Although the growth of graphene by chemical vapor deposition is a production technique that guarantees high crystallinity and superior electronic properties on large areas, it is still a challenge for manufacturers to efficiently scale up the production to the industrial scale. In this context, issues related to the purity and reproducibility of the graphene batches exist and need to be tackled. When graphene is grown in quartz furnaces, in particular, it is common to end up with samples contaminated by heterogeneous particles, which alter the growth mechanism and affect graphene’s properties. In this paper, we fully unveil the source of such contaminations and explain how they create during the growth process. We further propose a modification of the widely used quartz furnace configuration to fully suppress the sample contamination and obtain identical and clean graphene batches on large areas. Concerning the applications in electronics, the fabrication of low-cost, large-area, high-quality graphene still poses crucial challenges. Since graphene was first isolated by mechanical exfoliation1, different production methods have been explored, which can used for diverse application10. Among the many, chemical vapor deposition (CVD) is currently considered the optimal method for producing graphene for high-performance electronics and on a large scale, considering its accessible cost, wide scalability and the high crystalline quality achievable9. In a typical CVD process, carbon-bearing gases react at high temperatures (900–1100 °C) in the presence of a metal catalyst, which serves both as a catalyst for the decomposition of the carbon species and as a surface for the nucleation and accretion of the graphene lattice11. The details of the graphene growth process are articulated and still subject of intense study12, being several factors involved: (i) the metal catalyst with its surface morphology  and properties 19; (ii) the choice of carbon precursor20; (iii) the CVD parameters, especially temperature and pressure. Copper (Cu) and nickel (Ni) are at present the preferred metal substrates because of their favorable catalytic properties and low cost. Cu in particular is advantageous as it is easily removed after the growth, hence offering an efficient means for graphene transfer11. Methane is the most used carbon precursor11, although many other carbon sources have proven very efficient in later years: Among them ethanol is a valid choice for the growth of high-quality graphene at high temperature (>1000 °C)22. As a carbon precursor, ethanol is safer and easier to handle than methane, being liquid at STP. Advantages arise also from the weakly oxidizing ability of ethanol, which translates during the CVD process in an increased graphene growth rate (faster than in the case of methane), and hence in the formation of continuous graphene layers in just a few seconds23. Ethanol can yield large graphene crystals inside “sealed” Cu enclosures with a quality comparable to those obtained with methane precursor24. It also contains oxygen, whose beneficial effect in reducing the nucleation density and in improving the quality of the grown graphene has been recently recognized and explored. A pure oxygen flow prior to the growth or the use of so-called oxygen-rich copper substrates were proved to favor the growth of graphene of higher quality than pure hydrocarbons25. The presence of some amounts of carbon dioxide, a milder oxidant, was also found to suppress unwanted graphitic phases26.Graphene – a single atomic layer of carbon atoms tightly packed in a two-dimensional honeycomb lattice, chemically inert with super strength, and the ability to conduct heat and electricity better than any other known material – has been suggested for a variety of potential industrial applications, ranging from flexible electronics to high-speed computing, and from more efficient wind turbine blades to next-generation solar cellsm = 1084.62 °C). CVD can be performed either in cold-wall metal chamber reactors or in hot-wall reactors. The latter kind of reactors are the most widespread because of the steady-state, isothermal, clean gas flow conditions they offer, which can be achieved by simple and inexpensive experimental instrumentations. A hot-wall reactor usually consists of a quartz-tube furnace12, where by “quartz” the graphene community is commonly intending fused quartz or fused silica, i.e., amorphous SiO2. Differently from other kind of glasses, fused quartz contains no heterogeneous elements and then has higher working and melting temperatures27, with negligible vapor pressure at 1000 °C28. A tube-furnace made of electrically fused quartz of the highest grade can operate in vacuum up to 1160 °C. For these reasons most of the research focusing on the CVD growth of graphene on Cu has been carried out in hot-wall quartz furnaces. Although excellent in terms of crystalline quality, the graphene samples grown with this kind of CVD systems may often show dot-like contaminations on their surface. These contaminations appear as white particles (with various density) sitting on top of graphene in the reported scanning electron microscopy (SEM) images (either on Cu or on other transfer substrates), or are identifiable as round particle with height of a few nm in atomic force microscopy (AFM) images. The contaminations are found when graphene is grown in a CVD system with a quartz tube (either at low or atmospheric pressure), regardless of the carbon precursors36, although their density seem to be higher in case of oxygen-containing precursors36. No definitive explanation for this contamination issue has been so far presented, nor sufficient emphasis has been given, in our opinion, to such relevant and ubiquitous effect.Usually, CVD graphene is grown on Cu foils at high temperature , slightly below the metal’s melting temperature  as responsible for the contamination process: SiO2 is emitted from the quartz tube when Cu atoms (originating from the Cu substrates during heating) diffuse inside the tube walls at the α/β quartz phase transition temperature (573 °C)37. Other authors attributed the SiOx contamination to the presence of silicon (Si) in the bulk of the Cu substrates, which in their view would emerge during the growth process and contaminate the graphene surface38. A third group linked the presence of SiO2 clusters on graphene to the reaction of hot hydrogen with the quartz reactor39: During the annealing of the Cu substrate the hot part of the tube furnace would be etched by H2 gas, depositing SiO2 on the substrate. These studies propose possible contamination mechanisms but leave some questions unanswered. In the first case, it is unclear how the α/β transition may occur in a tube made of amorphous fused quartz. In the second, if the bulk of the Cu catalyst is the source of SiOx particles, it is difficult to explain how these particles can be found on top of graphene if they emerge from the underlying Cu substrate . Concerning the last study, the proposed mechanism is not supported by any other evidence or published record for the reaction of quartz with molecular hydrogen at 1000 °C.Only very recently, a few groups analyzed and discussed the nature of such contamination. Ruiz 21, which, as mentioned, has been observed to lead to a high level of contamination35. The presence of contaminants in the grown graphene is assessed by electron and scanning probe microscopy, and by energy dispersive X-ray spectroscopy. We ascertain the contamination mechanism and ultimately propose a novel design for a CVD system capable of completely eliminating the occurrence of contamination.In this paper we discuss the contamination issue and its effect on the production of graphene, presenting a practical solution to solve it and obtain clean graphene in a consistent and reproducible way. In our case study, we focus on graphene grown on copper foils using ethanol as liquid precursor39. At the time of that first observation, our quartz furnace had previously sustained hundreds of graphene growth processes (and hence a corresponding number of heating/cooling cycles). Heavy contaminations (a particle density up to 1014 cm−2) could be found in all the graphene samples, with the highest density in case of the longest growth processes (30 min). Figure 2, as visible in Fig. 2 showed a degree of contamination increasing with the working age of the tube. To further evaluate the impact of the quartz tube ageing on the sample pollution, we examined graphene grown in quartz tubes with an increasing number of operating hours, evaluated in terms of heating/cooling cycles. In general, when using quartz tubes with a few tens of cycles graphene appears clean of contamination upon SEM and AFM inspection. The contamination increases from negligible to noticeable, finally becoming very heavy; three broad ranges have been identified: <50 cycles, ~100 cycles, and >150 cycles. The contamination level was observed to worsen in case of longer (>30 min) and lower pressure (<1 mbar) process, not reported here.This study began after the observation of microscopic particles dispersed on the graphene samples grown with our CVD system, which has the typical configuration  detained by most of the CVD systems used in literature for graphene growthx particles, which can be thus deemed to be amorphous.Transmission electron microscopy (TEM) was employed to analyze the impurities. Figure 4 tetrahedra linked by shared ‘corner’ oxygen atoms. The crystalline polymorphs of quartz undergo a reversible phase transition at 573 °C, where the lattice structure changes from α- to β-quartz 41. Concurrently, the quartz lattice expands by a factor of 0.45%, with a density decrease from 2.65 g/cm3 to 2.53 g/cm3. As mentioned in the introduction, it has been suggested that these density fluctuations are responsible for the diffusion of Cu into the bulk of the quartz tube and the consequent displacement of SiO2, which is emitted and eventually deposits on graphene as nanoparticles37. This explanation is however incomplete, since pure amorphous fused silica does not undergo phase transitions within the usual CVD temperature range (up to ~1070 °C). During CVD, however, the quartz tube gets in contact with impurities and reactive elements and is subject to heating/cooling phases at varying pressure conditions: Therefore a more complex scenario involving “devitrification” should be taken into consideration. Devitrification is a recrystallization process occurring above ~1220 °C27 in fused silica via the nucleation and growth of metastable crystalline phases . Although even the highest graphene growth temperatures on Cu substrates are not sufficient to enable the devitrification of pure fused silica, the presence of impurities (such as Cu atoms) can trigger it even at temperatures around 600 °C42. The amorphous structure of fused silica could be further degraded by repeated exposure to oxidizing and reducing environments43, a conditions that is met during CVD and that may also contribute to a faster devitrification kinetics44. In order to ascertain and understand the possible structural evolution of the CVD quartz tube, we analyzed it by X-ray diffraction (XRD) after >150 graphene CVD growth processes. We concentrated the analysis on three tube parts held at different temperatures in the furnace during CVD, as detailed in Fig. Quartz has a framework structure of SiO42. The α-cristobalite phase formed by the devitrification is metastable and slowly transforms in β-quartz, and then back to α-quartz upon cooling to room temperature. We further confirmed such phase transformations by checking the evolution of the XRD spectrum of an analogous quartz tube part while heating in vacuum up to 900 °C, as shown in Figure x contamination of CVD graphene, as follows.The XRD pattern of the tube area held at the graphene growth temperature (part C) shows the broad halo typical of amorphous quartz. This pattern is coincident to that of the room-temperature areas of the tube (not shown). The tube part A shows an analogous amorphous quartz pattern but in this case two weak peaks ascribed to metallic Cu also appear. Conversely, the tube part B presents a completely different spectrum: two set of peaks ascribed to α-quartz and α-cristobalite are accompanied by a small amount of metallic Cu. This analysis sheds light on the evolution of the quartz phases due to the CVD processes. Although the hot tube zone reaches temperatures up to 1070 °C, no Cu atoms (sublimating from the foil substrate) condense on its inner surface and thus neither Cu diffusion nor devitrification take place. On the tube zone held up to 570 °C, Cu can condense and diffuse into the bulk, but the temperature is not high enough for the devitrification to occur. The situation is completely different for the tube zone kept at temperature up to 860 °C, where the Cu atoms are able to diffuse and promote the devitrification46) is fostered during CVD by the α/β quartz phase transitions induced by the devitrification. While diffusing within the bulk, the Cu atoms subtract oxygen from the quartz (silica) forming SiO, which is emitted as vapor into the tube. This happens since the SiO vapor pressure is two orders of magnitude or more higher than that of SiO247. The gaseous “desiliconization” is an established technique for the removal of silica from minerals and ores48. The emitted SiO vapor eventually condense and deposit as nanoparticles inside the CVD chamber, polluting the Cu substrates and the growing graphene. Such contamination issue is so serious and ubiquitous that we deem it partially responsible for the common choice in literature of the semi-sealed “copper enclosures” used as growth substrates in fundamental studies about the nucleation and growth of graphene25. Curiously, this contamination process has been even exploited as a source for the controlled assembly of SiOx nanoparticles, aiming at the formation of lateral heterostructures between SiOx and graphene49. Other than clearly being an unwanted contamination, these nanoparticles are expected to affect the graphene growth process, e.g., by acting as preferential nucleation sites and/or undesired secondary nucleation sites (giving rise to multilayered graphene in an otherwise self-limiting monolayer growth process)31. This situation is further confirmed by inspecting CVD graphene grown in our aged quartz tube  is considerably higher than that of quartz. Conversely, a tube made of alumina would be less proficient in a CVD furnace configuration due to many practical drawbacks with respect to one made of quartz: Alumina’s higher thermal conductivity would increase the heat flow along the tube axis; its higher sensitivity to thermal shocks could cause mechanical failures and its rough, microcrystalline surface would make vacuum head seals more difficult to realize. We avoided all these issues by using a larger quartz tube (60mm outer diameter - OD - and 55mm inner diameter - ID) fitted with high vacuum seals and a smaller coaxial alumina tube , bridging the two reactor cold ends and acting as a screen . In this geometry, the SiO vapors cannot travel directly from the inner quartz tube surface towards the hot samples since they would readily condense onto the colder alumina tube edges, nor Cu vapors could follow the opposite route to coat and degrade the quartz walls. Alumina is not impervious to copper, which at high temperature can diffuse into its bulk51, however the diffusion coefficient is considerably lower than that in SiO245; further, alumina is not subject to phase transitions at pressures and temperatures typical for CVD. In Figure In order to overcome the practical and cost limitations arising from replacing the quartz tubes every few growth processes, we developed a novel reactor configuration by adding an alumina tube coaxial to the tube furnace as a screen for Cu (from the substrates to the tube) and SiO (from the tube to the substrate) vapors contaminants during growth  and can efficiently act as an oxygen getter, possibly further reducing SiO vapors, and can thus guarantee an unpolluted, stable CVD gaseous environment. Figure We gained no direct evidence of a detectable contamination arising from the alumina boat, but it was subjected to cracking upon cooling after a few cycles. It has to be considered that the boat is not exposed to air while hot, unlike the furnace tube. Therefore, we deemed more appropriate to substitute it with a boat made of a refractory metal to further avoid possible contamination sources upon aging. As expected, the contaminants were observed to be fully suppressed by substituting the alumina boat with a tantalum (Ta) boat with a lid, built with an open “matchbox” geometry which allows the free circulation of the process gases over the copper foils, as schematized in Fig. 2 with a different degree of contamination depending on the CVD system used. The sample grown in our modified system is completely free of contamination. The comparison of the Raman spectra taken on the two samples  and promotes the undesired nucleation of secondary islands/layers53, as also suggested by Figure The AFM images in Fig. les Fig.  demonstr54.By using our modified CVD system it is possible to grow and transfer clean graphene over cm-large areas, as shown in Fig. Despite their widespread use, quartz tubes may not be the ideal choice for CVD furnaces dedicated to the growth of graphene on Cu substrates, since the presence of Cu vapors is expected to degrade the purity of the quartz tube. As Cu diffuses into the quartz, a phenomenon accompanied by its devitrification, SiO vapors are emitted from the tube walls and can severely contaminate the exposed growth surfaces. All these effects have a practical relevance in the CVD growth of graphene, since the ageing of quartz tubes and other components - such as sample holders - will introduce contamination and cause reproducibility issues in the graphene batches, with a large increase in the production cost if the quartz glassware must be often replaced. We here proposed a modified hot-wall CVD system based on a quartz tube furnace with an internal coaxial alumina screen tube and a tantalum sample holder. This CVD configuration successfully produce contamination-free graphene samples on Cu foils over extended period of time. The reactor and process presented in this work were used to perform more than 100 growth processes without the occurrence of heterogeneous sample pollution. Our novel system configuration coupled with fast graphene growth from ethanol CVD and cyclododecane assisted transfer provide a platform for the high-throughput production of pure graphene. This modified system would be also strategic for the CVD growth of large single-crystal graphene which is at present are actively sought after for electronic applications, since this process is usually performed in highly diluted carbon precursors over extended time  and it is possibly subjected to an extreme level of contamination.2 substrates) and cleaned by ultrasonication in acetone and ethanol (15 min each). The details of the CVD system can be found in ref. 2 (20/20 sccm) for 20 min at 1000 °C. Afterwards, ethanol (C2H5OH) diluted in Ar (0.1% in 20 sccm of Ar) is introduced in the tube with 100 sccm of H2 for 30 min to carry out the graphene growth. Finally the sample holder is rapidly extracted from the hot zone and cooled down to room temperature under Ar flow. A typical graphene growth process is a six step process as summarized by the temperature-time diagram in Fig. 8. In this work we compare graphene grown in a system with all the components made of quartz  to graphene grown in a modified system designed to avoid the close exposure of the samples to quartz surfaces.Graphene was synthesized on polycrystalline Cu foil  using low-pressure CVD. The Cu foil substrates were cut  with an optical microscope57 the presence of holes and cracks due to the transfer process was evaluated. XRD patterns were recorded on a PANalytical Empyrean X-ray diffractometer equipped with a 1.8 kW Cu Kα ceramic X-ray tube, PIXcel3D 2 × 2 area detector and operating at 45 kV and 40 mA. The diffraction patterns were collected in air at room temperature using Parallel-Beam (PB) geometry and symmetric reflection mode. High Temperature X-ray diffraction analysis (HTXRD) was performed using a Rigaku Smartlab system equipped with a 9 kW CuKα rotating anode (operating at 40 kV and 150 mA) and an Anton Paar DHS 900 domed hot stage, operating under vacuum conditions.The presence of contaminants and the general surface morphology were investigated using a Zeiss (formerly LEO) 1530 field emission SEM, both on as grown samples and after their transfer. The analysis of samples transferred onto grids (Ni mesh with holey carbon film) was performed with a FEI Tecnai F20 ST TEM to determine the film microstructure. EDX was employed to determine the nature of the contaminants. By the examination of the transferred samples onto a Si/SiO2 substrates) were collected with a Renishaw inVia confocal Raman microscope using an excitation line of 532 nm (2.33 eV) with a 50x objective lens, and an incident power of 1 mW on the samples (20 spectra collected and averaged for each sample).Raman measurements on the graphene sample (transferred on Si/SiOSupplementary Information


However, only little is known about sponge intron mobility, transmission, and origin due to the lack of a comprehensive dataset. We analyzed the largest dataset on sponge mitochondrial group I introns to date: 95 specimens, from 11 different sponge genera which provided novel insights into the evolution of group I introns.Mitochondrial introns intermit coding regions of genes and feature characteristic secondary structures and splicing mechanisms. In metazoans, mitochondrial introns have only been detected in sponges, cnidarians, placozoans and one annelid species. Within demosponges, group I and group II introns are present in six families. Based on different insertion sites within the Scleritoderma, Microscleroderma, Aciculites, Setidium). We demonstrated that group I introns in sponges aggregate in the most conserved regions of cox1. We showed that co-occurrence of two introns in cox1 is unique among metazoans, but not uncommon in sponges. However, this combination always associates an active intron with a degenerating one. Earlier hypotheses of HGT were confirmed and for the first time VGT and secondary losses of introns conclusively demonstrated.For the first time group I introns were detected in four genera of the sponge family Scleritodermidae  as an The online version of this article (doi:10.1186/s12862-017-0928-9) contains supplementary material, which is available to authorized users. Nephtys sp.  = 1.00 / bootstrap support [BS] = 98). As shown by Szitenberg et al. [Levantiniella levantinensis with respect to the Cinachyrella/Paratetilla/Amphitethya clade. Intron 723-bearing Tetillidae did not form a monophyletic group due to the position of L. levantinensis and the presence of a highly supported (PP 1.00/BS 100) clade of intron 714 bearing species consisting of C. sp. 1 (intron 714 only) and C. sp. 2 (with intron 714 only or in combination with intron 723). There were no genetic differences in cox1 between Cinachyrella sp. 2 bearing one (714) or two (714 and 723) introns.The relationships of major clades Fig.  were in g et al. , our anacox1 exon phylogeny -test). One single C. alloclada 1 sequence falls within the C. alloclada 3 clade. This position is regarded as artifactual due to an incomplete intron sequence as retrieved from degraded DNA.The intron 723 phylogeny (intron + LAGLIDADG) Fig.  broadly Agelas oroides and Cymbaxinella verrucosa intron sequences grouped within Spirophorina in a highly supported sister group to Cinachyrella introns. The intron + LAGLIDADG phylogenies for 870 and 714 were congruent with the exon phylogeny for all supported clades , Cinachyrella sp. 4 (PC944), Cinachyrella alloclada 2 (GW3920), Cinachyrella cf. providentiae (RMNH POR11228), and Cinachyrella cf. anomala (JX177887) , Setidium sp. 1 (HBOI 14-XI-02-3-008) and Scleritoderma sp. 2 (HBOI 25-X-95-1-010)  and Microscleroderma herdmani 3 (BMNH 1994.10.5.1)  Fig. . Both ta radiata . The intStupenda singularis forms a highly supported sister group relationship with a Fungi/Marchantiophyta clade. Cnidarian LAGLIDADG encoding sequences are only present in Hexacorallia and never revealed in the other subclasses Octocorallia and Ceriantharia. Within Hexacorallia, the sequences of intron 888 are monophyletic and include the orders Actiniaria (sea anemones), Scleractinia , Corallimorpharia  and Antipatharia  [The sponge LAGLIDADG sequences displayed phylogenetic affinities to four different clades Fig. . LAGLIDA corals) . NotablyPlakina/Tetilla and the Scleritodermidae are closely related to Hexacorallia (Zoantharia) LAGLIDADG sequences, but with considerable genetic distance. The genealogical affinities of intron 714 appear unresolved.LAGLIDADG (intron 870) of cox1 gene in a few sponge groups , encompassing 11 different sponge genera. In accordance with previous studies [cox1 gene were observed among different taxa  [cox1. Our conservation analysis showed that these two group II introns were located in conserved regions  , with twcox1 gene in demosponges has the lowest substitution rate of all mt protein coding genes [cox1), and the most conserved regions of this gene , which is in concordance with the already published data [Microscleroderma sp. 1 & 2 and Scleritoderma sp. 1 & 2 are also found to be in stage 1. A study comparing the length of DNA and RNA in combination with RT-PCR on a Cinachyrella intron 723 from Taiwan (probably Cinachyrella sp. 5) suggests that it can self-splice in vivo or in vitro [Aplysinella rhax, Microscleroderma sp. 3, Cinachyrella sp. 2, Setidium sp.1 (degenerated HEG) and Aciculites sp. 1 (short sequence and no HEG) respectively, which rebuts the suggested recent infection of intron 723 in sponges [Tetilla radiata, Plakina crypta and Plakina trilopha [Microscleroderma herdmani 1–3, Tetilla quirimure and Setidium sp. 1. Interestingly, both stage 3–4 intron 870  previously described [Plakinastrella sp.) co-occurs with a stage 1 intron 723. Similarly, all of the stage 3–4 intron 723  co-occur with stage 1 introns (either 714 or 870). Overall, two stage 1 introns never co-occur, one of the two is always degenerating. We can therefore hypothesize that the presence of two group I introns is unstable or that maybe the degeneration of one somehow enables the insertion of a different intron. More double-intron-bearing cox1 sequences are needed to study this further. Moreover, we noted that although Scleractinia  possesses intron 723 or 888, no evidence of double-intron cox1 sequences in this group is given, which applies for Cnidaria in general. Since double-intron cox1 sequences are also absent in Placozoa, sponges  are to date the only metazoans with double-intron cox1 sequences.Sponge group I introns consist of complex catalytic ribozymes (RNAs) that fold into a conserved three-dimensional core structure of ten helices. Within this structure, the sponge HEGs are found to be always located within the loop region of the catalytic domain  and Cinachyrella sp. 4  are sister to Cinachyrella alloclada 2–3/ Cinachyrella spp. from the Pacific whereas their intron 723 are sister to the intron of Levantiniella levantinensis  invaded the ancestor of Cinachyrella sp. 3 and Cinachyrella sp. 4. In addition, two Cinachyrella sp. 2 specimens from the Pacific (RMNH POR11161 and RMNH POR11187) group together with other conspecifics in the cox1 phylogeny; however, their introns are closely related to Cinachyrella species from the Pacific. Therefore, we hypothesize that those two Cinachyrella sp. 2 specimens from marine lakes and mangroves were reinfested by intron 723 after it was primarily lost, which would indicate a “secondary” HGT between C. sp. 2 and the Pacific Cinachyrella spp. . Another HGT event was found within the family Sceritodermidae: while Setidium sp. 1 is sister to Microscleroderma taxa in the intron phylogeny  are fairly divergent, the intron and the LAGLIDADG phylogenies both suggest that they are phylogenetically related to the Cinachyrella/Levantiniella introns. Indeed, the Agelasida introns share a more recent common ancestor with the Cinachyrella/Levantiniella introns, than with the Scleritodermidae , which Tetilla quirimure (MNRJ 17891)  [Tetilla japonica (JX177901), which is sister to Tetilla radiata (with a strongly supported node) does not posses intron 870. We therefore assume that Tetilla japonica secondarily lost intron 870, which would represent another case of mt intron loss in sponges. The structure of intron 870 in Microscleroderma herdmani 3 displays a reduced P6a and an additional P5d region  Fig.  and TetiM032742) . Remarkaion Fig.  comparedCinachyrella sequences provides conclusive evidence that introns were vertically transmitted due to 1) mostly congruent cox1 versus intron phylogenies and 2) similarity of secondary structures among closely related species. Introns 714, 723 and 870 have all undergone VGT, but this is especially apparent for intron 723 for which we have the largest sampling   is the dominant sponge-associated fungus, while nine more ascomycete species were found [Cinachyrella and other sponge taxa display a close relationship to ascomycete intron sequences, but also Viridiplantae (Chlorophyta and Streptophyta). The latter is not surprising, as intron of chlorophytes and Viridiplantae similarly have their origin from Ascomycota [cox1, fungus like Ophiocordyceps sinensis contains 21 group I introns within its cox1 region alone [The origins of group I introns has been debated for many eukaryotic organisms  includihina Sea ). This ire found . Indeed,comycota , 87. Theon alone .Cinachyrella species, as shown above. Intron 387 of Stupenda singularis is the only sponge LAGLIDADG sequence apparently unrelated to any coral LAGLIDADG in the data set, but is closely related to LAGLIDADG found in fungi and Marchantiophyta (liverworts). Liverworts are thought to have received their introns from fungi [Stupenda singularis LAGLIDADG 387 likewise suggests a fungal origin .Unfortunately, the origin of sponge intron 714 remains unresolved, since no supported relationship to any of the included taxonomic groups is given. Therefore, we cannot exclude the possibility that group I introns in sponges may originate from an undiscovered and/or un-sequenced sponge-associated symbionts, e.g., fungi, Archaea, Bacteria or dinoflagellates, since all are known to posses group I introns. In particular sponge bacterial symbionts, which can contribute to over 50% of the sponge biomass , 69, maycox1 regions decreases the possibility of intron elimination, because the removal must be specific in order to avoid any disruption of the protein function. The most widespread intron 723 is located at the most conserved site in the cox1 gene  and intron 870 , both in conserved cox1 regions. Intron presence in conserved cox1 gene regions has major consequences for other fields of science such as molecular taxonomy. In order to gain a better understanding of locations and conservation of the currently recommended barcoding primers [cox1 conservation profile line , as an i groups . We showCinachyrella samples were collected in Florida (U.S.A.) by snorkeling in the seagrass meadows adjacent to the Mote Marine Laboratory/Tropical Research Laboratory  and by scuba-diving on the Broward County reef located off Fort Lauderdale . More Cinachyrella spp. were collected in Indonesia by diving on reefs and snorkeling in mangroves and marine lakes in West Papua and East Kalimantan, Indonesia. The remaining material was obtained through collaborators or sampled in several museum collections . A detailed list of species origin including collector, voucher numbers and accession numbers, location and depth are provided in Additional file Cinachyrella cox1 sequence (including a group I intron) from Taiwan was manually copied from Hsiao [Cinachyrella australiensis and is identified as Cinachyrella sp. 5 based on our cox1 CDS phylogeny. One complete cox1 sequence of Microscleroderma sp. (USNM 1133739) with an intron was kindly provided by D. V. Lavrov . The higher level demosponge classification follows Morrow & Cárdenas [g et al.  were sucg et al.  on Tetilom Hsiao . This spCárdenas .Genomic DNA was isolated from the choanosome of the sponge tissue by using the NucleoSpin (Machery-Nagel) or the DNeasy (Qiagen) Blood and Tissue Kit according to the manufacturer’s protocol. An additional centrifugation step was added before transferring the lysate to the Spin Column in order to avoid any clogging of the membrane, caused by sponge spicules. Quantification of the isolated genomic DNA was performed using a NanoDrop 1000 Spectrophotometer (Thermo Scientific).cox1 was performed by using different primers and PCR conditions. Detailed information of primers used for each sample is provided in Additional file cox1 fragment was amplified using the primers LCO1490 [Cinachyrella samples with introns, we observed an additional non-specific band at position ~600 bp of bacteria and fungi cox1 fragments. Separation of double bands and PCR clean-up was performed using a modified freeze-squeeze method [Amplification of the partial  LCO1490  and COX1 LCO1490  and for  LCO1490 . For bote method  in whichcox1 sequence of the sponge Amphimedon queenslandica following Szitenberg et al. [http://megasun.bch.umontreal.ca/RNAweasel/ [www.geneious.com) with the following settings: translation Table 4  with start codons ATG, GTG, TTG and ATT [Trypanosoma [Insertion sites for each intron were ascertained in an alignment including other intron-bearing sequences , 43, 82.Aweasel/ . Initiat and ATT , minimum and ATT , 65, thepanosoma . We therCinachyrella alloclada (HM032738) for intron 723 and Tetilla radiata (HM032742) for intron 870 [http://unafold.rna.albany.edu/ [http://www.palaeontologie.geo.lmu.de/molpal/RRNA/index.htm). All secondary structures were visualized in RNAViz 2.0.3 http://rnaviz.sourceforge.net/ [In order to predict the secondary structures of group I introns, we manually converted the given secondary core structures into a dot-bracket notation including pseudoknot informations in square brackets. As secondary structure references, we used tron 870 . In ordeany.edu/  under thclada HM0738 for iany.edu/  (availabrge.net/ . Helix nrge.net/ .Geodia barretti and Alectona millari) were used as outgroups in Tetillidae phylogenetic analyses. Astrophorina has been established as the sister clade of Spirophorina in previous studies [Halichondria melanodocia and Axinella corrugata, which were already successfully used as outgroups in previous studies on the molecular phylogeny of the Tetractinellida .cox1 alignment (excluding intron(s)) of the Tetillidae phylogeny comprised 133 sequences (including the two outgroups), of which 76 were newly generated from this study. The alignment was 1177 bp long, of which 829 bp were constant, 62 bp were parsimony uninformative and 286 bp were parsimony informative. The final cox1 alignment of the Tetractinellida phylogeny constituted 82 sequences (including the two outgroups) of which 33 were newly generated from this study. In total the alignment comprised 1118 bp, of which 642 bp were invariant, 77 bp parsimony uninformative and 399 bp were parsimony informative.The final http://tree.bio.ed.ac.uk/software/figtree/.Phylogenetic tree reconstructions for both analyses were performed on a parallel version of MrBayes v3.2.4  and RAxMCinachyrella, we conducted phylogenetic analyses on separate datasets respectively including all sponge introns 723, 714 and 870. For the analysis of intron 723, we included 74 sequences of which 63 belong to the genus Cinachyrella. One taxon Aciculites sp. 1 (HBOI 26-IX-11-2-002), was excluded from this analysis, as no putative HEG were detected in the intron. The final intron 723 alignment was 1167 bp long, of which 488 bp were constant, 307 bp parsimony uninformative and 372 bp parsimony informative. The final intron 714 dataset included 13 sequences and was 946 bp long, of which 891 bp were constant, 49 bp were parsimony uninformative and 6 bp were parsimony informative. As an outgroup for both analysis we used the introns of Plakinastrella sp. (NC 010217), a species that belongs to a different sponge class  and P. crypta (HQ269352) which belong to a different sponge class  in the genus In order to test whether the incongruencies between the exon and the intron/HE phylogeny were significant, we performed a series of Shimodaira-Hasegawa (SH) tests  as impleIn order to investigate the evolutionary origins of the putative LAGLIDADG encoding introns  in sponges the newly generated sequences were added to the LAGLIDADG dataset by Huchon et al. . Additiocox1 protein alignment dataset compiled from the demosponge sequences from [A conservation profile was calculated from a ces from , complemces from . The finces from  using thces from ) but wit


The L1 stalk of the large ribosomal subunit undergoes large-scale movements coupled to the translocation of deacylated tRNA during protein synthesis. We use quantitative comparative structural analysis to localize the origins of L1 stalk movement and to understand its dynamic interactions with tRNA and other structural elements of the ribosome. Besides its stacking interactions with the tRNA elbow, stalk movement is directly linked to intersubunit rotation, rotation of the 30S head domain and contact of the acceptor arm of deacylated tRNA with helix 68 of 23S rRNA. Movement originates from pivoting at stacked non-canonical base pairs in a Family A three-way junction and bending in an internal G-U-rich zone. Use of these same motifs as hinge points to enable such dynamic events as rotation of the 30S subunit head domain and in flexing of the anticodon arm of tRNA suggests that they represent general strategies for movement of functional RNAs. Translocation of the tRNA on the ribosome is associated with large-scale molecular movements of the ribosomal L1 stalk. Here the authors identify the key determinants that allow these dramatic movements, and suggest they represent general strategies used to enable large-scale motions in functional RNAs. It has become clear from extensive studies using a wide range of biochemical, biophysical and structural approaches that the ribosome is a complex molecular machine, with many moving parts. By far the most dramatic dynamic events are associated with the step of the elongation phase of protein synthesis known as translocation23455781011Here we examine another prominent dynamic feature of the ribosome, the L1 stalk, which undergoes the largest-scale structural movements that have so far been observed for the ribosome, comparable to the largest excursions made by motor proteins141516171819206181920212221In classical-state ribosomes containing a vacant E site, the L1 stalk is found in an open conformation, with its head domain oriented away from the core of the ribosome  and 2a. 1619252621112293031In this study, we address several key questions. First, what is the functional role (or roles) of L1 stalk movement? Second, how are its movements coordinated with the numerous other dynamic events associated with ribosomal translocation? Third, what is the structural basis of L1 stalk movement—that is, where exactly does movement originate, and which structural features are responsible? And finally, are there common structural principles underlying the molecular movements observed for different functional RNAs?Our approach uses quantitative comparative structural analysis of an extensive database of 32 high- and medium-resolution X-ray and cryo-EM structures of ribosome complexes captured in intermediate states of translocation. As a frame of reference, we use a minimal model for translocation based on the four well-characterized functional states of the ribosome described above. Our findings point to a role for the L1 stalk in helping to coordinate movements of tRNA with movements of dynamic elements of the ribosome and in creating a possible checkpoint for the translocation process. Analysis of the trajectories of stalk movement between its different states shows that movement originates at the same RNA hinge points for transitions between each of the states. Most interestingly, the presence of similar structural motifs at the flexing points of other dynamic RNAs suggests that they represent a common set of strategies that are used for movement in many functional RNAs.We carried out a quantitative comparative structural analysis on a data set of 32 X-ray and cryo-EM structures of ribosome complexes from the Protein DataBank (PDB)The structure of the L1 stalk begins with the long 23S rRNA helix H76, which is connected at its base to the body of the 50S subunit through helices H75 and H79; at its distal end, helices H77 and H78 are connected by tertiary interactions to create the compact fold of the head domain, which binds ribosomal protein L1 . To idenTo quantify the magnitude and direction of L1 stalk movement, we applied the Euler–Rodrigues (E–R) methodThe magnitude of L1 stalk rotation values ranges from 0 to 30°  between 9265When the tRNA next moves into its chimeric hybrid pe/E state21112Contact between the head domain of the L1 stalk and the deacylated tRNA involves stacking of the non-canonical G2112–A2169 base pair of the stalk rRNA on the tertiary G19–C56 Watson–Crick pair on the elbow of the tRNA , as obse161926We localized the origin of L1 stalk movement by calculating the deviation of the axes of the helical elements of the stalk relative to their positions in the classical state  see ‘Me; a seconHelices H75 and H79, two of the three helices at the three-way junction, lie perpendicular to each other, but remain static during movement of the dynamic H76. U2092, at the first inflection point of the deviation plot, is positioned precisely at a sharp bend formed between nucleotides 2091–2093 in the linker between H75 and H76 . AlthougU2092 lies at the end of helix H75 but packs perpendicularly against the minor groove of H79 near the three-way junction , apparenAlthough hinging at the three-way junction dominates all L1 stalk transitions , a secon1519The 23S rRNA helix H68 is wedged in a complex network of minor groove interactions involving H75, H76 and the tRNA . H68 maiUniquely in the P/E hybrid-state ribosome, movement of the L1 stalk creates contact between complementary surfaces of its head domain and the 30S subunit , a transPlasmodiumet al.Although there are fewer available eukaryotic structures, the mechanics of L1 stalk movement are likely to be very similar . The len4344454647434449There are a handful of notable differences between the eukaryotic and prokaryotic L1 stalks. The head domain of the eukaryotic L1 stalk lacks the structural equivalent of bacterial helix H78. Nevertheless, we note that in recent high-resolution cryo-EM structures of the mammalian ribosome containing tRNA bound in the A/P and P/E hybrid states4647In this study, quantitative structural analysis of 32 X-ray and cryo-EM structures of ribosome complexes containing well-ordered L1 stalks shows that it occupies at least four distinct positions, corresponding to four well-characterized functional states of the ribosome  and 3. DIn the transition from the open to the closed state, a network of minor-groove interactions is formed around H68 of 23S rRNA. Inward movement of the stalk creates a new contact between the minor groove surface of the G-U-rich region of H76 and the minor groove of H68 at positions 1856/1886–1888, immediately adjacent to the fixed minor groove interaction between H75 and H68 . MovemenA critical aspect of RNA dynamics is that the range of motion of a dynamic element needs to be constrained, reducing the degrees of freedom in its trajectory to establish the precision of an associated functional process. Examination of contacts formed around the sites of flexing of the L1 stalk reveals several constraints on the range of L1 stalk movement. These include the unusual perpendicular packing of U2092 and A2198 against the minor groove surfaces surrounding the hinge point at the three-way junction  and the Interestingly, the head of the L1 stalk forms an extensive interaction with the 30S subunit uniquely in the hybrid (closed) state, through contact between complementary surfaces . Helices21228Our findings point to some emerging principles of RNA functional dynamics. Together with our previous analysis of the mechanism of rotation of the 30S subunit head domain2555657The possible role of Family A three-way junctions in RNA dynamics has been suggested previously based on comparative structural analysis39A second theme is the observed flexibility of RNA helices in regions containing multiple G-U wobble pairs and other weak helical features. This is seen for the L1 stalk at the G-U-rich region in the middle of helix H76 . PreviouA third strategy is the occurrence of non-canonical base pairs (most commonly purine–purine pairs) at the pivot point of hinging between coaxially stacked helices. In the case of the L1 stalk, hinging occurs at the junction of H76 and H79, where the non-canonical A2199–G2224 purine–purine pair at the base of H79 is stacked on the U2197–A2225 Hoogsteen pair at the base of H76 . In tRNA55562526A fourth example, stacking and sliding between the surfaces of bases in tertiary folds appears to be a common strategy employed by RNAs that contact the elbow of tRNA. Stacking interactions similar to that observed between the tertiary base pairs of the elbow of the translocating tRNA and the head of the L1 stalk have been observed in RNaseP and in the T-box riboswitchThe finding that similar structural features are found repeatedly at the origins of movement in prominent examples of functionally important RNA dynamics suggests that they represent general principles for enabling movement in RNA. Another emerging idea is that the most crucial aspect of RNA movement may be restriction of the degrees of freedom of dynamic elements, as exemplified by movement of the L1 stalk; thermal energy alone may be sufficient to enable all of the movements involved in ribosome translocation, as has been seen, for example, in intersubunit rotationE. coli or T. thermophilus and eukaryotic ribosomes from yeast and Plasmodium  rRNA, we used PyMolBacterial 23S rRNA: 173–269, 292–344, 372–542, 552–626, 657–926, 938–1053, 1108–1373, 1375–1412, 1425–1478, 1552–1579, 1587–1718, 1744–1859, 1882–1906, 1930–2088, 2227–2787.Plasmodium 28S rRNA: 16–114, 180–234, 270–433, 626–704, 788–1062, 1109–1228, 1282–1554, 1583–1622, 1653–1705,1780–1807, 1819–1948, 2101–2219, 2225–2249, 2273–2430, 2596–3150.Yeast and E. coli ribosome complex containing a vacant E site (the open conformation) as a reference structureWe calculated the magnitude of L1 stalk rotation using the E–R formula (see below) for each ribosome, relative to the position of the stalk in the X-ray structure of a classical-state http://rna.ucsc.edu/rnacenter/erodaxis.py.We applied the E–R method as described in ref. The E–R method describes any movement of a mobile domain as a simple rotational event about a single calculated axis (the E–R axis), with respect to a reference state. For a rigid body rotational event, applying the E–R formula generates an axis and a corresponding angle of rotation for the mobile domains. The structures being compared must be aligned on their static domains. Pymol generates a composite 4 × 4 transformation matrix containing a 3 × 3 the rotation (R) matrix and 3 × 1 translation (T) matrix, for this alignment. The angle (Θ) of rotation is derived from diagonal elements of the rotation matrix as:The direction of the axis is calculated as:I is a 3 × 3 identity matrix.The origin of the axis can be described by For purposes of quantifying stalk movement, the mobile domain is defined as helix H76 , while Helix H75  is the static domain.Positions of the helical axes for different L1 stalks were determined using Curves+All data generated or analysed during this study are included in this published article and its How to cite this article: Mohan, S. et al. Recurring RNA structural motifs underlie the mechanics of L1 stalk movement. Nat. Commun.8, 14285 doi: 10.1038/ncomms14285 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables and Supplementary ReferencesThe position of the L1 stalk correlates with the functional state of the ribosome. The position of the L1 stalk correlates with the functional state of the ribosome, which in turn, is closely correlated to the four binding states of the translocating tRNA from the P site to the E site. The L1 stalk occupies a distinct position in each state. Interpolations of the structural transitions through the four functional states were rendered using PyMol28 Network of minor-groove interactions around the 50S E site. Network of minor-groove interactions around the 50S E site. The 23S rRNA helix H68 is at the center of a complex network of minor groove interactions. H68 interacts with the static H75 at all times; however, this minor groove surface of H68 is flanked by two dynamic minor-groove interactions with the acceptor arm of the deacylated tRNA and H76 of the L1 stalk. H68 is also a point of contact with the 30S subunit though intersubunit bridge B7a. Interpolations of the structural transitions through the four functional states were rendered using PyMol28


Transgenic mouse models are indispensable tools to mimic human diseases and analyze the effectiveness of related new drugs. For a long time amyotrophic lateral sclerosis (ALS) research depended on only a few mouse models that exhibit a very strong and early phenotype, e.g. SOD1 mice, resulting in a short treatment time window. By now, several models are available that need to be characterized to highlight characteristics of each model. Here we further characterized the mThy1-hTDP-43 transgenic mouse model TAR6/6 that overexpresses wild type human TARDBP, also called TDP-43, under control of the neuronal Thy-1 promoter presented by Wils and colleagues, 2010, by using biochemical, histological and behavioral readouts. Our results show that TAR6/6 mice exhibit a strong TDP-43 expression in the hippocampus, spinal cord, hypothalamus and medulla oblongata. Apart from prominent protein expression in the nucleus, TDP-43 protein was found at lower levels in the cytosol of transgenic mice. Additionally, we detected insoluble TDP-43 in the cortex, motoneuron loss, and increased neuroinflammation in the central nervous system of TAR6/6 animals. Behavioral analyses revealed early motor deficits in the clasping- and wire suspension test as well as decreased anxiety in the elevated plus maze. Further motor tests showed differences at later time points compared to non-transgenic littermates, thus allowing the observation of onset and severity of such deficits. Together, TAR6/6 mice are a valuable tool to test new ALS/FTLD drugs that target TDP-43 expression and insolubility, neuroinflammation, motoneuron loss or other TDP-43 related downstream signaling pathways since these mice exhibit a later pathology as previously used ALS/FTLD mouse models. The transactive response DNA-binding protein 43  has been identified to play a crucial role in the development of neurodegenerative diseases, especially in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). TDP-43 protein has been recognized to be the main component of ubiquitinated inclusions in human ALS and FTLD samples ,2 and FTTARDBP gene mutations have been reported , most oview see ). The diview see . In ALS,view see ,12.Since 2009, a whole series of genetically altered TDP-43 mouse models were generated . The™, Germany) normal tap water ad libitum and a 50 mm square piece of pressed cotton (nestlets) as nesting material. Room temperature and humidity were kept at approximately 24°C and 40–70%, respectively. Before starting the experiments animals were group-housed, but since mice had to be single-housed for the first behavioral test, all animals were kept single housed from that time on to prevent fighting between male groups caused by regrouping. Single housing is shown to influence several aspects of the mouse phenotype like body weight, activity and anxiety but also striatal changes in dopamine and serotonin turnover levels  were weighed and homogenized for 20 seconds using the highest level of the Tissue Ruptor  in 5 volumes per weight RIPA buffer , 1x phosphatase inhibitor .Samples were further analyzed by Western blotting using antibodies against human-TDP-43 , total TDP-43 , or β-tubulin isotype III . Protein levels of tTDP-43, hTDP-43 and ß-tubulin were densitometrically quantified using Image J. For densitometric quantification, protein signals of tTDP-43, hTDP-43 and truncated TDP-43 were analyzed by Image J.™ Nuclear and Cytoplasmic Extraction Reagent . Fractionation was performed according to the manual. Samples were additionally analyzed by Western blotting using antibodies against total TDP-43 , human-TDP-43 , glyceraldehyde-3-phosphate-dehydrogenase  as marker for cytoplasmic fraction and HDAC3  as marker for nuclear fraction. Protein levels of tTDP-43, hTDP-43 and truncated TDP-43 were densitometrically quantified using Image J.Two mid brain and 2 cortex samples of 4 ntg, 4 TAR6 and 4 TAR6/6 mice at an age of 3 months were fractionated into cytoplasm and nucleus using NE-PERFive frozen brain samples of 3 months old ntg or TAR6/6 mice were thawed on ice and sonicated in 5x v/w RIPA buffer  containing protease and phosphatase inhibitor cocktails . Afterwards, samples were centrifuged at 100,000 g for 35 min at 4°C and supernatants collected as RIPA soluble fractions. The pellets were washed with RIPA buffer and sonicated in 2x (v/w) urea buffer . After centrifugation at 100,000 g for 35 min at 22°C the supernatants were taken as RIPA insoluble (urea soluble) fractions. Samples were further analyzed by Western blotting using antibodies against total TDP-43 , human-TDP-43 , or GAPDH  as loading control. Protein levels of tTDP-43, hTDP-43 and truncated TDP-43 were densitometrically quantified using Image J.Frozen brain hemispheres and paraffin-embedded spinal cords were cut sagittally or transversely, respectively. Unless indicated otherwise, a systematic uniform random set of five mounted 10 μm thick brain sections from five medio-lateral levels and three mounted 5 μm thick spinal cord sections per animal were used for immunofluorescent labeling.Sagittal sections were treated for 15 min with citrate buffer at 95°C and a 1 mg/ml sodium borohydride/PBS solution and subsequently blocked with 10% horse serum for one hour. Thereafter, sections were labeled with a human-TDP-43 antibody . Binding of the primary antibody was visualized using a highly cross-absorbed DyLight secondary antibody .Sagittal sections were treated for 15 min with citrate buffer at 95°C and a 1 mg/ml sodium borohydride/PBS solution and subsequently blocked with M.O.M. Blocking Reagent in 0.3% Triton X-100/PBS for one hour at room temperature. Thereafter, sections were labeled with a human-TDP-43 antibody , total TDP-43 antibody  and NeuN antibody  over night at room temperature. Binding of the primary antibody was visualized using highly cross-absorbed Alexa Fluor and DyLight secondary antibodies  for 1 hour at room temperature followed by nuclear staining with 4’,6-Diamidin-2-phenylindol  for 15 minutes.Sagittal sections were treated with a 1 mg/ml sodium borohydride/PBS solution followed by 1% hydrogen peroxide in methanol. To block unspecific labeling sections were treated with 10% donkey serum for 30 min. Further, sections were incubated with a GFAP  and CD11b antibody  for 1 h at room temperature. Binding of primary antibodies was visualized using highly cross-absorbed DyLight secondary antibodies  and a Cy3 secondary antibody . Cell nuclei were labeled by 4’,6-Diamidin-2-phenylindol (DAPI) counterstaining .Cervical and lumbar spinal cord sections of 3 months old animals were treated for 15 min with citrate buffer at 95°C and a 1 mg/ml sodium borohydride/PBS solution and subsequently blocked with 10% horse serum for one hour. Thereafter sections were labeled with a ChAT antibody . Binding of the primary antibody was visualized using a highly cross-absorbed DyLight secondary antibody . Cell nuclei were labeled by DAPI counterstaining .+ cells were counted manually (overlapping of ChAT and DAPI staining). For evaluation of TDP-43, GFAP and CD11b expression, regions of interest (ROI) were defined by individual delineation of the whole isocortex, spinal cord, hypothalamus and medulla oblongata. These brain regions were chosen after manual evaluation of TDP-43 expression on sagittal sections. Immunofluorescence signals were measured by adequate thresholding and morphological filtering  throughout the ROIs of all images using ImageProPlus Software (Version 6.2). Individual means were determined from three to five sections per animal. Group means were calculated by averaging the individual means. Analyzed parameter were immunoreactive area (IR) in percent of the ROI area and summarized object intensity in a.u./mm2.Grey-scale images for quantitative analysis were recorded with an AxioCam MRm camera mounted on a Zeiss Axio Imager.Z1 epifluorescence microscope at 10x magnification. Exposure time and additional setting details were kept constant within the same quantification. ChAT™ 4.03 . Graphs show group means and standard error of the mean (SEM). The significance level was set at p < 0.05. Group means were compared using Student’s t-test with Welch’s correction or a Two-way analysis of variance (ANOVA) with a subsequent post-hoc test. The utilized statistical tests and exact sample numbers are mentioned in the figure legends.Data analysis was performed in GraphPad PrismFor quantification of total, truncated and human TDP-43 protein levels, Western blot analyses of total brain, hippocampus and spinal cord tissue obtained from 1.5, 3 and 6 months old TAR6/6 and non-transgenic (ntg) littermates were performed .Total TDP-43 levels (tTDP-43) were analyzed in ntg and TAR6/6 mice . tTDP-43In addition to tTDP-43 and CTF-35, densitometric analyses of immunoreactive signals revealed high hTDP-43 levels in the central nervous system already at the age of 1.5 months in TAR6/6 mice . hTDP-43Furthermore, histological analysis of hTDP-43 expression levels in the hypothalamus, medulla oblongata and spinal cord grey matter, including anterior and posterior horn areas, revealed a high expression of hTDP-43 protein in TAR6, TAR6/6 and ntg mice already at the age of 1.5 months . We obseTo investigate the cellular localization of TDP-43 protein, midbrain and cortex tissue of 3 months old ntg, TAR6 and TAR6/6 mice was separated into cytoplasmic and nuclear fractions and analyzed by Western blot . tTDP-43Furthermore, it was analyzed whether overexpression of hTDP-43 enhances insolubility of TDP-43. For this purpose, brain samples of 3 months old TAR6/6 and ntg mice were separated into RIPA soluble and RIPA insoluble (UREA) fractions and analyzed by Western blot using antibodies against tTDP-43 and hTDP-43 . In the In order to visualize the subcellular localization of human and total TDP-43 protein as observed by cell fractionation and Western blotting, co-labeling of both proteins by immunofluorescence was performed  in 3 monNeuroinflammation has been described as one of the major hallmarks of neurodegenerative diseases (for review see ) and alsSince ALS pathology is accompanied by loss of motoneurons in the spinal cord, the number of choline acetyltransferase (ChAT) positive neurons was counted in cervical and lumbar spinal cord regions of 3 months old ntg and TAR6/6 mice . In bothTAR6/6 mice were tested for hind limb reflexes at the age of 1.5 and 3 months. Our results show that already at the age of 1.5 months TAR6/6 mice reached a significantly higher clasping score compared to ntg animals . This efTo test TAR6/6 mice for anxiety, mice were assessed in the elevated plus maze test. 1.5 and 4.5 months old TAR6/6 mice spent significantly more time in the open arms of the elevated plus maze compared to ntg littermates  indicatiTo validate that motor deficits are not caused by obesity of TAR6/6 mice, the first cohort of animals was weight once at 1.5 months of age and a second cohort from the age of 3.25 to 4.25 months. Analysis by Two-way ANOVA revealed a comparable body weight at young age but a significantly decreased body weight of TAR6/6 mice at the age of 3.25 to 3.75 months. With increasing age, this difference attenuates to comparable levels .Analysis of up to 12 months old hemizygous TAR6 mice in a similar test battery did not reveal significant behavioral abnormalities compared to ntg littermates .In summary, we observed a strong hTDP-43 overexpression followed by ALS/FTLD pathologies, such as enriched cytoplasmic and insoluble TDP-43, neuroinflammation as revealed by increased astrogliosis and microgliosis, loss of motor neurons in the spinal cord as well as pronounced motor abnormalities in homozygous TAR6/6 mice .To represent a suitable new transgenic animal model for compound testing it is essential that the model ensures a high construct and face validity. Since TDP-43 is well known to be a major factor in the development of human ALS and the here presented TAR6/6 mouse model overexpresses human wild type TDP-43 protein , the conBiochemical analysis of hTDP-43 levels showed a strong expression in the total brain, hippocampus and spinal cord of TAR6/6 mice and a slightly weaker expression in TAR6 mice. The expression pattern of hTDP-43 is comparable with evaluated tTDP-43 levels which are 1.5 to 2.5 fold increased depending on the tissue. The overexpression might seem to be rather weak but Ayala and colleagues previously showed that endogenous TDP-43 levels can be downregulated by binding of overexpressed TDP-43 in the 3’UTR region of the endogenous mRNA . We hypoet al. [et al. analyzed inflammation processes in the TAR6/6 mice by GFAP and Iba-1 staining. The group also revealed activation of microglia in 6 months old animals but additionally activation of astrocytes by GFAP at the age of 6 months in the motor cortex layer V and anterior horn of the spinal cord [Overall histological analyses showed similar expression levels of TDP-43 in different brain regions. We observed an increase of hTDP-43 in the hypothalamus, medulla oblongata and spinal cord up to an age of 3 months, followed by a slight decrease at the age of 6 months which was observed by Western blot in the spinal cord but not in brain and hippocampal tissue. The decrease in TDP-43 expression at higher age might be caused by a loss of hTDP-43 expressing neurons. The reduced ChAT labeling in the spinal cord of TAR6/6 samples thus indicates an increased motoneuron death which is accompanied by neuronal loss of motor cortex layer V and anterior horn neurons detected by analyzing Cresyl violet staining as described by Wils et al. . Furthernal cord . The obsnal cord , the SODnal cord , human Anal cord  or humannal cord . Neuroin89 [et al. [Although expression levels of hTDP-43 and inflammation processes in hemizygous TAR6 and homozygous TAR6/6 mice differ, our biochemical and histological data consistently indicate that TDP-43 is strongly enriched in the nucleus whereas immunoreactivity is much lower in the cytoplasm. Of note, we did not detect a shift of subcellular hTDP-43 from the nucleus to the cytoplasm. On the contrary, the genotypes display significantly increased nuclear hTDP-43 although in human ALS/FTLD samples the absence of nuclear TDP-43 has been described and correlated to crucial pathological features of ALS/FTLD . Further89 ,36 which [et al. . The fraIn another experiment, the influence of overexpressed hTDP-43 on the solubility of TDP-43 was investigated. In 3 months old TAR6/6 mice, tTDP-43 as well as hTDP-43 was highly enriched in the RIPA insoluble (UREA) fraction. Thus, insoluble TDP-43 was accumulated in brains of TAR6/6 animals and mimics one of the well described hallmarks of human ALS/FTLD pathology . The 35 To demonstrate a similarity of behavioral deficits between TAR6/6 mice and ALS patients, several specific tests were performed. Mice not only presented altered reflexes and muscle weakness but also strong deficits in motor coordination as shown with the Rota Rod test. These results are comparable with muscle weakness, gait disturbance, coordination problems as well as weakness in arm and leg muscles described in ALS patients (reviewed in ). BesideTAR4/4 mice, which were in detail characterized by Wils and colleagues, have an average survival of only 24 days while TAR6/6 mice live on average 6–7 months . This ex+ neurons in the spinal cord , medulla oblongata (B) and spinal cord (C) analyzed by immunofluorescent labeling of CNS samples.  1.5 months: ntg: n = 4; TAR6: n = 10; TAR6/6: n = 5; 3 months: ntg: n = 5; TAR6: n = 8; TAR6/6: n = 5; 6 months: ntg: n = 3; TAR6: n = 3; TAR6/6: n = 3. (C) n as in A, B exempt: 1.5 months: ntg: n = 3; TAR6: n = 11. (A-C) Two-way ANOVA followed by Bonferroni‘s post-hoc test. Mean+SEM. *significances between genotypes, #significances between age groups. *p<0.05, **p<0.01, ***p<0.001.(PDF)Click here for additional data file.S2 Fig(A) Cortical, (B) medulla oblongata, (C) spinal cord GFAP expression levels labelling reactive astrocytes and (D) CD11b expression levels labeling reactive microglia. Representative images of 1.5 months old  and 6 months old (D) animals. Cell nuclei are stained by DAPI.(PDF)Click here for additional data file.S3 Fig(A) Clasping behavior scored from 1–3 of 1.5 months old TAR6 mice . (B) Latency to fall in the wire suspension test in seconds of 1.5, 9 and 12 months old TAR6 mice. (C) Latency to fall from the rotating rod in the Rota Rod test of 1.5 months old TAR6 mice. (D) Nesting behavior score of 1.5 months old TAR6 and TAR6/6 mice (high score: perfect nest). (E) Time in seconds 1.5 months old TAR6 mice spent in the open arms of the elevated plus maze. (F) Body weight in gram of 1.5, 9 and 12 months old TAR6 mice.  TAR6: n = 11, ntg: n = 7; (E) TAR6: n = 11, ntg: n = 6. (D) TAR6: n = 9, ntg: n = 4, TAR6/6: n = 4.  1.5 months: TAR6: n = 11, ntg: n = 7; 9 months: TAR6: n = 15, ntg: n = 12; 12 months: TAR6: n = 15, ntg: n = 13; Mean+SEM. A: One sample t-test; C-E: unpaired t-test. B,F: Two-way ANOVA; A-F: non significant (p>0.05).(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Crosslinked gelatin nanofibers are one of the widely used scaffolds for soft tissue engineering. However, modifying the biodegradation rate of chemically crosslinked gelatin is necessary to facilitate cell migration and tissue regeneration. Here, we investigated the optimal electron beam (e-beam) irradiation doses with biodegradation behavior on changes in the molecular weight, morphology, pore structure, and cell proliferation profiles of electrospun nanofibrous gelatin sheets.The molecular weights of uncrosslinked gelatin nanofibers were measured using gel permeation chromatography. The morphology and pore structure of the gelatin scaffolds were analyzed by scanning electron microscopy and a porosimeter. Biodegradation tests were performed in phosphate-buffered saline solutions for 4 weeks. Cell proliferation and tissue regeneration profiles were examined in fibroblasts using WST-1 assays and hematoxylin and eosin staining.Crosslinked gelatin nanofiber sheets exposed to e-beam irradiation over 300 kGy showed approximately 50% weight loss in 2 weeks. Gelatin scaffolds exposed to e-beam irradiation at 100–200 kGy showed significantly increased cell proliferation after 7 days of incubation.These findings suggested that the biodegradation and cell proliferation rates of gelatin nanofiber scaffolds could be optimized by varying e-beam irradiation doses for soft tissue engineering. Gelatin, a protein derived from controlled hydrolysis of collagen, is potentially useful as a biomaterial because of its biological origin, biocompatibility, non-antigenicity, and biodegradability. Furthermore, the existence of unique sequences of amino acids and many functional side groups makes it easy to modulate its physicochemical properties –3; thus,Among reported physical crosslinking methods, ionizing irradiation treatment with γ-ray or electron beam (e-beam) radiation is operationally fast and does not require additives or severe conditions, which could deform the material structure –20. MoreAlthough some studies have assessed the effects of γ-radiation on gelatin, very few studies have investigated the effects of e-beam irradiation on gelatin –17. Accow/v) by stirring overnight at room temperature. Electrospinning was performed using a 10-mL polypropylene syringe with a 21-gauge needle. A high voltage of 15 kV was applied to the needle and stainless steel drum collector (drum diameter: 23 cm). The distance between the needle and collector was set as 12 cm. The flow rate of the gelatin-TFE solution was 2 mL/h. The electrospun sheets were placed in a vacuum oven overnight at room temperature to remove a residual solvent.Type B gelatin (250 bloom) was obtained from Geltech Co., Korea, and 2,2,2-trifluoroethanol (TFE) was purchased from Sigma-Aldrich (USA). Gelatin nanofiber sheets were fabricated using electrospinning of a gelatin-TFE solution. Gelatin was dissolved in TFE at 10% . The vapor crosslinking process was carried out for 6 h in a sealed desiccator with 25% glutaraldehyde (10 mL) in a petri dish [2 atmosphere at room temperature. The irradiation doses were 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, and 300 kGy. For crosslinked gelatin, irradiation of the e-beam was performed in air at room temperature. The doses were 100, 200, 300, 400, 500, and 600 kGy. For each treatment, the accelerating voltage of the e-beam energy and current were 1 MeV and 17 mA, respectively, and the dose rate of the e-beam was 8.33 kGy/s.E-beam irradiation of the gelatin nanofibrous sheets was performed using an Electron Beam Accelerator . Uncrosslinked and crosslinked gelatin samples were irradiated to investigate the effects of e-beam radiation. For uncrosslinked gelatin, irradiation of the e-beam was carried out in an air and NThe surface morphology of crosslinked and uncrosslinked gelatin samples was observed using a scanning electron microscope . Crosslinked samples were immersed in purified water and lyophilized to observe the fibrous structure of gelatin. Dry samples of gelatin were observed with 11 kV accelerating voltage after sputter coating with platinum. The diameters of nanofiber strands were determined using an image measurement software .The pore size and porosity of the gelatin nanofiber sheets were determined by multiple point Brunauer, Emmet and Teller (BET) gas adsorption measurement using NOVA 2000 and Autosorb-1-C instruments .3 as a solvent with a flow rate of 0.8 mL/min. The GPC calibration curve was obtained using pullulan standards.Changes in the molecular weight of each e-beam-irradiated uncrosslinked gelatin sheet were determined using gel permeation chromatography  at 40 °C with a refractive index detector. We used 0.02 N NaNOw/v) sodium bicarbonate solution  and 1.0 mL of 0.5% (w/v) in methanol. After incubation at 40 °C for 2 h, the solution was treated with 3 mL of 6 M HCl, and the temperature was increased to 60 °C for 1.5 h to solubilize gelatin samples. The absorbance of the resulting solution was measured at 345 nm using a spectrophotometer  after diluting with 4 volumes of deionized water. The degree of crosslinking was calculated using the following equation [The degree of crosslinking was determined using 2,4,6-trinitrobenzensulfonic acid  by calculating the primary amine content of crosslinked and uncrosslinked gelatin samples. Briefly, 2–4 mg of gelatin was placed in a conical tube with 1.0 mL of 4% (equation :\documenn = 3).where the subscripts c and u stand for the crosslinked and uncrosslinked gelatin, respectively  and incubated in a shaking water bath (37 °C with shaking at 0.15 m/s) for 1, 2, 3, or 4 weeks. After each designated period, samples were rinsed twice with purified water and lyophilized. Thereafter, the weight after the degradation test was measured, and the percent weight loss was calculated using the following equation:Wi is the initial weight of the gelatin sheet, and Wf is the weight of the gelatin sheet after the degradation test (n = 5).where n = 5).The hydrophilicity of the e-beam irradiated gelatin sheets was evaluated using a contact angle analyzer  by measuring the complete water absorption time from a deposited droplet of purified water onto the surface of the irradiated gelatin nanofiber sheets at 25 °C and 50% humidity. The results were expressed as the mean ± standard deviation  containing 10% fetal bovine serum  and 1% antibiotics . Seeded cells were cultured for 1, 3, 5, or 7 days under standard cell culture conditions. After the designated culture period, the culture medium was replaced with 1 mL of medium containing 10% WST-1 reagent. Subsequently, samples were incubated under standard cell culture conditions for 2 h, and 100 μL of the incubated solution was then transferred to a 96-well culture plate. A microplate reader  was used to measure the absorbance of formazan at 450 nm.Fibroblasts (NIH-3 T3 cells) were used to evaluate cell proliferation on e-beam-irradiated gelatin nanofiber scaffolds. Samples were cut into a disk shape (22 mm in diameter), immersed in 70% ethanol for sterilization for 24 h, and then rinsed with PBS for 24 h before seeding. Prepared gelatin fibrous sheets were placed in 12-well culture plates with glass rings to prevent floating. Cells were seeded at a density of 5.0 × 105 cells/cm2 with 15 mL cell culture medium. Cell-seeded gelatin scaffolds were rinsed with medium to eliminate detached cells and incubated for 3 weeks in a CO2 incubator, with daily changes in culture medium. After incubation, the tissue-scaffold constructs were fixed in 4% formaldehyde aqueous solution and processed for routine paraffin embedding and sectioning. The cell cultured gelatin scaffold sections were stained with hematoxylin and eosin (H&E) to evaluate migration into the gelatin sheet and observed under an optical microscope  equipped with a digital camera .Nonirradiated and irradiated (200 kGy) crosslinked gelatin scaffolds were used for tissue culture experiments at a density of 5.0 × 10t-tests. Results with p values of less than 0.05 were considered significant.Values are expressed as means ± standard deviations. Statistical analysis was performed using Student’s 2; Fig. Electrospun gelatin nanofibers showed an even cylindrical shape with an average fiber diameter of 443 ± 114 nm Fig. . Since g2 atmosphere or at the entire range of irradiation dosages in air gradually decreased in a dose-dependent manner. In contrast, the molecular weights of gelatin nanofibers irradiated at less than 60 kGy in an N2 atmosphere were increased in comparison with those of nonirradiated gelatin nanofibers.However, changes in the molecular weight (Mw) of gelatin fibers as a function of e-beam irradiation dose were observed Fig. . The molThe microstructures of crosslinked gelatin nanofibers with glutaraldehyde vapor following e-beam irradiation are shown in Fig. The degree of crosslinking after electron beam irradiation with varied irradiation doses  was, 40 ± 3, 37 ± 3, 35 ± 2, 27 ± 4, 22 ± 5, 16 ± 4%. The degree of crosslinking in nonirradiated crosslinked gelatin was 48 ± 4% and decreased as e-beam irradiation increased, reaching a minimum value of 15.5%. The results indicated that the high energy of e-beams promoted the cleavage of the chemical bonds of gelatin, including the site of crosslinking. However, the morphologies of crosslinked gelatin nanofibers after e-beam irradiation were not significantly different from those of nonirradiated crosslinked gelatin Fig. .Biodegradation behavior as a function of e-beam irradiation in a nonenzymatic aqueous system was determined using irradiated gelatin sheets with increasing radiation dosages. The percent weight loss of crosslinked gelatin sheets irradiated with 100, 200, 300, 400, 500, and 600 kGy after incubation in nonenzymatic PBS solution at 37 °C for 1, 2, 3, or 4 weeks is shown in Fig. Morphology as a function of incubation time is shown in Fig. To better understand the effects of e-beam irradiation on surface hydrophilic properties, the water absorption rate of nonirradiated and irradiated gelatin sheets was measured on a series of samples with increasing irradiation dosages Fig. . GeneralNIH3T3 fibroblasts were cultured on irradiated gelatin sheets to determine the compatibility and proliferation of the cells. WST-1 assays were utilized to evaluate the number of metabolically active cells cultured on e-beam-irradiated gelatin sheets Fig. . InitialHistological images of the cell-seeded gelatin nanofiber scaffolds after 4 weeks of culture are shown in Fig. 2 atmosphere are increased at under 60 kGy (Fig. In this study, we focused on not only nanofibrous morphology of gelatin sheet scaffolds but also degradation of gelatin molecules. For instance, we investigated optimum e-beam radiation for molecular degradation as a gelatin nanofiber sheet scaffold. Electrospun gelatin fibrous sheets treated with e-beam irradiation maintained fibrous networks without morphological deformation for both uncrosslinked and crosslinked gelatin Figs.  and 3. ECrosslinked gelatin nanofibers enabled modification of the polymeric molecular chain without microstructural deformation by irradiation with e-beams. The degree of crosslinking is mainly correlated with the lifespan of the biomaterials, and highly crosslinked biomaterials can resist both enzymatic degradation and hydrolysis because the degradation rate is generally related to molecular weight and crosslinking density. Accordingly, e-beam irradiation could be an effective method for adjustment of degradation rates in vitro and in vivo. Notably, amide bonds are more resistant to hydrolysis than ester bonds, and biodegradation of crosslinked proteins rarely occurs in vitro since in vitro conditions are different from the conditions observed in vivo. For this reason, scaffolds made of crosslinked proteins can be implanted into animals or can use enzymes for degradation.In crosslinked gelatin, high irradiation dosages decreased the crosslinking density and affected the biodegradation Figs.  and 5 anChanges in water absorption rates were correlated with crosslinking and irradiation. In collagenous materials, previous studies showed that the water absorption ability decreased after crosslinking because the reaction between the ε-amino group and functional group in a crosslinking agent could weaken protonation. As mentioned above, polymeric chain scission and decreased crosslinking after e-beam irradiation could cause increased water absorption ability. Another reason is the direct correlation with e-beam irradiation. After e-beam treatment, the polar component and surface tension of gelatin increased, and this enhancement was related to the decrease in surface hydrophobicity because of reorganization of hydrophilic groups or generation of charged groups on the surface.The scaffold should keep their physical and chemical structure before cell attachment and proliferation. Then, the scaffolds are degraded according to meet tissue formation. The immediate effect of e-beam irradiation and increased hydrophilicity after e-beam treatment on cell attachment and proliferation were not observed Fig. . HoweverMigration of proliferated cells into a scaffold is essential for cultivation of soft tissues. To facilitate migration of fibroblasts into the gelatin nanofiber scaffolds, biodegradation should be induced in a week to ensure the inner space of proliferated cells and transportation of nutrients, oxygen and wastes. The gelatin nanofiber scaffold treated with 300 kGy e-beam irradiation showed tissue migration compared to the control scaffold. Based on our SEM, weight loss, cell proliferation and tissue formation results, the 300 kGy e-beam radiation dose demonstrated appropriate condition for soft tissue engineering application.2 atmosphere were crosslinked by high-energy e-beam irradiation. In crosslinked gelatin, high irradiation dosages decreased the crosslinking density and affected the biodegradation and hydrophilicity of gelatin. Physicochemical properties were altered by e-beam irradiation, but the immediate effects of e-beam irradiation on cell attachment and proliferation were not observed. E-beam irradiation could modify the properties of both of uncrosslinked and crosslinked gelatin nanofibrous sheets by changing macromolecular chains without microstructural deformation. In particular, irradiation of crosslinked gelatin resulted in biodegradation without enzyme in PBS, and the lifespan of crosslinked gelatin could be modulated by adjusting the irradiation dosage, demonstrating its potential applications in tissue engineering.Electrospun gelatin fibrous sheets treated with e-beam irradiation maintained fibrous networks without morphological deformation for both uncrosslinked and crosslinked gelatin. The molecular weight data of uncrosslinked gelatin as a function of e-beam irradiation revealed that gelatin nanofibers irradiated at under 60 kGy in an N