Graphene has received significant attention due to its excellent mechanical properties, which has resulted in the emergence of graphene-based nano-electro-mechanical system such as nanoresonators. The nonlinear vibration of a graphene resonator and its application to mass sensing (based on nonlinear oscillation) have been poorly studied, although a graphene resonator is able to easily reach the nonlinear vibration. In this work, we have studied the nonlinear vibration of a graphene resonator driven by a geometric nonlinear effect due to an edge-clamped boundary condition using a continuum elastic model such as a plate model. We have shown that an in-plane tension can play a role in modulating the nonlinearity of a resonance for a graphene. It has been found that the detection sensitivity of a graphene resonator can be improved by using nonlinear vibration induced by an actuation force-driven geometric nonlinear effect. It is also shown that an in-plane tension can control the detection sensitivity of a graphene resonator that operates both harmonic and nonlinear oscillation regimes. Our study suggests the design principles of a graphene resonator as a mass sensor for developing a novel detection scheme using graphene-based nonlinear oscillators. Graphene has recently been attracting the scientific community due to its excellent electrical -3 and/orTo gain a detailed insight into the underlying mechanism of the vibration of a graphene resonator, an atomistic simulation such as molecular dynamics (MD) simulation has been widely utilized. For instance, Park and coworkers ,23 have The computational limitation of atomistic simulations in depicting the underpinning principles of experimentally observed mechanics of graphene resonators has led researchers ,25-27 toIn this work, we have studied the nonlinear vibration of a graphene resonator using a continuum elastic model, i.e., plate model. We have found that nonlinear oscillation is a useful avenue for improving the detection sensitivity of a graphene resonator and that the detection sensitivity of a graphene-based nonlinear oscillator is governed by both the actuation force (which determines the nonlinearity of vibration) and the size of a graphene resonator. It is shown that the nonlinearity of vibration for a graphene resonator can be tuned by an in-plane tension and that such in-plane tension can modulate the detection sensitivity of a graphene resonator that operates in both harmonic and nonlinear oscillations. In particular, an in-plane tension improves the dynamic frequency range and detection sensitivity of a graphene resonator that operated in harmonic oscillation, while an in-plane tension deteriorates the dynamic frequency range and sensing performance of a graphene-based nonlinear oscillator. Our study sheds light on a continuum elastic model for gaining insight into not only the underlying mechanisms of nonlinear vibration-based enhancement of the dynamic frequencies and sensing performance of a graphene resonator, but also the role of an in-plane tension in modulating the nonlinearity of a graphene resonator.UB and stretching energy US represented in the form [Graphene can be modeled as a plate whose mechanical deformation is attributed to the strain energy composed of bending energy the form κESh, and ν represent the bending rigidity, axial stretching modulus, thickness, and Poisson's ratio of a graphene, respectively, w(xyt) indicates the out-of-plane deflection of a graphene, x and y are the coordinates along the in-plane direction of a graphene, respectively, N0 is a constant axial tension (due to pre-strain) applied to a graphene, and the symbol Ω in an integrand indicates the surface integral. The strain energy can be related to a potential field prescribed to the atomic structure of a graphene, as has been elucidated in Cauchy-Born model [where rn model -34, suchi-th carbon atom for a graphene, r is the atomic coordinates of a graphene, and N is the total number of carbon atoms for a graphene. Here, it should be noted that when the elastic constants of a graphene (i.e. κ and ES) are determined from Equation 2, an axial tension N0 is assumed to be zero . As described in a literature [κ = 1.5 eV and ESh = 2,000 eV/nm2.Here, terature , the forT for a vibrating graphene. The kinetic energy T can be written asIn order to obtain the equation of motion, we need to know the kinetic energy ρ0 is the mass density of a graphene; the mass density ρ0 can be straightforwardly determined from a relation of ρ0 = NmC/S, where mC is the atomic mass of a carbon atom, and S is the surface area of a graphene. The equation of motion for a vibrant graphene can be obtained from the minimization of a Hamiltonian H defined as H = U + T − W, where W is the work done by an external force field such as actuation force. The variation of the Hamiltonian, δH, can be obtained as [where ained as ,35f is an actuation force per unit area for a graphene, a symbol δ indicates a variation, δw is a virtual out-of-plane deflection of a graphene, a Greek index indicates the coordinates, i.e., α = x (for α = 1) or y (for α = 2), and a repeated Greek symbol represents Einstein's summation rule. The equation of motion is therefore given bywhere w(xt). In this work, for theoretical convenience, we assume that axial force N0 is the biaxial loading represented in the form of N0 = N0(ex + ey), where ex and ey indicate the directional unit vectors in the x and y directions, respectively. Furthermore, the actuation force f is assumed to be in the form of f = f0cosΩt, where f0 is the amplitude of an actuation force, and Ω is a driving frequency. For solving the equation of motion given by Equation 5, we assume that the out-of-plane displacement, i.e., w(xyt), can be decomposed in the following form [Here, it should be noted that in-plane displacements are ignored in the governing equation given by Equation 5 since in-plane displacements are small in comparison with out-of-plane displacement ing form ,36-38z(t) indicates a time-dependent amplitude, and ψ(xy) represents the deflection eigenmode for a vibrant graphene. In our work, we presume that a monolayer graphene exhibits a rectangular shape and that all edges of a graphene are clamped. The deflection eigenmode that satisfies the clamped boundary conditions is represented in the formHere, a and b are the lengths of the graphene edges, respectively  as shown in Figure μ is given byFor a case in which atoms are adsorbed onto a graphene resonator, as shown in Figure δ(x) is the Dirac delta function, and Δm is the total mass of adsorbed atoms. For the case in which atoms are uniformly adsorbed onto a graphene , the inertia term μ is written aswhere m0 is the mass of atoms adsorbed onto the unit area of a graphene, while Δm is the total mass of adsorbed atoms onto the entire surface of a graphene. Based on inertia terms for both mass-adsorbed graphene (as described in Equation 10) and a bare graphene (represented in Equation 9.a), it is straightforward to compute the resonant frequency shift, Δω, of a graphene resonator due to the mass adsorption; Δω = ω(m + Δm) – ω(m), where ω(m) is the resonant frequency of a bare graphene resonator whose effective mass is given as m (with m = ρab), and ω(m + Δm) is the resonant frequency of a graphene resonator onto which the mass adsorption with the amount of Δm occurs. This frequency shift due to mass adsorption is typically negative since mass adsorption increases the overall mass of a resonator and, consequently, reduces the resonant frequency of a resonator with respect to that of a bare resonator.Here, Δp0 = 0.001 aN (where 1 aN = 10−18 N) in order to induce the harmonic oscillation of a graphene resonator. Moreover, we have taken into account the case in which an in-plane tension N0 is driven by pre-strain, such as N0 = EShϵ0/(1 − ν), where ESh, and ν indicate the stretching modulus, thickness, and Poisson's ratio of a graphene resonator, respectively, and ϵ0 is the pre-strain applied to a graphene resonator. With ϵ0 = 4 × 10−5, the resonant frequency of a graphene undergoing a harmonic oscillation is predicted as ω0 = 20.63 MHz, which is consistent with the experimentally measured resonant frequency of 19.8 MHz  = ∇ψ(xy) = 0 along all edges; this boundary condition is referred to as “fully clamped” boundary condition. In order to understand the effect of boundary condition on the resonant frequency of a graphene resonator, we have taken into account the deflection eigenmode in the form of ψ(xy) = sin(πx/a)⋅sin(πy/b), which satisfies the boundary condition as follows: ψ(xy) = 0 at all edges, but ∇ψ(xy) ≠ 0 along all edges. This boundary condition is referred to as “weakly clamped” boundary condition. As shown in Figure As described above, the resonant frequency of a graphene resonator predicted from a continuum elastic model is overestimated in comparison with that measured from experiment. This may be attributed to the boundary condition that we used in our simulation; in our simulation, we have utilized the fully clamped boundary conditions such as D = 250 nm, to which pre-strain with the amount of ϵ0 = 10−5 is applied. It is shown that when the amplitude of an actuation force is in the order of 0.01 fN, the graphene resonator undergoes harmonic oscillation. On the other hand, when a graphene resonator is actuated by the amplitude of an actuation force in the order of >0.05 fN, the graphene resonator experiences nonlinear vibration /Ω0, where ω is the resonance of a nonlinearly oscillating graphene, and Ω0 is the harmonic resonance of a graphene defined as Ω0 = (α/μ)1/2. Here, it should be noted that the dimensionless parameter θ represents the degree of nonlinearity for the resonance of a graphene. Figure θ) as a function of the amplitude of an actuation force. It is found that when a graphene resonator bears a pre-strain with the amount of 10−5, the amplitude of an actuation force in the order of 0.5 fN results in θ = 0.15, which indicates that the resonance behavior at an amplitude of 0.5 fN is close to the harmonic oscillation. As the amplitude increases, the dimensionless parameter θ significantly increases, indicating that the nonlinearity of graphene resonance can be induced by a large amplitude of actuation force. In particular, when the graphene resonator is actuated with an actuation amplitude of 5 fN, the parameter θ becomes θ = 0.75, indicating that the resonance behavior is highly nonlinear. This indicates that the nonlinear vibration of a graphene resonator can be easily observed even when the actuation amplitude is applied in the order of 1 fN. The nonlinear vibration of a graphene resonator actuated with an actuation amplitude p0 even in the order of 1 fN is attributed to the fact that the deflection amplitude of a graphene resonator is typically much larger than the thickness of a graphene resonator.Now, we have taken into account the nonlinear oscillation of a squared graphene resonator with a size of n Figure a. This iEven though graphene resonator has recently been extensively considered as a NEMS device for its applications in actuation, it has been barely employed as a mass sensor that enables highly sensitive atomic detection . The high detection sensitivity of a graphene resonator is attributed to the high-frequency dynamic range that is achieved due to high elastic stiffness and low mass density of a graphene resonator. In this study, we have scrutinized the resonant responses of graphene resonators, which undergo not only harmonic oscillation but also nonlinear vibration, to atomic adsorption onto the surface of a graphene resonator. Our study on the nonlinear response of a graphene resonator to mass adsorption is ascribed to our previous finding ,20,21 thD = 250 nm without any applied pre-strain . Figure p0 = 1 aN). On the other hand, when a graphene resonator is excited by a large amplitude of actuation force , the frequency shift due to mass adsorption is no longer proportional to the adsorbed mass. It is also found that as the amplitude of actuation force increases, the resonant frequency shift due to mass adsorption significantly increases, which indicates that nonlinear oscillation increases the detection sensitivity of a graphene resonator as anticipated. In order to gain a deep insight into the effect of nonlinear oscillation on the detection sensitivity of a graphene resonator, we have taken into account the resonant response of a graphene resonator to atomic adsorption with the amount of Δm = 10 ag. As shown in Figure m = 10 ag is critically dependent on the amplitude of actuation force. In addition, we have shown the aforementioned dimensionless parameter θ as a function of the amplitude of actuation force , the frequency shift of a graphene resonator with a size of D = 250 nm (without any pre-strain) due to atomic adsorption with the amount of 10 ag is in the order of 0.1 GHz. On the other hand, when an actuation amplitude is increased to 5 fN, which corresponds to highly nonlinear oscillation , the frequency shift due to mass adsorption (with the amount of 10 ag) increases by about sixfold . This clearly elucidates that the nonlinear vibration is useful in increasing the frequency shift of a graphene resonator due to mass adsorption, which highlights the nonlinear vibration that improves the detection sensitivity of a graphene resonator. We have considered a squared graphene resonator whose size is e Figure b. It is D) on the frequency shift of a graphene resonator due to mass adsorption, Δω, with respect to the actuation amplitude (p0). It is found that for a graphene resonator whose size is D ≥ 150 nm, the increase of actuation amplitude enhances the frequency shift of a graphene resonator due to mass adsorption, which is consistent with our conjecture that nonlinear vibration improves the detection sensitivity of a graphene resonator. On the other hand, for a graphene resonator whose size is D ≤ 100 nm, an increase in the actuation amplitude does not significantly amplify the frequency shift of a graphene resonator due to mass adsorption in comparison to that of a large-scale graphene resonator . This indicates that even though large actuation amplitude induces the nonlinear oscillation of a small-scale graphene resonator , the nonlinear vibration does not remarkably increase the frequency shift due to mass adsorption. This result suggests that the length scale of a graphene resonator plays a central role in not only the dynamic frequency range of a graphene resonator but also in the sensing performance of graphene-based nonlinear oscillators.We have also studied the effect of graphene size  as a function of actuation amplitude and in-plane tension. When the actuation amplitude is in the order of 0.01 fN, the vibration behavior of a graphene becomes almost harmonic oscillation regardless of in-plane tension, as anticipated. For a graphene resonator actuated by an actuation amplitude of 1 fN, the dimensionless parameter for a bare graphene resonator (i.e. N0 = 0) is in the order of 10, which indicates that the vibration behavior of a bare graphene is almost nonlinear oscillation. On the other hand, when an in-plane tension with the amount of 10 pN/nm is applied to a graphene resonator actuated by the amplitude of 1 fN, the dimensionless parameter is in the order of 0.5, which indicates that an in-plane tension reduces the nonlinearity of the resonance of a graphene. As in-plane tension increases, the nonlinearity is significantly reduced even up to the order of 10−1, indicating that the resonance behavior is almost harmonic oscillation. Our result suggests that an in-plane tension plays a central role not only in increasing the dynamic frequency range of a graphene resonator but also in inducing the transition from nonlinear vibration to harmonic oscillation.In order to validate our conjecture that an in-plane tension can play a role in the transition from nonlinear vibration to harmonic oscillation, as shown in Figure D = 200 nm, the resonance behavior of a graphene actuated by an amplitude of ≤0.3 fN is almost close to harmonic oscillation. On the other hand, the vibrational behavior of a 350-nm graphene resonator driven by even an amplitude of 0.1 fN becomes nonlinear oscillation. This indicates that the size of a graphene resonator determines the actuation amplitude that is required to induce the nonlinear vibration of a graphene resonator. Moreover, it is shown that the smaller the graphene resonator is, the smaller is the amount of an in-plane tension that can induce the transition from nonlinear oscillation to harmonic resonance. This suggests that an in-plane tension-driven transition from nonlinear vibration to harmonic oscillation is determined by the size of a graphene resonator.Figure m = 20 ag) as a function of the size of graphene as well as in-plane tension. It is interestingly found that for graphene resonators operating in both nonlinear oscillation and harmonic vibration, an in-plane tension-induced improvement of detection sensitivity of a graphene resonator is significantly dependent on the size of a graphene such that an in-plane tension is useful in increasing the detection sensitivity of a graphene resonator whose size is D ≈ 100 nm, whereas an in-plane tension is ineffective in enhancing the sensing performance of a graphene with a size of D > 300 nm in comparison with the detection sensitivity of a graphene resonator with a size of D = 100 nm. Our study sheds light on the important role of an in-plane tension on modulating not only the resonance behavior of a graphene resonator but also the detection sensitivity of a graphene resonator.Now, we have studied the role of an in-plane tension on the detection sensitivity of a graphene resonator that experiences both nonlinear vibration and harmonic oscillation Figure . For a gIn this work, we have studied the vibrational behaviors of graphene resonators as well as their sensing performance based on continuum elastic model such as plate model. It is shown that nonlinear vibration is useful in improving the detection sensitivity of a graphene resonator and that an in-plane tension is able to tune both the nonlinearity of the vibrating graphene resonators and their detection sensitivity. It should be noted that, in this work, our continuum model is only applicable to a monolayer graphene resonator. For modeling the multilayered graphene resonator, the interactions between graphene sheets have to be considered in the continuum modeling , which wThe authors declare that they have no competing interests.KE and C-WK designed the research. MDD performed the simulation. MDD, C-WK, and KE analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.Figure S1. The dependence of frequency shift due to mass adsorption of a graphene on the location at which atomic mass was adsorbed.Click here for file


We found that while a number of kinase inhibitors, particularly of the MAPK pathways modulated both TDP-43 and the global stress granule marker, human antigen R (HuR), multiple inhibitors were more specific to TDP-43 accumulation, including inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR, but often with different effects on HuR accumulation. This may indicate a potential interaction between TDP-43, hnRNP K and TIAR. CDK inhibitors were also found to reverse pre-formed TDP-43-positive stress granules and both CDK and GSK3 inhibitors abrogated the accumulation of C-terminal TDP-43 (219–414) in transfected cells. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partner hnRNP K. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation and may herald new opportunities for kinase modulation-based therapeutic intervention in ALS and FTLD.Abnormal processing of TAR DNA binding protein 43 (TDP-43) has been identified as a major factor in neuronal degeneration during amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). It is unclear how changes to TDP-43, including nuclear to cytosolic translocation and subsequent accumulation, are controlled in these diseases. TDP-43 is a member of the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family and is known to associate with cytosolic RNA stress granule proteins in ALS and FTLD. hnRNP trafficking and accumulation is controlled by the action of specific kinases including members of the mitogen-activated protein kinase (MAPK) pathway. However, little is known about how kinase pathways control TDP-43 movement and accumulation. In this study, we used an  Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset motor neuron disease that commonly strikes people between 40 and 60 years of age. During the disease, motor neurons in the spinal cord and brain degenerate, normally leading to death in 1–5 years. The progressive deterioration of patients with ALS results in enormous healthcare and social costs, yet little is known about the disease process and no long-term effective treatments exist. Frontotemporal lobar degeneration (FTLD) is a collective term for a group of neurodegenerative diseases associated with degeneration in the frontal and temporal lobes of the brain The majority of ALS cases are sporadic but ∼5% of patients have a familial mutation. In 2006, TAR DNA binding protein 43 (TDP-43) was identified as a major protein constituent within ubiquitinated neuronal inclusions in a large proportion of ALS and FTLD cases in vivo and how this may contribute to the disease process.TDP-43 has been found to associate with cytosolic RNA stress granule (SG) proteins. This may be an essential early step in pathological accumulation of TDP-43 It is well known that protein kinases control movement and accumulation of SG proteins including hnRNPs and HuR in vivoWhether additional kinases have a critical role in controlling TDP-43 nuclear to cytosolic trafficking and subsequent accumulation in the cytosol is not clear. Glycogen synthase kinase 3 (GSK3) has a central role in neurodegeneration due to its modulation of the microtubule-associated protein, tau In the present study we examined whether additional kinases are involved in accumulation of TDP-43 in SGs in cell culture. Using our established model of mitochondrial inhibition to induce TDP-43-positive SGs in SH-SY5Y cells, we screened 80 kinase inhibitors covering 35 different kinases (Tocriscreen kinase inhibitor toolbox) to determine the effect of kinase inhibition on TDP-43 localization to SGs.kinase inhibitor toolbox was from Tocris Bioscience .4′,6′ Diamino-2-phenylindole dihydrochloride (DAPI) was obtained from Invitrogen . N,N′-dimethyl-4,4′-bipyridinium dichloride (paraquat) and sodium arsenite, were from Sigma Aldrich . Tocriscreen Polyclonal TDP-43 antisera were purchased from Proteintech Group . Polyclonal antisera to hnRNP K were purchased from Abcam . Monoclonal antisera to hnRNP A1 were from Merck . TIA-1-related (TIAR) polyclonal antisera were from Cell Signaling Technology . Monoclonal antisera to HuR were obtained from Invitrogen .2 at 37°C.The cell lines used in this study were human neuroblastoma SH-SY5Y cell line and the human epithelial HeLa cell line. Cells were passaged and maintained in DMEM plus 5% FBS (HeLa cells) or DMEM/F12 plus 10% FBS (SH-SY5Y cells). To induce differentiation, SH-SY5Y cells were treated with 10 µM retinoic acid for 7 days. Differentiation was confirmed by morphological changes (neurite extension) and up-regulated expression of synaptophysin, tyrosine hydroxylase and VMAT2 2O and added at indicated concentrations and the medium was briefly mixed by aspiration. Incubations were performed for periods stated in individual experiments. Where indicated, cells were co-treated with kinase inhibitors at 10 µM from Tocriscreen kinase inhibitor toolbox stock solutions prepared at 10 mM in DMSO. See Undifferentiated cells were grown in 24 or 6-well plates or on 12 mm coverslips (for immunofluorescence) for 2–3 days before experiments (∼80% confluent). Where indicated, retinoic acid-treated SH-SY5Y cells were cultured for 7 days before experiments. Paraquat or sodium arsenite was prepared in dHCell lysates prepared in Phosphosafe Extraction Buffer at equal protein concentration were mixed with electrophoresis SDS sample buffer and separated on 12% SDS-PAGE Tris-Glycine gels. Proteins were transferred to PVDF membranes and blocked with 4% skim milk solution in PBST before immunoblotting. For detection of total TDP-43, membranes were probed with polyclonal antisera  against TDP-43. For detection of total and phospho-forms of extracellular signal regulated protein (ERK), CDK2 and p38, polyclonal antisera from Cell Signaling Technology was used at 1∶5,000. Secondary antiserum was rabbit-HRP at 1∶5,000 dilution. Blots were developed using GE Healthcare ECL Advance Chemiluminescence  and imaged on a Fujifilm LAS3000 imager . Expression of GAPDH was determined using antisera at 1∶5,000 for protein loading controls where necessary. Densitometric analysis of TDP-43 expression was performed using NIH Image J quantification of protein bands from at least three separate cultures.SH-SY5Y cells, or HeLa cells, were grown on 12 mm diameter coverslips and treated with stress inducers and kinase inhibitors as indicated. Cells were fixed with 4% w/v paraformaldehyde in PBS for 30 min and permeabilized with 90% chilled methanol for 5 min. After blocking for 1 h with 10% normal goat serum, cells were incubated with primary antibody for total TDP-43 , HuR (1∶50), TIAR (1∶40), hnRNP A1 (1∶200) or hnRNP K (1∶200) for 2 h at room temperature or overnight at 4°C. This was followed by labeling with secondary AlexaFluor or FITC goat anti-mouse or anti-rabbit antisera at 1∶500 for 2 h at room temperature or overnight at 4°C. After washing, the coverslips were incubated with DAPI at 0.5 µg/ml for 5 min and analyzed using a Leica inverted microscope with Zeiss Axiocam digital camera. Coverslips were examined blinded to the inhibitor treatment and the numbers of TDP-43, HuR, hnRNP K or TIAR-positive stress granules were manually counted in pre-determined multiple fields of view of two to three coverslips for each treatment. Where inhibitors increased or decreased stress granules, treatments were repeated twice. Stress granules were determined as positive if they were distinctly brighter than the surrounding cytoplasmic or nuclear immunofluorescence and/or were clearly separated from surrounding immunofluorescence to allow positive identification of a discrete structure. Results are presented as a percentage change compared to the number of stress-granule-positive cells from paraquat treatment alone. Images shown are representative of multiple fields and replicate or triplicate coverslips per experiment.Plus Midiprep DNA Purification System (Promega Corporation) as per manufacturer’s instructions. DNA was quantified and TDP-43 inserts were identified positively by digestion with BamHI and XhoI.Plasmid DNA corresponding to GFP-tagged full-length wild-type (WT) TDP-43 (pEGFP-TDP WT) or C-terminal fragments of TDP-43, (pEGFP-TDP 219–414) or empty expression vector pEGFP-C1 were prepared as described by Nonaka et. al. 5 cells per well in 24 well–plates on coverslips. Non-retinoic acid-treated cells were transfected 24 h after seeding with the pEGFP-C1 empty vector, pEGFP-TDP WT, and pEGFP-TDP 219–414 using Attractene (Qiagen) according to manufacturer’s instructions. Kinase inhibitors were added at 10 or 1 µM after 24 h. After a further 24 h incubation, cells were fixed with 4% w/v paraformaldehyde in PBS for 30 min. and permeabilized with 90% chilled methanol for 5 min. After washing, the coverslips were incubated with DAPI at 0.5 µg/ml for 5 min and analyzed using a Leica inverted microscope with Zeiss Axiocam digital camera. Expression of TDP-43 was determined by the EGFP-tagged construct SH-SY5Y cells were seeded at 2×10All data described in graphical representations are mean ± standard error of the mean (SEM) unless stated. Results were analysed using a two-way ANOVA and Dunnett post-hoc test.To examine the role of different kinase pathways in stress-induced accumulation of TDP-43, we exposed non-differentiated SH-SY5Y neurons to 1 mM paraquat overnight in the presence or absence of 80 different kinases inhibitors covering 35 separate kinases (Tocriscreen kinase inhibitor toolbox). Each inhibitor was used at a concentration of 10 µM, except where indicated, and coverslips were examined for the number of cells containing TDP-43- and/or HuR-positive stress granules. Paraquat-treated cells revealed robust formation of TDP-43 and/or HuR-positive stress cytosolic granules consistent with our previous studies . Stress Subsequently, we examined the effects of additional kinase inhibitors. The phosphoinositol-3-kinase (PI3K) inhibitor, LY294002 (#7) significantly reduced TDP-43-positive stress granule formation with no significant change to HuR-positive stress granules . InhibitMany additional kinase inhibitors either decreased or increased the number of both TDP-43 and HuR-positive stress granules. For example the MEK inhibitor, U0126 (#8) ; Raf inhFrom this screen, we identified that multiple inhibitors of particular kinases or known kinase pathways induced consistent effects on TDP-43-positive stress granule formation. As shown in To determine if the action of some key kinase inhibitors reflected the ability of the inhibitor to abrogate phosphorylation of its target kinase, we performed a dose-response analysis on TDP-43-positive stress granule accumulation compared to kinase phosphorylation. As shown in Inhibitors of some kinases showed a great deal of variation. Six inhibitors of EGFR were examined with a range of effects including no inhibition , and inhibition of TDP-43-positive stress granules only (#6 and #43) . SummariThe inhibition of TDP-43 stress granule formation by various kinase inhibitors could potentially reflect an inhibition of TDP-43 expression rather than inhibition of TDP-43 cytosolic accumulation. To investigate this, we measured the effect of selected inhibitors on TDP-43 expression in SH-SY5Y cells by western blot. Our initial broad kinase inhibitor screen  identifiNext we examined if selected inhibitors identified in our initial screen of SH-SY5Y cells treated with paraquat induced similar effects on TDP-43 in additional models of stress. This was performed to determine if the effects of the inhibitors were consistent in alternate forms of stress. Initially we examined the effect of these inhibitors in retinoic acid-treated neurons We previously reported that while JNK and ERK inhibitors were able to block formation of TDP-43-positive stress granules when added at the time of insult Finally, to obtain a further insight into the action of the selected kinase inhibitors on TDP-43 cytosolic accumulation, we examined the effect of the inhibitors on C-terminal TDP-43 219-414-GFP (CTF-TDP-43). C-terminal TDP-43 is the major accumulating form of TDP-43 in TDP-43 proteinopathies. We have previously shown that kinases may, at least partially, control C-terminal TDP-43 accumulation. Therefore, in this study we examined this further. As previously reported, non-differentiated SH-SY5Y cells transfected with CTF-TDP-43 revealed localized accumulation of TDP-43 within the cytosol without additional stress induction. Upon treatment with selected kinase inhibitors, we observed the inhibition of TDP-43 accumulation with LY294002 ; olomoucine ; GW5074 ; SB 203580 ; SP600125 ; SB 415286 ; SB 239063 ; and CGP 53353  . InteresThe accumulation of stress granule proteins such as hnRNPs, HuR and TIA-1 or TIAR are controlled by the action of protein kinases Due to the fact that most kinase inhibitors reveal only selectivity for particular kinase targets and are not specific for only one kinase, it can be problematic to identify a kinase associated with inhibition of TDP-43 accumulation based on a single inhibitor. However, for many of the kinase targets, there were multiple inhibitors tested and this can increase the likelihood that a specific target kinase is associated with TDP-43 accumulation. Using this approach we identified inhibitors of CDKs as a potentially important control point for TDP-43 cytoplasmic accumulation. We found that four inhibitors  blocked formation of TDP-43-positive stress granules without effect on HuR-positive stress granule formation. Two additional CDK inhibitors  blocked formation of both TDP-43 and HuR-positive stress granule formation while two  had no effect at the concentrations tested. Notably, CDK inhibitors also reversed the accumulation of TDP-43-positive stress granules when added for the final 6 h of incubation. This strongly suggests that while JNK, other MAPKs and GSK3 are associated with formation of TDP-43-positive stress granules, CDKs may be essential in maintaining their formation once established.The involvement of CDKs in modulating TDP-43 and additional stress granule protein accumulation is not well understood. Lian and Gallouzi reported that decreased levels of cyclin kinase inhibitor p21 in senescent cells was associated with increased stress granule formation Drosophila model. Microtubular integrity is also essential for stress granule dynamics GSK3 was also identified as a key kinase associated with formation of TDP-43-positive stress granules. Three inhibitors of GSK3 all reduced formation of TDP-43-positive stress granules and two of the three (SB 216763 and SB 415286) were specific for TDP-43 effects. GSK3 has an essential role in neurodegenerative disease and has long been a potential therapeutic target for treatment of Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia and ALS One of the intriguing aspects of kinase control of TDP-43 is that the latter has no known consensus sites for phosphorylation by these kinases. Hasegawa et al. in vitro model of TDP-43-positive stress granule formation to screen for the effect of kinase inhibitors on TDP-43 accumulation. We have found that while a number of kinase inhibitors, particularly of the MAPK pathways, modulated both TDP-43 and the global stress granule marker, HuR, multiple inhibitors also more specifically targeted TDP-43 accumulation, including inhibitors of CDKs and GSK3. Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR while differential effects were often observed on HuR accumulation. Further examination of selected inhibitors also revealed a high level of conservation of TDP-43-controlling kinase pathways in different cell types and stresses. Moreover, CDK inhibitors were found to reverse pre-formed TDP-43-positive stress granules rather than just preventing their initial formation. Inhibition of CTF-TDP-43 accumulation in transfected cells with CDK and GSK3 inhibitors suggests a role for modulation of C-terminal TDP-43 trafficking by the identified kinase groups. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partners, hnRNP K and/or TIAR. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation that is a hallmark of TDP-43 proteinopathies such as ALS and FTLD and may herald new opportunities for kinase modulation-based therapeutic intervention in these diseases.In summary, we have used an Figure S1Effect of selected kinase inhibitors on TDP-43 and HuR-positive stress granule formation. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight in the presence or absence of 10 µM LY294002 ; 10 µM U0126 ; 10 µM olomoucine ; 10 µM SB 415286 ; or 10 µM SB 239063 . Green = TDP-43, red = HuR, blue = DAPI. Righthand column shows merged images of TDP-43 and HuR. Arrows indicate stress granules common to both TDP-43 and HuR images. Closed arrowheads indicate HuR-specific stress granules. Open arrowheads indicate cytosolic diffuse TDP-43. Bar = 10 µm.(TIF)Click here for additional data file.Figure S2Effect of additional selected kinase inhibitors on TDP-43 and HuR-positive stress granule formation. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight in the presence or absence of 10 µM PD98059 ; 10 µM GW5074 ; 10 µM SB 203580 ; or 10 µM Arctigenin . Green = TDP-43, red = HuR, blue = DAPI. Righthand column shows merged images of TDP-43 and HuR. Arrows indicate stress granules common to both TDP-43 and HuR images. Bar = 10 µm.(TIF)Click here for additional data file.Figure S3Effect of additional selected kinase inhibitors on TDP-43 and HuR-positive stress granule formation. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight in the presence or absence of 1 µM aminopuvalonol A ; 10 µM HA 1100 ; 10 µM CGP 533353 ; 10 µM arcyriaflavin A ; or 10 µM ZM 447439 . Green = TDP-43, red = HuR, blue = DAPI. Righthand column shows merged images of TDP-43 and HuR. Arrows indicate stress granules common to both TDP-43 and HuR images. Closed arrowheads indicate HuR-specific stress granules. Bar = 10 µm.(TIF)Click here for additional data file.Figure S4Effect of selected kinase inhibitors on TDP-43 expression. SH-SY5Y cells were treated with paraquat (PQ) overnight in the presence or absence of 10 µM LY294002 ; olomoucine ; ZM 449829 ; GW 5074 ; SB 203580 ; SB 415286 ; arctigenin ; SB 239063 ; (1 µM) aminopurvalanol A ; TBB ; HA 1100 ; BIBX 1382 ; CGP 53353 ; arcyriaflavin A . Western blot analysis of TDP-43 expression was determined compared to GAPDH control. Representative image from three experiments. Dotted lines indicate removal of unrelated lanes.(TIF)Click here for additional data file.Figure S5Effect of selected kinase inhibitors on TDP-43 and hnRNP K-positive stress granule formation. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight in the presence or absence of 10 µM LY294002 ; 10 µM U0126 ;10 µM olomoucine ; 10 µM SB 415286 ; or 10 µM SB 299063 . Green = TDP-43, red = hnRNP K, blue = DAPI. Righthand column shows merged images of TDP-43 and hnRNP K. Arrows indicate stress granules common to both TDP-43 and hnRNP K images. Bar = 10 µm.(TIF)Click here for additional data file.Figure S6Effect of selected kinase inhibitors on TDP-43 and TIAR-positive stress granule formation. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight in the presence or absence of 10 µM LY294002 ; 10 µM olomoucine ; or 10 µM SB 415286 . Green = TDP-43, red = TIAR, blue = DAPI. Righthand column shows merged images of TDP-43 and TIAR. Arrows indicate stress granules common to both TDP-43 and TIAR images. Bar = 10 µm.(TIF)Click here for additional data file.Figure S7Effect of CDK inhibitors on hnRNP A1 accumulation in paraquat-treated cells. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight with or without 10 µM of the CDK inhibitors olomoucine (#12) or arcyriaflavin A (#45). Green = hnRNP A1, blue = DAPI. Righthand column shows merged images of hnRNP A1 and DAPI. Bar = 10 µm.(TIF)Click here for additional data file.Figure S8Stress granule inhibition by olomoucine (#12) CDK inhibitor in retinoic acid-treated and non-treated SH-SY5Y cells. SH-SY5Y cells were treated with retinoic acid or left un-treated as described in Methods. Cells were then treated overnight with 1 mM paraquat (PQ) in the presence or absence of 10 µM olomoucine. Green = TDP-43, blue = DAPI. Arrows indicate TDP-43-positive stress granules. Bar = 10 µm.(TIF)Click here for additional data file.Figure S9Stress granule inhibition by olomoucine (#12) CDK inhibitor in HeLa cells. HeLa cells were treated overnight with 50 µM sodium arsenite overnight in the presence or absence of 10 µM olomoucine. Green = TDP-43, red = HuR, blue = DAPI. Righthand column shows merged images of TDP-43 and HuR. Arrows indicate TDP-43-positive stress granules. Arrowhead indicates HuR-specific stress granule. Bar = 10 µm.(TIF)Click here for additional data file.Figure S10Effect of selected kinase inhibitors on reversal of pre-formed TDP-43 and HuR-positive stress granules. SH-SY5Y cells were treated with 1 mM paraquat (PQ) overnight and exposed to selected inhibitors for the final 6 h of incubation ; or 10 µM arcyriaflavin A . Green = TDP-43, red = HuR, blue = DAPI. Righthand column shows merged images of TDP-43 and HuR. Arrows indicate stress granules. Bar = 10 µm.(TIF)Click here for additional data file.Table S1List of kinase abbreviations and names.(DOCX)Click here for additional data file.Table S2Effect of kinase inhibitors on TDP-43 and HuR-positive stress granule formation induced by paraquat treatment in SH-SY5Y cells.(DOCX)Click here for additional data file.Table S3List of kinase classes associated with formation of TDP-43 and/or HuR-positive stress granules induced by paraquat treatment in SH-SY5Y cells.(DOCX)Click here for additional data file.Table S4Dose-response effect of representative kinase inhibitors on TDP-43-positive stress granules and inhibition of target kinase phosphorylation.(DOCX)Click here for additional data file.Table S5Effect of kinase inhibitors on TDP-43, hnRNP K, TIAR and HuR-positive stress granule formation induced by paraquat treatment in SH-SY5Y cells.(DOCX)Click here for additional data file.Table S6Comparison of kinases associated with formation of TDP-43, hnRNP K, TIAR and/or HuR-positive stress granules induced by paraquat treatment in SH-SY5Y cells.(DOCX)Click here for additional data file.Table S7retinoic-acid treated compared to non-treated SH-SY5Y cells.Effect of kinase inhibitors on formation of TDP-43-positive stress granules induced by paraquat treatment in (DOCX)Click here for additional data file.Table S8Effect of kinase inhibitors on formation of TDP-43 and HuR-positive stress granules induced by sodium arsenite treatment in HeLa epithelial cells.(DOCX)Click here for additional data file.Table S9reverse TDP-43 and/or HuR-positive stress granule formation induced by paraquat treatment when for the final 6 hr of 24 hr paraquat treatment (SH-SY5Y cells).Ability of kinase inhibitors to (DOCX)Click here for additional data file.


Complex host–pathogen interactions modulate differential responses to group A streptococcal (GAS) sepsis systemic disease ,2. We pr


An estimated 1500–3000 invasive Endometrial Stromal Sarcomas (ESS) cases annually occur worldwide. Before 2003, ESS was divided as low and high grade ESS based on mitotic activity. In 2003 the WHO changed the names, excluded mitoses and made nuclear atypia and necrosis the essential diagnostic criteria to distinguish ESS, Low Grade  and Undifferentiated Endometrial Sarcoma . We have evaluated in WHO2003 defined ESS-LG whether proliferation biomarkers predict recurrence. Using survival analysis, the prognostic value of classical mitosis counts  in haematoxyllin-eosin (H&E) sections, and immunohistochemical proliferation biomarkers (Ki-67 and PhosphoHistone-3 (PPH3)) were examined in 24 invasive endometrial stromal sarcomas. Three of 24 (12.5%) ESS-LG recurred. The MAI, PPH3 and Ki-67 were all prognostic . MAI values were >3 in the recurrent cases, but never exceeded 10 . Non-recurrent cases had 0≤MAI≤3. PPH3 and Ki67 counts can be easier to perform than MAI and therefore helpful in the diagnosis of ESS, Low Grade. In conclusion, in this small study of WHO2003 defined ESS-LG, high levels of proliferation as measured by MAI, PPH3 and Ki-67 are predictive of recurrence. Larger studies are required to confirm these results. Invasive endometrial stromal neoplasms are rare uterine tumors, accounting for 0.2 to 0.7% of all uterine malignancies and 15% of all uterine sarcomas During the past decade, the rules for what constitutes an Endometrial Stromal Sarcoma (ESS) and what not have changed. Before 2003, invasive endometrial stroma tumors were classified according to their mitotic count per 10 high power fields (HPF) as low (<10 per 10 HPF) or high grade (≥10 per 10 HPF) A number of tumors, whose mitosis counts were more than 10 but without nuclear atypia and necrosis, previously classified as high grade ESS, are now ESS-LG. On the other hand, some previously low grade ESSs are now UES. Because of changes in definition, studies on biologic and prognostic features before 2003 might have biases. Moreover, many studies since 2003 have still used the old classification. Thus, results of prognostic features in ESS-LG need to be thoroughly interpreted.Due to the rarity of ESS-LG and UES, therapies used vary widely from observation without additional treatment after different surgery, to hormone therapy, chemotherapy and radiation therapy, either alone or in varying combinations JAZF1/JJAZ1 and other fusions are not commercially available, it is important to have easy and widely available methods allowing pathologists to assess which patients with an ESS are at high recurrence risk.Regarding the etiology, several studies suggested that ESS-LG and UES have distinct cytogenetic profiles. Two zinc finger genes, JAZF1 and JJAZ1, at the sites of the 7p15 and 17q21 breakpoints were the first translocations identified in endometrial stromal nodules and low grade ESS. The presence of JAZF1-JJAZ1 markedly inhibited apoptosis and induced proliferation rates While WHO2003 ESS-LG tumors in general have a good prognosis and behave in a relatively indolent manner, late recurrences and distal metastases do occasionally occur. This has in recent years fostered a rebirth of studies of nuclear proliferation markers. The proliferation biomarker Cyclin D1 can be used as a simple immuno-histochemical surrogate biomarker for YWHAE-FAM22 ESS To further study this hypothesis, we have investigated WHO2003 defined ESS-LG with reasonably long term follow-up  This study was approved by the Institutional Review Board of the Obstetrics and Gynecology Hospital of Fudan University (FUOGH), Shanghai, China, where the patients were diagnosed and treated, and more recently by Regional Ethics Committee of Norway . Details for the patients have been described elsewhere Mitotic activity index (MAI) was assessed in H&E stained 4 µm thick paraffin sections. Following Good Laboratory Practice criteria, the Standard Operating Procedure for the assessment of the MAI was the same as described in details elsewhere for breast cancer Immunohistochemistry (IHC), antigen retrieval and antibody dilution were optimized prior to the study onset. To ensure uniformity, all sections were processed simultaneously. Four micrometer paraffin sections adjacent to the H&E sections used for histologic assessment were mounted onto Superfrost Plus slides , for PPH3 dried overnight at 60°C and for Ki-67 dried for one hour. Sections were deparaffinized in xylene and rehydrated in decreasing concentrations of alcohol. The Ki-67 and PPH3 antibodies have been described in detail before SPSS version 18  was used for the statistical analyses. For evaluating the variables' prognostic significance, Kaplan-Meier survival analysis was performed. Recurrence free survival and overall disease related survival were endpoints . For age, MAI, Ki-67 and PPH3 which are continuous variables, the optimal prognostic threshold of sensitivity and specificity were detected by Receiver Operating Curve (ROC) analysis using the MedCalc® program .The clinico-pathologic features for the subset of 24 patients for whom the tissue blocks were suitable for the current study were not statistically different from that of the full cohort of 68 ESS-LG patients presented elsewhere Median follow-up time for the 24 patients was 53 (range: 23–84) months. Three (12.5%) patients developed recurrences at 4, 23 and 35 months. The mean age of patients without and with recurrence was not different . 17 of the tumors were stage 1, 6 were stage 2, and 1 was stage 3.Two patients with stage 1 and one with stage 3 disease recurred and stage was prognostically not significant.Like for the full cohort of 68 patients, the variable in the current cohort of 24 patients that correlated strongest with recurrence was the Mitotic Activity Index with a threshold of 0–3 versus 4 or more per 10 HPF (these 10 HPF represent 1.59 mm2 at specimen level) (P = 0.001), PPH3 (P = 0.002) and Ki-67 (P = 0.03) showed comparable results and correlated with recurrence, as is also clear from a mitotic figure”, how and where to select fields of vision for mitosis counts and the wide variation of the area at specimen level of “10 high power fields of vision”. In spite of these influencing factors, mitoses can be well reproducible as was shown in the in the nationwide Dutch MMMCP protocol comparing mitosis counts in nearly 3500 consecutive breast cancers in 34 different pathology laboratories over a three years period. In that study the pathologists all used the same protocol and microscopic objective, resulting in a total measurement area of 1.59 mm2 at specimen level. However, other studies stating to have used “10 HPFs” have used objectives with a much larger diameter, resulting in a total measurement area of nearly 2.8 mm2Before 2003, Endometrial Stromal Sarcomas were traditionally divided as low and high grade, based on the mitotic counts (<10 versus ≥10 per 10 High Power Fields). The change by the WHO2003 of the definition and classification and nomenclature of “low and high grade ESS” into ESS, Low Grade and UES has a long history, going back to Evans in 1982 Nevertheless, about 10% of the WHO2003 ESS-LG still recurs and it would be of clinical significance to identify which cases do and which do not recur. Aided with the exact protocol available for assessment of Mitotic Activity Index and the immunohistochemical proliferation biomarkers Ki-67 and PPH3, a “diagnostic proliferation add-on” consisting of the MAI, Ki-67 and PPH3 could be of diagnostic value.Due to the rarity of ESS-LG and UES, the number of studies since 2003 on ESS-LG and UES is limited The purpose of this study therefore was to identify whether proliferation biomarkers of Endometrial Stromal Sarcomas, Low Grade can help to predict recurrence. All three proliferation biomarkers  significantly predicted recurrence, thereby strengthening the historically known fact (from before the WHO2003 definition) that high proliferation is associated with an increased risk on recurrence.The fact that all three proliferation features are prognostic is interesting as they represent different parts of the cell cycle and their expressions are only partly overlapping. This greatly strengthens the findings and underlines that their increased values in recurrent ESS-LG indeed reflect biological increased growth speed in the recurrent cases. It makes it also very unlikely that the increased proliferation detected is due to chance. Cells in their resting phase (G0-phase) show no activity for any of the three proliferation markers. Cells in the cell-division cycle or “cycling cells” are the cells preparing to divide. These cells all go through a sequence of identical phases  before they divide to form two identical daughter cells. The duration for the entire cell-division cycle process to go to completion varies greatly from one tissue to another, but generally takes 18–24 hours (sometimes shorter or longer). The M(itosis) phase, well known to pathologists as metaphases are visible during this phase, varies greatly but roughly accounts for approximately 4% of the cell cycle duration and typically is confined to the last hour of the cycle. Nearly all phases of cycling cells  express Ki-67. The PPH3 antigen is expressed nearly exclusively in the cells late G2-phase  and M-phase (mitotic figures). Therefore, the frequency of PPH3 and Ki-67 positive cells understandably is higher than that seen for the MAI. Yet, all three proliferation markers tell the same story: increased proliferation in ESS-LG is associated with a significantly increased chance on recurrence.In the past, the prognostic threshold for low and high grade ESS was 10, but we found that 4 or more mitoses per 10 HPF  were significant in ESS-LG. This is very similar to the one found by Abeler et al The cell proliferation index assessed by using MIB-1 antibody against the Ki-67 antigen is widely used and accepted because of its crisp contrast-rich staining pattern and being sympathetic to less than stellar managing of tissue acquisition and tissue handling. Many studies have found it to be diagnostically and prognostically useful and in a small series of 11 low grade ESS it successfully predicted the 2 patients who developed recurrent disease Other studies also found a relation between high-risk on recurrence and increased proliferation in ESS-LG Of course, the therapeutic consequences of increased recurrence rate in ESS-LG with elevated proliferation are a matter of further studies. Li In spite of these promising and biologically understandable results, the number of cases was small . On the other hand, the differences were highly significant (P<0.0001) in 2 of the 3 markers and the three different proliferation biomarkers all point into the same direction. The likelihood that the differences are due to chance, when there are no real differences, are therefore very low. Based on the national incidence rates of ESS in the whole of Norway, with less than 90 cases of ESS and UES in more than 30 years, the chances to soon get a large enough series of WHO2003defined ESS-LG, with long enough follow-up and rather homogeneous treatment, are slim. We call for international multicenter collaborative studies to validate the current results. The quality of the immuno-histochemical stainings should be carefully controlled, preferably by means of an external international Quality Control and Assurance system, such as NordiCQ In conclusion, in FIGO2009, WHO2003 defined ESS-LG, elevated levels of proliferation as measured by MAI, PPH3 and Ki-67 seem predictive of tumors that will recur. Larger independent studies are required to confirm these results.


In this work, we sequenced a 12 kbp-fragment of the mt genome of the katablepharid Leucocryptos marina. Among nine protein-coding genes included in the mt genome fragment, the genes encoding cytochrome b and cytochrome c oxidase subunit I (cob and cox1) were interrupted by group I introns. We further identified that the cob and cox1 introns host open reading frames for homing endonucleases (HEs) belonging to distantly related superfamilies. Phylogenetic analyses recovered an affinity between the HE in the Leucocryptos cob intron and two green algal HEs, and that between the HE in the Leucocryptos cox1 intron and a fungal HE, suggesting that the Leucocryptos cob and cox1 introns possess distinct evolutionary origins. Although the current intron (and intronic HE) data are insufficient to infer how the homologous introns were distributed to distantly related mt genomes, the results presented here successfully expanded the evolutionary dynamism of group I introns in mt genomes.Mitochondria are descendants of the endosymbiotic α-proteobacterium most likely engulfed by the ancestral eukaryotic cells, and the proto-mitochondrial genome should have been severely streamlined in terms of both genome size and gene repertoire. In addition, mitochondrial (mt) sequence data indicated that frequent intron gain/loss events contributed to shaping the modern mt genome organizations, resulting in the homologous introns being shared between two distantly related mt genomes. Unfortunately, the bulk of mt sequence data currently available are of phylogenetically restricted lineages,  Group I (gI) introns are a major class of introns found in bacterial genomes, mitochondrial and plastid genomes, and eukaryotic nuclear genomes http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome), as of January 2012. Thus, we can shed light on novel aspects in the evolution of gI introns in mt genomes by investigating the intron data from the lineages of which mt gene sequences have not been accumulated.Mitochondrial (mt) gene/genome data are potentially informative for inspecting the evolution of gI introns hosting HEs, as a number of gI introns has been identified in mt genomes Leucocryptos marina mt genome, and explored the evolutionary histories of these introns by combining their putative secondary structures, the intron positions, and the phylogenetic affinities of the intronic HEs.Katablepharida is a group of heterotrophic unicellular eukaryotes whose members widely distribute in aquatic environments Leucocryptos mt genome including NADH dehydrogenase subunit 11 (nad11), NADH dehydrogenase subunit 1 (nad1), NADH dehydrogenase subunit 6 (nad6), ATP synthase F0 subunit 6 (atp6), NADH dehydrogenase subunit 7 (nad7), cytochrome c oxidase subunit 2 (cox2), cytochrome c oxidase subunit 3 (cox3), cytochrome b (cob), and cytochrome c oxidase subunit 1 (cox1) genes in this order (cox1 and the 5′ terminus of nad11 are not available in this mt genome fragment). The intergenic spacer regions are short, ranging from 4–65 bp in length. Neither transfer RNA nor ribosomal RNA gene was found in the mt genome fragment determined in the current study. By the comparison between the cDNA and genomic sequences, two introns in this region, one in the cob gene and the other in the cox1 gene, were detected  with an E-value = 10−13]. On the other hand, the putative core region of the cox1 intron appeared to share sequence similarity to those of group IA1 introns . The two gI introns are also distinguishable from one another by the two following points: (i) The cox1 intron have two extra double helical domains, P7.1 and P9.1  An ORF occupies the loop region between P1 and P10 in the cob intron, while an ORF places in the loop region between P1 and P2 in the cox1 intron  residue-long and 267 aa residue-long polypeptides, respectively. The two intronic ORFs likely encode LAGLIDADG-type HEs, but no significant similarity was detected between their putative aa sequences by a BlastP search (bl2seq) with the default settings. Henceforth here, we designate the HE hosted in the cob intron as HELm-cob, while that hosted in the cox1 intron as HELm-cox1. HELm-cob and HELm-cox1 appeared to belong to distant superfamilies, LAGLIDADG_2 (pfam031611) and LAGLIDADG_1 (pfam00961), respectively.The ORFs hosted in the Lm-cob, and subjected to the maximum-likelihood (ML) and Bayesian phylogenetic analyses. In the unrooted phylogeny of this ‘LAGLIDADG_2’ alignment, HELm-cob and two HEs encoded in the cob introns of two green algae, Nephroselmis olivacea and Chlorokybus atmophyticus, grouped together with a BP of 98% and a Bayesian posterior probability (BPP) of 1.00  lied at phase-0 position of the codon corresponding to Gln138 in the Saccharomyces cerevisiae cob gene (GenBank accession number NC_001224), and (ii) hosted a HE belonging to LAGLIDADG_2 superfamily in the loop region between P1 and P2 as shown in Leucocryptos cob intron by intron positions, as the HEs hosted by the introns lying in the homologous positions were sporadically distributed in the LAGLIDADG_2 phylogeny . Thus, Leucocryptos cox1 intron and the forth intron in Rhizophydium cox1 gene likely derived from a single ancestral intron, which lied at phase-0 position of the codon corresponding to Thr93 in the S. cerevisiae cox1 gene, and hosted a LAGLIDADG_1-type HE in the loop region between P1 and P10 , being in close proximity to but apparently distinct from the homing position of the Leucocryptos and Rhizophydium introns (see above). One possibility is that HELm-cox1 and the Rhizophydium HE, and the Podospora and Dictyostelium HEs have evolved from a single ancestral HE and still recognize the identical nucleotide sequence (or very similar sequences), but the cleavage position altered after the separation of the two HE pairs. In any case, the evolutionary link between the cox1 introns in Leucocryptos and Rhizophydium, and those in Podospora and Dictyostelium can be assessed only after the enzymatic properties of the HEs hosted in the four cox1 introns are characterized.The clan of HEcob and cox1 genes discussed above have been vertically inherited from the common ancestor of katablepharids, green algae and fungi, but (ii) secondary intron loss occurred in other descendent lineages, as the HE sequences considered here unlikely represent the true diversity of LAGLIDADG_2-type or LAGLIDADG_1-type HE superfamily. The origins of the two gI introns found in Leucocryptos mt genome should be revisited after in-depth surveying introns and intronic HEs in the mt genomes of phylogenetically broad eukaryotic lineages, particularly those of close relatives of katablepharids, such as goniomonads and cryptomonads.Introns in organellar genomes are generally regarded as mobile genetic elements powered by intronic HEs, as ‘trans-genomic’ intron invasion have been accumulated in the literature Leucocryptos marina NIES-1335 and the haptophyte Chrysochromulina sp. NIES-1333 were purchased from the National Institute for Environmental Study (NIES). Leucocryptos was maintained in f/2 medium (http://mcc.nies.go.jp/02medium.html#f2) with Chrysochromulina as a prey at 20°C under 14∶10 hours of light∶dark cycles.The katablepharid Leucocryptos cells containing a small amount of the Chrysochromulina (prey) cells, and this sample was then subjected to DNA and RNA extractions by using Plant DNA Isolation Reagent (TaKaRa) and RNeasy Plant Mini Kit (QIAGEN), respectively. Total RNA was used for random hexamer-primed cDNA synthesis by Superscript II reverse transcriptase (Invitrogen). The experiments mentioned above were conducted by following the manufactures' instructions. The cDNA and total DNA were used as the templates for polymerase chain reactions (PCR) aiming at the amplification of gene transcripts and genome fragments, respectively.We harvested the cob, cox1, cox3, nad1, nad7, and nad11. PCR products were cloned into pGEM-TEasy vector (Promega). For each gene transcript, we completely sequenced eight clones and confirmed no sequence heterogeneity among clones, except the cob and cox3 transcripts. The cob and cox3 samples appeared to consist of two distinctive types of amplicons, one with and the other without in-frame TGA codons . We regarded the amplicons with in-frame TGA codons as the mt gene transcripts from the haptophyte (Chrysochromulina) prey cells for two reasons: Firstly, our preliminary phylogenetic analyses indicated that the two amplicons were distantly related to each other, and only the one with in-frame TGA codons displayed an intimate affinity to the haptophyte homologues  with the primer sets shown in mologues  & B. Secnad11 and nad1, nad1 and nad7, nad7 and cox3, cox3 and cob, and cob and cox1 with outwarded exact match primers designed based on the six mt gene transcripts initially determined (see above) as performed in previous works nad6, atp6, and cox2) were additionally found. Cloning and sequencing were performed as described above. The partial mt genome sequence was deposited to DNA Data Bank of Japan (GenBank/EMBL/DDBJ accession no. AB693966).We then amplified the mt genome fragments corresponding to the six transcripts with no in-frame TGA codon with exact-match primers (not shown). We also amplified the intergenic spacer regions between cob and cox1 genes in the Leucocryptos mt genome appeared to possess a single gI intron with a HE (see Results and Discussion). Both 5′ and 3′ splice sites were determined by comparing the cDNA and genomic sequences. Intron secondary structures were predicted using MFOLD Each of Leucocryptos cob intron (HELm-cob) was aligned with 29 HEs belonging to the LAGLIDADG_2 superfamily, which showed significant similarity to HELm-cob in TBlastN search against the GenBank nr database . We carefully assessed the alignments from the Blast search, and excluded redundant sequences and the sequences which produced very short alignments with HELm-cob. After manual refinement followed by the exclusion of ambiguously aligned positions, 183 aa positions were remained in the final ‘LAGLIDADG_2’ alignment. Pairwise aa identities and similarities ranged from 30 to 98%, and from 48 to 98%, respectively (Chlorokybus atmophyticus cob intron showed the highest aa identity (47%) to HELm-cob, while the ones hosted in the Millerozyma farinose and Chlorogonium elongatum cob genes showed the lowest aa identity (34%) to HELm-cob (see the upper triangular in Chlorokybus atmophyticus cob intron showed the highest aa similarity (65%) to the HELm-cob, while the one hosted in the Chlamydomonas incerta cob genes showed the lowest aa similarity (49%) to HELm-cob (see the lower triangular in The HE encoded in the ectively . The HE Leucocryptos cox1 intron (HELm-cox1) and 24 HEs belonging to the LAGLIDADG_1 superfamily, which can form global alignments with HELm-cox1 with TBlastN E-values<10−10. The final LAGLIDADG_1 alignment contains 191 unambiguously-aligned aa positions. Pairwise aa identities and similarities ranged from 14 to 95%, and from 34 to 97%, respectively (Rhizophydium sp. cox1 intron showed the highest aa identity (44%) to HELm-cox1, while the one hosted in the Flammulina velutipes cox1 gene showed the lowest aa identity (20%) to HELm-cox1 (see the upper triangular in Podospora anserina cox1 intron showed the highest aa similarity (62%) to HELm-cox1, while the ones hosted in the Blastomyces dermatitidis cox1 gene and Allomyces macrogynus cob gene showed the lowest aa similarity (41%) to HELm-cob . B. Schematic structures of Leucocryptos and Rhizophydium cox1 introns. Both introns harbor LAGLIDADG_1-type homing endonucleases in the region between P1 and P10 (shown as closed boxes).(TIF)Click here for additional data file.Figure S2Maximum-likelihood (ML) analyses of the COB and COX3 amino acid (aa) alignments.A. The ML phylogeny inferred from the COB alignment comprises 31 taxa with 368 unambiguously aligned aa positions B. The ML phylogeny inferred from the COX3 alignment comprising 26 taxa with 218 unambiguously aligned aa positions. Leucocryptos marina and Chrysochromulina sp. are highlighted by arrowheads. The haptophyte clade is shaded. Only ML bootstrap values equal to or greater than 50% are shown. Methods: The two aa alignments were separately analyzed with the ML method with the LG+Γ+F model by using RAxML ver. 7.2.1. The details of the ML and ML bootstrap analyses were same as described in Materials and Methods/Phylogenetic analyses of intronic HEs.(TIF)Click here for additional data file.Figure S3Amino acid (aa) sequence homology.A. Pairwise aa identity matrix of the 30 endonuclease (HE) sequences in the LAGLIDADG_2 alignment. We also recoded 20 aa characters in the HE sequences to six Dayhoff classes, and then made the identity matrix presented below diagonal. B. Pairwise aa identity matrix of the 25 HE sequences in the LAGLIDADG_1 alignment. We also provide the pairwise ‘Dayhoff-class’ identity matrix below diagonal. For each sequence, the GenBank accession no. is shown in brackets.(TIF)Click here for additional data file.


In this article, a new method for doping graphene using the conjugated organic molecule, tetracyanoquinodimethane (TCNQ), is described. Notably, TCNQ is well known as a powerful electron accepter and is expected to favor electron transfer from graphene into TCNQ molecules, thereby leading to p-type doping of graphene films. Small amounts of TCNQ drastically improved the resistivity without degradation of optical transparency. Our carrier doping method based on charge transfer has a huge potential for graphene-based TCFs.Transparent conductive films (TCFs) are critical components of a myriad of technologies including flat panel displays, light-emitting diodes, and solar cells. Graphene-based TCFs have attracted a lot of attention because of their high electrical conductivity, transparency, and low cost. Carrier doping of graphene would potentially improve the properties of graphene-based TCFs for practical industrial applications. However, controlling the carrier type and concentration of dopants in graphene films is challenging, especially for the synthesis of  Transparent conductive films (TCFs) are a class of extremely important components of modern technology for applications such as optical devices and solar energy utilization . Indium Meanwhile, graphene, a single atomic layer of carbon, has attracted greater attention as an alternative material of TCFs because of its high electrical conductivity and transparency . In addi2 [p-type films. With a view to improving the electrical properties of graphene-based TCFs, we propose a novel carrier doping method based on charge transfer from conjugated organic molecules. It is anticipated that liquid phase chemical interaction between graphene and conjugated organic molecules induces a high doping efficiency.The problem of high resistivity of chemically derived graphene-based TCFs  still re2 , and cha2 ,12. Howep-type doping of graphene films. Figure Tetracyanoquinodimethane (TCNQ) is well known as a powerful electron accepter and is expected to favor electron transfer from graphene into TCNQ molecules, thereby leading to 2SO4 and K2S2O8, and oxidized in KMnO4 and H2SO4. After centrifugation, the resulting graphite oxide was exfoliated into graphene oxide (GO) by ultra-sonication . Then, a GO aqueous dispersion was produced by centrifugation and dialysis to neutralize a pH. The morphology of GO synthesized by this procedure was characterized by Raman spectroscopy (excited by 532-nm Ne laser) [Chemically derived graphene was synthesized by the modified Hummer's method , a well-e laser) , optical2H4, a strong reductant, with NH3 to adjust pH [N,N-dimethylformamide (DMF) using an evaporator. RGO can be dispersed well in many kinds of organic solvents including DMF, while it is easily aggregated in aqueous solution because of its low electrostatic repulsion force. A RGO sample deposited on Au (10 nm)/SiO2 (90 nm)/Si substrate was prepared for the evaluation of the reduction state by x-ray photoelectron spectroscopy (monochrome Al Kα X-ray).A reduction step of GO into graphene plays an essential role to determine the electrical properties of the resulting graphene films. GO was reduced as follows: GO was dispersed in aqueous solution containing Ndjust pH . This waOur graphene films were deposited on glass substrates (Corning7059) by a spray-coat method at a substrate temperature of 200°C in an atmosphere containing the solvent vapor. The thickness of the films was controlled by varying the spray amounts. The optical transmittance was measured in the wavelength range from 250 to 2500 nm, and the sheet resistance was measured by van der Pauw method.Doping graphene via charge transfer by TCNQ molecules was carried out as follows. First, 0.01 g of TCNQ powder  was dissolved into 5 ml of DMF solvent. It is expected that TCNQ molecules in DMF are radicalized . Then, 52) were present in the GO aqueous dispersion as shown in Figure -1, referred to as the 2D band, which indicates about 25% of GO flakes were single layer of carbon as demonstrated in our previous article [Large GO flakes (over 30 × 30 μm article .The carbon 1s core level XPS spectra of GO, RGO, and graphite samples were shown in Figure 6 Ω/square with a transparency of 88% at 550 nm. Such a sheet resistance was the lowest obtained compared with previously reported chemically derived graphene films as deposited [Figure eposited . Post-aneposited  obtainedThe SEM images of individual doped graphene flakes indicate RGO flakes maintaining 2D structures after interaction with TCNQ molecules in liquid phase as shown in Figure 10 and 1.17 × 1012 cm-2 for the undoped and doped graphenes, respectively. These estimated values are similar to the reported values by Coletti et al. [Figure i et al. . They moThe authors developed a new and inexpensive fabrication method of chemically derived graphene-based TCFs and demonstrated a huge potential of doping effect via charge transfer by conjugated organic molecules. All of the fabrication steps including the reduction of GO and carrier doping were carried out in liquid phase. Therefore, this novel method proposed in this study does not require any vacuum system and is suitable for quantity synthesis. Furthermore, chemically derived graphene combined with the above doping technique could be a potential alternative to conventional transparent conductive materials.N,N-dimethylformamide; GO: graphene oxide; ITO: indium tin oxide; RGO: reduced graphene oxide; TCFs: transparent conductive films; TCNQ: tetracyanoquinodimethane.DMF: The authors declare that they have no competing interests.RI designed and conducted all experiments and characterisation and drafted the manuscript. MB helped in technical support for experiments and drafting the manuscript. Both YM and AS have read and approved the final manuscript.


In the course of sequencing the complete mitochondrial genome of the prasinophyte picoplanktonic green alga Prasinoderma coloniale CCMP 1220 , we uncovered two additional cases of trans-spliced group I introns. Here, we describe these introns and compare the 54,546 bp-long mitochondrial genome of Prasinoderma with those of four other prasinophytes . This comparison underscores the highly variable mitochondrial genome architecture in these ancient chlorophyte lineages. Both Prasinoderma trans-spliced introns reside within the large subunit rRNA gene (rnl) at positions where cis-spliced relatives, often containing homing endonuclease genes, have been found in other organelles. In contrast, all previously reported trans-spliced group I introns occur in different mitochondrial genes (rns or coxI). Each Prasinoderma intron is fragmented into two pieces, forming at the RNA level a secondary structure that resembles those of its cis-spliced counterparts. As observed for other trans-spliced group I introns, the breakpoint of the first intron maps to the variable loop L8, whereas that of the second is uniquely located downstream of P9.1. The breakpoint In each Prasinoderma intron corresponds to the same region where the open reading frame (ORF) occurs when present in cis-spliced orthologs. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns; we discuss the possible implications of this interesting observation for trans-splicing of group I introns.Organelle genes are often interrupted by group I and or group II introns. Splicing of these mobile genetic occurs at the RNA level via serial transesterification steps catalyzed by the introns'own tertiary structures and, sometimes, with the help of external factors. These catalytic ribozymes can be found in  These intron pieces flanked by exon sequences must interact at the RNA level to produce a functional intron structure that allows splicing to take place; the separate primary transcripts derived from the individual exon sequences are joined and ligated after assembly and splicing of the flanking intron sequences. While trans-spliced group II introns have been known for decades trans-spliced group I introns were reported in 2009 Trans-spliced group I introns are widely distributed across lineages and have been found within fungi trans-spliced group I introns are restricted to the cox1 and rns genes in mitochondria and examples of trans-spliced introns in the plastid have yet to be identified. All reported trans-spliced group I introns display bipartite RNA structures, with one instance hypothesized to use an helper RNA fragment to guide splicing trans-spliced introns whose secondary structures have been predicted, the junction between the 5′ and 3′ fragments, i.e. the breakpoint, is usually located in the loop subtending the base-paired region P8 (L8) cox1 introns found in the lycophytes Isoetes engelmannii/Selaginella moellendorffii are uniquely split in the L9 loop Group I and group II introns can be found in Prasinoderma coloniale CCMP 1220, a marine green alga belonging to the prasinophyte clade VI , and report the characteristics of its trans-spliced group I introns. The Prasinoderma mitochondrial introns are the first trans-spliced introns reported within the rnl gene. One of these introns also provides the first example of a trans-spliced group I intron split in the region downstream of P9.1. We show that the breakpoint in the bipartite RNA structure of each Prasinoderma intron corresponds to the same region that contains an ORF in cis-spliced relatives at the same cognate site. This correlation between the intron breakpoint and the ORF location in cis-spliced orthologs also holds for other trans-spliced introns.In the course of investigating the mitochondrial genome of Prasinoderma coloniale CCMP1220 belongs to one of the deepest prasinophyte lineages. The prasinophytes are paraphyletic, forming at least seven lineages  at the base of the Chlorophyta Prasinoderma represents the Prasinococcales (clade VI), a lineage that had not been previously sampled for organelle genome studies. The complete mitochondrial genome sequence of Prasinoderma was compared with those of four other prasinophytes representing three distinct lineages: Ostreococcus tauri (clades II) Micromonas pusilla (clade II) Nephroselmis olivacea (clade III) Pycnococcus provasolii III (clade V) Nephroselmis, are picoplanktonic prasinophytes, with Ostreococcus being the smallest free-living eukaryote described to date.Prasinoderma mitochondrial genome [GenBank:KF387569] maps as a 54,546 bp-long single circular molecule and features two copies of a large inverted repeat  encompassing 53% of the genome  is slightly higher than that found in genes (52.3%).The e genome . While iclade II . With annophytes  and alsoPrasinoderma mitochondrial genome encodes a set of 55 unique genes, 20 of which (including both rRNA genes) are present in the large inverted repeat. This gene repertoire is intermediate between those of the gene-rich genomes of Ostreococcus tauri, Micromonas pusilla and Nephroselmis olivacea (clades II and III) and that of the gene-poor genome of Pycnococcus provasolii (clade V)  . Unlike nococcus . These dPrasinoderma genome; this is the least densely packed mtDNA among the prasinophyte mitochondrial genomes sequenced so far -3′, 5′-trnR(ucg)-trnI(gau)-3′, 5′-trnS(gcu)-trnA(ugc)-trnT(ggu)-3′ and 5′-rps11-rps13-rpl6-rps8-rps14-rpl5-rpl14-rpl16-rps3-rps19-3′ clusters have been eroded only in the Pycnococcus lineage. The genes comprised in the latter ribosomal protein cluster are missing entirely from the Pycnococcus mtDNA In terms of synteny, the Prasinoderma and Nephroselmis harbor these elements, with two and four group I introns found in these mtDNAs, respectively. Both Prasinoderma group I introns and three of the Nephroselmis introns reside in the large subunit rRNA gene (rnl), whereas the remaining Nephroselmis intron is located in cob. Unlike their Nephroselmis counterparts, the two Prasinoderma mitochondrial introns, named hereafter Pr.rnl.1 and Pr.rnl.2, are discontinuous. Each of these introns is fragmented into two non-adjacent pieces, thus accounting for the three distinct coding regions observed for the rnl gene. The rnl a and rnl b exons are separated from one another by rns, a gene encoded on the same DNA strand as the latter exons; in contrast, the third piece of rnl lies on the opposite strand between cob and rps12  had revealed important variations at the level of mitochondrial genome size, gene content, gene density, and overall genome structure among lineages Prasinoderma, a representative of clade VI, also differs substantially from its counterparts . The Prasinoderma genome has retained more genes than its Pycnococcus homolog but has lost many compared to the Ostreococcus, Micromonas and Nephroselmis mtDNAs, yet its genome size is the largest known among prasinophytes. Moreover, contrary to the genomes of the other three picoplanktonic prasinophytes, which are very tightly packed with genes and lack introns, that of Prasinoderma is much less compact than the Nephroselmis genome and like the latter contains introns. The comparative data reported here thus highlight differences in the types and extent of mtDNA changes that accompanied cell reduction in clades I, V and VI although these three lineages all display a reduced gene content.Sequencing of the terparts , includiPrasinoderma mtDNA was not surprising given the low gene density of this genome; however, the discovery that both are trans-spliced group I introns in the rnl gene was very unexpected. To our knowledge, these introns are the first trans-spliced group I introns reported in the rnl gene. In green algae/land plants, trans-splicing of group I introns had previously been reported only for the mtDNAs of the parasitic trebouxiophyte HelicosporidiumIsoetes and Selaginellatrans-spliced group I introns, cox1 was the gene interrupted.The finding of two introns in the trans-spliced group I introns are still fairly new molecular oddities, we expect that these catalytic ribozymes will be encountered more often in the future. This is due to the dramatically improved DNA sequencing capabilities that allow sampling both in depth and coverage of previously uninvestigated lineages at an unprecedented pace. It is perhaps not surprising that all of the reported examples of trans-spliced group I introns are located in the well-known cox1 and rns and, in this study, in the rnl gene. The main reason is that these genes, in particular cox1 and rnl, are often rich in introns and contain numerous potential intron insertion sites. Moreover, because the products of these genes are well conserved and essential for mitochondrion function, their partial or total absence from annotations is looked upon with suspicion. In contrast, divergent genes are often hard to analyze and thus annotation errors are more likely to be left unnoticed. Finding introns in such genes either in cis or trans configuration can be far from trivial, and when these introns are further split into distinct pieces jumbled across a whole genome, the complexity of this task is compounded.While cis to a trans configuration is rather straightforward and implies one or more recombination events in a segment of the intron that is malleable enough to accommodate the disruption. Therefore, variable loops containing expanded stretches of DNA between conserved pairings are the most obvious targets for recombination. Accordingly, all of the trans-spliced group I introns reported so far that have cis-spliced orthologs are broken at the same variable region as the one featuring the ORF in their cis-spliced relatives  acting as a de facto maturase might be required to yield the productive structure necessary for splicing. Reliance on many nuclear-encoded splicing factors (at least 14) has been demonstrated for the tripartite trans-spliced group II intron found in the chloroplast of the green alga Chlamydomonas reinhardtiiPrasinoderma Pa.rnl.1 intron, it is intriguing that the two fragments linked to the flanking exons cannot form the typical secondary structure expected for a group IB intron .Aside from the DNA rearrangements discussed above, at least two major conditions must be met for successful events of B intron . Indeed,Prasinoderma has several unique characteristics, including the presence of two trans-spliced group I introns in the rnl gene. Sampling of other prasinophyte lineages should provide further insights into the range of mtDNA variations seen in these basal chloroplast lineages and could also help deepen our understanding of how trans-spliced introns arise.The comparative genome analysis presented here underscores the high variability in mtDNA architecture among prasinophyte lineages. The newly sequenced mitochondrial genome of the picoplanktonic green alga Prasinoderma coloniale strain CCMP 1220 was obtained from the Provasoli-Guillard National Center for Marine Algae and Microbiota . Prasinoderma cells were cultured in K medium Prasinoderma A+T-rich organellar DNA (700 bp fragments) was constructed using the GS-FLX Titanium Rapid Library Preparation Kit from Roche 454 Life Sciences . Construction of this library as well as 454 GS-FLX DNA Titanium pyrosequencing (one eight of a run) were carried out by the Plate-forme d′Analyses Génomiques . The resulting reads were assembled with gsAssembler 2.5 from the Roche GS Data Analysis Software package . Contigs were visualized, linked, edited and polished using the CONSED 22 package Prasinoderma mtDNA, repeats ≥30 bp were retrieved using REPFIND of the REPuter 2.74 program http://www.repeatmasker.org/) running under the Crossmatch search engine (http://www.phrap.org/).A shotgun library of Prasinoderma was extracted from cells ground in liquid nitrogen with the Qiagen RNeasy Midi kit  as described in Turmel et al rnl transcripts undergo trans-splicing and also to confirm the insertion positions of the trans-spliced introns, RT-PCR reactions were performed on the DNA-free RNA using the Qiagen One-Step RT-PCR kit with the following primers: 1) 5′-ACCAAACTGTCTTACGACGTTC-3′, 2) 5′-ATACTGAACCGGAGTTTCCTTG-3′, 3) 5′-CTTCAATTTCACCGAGTCCATG-3′, 4) 5′-ACAGGTCTCTGCAAAGTCGAAG-3′, 5) 5′-GTGAAGTCGCAGAAAATTGTGG-3′ (for their genomic locations, consult Total RNA from