Furthermore, the quantum capacitance increases very rapidly with the gate voltage for SLG, but this increase is much slowed down when n becomes greater. On the other hand, the magnitude of the EFM phase shift with respect to the SiO2 substrate increases with n at the sample bias of +2 V but decreases with n at -2 V. The difference in both quantum capacitance and EFM phase shift is significant between SLG and BLG but becomes much weaker between MLGs with a different n. The layer-dependent quantum capacitance behaviors of graphene could be attributed to their layer-dependent electronic structure as well as the layer-varied dependence on gate voltage, while the layer-dependent EFM phase shift is caused by not only the layer-dependent surface potential but also the layer-dependent capacitance derivation.The nanoscale electrical properties of single-layer graphene (SLG), bilayer graphene (BLG) and multilayer graphene (MLG) are studied by scanning capacitance microscopy (SCM) and electrostatic force microscopy (EFM). The quantum capacitance of graphene deduced from SCM results is found to increase with the layer number ( Graphene is drawing an increasing interest nowadays since its debut in reality  as it is2 layer. Although many novel methods have been used to fabricate graphene [C between the tip and the underlying semiconductor in response to a change in the applied ac bias ΔV could be obtained. The detailed operational modes of EFM and SCM have been reported elsewhere [The graphene samples were prepared by the mechanical exfoliation method  and depographene ,22, mechgraphene  is stillgraphene , tappinglsewhere . All the2 substrate. The profile of the marked line is shown in Figure 2 substrates are obtained in the same way. As the height of a graphene layer on top of graphene is close to the interlayer distance of graphite [h) as a function of the assigned layer number (n) by a straight line: h = nt + t0, as shown in Figure t = 0.37 nm which is in close agreement with the interlayer distance of graphite (approximately 0.335 nm) and the offset t0 = 0.15 nm which may be caused by the different interaction between tip-graphene and tip-SiO2 [0.37 + 0.15 = 0.52 nm, which is in agreement with the results of SLG reported in the literatures [h-n linear fitting results, area A is termed as SLG, area B as BLG, and area C (four-layer) and D (eight-layer) as MLG.Figure graphite ,25 we fieratures ,15. FromC/dV amplitude at sample DC biases of 0 V and +3 V are shown in Figure C/dV amplitude does vary with the number of graphene layers, and the differences between SLG, BLG, and MLG can be obviously observed from both images. As the ac voltage variation (ΔV) is kept constant in all measurements, the capacitance variation (ΔC) obtained by multiplying dC/dV amplitude with ΔV was adopted afterwards instead of the dC/dV amplitude. The line profiles of ΔC obtained on SLG and BLG are shown in Figure C values of SLG are slightly smaller than those of BLG, but at the DC bias of +3 V, the ΔC values of SLG are larger than those of BLG. Figure C with respect to the SiO2 substrate for SLG, BLG, and MLG obtained at 0 V and +3 V, respectively. The results show that at the DC bias of 0 V, the ΔC measured on graphene increases with n. The increase is fast when n increases from 1 to 4, and it slows down when n increases from 4 to 8. On the other hand, ΔC decreases with n for the case of +3 V DC bias. Moreover, the ΔC values measured on graphene layers are always smaller than those measured on the SiO2 substrate for both biases.SCM measurements were carried out on graphene with different layer numbers, and the images of dCtot) could be written as:As the capacitance measured on graphene is composed of two series capacitance: the quantum capacitance of graphene and the capacitance of the underlying oxide layer, according to the previous studies -16, the rs is the radius of the disk on which the nonstationary electron/hole charge is distributed). C'MOS and C'q are the unit area capacitance for tip/SiO2/Si structure and graphene, respectively. By considering the contact area, the capacitance measured on SiO2 substrate is Cq can be derived as:where Ctot and CMOS are the capacitances measured on the top of graphene layers and on the SiO2 substrate, respectively, but the ratio Atip/Aeff could not be obtained from the experiments. For FLG, the ratio was found to vary with the gate voltage, as well as the SiO2 thickness [2 existed; this ratio for FLG was approximately equal to 1 at the gate voltage of 0 V and changed slightly with the gate voltage, but its relation with n is not clear. As a rough approximation, we took Atip/Aeff = 1 for all grephene layers, thus the values of Cq can be calculated from Equation 2. The calculated values for different graphene layers at both DC voltages of 0 and 3 V are shown in Table In Equation 2, hickness -16. As rhickness , in the n. With +3 V bias applied, all quantum capacitance variations are much larger than their corresponding values at 0 V. The increase is mostly significant for SLG, which increased about 280 times. The increase magnitude, as shown in Table n. Therefore, the change of graphene quantum capacitance with the DC biases is dependent on n, resulting in the different layer-dependent quantum capacitances of graphene at 0 V and +3 V. Since SCM has been performed in the contact mode where the tip contacts with the surface, the DC bias applied between the tip and sample backside acts as the gate voltage. So our results indicate that the capacitance variations increase with the gate voltage for different graphene layers, and the increase magnitude decreases as n increases. In previous studies, both the SCM measurements on FLG [Atip/Aeff = 1 is used for different graphene layers, it may cause errors for the obtained Cq values, especially at a DC bias of +3 V. Nevertheless, the different quantum capacitance behaviors for graphene with different n are definite. As the quantum capacitance represents the density of states (DOS) at Fermi level [n [n, as shown in Table et al. that the work function could be tuned by the gate voltage, where they found that SLG showed larger work function changes with gate voltage than BLG did [EF) in graphene, which was different for SLG and BLG. Our results can be interpreted in the similar viewpoint. Different changes of EF with gate voltage for different graphene layers could result in different carrier density changes with gate voltage, so are the changes of the quantum capacitance with gate voltage.From Table s on FLG -16 and ts on FLG  showed tmi level ,27 and t BLG did . They exn at the sample biases of +2 V and -2 V at a lift height of 20 nm are shown in Figure 2 substrate is smaller than that between BLG and SiO2, while for the bias of -2 V, SLG has a larger phase shift with respect to SiO2 than BLG. Detailed correlations of the phase shift with n obtained at +2 V and -2 V are shown in Figure 2 substrate increases with n at +2 V but decreases with n at -2 V. In a previous report [et al. measured the EFM phase shifts on FLG ranged from 2 to 18 layers, and also observed the similar phase shift reverse for two-layer and three-layer graphene at tip biases of -2 V and +3 V. They suggested that the phase shift difference was related with the layer-varied surface potential. This suggestion is doubtful, since the phase shift of electrostatic force is composed of two factors, which could be written as [Meanwhile, the EFM results measured on graphene with different s report , Datta eitten as :k is the stiffness of the cantilever, Q is the quality factor, z is the tip-sample distance and C is the tip-sample capacitance. VDC is the applied bias, and Vsurf is the surface potential correlated with the difference between the tip and sample work functions (Vsurf = (Wtip-Wsample)/e). Hence, both surface potential and capacitance derivation (∂2C/∂z2) will contribute to the phase shift of electrostatic force. First let's estimate the surface potential contribution (Vdc - Vsurf)2 to the different phase shift between SLG and BLG. The work function different between SLG and BLG was reported by Yu et al. [2 substrate is about 5.0 eV and the same tip is applied , SLG should have a larger phase shift difference with respect to the SiO2 substrate than that of BLG for both biases. In other words, the difference in phase shift behavior between SLG and BLG could not only be attributed to their different surface potentials. Thus, the capacitance derivation should be another contribution to the phase shift. Our SCM results aforementioned do indicate that the quantum capacitance of graphene varies with n, and it is significantly dependent on the sample biases, which could be expected to induce different EFM phase shifts for different graphene layers at different samples biases.where u et al. , which iIn conclusion, the nanoscale electrical properties of graphene with different number of layers have been studied by SCM and EFM, and the layer dependences of capacitance variation and EFM phase shift are obtained. SLG, BLG, and MLG exhibit obvious differences in electrostatic force and capacitance behaviors. The different electrical properties obtained on different number of graphene layers could be mainly attributed to their different electronic properties.The authors declare that they have no competing interests.SHZ carried out the experiments. YL participated in the SCM and EFM studies. SHZ and XJY interpreted the results and wrote the manuscript. All authors read and approved the final manuscript.


Phylogenetic profiling has been widely used for comparing bacterial communities, but has so far been impossible to apply to viruses because of the lack of a single marker gene analogous to 16S rRNA. Here we developed a reference tree approach for matching viral sequences and applied it to the largest viral datasets available. The resulting technique, Shotgun UniFrac, was used to compare host-associated and non-host-associated phage communities , and revealed a profound split similar to that found with bacterial communities. This new informatics approach complements analysis of bacterial communities and promises to provide new insights into viral community dynamics, such as top-down versus bottom-up control of bacterial communities by viruses in a range of systems. The phylogenetic composition of bacterial communities is primarily determined by whether they are found in host-associated or free-living environments The lack of a single marker gene in viral genomes complicates phylogenetic profiling of viral communities, a powerful technique for studying microbial communities, and previous studies have focused on profiling viral gene functions We applied Shotgun UniFrac to 130 phage metagenomes from diverse environments. As observed with microbial communities, the primary factor separating metagenomes was whether they were derived from a free-living or host-associated environment. Host-associated environments vary more than a variety of free-living communities (considering only matches to the subset of viruses in the reference tree), and phage communities from the same host species tended to cluster .−23, one-tailed t-test; p<0.001, Monte Carlo t-test with 1000 iterations), suggesting stability in distal gut phage community membership over time.Our analysis also included 26 human feces phage metagenomes from 12 individuals with between 1 and 4 metagenomes per individual  (n = 6). Four overlapping metagenomes (Ocean (db:MG-RAST) and Ocean (db:CAMERA)), were used as controls to ensure that the source database did not affect the clustering results which is possible, for example, if one required preprocessing that the other did not.The number of input metagenomes by type were: Reclaimed water at discharge point (n = 1); Reclaimed water at point-of-use (n = 2); Freshwater stromatolite (n = 2); Hot Spring, Yellowstone National Park (n = 2); Potable water (n = 1); Saltern  (n = 5); Ocean (db:MG-RAST) (n = 4); Saltern  (n = 3); Northern Islands Coral (n = 4); Marine stromatolite (n = 1); Ocean (db:CAMERA) (n = 4); Freshwater (n = 4); Human feces (n = 80); Saltern  (n = 3); Healthy human lung (n = 2); Mosquito-associated (n = 3); Cystic fibrosis human lung (n = 3); Table S1OTU assignment statistics by metagenome.(XLS)Click here for additional data file.Table S2Description of metagenome types and sources.(XLS)Click here for additional data file.Table S3Full QIIME metadata mapping file.(XLS)Click here for additional data file.Table S4Full viral OTU table . These data were used in jackknifed weighted Shotgun UniFrac calculations .(XLS)Click here for additional data file.Table S5Viral OTU table sub-sampled to 200 sequences per metagenome. These data were used in weighted UniFrac calculations .(XLS)Click here for additional data file.


First-principles study on the electronic structure and transport property of the boron nitride sheet (BNC) structure, in which a triangular graphene flake surrounded by a hexagonal boron nitride sheet, is implemented. As the graphene flake becomes small and is more isolated by the boron nitride region, the magnetic ordering of the flake increases. When the BNC structure is connected to the graphene electrodes, the spin-polarized charge-density distribution appears only at the triangular graphene flake region, and the electronic structure of the graphene electrode is not spin polarized. First-principles transport calculation reveals that the transport property of the BNC structure is spin dependent. One of the key factors in the field of spintronics is the spin filter effect, which plays a fundamental role as the spin-polarized current source in devices such as spin-field-effect transistors and single solid-state qubits. The carbon-related nanostructures have recently been fabricated experimentally and explored theoretically to clarify magnetic ordering mainly in the zigzag edge of graphene -3. TheseIn the previous study , we inveAll calculations are performed in the framework of the density functional theory using the real-space finite-difference approach, which makes it possible to carry out the calculation with a high degree of accuracy by combining with timesaving double-grid technique and the direct minimization of the energy functional -11. The In the calculation for electron transport properties, we employ the computational model in which the graphene/BNC/graphene structure is sandwiched between the two graphene electrodes. The scattering wave functions from the left electrode are written as follows:Φ′s are the bulk wave functions inside the electrode and i is the index of the propagating waves from the electrode. The reflection coefficients r, transmission coefficients t, and the wave function in the scattering region ϕ are evaluated by the overbridging boundary-matching formula under the nonperiodic condition in the z direction [where irection ,15,16. Tirection :e, and h are a transmission coefficient matrix, the electron charge, and Planck’s constant, respectively.where T, k point used in the two-dimensional Brillouin zone integration is 16. For all the calculations in this paper, a repeating sheet model is separated by 17.0 bohr in each layer. The lattice constant is 2.67 bohr, which is obtained by the bond length of the graphene sheet. Structural optimization is performed until the remaining forces are less than 0.08 eV/bohr. Table n↑(r)−n↓(r) of the BNC structures. The BNC sheet with the smallest graphene flake is found to be the largest magnetic moment, and the spin-polarized charge-density distribution accumulates at the graphene flake region.In order to investigate the effect of the size of graphene flakes on the magnetic orderings, we first consider the three BNC structures under periodic boundary conditions for all directions. Figure n↑(r)−n↓(r). From Table At the next step, for the purpose of investigating the effect of the distance between the graphene flakes on the magnetic moments, the other three models are investigated. Figure p↑ and 2 p↓ orbitals of the boron atom (1.60 eV) is smaller than that of the nitrogen atom (3.88 eV) in the local spin density approximation [By comparing the other BNC structures investigated in a previous study , where tximation , resultix direction is performed by the equidistant sampling of four k points. The calculated magnetic moment of the graphene/BNC/graphene structure is found to be 1.14 μB. Figure For the investigation of the electron transport properties of the BNC structure, the electrodes have to be positioned at both sides of the BNC structure. Since the graphene structure is employed as the electrode in our study, we need to take into account whether the magnetic moments of the BNC structure are retained after the BNC structures are sandwiched between the graphene electrodes. Figure It is important to evaluate the spin transmissions quantitatively toward the application of a spin-filter material. Based on the results in the previous subsection, the spin-polarized transport property of the graphene/BNC/graphene structure is investigated. Figure s is donated by σs(EF). The spin-polarization ratio of the graphene/BNC/graphene structure is found to be approximately 0.95 at the Fermi level, which is comparable to that obtained with ferromagnetic tunnel junctions using a transition metal [P(EF) in the present study is smaller than that in the previous study [to characterize the spin polarization of the electron current, where the conductance of spin on metal . Howeverus study  due to tus study . In the us study .We have investigated the magnetic ordering and transport property of the BNC structure suspended between the graphene electrodes by first-principles calculations. The magnetic moment of the BNC structure under the conventional periodic boundary conditions increases as the size of the graphene flake becomes small and the spin-polarized charge-density distribution accumulates at the graphene flake region. It is also found that the spin-polarized charge-density distribution is preserved at the graphene flake when the BNC structure is connected to the graphene electrodes. The magnetic moment is smaller than that of the BNC structures examined in the previous study  because The authors declare that they have no competing interests.TO (T Ota) carried out preliminary calculations and drifted the manuscript. TO (T Ono) developed the computational code, implemented the calculations, and completed the manuscript. Both authors read and approved the final manuscript.


The results reveal that the obtained films exhibit improved mechanical properties with the enhancement of tensile strength and Young's modulus by as high as 82% and 136%, respectively. The electrical properties of graphene-Ag composite films were studied as well, with the sheet resistance of which reaching lower than approximately 600 Ω/□. The graphene-Ag composite films can be expected to find interesting applications in the area of nanoelectronics, sensors, transparent electrodes, supercapacitors, and nanocomposites.In this paper, we have reported that paper-like graphene-Ag composite films could be prepared by a facile and novel chemical reduction method at a large scale. Using ascorbic acid as a reducing agent, graphene oxide films dipped in Ag Many operties ,6, fieldoperties , sensorsoperties -10, tranoperties -14, and operties -18, havein situ thermal reduction [Large-scale preparation of paper-like graphene films has aroused much attention for their unique mechanical and electrical properties ,16,19-22eduction . As a reeduction  and as aeduction .in situ reduction of graphene oxide films together with Ag+ by ascorbic acid. The mechanical and electrical properties of the obtained graphene-Ag composite films are also investigated.Although many efforts and applications have been achieved for these novel carbon films, it is still a great challenge to develop a novel method to prepare the films at a large scale. Herein, we report a new method to prepare graphene-Ag composite films with excellent and improved properties, which are fabricated by the large-scale assembly of graphene oxide films, followed by The natural graphite powder (carbon content 99.999%) in the experiment was purchased from Qingdao Tianyuan Carbon Co. Ltd, Qingdao, China. Other solvents and reagents were of analytical reagent grade and used as received.3 aqueous solution , and the graphene oxide films were soaked in the mixed solution for 5 h. The schematic illustration of two chemical synthesis routes is described in Figure Graphene oxide was synthesized through the modified Hummers method  as stateλ = 1.5418 Å) and 2θ from 5° to 85°. The Raman spectra were obtained using a Senterra R200-L Raman spectrometer  with a 514-nm line of laser source. Fourier transform infrared (FTIR) spectra were recorded using a Vertex 70 vacuum FTIR spectrometer (Bruker Optik GmbH) and scanned from 4,000 to 400 cm−1 with KBr as background. Thermogravimetric analysis  was performed under a highly pure nitrogen atmosphere with a heating rate of 1°C to 10°C/min from 30°C to 700°C. The films with 5-mm width and 4- to 5-cm length were measured by dynamic mechanical analysis  at the room temperature. A four-probe detector  was used to measure the sheet resistance of the films.Atomic force microscope (AFM) image was taken with the Multimode Nanoscope V scanning probe microscopy system  using tapping mode with Picoscan v5.3.3 software. The morphology of the films were observed using a scanning electron microscope (SEM) using a Carl Zeiss ULTRA 55  with energy dispersive X-ray (EDX) spectrometry mode. The crystal structures of the films were examined by X-ray diffraction  with Cu Kα  and G band . The G band represents the plane vibrations with E2g symmetry and is mainly sensitive to the configuration of sp2 sites, while the D band is related to the breathing mode of κ-point phonon of A1g symmetry. As the graphite was oxidized into graphene oxide, the G band is broadened and the D band increases substantially, indicating the decrease in size of the in-plane sp2 sites, possibly because of the extensive oxidation and ultrasonic exfoliation [ID/IG intensity ratio of graphene is observed compared to that of the graphene oxide. Finally, when AgNO3 was used, the increase of D band also occurred. This change suggests an increase in the average size of the sp2 sites upon reduction of graphene oxide, which indicates that the reduction reaction has taken place and agrees well with the Raman spectrum of the graphene oxide reduced by hydrazine as reported by Stankovich et al. [The Raman scattering signals were measured on the graphite powder , the peaks seem to be similar with those of samples c and d since a few existing Ag particles do not block the reaction. However, when the amount of AgNO3 is excessive as 20 mg (sample h) and 300 mg (sample i), all peaks become stronger again, which means that the side effects will arise gradually as the amount of AgNO3 increases.FTIR is used to characterize the functional groups in the films shown in Figure on peaks ,16. As f3 from 2 to 300 mg under nitrogen atmosphere. In the left image of Figure 3 is appropriate (no more than 10 mg), the TGA curves of graphene-Ag composite films exhibited a mass loss at a temperature lower than 600°C, slightly lesser than that of graphene reduced only by ascorbic acid. However, when the amount of AgNO3 is 20 mg and 300 mg, the TGA curves of the composite films turned out to have the same trend as that of graphene oxide. The right image of Figure 3 is increased, and more than 15% weight is left at 690°C as the AgNO3 is excessive up to 300 mg. We can also find that the residue weight of samples i and j has a little difference with the EDX results. It may be due to the excessive Ag particles which aggregated and deposited nonuniformly on the surface of the graphene-Ag composite films.Thermogravimetric analysis has also been performed. Figure The mechanical properties of graphene oxide films and graphene films have also been studied, as shown in Figure 3 is no more than 10 mg, the sheet resistance decreases; on the other hand, when the amount of AgNO3 is 20 mg, the sheet resistance increases. When the optimal amount of AgNO3 is 10 mg, a minimum sheet resistance of approximately 600 Ω/□ for graphene-Ag composite films is obtained. It can be found that the conductivity of the resultant graphene-Ag composite films can be improved greatly via the uniform decoration of Ag particles.Finally, the sheet resistance of these films was measured using the four-probe detector as shown in Figure + aqueous solution, Ag particles can be uniformly distributed on the surface of graphene films using ascorbic acid as a reducing agent. The morphology of the composite films can be maintained during the reduction process. The obtained films have been characterized by AFM, SEM, XRD, Raman, FTIR, TGA, DMA, and a four-probe detector. The results show that the obtained films exhibit improved mechanical properties with the enhancement of tensile strength and Young's modulus by as high as 82% and 136%, respectively. The electrical properties of the graphene-Ag composite films were studied as well, with the sheet resistance of which reaching lower than approximately 600 Ω/□. The composite films hold a great potential for applications in the fields of nanoelectronics, sensors, transparent electrodes, supercapacitors, and nanocomposites.In summary, we have demonstrated that graphene-Ag composite films are fabricated in a large scale using a facile chemical reduction method. The graphene oxide sheets can be easily assembled to form free-standing graphene oxide films during the volatilization process on PTFE hydrophobic substrate. After dipping the graphene oxide films into the AgThe authors declare that they have no competing interests.RGG carried out the sample preparation, participated on its analysis, performed all the analyses except AFM and FTIR analyses, and wrote the paper. NTH also wrote the paper and analyzed the samples. JC performed the FTIR analysis. QRZ participated on the AFM analysis and proof corrections. ZY, YJS, LYZ, and YFZ participated in the study guidance and paper correction. All authors read and approved the final manuscript.


Ubiquitin-immunoreactive neuronal inclusions composed of TAR DNA binding protein of 43 kDa (TDP-43) are a major pathological feature of frontotemporal lobar degeneration (FTLD-TDP). In vivo studies with TDP-43 knockout mice have suggested that TDP-43 plays a critical, although undefined role in development. In the current report, we generated transgenic mice that conditionally express wild-type human TDP-43 (hTDP-43) in the forebrain and established a paradigm to examine the sensitivity of neurons to TDP-43 overexpression at different developmental stages. Continuous TDP-43 expression during early neuronal development produced a complex phenotype, including aggregation of phospho-TDP-43, increased ubiquitin immunoreactivity, mitochondrial abnormalities, neurodegeneration and early lethality. In contrast, later induction of hTDP-43 in the forebrain of weaned mice prevented early death and mitochondrial abnormalities while yielding salient features of FTLD-TDP, including progressive neurodegeneration and ubiquitinated, phospho-TDP-43 neuronal cytoplasmic inclusions. These results suggest that neurons in the developing forebrain are extremely sensitive to TDP-43 overexpression and that timing of TDP-43 overexpression in transgenic mice must be considered when distinguishing normal roles of TDP-43, particularly as they relate to development, from its pathogenic role in FTLD-TDP and other TDP-43 proteinopathies. Finally, our adult induction of hTDP-43 strategy provides a mouse model that develops critical pathological features that are directly relevant for human TDP-43 proteinopathies.The online version of this article (doi:10.1007/s00401-012-0979-3) contains supplementary material, which is available to authorized users. TARDBP gene on human chromosome 1, is a major pathological component of the neuronal inclusions associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP) [TARDBP mutations have been identified in ALS, but they only account for 4 % of familial and 1.5 % of sporadic cases [TARDBP sequence variants [C9ORF72 gene have been identified as the most common genetic abnormality in familial FTLD-TDP and ALS [TARDBP mutations is also found in hippocampal sclerosis of the elderly, as well as a subset of patients with Alzheimer’s disease, Parkinson’s disease or other neurodegenerative disorders [TAR DNA binding protein TDP-43), encoded by the TLD-TDP) . The neuTLD-TDP) . TARDBP ic cases . FTLD-TDic cases , 12, butvariants , 20. Rec and ALS , 30. TDP, encodedisorders , 29, 37,TDP-43 plays a role in transcription and splicing regulation, with the number of target genes constantly growing. Other functional roles that are not well characterized include microRNA processing, RNA transport, cell division, and apoptosis . It is cWT that conditionally expresses TDP-43 under the control of the tetracycline conditional system of gene regulation [Given evidence that TDP-43 may be critically involved in both development and neurodegeneration, we designed a transgenic mouse model, termed iTDP-43gulation . These tAll mice were utilized with approval and in accordance with the Mayo Clinic Institutional Animal Care and Use Committee and the University of Florida Institutional Animal Care and Use Committee.WT mice were generated similarly to a previously described protocol [BsrBI digestion, gel purified followed by β–agarase digestion (NEB), filtration and concentration. The modified TDP-43 transgene was injected into the pronuclei of donor FVB/NCr embryos (Charles River). These responder mice were bred with 129S6 mice (Taconic) with the tetracycline transactivator (tTA) transgene downstream of calcium-calmodulin kinase II alpha (CaMKIIα) promoter elements [WT transgenic mice with forebrain hTDP-43 expression. For TDP-43 transgene suppression studies during development, doxycycline water (1.5 g/l doxycycline and 4 % sucrose) was placed in breeding cages for 2 days in addition to doxycycline diet , which remained in the breeding cage until pups were weaned at 21 days. Doxycycline, a tetracycline derivative, binds tTA and prevents transgene expression. At 21 days, weanlings were placed in cages with regular chow to allow hTDP-43 transgene expression to commence.iTDP-43protocol . Full leprotocol , was inselements  to produAfter euthanasia via cervical dislocation, brains were harvested and divided along the midline. The right hemisphere was flash-frozen on dry ice, while the left hemisphere was drop fixed in 10 % neutral buffered formalin for histological analyses. Brains were embedded in paraffin and cut into 5-μm sagittal sections. Tissues were immunostained with monoclonal antibodies to TDP-43, pS403/404-phosphorylated TDP-43, ubiquitin, ionized calcium-binding adaptor molecule 1 (Iba1), glial fibrillary acidic protein (GFAP), and cytochrome C oxidase subunit IV (COX-IV), using the DAKO Autostainer  with DAKO Envision+ HRP System. For cleaved caspase 3 and neuronal nuclear antigen (NeuN) double labeling, the DAKO Envision G2 Double Stain System was used. The peroxidase labeling of cleaved caspase 3 was visualized with diaminobenzidine (DAB), and the alkaline phosphatase labeling of NeuN was detected with Vector Blue alkaline Phosphatase Substrate Kit III . See Supplementary Table 1 for a complete list of primary antibodies used. Hematoxylin and eosin (H&E) staining was performed by standard procedures.g for 20 min at 20 °C. Supernatant protein concentration was determined by BCA assay (Pierce). Approximately, 50 μg protein was loaded onto 10 % Tris–glycine polyacrylamide gel (Novex). After electrophoresis, gels were transferred to a nitrocellulose membrane for 2 h at 200 mA constant current. Membranes were blocked with 5 % milk in Tris-buffered saline and 0.1 % Triton X-100  for 1 h and probed overnight at 4 °C with one of the following primary antibodies in 5 % TBS-T: mouse monoclonal TDP-43 antibody recognizing human TDP-43, rabbit polyclonal TDP-43 antibody recognizing total TDP-43, and mouse monoclonal glyceraldehyde-3-phosphate dehydrogenase antibody (GAPDH). See Supplementary Table 1 for a complete list of primary antibodies used. The membrane was washed 5 times for 5 min in TBS-T and then incubated in the appropriate secondary antibody for 1 h. The membrane was again washed 5 times for 5 min in TBS-T. ECL reagent was added for 2 min, and the membrane was exposed to X-ray film. For reprobing, the membrane was stripped with 70 mM SDS in Tris HCl (pH 6.8) with 0.7 % BME for 15 min at 55 °C before processing as described above.Brains were weighed and homogenized in lysis buffer  at 1 ml/100 mg. Samples were sonicated and centrifuged at 16,200Total RNA was isolated from dissected hippocampus and cortex using TRIzol reagent (Invitrogen) and Pure Link RNA Mini Kit (Invitrogen), and 2 μg were used to synthesize cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). All samples were run in triplicate on the BioRad CFX384 Real-Time PCR Detection System. A dilution series was used to construct a standard curve for each primer pair from which normalization and subsequent fold change calculations were performed. Primer sequences for qPCR were mouse Tardbp F (AAAGGTGTTTGTTGGACGTTGTACAG), mouse Tardbp R (AAAGCTCTGAATGGTTTGGGAATG), Gapdh F (CATGGCCTTCCGTGTTCCTA) and Gapdh R (CCTGCTTCACCACCTTCTTGAT). Single PCR products were verified by melt curve analysis.The ApopTag Peroxidase In Situ Apoptosis Detection Kit (Chemicon) was used to detect DNA fragmentation by labeling the free 3′-OH termini. Staining was performed according to the manufacturer recommendations. TUNEL-positive cells were visualized by the chromogen 3′-3′ diaminobenzidine (DAB) in sections that were counterstained with methyl green.Paraffin sections of brain tissue were deparaffinized and rehydrated in a graded series of alcohols. Tissue was blocked in DAKO Protein Block for 1 h then incubated with primary antibodies overnight at 4 °C. See Supplementary Table 1 for a complete list of primary antibodies used. After washing, tissue was incubated in Alexa Fluor 488 and Alexa Fluor 568 secondary antibodies  for 1 h at room temperature. Vectashield with DAPI (Vector Laboratories) was used to stain nuclei. Images were captured using an Axio Imager.Z1 microscope (Zeiss).Three samples per group were chosen and tissue sections approximately 2.0–2.1 mm from the midline were used to quantify the total number of neurons (labeled by NeuN) and the number of neurons containing phospho-TDP-43 and ubiquitin inclusions in addition to neurons with caspase 3 immunoreactivity. Immunofluorescent staining was performed as described above. Images were captured from different cortical regions  per sample after scanning slides at 40× with ScanScope (Aperio). Images were analyzed with ImageScope software (Aperio) for quantification.WT mice, wild-type human TDP-43 (hTDP-43) cDNA was placed behind a minimal CMV promoter with tetracycline operator sequences that effectively blocked human hTDP-43 expression in single transgenic mice (TDP-43WT). By crossing these mice with mice expressing the tetracycline transactivator (tTA) under the CaMKIIα promoter [WT mice that expressed hTDP-43 in the cortex, hippocampus, olfactory bulb, and striatum, which is the expected expression pattern for this CaMKIIα promoter  and hippocampus  of iTDP-43WT mice (17d) in response to the overexpression of hTDP-43  of age, while 80 % of iTDP-43WT mice from line 17d survived through the same time point . These data demonstrate that survival is dose dependent with respect to hTDP-43 expression.Only 30 % of iTDP-43WT mice and found that they had striking forebrain atrophy regardless of phenotype . We determined that the cells undergoing apoptosis were neurons by double immunostaining for cleaved caspase 3 and the neuronal marker NeuN  as well as their anatomical location in nerve cell layers of cortex and hippocampus. Overall, these results suggest that even moderate levels of wild-type hTDP-43 overexpression are extremely toxic to developing cortical and hippocampal neurons, causing severe and early neuronal loss through apoptosis.Neuronal loss is a prominent feature of TDP-43 proteinopathies such as FTLD-TDP and ALS. We examined the brains of iTDP-43ype Fig. ; howeverize Fig. a–d. Whilize Fig. e, f. PosWT mice of both founder lines, regardless of phenotype, had a general increase in ubiquitin immunoreactivity predominantly located in neuronal perikarya and in neuritic processes compared to NT mice  [WT mice were composed of abnormal mitochondria , similar to that observed in the spinal cord and brainstem of TDP-43PrP mice. Co-localization experiments demonstrated neurons with mitochondrial aggregates often had increased ubiquitin immunoreactivity . In summary, the pathological profiles of iTDP-43WT mice with hTDP-43 overexpression during early development is complex and exhibits only limited features observed in human TDP-43 proteinopathies.Histological examination was performed on both founder lines. We compared symptomatic 5a and 17d mice to 17d mice without an observable phenotype at 2M. Approximately 11 % of cortical neurons in symptomatic mice from the 5a line contained small (<1 μm) punctate cytoplasmic inclusions when probed with phosphorylation-specific TDP-43 antibodies that were absent in NT mice  , 43. UltWT mice may be due to the impact of hTDP-43 overexpression in the developing forebrain as opposed to the TDP-43 mediated proteinopathy. We utilized the conditional nature of this model to suppress hTDP-43 expression during early development with the introduction of doxycycline into the diet . We focused our suppression studies on line 5a, as this line had the most severe phenotype. hTDP-43 expression was suppressed in iTDP-43WT mice from line 5a from conception until the mice reached weaning age (21d), when cortical neurons have normally matured and display extensively developed dendritic arborizations and synaptogenesis has peaked [WT.Given that hTDP-43 has been proposed to play crucial roles in development, we sought to determine what features of the phenotype observed in iTDP-43s peaked , 24. At WT mice (n = 16) showed reduced spontaneous activity, weight loss, or poor grooming, which were the phenotypes observed in 70 % of 5a iTDP-43WT mice with continuous hTDP-43 expression. In addition, diTDP-43WT mice had no decrease in survival, with the oldest animal without an observable phenotype at 12M. To directly compare pathology between diTDP-43WT and symptomatic iTDP-43WT 5a mice, we aged a cohort of diTDP-43WT mice for 24d after induction of the hTDP-43 at weaning . We chose this duration of expression because 70 % of 5a iTDP-43WT mice with continuous expression of hTDP-43 are moribund and must be euthanized by postnatal day 24; therefore, both groups of mice expressed the transgene for approximately the same length of time. We also aged additional cohorts of diTDP-43WT mice following induction of hTDP-43 expression to 5.5M and 11M to determine how the impact of TDP-43 overexpression in the mature forebrain advances with age  mice were down-regulated in response to overexpression of hTDP-43  over iTDP-43WT mice that were matched for duration of hTDP-43 expression. The results suggest that the brain atrophy observed in young iTDP-43WT mice is due to overexpression of hTDP-43 in the developing forebrain. In contrast, induction of hTDP-43 expression within more mature weanling forebrains yields a progressive loss of brain weight that worsens with aging  and further declining by 11 months (P = 0.0003). Although no overt neurodegeneration was observed in the diTDP-43WT cortex at 45 days  pS409/410-TDP-43 inclusions were predominant and co-localized with ubiquitin 94 % of the time in 45d diTDP-43WT mice , likely reflecting the selective loss of those neurons. Diffuse cytoplasmic hTDP-43 immunoreactivity increased with age in diTDP-43WT mice . diTDP-43WT mice showed a moderate increase in activated microglia with age . diTDP-43WT (−10M + 24d) mice formed large pS409/410-TDP-43 inclusions that co-localized with ubiquitin in >90 % of the inclusion bearing neurons, similar to that previously observed in the diTDP-43 mice at 45d . This result demonstrates that pTDP-43 inclusions can readily form in adult neurons and that the loss of inclusions observed in 5.5M and 11M diTDP-43WT mice following hTDP-43 induction at weaning does not reflect a general inability of aged mice to form inclusions. Histological examination of sagittal H&E sections revealed apoptotic bodies, mainly in the dentate gyrus of the diTDP-43WT (−10M + 24d) mice . Futhermore, moderate microgliosis and astrocytosis were observed in diTDP-43WT (−10M + 24d) mice . These neuropathological features were elevated over that observed in NT controls . In total, these preliminary results suggest that hTDP-43 induction in both young adult mice (45d) and older adult mice (−10M + 24d) demonstrate characteristic features of FTLD-TDP.Because FTLD-TDP affects patients in mid to late life, we sought to validate our observations from diTDP-43WT mice does survive until at least 12M; however, it is not uncommon for transgenic mice to show variable penetrance and survival rates, even when on an inbred background [We have demonstrated that expression of hTDP-43 during early development yields a severe and complex phenotype (see Supplementary Table 2 for summary), including aggregation of phosphorylated TDP-43 and gliosis; however, these features are also accompanied by early lethality, extensive neuronal loss at an early age, TDP-43 inclusions that lack ubiquitin immunoreactivity and mitochondrial abnormalities that are not typical of human TDP-43 proteinopathies. A subset of the iTDP-43ckground , 11, 14.WT mice prevented early death, spontaneous movement deficits, severe neurodegeneration and mitochondrial abnormalities. While the loss of these features is interesting, it is rather the gain of the salient features of FTLD-TDP in diTDP-43WT mice over that observed in iTDP-43WT mice that is the most compelling finding of our study. diTDP-43WT mice showed slowly progressing neurodegeneration, progressive gliosis and punctate cytoplasmic TDP-43 inclusions with ubiquitin immunoreactivity—each of these features closely mimics that observed in human TDP-43 proteinopathies and is not akin to that observed in iTDP-43WT mice (see Supplementary Table 2 for summary). TDP-43 inclusions were primarily in cortical and hippocampal neurons, a distribution that correlates with the brain regions with highest hTDP-43 expression.hTDP-43 induction in the more mature forebrain of weaned diTDP-43We and others have previously shown that endogenous mTDP-43 is down-regulated in response to hTDP-43 overexpression , 33, 43.WT mice and other transgenic TDP-43 models continually expressing hTDP-43 [WT mice continually expressing hTDP-43 occurs in the developing forebrain and the severe brain atrophy appears not to progress in the iTDP-43WT animals who live beyond 2 months of age. To determine whether TDP-43 had an impact on early brain development, we exploited the conditional nature of our model by inducing hTDP-43 expression later in development after weaning. We demonstrated that all diTDP-43WT developmentally suppressed mice survived without early lethality of continually expressing iTDP-43WT mice. They did not have an overt phenotype up to 12M, the oldest age examined. This result is consistent with the recent report by Igaz and colleagues [WT model. Igaz and colleagues generated a high expressing line (8- to 9-fold) with a defective nuclear localization signal (hTDP-43ΔNLS) as well as a low expressing (0.4- to 1.7-fold) wild type line (hTDP-43-WT) where the transgene was suppressed until postnatal day 28 with subsequent aging to 6M without lethality. In contrast to the current report, Igaz and colleagues did not explore the differences between hTDP-43 induction in the developing brain when compared with the mature brain; therefore, it is difficult to determine if continuous expression in their model would have produced the severe phenotype and early lethality we observed in continuously expressing mice. On the other hand, a constitutive model over-expressing wild type TDP-43 at twofold higher than endogenous levels under the CaMKIIα promoter has been reported to have reduced survival though not at the early time points that we have described [Early lethality observed in our iTDP-43 hTDP-43 , 41, 43  hTDP-43 , where clleagues , who desescribed .WT mice developed frequent neuronal perikaryal mitochondrial aggregates in the cortex, particularly in layer V, and occasionally in the hippocampus. Our group and others have previously generated transgenic models with continuous overexpression of TDP-43 that develops mitochondrial aggregates [WT model were much smaller than those observed in the brainstem and spinal cord of our previously reported constitutive TDP 43PrP mice [WT mice following post-weaning induction of hTDP-43 in mature forebrain. Interestingly, Igaz et al. also did not report the occurrence of mitochondrial clusters when they induced TDP-43 overexpression at P28 in their inducible TDP-43 mice. Given our results, we suggest that Igaz et al. did not report mitochondrial clustering because they only expressed the TDP-43 transgene in their mice from P28 onward. Our data suggest that the mechanism through which TDP-43 regulates mitochondrial biology occurs during early brain development, when the brain appears to be unusually sensitive to hTDP-43 overexpression. Our current iTDP-43WT lines combined with the hTDP-43 post-weaning induction strategy will likely be useful in elucidating how TDP-43 impacts mitochondrial dynamics.In the present study, iTDP-43gregates , 43. ThePrP mice , 43. We WT mice but did not observe in diTDP-43WT mice. The severe neuronal loss and atrophy in iTDP-43WT mice were accompanied by comparable microgliosis and astrogliosis. In contrast, diTDP-43WT mice at earlier ages had equivalent brain weights as NT mice, but they had a progressive decrease in brain weight with increasing age—a degenerative phenotype that is similar to the later onset, progressive neuronal loss and gliosis that are features of human TDP-43 proteinopathies. Igaz and colleagues previously reported that post-weaning (P28) induction of hTDP-43ΔNLS (8- to 9-fold) or hTDP-43-WT (0.4- to 1.7-fold) resulted in progressive neuronal loss, which suggests that TDP-43 dysregulation in the mature forebrain results in progressive neurodegeneration regardless of expression level [WT mice. These results strongly indicate that during early development neuronal populations are highly sensitive to even moderate (2- to 3-fold) levels of hTDP-43 overexpression. The underlying mechanism through which this developmental sensitivity to TDP-43 remains to be determined, but we have identified the developmental window of selective neuronal vulnerability and, therefore, we can postulate which developmental milestones might be affected. The timing of neuronal loss during the third postnatal week suggests that neurogenesis and synaptogenesis are not markedly affected by hTDP-43 overexpression. Instead, the neuronal loss occurs when synaptic pruning is ongoing [The early, severe neuronal loss and atrophy, with near total ablation of the hippocampus was perhaps the most striking phenotype that we observed in iTDP-43on level . Further ongoing  and dend ongoing , 24. Con ongoing . Thus, TWT mice develop cytoplasmic inclusions that have phosphorylated TDP-43, these inclusions only occasionally had ubiquitin immunoreactivity. In contrast, 45-day-old diTDP-43WT mice developed distinct, punctate TDP-43 inclusions in neuronal perikarya and cell processes, which also had ubiquitin immunoreactivity. In this respect, TDP-43 aggregates in diTDP-43WT mice are similar in appearance to TDP-43 neuronal cytoplasmic inclusions in FTLD-TDP. Interestingly, inclusions in diTDP-43WT mice decrease in size and number with age, suggesting that neurons containing these inclusions may be preferentially lost. The hTDP-43ΔNLS model reported by Igaz and colleagues also had decrease in inclusions with time; however, the number of inclusions at peak was rare, ranging from <1 to <0.1 % depending on line [WT mice. Importantly, human and mouse TDP-43 are highly homologous [While iTDP-43 on line . In cont on line . Our res on line . Withoutmologous . It woulWT mice with forebrain expression, we sought to recapitulate features of FTLD-TDP and other TDP-43 proteinopathies, but the conditional nature of the model has also allowed us to determine the impact of TDP-43 induction at different developmental periods on the ensuing pathologic phenotype. Consequently, we report for the first time that the developing brain is more sensitive to hTDP-43 overexpression than more mature brain, despite having the same ability to autoregulate endogenous mTDP-43 levels. In addition, the early, severe neuronal loss and brain atrophy in iTDP-43WT mice probably has a different pathogenesis from the progressive neurodegeneration observed in diTDP-43WT mice when expression of transgene is delayed. Moreover, the present results suggest that ubiquitinated, phospho-TDP-43 aggregates may themselves be neurotoxic in mature neurons. Finally and most critically, the timing of hTDP-43 overexpression certainly affects the integrity of model phenotype as it relates to FTLD-TDP. The availability of this new TDP-43 model system provides the field with a more pathologically similar transgenic mouse model for FTLD-TDP as well as a system in which the role of TDP-43 in development versus disease can now be distinguished.In generating iTDP-43Supplementary material 1 Supplementary material 2 (DOCX 18 kb)Supplementary material 3 (PPT 97 kb)Below is the link to the electronic supplementary material.


This compound has received worldwide attention due to its extraordinary physical and chemical properties. Duo to the widespread application of geraphenes, concerns are raising about its environmental safety or the safety protocols for handling and waste of graphene-based materials. The generation of reactive free radicals, adsorption of important biomolecules, and physical toxicity of graphene also matter. Hereby we criticize the concerns on the toxicity of graphenes to provide some perspective on the potential hazards of future development in graphene-based biomaterials.Graphene, the new allotrope of carbon is a single layer of monocrystalline graphite with sp Different oxidative treatments resulted in production of graphene oxide with varying atomic C/O ratio which has an influence on toxicity profile of the graphene oxide[in vivo applications of graphene should be studied in details[Graphene, a two-dimensional carbon sheet with single atom thickness has recently received significant interest due to its unique mechanical and electrical properties. Graphene is grown via chemical vapor deposition from carbon-containing gases on the surface of catalytic metals including Co, Pt, Pd, Ni, and Fe. Geraphestration. The menstration. Howeverstration,6, the pstration. Investistration. When testration. Particlstration. The funstration. The effstration. Graphenstration. The intstration. The toxstration. The cytne oxide. Dimensine oxide proposed details. The uniThe authors declared that they have no competing interests.Authors contributed equally to the paper. Authors read and approved the final manuscript.


Twisted few layer graphene (FLG) is highly attractive from an application point of view, due to its extraordinary electronic properties. In order to study its properties, we demonstrate and discuss three different routes to in situ create and identify (twisted) FLG. Single layer graphene (SLG) sheets mechanically exfoliated under ambient conditions on 6H-SiC(0001) are modified by (i) swift heavy ion (SHI) irradiation, (ii) by a force microscope tip and (iii) by severe heating. The resulting surface topography and the surface potential are investigated with non-contact atomic force microscopy (NC-AFM) and Kelvin probe force microscopy (KPFM). SHI irradiation results in rupture of the SLG sheets, thereby creating foldings and bilayer graphene (BLG). Applying the other modification methods creates enlarged (twisted) graphene foldings that show rupture along preferential edges of zigzag and armchair type. Peeling at a folding over an edge different from a low index crystallographic direction can result in twisted BLG, showing a similar height as Bernal (or AA-stacked) BLG in NC-AFM images. The rotational stacking can be identified by a significant contrast in the local contact potential difference (LCPD) measured by KPFM. Twistedy others –11. For y others .post-preparation treatment. Note that in some cases the origin of the foldings can not be uniquely identified as method (ii) or (iii) as AFM imaging involves strong tip–sample interactions.In order to study these properties experimentally, (few layer) graphene can be produced in various ways. The growth of graphene on metals followed by transfer to another substrate as well as epitaxial growth on SiC, both have a potential for mass production if technological shortcomings can be overcome. However, exfoliation from graphite still results in graphene flakes of highest quality –2, whichRupture and folding result in a system where we can study and compare properties of graphene in several thicknesses, sheet orientations, edges –19 and s24+ ions under 1.5° grazing incidence. The ion fluence is adjusted to 5–10 ions/μm2.Graphene is exfoliated from a HOPG crystal  under ambient conditions on an as delivered Si-rich 6H-SiC0001) substrate  applying a well known recipe [ substrat−11 mbar. Force sensors used are NCH Si cantilevers  with the exception of the measurements presented in f0300 kHz. To obtain correct height measurements, KPFM [Irradiated samples are initially inspected by tapping mode atomic force microscopy performed under ambient conditions using a Dimension 3100 AFM  and NCHR cantilevers . NC-AFM images are obtained using a well characterized –22 UHV 7ts, KPFM –24 imagits, KPFM , the samts, KPFM .−1 combined with the quality of the single Lorentzian fit are characteristic for the presence of SLG [After mechanically exfoliating graphene on the 6H-SiC0001) substrate, a flake is selected that has a width of 2–3 μm and a length of ≈40 μm. The flake shows straight parallel edges and the apparent thickness of the SLG flake is 0.7 ± 0.4 nm. Since a thickness measurement of SLG flakes is not straightforward and thickness values reported in literature range from 0.35 nm to ≈1 nm  substrat–29, we ue of SLG –31.2 areas free of steps. The roughness can be characterized by the standard deviation σ of the height distribution [Mechanically exfoliated graphene is known to adapt to its substrate like a carpet . The Kelribution  shown inTo create FLG including layers with twisted stacking, the exfoliated graphene is exposed to SHI irradiation (i) followed by a combination of line scanning the sample with an AFM tip (ii) and severe heating (iii). The tapping mode AFM survey image shown in Next, we investigate the graphene layer, its rupture, foldings and stacking in more detail by Kelvin compensated NC-AFM imaging, demonstrating the identification of (twisted) FLG. To study the properties of rupture and foldings in the SLG originating from treatment with methods (i), (ii) and (iii) in detail, we analyze images taken in the areas A, B and C marked by the boxes in The folding marked by A in To understand the role of the preferential directions of rupture, we start by identifying the edge of the large SLG flake, marked by the white line at the bottom of the image. The edge resulting from exfoliation is expected to have a preferential direction being either a zigzag or an armchair edge. Therefore, the vast majority of edges formed by rupture due to post-preparation treatment are found to be aligned at 30° and 60° with respect to the SLG flake edge, exemplified at the bottom of Assigning the SLG flake edge, being of either zigzag or armchair structure, the dashed lines enclosing an angle of 30° with the SLG flake edge, therefore, correspond to the other type edges. However, the edge over which the folding on the right took place, marked by the arrow in However, by the use of KPFM, one is able to clearly discriminate between different rotational stackings. In In summary, we demonstrate different routes to rupture and folding of SLG on a 6H-SiC(0001) substrate as well as experimental techniques to identify and discriminate the resulting (twisted) BLG and TLG structures. Rupture and folding of SLG on a 6H-SiC(0001) can be induced by scanning and contacting with an AFM tip and by severe heating resulting in foldings with (AA- or) Bernal-stacking and twisted stacking. SLG sheets rupture along preferential edges of zigzag and armchair type, even when peeling an existing folding is performed under an angle resulting in a twisted stacking. While the (AA- or) Bernal-stacked BLG and twisted BLG can not be discriminated by their height, they can clearly be distinguished by the difference in their LCPD.


Species richness is the most commonly used but controversial biodiversity metric in studies on aspects of community stability such as structural composition or productivity. The apparent ambiguity of theoretical and experimental findings may in part be due to experimental shortcomings and/or heterogeneity of scales and methods in earlier studies. This has led to an urgent call for improved and more realistic experiments. In a series of experiments replicated at a global scale we translocated several hundred marine hard bottom communities to new environments simulating a rapid but moderate environmental change. Subsequently, we measured their rate of compositional change (re-structuring) which in the great majority of cases represented a compositional convergence towards local communities. Re-structuring is driven by mortality of community components  and establishment of new species in the changed environmental context. The rate of this re-structuring was then related to various system properties. We show that availability of free substratum relates negatively while taxon richness relates positively to structural persistence . Thus, when faced with environmental change, taxon-rich communities retain their original composition longer than taxon-poor communities. The effect of taxon richness, however, interacts with another aspect of diversity, functional richness. Indeed, taxon richness relates positively to persistence in functionally depauperate communities, but not in functionally diverse communities. The interaction between taxonomic and functional diversity with regard to the behaviour of communities exposed to environmental stress may help understand some of the seemingly contrasting findings of past research. While the concern about the consequences of taxon loss has spurred a burst of studies on the relation between diversity, both as driver and as response, with ecosystem functioning and compositional stability ] and the national ethics regulations of the participating countries.In eight different biogeographical regions (see below), we translocated over 500 natural fouling communities among two sites within a region and assessed the relation between the rate of compositional change following the translocation and the three system properties chosen, i.e. unoccupied substratum, functional richness, and taxonomic richness.Two suitable experimental sites were selected in each of the 8 biogeographic regions Tasman Sea , South West Atlantic (Brazil), South Pacific West , North West Pacific (Japan), West Pacific , South East Pacific (Chile), North Sea (England), and Baltic Sea (Finland) . Within Two to four months prior to the treatment (see below) in each site of each region 48 roughened PVC panels (15×15 cm) were suspended vertically in a water depth between 0.5 and 1 m. The experiments in different hemispheres were run during the respective spring/early summer to ensure comparability. In contrast to artificially assembled communities, the taxa within these communities co-occur and interact naturally.On the day of treatment all remaining communities (variable numbers were lost at the different sites) were taken to the lab in cooler boxes. Taxon identity (to the lowest possible level) and abundance, number of functional groups, and percentage of unoccupied substratum per panel were assessed. In the following we will use the term taxon richness rather than species richness since many of the organisms could not be identified to species level for lack of appropriate identification keys regarding certain groups or in a given region. Functional richness was measured as the number of groups (containing one or more taxa) which, as adults, differ in at least one trait within four functional categories considered of ecological importance, namely body size, growth form, trophic type, and whether solitary or colonial  [39]. ByAfter this characterization of the communities, half of the communities were translocated between the two sites within a region while the other half was back-transferred to their site of origin to control for transfer effects without site change. After the transfer, the communities transplanted from site A to site B were considered “introduced” (to B) while the communities from B and back-transplanted to B were considered “resident” to B. This treatment was done in both directions, so that each site possessed a batch of introduced and resident communities. Random pairs of introduced and resident panels were suspended side by side (distance ca 10 cm) at the same place and depth where the resident communities had assembled over the past months. The response of the communities to the treatment (translocation or back-transplantation) was assessed by comparing the rates of their compositional changes during the following six to 12 weeks. The mean rate of compositional convergence within each pair of translocated and resident community over the first three weeks following translocation was expressed as the slope of the convergence curve during this time. This period was chosen, because during the phase immediately following translocation restructuring is most directly influenced by the environmental change imposed and the community properties as assessed on the day of translocation. Both influences will fade with convergence. Additionally, convergence rates (see below) are generally linear during this initial phase turning to asymptotic in a later phase. Rates of change were quantified as changes in similarity (assessed by Bray-Curtis using the software Primer® on untransformed data of species abundances per panel) to a reference community per unit time. To assess the rate of compositional change of a given community after transfer or back-transfer, its structure three weeks after the treatment was compared to its own structure on the day of transfer. The acceleration of these change rates in transferred as compared to non-transferred communities of the same origin  was used to quantify the impact of stress associated with the change of environmental conditions. The capacity of structural persistence of the transferred communities under the imposed stress was quantified by the speed of their convergence towards resident communities at the new site, i.e. the decrease of initial dissimilarity between introduced and resident communities in a given site. As (inverse) metric of persistence we preferred convergence towards resident communities over structural change as compared to original structure (i.e. self-similarity) because the former depends more directly on mortality of introduced species and invasion of resident species. Slower rates of convergence reflect a longer persistence of the original community characteristics and, thus, indicate higher resistance to the imposed environmental change. Consequently, our measure of community stability (persistence) is the inverse of the mean convergence rate within pairs of introduced and resident communities.Effects of various predictors on the speed of convergence between transplanted and resident fouling communities were analysed using a mixed-effects-model The translocation represented an environmental change, the impact of which decreased over time as more and more introduced taxa were replaced by resident taxa. As is typical for all natural communities, both the introduced and the resident communities continued to change in composition after the day of translocation as a result of succession, seasonality and/or stochastic events. However, averaged over all communities, introduced communities changed faster by 29% relative to the background dynamics assessed in the resident communities of the same provenance . The accelerated re-structuring was driven by two processes: (i) mortality under the new conditions; and (ii) recruitment by taxa (“invasion”) belonging to the local species pool of the target site but previously not present in the introduced community. Mortality could have been caused by intolerance towards the new abiotic conditions, lack of conspecific recruits, or sensitivity towards new biotic threats such as parasites or consumers. Circumstantial evidence suggested that predation (mostly by fishes), at least in some regions, was heavier on introduced than on resident communities, but this difference was not rigorously quantified. At all sites, the re-structuring provoked a convergence of introduced communities towards local resident communities.2 = 0.017, p = 0.76) or to the change imposed . In contrast, the speed of convergence related strongly to regional mean temperature . This could merely have reflect the well known trend of metabolic rates of poikilotherms being faster and generation times being shorter in warmer regions (e.g. Convergence rates varied among sites and regions between 0.1% and 13.5% of similarity (Bray-Curtis) increase per week. Communities in Malaysia and New Zealand changed rapidly, while those at other sites appeared more persistent . The meaons e.g. . This efMost of these relationships have been studied before, but rarely simultaneously in a single experiment and never with a similar generality for different biogeographic regions but see . The strSpecies richness has been postulated to facilitate In the present study, taxon richness related negatively to convergence rate (i.e. enhanced persistence) in six of the 16 sites  whereas it related positively to convergence rate in only one New Zealand site . This enThe interaction between taxon richness and functional richness indicates that the impact of the former on structural persistence under stress (i.e. convergence rate) depends on the level of the latter. Indeed, the slope of the regression between convergence rate and taxon richness increased from negative (“enhancing persistence”) to positive (“reducing persistence”) with increasing functional richness of the experimental sites , while iAt present we can only speculate about the interaction between taxon richness and functional richness regarding the compositional stress resistance of benthic communities. It should be noted that the selection of functional traits is always based on expert guessing and it cannot be excluded for our approach that a different choice might have produced a stronger (or weaker) effect of functional diversity. Meanwhile, the interaction detected suggests that the different combinations of functional richness and taxon richness encountered in this study represent different positions on the continuum between complementarity and redundancy and offer some room for interpretation. The extremes of this continuum would be i) 1 species per functional group when functional richness is high relative to species richness and ii) many species in only 1 functional group when functional richness is minimal. Under scenario (i) all species are functionally different resulting in maximum functional complementarity. Under scenario (ii) all species are functionally similar resulting in minimal functional complementarity and maximal functional redundancy. Functional complementarity is thought to enhance resistance to invasion Our initial hypothesis was partially confirmed. Available substratum in most sites destabilized communities as expected, however, the effect of functional richness is more indirect than expected, i.e. it modulates the strength of the stabilizing effect of taxonomic richness. The variation in responses between geographic locations illustrates the complexity of how the relationships between taxonomic and functional richness help communities persist. We conclude that the drivers of compositional persistence in marine fouling communities exposed to environmental change (i.e. one aspect of stability) are multivariate and interactive. Considering only single community properties in diversity-stability studies must forcibly produce variable results in different settings.Figure S1Average convergence rates per region depicted against the total taxon richness in the same region. CR = convergence rate, SE = standard error, Aus = Australia (Tasmania), Bra = Brazil, Chi = Chile, Fi = Finland, GB = England, Jp = Japan, Mal = Malysia, NZ = New Zealand.(TIF)Click here for additional data file.Figure S2Average convergence rate depicted against mean available substratum. For clarity, only site means without scatter bars are shown. Slope and 95% confidence interval depicted.(TIF)Click here for additional data file.Figure S3Re-structuring and available substratum. Convergence rates (between paired panels) depicted against available substratum on introduced panel, stratified by region and site. Red lines indicate regression lines. Black squares indicate cases of significant (p<0.05) regressions.(TIF)Click here for additional data file.Figure S4Re-structuring and richness. Convergence rates (between paired panels) depicted against taxon richness on introduced panel, stratified by region and site. Red lines indicate regression lines. Black squares indicate cases of significant (p<0.05) regressions. Functional richness per panels is indicated as a colour gradient from blue (low) to red (high).(TIF)Click here for additional data file.Table S1List of taxa and their functional traits. In the majority of cases the “taxa” were individual species but could not be identified to a lower taxonomic level due to the lack of appropriate keys in several regions. Abbreviations of functional traits are as given in (DOCX)Click here for additional data file.Table S2Regression slopes  between convergence rate (CR) and taxon richness (TR) and between convergence rate (CR) and available substratum (Substr) stratified by sites and regions. n = number of pairs analysed per site.(DOCX)Click here for additional data file.


Heterologous gene expression is dependent on multistep processes involving regulation at the level of transcription, mRNA turnover, protein translation, and posttranslational modifications. Codon bias has a significant influence on protein yields. However, sometimes it is not clear which parameter causes observed differences in heterologous gene expression as codon adaptation typically optimizes many sequence properties at once. In the current study, we evaluated the influence of codon bias on heterologous production of human papillomavirus type 16 (HPV-16) major structural protein L1 in yeast by expressing five variants of codon-modified open reading frames (OFRs) encoding HPV-16 L1 protein. Our results showed that despite the high toleration of various codons used throughout the length of the sequence of heterologously expressed genes in transformed yeast, there was a significant positive correlation between the gene's expression level and the degree of its codon bias towards the favorable codon usage. The HPV-16 L1 protein expression in yeast can be optimized by adjusting codon composition towards the most preferred codon adaptation, and this effect most probably is dependent on the improved translational elongation. The production of functional proteins in heterologous hosts is an important issue of modern biotechnology. However, often it is difficult to generate recombinant proteins outside their original context. Protein expression is dependent of multistep processes involving regulation at the level of transcription, mRNA turnover, protein translation, and posttranslational modifications leading to the formation of a stable product . CurrentNonetheless, the codons that are adapted to the efficient elongation of endogenous genes may not always correspond to the efficient codons for heterologous genes, because overexpression often causes amino acid starvation what leads to changes and disbalance of charged tRNA pools . It was Despite accumulating information about the impact of codon bias on the heterologous gene's expression, it is still unclear what approach for codon adaptation should be used designing putative transgene sequences as these approaches have not been systematically compared against each other. The codon adaptation typically optimizes many sequence properties at once, and in most cases, it is difficult to determine which parameter causes the observed differences in heterologous gene expression.It was shown that papillomavirus late mRNAs may not be efficiently translated in undifferentiated cells due to a mismatch of codon usage and tRNA availability . OptimizIn the current study, we aimed to evaluate the influence of codon bias on heterologous generation of HPV type 16 (HPV-16) major structural protein L1 in yeast by expressing five different codon-modified open reading frames (OFRs) encoding HPV-16 L1 protein.http://www.kazusa.or.jp/codon/) for overall yeast S. cerevisiae proteins (L1-Sc), plant proteins , and mammalian proteins  or adapted for expression in E. coli (L1-Ec) allowing some deviations from the strictly optimized codon usage. The genes encoding L1-Sc and L1-Ec of the HPV-16 were synthesized in GenScript . The genes encoding L1-Pv, L1-Pl, and L1-Hm -dATP  by random priming of the DNA probe using a DecaLabel DNA Labeling Kit (Thermo Scientific Fermentas) according to the manufacturer's instructions. The blots were washed extensively at 65°C and L1 and PGK1 transcripts were identified by phosphorimaging.Total yeast RNA was isolated 4 hours after induction by the method described earlier . The Nor2, 0.001% Trition X-100 in 10 mM Tris/HCl-buffer, pH 7.2) and 1 mM PMSF. An equal volume of glass beads was added and the cells were lysed by vortexing at high speed, 8 times for 30 sec, with cooling on ice for 30 sec between each vortexing. Then an equal volume of 2 × SDS-PAGE sample buffer  was added directly to the same tube, mixed and boiled immediately at 100°C for 10 minutes. 4–10 μL of the prepared whole cell lysate was loaded onto SDS-polyacrylamide gel (up to 20 μg protein in each lane) and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) was run in SDS-Tris-glycine buffer. Western blot analyses were performed according to methods described previously [10–20 mg of yeast cell pellets was resuspended in 10 volumes (vol/wt) of DB150 buffer , plant , and Homo sapiens cells (L1-Hm) or adapted for expression in E.coli (L1-Ec) allowing some deviations from the strict usage of high-frequency codons to reflect the overall codon usage in the target genome. Few deviations from the strict usage of optimized codons were made also to allow the insertion or removal of recognition sites for restriction endonucleases. The majority of codons in used OFRs were modified as compared to the native HPV-16 L1 gene (L1-Pv): in L1-Sc ORF, 46.3%; in L1-Pl ORF, 51.1%; in L1-Ec ORF, 60.2%; and in L1-Hm ORF, 78.6% codons were modified while the encoded protein sequence remained unchanged (The codons in the authentic L1 ORF of HPV-16 isolate 114/K (L1-Pv) were optimized for either nchanged . All upsTo determine whether the alterations of the primary sequence of the L1 mRNAs introduced by the codon changes also affected the state and the level of transcripts, total yeast RNA was isolated from yeast cells 4 h after induction and the L1 mRNA transcription was analyzed by Northern blot. The transcripts of all five HPV-16 L1 ORFs were detected in the respective transformed yeast cells . DespiteIn the next step, the production of heterologous HPV-16 L1 protein in transformed yeast was analyzed by both SDS-PAGE and Western blot . The HPVS. cerevisiae optimized codons, was the most successful with clearly detectable protein band in both SDS-PAGE and Western blot  sequences were comprised of similar low GC content  even lower than that found in authentic HPV-16 L1-Pv ORF (GC content of 38.7%). Meanwhile, both human- and E. coli-adapted L1 ORFs (L1-Hm and L1-Ec) had significantly higher GC content . Thus, it was assumed that the efficiency of translation was not influenced by the differences in the GC content of mRNA. Most likely, the effective translation of studied HPV-16 L1 mRNAs was related to the codon usage and the availability of tRNA pool recognizing synonymous codons rather than the mRNA primary structure. Moreover, the usage of GGA (Gly), CUU (Leu) and TCA (Ser) codons as a single opportunity distinguished the L1-Pl construct from the most efficient L1-Sc and other constructs (http://www.kazusa.or.jp/codon/) these codons GGA (Gly), CUU (Leu), and TCA (Ser) are moderately used. However, in highly expressed yeast genes CUU (Leu) codons are not used but few GGA (Gly) and TCA (Ser) could be found [E. coli [The plant and nstructs . In the nstructs . Althougbe found , 21. PreIn conclusion, the HPV-16 L1 protein expression in yeast can be optimized by adjusting codon composition towards an efficient codon usage and this effect most probably is dependent on the improved translational elongation.


On the oxygen-terminated SiO2 surface, atomic structure reconstruction occurs at the graphene/SiO2 interface to eliminate the dangling bonds. The interface interaction is 77 meV/C atom, which indicates that van der Waals force dominates the interaction, but it is stronger than the force between the graphene layers in graphite. The distance between graphene and the SiO2 surface is 2.805 Å, which is smaller than the 3.4 Å interlayer distance of graphite. In addition, the SiO2 substrate induces p-type doping in graphene and opens a small gap of 0.13 eV at the Dirac point of graphene, which is desirable for electronic device applications.In this work, the graphene/α-SiO Graphene, a single two-dimensional layer of graphite in hexagonal structure, is the starting point for many nanographite devices with promising electrical properties . After t2] are widely used as a dielectric medium in electronic devices. The electrical properties of graphene can be modified using electrical gates, substrates, and chemical species such as atoms and molecules  calculations with considerations on surface reconstruction of SiO2. The interfacial atomic structure and the effects of the substrate on corresponding electronic structure of graphene will also be studied.In this work, we investigate the graphene/α-SiO2(0001) surface using the first principles  and the Si-O-Si bond angle [α1] in different structures; d1bulk >d1clean >d1A and α1bulk >α1clean >α1A are found. In light of Figure d1 is affected by both l0 and α1. The former brings out d1bulk <d1clean, and the latter leads to d1bulk >d1clean. Therefore, the effect of α1 on d1 is stronger than that of l0. Comparing structure A with the clean SiO2 surface slab, both l0 and α1 lead to the decrease of d1, where d1 and l0 of structure A shrink to about 4%.In addition, Table 2 surface is around 20 meV/C atom with interface distance d0 = 3.29 Å and that on a hydrogen passivation O-terminated SiO2 surface is 0.13 eV/C atom with d0 = 2.58 Å [2 surface, but it is smaller than that on the hydrogen-passivated O-terminated SiO2 surface. Note that d0 in structure A is 2.820 Å, which is smaller than 3.14 Å in graphite and graphene laid on the Si-terminated SiO2 surface, but it is larger than that on hydrogen-passivated O-terminated SiO2 surface. It is known that the binding energy is inversely proportional to d0. The system of graphene on hydrogen-passivated O-terminated SiO2 surface has the smallest d0 and the strongest binding energy. A previous study implied that the C-C bond is weakened through the strengthening of bonds to the substrate [2 with structure A is stronger than that between the bulk graphite layers as shown previously.Quantitative experimental data for the interaction strength of graphene/substrate interface is very limited. The first principle calculations showed that the binding energy of graphene on a Si-terminated SiO= 2.58 Å . In the 2(0001) slab, and graphene/SiO2 interface, are represented in Figure 2 slabs. This band structure shows the crossing of π and π* bands at the K point and also at the Fermi level, which agrees with a well-known result that graphene is semimetallic with a 0 bandgap. The band structure of the interface shows that the bands of graphene layer are open with a 0.13-eV gap at the K point. It is interesting to note that the Fermi level is lowered with the amount of transferred charges. Thus, the charge should be transferred from graphene to the substrate in structure A. As a consequence of the charge transfer, the SiO2 substrate induces p-type doping in the graphene. Except for these variations, the band structure of interface is almost identical with the sum of band structure of the free graphene monolayer and SiO2 slab, as shown in Figure On the other hand, in order to understand the effect of substrate on the graphene electrical properties, the band structure for three different systems in Figure e to the SiO2 substrate where e denotes one electron charge. This is consistent with the result of the band structure where the p-type-doped graphene induced by the substrate is found.The charge transfer and atomic charge can be obtained using the Mulliken analysis; it is shown in Table 2 surface are -0.448 and -0.424 e, respectively, for structure A. Those in the clean SiO2(0001) slab are -0.396 and -0.293 e, and in bulk SiO2 are both -0.781 e. On the other hand, the charges of Si15, which binds with O4 and O8, are 1.681, 1.510, and 1.562 e in structure A, clean SiO2 slab, and bulk SiO2, respectively. It is known that the bond strength is in proportion with the multiplication absolute value of changes of the O and Si atoms. Therefore, the interaction between O4 and Si15, and O8 and Si15 are the strongest in bulk SiO2, followed by that in structure A, and the weakest was that in the SiO2 slab. These also agree with the results in Table l0 at the surface or interface is l0bulk <l0A <l0clean.In addition, the electron charges of O4 and O8 atoms at the SiOn SiO2000 slab are2(0001) interface with an oxygen-terminated SiO2 surface. After geometry relaxation, a stable structure with a distance between graphene and the SiO2 surface, d0 = 2.805 Å, was observed. In addition, the structure reconstruction of the SiO2 surface took place where the two O atoms binding on a particular Si atom form a covalent bond on the surface to eliminate the surface dangling bonds. Simulation results indicate that the interface interaction is 77 meV/C atom, which indicates that the van der Waals force dominates the interaction. Furthermore, the SiO2 substrate induces p-type doping in graphene and open a small gap of 0.13 eV at the Dirac point of graphene.In conclusion, DFT calculations were employed to study the graphene/α-SiOThe authors declare that they have no competing interests.ZA did the calculation and drafted the manuscript. ZW, MJ, and SL co-drafted the manuscript. All authors read and approved the final manuscript.


To examine whether adeno-associated virus (AAV) vector-mediated overexpression of growth-associated protein-43 (GAP-43) has protective or deleterious effects on retinal ganglion cell (RGC) survival in laser-induced chronic intraocular pressure (IOP) elevation injury.cistronic AAV vector encoding GAP-43 and GFP (AAV-GAP-43). Two weeks later, experimental chronic glaucoma was induced in the injected eyes by scarring the trabecular meshwork with a diode laser. IOP was measured with an impact (rebound) tonometer. Survival of RGCs was estimated after 3 weeks of IOP elevation by quantifying β-III tubulin+ neurons in retinal whole mounts. The transfection efficiency of target genes was assessed with direct view of GFP and western blot analysis of GAP-43.Adult Fischer 344 rats received unilateral intravitreal injection of either normal saline, AAV-green fluorescent protein (AAV-GFP), or a bi+ immunostaining revealed that, compared to uninjured eyes , 3 weeks of laser-induced IOP elevation led to a 60% decline in RGC survival (496±136 cells/mm2). Transfection with control vector AAV-GFP by itself did not have a significant effect on RGC viability (468±124 cells/mm2). Overexpression of GAP-43 in RGC cell bodies and axons via bicistronic AAV-GAP-43 led to more severe RGC death (260±112 cells/mm2) in IOP elevated eyes, an 80% loss of the total RGC population.Quantification of β-III tubulinOverexpression of GAP-43 aggravated RGC death in experimental chronic IOP elevation injury. GAP-43 was upregulated in RGCs regenerating after optic nerve injury. Thus, the finding that this same protein is deleterious to RGC viability after chronic IOP elevation may aid in understanding the mechanisms involved in RGC loss in glaucoma and how best to treat this condition. Glaucoma is the second most common cause of blindness worldwide, and is becoming increasingly prevalent as people live longer ,2. The tIt has been proposed that modifications within the axonal microarchitecture and disturbances of axonal transport play important roles in apoptotic RGC death ,8, causeGrowth-associated protein-43 (GAP-43) is a protein kinase C substrate concentrated in growing axons, growth cones, and synapses in association with actin cytoskeleton -19. It wWe set out to determine if there is a direct effect of increased GAP-43 expression on RGC survival in experimental glaucomatous injury. By using adeno-associated virus (AAV) vectors -33, currcistronic AAV vector, the GFP gene was therefore expressed after the transcription of the upstream GAP-43 transgene, thus allowing direct identification of transduced RGCs in injected eyes [9 TU/ml. To verify the translation efficiency of transgene GAP-43 encoded in the vector plasmid, HEK 293T cells were transfected directly with the vectors. Twenty-four hours later, cells were fixed with paraformaldehyde and immunostained with antibodies against GFP and GAP-43. GFP was colocalized with GAP-43 protein in the majority of cells.Linear structures of the AAV-2 vector plasmid pTR-UF12.1 used in this study are shown in ted eyes -33. AAV-ted eyes ,36. BrieYoung adult (8- to 10-week-old) Fischer 344 (F344) rats were used in this study. Rats were housed under standard conditions with a 12 h:12 h light-dark cycle. All experiments conformed to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Hospital Ethics Committee. All possible measures were taken to minimize suffering and limit the number of rats used. All surgery was performed under anesthesia of a 1:1 mixture (1.5 ml/kg) of ketamine (100 mg/ml) and xylazine (20 mg/ml).Intraocular injection procedures were similar to those described previously ,38. BrieChronic IOP elevation was achieved by scarring the trabecular meshwork with a 532 nm diode laser. Two weeks after the intravitreal injection of viral vectors or saline, animals were anesthetized as described above. Laser energy was delivered to the trabecular meshwork through a slit lamp on 50–70 spots at 100 µm at a power of 0.7 W and 0.7 s duration. IOP was measured in each eye with the TonoLab , an impact (rebound) tonometer. Using this impact tonometer, IOP was measured in awake and nonsedated animals. Readings were taken before and 1, 3, and 7 days after laser treatment. Measurement of IOP was always performed in the morning between 9:00 and 12:00. Six measurements were taken from each eye and averaged. A second laser treatment was given 1 week later if the IOP difference between the laser and control eye was less than 8 mmHg. IOP was measured weekly for 3 weeks after experimental glaucoma was induced.2) at a fixed distance from one another, in a pattern of grid intersections, was counted. Forty to fifty fields were sampled per retina. The average density of positive cells per field was determined, and the total number obtained by multiplying this figure by the retinal area. Data from different groups were statistically analyzed using the Bonferroni test after one-way analysis of variance (ANOVA) [The procedures for tissue preparation and immunohistochemistry were also similar to those reported previously ,38. The  (ANOVA) .2HPO4·12H2O 2.9 g,NaH2PO4·2H2O 0.3 g, NaCl 9.0 g) and blocked with 10% normal goat serum  and 0.2% Triton  for 1 h. The sections were then immunoreacted with the syntaxin antibody  overnight at 4 °C. Cy3  was used as the secondary antibody for 1 h at room temperature. To identify the location of positive staining in the retina, 4',6-diamidino-2-phenylindole  was added at the same time as the secondary antibody to stain the nuclei of all cells, thus revealing cellular layers of the retina. Retinal section slices were examined under a fluorescent confocal microscope .Transfected cells, mostly RGCs and some displaced amacrine cells, were reported mainly located in the ganglion cell layer ,32. To i+ and GAP-43+ in each retina was obtained in the same way described above. Data from the different groups were statistically analyzed using the Bonferroni test following one-way ANOVA [Immunostaining of whole-mount retinas was essentially as reported previously -42. Afteay ANOVA ,40.Western blot experiments were applied to determine the expression levels of GAP-43 5 weeks after viral-vector transfection. After euthanasia with a lethal overdose of anesthesia, optic nerves were quickly dissected out and homogenized using lysis buffer containing 1% NP-40 , 0.25% sodium deoxycholate (Sigma-Aldrich), protease inhibitor cocktail , Phosphate Stop, and 1 mM phenylmethlsulfonyl fluoride . Protein concentration was determined using the Bio-Rad  protein assay reagent. Approximately 40 µg of protein was loaded and separated in a 10% acrylamide-Bis solution (Bio-Rad) gel. The protein was then transferred onto the nitrocellulose membrane  followed by washing in Tris-Buffered Saline Tween-20 (TBST) and blocking in 5% non-fat milk at room temperature for 1 h. To label the specific protein, the membrane was incubated with anti-GAP-43 antibody  in 0.5% milk/TBST at 4 °C with shaking overnight. After 2-min washing with TBST five times, horseradish peroxidase–conjugated (HRP) secondary antibody  was added to the membrane and shaken at room temperature for 1 h, followed by washing in TBST. The labeled proteins were detected using the enhanced chemiluminescence (ECL) reagent (Amersham Biosciences). Light signal from the membranes was detected and captured with the Molecular Imager ChemiDoc XRS System (Bio-Rad). Relative signal densities presenting amounts of target proteins were measured by calculating the band densities with Quantity One 4.6.2 (Bio-Rad), followed by normalization against the housekeeping protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH).IOP changes were similar to those seen in previous studies ,45. Twencistronic AAV-GAP-43-GFP vector and the control AAV-GFP were injected into eyes of non-injured animals containing a normal RGC population; GFP+ cells were either directly quantified by examination of retinal whole mounts or observed in retinal cryosection slices with fluorescence confocal microscopy. TUJ1 immunohistochemistry of retinal whole mounts  were transfected by the viral vector . Whereas other GFP+ transfected cells shown in the field of the white box were syntaxin negative (white arrows). Transfection efficiency was determined only in the identified β-III tubulin+ . The transfection efficiency of the target gene was also assessed with western blot analysis of the expression of the GAP-43 protein in the optic nerve, since GAP-43 mainly exerted biologic effects by regulating F-actin polymerization in axons [To assess the efficiency of transgene expression, bie mounts  showed tere RGCs ,32,46, htubulin+  RGC popuin axons . Blottin+ RGCs in retinal whole mounts in intact rats is shown in 2 (+ RGCs was undertaken 3 weeks after laser-induced IOP elevation. A significant (***p<0.001) decline in RGC survival was observed; nearly 60% of the RGCs had died, and only 496±136/mm2 (To ensure that the experimental chronic IOP elevation model in the rat was successfully established, the surviving RGC densities in normal saline versus IOP elevation–only groups were quantified. The representative appearance of β-III tubulinown in 2 , which i±136/mm2  RGCs sur2 (2, 2, **p<0.01; 2. That is, with overexpression of GAP-43 in cell bodies and axons, RGCs were more vulnerable to laser-induced chronic high IOP injury, leading to 80% RGC loss.Effects of overexpression of GAP-43 on RGC survival in animals with or without laser-induced IOP elevation were examined. In rats without IOP elevation, AAV vectors carrying exogenous gene GAP-43 were injected intravitreally 5 weeks before being euthanized. The average number of surviving RGCs in this group was 1,019±129/mm2 , not sig2 2,  was clos+ RGCs in the peripheral versus central retina. The average ratio in intact retinas was 1.25±0.22 (Based on the observations that axonal injury at different distances from the cell body results in different rates of RGC death, specifically, RGCs die largely and rapidly when the ON is transected close to the posterior eye pole, and is delayed as the distance from the transection site to the ON head increases ,51, we h.25±0.22 , showingIn the present study, we used a gene transfection approach in rats to assess the direct effects of GAP-43 overexpression on adult RGC survival in a laser-induced chronic IOP elevation model. There was a substantial increase in expression of GAP-43 in the optic nerve that appeared to be associated with greater RGC loss in IOP-elevated eyes. These new data indicating GAP-43 as a negative modulator of RGC survival provide a new perspective for interpreting the thus far unexplained loss of neurons overexpressing GAP-43 ,28,29.After laser-induced IOP elevation, GAP-43 expression was significantly upregulated but lasted no longer than 1 week . With viAfter AAV-GAP-43-GFP injection, only about 25% of the RGCs expressed the target protein; thus, RGC loss would not have been so extensive if overexpression of GAP-43 affected only those RGCs transfected with the vector. GAP-43 has been reported to play crucial roles in regulating synaptic plasticity ,58, whicAccording to the observation that optic nerve injury closer to the ONH resulted in more rapid and severe RGC loss ,51, RGCsIn conclusion, we demonstrated that AAV mediated overexpression of the axonal growth–associated protein GAP-43 in RGCs and severely aggravated RGC death in experimental glaucomatous injury. Thus, in addition to known positive biologic actions on promoting axonal growth, GAP-43 also exerts negative effects on RGC survival in high IOP conditions. This finding thus adds an important aspect to our existing understanding of RGC loss in glaucoma. Specific mechanisms involved thus far have not been well explained. More detailed investigations need to be conducted to obtain full understanding of the molecular pathological processes of glaucoma and therefore possibly provide better treatments.


The pandemic influenza A(H1N1)pdm09 virus was first detected in India in May 2009 and continued to circulate in the postpandemic period. Whole-genome sequence analysis of postpandemic A(H1N1)pdm09 viruses showed the circulation of clade 6 and clade 7 viruses. The hemagglutinin (HA) gene showed increased diversity compared with that in the pandemic phase. After the first reported case of infection with the influenza AH1N1)pdm09 virus in India N1pdm09 v of 31 whDuring the study period, 6,836 samples were analyzed, and of the 1,675 real-time PCR positives, 1,036 (61.8%) were A(H1N1)pdm09, 242 (14%) were A(H3N2), and 397 (23%) were type B positive. Isolation was carried out with MDCK cells. Thirteen A(H1N1)pdm09 isolates from a period of unusual activity  were selected for whole-genome sequencing. All the eight influenza gene segments were amplified in an overlapping manner by one-step conventional reverse transcriptase PCR (RT-PCR) using the whole-genome primers recommended by the WHO and the CDC pdm09 viruses of clade 7 (D97N mutation) and clade 6 (A197T mutation) were in circulation. A single isolate from 2013 possessed D222N (D239N) in the receptor binding region of the hemagglutinin (HA) molecule . The HA N1pdm09 vContinued molecular surveillance and whole-genome sequencing are important for understanding significant evolutionary changes in this pandemic virus.KF280652 to KF280755; accession numbers are listed in The whole-genome sequences of 13 Indian A(H1N1) pdm09 isolates from the period 2011 to 2013 have been deposited in GenBank under accession no.