We analyzed femoral nerve regeneration in C57BL/6J mice that received this antibody in a hydrogel filled conduit connecting the cut and sutured nerve before its bifurcation, leading to short-term release of antibody by diffusion. Video-based quantitative analysis of motor functions showed improved recovery when compared to mice treated with conduits containing PBS in the hydrogel scaffold, as a vehicle control. This improved recovery was associated with attenuated motoneuron loss, remyelination and improved precision of preferential motor reinnervation. We suggest that function-triggering L1 antibodies applied to the lesion site at the time of injury over a limited time period will not only be beneficial in peripheral, but also central nervous system regeneration.L1 is among the few adhesion molecules that favors repair after trauma in the adult central nervous system of vertebrates by promoting neuritogenesis and neuronal survival, among other beneficial features. In the peripheral nervous system, L1 is up-regulated in Schwann cells and regrowing axons after nerve damage, but the functional consequences of this expression remain unclear. Our previous study of L1-deficient mice in a femoral nerve injury model showed an unexpected improved functional recovery, attenuated motoneuronal cell death, and enhanced Schwann cell proliferation, being attributed to the persistent synthesis of neurotrophic factors. On the other hand, transgenic mice over-expressing L1 in neurons led to improved remyelination, but not improved functional recovery. The present study was undertaken to investigate whether the monoclonal L1 antibody 557 that triggers beneficial L1 functions  The neural cell adhesion molecule L1 is a glycoprotein of the immunoglobulin superfamily expressed in most, if not all, neurons in the central and peripheral nervous systems of mammals. During development, L1 is targeted to the surface of developing neurites and growth cones and mediates axonal outgrowth, fasciculation and guidance as well as neuronal migration and survival in vitroIn vitro, application of recombinant L1 to Schwann cells increased their motility, and, in vivo, virally L1-transduced Schwann cells myelinated regrown/sprouted axons after spinal cord injury, while retaining their motility In the peripheral nervous system, L1 is expressed in axons and Schwann cells during embryonic and early postnatal development, and remains expressed by non-myelinating Schwann cells in the adult L1 monoclonal antibody 557 (L1 Ab 557) was prepared against L1 from adult mouse brain in Lou×Sprague-Dawley F1 hybrid rats as described Application of peptides and proteins via filling of a conduit is sufficient to deliver a protein or peptide of interest to the injury site and has been shown in several studies performed in our group All experiments were conducted in accordance with the German and European Community laws on protection of experimental animals, and all procedures used were approved by the responsible committee of The State of Hamburg (TvG 05/65). Experiments were carried out and the manuscript prepared following the ARRIVE guidelines for animal research.ad libitum, and an artificial 12 h light/dark cycle.Before and after the experiments, three-month-old C57BL/6J mice were kept under standard laboratory conditions, with food and tap water Surgery was performed as described pre, X den and X reinn are values prior to surgery, during the state of denervation (7 days after injury), and at any given time-point during reinnervation, respectively.Analysis was performed over a time-period of 12 weeks using a quantitative video-based approach  A third parameter, the limb protraction length ratio PLR, , was evaTwelve weeks after nerve transection the animals were anaesthetized with fentanyl, droperidol and diazepam for retrograde labeling of regenerated motoneurons Seven days after retrograde labeling the mice were anesthetized by intraperitoneal injection of 16% sodium pentobarbital solution  and transcardially perfused with 4% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.3. The lumbar spinal cords were removed, post-fixed overnight at 4°C in the perfusion solution and then immersed in 15% sucrose solution in 0.1 M cacodylate buffer, pH 7.3, for one day at 4°C. Thereafter the tissue was frozen for 2 min in 2-methyl-butane  pre-cooled to −30°C. For sectioning, spinal cord segments were attached to a cryostat specimen holder using TissueTek . Serial cross-sections of 25 µm thickness were cut on a cryostat  and attached to SuperFrostPlus glass slides (Carl Roth). The sections were air-dried for at least 1 h at room temperature and mounted in anti-fading medium .For the analysis of re-myelination, femoral nerves were dissected from the animals fixed by perfusion and post-fixed in 1% osmium tetroxide  in 0.1 M sodium cacodylate buffer, pH 7.3, for 1 h at room temperature, dehydrated and embedded in resin according to standard protocols. One µm-thick cross-sections from the motor and sensory nerve branches were cut at a distance of approximately 3 mm distal to the bifurcation and stained with 1% toluidine blue/1% borax in distilled water for analysis of axons in the regenerated nerve branches.Serial cross-sections of spinal cords were examined under a fluorescence microscope  with appropriate filter sets. Each section was examined using a 40× objective by focusing through the section thickness starting from the top surface. All profiles, except those visible at the top surfaces of sections, were counted Immunofluorescence staining was performed as described For analyses of perisomatic terminals, spinal cord sections containing retrogradely labeled motoneurons (see above) were freed from the coverslips and mounting medium by soaking in PBS. Antigen retrieval was performed by immersion into 0.01 M sodium citrate solution, pH 9.0, heated to 70°C in a water bath for 30 min. Blocking of non-specific binding sites was then performed using PBS containing 0.2% Triton X-100 , 0.02% sodium azide  and 5% normal goat or normal donkey serum  for 1 h at room temperature. Incubation with primary antibodies against VGAT or ChAT , dilutedhttp://fiji.sc/Fiji) and the LSM image browser (Zeiss) was used to select the image with the largest cell body cross section.Sections containing retrogradely labeled motoneurons and additionally stained for ChAT and VGAT were used to estimate soma areas and motoneuron perisomatic coverage Total numbers of myelinated axons per nerve were estimated in semithin cross-sections on an Axioskop microscope (Zeiss) equipped with a motorized stage and Neurolucida software-controlled computer system  using a 100× oil objective All numerical data are presented as group mean values with SEM. Parametric or nonparametric tests were used for comparisons, as indicated in the text and figure legends. For parametric tests, the mean was used as a representative value if two or more measurements per parameter and animal were performed. Thus, for all these comparisons the degree of freedom was determined by the number of animals. Analyses were performed using the SYSTAT 9 software package . The threshold value for acceptance of differences was 5%. The size of each sample  is indicated in the figures or their captions.t test, Transection of the femoral nerve induces changes in the heels-tail angle (HTA) and foot-base angle (FBA)  as well In addition to ground locomotion, we also evaluated the animals' ability to extend the knee joint during movements without body weight support using the “pencil” test versus 5.4±2.5) . Thus, motoneuron loss and/or failure of axonal regrowth are reduced and precision of preferential motor reinnervation is improved in mice treated with L1 Ab 557.To analyze the numbers of neurons having regrown into the appropriate branch, retrograde labeling of motoneurons was performed at 12 weeks after injury. The total number of motoneurons retrogradely labeled through the motor or the sensory branches, or through both branches of the femoral nerve, was reduced after treatment with PBS compared with non-injured mice and mice treated with L1 Ab 557. The total number of motoneurons in L1 Ab 557 treated mice was similar to that in non-injured mice and displayed preferential motor reinnervation (PMR) when compared with the PBS treated mice . In everWe next analyzed non-injured and regenerated nerves histologically to assess axonal numbers, axonal diameters and degree of myelination 12 weeks after injury. In both motor and sensory femoral nerve branches, the total number of myelinated axons was similar in both experimental groups compared with non-injured mice . However+) terminals and inhibitory  terminals at motoneuron perikarya were measured as indicators for successful recovery at 12 weeks after injury. Somata of motoneurons were larger in L1 Ab 557 treated mice or non-injured mice compared with PBS treated mice (Soma sizes of motoneurons and levels of cholinergic (ChATted mice .+) terminals at motoneuron perikarya were increased in mice treated with L1 Ab 557 compared with the PBS group or mice without injury (+) terminals were decreased in the PBS group as compared with mice treated with L1 Ab 557 and the non-injured group, which were similar  terminals and modulatory cholinergic (ChAT+) terminals at motoneuron perikarya. Cholinergic synaptic input to motoneuron perikarya modulates the excitability of motoneurons and plays an important role in the control of locomotion +) boutons correlate with maintenance of excitatory synapses which contribute to improved recovery after injury in vitro+) terminals, similar in extent to the non-injured situation. Interestingly, L1 activation is necessary for its interaction with the cytoskeletal linker molecule ankyrin With reinnervation, motoneuron cell bodies recover synaptic input In vivo, L1 overexpressing embryonic stem cells, neural stem cells or radial glial cells not only enhanced locomotor recovery after spinal cord injury, but also rescued imperiled host motoneurons and parvalbumin-positive interneurons Our findings also agree with observations in a central nervous system injury paradigm, where treatment of the severed spinal cord with Fab fragments from L1 Ab 557 enhanced regeneration th immunoglobulin domain is deleted, indicate that interactions of L1 on axons with integrins on Schwann cells may be essential for ensheathment and may affect myelination in vitroIn the PNS, L1 is detected on both non-myelinating Schwann cells and non-myelinated sensory axons of adult mice. Non-myelinating Schwann cells show a reduced association with axons in L1 knock-out mice, and the non-myelinated sensory axons undergo degeneration In the present study we observed better myelination in the sensory branch of the femoral nerve of mice treated with L1 Ab 557, while the myelination in the motor branch did not differ from the PBS treated group. This finding might be related to a more pronounced expression of L1 by sensory but not motor axons 2+ or inositol-1,4,5-trisphosphate in vitro paradigm 2+-channel blockers and pertussis toxin. Partial inhibition of neurite outgrowth in these experiments suggests that different molecular mechanisms are involved 2+ and G-protein independent second messenger cascades or even modes of signal transduction independent of second messenger systems. For instance, application of L1 Ab 557 leads to sumoylation and proteolytic processing of L1 in vitroin vivo.The mechanisms by which L1 Ab 557 triggers neurite outgrowth are noteworthy in that the epitope recognized by this antibody recruits second messenger systems in neurons and Schwann cells, such as CaStudies related to regeneration in the presence of L1 antibody have to be taken with caution with regard to systemic application, since L1 is not only expressed in the nervous system but also, for instance, by cells of the immune system, skin, kidney and intestine and application of function triggering L1 antibodies could lead to stimulation of L1-dependent functions in these cells, thereby inducing side effects, which could be avoided by local antibody application. Our study with direct application of beneficial function-triggering L1 antibody indicates favorable effects on recovery and may encourage designing novel, clinically feasible strategies to improve functional outcomes after nervous system injury, for instance by construction of biodegradable conduits and particles that allow controlled release of L1 function triggering reagents, not only the peripheral but also central nervous system.


The crystal structure of the TDP-43 RRM1 domain containing the D169G mutation in complex with DNA along with molecular dynamics simulations reveal that the D169G mutation induces a local conformational change in a β turn and increases the hydrophobic interactions in the RRM1 core, thus enhancing the thermal stability of the RRM1 domain. Our results provide the first crystal structure of TDP-43 containing a disease-linked D169G mutation and a disease-related mechanism showing that D169G mutant is more susceptible to proteolytic cleavage by caspase 3 into the pathogenic C-terminal 35-kD fragments due to its increased stability in the RRM1 domain. Modulation of TDP-43 stability and caspase cleavage efficiency could present an avenue for prevention and treatment of TDP-43-linked neurodegeneration.The RNA-binding protein TDP-43 forms intracellular inclusions in amyotrophic lateral sclerosis (ALS). While TDP-43 mutations have been identified in ALS patients, how these mutations are linked to ALS remains unclear. Here we examined the biophysical properties of six ALS-linked TDP-43 mutants and found that one of the mutants, D169G, had higher thermal stability than wild-type TDP-43 and that it was cleaved by caspase 3 more efficiently, producing increased levels of the C-terminal 35 kD fragments (TDP-35)  Protein misfolding can lead to the formation of intra or extracellular accumulation of aggregated protein and is frequently associated with various pathological conditions such as neurodegeneration. One example is TDP-43, which binds RNA and DNA and functions in mRNA splicing, translation regulation and transport2146TARDBP, the gene encoding TDP-4312TARDBP have been identified in familial and sporadic ALS, most of which are located in the C-terminal G-rich region with only two exceptions to-date, A90V in the NTD and D169G in the RRM11314151617181920212223242526272829303132333435368TDP-43 consists of an N-terminal domain (NTD) and two tandem RNA recognition motifs, RRM1 and RRM2, followed by a C-terminal glycine-rich region (G) . It form8910ALS-linked mutants have been suggested to be more prone to aggregation and more potent in inducing cell death than wild-type TDP-43: Compared to the latter, Q331K and M337V accelerate spontaneous TDP-43 aggregation4045However, it is still unknown why some ALS-linked mutants exhibit increased stability with longer half-lives and how these properties contribute to protein accumulation and disease onset. Progranulin-mediated cleavage of TDP-43 by caspases 3 and 7 may generate the 25 kD (TDP-25) and 35 kD (TDP-35) C-terminal fragments4950515249545556Here, we provide biochemical, structural, simulation, and cellular data to show that compared to wild-type TDP-43, the ALS-linked D169G mutation in TDP-43 generates a more stable protein that is cleaved by caspase 3 more efficiently, producing increased levels of TDP-35, but not TDP-25. The crystal structure of the RRM1 domain containing the D169G mutation, along with molecular dynamics simulations, provides a molecular basis as to how this mutation increases protein stability. We suggest that TDP-35 bearing the RRM1 domain is stabilized by the D169G mutation and is thus the favored product in caspase digestion of TDP-43. This result provides the first crystal structure of TDP-43 containing a disease-associated mutation and the working mechanism showing that the D169G mutation renders TDP-43 more susceptible to production of the pathogenic C-terminal fragment TDP-35, thus linking it to ALS.E. coli. However, the recombinant full-length TDP-43 was highly unstable and degraded within hours of its expression, suggesting that the C-terminal G-rich tail destabilizes TDP-43. We further expressed the His-tagged truncated TDP-43 with a shorter C-terminal G-rich tail and identified a more stable truncated form of TDP-43 (residues 1–330), which could be analyzed in biochemical assays within 24 hours after its expression and purification. This TDP-43 (residues 1–330) is referred to as N-RRM12-G, as it had complete NTD, RRM1 and RRM2 domains, and a C-terminal glycine-rich tail of about 65 residues  and the corresponding D169G mutant with caspases 3, 4, 6 and 7, since it has been suggested that TDP-35 is generated mainly through caspase 3/7-mediated cleavage at D89–A90in vitro digestion experiments  of the wild-type protein  by caspase 3 than wild-type TDP-43 (~20%), likely because A90 is an important determinant for caspase 3 cleavage being right next to the cleavage site of D89–A90 . LikewisTo reveal the molecular basis for the increased stability rendered by the D169G mutation, we co-crystallized RRM1 with DNA and determined the crystal structures of the wild-type RRM1 and RRM1-D169G mutant in complex with a single-stranded DNA, 5′-GTTGAGCGTT-3′ (see RRM1 sequence in 815 DNA (Kd of 20.6 ± 1.8 nM vs. 14.2 ± 1.7 nM)The overall structure of RRM1-D169G mutant is similar to that of wild-type RRM1, nevertheless, a small local difference was observed in the β turn (Turn6) containing the D169G mutation. Superposition of the two proteins yielded an average root-mean-square deviation (RMSD) of 0.39 Å for 71 Cα atoms. Both wild-type RRM1 and D169G mutant exhibit similar affinity for single-stranded (TG)To understand why a local conformational change in Turn6 in the TDP-43 RRM1 is linked to the increased stability, the crystal structures of the RRM1 domain of the wild-type and D169G mutant were subjected to multiple molecular dynamics simulations at a mean temperature of 300 K and pH 7 for a total sampling period of at least 200 ns (see Methods). The simulation protocol adopted maintained the overall protein structure, as evidenced by an average RMSD of 1.0 Å (wild-type) and 0.9 Å (D169G mutant) for the protein backbone atoms from those in the initial structures. To detect subtle conformational changes, the Cα–Cα and SC–SC (where SC denotes the center of mass of the side chain) distances between wild type RRM1 and RRM1-D169G mutant were compared. Furthermore, the pairwise interactions between all residues were decomposed into van der Waals and electrostatic contributions.The simulation analyses suggest that the D169G mutation results in enhanced packing  interactions between Ile168 and several residues involved in the hydrophobic folding of the domain: The D169G mutation enabled the Turn6 backbone to move closer to Loop1: compared to wild-type D169, the Cα–Cα distance between G169 and the residues located on Loop1 and on the N-terminal part of Helix 1 was reduced by roughly 1 Å on average as compared to D169 . This inTo further confirm that Ile168 is a key residue in the inner hydrophobic core of RRM1 domain, we mutated Ile168 into alanine and generated two mutants, RRM1-I168A and RRM1-I168A/D169G. However, these two mutants could not be expressed, indicating that Ile168 is indeed crucial for protein folding. To assess the importance of the hydrogen-bonding interactions between D169G and Thr115 for protein stability, we also mutated Thr115 to alanine in RRM1. Thr115 is located in Loop1 whereas D169G is located in Turn6 in RRM1. The RRM1-T115A mutant could be purified and its secondary structure was similar to that of wild-type RRM1 as revealed by CD . Howeverin vivo, we generated the mouse neuroblastoma (Neuro2a) stable cell lines constitutively expressing the wild-type TDP-43 and the D169G mutant, using the N390D mutant as the positive control. The ALS-associated N390D mutant is more stable and has a higher steady-state level in Neuro2a cells, and it causes higher apoptotic death rate than wild-type TDP-43in vivo result shows that TDP-43 with the D169G mutation was cleaved more efficiently into TDP-35, but not TDP-25. This result is consistent with a recent studyTo further examine the stability and cleavage pattern of TDP-43 in vivo study also shows that expression of the D169G TDP-43 mutant generates a higher level of accumulated TDP-35 than wild type and TDP-35 with the D169G mutation is more stable than wild-type TDP-35.Several TDP-43 mutants have been shown to exhibit an increased protein half-life in different cell models434744In this study we tested six ALS-linked TDP-43 mutants and found one mutant D169G with significantly higher thermal stability than the wild-type protein. The crystal structure and MD simulations further reveal that the higher stability of D169G compared to wild-type TDP-43 is due to a local conformational change in the β turn region containing the D169G mutation and a reinforcement of the hydrophobic interactions in the core of RRM1. Although mutation of D169 to G in TDP-43 has been reported to result in enhanced protein stability44in vitro, and the expression of the D169G TDP-43 mutant in mouse neuroblastoma cells consistently generated higher levels of TDP-35 than that of the wild-type TDP-43  in Alzheimer’s disease, as both proteins are cleaved before aggregation. APP is endoproteolytically cleaved by β and γ secretases to generate Aβ peptides that are the primary components of amyloid deposits in the brain48249TARDBP gene and inserted into the BamHI/HindIII sites in the pQE30 vector (Qiagen) for the expression of the N-terminal His-tagged recombinant proteins. The constructs for the single-point mutants of A90V, D169G, G287S, G294A, G298S and A315T and other mutations for structural study  were generated by QuikChange® Site-Directed Mutagenesis Kit (Agilent Stratagene). All of the plasmids were transformed into Escherichia coli M15 strain for overexpression. Small cell culture of ~25 ml was incubated overnight and then added into 1 liter of LB medium with 50 μg ampicillin for cell growth for about 3–4 hours. The cell culture was cooled down to 18 °C and then induced by adding 0.8 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) and 50 μg ampicillin for 20 hours at 18 °C. The cells were suspended in 50 mM phosphate buffer (pH7.5), 100 mM NaCl and 10 mM β-mercaptoethanol (βME) and lysed by microfluidizer  and centrifuged at 12,000 rpm for 30 minutes. The supernatant of cell extracts were applied onto an equilibrated nickel-nitrilotriacetic acid (Ni-NTA) affinity column (Qiagen) with 50 mM phosphate buffer (pH7.5), 100 mM NaCl and 10 mM βME. The N-terminal His-tagged proteins were eluted by a gradient buffer containing 50 mM sodium phosphate buffer (pH 7.5), 100 mM NaCl, 0–1.0 M imidazole and 10 mM βME. The eluted proteins were then purified by the HiTrap heparin column  and eluted by a gradient buffer containing 50 mM phosphate buffer (pH 7.5), 0.1–1.0 M NaCl and 10 mM βME. The purified proteins (or mutants) were dialyzed against the buffer containing 50 mM phosphate buffer (pH 7.5), 100 mM NaCl and 10 mM βME for further biochemical assays, or the buffer containing 50 mM HEPES buffer (pH7.5), 100 mM NaCl and 10 mM βME for crystallization and caspase digestion assays.The cDNA of RRM1 (residues 101–191), N-RRM1 (1–191), N-RRM12 (1–265) and N-RRM12-G (1–330) were amplified from the human 2 . dmol−1. Thermal denaturation measurements were performed by monitoring the change in CD signals at wavelength 218 nm from 20 °C to 85 °C in 1 degree intervals with 0.5 minute equilibration time.All far-UV CD spectra were recorded on the Aviv Circular Dichroism Spectrometer Model 400. Protein samples were diluted with 50 mM sodium phosphate buffer (pH 7.5), 100 mM NaCl and 10 mM βME to give the appropriate protein concentrations (~20 μM) and a total volume of 300 μl for the measurement in a quartz cell with 1-mm path length. The CD spectra were scanned ranging from 260 to 200 nm three times at 25  °C. The final spectra were represented by mean residue ellipticity [θ] (MRE) in deg . cm® 480 system (Roche) in triplicate using the protocols described previously4 buffer, pH7.5, 100 mM NaCl and 10 mM βME) was incubated at room temperature for 10 minutes. The 125X SYPRO Orange (Invitrogen) dye (1 μl) was then added to the protein and loaded on a multiwell plate (MultiWell Plate 96 White (Roche)) and sealed with LightCycler® 480 Sealing Foil (Roche). The temperature was increased from 20 to 85 °C at a heating rate of 0.012 °C/second and the emitted fluorescence at 568 nm (excited at 483 nm) was recorded every 0.02 °C. The melting temperature (Tm) was calculated by the LightCycler® Protein Melting Analysis.The differential scanning fluorimetry experiments were carried out by the LightCyclert-test which associated comparisons between the wild-type and mutant proteins.The TDP-43 (N-RRM12) (5 μg) and its mutants were incubated respectively with human active recombinant caspase 3 (BD Phamingen), caspase 4 (BioVision), caspase 6 (BioVision), and caspase 7 (BioVision) in a buffer containing 50 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM DTT, 1 mM EDTA, 10% glycerol and 0.1% CHAPS at 37 °C for 2 hours . The reactions were stopped by heating the mixture at 95 °C for 5 minutes, and the digested protein samples were resolved in 15% Tricine-SDS-PAGE with Coomassie blue staining. The data obtained from three independent experiments were shown as the mean standard deviation (±S.D.). The differences between the variants were appraised by the ANOVA test. Statistical significance P values were determined by an unpaired two-tailed Student’s 3COONH4, pH 5.0 and 15% v/v Jeffamine ED-2001, pH 7.0.The purified human RRM1 and RRM1-D169G were mixed with the single-stranded DNA with a sequence of 5′-GTTGAGCGTT-3′ in a molar ratio of 1:1.2. The crystals of RRM1-DNA complex were grown by hanging drop vapor-diffusion method at room temperature by mixing 1 μl of protein-DNA solution with 1 μl of reservoir solution containing 0.12 ammonium acetate, 20% PEG 3350 and 0.08 BIS-TRIS pH 5.5. The crystals of RRM1-D169G/DNA complex were grown in the reservoir solution containing 0.05 M CH522 with two complexes per asymmetric unit, whereas the RRM1-D169G/DNA complex was crystallized in the trigonal space group P3221 with four complexes per asymmetric unit. The two complex structures were solved by molecular replacement using the wild-type RRM1 structure (PDB entry code: 4IUF) as the search model. The crystal structures were refined by PHENIX and the data collection and refinement statistics are summarized in The X-ray diffraction data of hRRM1/DNA were collected by Rigaku FR-E + SuperBright X-ray generator with Saturn 944 + CCD detector. The diffraction data of RRM1-D169G/DNA were collected at SPXF beamline BL15A1 at NSRRC (Taiwan). The diffraction data were processed and scaled by HKL2000. The RRM1/DNA complex was crystallized in the hexagonal space group P6The crystal structure of the wild-type RRM1 reported previously (PDB entry code: 4IUF) and RRM1-D169G determined in this study (PDB entry code: 4Y00) were subjected to molecular dynamics simulation. All simulations were performed using the OpenMM5.2 CUDA version of CHARMM version 37, and the CHARMM36 all-atom parameter set. The systems were first submitted to a 30-ps heating phase (from 0 to 300 K), followed by a 500-ps equilibration and a 20-ns production phase in the NPT ensemble. Simulation analysis was performed at room temperature (300 K) with an integration step set to 2 fs, except for the heating phase for which a 1-fs time step was used. Constant pressure was ensured by the use of a Langevin piston with a friction coefficient of 10 ps-1 at a reference pressure of 1 bar. Coordinates were saved every picosecond. All non-bonded interactions were truncated at 16 Å, van der Waals interactions were switched at 12 Å and electrostatic interactions were treated via the particle mesh Ewald summation method. To increase the sampling, several replicates of 20 ns each were performed, rather than a single long-trajectory. As four copies of RRM1-D169G were present in the crystal structure, 3 replicates of each copy were simulated. As for the wild-type RRM1, only one copy was found in the crystal structure, 10 replicates were performed. Therefore, in total, at least 200 ns of sampling was obtained for each form.The cDNA of the wild-type TDP-43, D169G and N390D mutants were cloned into the XhoI/ SacII sites of a pcDNA3.1-Myc/His (B) vector (Invitrogen) with a C-terminal Myc/ His tag. Mouse neuroblastoma (Neuro2a) cells were cultured in Modified Eagle Medium with 10% heat-inactivated fetal bovine serum (Invitrogen), 1% penicillin-streptomycin (Invitrogen) and 1% sodium pyrubate (Invitrogen). For generating stable cell lines, cells were subcultured and transfected in 6-well plates with TDP-43 constructs by selection in 500 mg/ml G418 (Promega) for two weeks and limiting dilution was used to generate monoclonal cell lines.For detecting the TDP-35 protein degradation, stable cells expressing wild-type TDP-43 and mutant (D169G and N390D) constructs were treated with cycloheximide . At various time points thereafter, the transfected cells were lysed and the amounts of the TDP-43 proteins were measured by Western blot analysis using the anti-His antibody (Proteintech). Cells were lysed in RIPA buffer  supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Sigma). The protein concentrations of the lysates were measured using the Bio-Rad protein assay reagent on a Beckman Coulter DU-800 machine. The lysates were then resolved by 12% SDS–PAGE and immunoblotted with the anti-His antibodies. Quantification of the immunoblot band intensities was performed with use of the Image J software (NIH) as described previouslyHow to cite this article: Chiang, C.-H. et al. Structural analysis of disease-related TDP-43 D169G mutation: linking enhanced stability and caspase cleavage efficiency to protein accumulation. Sci. Rep.6, 21581; doi: 10.1038/srep21581 (2016).


We evaluate the damage level and the influence of the HfO2 thickness on graphene. The results indicate that in the case of Al2O3/graphene, whether 1L or FL graphene is strongly damaged under our process conditions. For the case of HfO2/graphene, μRS analysis clearly shows that FL graphene is less disordered than 1L graphene. In addition, the damage levels in FL graphene decrease with the number of layers. Moreover, the FL graphene damage is inversely proportional to the thickness of HfO2 film. Particularly, the bottom layer of twisted bilayer (t-2L) has the salient features of 1L graphene. Therefore, FL graphene allows for controlling/limiting the degree of defect during the PE-ALD HfO2 of dielectrics and could be a good starting material for building field effect transistors, sensors, touch screens and solar cells. Besides, the formation of Hf-C bonds may favor growing high-quality and uniform-coverage dielectric. HfO2 could be a suitable high-K gate dielectric with a scaling capability down to sub-5-nm for graphene-based transistors.Based on micro-Raman spectroscopy (μRS) and X-ray photoelectron spectroscopy (XPS), we study the structural damage incurred in monolayer (1L) and few-layer (FL) graphene subjected to atomic-layer deposition of HfO Most of the reports are related to the latter method, because plasma is rather aggressive  and generally etches graphene11151823Although physical vapor deposition (such as magnetron sputtering), widely used in the semiconductor industry, can provide high deposition rates and preserve film stoichiometry, it generates extensive damage in graphene from high energy sputtered atoms62) and Al2O3 dielectrics on graphene by PE-ALD. In our process, the graphene samples are placed away from the plasma source for outside of the glow discharge. The remote oxygen plasma with low ion bombardment avoids fast etching of graphene. Simultaneously, the reaction between graphene and the precursor still guarantees physical/chemical modification of graphene. Based on micro-Raman spectroscopy (μRS) and X-ray photoelectron spectroscopy (XPS), we study the structural damage induced in 1L graphene underlying HfO2 and Al2O3 upon different oxygen plasma power levels. We evaluate the damage levels in AB-stacked bilayer (AB-2L), twisted bilayer (t-BL) and trilayer (3L) graphene (hereafter they are collectively referred to as FL) underlying HfO2 and Al2O3 for a fixed oxygen plasma power. We also investigate the influence of the HfO2 thickness on graphene with various layer numbers. The results indicate that in the case of Al2O3/graphene, both 1L and FL graphene are strongly damaged under the present process conditions. In the case of HfO2/graphene, μRS analysis clearly shows that FL graphene presents much less disorder than 1L graphene. Moreover, the FL graphene damage decreases with the number of layers. Our results also reveal that an inverse dependence of FL graphene damage with increasing the thickness of HfO2 film. FL graphene allows for controlling/limiting the defect formation during the PE-ALD HfO2 process. Therefore, it could be a good starting material for applications such as graphene-based transistors and sensing devices26In this work, we use mild plasma conditions to directly grow hafnium oxide  is 1.9, and the full width at half maximum (FWHM) of the 2D peak is 31 cm−1. These figures of merit first confirm the presence of as-transferred 1L graphene at the probed locations. At the exact same positions, two new peaks (defect-activated peaks) appear after PE-ALD HfO2 and PE-ALD Al2O3. Namely the D peak located at ~1356 cm−1 and the D’ peak (~1620 cm−1) at the right shoulder of the G mode. The D peak in sp2 graphene is activated by a double resonance Raman process in the presence of disorder and defects and is related to the breathing modes of carbon atoms in the vicinity of the K point in the Brillouin zone  to make the measurement inside the crystalline region (in the center of hexagon) with a Raman laser spot diameter of about 1 μm, the boundary of the hexagons does not contribute to the spectrum here. After the dielectric depositions, the intensity of D peaks becomes very strong. This indicates that the dielectric depositions break the symmetry of the graphene lattice and induce structural defects in graphene. Moreover, the positions of the G peaks are slightly shifted, the FWHM of 2D peaks are broadened and the D’ peaks are separated from the G peaks. These characteristics indicate that graphene is disordered, but it is not completely etched and then still optically visible. We therefore use the area ratio AD/AG between the integrated intensities of the D and G peaks to quantify the amount of disorder (i.e. the AD/AG increases with increasing amount of disorder at low defect concentration range). The area ratio is preferred over the individual intensities since it accounts for variations in peak position, intensity and linewidthμRS is a nondestructive method and is employed here to assess the structural damage in graphene underlying dielectric films. It is worth noting that a laser wave length of 514 nm was used in all the measurements except for the case by special description. 2 and PE-ALD Al2O3, respectively. The corresponding Raman spectra of 1L graphene underlying the two dielectrics are shown in AD/AG ratios are very similar for the different power levels , implying that the amount of generated disorders in graphene is not correlated to the plasma power levels in this range of powers. For ALD Al2O3 on graphene, Lim et al. used nitrogen plasma to pretreat grapheneTo investigate the damage in graphene subjected to different oxygen plasma power levels, we reduce the oxygen plasma power from 300 to 200 W and 150 W for PE-ALD HfO2 and Al2O3 with a thickness of about 5 nm, respectively. The Raman spectra of as-transferred graphene with various layer thicknesses (black curves) are also shown at the bottom of the figure as references. In the case of Al2O3/graphene (red curves), all the AD/AG ratios are large and all the D’ peaks clearly separate from G peaks. These features indicate that all kinds of graphene are damaged during PE-ALD Al2O3. In the case of HfO2/3L graphene, the intensity of the D peak becomes very weak and the D’ peak even almost disappears  is similar to that of the Al2O3/graphene case. These results point out that in PE-ALD HfO2, FL graphene is less damaged than 1L graphene and the damage level of graphene decreases with the number of layers.The layer number is first identified by the color contrast of graphene under optical microscopy, followed by μRS measurements. 2 thickness on graphene. 2 with different thicknesses , respectively. It can be seen from 2 thicknesses, the AD/AG ratio of 1L graphene dramatically increases compared with that of as-transferred graphene. However, the changes in the Raman spectra of FL graphene are less drastic. Surprisingly, all the AD/AG ratios decrease with increasing the thickness of HfO2 film  gratings and three excitation laser energies in the visible range, namely 514.5 nm (2.41 eV), 488 nm (2.54 eV) and 633 nm (1.92 eV). We do not find any peak related to HfO2 or Hf-C in the measurement range from 0 to 4000 cm−1. This implies that the HfO2 film is amorphous due to the low growth temperature.Finally, to identify if the HfOex situ XPS measurements to evaluate the impact of PE-ALD dielectrics on graphene. Four samples are sputtered with an Ar+ gun to perform a depth profile: 5.5-nm-thick Al2O3 and 5.9-nm-thick HfO2 are deposited either on silicon (as references) or on graphene/SiO2/silicon stacks . Core level spectra are recorded from carbon (C 1s), oxygen (O 1s), hafnium (Hf 4f), aluminum (Al 2p), and silicon (Si 2p). The elemental composition of the dielectrics obtained from 20-nm-thick reference layers can be estimated from the ratios of the integrated intensities of the XPS spectra: [O/Hf] = 2.15 ± 0.1 and [O/Al] = 1.47 ± 0.05. These results testify to the good quality of the dielectrics. We now focus on the C 1s atomic concentration profile and spectra of each sample. 2O3/silicon and HfO2/silicon samples, respectively. In both cases, a small amount of carbon (2% on average) is found in the profiles (except for a ~15% concentration corresponding to adventitious carbon on top of the dielectric layers). Moreover, a slight increase of the carbon concentration is observed when approaching the interface between the dielectric and silicon, most likely originating from residual contamination on silicon before PE-ALD. 2O3/graphene and HfO2/graphene samples, respectively. We can clearly identify the presence of graphene between the dielectric and the SiO2/silicon substrate. 2O3/graphene and HfO2/graphene samples at the maximum of the carbon profiles, respectively. The main peak at 284.5 eV in both spectra corresponds to graphene. Strikingly, in contrast to the Al2O3/graphene sample, the HfO2/graphene sample displays an additional peak at 281.5 eV. This peak can be attributed to the formation of the metallic carbide Hf-C. Consequently, the HfO2/graphene profile in et al.+ sputtering of ALD HfO2 induces the formation of Hf-C (at an ion-gun energy of 2000 eV), amounting to 1% of the total composition. To ascertain that the observed Hf-C peak is not related to sputtering (the Ar+ gun is operated on purpose at the very low energy of 200 eV in the hope of preventing Hf-C formation), we have performed an additional experiment. A 1L graphene is transferred onto an HfO2/SiO2/Si substrate and next subjected to sputtering in the same conditions as before. In the corresponding C 1 s spectrum, the carbide-related peak occurs as well, most likely due to intermixing between Hf and C during the erosion, resulting in Hf-C bonding. This means that, from the ex situ XPS analysis only, we cannot conclude if Hf-C forms during the PE-ALD process or during the erosion process or both. Nonetheless, as opposed to Al-C, this illustrates how easy it is to form Hf-C, since the Ar+ sputtering is operated at a very low energy of 200 eV. To further elucidate where Hf-C originates from requires an ALD apparatus fitted with an in situ XPS analyzer.We carry out 2O3/graphene/SiO2/Si , the Al2O3 film starts to nucleate and then grow. Unfortunately, 1L graphene, and even FL graphene, have been degraded during the pretreatment. On the contrary, the XPS results have demonstrated that the Hf-C bonds are quite easily formed, whatever the origin of the bonding. We hypothesize that, during the initial TDMA-Hf precursor pulse, a chemical reaction occurs between the Hf atoms and the C atoms constituting graphene to form Hf-C (its formation temperature is 25 °C)2 growth. Subsequently, the coverage of the first HfO2 layer protects the underlying graphene layer from damage during the following deposition cycles. This may also explain why HfO2 rather than Al2O3 can be directly grown on graphene without out-of-plane covalent functional groups, in low-temperature thermal ALD process41Although the optical image of the Al2/Si see  looks si2/Si see , all theAD/AG ratio on the number of graphene layers is not clear. It may be due to the interlayer interactions between the HfO2 layer and graphene or a direct consequence of the HfO2 deposition process or both. Previous works about the impact of the graphene thickness on its physical properties suggest that graphene’s rigidity may increase with increasing number of layers2 substrate, it conforms more easily to the surface morphology of the underlying substrate, thereby more prone to deform compared with FL graphene. Oxygen plasma modification of 2L and FL graphene has been studied in the literature. Calculation resultset al.462/graphene  made from 2L graphene is strongly suppressed compared with 1L graphene transistors532 and Al2O3) deposited by remote PE-ALD. Our results show that FL graphene is less damaged than 1L graphene; the damage level of FL graphene decreases not only with the number of graphene layers but also with the thickness of HfO2. Interestingly, the Raman spectrum of t-2L graphene underlying HfO2 presents the features of that of as-transferred 1L graphene. XPS measurements indicate that Hf-C is easily formed. After coverage by the first HfO2, the bottom graphene layer has an additional protection. The oxygen plasma pulse in PE-ALD is less reactive to FL graphene than 1L. Graphene rigidity increases with the number of graphene layers. Moreover, it also increases with the thickness of HfO2. These may be the reasons of FL graphene less damaged in PE-ALD HfO2. Therefore, FL graphene, more particularly, t-2L graphene allows for controlling/limiting the defect formation during the PE-ALD HfO2 process and might be a prospective material for applications such as graphene-based transistors and sensing devices. It appears that the thickness of PE-ALD HfO2 can be arbitrarily scaled down to 5 nm. Our results open up direct perspectives for FL graphene and HfO2 gate dielectric in graphene-based transistor applications.We have investigated the structural damage in graphene underlying dielectrics  by the usual method based on PMMA, after etching the copper foil in ammonium persulfate.Graphene is synthesized by APCVD with dilute methane (5% in argon) as hydrocarbon precursor on copper foils (Alfa Aesar #13382). The samples are grown at 1000 °C for 1 h under flows of 500 sccm of argon, 20 sccm of hydrogen, and 0.2 sccm of dilute methane. Graphene is then transferred onto 300-nm-thick SiO2 and Al2O3 films are deposited on graphene/SiO2/Si stacks, by PE-ALD  at 250 °C. The plasma source, inductively coupled at 13.56 MHz, is far away from the samples. It is very important to note that the distance between the plasma source and sample location is larger than 40 cm since the type and concentration of the reactive species  strongly depend on this distance. Outside of the glow discharge, only long lifetime radicals are present while ions and electrons recombine quickly.HfOin situ ellipsometry from reference films directly deposited on silicon substrates. The composition of the dielectric films is characterized by XPS. In order to investigate the damage level of graphene upon different oxygen plasma power levels, HfO2 and Al2O3 films are deposited on graphene/SiO2/Si stacks with nominal 300 W and with reduced oxygen plasma power of 200 and 150 W, respectively.In order to remove the native stress and polymethyl methacrylate (PMMA) residues from growth and transfer, the graphene samples on the SiO2/Si substrate first are annealed at 250 °C for 2 h in the deposition chamber under pressure of 80 mtorr (argon gas). During both dielectric film depositions, the pulse duration of the oxygen plasma (oxidant precursor) is 10 s for each cycle. The flows of the oxygen plasma and the argon carrier gas are 20 and 200 sccm, respectively. The metallic and oxidant precursor pulses are separated by a short argon purge of 5 s. To obtain uniform dielectric films and avoid graphene etching, the metallic precursors are first pulsed on the graphene surface. The metallic precursors can adsorb or react with carbon to form the related metal oxides following the first oxidant precursor pulse. In contrast, if the first pulse is a single cycle of oxygen plasma, the graphene surface is possibly damaged or non-uniform dielectric films are formed. The other parameters related to the metallic precursors and the final thickness of both dielectric films are listed in A LabRam HR 800 confocal laser system from Horiba Jobin Yvon was used for the acquisition of the Raman spectra. The measurements are performed at room temperature with a laser wavelength λ = 514 nm in backscattering geometry. The laser beam is focused on the center of hexagons and a 100 × objective (NA = 0.95) is used to collect the signal. The incident power is kept below 1 mW. Low resolution (150 g/mm) and high resolution (1800 g/mm) gratings are used for the measurements.Kα1,2 x-ray source and a hemispherical deflector analyzer. The spectra are recorded at constant pass energy . A flood gun (low energy electrons and Ar ions) is used during all the measurements. During the sputtering, the Ar+ ion gun is operated at a low energy (200 eV), with an erosion time of 5 s per cycle, and the analysis is done in snapshot mode. The XPS data are treated with the Avantage software. High resolution spectra are fitted by Gaussian-Lorentzian lineshapes with an Avantage “smart” background .A ThermoFisher Scientific K-alpha spectrometer is employed. It is equipped with a monochromatized Al How to cite this article: Tang, X. et al. Damage evaluation in graphene underlying atomic layer deposition dielectrics. Sci. Rep.5, 13523; doi: 10.1038/srep13523 (2015).


Carriers with energy higher than the Schottky barrier between graphene and WSe2 can be emitted over the barrier, thus creating photocurrent. We experimentally demonstrate that the photo-thermionic effect enables detection of sub-bandgap photons, while being size-scalable, electrically tunable, broadband and ultrafast.Finding alternative optoelectronic mechanisms that overcome the limitations of conventional semiconductor devices is paramount for detecting and harvesting low-energy photons. A highly promising approach is to drive a current from the thermal energy added to the free-electron bath as a result of light absorption. Successful implementation of this strategy requires a broadband absorber where carriers interact among themselves more strongly than with phonons, as well as energy-selective contacts to extract the excess electronic heat. Here we show that graphene-WSe The detection of low-energy photons may be enabled by devices that make use of the excess thermal energy from photoexcited carriers as a result of light absorption. Here the authors demonstrate a vertical graphene-WSe2-graphene heterostructure that takes advantage of the photo-thermionic effect. ΦB  even under continuous-wave (CW) excitation1819Graphene, which has recently emerged as an excellent platform for converting photons into hot carriers2/graphene van der Waals heterostructures to detect low-energy photons (with a wavelength up to 1.5 μm) through photo-thermionic (PTI) emission. Te compared with the temperature of the lattice Tph and the environment T0; carriers within this distribution with an energy larger than the Schottky barrier height ΦB at the graphene/WSe2 interface can be injected into the WSe2 and travel to the other graphene layer. The number of carriers with sufficient energy scales with kB is the Boltzmann constant.Here, we use graphene/WSe2—a transition metal dichalcogenide with a bandgap Eg∼1.3 eV—provides an energy barrier between the two graphene sheets with low interfacial defects and reduced Fermi-level pinning. The active device  that enables control of the Fermi energy μ and thereby ΦB of (mainly) the bottom graphene. All measurements presented in the main text are obtained from one particular device comprising a 28-nm-thick WSe2 flake (see 2 flakes of various thicknesses (L=2.2–40 nm) and obtained similar results, consistent with the PTI effect . Signatures of this mechanism are readily visible in the data presented in 2 (Ephoton<Eg). That is expected from a thermally driven photocurrent, given the uniform absorption of graphene in the visible, near-infrared range and the fact that kBTe is independent of Ephoton for constant power15P (P (see Methods). In contrast, the photocurrent in the above-bandgap regime (Ephoton>Eg) varies strongly with Ephoton and scales linearly with P. This photoresponse is characteristic of light absorption in WSe2 and transfer of photoexcited carriers to the graphene electrodes, a process driven by the potential drop across the WSe2 layer2324The PTI process is driven by the light-induced increase of the thermal energy of the electron gas  devices. We note that multi-photon absorption followed by charge transfer could also lead to a superlinear power dependence, but the laser intensity required to induce significant two-photon absorption in either graphene or WSe2 is at least 1–2 orders of magnitude higher than the one used in our experiment   refs , 27. Simt between two sub-picosecond laser pulses generated by a Ti:sapphire laser  and gate voltage (VG). Applying these voltages is necessary in order to generate a finite photocurrent, as it breaks the symmetry of our device, which is composed of two G/WSe2 Schottky barriers with opposite polarity. As the infrared photocurrent maps (Ephoton=0.8 eV) in VB essentially controls over which of the two Schottky barriers hot carriers are injected: for VB=−0.6 V (2 layer (GT/WSe2), whereas the interface with the bottom graphene (GB/WSe2) is photoactive for VB=+0.6 V , the device reaches a responsivity of up to 0.12 mAW−1 at wavelength λ=1,500 nm, which, for 0.5% light absorption in grapheneOne of the hallmarks of thermionic emission is its exponential dependence on the Schottky barrier height. In our device, the gate voltage Te and ΦB, and on the carrier injection time τinj. The values for ΦB(VG) are extracted from temperature-dependent dark current measurements  and laser power. This two-dimensional fit yields a carrier injection time τinj=47±10 ps and a thermal conductance Γ=0.5±0.3 MWm−2 K−1. This value of τinj is almost identical to the one found for ideal G/Si Schottky barriersΓ matches the predicted thermal conductance of G/hBN interfaces due to electron coupling with SPP phononsT0=230 and 330 K) and the analysis yields very similar results  metal filmsΦB. Indeed, we find that the PTI efficiency increases by one order of magnitude (up to 20%) by extrapolating the IQE to higher Te  or lower ΦB , with the important differences that for PETE the photoexcited carriers are in thermal equilibrium with the lattice of a hot semiconductor and are emitted over a vacuum energy barrier3411f=40 MHz and pulse duration dt=100 ps) with a microscope objective (Olympus LCPlanN × 50) on the device. The photocurrent is measured using a preamplifier and a lock-in amplifier synchronized with a mechanical chopper at 117 Hz.The heterostructures are fabricated the same way as described in ref. J thermionically emitted over a Schottky barrier of height ΦB at temperature T isIn the reverse-bias regime, the Schottky barrier model based on the Landauer transport formalisme0 is the elementary charge, kB is the Boltzmann's constant, ħ is the reduced Planck's constant, vF is the graphene Fermi velocity, Φ0 is the band offset at the G/WSe2 interface (0.54 eV) and τinj is the charge injection time. In our experiment, the thermionic photocurrent (PC) we measure is produced by the increase of electronic temperature ΔT=Te−T0 upon illumination of the device with a quasi-CW laser at λ=1,500 nm. Hence, it follows that the photocurrent is A is the area of the laser beam (laser spot size of 1.75 μm), D is the duty cycle (D=dt·f=0.04%) and T0 is the ambient temperature. Using these equations and assuming T0≫ΔT, one can show that α is the light absorption in graphene (0.5%), ηheat is the fraction of absorbed energy that is transferred to the electron bath (∼70%)  ref. , P is thstep see .The data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Massicotte, M. et al. Photo-thermionic effect in vertical graphene heterostructures. Nat. Commun. 7:12174 doi: 10.1038/ncomms12174 (2016).Supplementary Figures 1-7, Supplementary Notes 1-7 and Supplementary References


Chemical vapour deposited graphene rather than exfoliated or epitaxial graphene is used, because layer transfer methods are easily applied in fabrication. We employ a method of doping to increase the layer conductivity, and our analysis shows that the doped chemical vapour deposited graphene has good optical properties in the mid-infrared range. We therefore design the metamaterial for mid-infrared operation; our characterization with an infrared ellipsometer demonstrates that the metamaterial experiences an optical topological transition from elliptic to hyperbolic dispersion at a wavelength of 4.5 μm.While metal is the most common conducting constituent element in the fabrication of metamaterials, graphene provides another useful building block, that is, a truly two-dimensional conducting sheet whose conductivity can be controlled by doping. Here we report the experimental realization of a multilayer structure of alternating graphene and Al The use of graphene can provide another useful building block for metamaterials. Here, Chang et al. have explored the experimental realization of a mid-infrared hyperbolic metamaterial, in which the role of the metal in providing a conducting layer is taken over by graphene. Many interesting applications have been enabled by HMMs. For example, the spontaneous emission rate of quantum emitters can be modified if they are brought close to a HMMk-modes supported by HMM are exploited to achieve sub-diffraction-limited images using a hyperlens894Hyperbolic metamaterials (HMMs) are artificially structured materials designed to attain an extremely anisotropic optical response, in which the permittivities associated with different polarization directions exhibit opposite signs12k-modes1426293031In this paper, we explore the realization of a particular HMM, in which the role of the metal in providing a conducting layer is taken over by graphene1314151617181920221314151617181920The graphene-dielectric multilayer structure can be homogenized and viewed as a metamaterial using the effective medium approximation (EMA). The effective out-of-plane and in-plane permittivities of this metamaterial can be derived by taking the long-wavelength limit of the Bloch theory131415ɛd is the permittivity of the dielectric layer, d is the dielectric thickness and σ is the optical conductivity of graphene. Z0 is the vacuum impedance. Here graphene, as a 2D material, is treated as an infinitely thin layer described by its in-plane sheet conductivity. As indicated by ɛeff,⊥ is the same as the constituent dielectric and is always positive. On the other hand, the real part of ɛeff,|| becomes negative ifHere k-modes, which can be traced back to the coupled plasmon modes in the graphene-dielectric multilayer structureWhen this criterion is satisfied, the isofrequency surface becomes a hyperboloid and we obtain HMM. Such an isofrequency surface allows the existence of propagating high 2 V−1 s−1). As a result of the lower crystal quality, the stronger carrier scattering in typical polycrystalline CVD graphene enhances the free-carrier absorption at THz frequencies, which can be understood from the theoretical optical conductivity of graphene343536While most previous theoretical work has concentrated on using high-mobility graphene that may be obtained from mechanically exfoliated or epitaxially grown samples, we use CVD graphene because it is the most realistic choice for practical fabrication of a multilayer structureσ0 equals to e2/(4ℏ), EF is the Fermi energy relative to the Dirac point and γ is the intraband scattering rate. In this expression, the first two terms correspond to interband transitions, while the third term is the Drude-like intraband conductivity. ℏω>2EF because of interband transitions. On the other hand, at low frequencies when ℏω≲ℏγ, graphene also exhibits a large loss because of the intraband free-carrier absorption enabled by scattering. Because CVD graphene typically has a ℏγ of tens of meV, it is a lossy material at THz frequenciesEF can turn off the interband absorption by the Pauli blocking and increase the Imσ required for achieving negative ɛeff,||. Furthermore, doping can also suppress the intraband scattering by screening charged impurities37where Because graphene is the key building block of the metamaterial, it is important to have an accurate measurement on the optical conductivity of the actual CVD graphene layers used to fabricate the sample. Although the theoretical optical conductivity given by 2 substrates by the standard PMMA transfer method. Even without chemical treatment, unintentionally doped CVD graphene is p-type because of adsorbed gas molecules and residual ammonium persulfate from the transfer process4042a priori theoretical expression like In our previous work, we have developed a technique based on ellipsometry to measure the optical conductivity of truly 2D materialsσ(ω) by the model given by EF and γ being the only two unknown fitting parameters. We also show in the same figure the spline-fitted conductivity obtained from the same set of data. It is apparent that the resulting conductivity based on EF=460 meV and ℏγ=23 meV. A mobility of ∼2,000 cm2 V−1 s−1 can be calculated from these numbers using the relationship μ is the mobility and VF is the Fermi velocity.Although the spline-fitted conductivity of actual CVD graphene sample shown in In the mid-infrared range, the optical conductivity is mostly determined by intraband transitions, which are described by the Drude-like term in 442O3. The CVD graphene is transferred by the PMMA method and doped with Tris (4-romophenyl) ammoniumyl hexachloroantimonate (‘magic blue'). The Al2O3 dielectric layer is grown by atomic layer deposition (ALD). We choose Al2O3 as the dielectric material, because it has negligible loss at the mid-infrared wavelengths up to 8 μm. The dielectric thickness is chosen to be ∼10 nm to create an optical topological transition in the mid-infrared range.We have fabricated the multilayer structure shown in k0sinθ) associated with the free-space plane waves is very small . We are therefore probing the low k-modes of the metamaterial, ensuring the validity of the long-wavelength approximation. Although the long-wavelength approximation is evidently satisfied for our metamaterial (d/λ<1/300 in our case), we still need to confirm the validity of the EMA with a rigorous transfer-matrix calculation, since the EMA is derived for an infinite periodic system, while our metamaterial has only five periods. In rp/rs=(tanΨ)eiΔ, where rp and rs are the reflection coefficients for p and s light, respectively. Numbers used in the simulation are chosen according to measured material properties of the individual layers. As demonstrated by k-regime probed by ellipsometry, can be accurately treated as a metamaterial with the effective permittivities given by k-regime, even one period of the graphene-dielectric unit cell can be homogenized by the same EMA formula given by k-regime is where the real interest of HMM lies, and as discussed in k optical properties depend on the number of unit cells in the metamaterial. The five-period structure in our experimental realization of graphene HMM is chosen to create desirable high k optical properties.To characterize the metamaterial, we use infrared ellipsometry, which is appropriate to probe the effective permittivity of a metamaterial, since it measures the sample with free-space plane waves and the transverse wave vector . Furthermore, as shown by EF and γ. As a result of this independent knowledge of the sample, only two unknowns, EF and γ, are sufficient to fit the experimental data of the multilayer metamaterial.The results of infrared ellipsometry, ellipsometric angles Ψ and Δ for our HMM sample, are shown in 2O3. As shown by EF is 365 meV, and the extracted ℏγ is 41 meV. The extracted EF is lower than the value we typically obtain from chemically doped monolayer CVD graphene, because some dopants are lost in the ALD process because of the vacuum environment and the elevated temperature. The obtained scattering rate ℏγ is higher than the value of graphene on CaF2 substrate shown in 2O3 increases the carrier scattering.The fitted results of the ellipsometric angles Ψ and Δ are plotted as the blue dash lines in EF and γ. ɛeff,⊥ is always positive because it equals the permittivity of Al2O3. On the other hand, the real part of ɛeff,|| changes from positive to negative at 4.5 μm indicating an optical topological transition from an elliptical metamaterial to a HMM. This graphene metamaterial is therefore a transverse epsilon-near-zero metamaterial at the wavelength of 4.5 μm  and transferred to the substrate using the standard PMMA transfer technique332O3 dielectric layer is deposited by the ALD at 150 °C using trimethylaluminium as the Al precursor and H2O as the oxygen precursor. The number of cycles used in the ALD process is calibrated to grow ∼10 nm of Al2O3 on graphene, with the thickness characterized by an ellipsometer (Woollam M-2000). The procedure is repeated to fabricate five periods of the graphene-Al2O3 unit cell. We have also confirmed that the chemical doping with Tris (4-bromophenyl) ammoniumyl hexachloroantimonate does not affect the Al2O3 layer and the substrate. We have found that although nitric acid can also p-dope graphene effectively372O3 layer. The substrate is wedged to avoid back side reflection in the ellipsometry measurement. We also characterize the graphene-dielectric multilayer structure with the Woollam M-2000 ellipsometer after depositing each Al2O3 layer and after transferring each graphene layer. With the acquired ellipsometry data, we extract an average Al2O3 thickness of 10.4 nm.The graphene-dielectric multilayer structure with five periods is fabricated on a CaF2O3, we have prepared a sample with ALD-grown Al2O3 film on a CaF2 wedge. We measure the sample with both ellipsometers, and fit the refractive index of Al2O3 with the Sellmeier equation. The measurement of the effective permittivities of the graphene-dielectric multilayer structure is performed by the IR-VASE ellipsometer with the same settings described above.The optical conductivity of monolayer graphene is measured by the ellipsometric analysis method described in ref. How to cite this article: Chang, Y.-C. et al. Realization of mid-infrared graphene hyperbolic metamaterials. Nat. Commun. 7:10568 doi: 10.1038/ncomms10568 (2016).Supplementary Figures 1-6, Supplementary Notes 1-5 and Supplementary References.


The solvent-evaporation method produced spherical TPCA-1-loaded PLGA microparticles characterized with a mean diameter of 2.4 ¼m and loading efficiency of 80%. Retrobulbar administration of the TPCA-1-loaded PLGA microparticles maintained a sustained drug level in the retina during the study period. No detectable TPCA-1 level was observed in the untreated contralateral eye. The anti-CNV effect of retrobulbarly administrated TPCA-1-loaded PLGA microparticles was assessed by retinal fluorescein leakage and isolectin staining methods, showing significantly reduced CNV development on day 7 after laser injury. Macrophage infiltration into the laser lesion was attenuated as assayed by choroid/RPE flat-mount staining with anti-F4/80 antibody. Consistently, laser induced expressions of Vegfa and Ccl2 were inhibited by the TPCA-1-loaded PLGA treatment. This TPCA-1 delivery system did not cause any noticeable cellular or functional toxicity to the treated eyes as evaluated by histology and optokinetic reflex (OKR) tests; and no systemic toxicity was observed. We conclude that retrobulbar injection of the small-molecule IKK2 inhibitor TPCA-1, delivered by biodegradable PLGA microparticles, can achieve a sustained and controllable drug release into choroid/retina and attenuate laser-induced CNV development without causing apparent systemic toxicity. Our results suggest a potential clinical application of TPCA-1 delivered by microparticles in treatment of CNV in the patients with age-related macular degeneration and other retinal neovascularization diseases.The inhibition of NF-κB by genetic deletion or pharmacological inhibition of IKK2 significantly reduces laser-induced choroid neovascularization (CNV). To achieve a sustained and controlled intraocular release of a selective and potent IKK2 inhibitor, 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) (MW: 279.29), we developed a biodegradable poly-lactide-co-glycolide (PLGA) polymer-delivery system to further investigate the anti-neovascularization effects of IKK2 inhibition and  Inflammation in retina is associated with several eye diseases, such as age-related macular degeneration (AMD)  and diabin vivo and is therefore able to attenuate laser-induced CNV formation in vivo -5-(4-fluorophenyl)-3-thiophenecarboxamide  was purchased from Tocris Bioscience .TPCA-1-loaded PLGA microparticles were fabricated using a solvent-evaporation method , 22. PLG20 with 0.1% TFA for 5 minutes, immediately followed by a 30-minute linear gradient to 100% acetonitrile. Acetonitrile (100%) was maintained for 5 minutes before returning to 100% H2O/TFA conditions. Spectrophotometric data was collected from 200–800 nm, and the baseline was monitored at 260 nm and 280 nm. An extracted chromatogram at 310 nm was used for calibration purposes. A standard curve was created using the peak area generated by a set of standard solutions ranging from 500 μM to 0.1 μM. Authentic TPCA-1 eluted at approximately 21 minutes. As an additional check to confirm the identity of the peak presumed to be comprised of authentic TPCA-1 fractions of the highest concentration, calibration standards were collected and submitted for mass spec analysis using an Agilent Technologies 6224 TOF-LC/MS system equipped with 1260 series Infinity HPLC/Auto sampler/UV-vis detector and Mass Hunter software. Lyophilized unknown samples were dissolved in acetonitrile, spun down, and aliquots were placed into sample bottles. The samples were then run on the HPLC, and the results were evaluated in comparison with the previous linear calibration.The TPCA-1 content in the DCM solvent-extracted samples was quantitated with a Waters Alliance e2695 HPLC/Auto-sampler system equipped with a Waters 996 UV-vis photodiode array detector operated by Empower software (Waters) with a μRPC C2/C18 ST 4.6/100 column  equilibrated with water/acetonitrile. The flow rate was set to 0.5 mL/min. Initial injection conditions were 100% Hg for 10 min, and the TPCA-1 levels were then measured by injecting 150 μL of 1:500-diluted supernatant into the HPLC as described above.The loading efficiency of TPCA-1-loaded PLGA microparticles was determined by the extraction and quantification of free relative to encapsulated TPCA-1. PLGA/TPCA-1 microparticles (10 mg PLGA/1 mg TPCA-1) were briefly suspended in 1.5 mL of methylene chloride and incubated at room temperature on a shaker. The resulting solution was mixed with 1.5 mL acetonitrile. This solution was mixed on a vortex for 1 min and centrifuged at 18,000 x Mice were anesthetized with an intra-peritoneal injection of ketamine hydrochloride (0.0613 mg/g body weight) and xylazine hydrochloride (0.0245 mg/g body weight). The anesthetized mouse was positioned on its side under a stereo microscope, and the mouse head was restrained with thumb and middle finger. The needle was inserted at 45° to the eye, from the lateral to the medial canthus. The tip of the needle was positioned behind the eye globe, and 100 μl of PLGA microparticle suspension (10 mg PLGA in PBS) or TPCA-1-loaded PLGA microparticle suspension (10 mg PLGA/0.8 mg TPCA-1 in PBS) was gradually injected.Eight-week-old C57BL/6J mice received 100 μl of TPCA-1-loaded PLGA microparticle suspension (10 mg PLGA/0.8 mg TPCA-1 in PBS) in one eye. At 1, 7, 14, or 21 days after injection, both eyes were enucleated and washed at least four times in PBS. The anterior segment, lens, and sclera tissue were removed, and the remaining choroid/RPE/retina complex was retained, homogenized in DCM, and centrifuged at 12,000 RCF/4°C to remove tissue debris. The supernatant was used for quantitation by HPLC as described above.The procedure was performed as previously described . Laser pVisual function was assessed using a non-invasive OptoMotry© optokinetic testing system , following the procedures described previously .Seven days after laser photocoagulation, fluorescein angiography was performed using a stereo microscope  as previously described [For isolectin IB4 staining, laser-injured eyes were enucleated and fixed with ice-cold 4% paraformaldehyde at 4°C overnight. The anterior segment, lens, and neural retina were removed, and the remaining eye cups with attached RPE layer were washed with 1xPBS and incubated with Alexa Fluor 568-conjugated isolectin GS-IB4  overnight at room temperature. The eye cups were washed with 1xPBS and flat-mounted with a radial cut. The images were taken under a fluorescence microscope (Carl Zeiss MicroImaging GmbH) immediately after the staining procedure .Following enucleation, eyes were immediately fixed in 4% paraformaldehyde at 4°C overnight. Using a dissecting microscopy, the posterior eye cup with attached RPE was separated for macrophage staining of the choroid/RPE, washed with 1xPBS, and incubated with rat anti-F4/80 antibody  overnight at 4°C. The eye cups were washed with 1xPBS and flat-mounted with a radial cut. The images were taken under a fluorescence microscope (Carl Zeiss MicroImaging GmbH) immediately after the staining procedure.The area of GS-IB4-stained CNV was measured using AxioVision software (Carl Zeiss MicroImaging GmbH). All data were collected as the total CNV area per eye per mouse and are presented as the relative CNV area compared with control mice. Data was expressed as mean±SD, and statistical analysis was performed using a paired Student’s t-test.Tissues were fixed in 10%-buffered formalin and embedded in paraffin. Serial sections (5 μm thick) of eyes, liver, and spleen were generated. Sections were deparaffinized, rehydrated with distilled water, and stained with hematoxylin and eosin (H&E). Sections were observed under a bright-field microscope.6 cells in 50 μL of staining buffer (1xPBS containing 3% BSA and 0.1% sodium azide) were incubated at 4°C for 30 min with fluorochrome-conjugated mAbs; to stain apoptotic cells with annexin V, cells were resuspended in an annexin V binding buffer  and incubated with rat anti-TCRbeta and PE-labeled-annexin V reagent . Ab-stained cells were analyzed on a four-color BD FACSCalibur cell analyzer (BD Biosciences), and the data were analyzed with CellQuestPro 5.1.1 software (BD Biosciences). The following antibodies were used: rat anti-mouse CD16/32 blocking Ab (clone 2.4G2) obtained from BD Biosciences ; rat anti-mouse TCR-β-FITC (H57–597), CD8a-PE (53–6.7), CD4-APC (GK1.5), and PE-conjugated rat IgG2a or IgG2b isotype Ab controls purchased from eBiosciences .Flow cytometry was performed as previously described , 25. BriThe total RNA from the RPE/Choroid tissues of laser injury eyes were extracted using TRIzol reagent (Invitrogen). The A260/A280 ratio of all RNA samples was >2.0, as measured by Nanodrop. Double-stranded cDNA was reverse-transcribed using random primers and the SuperScript VILO cDNA synthesis kit (Invitrogen).Taq polymerase at 95°C, followed by 40 cycles of amplification . The comparative threshold cycle (CT) method normalized to Gapdh was used to analyze relative changes in gene expression. The qPCR primer sequences for Gapdh have been published elsewhere [Vegfa (ID 6678563a1) andCcl2 (ID 141803162c1) were designed based on the online PrimerBank database .Real-time qPCR was performed in a SYBR green-based PCR reaction mixture on a MX3005p system , programmed with a 10-minute initial hot-start activation of lsewhere . The prihomogeneous TPCA-1-loaded PLGA microparticles were generated with an average diameter of ~2.4 μm  TPCA-1 by HPLC with UV detection at 310 nm. Encapsulation was further confirmed by directly checking the amount of TPCA-1 loaded into microspheres by dissolving the polymer complexes in 100% ethanol. Our preliminary study shows that TPCA-1 encapsulation efficiency was approximately 80%  under thin vivo, TPCA-1-loaded PLGA (10 mg of microparticles loaded with 0.8 mg TPCA-1 suspended in 100 μL PBS) microparticles were unilaterally retrobulbar-injected into C57B/L6 mouse eyes using a 30-gauge needle. Mice were euthanized at 1, 7, 14, or 21 days after injection, and the TPCA-1 level in the treated and untreated homogenized retinal lysate was determined by HPLC after extraction with dichloromethane. A constant release of TPCA-1 was measured in the retinas as late as 21 days after TPCA-1-loaded PLGA retrobulbar injection  AMD contributes to only 10% of all AMD afflictions, it accounts for 90% of the severe vision loss caused by AMD. The current treatment for wet AMD is blocking VEGF activity in the eye to reduce CNV leakage and growth. However, a majority of patients are unresponsive to the treatment, and CNV may progress even during the treatment –38. DeveAs a chronic retinal disease with a slow, progressive pathogenic process leading to choroid neovascularization and consequent blindness, AMD requires long-term treatment with anti-angiogenic molecules. The development of a drug delivery system that reduces the frequency of operational intervention and drug dose is in demand. Microparticles provide an alternative to frequent intravitreal or periocular injection, as they release the drug sustainably for several weeks or months.In the present study, the potential for sustained release of TPCA-1 into retina using PLGA microparticles as therapeutic drug carriers in the treatment of laser-induced CNV was investigated. The biodegradable PLGA microparticles gradually degrade and release the packed drug until degradation is completed. Microparticles offer higher drug loading and a longer time for drug release than nanoparticles, which reduces administration frequency and makes microparticle a better option. In general, the higher the homogenization speed and/or the ratio of internal PLGA to polyvinyl alcohol are used, the smaller the microsphere is generated. By adjusting the PLGA molecular weight and polymer size, high encapsulation efficiency, low initial release, and sustained second-phase release (more than 1 month) can be achieved. This result suggests that small amount of TPCA-1 is progressively released from the carrier microparticles. Importantly, our results also show that TPCA-1 reaches the subretinal space after it is released from particles injected into the retrobulbar space. This indicates that TPCA-1-loaded PLGA can be injected periocularly, which will reduce the risks associated with intraocular injection. TPCA-1/PLGA retrobulbar treatment not only resulted in a significant reduction of laser-induced CNV formation, but also effectively halted the recruitment of macrophages into laser lesions at 24, 48, and 72 hours post laser injury.Ikk2 in the retina does not cause detrimental effects to retinal development and vision function [Safety is a major concern when tampering with NF-κB signaling, as this pathway plays key roles in many biological processes, particularly in innate immunity and cell survival –16. The function . RPE celfunction . MicropaIn summary, the IKK2 inhibitor TPCA-1 can be slowly released into the posterior segment of the eye from microparticle carriers injected into the retrobulbar space and prevent laser-induced CNV formation, which indicates its potential for treatment of wet AMD and other retinal vascular disorders using this inhibitor. This finding also suggests that local IKK2 inhibition for treatment of other chronic and genetic angiogenesis diseases should be reconsidered.


Pandemic influenza A(H1N1)pdm09 emerged in Thailand in 2009. A prospective longitudinal adult cohort and household transmission study of influenza-like illness (ILI) was ongoing in rural Thailand at the time of emergence. Symptomatic and subclinical A(H1N1)pdm09 infection rates in the cohort and among household members were evaluated.A cohort of 800 Thai adults underwent active community-based surveillance for ILI from 2008–2010. Acute respiratory samples from ILI episodes were tested for A(H1N1)pdm09 by qRT-PCR; acute and 60-day convalescent blood samples were tested by A(H1N1)pdm09 hemagglutination inhibition assay (HI). Enrollment, 12-month and 24-month follow-up blood samples were tested for A(H1N1)pdm09 seroconversion by HI. Household members of influenza A-infected cohort subjects with ILI were enrolled in household transmission investigations in which day 0 and 60 blood samples and acute respiratory samples were tested by either qRT-PCR or HI for A(H1N1)pdm09. Seroconversion between annual blood samples without A(H1N1)pdm09-positive ILI was considered as subclinical infection.The 2-yr cumulative incidence of A(H1N1)pdm09 infection in the cohort in 2009/2010 was 10.8% (84/781) with an annual incidence of 1.2% in 2009 and 9.7% in 2010; 83.3% of infections were subclinical (50% in 2009 and 85.9% in 2010). The 2-yr cumulative incidence was lowest (5%) in adults born ≤1957. The A(H1N1)pdm09 secondary attack rate among household contacts was 47.2% (17/36); 47.1% of these infections were subclinical. The highest A(H1N1)pdm09 secondary attack rate among household contacts  occurred among children born between 1990 and 2003.Subclinical A(H1N1)pdm09 infections in Thai adults occurred frequently and accounted for a greater proportion of all A(H1N1)pdm09 infections than previously estimated. The role of subclinical infections in A(H1N1)pdm09 transmission has important implications in formulating strategies to predict and prevent the spread of A(H1N1)pdm09 and other influenza virus strains. Pandemic influenza A(H1N1)pdm09 emerged in North America in March 2009 and rapidly spread to other parts of the world, eventually replacing previous seasonal H1N1 virus strains. Several studies from different countries established a pattern of higher A(H1N1)pdm09 infection rates in school-age children relative to young adults, with older adults having the lowest infection rates In Thailand, like other tropical countries, influenza activity tends to occur in a biphasic seasonal pattern (June to August and January to March) with sporadic infection throughout the year At the time of pandemic onset in 2009, a prospective longitudinal cohort study was ongoing in Kamphaeng Phet, Thailand in which 800 adults (age range 20–85 years) were undergoing active surveillance for influenza-like illness (ILI) from 2008 to 2010. The study also included a household transmission component triggered by confirmed influenza virus (IFV) infections in the cohort The study was approved by the institutional review boards of the Thai Ministry of Public Health (MOPH), Walter Reed Army Institute of Research (WRAIR), Naval Medical Research Unit No. 2 (NAMRU-2), US Army Medical Research and Materiel Command, University of Iowa, and University of Florida. Written informed consent was obtained from all study subjects (or their parents if applicable) and assent was obtained from children older than seven years.Although the study was designed to focus chiefly upon studying subclinical avian influenza infections, reviewing IRBs approved the study to include all influenza A infections.Approximately 800 adult subjects living in Kamphaeng Phet province (KPP), Thailand, a rural province located about 350 km north of Bangkok, were enrolled in a prospective longitudinal cohort study. Details of study methods have been previously published Annual follow-up visits at 12 and 24 months after enrollment were performed in 2009 and 2010 during which clinical questionnaires were administered and blood samples were obtained. The questionnaires assessed changes in demographic or health status during the preceding year. Serological testing of the enrollment and annual follow-up blood samples was conducted by influenza HI to monitor for changes in influenza antibody titers over time. An IFV infection was considered to be subclinical if IFV HI seroconversion occurred between annual blood samples without an IFV-positive ILI detected during the intervening surveillance period.When a cohort subject developed an ILI in which an acute respiratory sample tested positive for influenza A by qRT-PCR, that cohort subject’s household members ≥6 months old were enrolled in a household transmission investigation. A blood sample was collected at enrollment (day 0) from each household member . If a contact subject developed ILI during the following 60 days, acute respiratory samples  were additionally collected from that contact subject for qRT-PCR for influenza A and B. A questionnaire was administered to each contact subject to assess the degree of contact with the cohort subject and identify any illnesses during the preceding 7 days. Weekly follow-up visits were conducted with the contact subjects for 60 days after initiation of the household investigation. At day 60, a convalescent blood sample was collected from each contact subject.Blood samples were transported at room temperature and respiratory swabs were transported in Micro Test M4RT Viral Transport Media  at 4°C within the same day to the field laboratory at Kamphaeng Phet-AFRIMS Virology Research Unit (KAVRU) in KPP, Thailand, where qRT-PCR for IFV was performed. Serological testing by HI was performed at the University of Florida Global Pathogens Laboratory in Gainesville, Florida, USA.Enrollment and annual follow-up serum samples were tested using the World Health Organization (WHO) or US Centers for Disease Control and Prevention (CDC) HI protocol as previously described The procedures have been previously described Infection with A(H1N1)pdm09 was considered to have occurred if (a) a respiratory sample was positive for A(H1N1)pdm09 by qRT-PCR, or (b) a 4-fold or greater increase in HI titer (seroconversion) against A(H1N1)pdm09 occurred between paired annual sera or paired ILI sera, with a second titer of at least 1∶40. If paired sera were not available, a single high titer of at least 1∶40 was considered to indicate recent infection.Risk factors for bivariate associations with HI results were initially examined using binary logistic regression and proportional odds modeling Between April and October 2008, field staff enrolled a total of 800 adults (100 from each of 8 sites). Details of participant demographics at enrollment have been previously described Seroprevalence of elevated HI antibodies against A(H1N1)pdm09 increased from 1.7% and 1.3% in 2008 and 2009, respectively, to 10.3% in 2010 . Based oFrom January 2009 to December 2010, 53 cohort subjects met the case definition for ILI; 10 of these subjects were confirmed to have A(H1N1)pdm09 infection by qRT-PCR. Most  of these subjects were born after 1957. Prior to ILI occurrence, none of the 10 subjects had pre-existing A(H1N1)pdm09 antibody. Only 3 of these subjects had A(H1N1)pdm09 seroconversion in the paired ILI sera. Among the remaining 7 subjects with A(H1N1)pdm09-positive qRT-PCR, 5 were seronegative in both of the paired ILI sera and 2 did not have sufficient paired sera for HI testing.When comparing A(H1N1)pdm09 seroconversions from the enrollment and annual follow-up blood samples and the qRT-PCR data from ILI acute respiratory samples, 5 subjects had A(H1N1)pdm09 infection by both qRT-PCR and HI, 5 by qRT-PCR only, and 74 by HI alone. Among these 84 cohort subjects with A(H1N1)pdm09 infection, only 14 subjects reported ILI . Most  of the A(H1N1)pdm09 infections among the cohort subjects were subclinical. The proportion of subclinical infections among cohort subjects born after 1957 was higher than those born ≤1957 .Using binary logistic regression analysis, several covariates were associated with an increased risk for seroconversion against A/Mexico/4108/2009(H1N1) . Cohort Thirty-six contact subjects (age range 1–70 years) living in the same household as the 10 cohort subjects with A(H1N1)pdm09-positive qRT-PCR were enrolled. Paired day 0 and 60 sera from these contact subjects were evaluated by HI against A(H1N1)pdm09 [A/Mexico/4108/2009(H1N1)]; three contact subjects missed their day 60 blood draw. Of the 36 contact subjects, 6 had ILI of which all were A(H1N1)pdm09-positive by qRT-PCR . Five ofOur report is one of the few combined prospective longitudinal cohort and household transmission studies conducted during the A(H1N1)pdm09 pandemic that describes the epidemiology of A(H1N1)pdm09 in a tropical region. The proportion of A(H1N1)pdm09 infections in our cohort that were subclinical was very high (83.3%). This proportion was higher than some prior estimates (approximately 50%) from a few comparable studies Cohort subjects born ≤1957 (age ≥52 years) were less likely to have A(H1N1)pdm09 seroconversion, comparable to the findings from other studies The cumulative incidence of A(H1N1)pdm09 infection by qRT-PCR and HI seroconversion in the cohort from January 2009 to December 2010 was 10.8% with an annual incidence of 1.2% and 9.7% in 2009 and 2010, respectively. Previous studies estimated the incidence of A(H1N1)pdm09 infection among adults to range from 6% to 13% during the early pandemic (May to September 2009) The A(H1N1)pdm09 secondary attack rate (SAR) among household members of cohort subjects was 47.2% . Previous A(H1N1)pdm09 household transmission studies conducted during the early pandemic (April to August 2009) reported a wide range (4% to 45%) of SARs Our study had some limitations. Only adults over 20 years of age were enrolled in the cohort. Since children are at high risk of A(H1N1)pdm09 infection Our study was unique in evaluating the 2009 influenza pandemic in several ways. First, it combined a prospective longitudinal cohort study undergoing active ILI surveillance with a household transmission component. Second, it was already fully operational at the outset of the pandemic. Third, it continued operating for the full duration of the pandemic. Keeping these aspects of the study in mind, our results demonstrate that subclinical A(H1N1)pdm09 infection occurred frequently among Thai adults in this rural population. The role of these subclinical infections in A(H1N1)pdm09 transmission has important implications in formulating strategies to predict and control the spread of A(H1N1)pdm09 and other IFV strains. For instance, if a large portion of viral infections are subclinical, hospital-based surveillance will not well-represent a population’s influenza ecology. Hence, public health officials should consider these large numbers of subclinical infections in developing future influenza surveillance and control programs. The effort to capture subclinical data need not be as extensive and expensive as this cohort study. For example, subclinical infection data might be collected through modest household transmission studies, where secondary attack rates  among persons in the same household as index cases may be used as a surrogate for general population data.


Upon intercalation, transport measurements reveal a change in the carrier type from n- to p-type, accompanied by a more than three-fold increase in carrier mobility, up to μh ≈ 4540 cm2 V−1 s−1. On a local scale, Kelvin probe force microscopy provides a complete and detailed map of the surface potential distribution of graphene domains of different thicknesses. Rearrangement of graphene layers upon intercalation to (n + 1)LG, where n is the number of graphene layers (LG) before intercalation, is demonstrated. This is accompanied by a significant increase in the work function of the graphene after the H2-intercalation, which confirms the change of majority carriers from electrons to holes. Raman spectroscopy and mapping corroborate surface potential studies.We investigate the local surface potential and Raman characteristics of as-grown and  In the present work graphene growth is achieved by the chemical vapor deposition (CVD) method. In this method uniform graphene films are grown by carbon deposition, introduced by a hydrocarbon source, on the Si(0001) side of the SiC wafer778102-intercalated graphene78102-intercalation on the local electronic and structural properties of the QFSG. The verification of number of graphene layers was achieved using Raman spectroscopy and mapping, whereas a detailed image of the surface potential of the layer structure was constructed using frequency-modulated Kelvin probe force microscopy (FM-KPFM)n + 1)LG, where n is the number of graphene layers (LG) before intercalation). This is accompanied by a considerable increase of work function upon intercalation, which is evidence for the change of the carrier type from electrons to holes with the Fermi level straddling either side of the Dirac point as a function of H2-intercalation.While several groups have investigated the structural properties and electronic band structure of Hne ≈ 1.8 × 1012 cm−2 and μe ≈ 1370 cm2 V−1 s−1, respectively.Based on the van der Pauw measurements on the as-grown sample, the carrier concentration and electron mobility were determined as –1–1). Moreover, the 2D peak at the terrace edge is blue-shifted  towards higher wavenumbers by ~33 cm–1 compared to 1LG  is much broader (FWHM = 75 cm–1) than that of 1LG and 2LG, possibly indicating the presence of 3LG. There are some reports in the literature showing that fitting the line shape of graphene with 6 Lorentzian components is an indication of 3LGTo investigate the layer structure of the as-grown graphene sample, Raman spectroscopy and mapping were employed . G peak d to 1LG . This pe−1) within the terrace are visible in −1) have been measured and presented in 2015202, using these studies as a reference to determine the doping and charge inhomogeneities in CVD graphene on SiC would be inaccurate, since the interactions between graphene and supporting substrate are different. Thus, the combination of strain and charge carrier coupling may be the origin of the fluctuations of 2D and G peak positions on the terracesSome small variations of the 2D peak shift  between 1LG and 2LG to be ΦTip = 4.52 ± 0.05 eV . The decrease in work function (increase of electron carrier concentration) with increasing number of layers confirms the n-type character of the as-grown sample. Having correlated the Raman characteristics with the surface potential maps, it is then possible to conclude that the SiC terraces are indeed covered with 1LG, whereas thicker graphene (2–3LG) grows at terrace edges, resulting in lower local work function.For the as-grown sample, we report the difference in the surface potential  was measured using Hall effect in the van der Pauw geometry, where the hole carrier concentration and mobility are nh≈1.5 × 1013 cm−2 and μh ≈ 4540 cm2 V−1 s−1 (i.e. more than three times greater than the as-grown sample), respectively. The transformation of the graphene from an electron to hole-doped material is a fingerprint for the successful intercalation of the sample.The –1). In addition, the peak is broader with a FWHM of 58 cm–1 15–1 and can be fitted with six Lorentzians2-intercalation, we prove that the IFL, which was under the 1LG in the case of the as-grown sample, is now transformed into the new first graphene layer. This results in the general rearrangement of the graphene layers to (n + 1)LG, where n is the number of layers before intercalation.−1  is still observed . Compare2-intercalation on the local electronic properties of QFSG. The map of the surface potential and the relevant histogram  has also been examined experimentally and theoretically9With the annealing of the sample in hydrogen environment at temperatures around 1100 °C, the Hex-situ H2-intercalation of CVD grown graphene on 4H-SiC(0001) using bulk transport, local surface potential and Raman spectroscopy and mapping. Transport measurements on the as-grown sample demonstrated n-type doping with μe ≈ 1370 cm2 V−1 s−1. Following the ex-situ intercalation, the graphene conductivity switched to p-type, which along with a significant increase in mobility, μh ≈ 4540 cm2 V−1 s−1, is indicative of successful intercalation. The FM-KPFM measurements of the as-grown sample revealed that SiC terraces were covered by predominantly 1LG and additionally decorated with 2LG islands and 2LG/3LG edges. The work function measurements also indicated an increase in the electron concentration (i.e. decrease in work function) as the number of layers increased. On the contrary, the H2-intercalated sample exhibited increase of the work function as the number of graphene layers increased. This is the ultimate proof that the Fermi energy crossed the charge neutrality point from n- to p-type upon intercalation. In addition, for the first time a high-resolution image of the surface potential of intercalated graphene was constructed, which provided a detailed understanding of the layer structure and its transformation upon decoupling from the substrate. The Raman studies proved that upon intercalation the 1LG has been transformed into 2LG and, in general, the as-grown layers (n) have been transformed into (n + 1)LG, as followed by conversion of the IFL into 1LG.We investigated the effects of Thus, using local, layer-resolving techniques, we demonstrate successful transformation of graphene covalently bound to the substrate into QFSG with superior electronic properties. QFSG is one of the preferable candidates for high-speed electronics, as the decoupling of the IFL from the SiC substrate increases the mobility dramatically, while maintaining the excellent intrinsic electronic and topographic structure.H-SiC (0001) substrates (Cree) 10 × 10 mm2 size were cut out from 4” wafer and etched in hydrogen at 1600 °C prior to the epitaxy process. Graphene growth was controlled by Ar pressure, Ar linear flow velocity and reactor temperature. The process relies critically on the creation of dynamic flow conditions in the reactor, which control Si sublimation rate and enable the mass transport of hydrocarbon to the SiC substrate. Tuning the value of the Reynolds number enables formation of an Ar boundary layer, which is thick enough to prevent Si sublimation and allowing diffusion of hydrocarbon to the SiC surface, followed by CVD growth of graphene on the SiC surface. The ex-situ intercalation of hydrogen on the same sample was achieved by annealing the sample in molecular hydrogen at temperature of 1100–1200 °C and reactor pressure of 900 mbar. Cooling down in H2 atmosphere keeps hydrogen atoms trapped between graphene and substrate. Prior to unloading the sample, the process gas was changed back to argon6For this study, graphene samples were grown by CVD method at 1600 °C under an argon laminar flow in an Aixtron VP508 hot-wall reactor. Semi-insulating on-axis oriented 4ex-situ hydrogen intercalated samples were characterized using Hall effect measurements in the van der Pauw geometry in ambient conditions.The mobility and carrier concentration of the as-grown and 2 were obtained using a Horiba Jobin-Yvon HR800 system in order to investigate the structure of graphene samples. The 532 nm wavelength laser (5.9 mW power) was focused through a 100 × objective lens onto the graphene sample. The spectral resolution was 1.59 cm–1. The Raman spectra were initially obtained for a reference SiC substrate, which is then used to subtract the substrate related signal, allowing effective separation of the Raman peaks originating only from the graphene. The Raman maps were constructed by mapping G and 2D peak intensity, shift and FWHM of 3025 individual spectra with XY resolution of 0.2 μm. The G peak (~1582 cm–1) originates from the first order scattering process due to the double degenerate phonon mode vibrations at the center of Brillouin zone28–1) originates from the double resonance scattering process near the K point. The 2D peak exhibits a dispersive behavior. A characteristic feature of increasing number of graphene layers on SiC is the blue shift of the 2D peakAB stacking) and six Lorentzians, respectively28Raman maps of 10 × 10 μm G peak ~82 cm–1 o G peak ~82 cm–1 ok ≈ 0.4–1.2 N m−1 were used in single-pass tapping mode. In FM-KPFM, the cantilever is oscillating at its mechanical resonant frequency f0 ≈ 300 kHz, in addition to a much lower AC frequency voltage, fmod ≈ 5 kHz and Vmod ≈2-5 V, to induce a frequency shift (f0 ± fmod). The f0 ± fmod side lobes are monitored by a PID feedback loop, which applies a compensation DC voltage to minimize the side lobes, thus acquiring the surface potential map. Since FM-KPFM detects the force gradient using the frequency shift, it can achieve spatial resolution of <20 nm, which is limited only by the tip apex diameter3234UCPD)] with great accuracy for the number of graphene layers. To further investigate the surface potential of graphene samples, the work function of the PFQNE-AL tip (ΦTIP) was calibrated against a gold reference sample using the approximation ΦTip ≈ ΦAu + eUCPD .