Drosophila melanogaster to examine the roles that capping, splicing, cleavage/polyadenylation, and telescripting  might play in shaping exon-intron architecture in protein-coding genes. Our findings suggest that the distance between subsequent internal 5′ splice sites (5′ss) in Drosophila genes is constrained such that telescripting effects are maximized, in theory, and thus nascent transcripts are less vulnerable to premature termination. Exceptionally weak 5′ss and constraints on intron-exon size at the gene 5′ end also indicate that capping might enhance the recruitment of U1 and, in turn, promote telescripting at this location. Finally, a positive correlation between last exon length and last 5′ss strength suggests that optimal donor splice sites in the proximity of the pre-mRNA tail may inhibit the processing of downstream polyadenylation signals more than weak donor splice sites do. These findings corroborate and build upon previous experimental and computational studies on Drosophila genes. They support the possibility, hitherto scantly explored, that mRNA-associated processes impose significant constraints on the evolution of eukaryotic gene structure.mRNA-associated processes and gene structure in eukaryotes are typically treated as separate research subjects. Here, we bridge this separation and leverage the extensive multidisciplinary work on  Protein-coding genes in eukaryotes have a paradoxical structure. They contain sequences that need to be removed from precursor messenger (pre-m)RNAs to enable the synthesis of functional proteins. These intragenic noncoding sequences, known as spliceosomal introns , a nonprWhile several genetic and epigenetic factors influence intron recognition and splicing . The 5′sRNA splicing is not an isolated process in the cell. Rather, it interacts tightly with several co- and post-transcriptional processes, including mRNA capping, and 3′ end cleavage/polyadenylation, among others. It is well established that these molecular interactions are instrumental for the accurate processing of nascent transcripts . What isFirst, empirical evidence shows that at the pre-mRNA 5′ end the cap-binding complex (CBC) enhances the association between U1 and the cap-proximal 5′ss in human and yeast .Drosophila) that 5′ss-bound U1 protects pre-mRNAs from premature cleavage/polyadenylation at nearby polyadenylation signals (PAS) . Our working dataset contains 7256 protein-coding genes with at least two introns , for a total of 35,896 introns.We extracted information about gene structure using in-house python (python.org), bash (shell language), and R scripts (r-project.org). We randomly selected one isoform (rather than focusing only on the longest isoforms), and considered only protein-coding genes. Our dataset includes constitutively and alternatively spliced introns that contain no nested genes and whose size is equal to, or greater than, 32 nt . Finally, to focus on RNA splicing mediated by the major spliceosome, we discarded introns with noncanonical splice sites , we estimated subsequent medians of 2000 observations for the remaining variables, using a step size of 1. So, each point on the graph is supported by exactly 2000 observations. Sliding-window analyses were conducted using default functions  in R. All the reported correlation coefficients are based on raw data.We used a sliding-window approach to illustrate the trend of the examined correlations, similar to that described in e.g., the sense DNA strand, Ns) is equal to the number of their reverse complements on the opposite strand . The degree of DSA supplies a measure of the selective pressure exerted on a motif of a particular DNA strand  using published protocols  equivalent numbers of introns and exons. Any intron in a group had the same probability to be chosen, and to be associated with any following (second) exon. A nonparametric two-sample Kolmogorov-Smirnov test was used to measure the level of discrepancy between the actual and the random size distributions. Kolmogorov-Smirnov P-values were estimated for each of the 100,000 simulations. An overall unique P-value was estimated as N/100,000, i.e., the number of times that the test was nonsignificant  divided by the number of simulations.We tested the hypothesis that the size of the introns at specific positions , and that of their next exon, are nonrandomly associated. To this end, we first partitioned our dataset into three groups, https://github.com/GildasLepennetier/GildasSources.The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article. All of the scripts are publicly available through GitHub at At the pre-mRNA 5’ end, the CBC enhances the association of U1 with the cap-proximal 5′ss . Separate analyses of first, internal, and last introns, however, reveal that the positive relationship between 5′ss strength and intron size is relatively weak, though statistically significant, at the gene 5′ end . Moreover, the association between first intron size and the 5′ most 5′ss even becomes negative when introns are near the CBC   . AlthougP < 0.001) (P < 0.001). In contrast, the strength of 5′ss and the size of the upstream exon are not correlated in the gene body , and they are only weakly correlated at the gene 3′ end . These results suggest that the putative splicing-enhancing effects of the CBC in Drosophila might vanish with distance. Our observations hint at a maximum interval over which the putative splicing-enhancing effects of the CBC might extend . Beyond this distance, both the strength of first 5′ss and first exon length are tightly and positively coupled with first intron size.It has also been reported that the splicing-enhancing effects of the CBC may be distance dependent (< 0.001) . The rob< 0.001) —whereas Drosophila, as well as human and mouse, 5′ss-bound U1 counteracts premature termination by cleavage/polyadenylation at the nearby PAS, a process known as telescripting  (P < 0.001) (P < 0.001) (P < 0.001), and 2) weakly increases when second exons are larger than 200 nt . These observations demonstrate that intron-exon structure may differ dramatically at the gene 5′ end. They reveal constraints that fall within the action range of telescripting in Drosophila, i.e., the sizes of introns and their next exon covary such that the median length of the IpE unit is ≤ ∼500 nt  the size of first IpE units, and 2) the relationship between the size of introns to their next exon within these units. We examined first IpE units whose 5′ end donor splice site resides either within or outside the 5′-most 250 nt separately . This partition might help uncover putative interactions between the processes of capping and telescripting. We found that the size of cap-proximal IpE units is uniform (median: 553 nt) irrespective of the strength of the upstream 5′ss  . In cont< 0.001) . Further< 0.001) , the rel< 0.001) . More spUnder a model where selection pressure keeps two subsequent 5′ss at a distance that minimizes the risk of premature cleavage/polyadenylation, the CBC-enhanced recruitment of U1 should produce two effects: 1) it should compensate for the weak strength of cap-proximal splice sites, and 2) it should maximize the protective effects of telescripting at the gene 5′ end. Under this same model, large (>> 500 nt) IpE units are at major risk of premature cleavage/polyadenylation, unless additional protective features come into play (see below).e.g., when exon size is ≤ 400 nt: 14,428 observations; Kendall’s tau = –0.07, P < 0.001), whereas they are positively, albeit very weakly, correlated beyond this range   and 735 nt (3′-end), Kruskal-Wallis test, P < 0.001]. Finally, the size distribution of internal IpE units differs significantly from a set of distributions generated by randomly sampling introns and exons from our dataset (P < 0.001) . These observations suggest that there are constraints on the internal intron-exon structure of protein-coding genes in D. melanogaster that approximate the action range of telescripting in this species  . AdditioThe median size of internal IpE units increases alongside the strength of the 5′-end 5′ss, ranging from 294 nt (weak 5′ss), through 328 nt (weak to moderate) and 374 nt (moderate to strong), to 513 nt (strong 5′ss) . This trDrosophila introns is indeed negative (–0.06) , and ∼4-fold more negative compared to the DSA value of two AATAAA anagrams, TAAAAA, and AAAAAT (data not shown). The average DSAAATAAA is also > 3-fold more negative at the gene 3′ end than at the 5′ end . Finally, levels of counter-selection of intronic AATAAA decrease  when the strength of the upstream 5′ss increases (Table S2). These patterned variations suggest that the risk of premature polyadenylation and/or the efficiency of telescripting might differ between the gene 5′ end and 3′ end, and that the strength of the 5′ss may indeed bear on the action range of telescripting.We found that the average DSA of AATAAA within  (–0.06) . In intrAATAAA = 0.21)—where it may serve to regulate gene expression . As rationalized above, the excess of cryptic PAS at the gene 5′ end may reflect efficient U1-mediated silencing of polyadenylation in this region. Elegant work by We also examined the degree of DNA strand asymmetry of AATAAA in exons . AATAAA AATAAA in first or second exons and the size of the intervening intron . This observation provides some ground to speculate on why shielded PAS at the gene 5′ end might be favored (see Discussion).Finally, we detected a positive relationship between DSASplicing can be problematic for accurate 3′ end formation in that the suppressive effects of 5′ss-bound U1s might perturb the accurate processing of downstream cleavage and polyadenylation signals, particularly when the U1-bound 5′ss is very close to the polyadenylation site . Nowhere else along the gene do we detect a significant correlation between exon size and upstream 5′ss strength . This indicates that increasing exon length significantly contributes to the positive coupling between the size of last  IpE units and the strength of the 5′-end 5′ss , then, in order for accurate mRNA 3′ end processing to take place, progressively stronger last-intron 5′ss should be coupled with increasingly large next exons.Furthermore, we found that the strength of the last 5′ss is significantly and positively correlated with the size of the last exon (after correcting for last intron size) , end 5′ss . This obP < 0.001). This observation is surprising given the exceedingly small size of last introns in Drosophila , in line with previous findings (Table S2). This negative selection notwithstanding, about 4% (n = 1417) of the surveyed introns  contain at least one GGTAAG, and the motifs GGTAAG and AATAAA co-occur more frequently 1) in > 500-nt introns compared to smaller introns , and 2) downstream from, rather than within, the first 500 nt, in ∼1000-nt introns . Finally, we observed that the AATAAA motif resides preferentially toward the intron 3′ end, downstream (rather than upstream) from GGTAAG . Taken together, these findings lend some support to the hypothesis that U1 binding to cryptic 5′ss in Drosophila’s large introns may help suppress cleavage/polyadenylation at otherwise unshielded cryptic PAS.We found that the 5′ss-related GGTAAG motif is counter-selected in introns and exons , a distant relative of D. melanogaster (∼10 million yr of sequence divergence). These analyses corroborate virtually all of the relationships and trends that we document for D. melanogaster . Two discrepancies, which we detected, do not seem to have significant consequences for our overall conclusions. First, we found no significant negative association between the size of cap-proximal first introns and the 5′ most 5′ss for D. yakuba . Second, for D. yakuba the size of cap-proximal IpE units may vary depending on the strength of the upstream 5′ss (rather than being uniform as is for D. melanogaster). The magnitude of this variation in size is marginal however .To assess the validity of our study, we replicated most of our analyses on the annotated genome of Drosophila. The principal conclusions of this study are summarized in We have presented several findings which suggest that interacting mRNA-associated processes, including capping, splicing, cleavage/polyadenylation, and telescripting might play a role in molding the structural properties of protein-coding genes in Drosophila genes that, in theory, maximize telescripting effects, thus minimizing the risk of premature cleavage/polyadenylation in nascent transcripts. At the gene 3′ end, on the other hand, our observations suggest that telescripting effects are often circumvented, so that the process of 3′ end formation is facilitated.We have detected size constraints in the internal regions and at the 5′ end of AATAAA might also indicate that intron-bound cleavage/polyadenylation factors perturb splicing, e.g., intron-bound cleavage/polyadenylation factors might disturb the recruitment of U1 at the upstream canonical donor splice site. If so, then weakly counter-selected intronic polyA sites would preferentially occur downstream of strong (rather than weak) donor splice sites, which facilitate the recruitment of U1. This scenario is consistent with previously proposed models that invoke competing splice and cleavage and polyadenylation reactions  is more pronounced in IpE units with weak 5′-end 5′ss compared to IpE units with strong 5′-end 5′ss. This observation may indicate that strong 5′ss protect pre-mRNAs from premature termination more efficiently than weak 5′ss. In line with this scenario, introns with a weak 5′ss are more likely to undergo alternative cleavage/polyadenylation compared to introns with a strong 5′ss , and rather strong at the gene 3′ end . It follows that, whereas splice site strength and intron size are overall positively correlated within a certain intragenic location, across intragenic regions this relationship may disappear . We interpret this pattern of longitudinal decoupling between intron size and splice site strength as indicating that splicing in Drosophila is assisted at the pre-mRNA 5′ end but not at its 3′ end.Our analyses reveal significant variations in the average splice site strength along the genes of Drosophila genes is that splicing efficiency differs among introns, and that first introns are more prone to be inefficiently spliced, at least in part because of their relatively weak 5′ splice sites. This view provides an additional explanation for why first introns in Drosophila are retained more frequently than others . This selection for last exon expansion would have the beneficial effects of keeping the 3′ termination signals and the upstream 5′ss-bound U1 separated by a distance that is sufficiently large to circumvent telescripting effects and, thus, to facilitate both cotranscriptional splicing and mRNA 3′-end formation. Should telescripting effects not be adequately circumvented, splicing at the mRNA tail could still be completed post-transcriptionally, which is consistent with the observed excess of post-transcriptional splicing at the mRNA 3′-end compared to the upstream regions  complicates attempts to draw conclusions concerning underlying molecular dynamics, and might yield erroneous interpretations. In consequence, we cannot definitively rule out the possibility that some of the evidence presented in support of the proposed role of mRNA-associated processes in molding gene structure may be inaccurate. With this caveat in mind, we attempted to eliminate false signals whenever possible, using partial correlations or control sequence motifs. Not only do our conclusions withstand this scrutiny, they are also sustained by the coherence of the detected patterns. These patterns are in accord with U1-dependent definition  of the variables that we have investigated in this study , and potentially extend the repertoire of these traits with novel hypotheses that can be verified experimentally. In one example, the degree of DNA strand asymmetry of cryptic PAS motifs might serve as a simple but powerful tool to detect and measure in silico the efficiency of telescripting effects within the intragenic regions of eukaryotic species.The features and the relationships that we document support the idea that intracellular processes in conjunction with population-genetic forces may shape gene structure in Drosophila may extend to other eukaryotes is an open question that warrants further investigation. Many eukaryotic species, such as humans for example, have considerably longer introns compared to Drosophila and there is compelling experimental evidence that in human and mouse U1 telescripting effects extend for ∼1000 nt (rather than 500 nt as in D. melanogaster). Human and mouse are therefore excellent candidate systems to examine. These investigations would also furnish a test of the validity and the generalizability of the patterns observed for Drosophila.The extent to which the proposed effects of mRNA-associated processes in e.g., distinct levels and types of DNA methylation (We anticipate that much will be gained from examining the features that distinguish fruit flies from other species. Between-species differences in gene structure might flag different properties of the intracellular environment (hylation , and/or


We examine the unusual dispersion and attenuation of transverse electromagnetic waves in the few-THz regime on nanoscale graphene and copper transmission lines. Conventionally, such propagation has been considered to be highly dispersive, due to the RC time constant-driven voltage diffusion below 1 THz and plasmonic effects at higher optical frequencies. Our numerical modeling across the microwave, THz, and optical frequency ranges reveals that the conductor kinetic inductance creates an ultra-broadband linear-dispersion and constant-attenuation region in the THz regime. This so-called LC region is an ideal characteristic that is known to be absent in macro-scale transmission lines. The kinetic-LC frequency range is dictated by the structural dimensionality and the free-carrier scattering rate of the conductor material. Moreover, up to 40x wavelength reduction is observed in graphene transmission lines. Transmission lines, one of the simplest forms of single-mode electromagnetic waveguides, are widely used for signaling in digital, analog, and optoelectronic systems. Transmission lines generally consist of two or more parallel conductors that guide a transverse electromagnetic (TEM) wave over many wavelengths1368910Two recent advancements have made it intriguing to examine the dispersion of TEM waves in nanoscale transmission lines: state-of-the-art fabrication processes have allowed the dimension of transmission lines to fall below the skin depth; graphene has emerged as a practical conductor with large kinetic inductance1314151617212224252427236–fold variation in dimensions (nm through cm) and 104–fold variation in frequency (10 GHz–100 THz), we examine the conditions for the kinetic inductance to produce a surprisingly broadband LC region for small-scale transmission lines. Investigating both graphene and copper conductors, we develop a map of the performance regions for 2D and 3D materials, contrast the significant differences in their scaling laws, and explain dominant physical mechanisms that limit their performance.In this article, we carry out a first-principle numerical and analytical study on the effect of material kinetic inductance on the propagation and attenuation of the transverse electromagnetic modes in nanoscale and microscale transmission lines. We explore the potential of the kinetic inductance in providing a usable broadband LC response for signaling, transducing and sensing applications. Over a vast space, spanning 10t, and the graphene ribbons are treated as an infinitesimally-thin surface conductor. At THz frequencies and below, both materials are accurately modeled with a Drude conductivityAmong various transmission line structures, we focus on the coplanar stripline (CPS) shown in 30σ0 corresponds to the bulk DC conductivity for copper and surface conductivity for graphene. For the purposes of this paper, graphene has been modeled as an infinitesimally-thin conducting surface. Scattering due to surface roughness, finite temperature effects and spatial dispersion are neglected. The effective surface conductivity  and spatial phase evolution (β), respectively. The circuit parameters C, R, R, but at low enough frequencies where R, R and The underlying physical mechanisms leading to frequency-dependent attenuation in transmission line modes are captured by the circuit parameters in the solution to the telegrapher’s equations. Since we limit our discussion to lines with lateral dimensions that are much smaller than the free-space wavelength, this analytical model is accurate and yields comparable results to ab-initio solutions to Maxwell’s equations1In contrast to the Faraday inductance, the kinetic inductance stems from a material constant, the complex-valued conductivity. The kinetic inductive impedance is proportional to the imaginary part of the reciprocal of the conductivity, while the resistive impedance relates to the real part. Physically, they contribute to the power stored or dissipated, respectively, in the conduction currents:J is the surface or volume current density produced by an electric field, E. From this equation follows an important relation between the kinetic inductance and the resistance:A (or width w in the case of graphene), but unlike the resistance, it is related to the imaginary part of the inverse conductivity, This ratio reduces to a material constant and hence, may be frequency dependent but is completely independent of the geometry of the transmission line. Therefore, similarly to the resistance, the kinetic inductance is also inversely proportional to the current carrying area, R and increases with current crowding ; lines with a dominant kinetic inductance suppress current crowding because current favors a distribution of lowest total impedance.When the conductivity is represented by a Drude model, m and Nanoscale lines are where the kinetic inductance typically exceeds the Faraday inductance. Generally, the Faraday inductance of a transmission line is scale-invariant, for 3D conductors andfor 2D conductors. For the transmission line configurations chosen in this paper, w < wK and ωoperating > Γ.In lines with conductors that are smaller than the thresholds given in w = 20 nm to 500 nm. The conductor separation (as well as the copper conductor thickness) are equal to w/2, i.e. below the threshold predicted by w = 20 nm and w = 500 nm graphene CPS lines have an LC onset frequency that is exactly equal to the intrinsic scattering rate of graphene (1 THz). In the w = 500 nm copper CPS, although R and R and First-principle modeling with Maxwell’s equations, via the finite element method (see Methods section), confirms the above conditions through the inductive and resistive impedances  that havR in w = 50 nm, exhibits a clearly dominant kinetic inductance while the w = 100 nm CPS lies on the threshold that was predicted by The above line classifications are also reflected in the spectra of ne CPS’s , regardlThe high frequency boundary of the LC region in nanoscale transmission lines is determined by a different set of mechanisms than in micro and macroscale transmission lines. In copper and other metals, for frequencies below their intrinsic scattering rate, Γ the skin depth is collisional and scales as not defined by the skin effect. At high frequencies, as In nanoscale graphene lines, the upper frequency limit for the LC region is mon band , the selFor graphene  and coppw−2 dependence in the copper CPS and a more gradual w−1 dependence in the graphene CPS. At very low frequencies in the RC region, the current distribution is highly uniform and the mode energy is concentrated predominantly in the region between the conductors (The lower bound of the LC region (the dark red curve), nductors .w−2 geometric dependence of the LC region onset. In copper at frequencies above the intrinsic scattering rate, this boundary becomes a vertical asymptote as the skin depth converges to its constant collisionless valuew−0.5 dependence, while below the graphene scattering rate, it closely follows the RC-LC boundary. This strongly suggests that graphene ribbons can exhibit edge-current crowding whenever their widths exceed 100 μm.The upper bound of the LC region (the dark green curve) is defined by the onset of the skin or edge effect region where the current shifts strongly towards the surface of the conductor . For theThe third boundary for graphene (the dark blue curve) separates the skin/edge effect region from the plasmonic region. In the plasmonic region the transmission line becomes multi-moded since the geometry is capable of supporting individual and hybridized ribbon plasmons. For graphene, this boundary occurs when the wavelength of an infinitely-wide-graphene plasmon falls below twice the ribbon width, and runs parallel to the LC-edge-effect boundary. This implies that the observed edge effect is associated with the transition from quasi-TEM modes to ribbon plasmons where the field concentrates highly on the graphene and the current becomes non-uniform . In the In copper, the cutoff for guided plasmonic modes lies at much higher frequencies for a given waveguide size, or much larger waveguide sizes at a given frequency, due to copper’s larger DC conductivity. However, at microwave frequencies, the more practical boundary is the one that separates single-mode (TEM) operation form multi-mode (TEM+TE/TM) operation. If we contrast the overall performance of graphene transmission lines to their copper counterparts, we observe that graphene lines can provide a much lower onset frequency for the kinetic LC region. Furthermore, graphene’s LC region is supported in much larger lines, up to widths of about 100 μm. For larger structures, graphene’s reduced dimensionality also results in a more gradual shift towards a Faraday inductance-dominated LC response.High quality graphene with a sub-THz scattering rate would be extremely valuable for realizing nanoscale traveling-wave transducersEF = 0.4 eV, see β than those of the infinitely-wide graphene films, and therefore experience even higher cut-off frequencies. Thus, in the spectral range between 1 and 10 THz, the graphene TEM mode provides the optimal combination of wavelength-reduction, linear phase propagation, and constant attenuation.Finally, it is worth pointing out that in comparison to graphene plasmons, which are known for their exceptional wavelength reduction21In conclusion, our analysis of graphene and copper transmission lines has revealed that the well-known TEM modes enjoy a previously unnoticed broadband constant-attenuation regime, when the lateral dimensions are reduced to the nanoscale for both 2D and 3D conductors. This so-called kinetic-LC regime is induced by a predominant kinetic inductive impedance. The threshold dimension for this regime in a 3D line is defined by the collision-less skin depth of the conductor, about 100 nm for copper. Graphene transmission lines are remarkable in that their kinetic-LC region extends to relatively large micron-scale widths. The onset frequency of the kinetic-LC region is fixed at the free-carrier scattering rate, a material property, and therefore high-quality graphene with a low scattering rate would be desirable for THz/sub-THz applications. The upper frequency cutoff, on the other hand, depends on the material dimensionality: 2D graphene lines exhibit a width-dependent upper cutoff induced by plasmonic edge effects, whereas 3D lines have no upper cutoff in the spectral range in which the Drude model is valid. Although graphene lines exhibit constant attenuation at larger dimensions, copper lines of comparable widths provide lower attenuation. However, graphene’s large kinetic inductance drastically reduces the mode wavelength by 10–40 fold, approaching 1 micron at 10 THz. This deep subwavelength operation and the inherent tunability of graphene open up new opportunities for conventional signaling and sensing applications, as well as emerging applications involving metamaterials.z) being taken into account by replacing k as the eigenvalue. The circuit parameters .


Graphene films grown by vapour deposition tend to be polycrystalline due to the nucleation and growth of islands with different in-plane orientations. Here, using low-energy electron microscopy, we find that micron-sized graphene islands on Ir(111) rotate to a preferred orientation during thermal annealing. We observe three alignment mechanisms: the simultaneous growth of aligned domains and dissolution of rotated domains, that is, ‘ripening'; domain boundary motion within islands; and continuous lattice rotation of entire domains. By measuring the relative growth velocity of domains during ripening, we estimate that the driving force for alignment is on the order of 0.1 meV per C atom and increases with rotation angle. A simple model of the orientation-dependent energy associated with the moiré corrugation of the graphene sheet due to local variations in the graphene–substrate interaction reproduces the results. This work suggests new strategies for improving the van der Waals epitaxy of 2D materials. Understanding the factors that control island orientation in the growth of two-dimensional materials is likely to improve their quality. Here, using low-energy electron microscopy, the authors show that post-nucleation annealing of 2D materials can improve rotational order. Attempts to grow large, structurally pristine two-dimensional 2D) materials such as graphene and transition metal dichalcogenides are often frustrated by the fact that different rotational variants nucleate and grow, leading to a polycrystalline film1D materia34491112Here we use low-energy electron microscopy (LEEM) to observe in real time the evolution of graphene domains during thermal annealing. We show that large, micron-scale graphene domains on Ir(111) can reorient themselves to a preferred orientation by three mechanisms after nucleation. We estimate that domains aligned with the Ir(111) lattice are energetically favourable by as little as ∼0.1 meV per C atom. A simple model of the orientation-dependent energy associated with the moiré corrugation of the graphene sheet due to local variations in the graphene–substrate interaction reproduces the results. These results provide valuable guidance for the synthesis of 2D materials on current and future substrates: annealing immediately after nucleation will improve quality and this effect will be greater for substrates that induce larger corrugations in the 2D sheets.The first mechanism identified, shown in c, relative to the equilibrium adatom concentration for the graphene domain, ceq. Growth occurs when c>ceq and dissolution occurs when c<ceq. For an ideal adatom lattice gas, the chemical potential difference associated with growth or dissolution is Δμ≈kT ln c/ceq, where k is the Boltzmann constant and T is the absolute temperatureWe now turn to the cause of the simultaneous growth and dissolution. In general, growth or dissolution is dictated by the surface adatom concentration adjacent to the island, ceq compared with the R12 domain, which results in the growth of the R4 domain after the transformation, while the remaining R12 domain continues to dissolve.The relative chemical potential of different rotations can be immediately determined by combining the experimental results, as shown in The simultaneous growth and dissolution, that is, ‘ripening', is sensitive to temperature. As one sees in What is the magnitude of the driving force for a rotated domain RX to realign itself to R0? To estimate it, we use the temperature-controlled ripening experiments described above to determine the differences in equilibrium adatom concentrations between different rotations. From these adatom concentrations, we then extract the difference in chemical potentials of the islands. In previous work, the velocity of R0 domains was precisely determined as a function of the adatom concentration and temperature17ceq,R0 is the equilibrium adatom concentration for the R0 island, n is the cluster size for attachment to a kink and is equal to four, and B(T)=exp/T) nm s−1 is the kinetic coefficientA(t) plot of where −1 , where the error is from the s.d. of the dA/dt line fit. When the R8 domain was stationary from 2,800 to 3,200 s , the R0 velocity decreased to 144±36 nm h−1. Using When the R14 domain was stationary, the R0 velocity was 540±36 nm hB(T), the absolute scale of the chemical potential may be a factor of two larger or a quarter smaller.Five temperature-controlled experiments were conducted in the same manner. The estimated differences in chemical potential relative to R0 are shown in These measurements allow us to estimate the difference in chemical potential that cause the observed grain boundary motion in ab initio techniques such as density functional theory (DFT) to compute directly. To understand the origin of the orientation-dependent chemical potential differences and the extent to which they can be generalized, we make a model of the energy differences due to the corrugation of the graphene sheet, which is known to significantly change with orientation. In incommensurate, undistorted moirés, all equivalent positions of C atoms with respect to the substrate are occupied with equal probability, independent of the layer's rotation41920These small energy differences seem beyond the capabilities of The reason for this corrugation is that the preferred distance of the graphene sheet from the substrate varies locally, depending on the lateral position of the C atoms with respect to the substrate . To maximize its binding to the substrate, the graphene sheet flexes slightly to follow a contour of minimum C-Ir potential. Due to the bending rigidity of the graphene, the C atoms are further away from their optimal positions as the wavelength of the corrugation shortens. So the short-wavelength corrugations observed experimentally for rotated graphene would lead to an energy cost.z0 of C atoms from the Ir(111) substrate is represented by a 2D sinusoid, which has opposite extrema at the atop sites and hollow sites. We take the force on each C atom to be proportional to how far the atom is displaced from the preferred distance and assume that the spring constant k is uniform. In the relaxed configuration, this force is balanced by the forces caused by bending the graphene sheet. In the continuum limit, the energy cost Eb of periodic corrugations is2324To estimate the magnitude of this effect, we construct a simple model for the observed corrugations. For simplicity, we assume that the graphene is incommensurate with respect to the Ir substrate, that is, that the possible energy reduction due to locking into a (higher order) commensurate lattice is not sufficient to overcome the energy cost of straining the graphene from its preferred lattice constant. We assume that the preferred distance λ is the mean bending rigidity, ∼1.4 eVK is the mean curvature. Our model approximates the local curvature Ki at a given atom i as being proportional to the height difference Δzi between atom i and the average of its surrounding three nearest neighbours. This gives a force on each atom equal to d is the C–C distance in graphene , we fit these two parameters to our DFT calculations for R0 shown in k to exactly reproduce the amplitude of the corrugation of the relaxed sheet and the root mean square of the forces on the flat sheet. Given these parameters, we predicted the corrugation for domains rotated by 7.5, 15 and 30 degrees. Height profiles extracted from our moiré corrugation model along the close-packed zigzag direction are shown in This model has only two parameters, the amplitude of the sinusoidal preferred C atom distance and the spring constant The agreement between this prediction and DFT calculations is striking. It reproduces the position of all C atoms in the profiles in Our results have significant implications on the nature of rotational disorder of the graphene islands on various substrates. Scanning tunnelling microscopy shows that graphene on Au(111)272829In summary, we have identified the energetically preferred graphene orientation on Ir(111). Domains can evolve to this orientation during thermal annealing by three distinct mechanisms. The driving force for the alignment can be as small as 0.1 meV per C atom, much less than the total binding strength of graphene to metal surfaces. We propose that the origin of the preferential alignment is caused by the varying degree to which C atoms can attain the preferred distance from the substrate: the graphene-bending rigidity prevents the sheet from following the short-wavelength corrugations inherent in highly rotated domains. Since graphene corrugations of similar size are ubiquitous, the driving force for the coarsening on Ir reported here is expected to be similar in other systems. Despite this small driving force for rotational alignment, our results indicate that post-nucleation annealing of 2D materials can still improve rotational order. This strategy means that high quality 2D materials can be grown on more substrates than is now recognized. Our understanding of the driving force for reorientation can guide the search for new substrates.Graphene islands were nucleated by heating the Ir substrate between 800 and 900 °C and introducing ethylene gas into the LEEM chamber. After nucleation, the ethylene flow was turned off. The temperature was measured by a W-Rh thermocouple welded directly to the substrate.The domain orientation relative to the Ir(111) lattice was identified by selected-area LEED. The moiré LEED spots are sensitive to small changes in domain orientation and were used to determine the orientation to within ±0.5°. The distance between each pair of moiré superstructure LEED spots was measured and normalized by the distance between the corresponding pair of first-order graphene LEED spots. This ratio was computed for the three pairs of spots per LEED pattern and then averaged. Over 47 LEED patterns were analyzed and orientations were assigned based on this ratio. This ratio is easily determined using a simple vector model and there was excellent agreement between the experimental and expected ratios.A, and perimeter, p, with time: ɛave=(1/pave)dA/dt, where dA/dt is the slope of a line fit to the area versus time data for the time interval in question, and pave was the average perimeter over the time interval.The average R0 growth velocity was computed by measuring island area, All DFT calculations were performed with the optB86b-vdW functional with spin-polarization and using the projector-augmented-wave GW pseudopotentials supplied by VASP. A cutoff energy of 400 eV for the plane-wave basis and the Brillouin zone was sampled using 1 × 1 × 1 and 2 × 2 × 1 k-point grids. The optB86b-vdW exchange–correlation functional was employed to account for van der Waals interactions that are required for a qualitative description of the intermolecular interactions between the graphene sheet and the Ir(111) surface. The functional is of the form: a=b=c=3.843 Å; the lattice of the surface was fixed during all simulations. The Ir(111) slab consisted of three atomic layers, which helped minimize computational cost and allowed for the full graphene moiré to be modelled. Five different graphene orientations were considered: R0 , R7.5 , R15 , R22.5  and R30 . Geometry optimizations were performed until all forces were <0.05 eV Å−1; the total energy was converged to within 1 × 10−6 eV for both k-point samplings.The Ir(111) surface was constructed from the optimized face-centred cubic structure, The binding energies per C atom of the systems were determined by the following equation:EG/Ir111 is the total energy of the system  substrate), EG and EIr(111) are the total energies of the optimized isolated graphene layer and the Ir(111) surface, and N is the total number of C atoms. The graphene deformation energies (bending energies) were determined from the difference between the single-point energy of the optimized graphene sheet on the Ir(111) surface and the fully optimized graphene sheet within the same unit cell.where E/Δk). For the deformation energies, ΔE/Δk is much smaller than the magnitude of the values. On the other hand, the relative binding energies between the different orientations  are smaller than ΔE/Δk. On the basis of this analysis, it is concluded that the predicted ordering of the deformation energies is statistically significant, but the relative binding energies between orientations is not significant. As a result, the orientation with the greatest binding energy per C atom cannot reliably be determined from these results. Increasing k-space much further is computationally intractable; moreover, each system has different lattice dimensions that affect the relative k-point spacing. Computationally determining differences of one-tenth of a meV is difficult given the various numerical approximations used within DFT .Two k-point grids were used to compute graphene deformation energies and binding energies per C atom. The larger 2 × 2 × 1 k-point grid was used to test whether the predicted energies from the 1 × 1 × 1 k-point grid were converged with respect to k-space. The results indicate that the computed graphene deformation energies are converged; however, the binding energies are not. This conclusion was reached by comparing the difference in energies with respect to k-space .Supplementary Figure 1, Supplementary Table 1, Supplementary Notes 1-3 and Supplementary ReferencesObservation of three graphene domain reorientation mechanisms using LEEM


In concert with experiments, multiscale calculations reveal that defect complexes in graphene are prerequisite for low-temperature hydrogenation, and that the hydrogenation of defective or functionalized sites at strained domain boundaries plays a beneficial role in improving rate capacity by opening gaps to facilitate easier Li penetration. Additional reversible capacity is provided by enhanced lithium binding near hydrogen-terminated edge sites. These findings provide qualitative insights in helping the design of graphene-based materials for high-power electrodes.Atomic hydrogen exists ubiquitously in graphene materials made by chemical methods. Yet determining the effect of hydrogen on the electrochemical performance of graphene remains a significant challenge. Here we report the experimental observations of high rate capacity in hydrogen-treated 3-dimensional (3D) graphene nanofoam electrodes for lithium ion batteries. Structural and electronic characterization suggests that defect sites and hydrogen play synergistic roles in disrupting  The commercial applications of graphene materials for energy storage devices, including lithium ion batteries (LIBs) and supercapacitors12345181112Earlier studies on various carbon materials  generally pointed at an increased lithium storage capacity with increasing hydrogen content1520To investigate the involvement of hydrogen and hydrogenated defects in the lithium storage ability of graphene, we apply various heat treatment conditions combined with hydrogen exposure and interrogate the electrochemical performance of 3D graphene nanofoam (GNF) electrodes. Our studies use self-assembled 3D graphene nanofoams due to their numerous potential applications, including hydrogen storage, catalysis, filtration, insulation, energy sorbents, capacitive desalination, supercapacitors, and LIBs2 environment. The original 3D structure of these three GNFs can be seen in 2 + Ar) at 400 °C (Methods), we note that the D/G band intensity ratio of graphene (GNF-1050C-H) is marginally changed (1.48 ± 0.03). However, a blue-shift of E2g2 mode  is observed in GNF-1050C-H Our electrode materials are 3D GNFs . The increased D/G band ratio at higher annealing temperatures is counterintuitive but suggests that the GNF-1050C and the GNF-1600C belong to Stage II and Stage I defect region materials with an estimated graphene domain size of 1.4 nm and 10.4 nm1826reatment . Elasticd sample .K-edge X-ray adsorption spectroscopy (XAS), sp2 carbon materials, including a sharp resonance at ~285.4 eV that is attributed to C(1s) → (C-C) π* transitions, a feature at ~291.5 eV arising from a core-hole (σ) exciton state2 environment at 400 °C. The GNF-1050C-H spectrum exhibits a slight reduction in intensity of both the C(1s) → (C-C) π* resonance and the C(1s) → (C-C) σ* resonance at ~292.5 eV with respect to the GNF-1050C data. In contrast, any changes in the intensity of the C(1s) → (C-H) σ* resonance fall within experimental error. The diminished intensity of the C-C π* and σ* resonances suggests a reduction in the overall sp2 character of the GNF2. In light of this assignment, the absence of any accompanying intensity changes in the C(1s) → (C-H) σ* is important because it eliminates possible mechanisms by which the proportion of sp2 carbon could decrease. In particular, the addition of molecular hydrogen across two adjacent sp2-hybridized carbon atoms (i.e. to yield two sp3-hybridized carbons) is omitted as a viable mechanism for the reduction in sp2 character because it would necessitate the observation of a corresponding increase in intensity of the C(1s) → (C-H) σ* resonance at ~ 289.5 eVsp2 hybridized carbon within the GNFs. This assignment is further supported by the observed sharpening and intensity increase in the core-hole exciton feature upon hydrogenation. Both features are characteristic of an increase in domain size and crystallinity of the GNF-1600C sample, consistent with Raman signatures.The electronic structure of three GNF samples  is investigated by carbon 2-treated sample  has substantially better rate capacity compared to the GNF-1050C sample, whereas the GNF-1600C has the worst rate performance (the voltage profiles of three samples at the 1st and 5th cycle can be seen in GNF-1050C-H-CGNF-1050C)/CGNF-1050C × 100%, where CGNF-1050C-H and CGNF-1050C are the delithiation capacities of H2-treated and reference GNF-1050C graphene samples, respectively. In 2 treatment. At lower rates, δ increases sharply before generally saturating at higher rates. The maximum enhancement of ~43% is observed at the charge/discharge rate of 200 mA/g. This impressive improvement of rate capacity in H2-treated sample is surprising, given the fact that the overall hydrogen content in GNF-1050C-H was only slightly increased , suggestive of easier intercalation processes; (2) higher temperature annealing without H2 leads to higher graphitization of graphene and thus stronger lithium intercalation peaks. However, the same higher temperature annealing also leads to the disappearance of multiple lithium reaction peaks in the voltage range of 1–3 V, causing low capacity in higher temperature annealed graphene materials , but the highest first-cycle Coulombic efficiency (~39.0%). The observation of a lower Coulombic efficiency in the less-defective GNF-1600C  follows a power law relation with the sweep rate (ν), we have32More information related to lithium storage kinetics of our 3D GNFs is revealed by cyclic voltammetry (CV) data shown in i) response at a given voltage (V) as a linear sum:where a and b are materials-dependent variables. By using a ν range of 0.1–2 mV/s, we derive a b-value of 0.72 for the GNF-1050C, which increases to 0.75 for the GNF-1050C-H but decreases to 0.70 for the GNF-1600C, 2ν1/2 describes diffusion-controlled (battery-like) contributions and c1ν encompasses remaining contributions that behave in a capacitive fashion. We emphasize that the “capacitive” contribution (c1ν) could encompass a variety of possible processes that are not diffusion-controlled, including double layer capacitance, pseudocapacitance, and general kinetically fast charge storage at accessible sites. By determining c1 and c2, the individual contributions of diffusion-controlled and capacitive mechanisms within the independent model can be estimated at each sweeping rate, as illustrated in 2-treated sample compared with the other two samples. For example, at the highest sweeping rate of 2 mV/s, we find that 66.4% of lithium storage comes from capacitive mechanisms in GNF-1050C-H versus 60.5% and 59% for GNF-1050C and GNF-1600C, respectively. At a fixed scan rate of 0.2 mV/s, we observe a similar trend for three materials in the voltage window of 0.25–3.2 V, 2 treatment, which is one of the main causes why GNF-1050C-H has the highest rate capacity.Here, c2 treatment is not directly discernible. However, as previously discussed, no double layer capacitive enhancement is observed in the equivalent circuit models of + ions simultaneously. The Nyquist plot in sp2 carbon character, as expected from disruptions in ring structures or loss of surface carbon. At the same time, the Raman data  cleavage of strained C-C bonds; (2) etching via successive reduction and hydrogenation of energetic carbon ; (3) reduction and hydrogenation of oxygen- or nitrogen-containing functional groups . In each case, the process is accompanied by H-termination of dangling carbon bonds. The broken bonds alleviate local strain and open up H-passivated nanopores and edges in the framework without creating additional traps, thus improving the accessibility of Li to regions that were previously kinetically restricted . This mechanism is illustrated schematically in E2g2) Raman shift  at defective sites on the graphene surface at 400 °C. According to a “hot hydrogen” model described in Ref. 2 molecules at the tail of the thermal distribution collide with the graphene surface, the proximity of their approach alleviates the need to overcome the H2 high dissociation barrier (~4.12 eV)2 near defective sites may also assist in low-temperature dissociation352 on defective binding sites need only be on the order of the bond energy difference between H in H2 and in defective graphene. Using relevant DFT energetics, combined with key information about our experimental conditions . Appreciable surface diffusion of atomic H can also be activated at 400 oC , ΔE may include strain energy dissipation (ΔEstrain) arising from the relaxation of highly curved or strained configurations at the attack sitesE = EB + ΔEstrain. The existence of such strained regions in our graphene samples is supported by direct experimental observation of rippled/twisted structures (Estrain could account for ~0.07–0.42 eV per binding site (based on the strain energy of a rippled graphene sheet induced by 5% to 20% compressive strainEB > 3.53 eV (assuming 5% strain) or >3.18 eV (assuming 20% strain) are possible candidates for dissociative hydrogenation  would be released at low pressure or elevated temperatures, effectively conserving the total amount of hydrogen in the system; this may explain the largely similar H concentrations before and after hydrogen treatment detected in high vacuum conditions (<~10−6 Torr) by ERDA and implied in the XAS results. To conclude, we find that functionalized edges, multi-vacancy complexes, and strained non-hexagonal rings — likely to be concentrated at strained domain boundaries — all constitute probable sites for dissociative hydrogenation.+ that could otherwise lead to irreversible trapping. We point out that increases in the interlayer spacing upon insertion and binding of surface H between graphene layers could further aid intercalation near the perforations, as explored theoretically in To test the degree of kinetic enhancement associated with hydrogenation of multi-vacancy complexes and domain boundaries according to the mechanism in + binding is too weak for practical use as an anode. Interestingly, the addition of hydrogen enhances the adsorption of Li+ in the hexagonal rings nearest the hydrogen  while maintaining reversibility under practical cycling conditions. The introduction of curvature would further stabilize such sites. We emphasize that these mechanisms can only add reversible capacity from local sites nearest the hydrogen, and would therefore have a negligible effect on overall kinetics. Accordingly, they cannot represent the dominant mechanism for our reported high-rate capacity increase, but may nonetheless provide a measureable contribution.Although the dominant effect of hydrogenation is improved kinetics associated with enhanced surface capacitive behaviour  and hydrogen are found to play an integral role in helping enhance the lithiation kinetics, leading to high rate capacity in hydrogen treated graphene materials. This behaviour appears universal in all hydrogen treated samples. Among several beneficial roles of hydrogen, the binding of hydrogen along domain boundaries is considered as an effective pathway to help achieve high-rate performance 3D GNF electrodes, as it can substantially lower the barrier for Li2 in a critical point drier overnight. The drier was heated to 50 °C and the pressure was adjusted to maintain ~1500 psi to exceed the critical point of CO2. The gel was held at this state for 2 h, after which the drier was slowly vented to ambient pressure to remove dried gels. The obtained dried gels were placed in a tube furnace for carbonization at 1050 °C for 4 h in N2. The GNF sample was further annealed at 400 °C in 4 at% H2/Argon for 24 h to obtain GNF-1050C-H, or pure H2 for 4 h to obtain GNF-1050C-H2. To increase crystallinity, GNF-1050C was further annealed at 1600 °C in argon, 2000 °C in helium, and 2500 °C in helium for 4 h.GNFs were synthesized by gelation of graphene oxide followed by supercritical drying and high temperature pyrolysis. 400 mg single-layer graphene oxide powder  were suspended in 20 mL deionized (DI) water (resistivity of 18.2 MΩ). After adding 4.22 ml concentrated ammonium, the suspension was sonicated overnight at 15 °C. The obtained slurry was poured into a rubber mould (diameter of 12.5 cm and thickness of 1 mm) and sealed between glass slides. The slurry-filled mould was immersed in DI water and placed in an oven for gelation at 80 °C. The wet gel was washed in DI water, followed by acetone. The acetone was exchanged with liquid COScanning electron microscopy (SEM) was conducted using a JOEL JSM-7401F field-emission SEM under secondary electron imaging (SEI) mode with an accelerating voltage of 20 kV and a beam current of 20 mA. The cross sectional GNFs with fresh fracture surface was examined. Transmission electron microscopy (TEM) was performed on a Philips CM30 field-emission TEM. Samples were prepared by manually crashing GNF samples onto a Lacey carbon supported copper grids .2 flow for 24 h to remove moisture.The porosimetry was determined by Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods in nitrogen adsorption/desorption experiments using an ASAP 2020 Surface Area Analyser (Micromeritics Instrument Corporation). GNF samples with mass of 10 mg to 20 mg were preheated at 300 °C under NRaman spectra were collected using three laser excitation wavelengths, 473 nm, 532 nm, and 633 nm. The spot size was 1.0–2.5 μm, and the laser beam power was 1–2 mW. Here 0, and the absorption edge step. I0 was measured concurrently with the XAS signal via the drainage current from a gold grid located upstream of the experimental sample and the absorption edge step was taken as the difference in absorbance in the pre-edge (280 eV) and post-edge (330 eV) regions of the XAS spectrum.XAS measurements were performed at beamline (BL) 8.0.1.1 of the Advanced Light Source, Lawrence Berkeley National Laboratory. Carbon K-edge XAS data was recorded in the total electron yield (TEY) mode over a spectral range of 280–330 eV, with the sample angled at 45°with respect to the incident x-ray beam. Energy calibration of BL8.0.1.1 was achieved via reference to the C(1s) → π* resonance for a freshly cleaved sample of highly oriented pyrolytic graphite (285.38 eV)XRD was carried out using Bruker AXS D8 ADVANCE X-ray diffractometer with Cu radiation source operated at 40 kV and 40 mA. 2θ scan was conducted from 10° to 90° with 0.02° steps and 2 s counting time per step.2 environment. The H-content in GNF was determined by elastic recoil detection analysis (ERDA)4He+ ions. During the ERDA experiment, the sample normal direction was tilted by 70° with respect to the incident He beam. H atoms recoiled into a surface barrier detector at 150° were measured. The detector is covered with a 13-um thick carbon foil that was used to filter out the forward-scattered He ions. The oxygen content was measured by Rutherford backscattering spectrometry (RBS) with a 2 MeV 4He+ beam incident normal to the sample surface. The detector located at 164° from the incident beam direction was used to register backscattered He ions. Both ERDA and RBS spectra were analysed with the RUMP code1H 4He reaction from Ref. Sample was mounted on a Si piece. Mounting and sample transfer were completed in NSwagelok-type Half cells were assembled in an Argon-filled glove box with GNFs as the working electrodes, lithium as the counter electrodes, two layers of Celgard 3501 porous polypropylene films as the separators, and 1 M LiPF6 in 1:1:1 (in volume) of ethyl carbonate (EC)/diethyl carbonate (DEC)/dimethyl carbonate as electrolyte. Galvanostatic charge/discharge experiments were performed on a Maccor 4304 Battery cycler (voltage window 0.01–3.5 V)43E. Within the model discussed in Ref. We consider a graphene surface consisting of only one type of defective binding site where the energy change upon H binding is given by Δhot’ hydrogen gas molecule. The moles of T is calculated by 2 molecules flowing into the chamber and ED = |ΔE|–|EB|. The total energy variation upon dissociative hydrogenation is computed as follows:where β is the extent of reaction and 2 molecules, respectively, at the standard state, and N0 is Avogadro’s number. where k-point mesh. Periodic boundary conditions were used, with a 20 Å vacuum layer inserted perpendicular to the planes and a dipole correction applied. Gaussian smearing was used with a smearing width of 0.03 eV. Forces were converged to <1 × 10−2 eV/Å. For calculation of surface diffusion barriers  above the largest opening in the graphene sheet at a distance of 1.72 Å ; and (2) in the plane of graphene at the centre of the largest opening.We use density functional theory implemented in VASPbarriers , the nudHow to cite this article: Ye, J. et al. Universal roles of hydrogen in electrochemical performance of graphene: high rate capacity and atomistic origins. Sci. Rep.5, 16190; doi: 10.1038/srep16190 (2015).


TAR DNA-binding protein 43 (TDP-43) is normally a nuclear RNA-binding protein that exhibits a range of functions including regulation of alternative splicing, RNA trafficking, and RNA stability. However, in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP), TDP-43 is abnormally phosphorylated, ubiquitinated, and cleaved, and is mislocalized to the cytoplasm where it forms distinctive aggregates. We previously developed a mouse model expressing human TDP-43 with a mutation in its nuclear localization signal (ΔNLS-hTDP-43) so that the protein preferentially localizes to the cytoplasm. These mice did not exhibit a significant number of cytoplasmic aggregates, but did display dramatic changes in gene expression as measured by microarray, suggesting that cytoplasmic TDP-43 may be associated with a toxic gain-of-function. Here, we analyze new RNA-sequencing data from the ΔNLS-hTDP-43 mouse model, together with published RNA-sequencing data obtained previously from TDP-43 antisense oligonucleotide (ASO) knockdown mice to investigate further the dysregulation of gene expression in the ΔNLS model. This analysis reveals that the transcriptomic effects of the overexpression of the ΔNLS-hTDP-43 transgene are likely due to a gain of cytoplasmic function. Moreover, cytoplasmic TDP-43 expression alters transcripts that regulate chromatin assembly, the nucleolus, lysosomal function, and histone 3’ untranslated region (UTR) processing. These transcriptomic alterations correlate with observed histologic abnormalities in heterochromatin structure and nuclear size in transgenic mouse and human brains. TDP-43 is a member of the heterogeneous ribonuclear protein group and binds to both DNA and RNA ,2. It haBoth a cytoplasmic gain of function and a nuclear loss of function of TDP-43 are possible disease mechanisms, as neurons with cytoplasmic TDP-43 aggregates are also associated with a loss of normal nuclear TDP-43 protein ,12. We pIn this study, we performed RNA-sequencing of ΔNLS-hTDP-43 transgenic mice brains to further study the transcriptomic effects of the gain of cytoplasmic TDP-43 in this model. This revealed significant changes in multiple molecular pathways, including confirmation that cytoplasmic TDP-43 expression alters transcription related pathways and histone transcript levels. Supporting a gain of cytoplasmic function mechanism, biochemical analysis of nuclear and cytosolic fractions of the ΔNLS-hTDP-43 mice brains showed that total nuclear TDP-43 expression is largely unaltered, suggesting that the exogenous human TDP-43 is able to compensate for autoregulatory reduction of endogenous nuclear mTDP-43. The transcriptomic changes were compared with RNA sequencing data from mice with reduced levels of TDP-43 after injection with an ASO against TDP-43 , revealiAnimal care and performed procedures were in accordance with the NIH Guide for the Care and Use of Experimental Animals and approved by the University of Pennsylvania Institutional Animal Care and Use Committee (Permit Number: 803385).Inducible transgenic mice overexpressing mutant ΔNLS-hTDP-43 were generated as described previously . BrieflyATCTCGTATGCCGTCTTCTGCTTG) trimmed using cutadapt [Mus musculus genome build using Bowtie (version 0.12.7 with options—time -k 100—best—strata -v 2 -p 3) [Raw reads from both RNAseq experiments had their 3’ Illumina v1.5 smRNA adaptors   and were 2 -p 3)  tool aga 2 -p 3)  tool. Th 2 -p 3)  to compu 2 -p 3) , which w 2 -p 3) . Splicin 2 -p 3)  followin 2 -p 3) , where t 2 -p 3) . For bot 2 -p 3) .For pathway analyses, lists of refSeq IDs were obtained through R analysis and uploaded to NIH DAVID ,26 for aPreviously published microarray data  is availhttps://github.com/alexamlie/bed_statistics.The processed peaks from the CLIP sequencing data were directly obtained from the authors, as analyzed in . A custo1/n reads in each of its mapped locations, where n is the number of times that read mapped. To normalize, we chose 50 random unchanging genes, defined as having an adjusted DESeq2 p-value of greater than 0.5, and mapped them with up to 10 multimappings as well. Then, we took the geometric mean of the unchanging gene coverage for each sample, and converted these means into normalization factors by dividing them all by the mean of the geometric means of the nontransgenic samples. All histone genes with no coverage were converted to 0.1, the lowest possible coverage, and histone values were divided by the normalization factors. Finally, we took the separate geometric mean in each sample for the normalized canonical and variant histone genes. The changes in canonical and variant histones were quantified with 2-sided t-tests between the 4 bigenic and 4 nontransgenic normalized geometric means.To account for the fact that histones are grouped in highly repetitive clusters, we remapped and allowed for up to 10 multimappings for the 50 canonical and variant histone genes of interest, weighting each read as counting for 2, 1 mM DTT, 10 mM HEPES-KOH, pH 7.4 with protease inhibitors; 5:1 v/w). After a 10 minute incubation at 4°C, 1.0 M sucrose in hypotonic buffer (0.36:1 v/w) was added, and samples were Dounce homogenized with an additional 15 strokes. Aliquots were removed as the total fraction. Homogenates were then centrifuged at 1,000xg for 10 minutes at 4°C. 120 μL of the supernatant was taken and spun at 100,000xg for 30 minutes at 4°C. The supernatant from this spin represented the cytosolic fraction. The remaining supernatant and pellet from the 1,000xg spin were then taken, and 1.77 volumes of 2.3 M sucrose in TKM  was added to raise the homogenate to 1.6 M sucrose. Additional 1.6M sucrose in TKM was then added to obtain ~4 mL of homogenate, which was overlaid on 3 mL of 1.8M sucrose in TKM followed by ultracentrifugation at 100,000xg for one hour at 4°C. The pellets were resuspended in 30 μL of PBS with protease inhibitors, and nuclei were counted using a hemocytometer. Finally, based on the nuclei counts, nuclei were diluted in PBS with protease inhibitors so that all samples contained the same concentration of nuclei. Sample buffer was added to all fractions followed by sonication and freezing at -80°C until SDS-PAGE analysis. Equal volumes of lysate were analyzed by electrophoresis on a 10–17% discontinuous step gradient tris-glycine polyacrylamide gel followed by transfer to nitrocellulose.Immediately upon sacrifice, cortices from mouse brains were dissected, weighed and frozen at -80°C. Total vs. nuclear vs. cytosolic fractions were prepared from cortex tissue as follows. Tissue was homogenized using 8 strokes of tight-fitted (B) Dounce homogenizer at 4°C in hypotonic buffer .RNA was extracted as described previously  from theThe antibodies used for immunoblotting were rabbit polyclonal C1038 antibody against total TDP-43 , rabbit Mouse brains and human brain tissue were fixed and processed into 6 μm sections as previously described . To stud+-selected RNA sequencing on right cerebral cortex from bigenic mice  and nontransgenic  control mice. This yielded mean values of 51,551,711 raw reads and 31,146,050 uniquely mapped reads per sample, with an average of 1.262x coverage of the protein-coding segment of the genome and 0.496x coverage of the entire genome. Differentially expressed genes were identified using DESeq2 [Mus musculus mm9 genome annotation) meeting a minimum read threshold of 10 reads per million in at least one sample as being significantly differentially expressed. Of these genes, 2,166 were upregulated in the ΔNLS mice compared to the control mice, and the remaining 2,155 were downregulated , were analyzed with the same pipeline used for ΔNLS-hTDP-43 mice. Technical replicates were combined to analyze biological replicates of control ASO injected mice  versus TDP-43 ASO injected knockdown mice . Across these samples, we found a mean of 30,794,457 total reads, with a mean of 29,132,557 uniquely mapping. We were able to map more reads than the original study , which uWe found 11,916 expressed genes in this dataset using the same minimum read count threshold as earlier. We first compared the expression of the 10,345 genes meeting the minimum read count threshold in both datasets (ΔNLS-hTDP-43 versus ASO) to check whether the transcriptomic changes due to ΔNLS-hTDP-43 expression versus ASO knockdown of TDP-43 were generally similar. This analysis demonstrated a complete lack of correlation between the two sets of mice , the two experiments revealed vastly different effects on the expression levels of these transcripts. In fact, only 495 and 202 were significantly down- or up-regulated in both datasets, respectively . Thus, tWe described above that ΔNLS-hTDP-43 mice exhibit a low number of splicing changes. To demonstrate that this was not due to a lack of power to detect splicing changes in our analysis pipeline, we performed an identical DEXSeq analysis on the RNA sequencing data from the TDP-43 ASO knockdown experiment. Remarkably, 1,029 out of 250,620 exons were significantly differentially spliced upon ASO knockdown, corresponding to 752 unique genes . The preNext, to compare the enriched pathways in the two experiments, we performed an identical pathway analysis procedure on the ASO data. In contrast to the 8 significant pathways identified in the ΔNLS-hTDP-43 experiment, we found 64 significantly enriched pathways among the differentially expressed genes  while three variant histone transcripts contained TDP-43 binding sites , suggesting that the majority of TDP-43’s effects on histone transcripts are due to indirect interactions.To determine whether these changes in canonical histone expression were due to direct interactions with TDP-43, we used High Throughput Sequencing of RNA isolated by Cross-Linking and Immuno-Precipitation (HITS-CLIP) data of TDP-43 RNA binding sites from a previous study to see whether TDP-43 directly binds to canonical histone transcripts . AnalysiWe next hypothesized that aberrant polyadenylation of histone transcripts could be due to a dysregulation of transcripts that regulate histone 3’ UTR processing. We manually generated a list of such genes involved in histone 3’ UTR processing . AnalysiSmn), another protein implicated in motor neuron degeneration, is also known to regulate histone 3’ UTR processing [Smn splicing is regulated by TDP-43 [2(fold change) = -0.34, adjusted p-value = 0.001034), further supporting the hypothesis that the transgene overexpression leads to aberrant histone 3’ UTR processing.Notably, survival of motor neuron , and several transcripts that regulate histone 3’ UTR processing . To test whether the observed changes in canonical histones were due to aberrant polyadenylation, we used both an oligo-dT primer, which selects for polyadenylated RNA transcripts, and random hexamer primers, which have no such selection bias. RT-qPCR measurements using either oligo-dT priming or random hexamer priming validated our RNA-seq data for transcripts which regulate histone 3’ UTR processing + selection step  relative to nuclei of adjacent uninvolved neurons (57.56 μm2)  . Compari.56 μm2) . Thus, n+ selected RNA sequencing data and elucidating a specific role for TDP-43 in the previously reported dysfunction of the chromatin assembly pathway [The integrative analysis of the molecular phenotype of the ΔNLS-hTDP-43 expressing mouse performed in this study revealed varied effects of the cytoplasmic overexpression of TDP-43. Transcriptomic analysis using RNA sequencing technology revealed broad effects of the transgene overexpression, including upregulation of genes related to transcription, chromatin assembly, the nucleolus and protein metabolism, as well as downregulation of genes related to synaptic activity and posttranslational modification. We identified specific dysregulation in histone 3’ UTR processing genes due to cytoplasmic TDP-43 overexpression, coincident with the surprising overrepresentation of canonical histone transcripts in the poly(A) pathway . Defects pathway .In the broader context of TDP-43 biology, this analysis suggests that a gain of cytoplasmic TDP-43 function affects nuclear dynamics. Specifically, the presence of increased cytoplasmic levels of TDP-43 protein leads to aberrations in histone 3’ UTR processing gene transcription, resulting in defects in histone processing such as irregular polyadenylation that contribute to defects in the chromatin assembly process. Several lines of evidence support this claim, including the presence of aberrantly polyadenylated canonical histone transcripts in the RNA sequencing data, PCR validation of increases in polyadenylated canonical histones, and histological validation of defects in chromatin assembly.Although we had previously hypothesized that the toxic effects of the ΔNLS-hTDP-43 overexpression were caused by dysregulation of endogenous mTDP-43, therefore mainly representing a loss of function , our comThe lack of transcriptomic similarity with the ASO knockdown, the unchanged level of nuclear total TDP-43 protein, and the marked increase in cytoplasmic TDP-43 protein levels altogether suggest that the widespread degeneration and transcriptomic changes observed in the ΔNLS-hTDP-43 expressing mice may be mainly due to a toxic gain of cytoplasmic TDP-43 function. This is further supported by the paucity of splicing changes upon expression of ΔNLS-hTDP-43, implying that the exogenous hTDP-43 seems to be able to mostly compensate for the disruption of endogenous mTDP-43 nuclear splicing regulatory activity. It is important to note that cytoplasmic TDP-43 inclusions are very rare in these mice, suggesting that the effects observed in this model seem to be due specifically to a gain of cytoplasmic function as opposed to toxic aggregation. Furthermore, comparison of the transcriptomic results with HITS-CLIP data of TDP-43 RNA binding partners showed that six out of the eight significantly changing pathways were significantly enriched for TDP-43 binding targets (data not shown). This suggests that many of the transcriptomic effects in these mice involve the direct interactions of TDP-43 with its RNA targets, as opposed to indirect effects arising from the cytoplasmic mislocalization.This analysis also confirmed our previous report of ΔNLS-hTDP-43 affecting chromatin assembly genes. Our RNA-seq, HITS-CLIP and RT-PCR analyses showed that TDP-43 interacts with and alters the expression of several transcripts involved in histone 3’ UTR processing, suggesting that canonical histone dysregulation may be a mechanism by which chromatin assembly pathways and nuclear dynamics are disrupted. These transcriptomic results were supported by our histological findings which showed irregular chromatin structure in neurons expressing ΔNLS-hTDP-43.Aberrant histone transcript polyadenylation has been shown to affect translation of histone transcripts. Knockdown of stem loop binding protein, the protein that binds the stem loop of histones and facilitates proper histone transcript processing and trafficking, leads to aberrantly polyadenylated histone transcripts which are retained in the nucleus and therefore not translated . HoweverTogether, this study further supports the importance of the role of TDP-43 in transcriptional regulation and identifies a specific effect of the overexpression of cytoplasmic TDP-43 on nuclear dynamics and misprocessing of the 3’ end of histone transcripts. Although this model artificially induces a gain of cytoplasmic function, the results observed here provide an insight into the consequences of pathological cytoplasmic accumulation of TDP-43. The question of whether the pathological effects of TDP-43 in ALS and FTLD-TDP are caused by a nuclear loss of function or a cytoplasmic gain of function is still uncertain , but thiS1 TableExon IDs include 3 digits because some annotated genes include hundreds of exons. NA gene symbols represent exons from unannotated genes, and both unannotated genes and exon IDs are directly from the Ensembl mm9 genome annotation .(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableEntries with the same gene cluster entry represent different members of highly similar clusters. Type c = canonical, v = variant.(XLSX)Click here for additional data file.


In 2012, 20% of residential care communities used EHRs. Greater proportions of communities with larger numbers of beds used EHRs compared with communities with fewer beds. Communities with >100 beds (36%) were more than three times as likely as communities with 4–10 beds (11%) to use EHRs.Source: National Study of Long-Term Care Providers, 2012. Available at http://www.cdc.gov/nchs/nsltcp.htm.


However, probing such buried interfacial processes is experimentally challenging. Infrared spectroscopy is sensitive to molecule vibrational signatures, yet to approach the interface three stringent requirements have to be met: interface specificity, sub-monolayer molecular detection sensitivity, and electrochemically stable and infrared transparent electrodes. Here we show that transparent graphene gratings electrode provide an attractive platform for vibrational spectroscopy at the electrolyte/electrode interfaces: infrared diffraction from graphene gratings offers enhanced detection sensitivity and interface specificity. We demonstrate the vibrational spectroscopy of methylene group of adsorbed sub-monolayer cetrimonium bromide molecules and reveal a reversible field-induced electrochemical deposition of cetrimonium bromide on the electrode controlled by the bias voltage. Such vibrational spectroscopy with graphene gratings is promising for real time and  Understanding of electrolyte-electrode interfaces is limited due to the lack of suitable probing techniques. Here, the authors present a vibrational spectroscopy based on graphene gratings, which enables sensitive and interface-specific detection of molecular vibrations at electrolyte-electrode interfaces. In this paper, we report a new technique based on graphene gratings that overcomes these difficulties, which enables sensitive and interface-specific detection of molecular vibrations at the electrolyte/electrode interface. An infrared spectroscopy technique that overcomes all these difficulties will be a promising method for sensitive interface detection of molecules at the electrolyte/electrode interface.The electrolyte/electrode interface is of great interest for both fundamental science and applied technology: a rich variety of electrical field-dependent physical1246713410111314Graphene is an attractive electrode for studies of electrolyte/electrode interfaces. It is stable and transparent to infrared light, and is being actively explored for applications in supercapacitors, batteries202 vibration peaks of polymer residue on graphene surface within seconds and the new spectroscopy enhances the relative contrast by ∼50 times compared with conventional absorption spectroscopy. Using the surfactant cetrimonium bromide (CTAB) as a model system, we observe CTAB adsorption on graphene and the reversible deposition/dissolution of CTAB at the graphene electrode with new diffraction vibrational spectroscopy.In this paper, we report a new vibrational spectroscopy technique based on diffraction of monolayer graphene gratings. This method overcomes the difficulties of probing buried interface with infrared, and enables sensitive and interface-specific detection of molecular vibrations spectra at the electrolyte/electrode interface. The advantages are demonstrated by detecting CH−1 and a repetition rate of 150 KHz. The infrared radiation is incident on a graphene grating electrode, which forms an electrochemical cell together with a platinum counter electrode and the aqueous electrolyte , where Ein and Emol are the electrical field of incident light and molecular radiation, respectively, and |Emol|2 is omitted because |Emol|≪|Ein|. The relative intensity change due to light absorption is therefore determined by ΔIt/It≈2·Re(Ein*·Emol)/|Ein|2. For a monolayer of material, |Emol| from molecular vibration is typically <10−3 of Ein  and the periodic modulation of molecular species within the electrical double layer (Emol) (Id/Id≈2·Re(Eg*·Emol)/|Eg|2. Because the diffracted electrical field from the graphene grating (Eg) is about 1% of the incident electrical field Ein  . TherefoEin refs , 25, the2 stretching modes  at the electrolyte/electrode interfaces in an electrochemical cell. The spectra of the diffracted infrared light from the graphene grating are taken with a two-dimensional InSb infrared camera.We demonstrate the capability of this vibrational spectroscopy based on graphene gratings by investigating CHEg from the graphene grating electrode using 12 mM NaCl solution as the electrolyte. −1 as a function of the bias voltage from 1.1 to −0.4 V (black squares), which is normalized by the intensity value at the charge neutral point (CNP) of graphene. The diffraction intensity at 3,000 cm−1 contains contribution mainly from the graphene grating because this frequency is away from any molecular vibration resonances in this system. The bias voltage of the electrochemical cell acts as an electrical gate that controls carrier concentration in graphene. At Vbias=0 V graphene is strongly p-doped in the NaCl solution, and it becomes charge neutral at Vbias=1.0 V. We observe that the diffraction intensity shows a local maximum at the CNP, which decreases upon hole doping to ∼10% of the CNP signal at Vbias=0.5 V. Afterwards, the diffraction signal starts to increase with further hole doping. This non-monotonically change in infrared diffraction from the graphene grating originates from the gate-dependent complex optical conductivity in graphene2021Ig is proportional to (Re(σg))2+(Im(σg))2, where Re(σg) and Im(σg) are the real and imaginary parts of graphene conductivity σg, respectively. σg), Im(σg) and |σg|2 at 3,000 cm−1 as a function of the Fermi energy EF. Near the CNP, Re(σg) from interband transitions dominates the diffraction signal. Re(σg) decreases significantly when 2|EF| equals the incident photon energy due to Pauli blocking, and accounts for the reduced diffraction intensity from carrier doping initially. However, at high doping levels Im(σg) from intraband transitions starts to dominate, which ultimately leads to diffraction intensity increasing at high |EF|. The solid line in First, we examine the diffraction signal −1) from as-prepared graphene electrodes in the 12 mM NaCl solution at different bias voltages. Id(ω)/IdCNP(ω), where IdCNP(ω), the spectrum at CNP, is used as the reference. We observed two sharp resonances at 2,916 and 2,848 cm−1 on top of a broad background signal. The broad background arises from the graphene electrode, which first decreases and then increases with higher hole doping controlled by Vbias. The two resonances at 2,916 and 2,848 cm−1 correspond to the anti-symmetric and symmetric stretching vibrations of the methylene group (-CH2-), respectively. A weak resonance at 2,960 cm−1 corresponding to methyl group (-CH3) is also present. The appearance of CH2 vibration peaks indicates that there are hydrocarbons on top of as-prepared graphene in NaCl solution, presumably due to the residue from the polymer transfer and/or photolithography processes. The diffraction spectra in 2 vibration signals, and their understanding requires quantitative description of diffracted electrical field from both graphene and CH2 vibrations at different bias voltages. The normalized diffraction intensity is described byNext we show the infrared diffraction spectra 15N(CH3)3Br) as a test system. CTAB have been widely used to assist DNA extractionThe vibrational spectroscopy based on graphene grating, with its high sensitivity and interface specificity, enables us to −1 from the graphene grating in 11 mM CTAB solution. Compared with graphene in NaCl solution, the voltage dependence shows a similar behaviour except that the CNP is strongly shifted to Vbias=0.2 V. Apparently CTAB largely cancels the strong p-doping in as-prepared graphene, presumably due to the adsorption of cation CH3(CH2)15N(CH3)3+ ( abbreviated as CTA+) that induces a significant amount of electrons in graphene.We plot in 2 vibration frequencies from the CTAB solution/graphene electrode interface at different bias voltages are shown in Vbias=0.2 V is used as the reference. The spectra are reproducible in many cycles when the bias voltage is varied between −1 and 1.5 V, and there is a finite hysteresis when scanning direction switches. The bias-dependent spectra are quite similar to that in NaCl solution  and 2,944 cm−1 (blue arrows). The evolution of these spectra features as a function of electrical potential and their fittings are shown in When d −1.2 V , the evo−1 is associated with the head group of CH3-(N+) in crystalline CTAB31Vbias reaches −1.5 V, a new electrochemical process is triggered to deposit highly ordered CTAB on the graphene electrode. This deposition process can be monitored in real time with our diffraction spectroscopy. When the bias voltage is further increased to positive, the deposited CTAB systematically modify the diffraction spectra. At the CNP with Vbias=0.2 V, the normalized spectrum shows distinct features rather than a constant. When Vbias is further scanned beyond 1 V, however, the features associated with the deposited CTAB layer become weaker and then disappears, and the spectral shape becomes similar to those in Vbias=−1.5 V in the negative scan, coincidence with the start of CTAB deposition, and a peak around Vbias=1 V in the positive scan, coincidence with the disappearing of CTAB deposition. After the Vbias reaches 1.5 V, most of the deposited CTAB is dissolved, and the graphene electrode recovers to its initial status. When Vbias is scanned from 1.5 to −1.5 V afterwards, the diffraction spectra display the same behaviour as that in Previous studies of CTAB have shown that a new vibrational resonance around 2,943 cmVbias=−1.5 V, it can potentially oxide Br− into Br2 at the electrode. In this case, one may expect the corresponding CTA+ cation reduction and deposition in the platinum counter electrode. However, we observe the deposition of CTAB layers at the graphene electrode. Further theoretical investigation will be required to understand microscopically the unconventional deposition processes taking place at the interfaces.The origin of the observed electrochemical deposition and dissolution of CTAB layers is not clear yet. When the graphene electrode is acting as an anode at in situ monitoring of molecular vibrations at the electrochemical interface. In this proof-of-principle study we used CH2 vibrations as an example, but the technique can be readily applied to other functional groups at different infrared frequency ranges. The diffraction spectroscopy enhances the relative contrast by ∼50 times compared with conventional absorption spectroscopy. Currently we demonstrate a sub-monolayer detection sensitivity, which is mainly limited by our laser fluctuation. With improved laser stability and/or balanced detection, we envision that the technique can probe as low as about 1% of a monolayer coverage and provides a powerful tool to investigate fundamental processes at the electrolyte/electrode interfaces in active electrochemical cells.We demonstrated a vibration spectroscopy at the electrolyte/electrode interfaces using graphene grating electrodes. Its high detection sensitivity and interface specificity enables fast and In this experiment, graphene is grown on Cu foil by chemical vapour deposition method−1 is collimated and sent to graphene at normal incidence. The first-order diffraction light is around 25.9° to the normal. The diffraction light is sent to a liquid nitrogen-cooled InSb camera with128 × 128 pixel arrays. The wavelength is then calibrated by absorption of thin film CTAB with its Fourier transform infrared spectrum. The detailed spectroscopy design is shown in The infrared radiation is generated by a femtosecond laser source. Specifically, an amplified femtosecond laser system  delivers laser pulses at 1,026 nm with a pulse duration of 260 fs and a repetition rate of 150 KHz. The laser amplifier pumps a broadly tunable optical parametric amplifier  covering wavelengths from 600 to 2,200 nm. The mid-infrared wavelengths are generated by mixing the pump laser  and the optical parametric amplifier output through difference frequency generation. The output infrared beam at around 3,000 cmHow to cite this article: Bie, Y.Q. et al. Vibrational Spectroscopy at Electrolyte/Electrode Interfaces with Graphene Gratings. Nat. Commun. 6:7593 doi: 10.1038/ncomms8593 (2015).Supplementary Figures 1-6, Supplementary Table 1, Supplementary Notes 1-3 and Supplementary References


Moreover, TAdV-3 does not grow well in cell culture, thus detailed structural studies of the infectious particle is particularly challenging. To develop a better understanding of virus-host interactions, we performed a comprehensive proteomic analysis of proteinase K treated purified TAdV-3 virions isolated from spleens of infected turkeys, by utilizing one-dimensional liquid chromatography mass spectrometry. Our analysis resulted in the identification of 13 viral proteins associated with TAdV-3 virions including a novel uncharacterized TaV3gp04 protein. Further, we detected 18 host proteins in purified virions, many of which are involved in cell-to cell spread, cytoskeleton dynamics and virus replication. Notably, seven of these host proteins have not yet been reported to be present in any other purified virus. In addition, five of these proteins are known antiviral host restriction factors. The availability of reagents allowed us to identify two cellular proteins  chain and haemoglobin) in the purified TAdV-3 preparations. These results represent the first comprehensive proteomic profile of TAdV-3 and may provide information for illustrating TAdV-3 replication and pathogenesis.Turkey adenovirus 3 (TAdV-3) causes high mortality and significant economic losses to the turkey industry. However, little is known about the molecular determinants required for viral replication and pathogenesisThe online version of this article (doi:10.1186/s13567-015-0214-z) contains supplementary material, which is available to authorized users. Siadenovirus A [Hemorrhagic enteritis (HE) is an economically important disease of turkeys characterized by depression, splenic enlargement, intestinal haemorrhages and sudden death . The disovirus A . Oral inovirus A . Althougovirus A -6.The genome of TAdV-3 is 26,263 bp . AlthougAdenoviridae family including TAdV-3 has not been reported so far.In recent years, mass spectrometry (MS) based proteomic characterization has revealed important insights into viral replication, tropism and virulence for a number of different enveloped viruses -14. In cHere, we report the protein composition of the purified TAdV-3 particles by performing a comprehensive proteomic analysis utilizing liquid chromatography-mass spectrometry (LC-MS/ MS). Our analysis resulted in successful identification of 13 viral structural proteins and 18 host-incorporated proteins. Moreover, incorporation of two host proteins in purified virions was verified by Western blot analysis using available immunological reagents.All turkey procedures were approved by University Committee of Animal Care and Supply (protocol # 19940211) at the University of Saskatchewan, Saskatoon, Canada according to guidelines set by the Canadian Council of Animal Care.Day-old Hybrid poults obtained from Chinook belt Hatcheries, Calgary, Canada were housed in isolation rooms throughout the experiments. The avirulent TAdV-3 isolate (pheasant origin) was passaged in sero negative turkeys by oral inoculation and purified from crude spleen extracts, as described earlier .The TAdV-3 virions were purified as previously described . The pro2 gradient purified TAdV-3 virions (proteinase K treated or untreated) at EM facility at Biology department, University of Victoria, BC, Canada, as described [2 gradient purified virus was first applied onto carbon and formvar coated grids, washed with H20 and stained with 2% aqueous phosphotungstic acid. The specimens were photographed using a charge-coupled device camera .Electron microscopy was performed on CsClescribed . BrieflyProduction and characterization of anti-TAdV-3 serum and monoclonal antibodies (MAbs) recognizing TAdV-3 hexon 15G4) and fiber (87–03) proteins has been described earlier  and fibe. ChickenProteins from purified TAdV-3 were separated by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis (PAGE) on 10–15% or 4–15% precast gradient gels (Bio-Rad),transferred to nitrocellulose membrane and probed with protein specific antibodies as described previously .2 gradient purified virion-enriched (proteinase K treated or untreated) samples were diluted with 200 mM ammonium bicarbonate prior to reduction with 200 mM dithiothreitol and incubated 30 min at 37 °C. Cysteine sulfhydryl groups were alkylated with 20 μL of 100 mM iodoacetamide (30 min at 37 °C in darkness). Each sample was digested with 5 μg of trypsin (Promega) at 37 °C for 16 h [Proteins from CsClfor 16 h ,35. FinaThe peptide mixtures were separated by on-line reverse phase chromatography using a EASY-nLC II system (Thermo Scientific) with a reversed-phase Magic C-18AQ pre-column  and reversed phase nano-analytical column Magic C-18AQ  at a flow rate of 300 1/min. The resulting peptides were analyzed by the chromatography system, which was coupled on-line with a LTQ OrbitrapVelos mass spectrometer  equipped with a NanosprayFlex source (Thermo Fisher Scientific) as described previously ,35. The Raw files were analysed with Proteome Discoverer 1.4 software suite (Thermo Scientific). Parameters for the spectrum selection to generate peak lists of the collision-induced “dissociation (CID) spectra were activation type: CID”; . The peak lists were submitted to an in-house Mascot 2.3 server against “the following databases”: Uniprot_Trembl 20111103  and Uniprot-Swissprot 20110104  all species taxonomy.Gallus gallus (chicken)) database to detect viral and host proteins. Only sequences identified with a mascot score value greater than 30 were considered as significant. Protein identifications were accepted when the peptide probability was greater than 95.0% [Database search parameters were as follows: precursor tolerance 8 ppm; MS/MS tolerance 0.6 Da; Trypsin enzyme 1 missed cleavages; Fourier Transform Ion Cyclotron Resonance (FT-ICR) instrument type; fixed modification: carbamidomethylation (C); variable modifications: deamidation ; oxidation (M). The Decoy database Percolator settings: Max delta Cn 0.05; Target FDR strict 0.01, Target FDR relaxed 0.05 with validation based on q-Value. Additional virus only species searches were also performed with tolerances previously mentioned. All data were also searched against NCBI  containing capsid and the lower band  containing complete infectious viruses . This method eliminates the problems reported with proteins that either enter gel poorly or extracted inefficiently from the gel slices. Our LC-MS/MS analysis revealed a total of 15 virus-encoded proteins packaged in the purified TAdV-3 virions. This included 13 proteins, which have been detected in human adenovirus 5 (HAdV-5) virions  Table 1.Table 1T2 purification. The proteinase K treated and untreated, purified virions were then analysed by Western blotting. Proteinase K treatment degrades fiber protein protruding from the capsid but does not degrade hexon protein not protruding from the capsid. As seen in Figure 2 density gradient purification  and subjected to another round (third round) of CsCl0 μg/mL a2 density gradient purified TAdV-3 virions identified eleven virus-encoded proteins  previously reported to be in other adenoviruses  peptides showed 70% identity to turkey collagen alpha-1(VI) and chicken collagen alpha-1(VI)  , which aThe proteomic analysis of proteinase K treated purified virions identified eleven cellular proteins incorporated in TAdV-3, which have been identified in other viruses Table . In addiAtadenovirus genus and purified HAdV-5, a prototype of Mastadenovirus genus [Siadenovirus genus) and origin of cells used for virus cultivation [Though earlier reports have demonstrated the packaging of viral  or non vus genus . The absus genus . Alternativation .The host proteins packaged inTAdV-3 are known to play important roles in enhancing the cell-to-cell spread of virus, transcription and virus replication  into TAdV-3 virions is particularly intriguing. All of these host defence factors have been implicated in establishing antiviral environments. Recent studies have implicated PML in maintaining host antiviral defence and revealed different strategies developed by viruses to disrupt PML nuclear bodies -46. In aAlthough TAdV-3 grows efficiently in spleen of infected turkey, virus grows poorly in primary or established cell lines. It is tempting to speculate that integration of certain established antiviral host restriction factors into viral particles may play a role in determining TAdV-3 replication “in vitro”. Additional studies need to be performed in order to investigate whether these proteins are functionally required for virus entry, replication and pathogenesis. Future availability of reagents and a reliable cell culture system to grow TAdV-3 should make it possible to determine the role of individual host restriction factor in TAdV-3 replication.