin. Control specimens were processed with the identical protocol minus the primary antibody to produce the negative controls.Data are expressed as the mean ± S.D. Experiments were performed three times. Paired evaluations were made for experimental and control conditions within each experiment, and significance was determined by Student's t test. Significance level was set at p<0.05.We have previously found that normal fibroblasts undergo apoptosis during their interaction with and contraction of polymerized collagen matrices Active FoxO3a can promote apoptosis. Since FoxO3a function is abnormally low in IPF fibroblasts, we next examined whether up-regulation of FoxO3a expression in IPF fibroblasts alters their morphology and viability when they interact with polymerized collagen. When FoxO3a was overexpressed in IPF fibroblasts on collagen matrix, they adopted a round appearance and apoptotic cells were readily apparent whereas IPF fibroblasts treated with GFP empty vector maintained their fibroblast morphology . The perRecent studies have demonstrated that caveolin-1 (cav-1) regulates the Fas-mediated apoptotic pathway Prior studies indicate that the cav-1 promoter contains putative FoxO consensus binding sites (5′-GTAAA(C/T)A-3′) To further verify whether FoxO3a is responsible for low cav-1 protein expression in FoxO3a −/− cells, we re-constituted wild type FoxO3a in FoxO3a−/− cells, and cav-1 expression was measured. Cav-1 protein level was high when wild type FoxO3a was expressed compared to that of cells expressing GFP , dominant negative FoxO3a (DF) or GFP in IPF fibroblasts cultured on polymerized collagen and measured cav-1 levels. Cav-1 protein expression increased in IPF fibroblasts in which wild type FoxO3a was over-expressed fibrolasts in the fibroblastic foci in vivo cell environment. Pepsin extracted process removes nonhelical telopeptides situated at the N- and C-terminal ends of native collagen molecules Although we found that IPF fibroblasts display an apoptotic resistant phenotype on type I polymerized collagen, it is possible that the pepsin extracted collagen that we used for our studies may not fully simulate the polymerized cross-linked collagen that exists in the Although we focused on the role of FoxO3 in regulating IPF fibroblast function, FoxO3a is also thought to be involved in aging. Since IPF is a disease of older individuals with a mean age of ∼65 years In summary, we demonstrate that IPF fibroblasts display an apoptotic resistant phenotype on polymerized collagen. The mechanism for this resistance involves inactivation of FoxO3a by Akt which down-regulates cav-1 expression . Decreas


The deduced electrical noise equivalent power is 1.2 A bolometer is a device that makes an electrical resistive response to the electromagnetic radiation resulted from a raise of temperature due to heating. The combination of the extremely weak electron-phonon interactions along with its small electron heat capacity makes graphene an ideal material for applications in ultra-fast and sensitive hot electron bolometer. However, a major issue is that the resistance of pristine graphene weakly depends on the electronic temperature. We propose using disordered graphene to obtain a strongly temperature dependent resistance. The measured electrical responsivity of the disordered graphene bolometer reaches 6 × 10 One of many astonishing properties of graphene is its weak electron-phonon (e-p) coupling. In normal conductor, e-p scattering quickly dominates as the temperature increases, resulting in a diminishing carrier mobility. In contrast, e-p scattering in graphene is negligible even at room temperature1346791011121314151819This present work demonstrates a new approach for implementing a graphene HEB. The main key is to drive the electronic system into the strong localization regime by adding disorder. Note that disorder here in general denotes the scattering potentials of various forms, such as lattice defects, charge impurities, etc. The divergence of the resistance at low temperature will allow for sensitive temperature measurement. The high resistance, together with an as-grown Boron Nitride (BN) tunneling barrier between graphene and electrical contacts, also reduces thermal dissipation out of the electron gas via diffusion. The bolometer fabricated with such method is found highly sensitive and with a very low background noise. Moreover, this approach does not require graphene with high mobility. The device structure with merely a graphene bar in contact with two electrodes can be easily fabricated with standard photo-lithography technique and extended into a 2D detector array.D peak in the Raman spectrum in Fig. 1(a)23The samples used in this study were Graphene/BN bilayer grown by CVD methodμm wide BN/Graphene ribbon on the metal electrodes, see R against T−1/3 in a semilog scale, where a linear dependence spans from the lowest temperature of 1.5 K to 300 K. The data suggest that electrons are strongly localized and the transport is dominated by the so-called variable range hopping (VRH)ρ(T) ∝ exp[(T0/T)1/3]. Here, T0 is a characteristic temperature, T0 = 12/πkBg(EF)ξ2, where kB is the Boltzmann constant, g(EF) the density of states at the Fermi level EF, ξ the localization length. We have performed a linear fit for the ln R versus T−1/3 plot. The slope of the fit yields a localization length ξ of 50 nm. Both the Raman results and the transport data indicate that the graphene film is disordered. The sharp increase of the resistance at low temperature due to localization is the key ingredient of our bolometer, as the responsivity of our bolometer is proportional to the slope of the R – T curve, dR/dT. R/dT as a function of temperature. At 2 K, dR/dT reaches 22 kΩ/K. Moreover, a high electrical resistance means a high thermal resistance, which prevents heat from dissipation via diffusion and also helps the responsivity.Our bolometer devices consist of a 5 Idc to generate heat in the electron gas. As shown in V/I = R0 exp(−eEl/BTk) when eEl/BTk < 1R0 is the zero bias resistance, E the electric field, e the electron charge and l a fraction of the hopping distance mr, i.e., l = 0.18mr. Since mr = ξ(T0/T)1/3, we have l = 29 nm at 1.57 K. A fit of the data to an exponential field dependence is very poor, see To see how the bolometer responses to heating, we apply a d.c. bias current Te will be determined by the strength of the e-p scattering and nearly uniform along the sample. We now show that this is indeed true in our devices. For a uniform temperature, Te at different biases can be obtained from the resistance R based on the R – T curve. We have measured R – Idc in magnetic fields of 0, 1, 5 T . It linearly follows ρ. The highest value in our experiment reaches 6 MΩ/nW. The corresponding responsivity of the device, which is 2.5 μm long and 5 μm wide, is 6 × 106 V/W at an excitation current of 2 nA.The responsivity of a graphene HEB depends mainly on two factors. One is the e-p coupling strength, which determines the amplitude of the temperature increase in response to a certain power input. The other is the voltage response to the temperature change, which is the sensitivity of graphene as a thermometer. In i.e., graphene/BN/SiO2/Si, the transfer matrix of the structure is deducedV at different temperatures are plotted in To test the actual bolometric response of the device, we employed a red LED to illuminate the device. The LED emits light with a peak light wavelength of 650 nm. Since the whole device is under uniform and constant illumination, photovoltaic and photothermoelectric signals will not be picked up by the lock-in (See Method). Considering a multilayer structure, R – Vdc plot into a Te – P plot and then numerically compute the derivative dTe/dP, as plotted in Te, which is expected by theoretical work on e-p scattering in graphene. By fitting the linearity, we have Tph is the phonon(lattice) temperature, while TBG is Bloch-Grüneisen temperature, defined as kBTBG = 2kFvph, where kF is the Fermi wave vector, vph the speed of soundvph = 2 × 104 m/s, TBG ≈ 102 K, much higher than Te and Tph. Considering a deformation potential D = 18 eVvF = 106 m/s, we find that for a graphene film with an area of 12.5 μm2, We have shown that the thermal dissipation pathway is mainly dominated by e-p scattering. Therefore, the thermal resistance due to e-p scattering can be computed by τ of the bolometer can now be estimated by ρ ≈ θCeR, where eC is the electronic heat capacity. −20 J/K, resulting in τ ≈ 1.4 nS. Apparently, the intrinsic speed of graphene bolometer is extremely fast. However, in practical application, the actual response time is also limited by the RC time constant of the whole circuit, which is τ = RC. Here, R and C are the circuit resistance and capacitance, respectively. Take the sample above as an example. Considering the total area of four metal electrode pads and the graphene sample, the capacitance to the back gate is estimated to be 7 pF. The total capacitance of our measurement cables is roughly about 250 pF, while the total resistance, including the sample resistance and two contact resistances, is 94 kΩ. We then have τ ≈ 24 μs. Therefore, it is important to minimize the parasitic capacitance of the bolometer circuit and the resistance of graphene in order to approach the intrinsic speed of graphene HEBs.Having known the value of the thermal resistance, the response time θR ≈ 58 K/nW at 1.5 K. Thus, NEP by the thermal fluctuations is R = 54 kΩ plus the total contact resistance of 40 kΩ. The corresponding NEP is the noise divided by the responsivity, Besides the responsivity, noise equivalent power (NEP) is a key factor that determines the performance of a bolometer. There are three noise sources, the measurement circuit noise, the thermal fluctuations and the Johnson noise6 × 0.027 ≈ 1.6 × 105 V/W and a NEP of When estimating the responsivity and NEP of our devices, we have used the absorbed power. Although this gives the ultimate performance of the device, it is useful to discuss the optical responsivity and NEP, estimated by taking the incident radiant power as the input. Taking into account the 2.7% absorptance of the device, we get an optical responsivity, 6 × 10e.g., the ratio of the device area to the contact area. In fact, for disordered graphene, the resistivity is relatively high so that the heat dissipation through diffusion can be substantially reduced. As seen in the We now discuss the role of the BN layer. As we have explained, it increases the contact resistance, therefore reduces thermal dissipation by diffusion. As a result, the temperature is uniform along the device, which allows for straightforward derivation of the electron temperature and the thermal conductance and analysis of e-p scattering. However, the temperature uniformity is not a requirement for our bolometer, although it is helpful to gain the responsivity by suppression of heat dissipation into contacts. In addition to increasing the contact resistance, it can be achieved by enhancing the graphene resistivity or by modifying the device geometry, 2/Si substrate. Similar 2D array is the basic structure for 2D bolometric imaging, where each element works independently to provide a pixel. The graphene ribbon is 5 μm long and 2 μm wide. The fabrication procedure is similar to the one for making an individual device (see the Method). Although the e-beam lithography was used for this demonstration, photo-lithography is well suited for fabrication of the array. Note that the structure of the detector array has not been optimized, i.e., the optical fill-factor is very low. We want to point out that the simplicity of the device structure and the aspect ratio dependent responsivity and resistance, offer significant flexibility in device design to meet applications.The basic structure of a disordered graphene HEB is comprised of only a graphene ribbon and two electrodes. It can be easily expanded to a detector array using conventional photo-lithography technique. In e.g., SdH oscillations, superconducting tunnel junctions and dual-gated bilayer graphene, disordered graphene provides a rather simple, yet effective scheme to implement a graphene HEB. The bolometer has an electrical responsivity of 6 × 106 V/W, over an order of magnitude higher than other approaches135 V/W. This value is much higher than 1 × 103 V/W for the dual-gated bilayer graphene deviceCompared with previous approaches, The responsivity of disordered graphene HEB rivals Si bolometers and composite bolometersh-BN samples in this work were synthesized via low pressure chemical vapor deposition on 25 um Cu foils (purchased from Alfa Aesar)h-BN was initially grown on Cu foil at 1000°C, using ammonia borane  as the precursorh-BN was achieved by introducing carbon sources into the system. Depending on the graphene growth parameters, i.e., growth temperature and carbon source inlet flow rate, the quality of graphene can be controlled. In order to obtain defective graphene, low temperature (800°C) and high carbon source flux were chosen to increase densities of defects and nucleation in graphene film. The bolometer devices were fabricated by e-beam lithography. 20 nm Ti/50 nm Au electrodes were first deposited on Si/SiO2 (300 nm) substrates. The graphene/BN films were then transferred onto the substrateBilayer structure of defective graphene/X.S.W. conceived the experiment. X.S.W. and D.P.Y. supervised the experiment. Q.H. fabricated devices and performed low temperature measurements. T.G. synthesized BN/graphene samples. Y.F.Z. and Z.F.L. supervised sample synthesis. X.S.W., Q.H., Y.C. and G.R.L. wrote the manuscript. X.S.W., Y.C. and G.R.L. performed data analysis. R.Z. and J.H.C. helped with the experiment. All authors participated in data discussion and reviewed the manuscript.Supplementary Info


Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that plays an important role in feeding behavior. It activates two G-protein-coupled receptors, MCHR1 and MCHR2, of which MCHR1 is the primary regulator of food intake and energy homeostasis in rodents. In mammalian cells transfected with MCHR1, MCH is able to activate multiple signaling pathways including calcium mobilization, extracellular signal-regulated kinase activation, and inhibition of cyclic AMP generation through Gi/o- and Gq-coupled pathways. Further evidence suggests that MCHR1 is regulated through posttranslational modifications, which control its intracellular localization and provide appropriate cellular responses involving G-protein signaling. This review summarizes the current data on the control of MCHR1 function through glycosylation and phosphorylation, as related to cell function. Especially, a series of mutagenesis study highlights the importance of complete glycosylation of MCHR1 for efficient trafficking to the plasma membrane. The G-protein-coupled receptor (GPCR) gene family is one of the largest families in the mammalian genome. GPCRs are typical heptahelical receptors composed of an extracellular N-terminus, an intracellular C-terminus, and seven transmembrane bundles connected by three intracellular loops and three extracellular loops. Activation of GPCRs induces second messenger responses that change the biochemical properties of the recipient cell and can modulate not only its electrophysiological responsiveness, but also its transcriptional activity. Thus, the diverse signaling and wide array of functions have allowed GPCRs to be employed in the physiological regulation of nearly all biological functions. These features coupled with ligands that are chemically highly specific for the receptors have resulted in the extensive utilization of GPCR-targeted drug design. Furthermore, the dynamic posttranslational modifications may provide tissue-specific functions, since distinct cellular environments or agonists can mediate different effects on receptor signaling or regulation of a number of GPCRs –5. ConseMCH gene is overexpressed upon fasting and in ob/ob leptin-deficient mice  was SLC-1/GPR24, an orphan GPCR, by applying orphan receptor strategies and reverse pharmacology –19. MCHRXenopus tropicalis, information for four MCH receptors has been obtained (A second MCH receptor (MCHR2) was subsequently identified by six groups using human genomic sequence searches . It sharobtained . Phylogeobtained . Therefoin vivo potency in the rodent. Comprehensive review of desired selectivity and efficacy of MCHR1 antagonist in vivo should consult some recent reviews  localized in the C-terminal cytoplasmic tail. The insertion of palmitate into the cytoplasmic leaflet of the plasma membrane can create a fourth loop named helix 8, thereby profoundly affecting the GPCR structure and consequently the interactions with intracellular partner proteins. Although a putative palmitoylation site has not been identified in mammalian MCHR1 , the exiXenopus, mouse, and human orthologs , six for protein kinase C , and one for protein kinase casein kinase 2 (Ser/Thr–X–X–Asp/Glu: T342) Figure . Howevers Figure , while ts Figure .The MCH-MCHR1 system has integral roles in many cellular events, and represents an important therapeutic target. For example, it may be targeted in strategies for developing treatments against obesity and mood disorders, and possibly also for inflammatory diseases. Therefore, the regulation of the MCH system by posttranslational modifications remains an emerging area at the nexus of endocrinology with important implications for drug development. However, our understanding of the regulation of MCHR1 signaling by glycosylation and phosphorylation is limited Table , 57, 58.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TAR-DNA binding protein 43 (TDP-43) is a heterogeneous ribonucleoprotein (hnRNP) identified as a major protein constituent of cytosolic inclusions in spinal cord of patients with motor neuron disease. The inclusions are formed by movement of TDP-43 from its predominantly nuclear localization to the cytosol, followed by accumulation of ubiquitinated and phosphorylated C-terminal TDP-43 fragments. The mechanisms controlling TDP-43 trafficking and accumulation are not well known, however, it has been demonstrated previously that kinases control movement and accumulation of hnRNPs. Therefore, we investigated whether kinases also controlled TDP-43 accumulation during cell stress.Neuronal cultures were treated with the mitochondrial inhibitors paraquat or sodium arsenite, or transfected with C-terminal TDP-43 or full length TDP-43 constructs and examined for kinase control of TDP-43 localization.We found that stress induction induced robust cytosolic accumulation of C-terminal TDP-43 into RNA stress granules, some of which progress to ubiquitinated inclusions. Inhibitors of c-Jun N-terminal kinase (JNK) and cyclin-dependent kinase 2 (CDK2) specifically inhibit and/or reverse the cytosolic accumulation of TDP-43 with little effect on ubiquitous stress granule formation. Our studies have shown active CDK2 co-localizes with accumulated TDP-43 and heterogeneous ribonucleoprotein K (hnRNP K). CDK2 inhibition blocks phosphorylation of hnRNP K, preventing its incorporation into stress granules. Due to Interaction between hnRNP K with TDP-43, the loss of hnRNP K from stress granules prevents accumulation of TDP-43. We have also determined that specific C-terminal mutations to TDP-43 alter expression of hnRNP K during cell stress. Preliminary studies indicate that this may involve altered hnRNP K phosphorylation.Due to the recently identified role for hnRNPs in motor neuron disease and frontotemporal dementia, further investigation of the association between hnRNP K and TDP-43 is warranted. Understanding how kinase activity modulates TDP-43 accumulation may provide new pharmacological targets for disease intervention.