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


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


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


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


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


Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Candida albicans and Aspergillus niger. During simulation relative humidity under the facepiece increased after 7 min of usage to 84%–92% and temperature increased to 29–30 °C. S. aureus survived the best on filter materials with 40%–200% moisture content. A decrease in survivability was observed for E. coli and C. albicans when mass humidity decreased. We found that B. subtilis and A. niger proliferated for 48–72 h of incubation and then died regardless of the moisture content. In conclusion, our tests showed that the survivability of microorganisms on filter materials depends on the amount of accumulated moisture and microorganism type.Bioaerosols are common biological factors in work environments, which require routine use of filtering respiratory protective devices (FRPDs). Currently, no studies link humidity changes in the filter materials of such devices, during use, with microorganism survivability. Our aim was to determine the microclimate inside FRPDs, by simulating breathing, and to evaluate microorganism survivability under varying humidity conditions. Breathing was simulated using commercial filtering facepiece respirators in a model system. Polypropylene melt-blown nonwoven fabrics with moisture contents of 40%, 80%, and 200%, were used for assessment of microorganisms survivability. A modified AATCC 100-2004 method was used to measure the survivability of ATCC and NCAIM microorganisms:  Biological factors constitute a very important problem of occupational medicine and public health. The threat posed by biological agents is increasingly seen as a global issue, mainly due to the rapid spread of microorganisms over time, and across work and living environments due to modern pace of work and life. It is estimated that, at least, several hundred million people worldwide are exposed to these factors in processes related to their work environment ,2. This In the work environment, biological agents occur most often in the form of bioaerosols. Therefore, it is common to use personal protective equipment (PPE), particularly, disposable and reusable filtering respiratory protective devices (FRPDs). The lack of adequate protection of the respiratory system in workers exposed to biological agents can lead to asthma, allergic alveolitis, organic dust toxic syndrome (ODTS), byssinosis, chronic bronchitis, allergic rhinitis, irritation of mucous membranes, sick building syndrome (SBS), and certain infectious diseases and cancer ,24,25,26There are many theories and studies on the selection of FRPDs in relation to biological hazards ,28,29. Ii.e., biocidal properties of filter materials for the construction of reusable FRPDs. The materials are designed to protect against proliferation of microorganisms, deposited on them during FRPD use, so that the respirator itself is not a source of infection for the user. Furthermore, it is important to carry out studies on the survivability of microorganisms on filter materials under conditions similar to those prevailing in the work environment where FRPDs are typically used. This makes it possible to adequately evaluate the extent to which FRPDs protect against bioaerosols in the “worst-case” scenario.Previous studies have tested the survivability of microorganisms on filter materials, which constitute the basic structural material of FRPDs ,37,38,39Currently, no studies link humidity changes occurring in filter materials during FRPD use with the survivability of the deposited microorganisms. Therefore, the aim of this study was to determine the microclimate inside the FFRs during simulated breathing, and to evaluate the survivability of different types of microorganisms deposited on the filter materials of the FRPDs under varying humidity conditions.2 ± 5% and 80 g/m2 ± 5%, the three middle layers of nonwoven fabric had a total surface weight of 90 g/m2 ± 15% and the inner layer of nonwoven had a surface weight of 17 g/m2 ± 5%.The level of humidity in the FRPDs during breathing cycles was measured using commercial reusable FFRs, consistent with the requirements of a harmonized standard  under EU3 ± 10%. To test the survivability of microorganisms in the FRPDs, polypropylene melt-blown nonwoven fabrics were used, as they were considered responsible for catching harmful microorganisms from a stream of contaminated air. Commercially-produced fabrics were used to construct the above FFRs. On average, the surface weight of each nonwoven fabric sample was 30 g/mThe test stand used to simulate breathing in our experiments is shown in The test stand consisted of a breathing machine, a Sheffield dummy head on which an FFR was mounted, and a saturator located in the exhalation line between the dummy head and the breathing machine. The breathing machine was set at 25 cycles/min and 2 L/stroke, which corresponded to 25 inhalations and 25 exhalations per minute with respiratory volume of 2 liters. The airflow pattern of the breathing machine was sinusoidal. The saturator was set at a temperature exceeding 37 °C, so that the temperature of the air exhaled from the dummy head’s mouth, after passing through the system, was 37 ± 2 °C. The relative humidity and temperature of the environment was monitored in the respiratory space between the dummy head and the tested FFRs. Integrated temperature and humidity data logger 1-Wire® Hygrochron™  was used. The temperature and relative humidity of the environment were 23–25 °C and 47%–55%, respectively.i.e., potential maximum protection factor calculated using the maximum percentage inward leakage permitted for FFRs of third protection class according to European standards [The study was conducted according to the following procedure: an FFR was placed on the dummy head, the simulation of breathing was carried out for 3.5 h, the FFR was taken off the dummy head, a one-hour break in the use of FFR was simulated , the FFR was again placed on the dummy head, the simulation of breathing was carried out for another 3.5 h, and the FFR was taken off the dummy head. Two series of simulations were performed. Each series was conducted using a new FFR. The accuracy of fit was based on the determination of a nominal protection factor, tandards . HoweverEscherichia coli 10536, Staphylococcus aureus 6538, Candida albicans 10231, Aspergillus niger 16404, and one, Bacillus subtilis 01644, was obtained from the National Collection of Agricultural and Industrial Microorganisms (NCAIM). The strains were selected based on their taxonomic classification . The strains were also characterized by their varying survivability in the environment i.e., whether they produced endospores (B. subtilis), spores (A. niger), or just vegetative cells . Moreover, according to Directive 2000/54/ EC [The study involved testing five strains from pure culture collections. Four were obtained from the American Type Culture Collection (ATCC), 0/54/ EC , the speThe microorganisms were stored on agar slants: bacteria on TSA , and molds and yeasts on MEA  at a temperature of 4 °C. Bacteria were activated in TSB medium  and fungi in MEB medium  at a temperature of 30 °C for 24 h (bacteria), 27 °C for 24 h (yeasts) and 72 h (moulds).®80. The number of microorganisms in the inoculum was determined using the plate count method and a Thom chamber. The average density of the suspension of microorganisms ranged from 1.3 × 108 to 1.3 × 1010 cfu/mL (bacteria) or from 3.9 × 107 to 6.5 × 107 cfu/mL (fungi). Verifying the density of the inoculum in the Thoma cell counting chamber, in the inoculum approximately 20% of all microorganism cells constituted to B. subtilis spores and 99% to A. niger spores. Removal efficiency of the tested microorganisms was 52.2% ± 4.1%.A microorganism inoculum was prepared. Bacteria and yeast colonies were transferred into 10 mL of TSB and MEB medium, respectively, and incubated in conditions as described above. In the case of mold, colonies from MEA slants were washed using 10 mL distilled water with 0.01% of TweenThe survivability of microorganisms on filter materials was measured using a modified quantitative method, AATCC 100-2004  with the2 each. Three levels of the mass humidity of the materials  were determined. Humidity levels of 40% and 80% were determined according to the results of the breathing simulation and corresponded to the estimated humidity of the filter material during a break in the usage of respirators and during breathing with a half mask. Humidity 200% was chosen as a model of very high humidity of the filter material (worst-case scenario). Three levels of the mass humidity of the materials were determined by applying sterile distilled water onto the samples. Sterile distilled water was applied homogenously onto the samples using a pipette with sterile tips for the distribution of small water droplets over the whole sample. Mass humidity (H), defined as the amount of moisture accumulated in the filter material, was determined by a gravimetric method  and calculated using the formula:2 (average mass mm = 0.0125 g).The tests were conducted for samples of filter materials with a surface area of 4 cmNext, 10 µL of standardized inoculum of microorganisms was applied evenly in the same way using pipette with sterile tips. Filter material samples were prepared in two independent repetitions of the experiment and placed in a climatic chamber  at 28 °C and relative humidity RH of 80%. The samples were collected at 0 (immediately after inoculum application) 8, 24, 48, 72, and 120 h.0 to 105 in three repetitions). This resulted in a 30 repetitions for one microorganism, for each humidity level and for each incubation time.The samples were then placed in 50 mL of sterile saline (0.85% NaCl) and shaken for 10 min  to wash out the microorganisms from the tested materials. Bacteria were incubated at 30 °C for 24–48 h on TSA medium and fungi on MEA medium at 27 °C for 3–5 days. The number of microorganisms (cfu/sample) was determined in two independent experiments using the plate count method ; N0: at time t = 0 h; tN: after a certain period of incubation .The survival rate of microorganisms on the filter material after different incubation periods (S) was determined using the formula:p < 0.05. All data were analyzed using the statistical software program Origin 6.1.The arithmetic mean and standard deviation for the number of microorganisms on the surface of the tested materials were calculated. Differences between the number of microorganisms after a particular incubation time and the control samples were analysed using one-way analysis of variance (ANOVA). Differences were considered significant at The results of temperature measurements performed directly under the facepiece are shown in In the first and second series of simulated breathing cycles, a microclimate with similar temperature was created inside the FFRs. After one hour of breathing, the temperature in the FFR under the facepiece reached 29–30 °C. Differences between the two series were not statistically significant. The temperature inside the FFR was stable and lower than the normal body temperature of a healthy worker. The microclimate temperature inside the FFR was only slightly affected by breaks in the use of the equipment.The values of relative humidity in the space directly under the facepiece are shown in The values of relative humidity inside the FFR microclimate obtained during the two series of simulations show that the start of simulation is followed by an immediate sharp increase in relative humidity up to 84%–92% (first and second series) inside the respirator. The relative humidity within the entire simulated breathing time ranged from 80% to 92%. When the simulation of breathing was started again after a one-hour break, the relative humidity inside the FFR increased again to 91% (first series) and 69% (second series). The difference in relative humidity changes inside the FFR (22%) between both series may result from the course of moisture condensation on the surface of the filter materials, and the rate of migration of the collected liquid to various structural layers of the FFR. This can be attributed to differences in the production of particular FFRs and in head adjustments in the successive phases of series I and II. The phenomena accompanying moisture deposition on filter materials were described by Raynor and Leith . They foThe amount of water accumulated on the filter materials has a strong impact on the pressure drop on these materials (breathing resistance) ,49,50. TBoth, literature data and results of the breathing simulations in the FFR confirm the fact that the conditions prevailing inside the respirators are favorable for the continuous gathering of moisture from the exhaled air. This study shows that it is maintained as long as the FFR is used even if its operation is interrupted. These conditions may affect the survivability of microorganisms deposited on the filter materials during the use of FFR at the workplace and during breaks.S. aureus and B. subtilis under the three mass humidity conditions, for E. coli at a mass humidity of 80%, and for C. albicans at a mass humidity of 80% and 200%. In the following incubation times, a decrease in the number of E. coli cells was observed compared to the number of cells deposited on the material at the time t0. The number of cells of the remaining bacteria and yeast increased significantly within 24 h—five days of incubation relative to t0. In general there were no statistically significant changes in the numbers of the mold A. niger in all tested incubation times and mass humidity conditions.The number of microorganisms on the tested nonwoven fabric after eight hours of incubation varied significantly for The dynamics of changes in the number of microorganisms in subsequent incubation times on the tested filter material is expressed as the survival rate. This parameter depending on the accumulated moisture in the filter materials is shown in Staphylococcus aureus  argue that storing respirator filters in high humidity conditions results in heavy microbial contamination, particularly when the filter material is biodegradable. Filters exposed to microorganisms present in an agriculture barn and in a wastewater treatment plant were stored at 98% RH for up to 35 days and tested. After the storage, the number of bacteria including actinomycetes was 1–3 orders of magnitude higher, and the concentration of fungi was larger or smaller, depending on the tested respirator filter [et al. (1994) indicate a strong correlation between the visual growth of mold Stachybotrys chartarum and cellulose content in filters at RH 100%. It is also shown that at high humidity, S. chartarum is capable of producing mycotoxins on the respirator filter [Pasanen r filter . In other filter .et al. [Micrococcus luteus and Escherichia coli declined within one hour after collection on ventilation filter media when no nutrients were present and the test was performed at low relative humidity (30%–60%). The study also suggests that bacteria cannot generally use modern filter materials as nutrients. For surviving, the bacteria use the more organic substances contained in the filtered air, coming from the user of the filtering device.The effect of humidity on survivability of microorganisms on the filter material was confirmed by Maus et al.  who demoet al. [Bacillus subtilis and Pseudomonas fluorescens. The bacteria were aerosolized and loaded on the respirator filters under three nutritional conditions: water, saliva, and TSB with 0–13 days of incubation. The authors showed that P. fluorescens cells couldn’t survive for more than three days on the respirator filter, while B. subtilis spores can remain viable for more than thirteen days. Wang et al.  showed tet al. [B. subtilis subsp. niger and Staphylococcus epidermidis showed high survivability and Mycobacterium abscessus demonstrated lower survivability.Brosseau et al.  investigE. coli (0%–0.1%) (depending on the moisture content) and A. niger (46%–80%). The highest survival rate was observed for bacteria S. aureus . As for other microorganisms, the rate ranged from 483% to 1632%  can be collected from the filter materials. Therefore, it is important to study the whole microorganism population directly from the material sample. In this way we can assess the biodiversity of used FFRP at workplaces.et al. [Johnson et al.  studied The results obtained by breathing simulation presented in this paper indicate that biocides that exhibit antimicrobial efficacy even at a moisture content of 40% should be used in reusable FFRs, practically from the very start of their use. S. aureus and B. subtilis, and the yeast C. albicans for five days. The environment, however, is not favorable for the development of E. coli and mold A. niger. This indicates that in workplaces where FFRs are used to protect workers against bioaerosols is necessary to develop and comply with appropriate rules for limited re-use and storage of FFRs, such as those defined in the guidance for healthcare institutions to protect health care workers from risks of exposure to infectious respiratory illnesses [The simulation of breathing shows that moisture content and temperature after 7 min of usage increased to 84%–92% and 29–30 °C, respectively. This condition is maintained as long as the FFR is used. This points to the fact that even with short-term use of the FFR favorable conditions for rapid growth of microorganisms deposited on the filter material can occur. The survivability of microorganisms on the filter material depends on the microorganism species and moisture content. At mass humidity ranging from 40% to 200%, the filter material is an appropriate environment for increase of survivability of llnesses . It is p


Single-Si-nanowire (NW)-based DNA sensors have been recently developed, but their sensitivity is very limited because of high noise signals, originating from small source-drain current of the single Si NW. Here, we demonstrate that chemical-vapor-deposition-grown large-scale graphene/surface-modified vertical-Si-NW-arrays junctions can be utilized as diode-type biosensors for highly-sensitive and -selective detection of specific oligonucleotides. For this, a twenty-seven-base-long synthetic oligonucleotide, which is a fragment of human DENND2D promoter sequence, is first decorated as a probe on the surface of vertical Si-NW arrays, and then the complementary oligonucleotide is hybridized to the probe. This hybridization gives rise to a doping effect on the surface of Si NWs, resulting in the increase of the current in the biosensor. The current of the biosensor increases from 19 to 120% as the concentration of the target DNA varies from 0.1 to 500 nM. In contrast, such biosensing does not come into play by the use of the oligonucleotide with incompatible or mismatched sequences. Similar results are observed from photoluminescence microscopic images and spectra. The biosensors show very-uniform current changes with standard deviations ranging ~1 to ~10% by ten-times endurance tests. These results are very promising for their applications in accurate, selective, and stable biosensing. Silicon nanomaterials widely known as one of the most biocompatible materials have been successfully applied to various kinds of bionics fields such as imaging cells, biosensors, cancer theranostics, bio-integrated electronics, and drug deliveries1234567910111213141112Recently, there have been several reports on field-effect-transistor (FET)-based electrochemical DNA sensors employing conducting polymers and single planar Si NW91114Here, we first report remarkable sensitivity and selectivity of the graphene/surface-modified-Si-NWs vertical-junction diodes in detecting the hybridization of oligonucleotides. Conventional nucleic-acid-based diagnostics by using real-time polymerise-chain-reaction facilities require large volume of space for set up, which makes it impossible for the technique to be realized into into portable devices and nano-scale information processing systems. In contrast, the approach for DNA diognostics by employing graphene/Si-NWs biosensors can extremely reduce them to be portable and integrable.The principal processes and mechanism for the selective and precise DNA detection by graphene/Si-NWs vertical-junction diodes are described as follows. A probe sequence  and a target sequence, which is complementary to the probe , and dummy single strand oligonucleotide (tct tgc aca agt tta aga ggg aaa gga) (d-ss oligonucleotide) are employed in this workρ = 1∼10 Ω cm) before MaCE. The Au film-loaded p-type Si substrate was immersed in a mixture solution of hydrofluoric acid (HF) and hydrogen peroxide (H2O2)  for around 3 min at room temperature (RT) to produce Si NWs. The prepared sample was washed out by deionized (DI) water and was subsequently moved into aqua regia  to remove the Au film selectively. Si NWs were cleaned in DI water again. The fabrication procedures of graphene/Si-NWs biosensors are schematically illustrated in 212SO4:H2O2 = 3:1) for the passivation of Si NW surface with amine group and glutaraldehyde, thereby forming the hydroxide (-OH)-terminated Si NWs that are easily reactive with amine (-NH2) groups, as shown in 26In previous studies, amine groups and glurataldehyde were employed for biomolecule immobilization on silicon surfaces23242 substrate shows the D/G ratio of ~0.1 and the G/2D ratio of ~0.47, indicating high-quality monolayer graphene -coated chemical-vapor-deposition (CVD) graphene was transferred onto tips of surface-modified Si NWs, dried by nitrogen gun, and baked on the hotplate at 75 °C for 4 h. The graphene sheet should not cover the entire Si NW tips to secure some part of them, on which the DNA solution can be dropped for DNAs to be spread on the entire surface of Si NWs. The Raman spectrum of graphene on the SiOgraphene 29. Then,I-V curves (I-V curves were characterized for various molar fractions of each oligonucleotide under a forward bias (I-V curves). Sensor performances were analyzed at 1 V, which belongs to the active bias region. As shown in p) of p-ss oligonucleotide increases from 10 up to 500 nM, and above 500 nM, it saturates. The current saturation might come from almost full coverage of the p-ss oligonucleotide on the surface of Si NWs. We studied the effect of the t-ss oligonucleotide mole fraction (nt) on the sensing properties for the device treated by p-ss oligonucleotide at a np of 1000 nM. For the positive control experiments, the solutions containing t-ss oligonucleotide with various mole fractions were dropped on the device. As shown in t varies from 0.1 to 500 nM, and above 500 mM, the current almost saturates, which can be similarly understood as in the variation of np. The current is enhanced by 0.1 mA (~19% increase) even at an extremely-low mole (0.1 pM) of t-ss oligonucleotide, indicating very-sensitive biosensing. The current increases to ~0.76 mA at the saturation point, corresponding to ~120% enhancement. For negative control experiments, solutions containing d-ss oligonucleotide with various mole fractions (nd) were dropped on the device. This resulted in the decrease of the current. For example, the current decreases by ~4 and ~30% at nd = 10 and 40 nM, respectively. However, by further increase of nd, no significant nd dependence is observed, as shown in The graphene/Si-NWs diodes show typical asymmetric V curves . The conV curves . The I-VThe increase of the current can be explained by surface-doping effect of DNAs. When the p-ss oligonucleotide is attached on the surface of Si NWs, the DNA oligonucleotide, acidic bio-species, attract electrons from Si NWs9/cm2) and thin diameter (~50 nm) of the Si NWs are very beneficial to the generation of the large charge carriers from the surface of Si NWs.The extremely-high sensitivity of the graphene/Si NW biosensors can be understood by following two factors. As one factor, graphene and vertically aligned Si NW arrays act as the efficient carrier collector and the active material, respectively. Graphene can be well contacted with Si NW tips without any other coverage of side surfaces of Si NWs, which makes the sensors more efficient than shown in the previous reportsThe endurance properties of the graphene/Si NWs biosensors were studied to check their detection repeatability for the t-ss oligonucleotide. Double stranded oligonucleotide is negatively charged because it possesses the negatively charged phosphate groups. When sodium chloride is added to the solution of double stranded oligonucleotide, the sodium and chloride ions are separated in the solution. Therefore, the positive sodium ions are attached to the negative charged phosphate group of the double stranded oligonucleotide, thereby neutralizing the negative charges. Eventually, the double stranded oligonucleotide is dehybridized, resulting in the generation of the two single stranded oligonucleotides. The hybridized oligonucleotides were detached from the surface of Si NWs by washing out the device in a solution of sodium chloride at 10 mM and subsequently drying it in the air, resulting in denaturation of the double stranded oligonucleotides. 1000 nM p-ss oligonucleotide was then dropped on the device, and the current was measured to check whether the combined double stranded p- and t-ss oligonucleotides were well dehybridized and t-ss oligonucleotide was removed with almost no damage on the device. Subsequently, 0.1 nM t-ss oligonucleotide was dropped to be hybridized to p-ss oligonucleotide on the surface of Si NWs, which resulted in the increase of the current to the level recorded in the previous measurement. These consecutive processes were repeated for ten times. As shown in PL microscopy and spectroscopy were performed to check whether the t-ss oligonucleotide is well hybridized to the p-ss oligonucleotide-decorated Si NWs. The p-, t-, and d-ss oligonucleotides have their-own distinguished fluorescence labels of red, orange, and green colors, respectively. For labelling, different fluorophores are attached to the oligonucleotides. To clarify the origin of each color from the PL images, the PL spectra were measured for the Si NWs decorated with the three different oligonucleotides species by using a visible laser (λ = 488 nm) as the excitation source. 303132We demonstrated selectivity, accuracy, and stability of graphene/surface-modified vertical-Si-NWs biosensors for specific oligonucleotides sensing, originating from the unique properties of vertically-aligned Si NWs, including large surface area. The graphene in the biosensors acted as a role of carrier collecting layer on the Si-NW arrays, resulting in the dramatic enhancement of the sensitivity. The p-ss oligonucleotide as DNA receptors and the t-ss oligonucleotide as DNA donors, having complementary nucleotide sequences to the p-ss oligonucleotide, and d-ss oligonucleotide as negative control DNAs, having mismatched nucleotide sequences with those of p-ss oligonucleotide, were employed to detect the DNA-DNA hybridization between specific oligonucleotides with the graphene/Si-NWs diode-type biosensor by analyzing the electrical and PL responses. The biosensors showed the responsivities of 19 to 120% for 0.1-pM to 1000-nM t-ss oligonucleotide, which is remarkably high compared to those previously obtained from single Si-NW device. The excellent reusability of the biosensors was confirmed by ten-times endurance tests. The PL microscopic image and spectra of the DNAs with PL markers also confirmed the mechanism of the selective DNA sensing by the graphene/Si-NWs biosensors.Our approach utilizes graphene and vertically-aligned Si NW arrays for the building block of the prototypic biosensors, which enables the electrically-sensing DNA devices with outstanding sensitivities to be extremely reduced. The achievement of high-performance graphene/Si-NWs biosensors in view of the detectable molar fraction and the current sensitivity, as compared to the previously-reported graphene-based DNA-sensing performances183PO4 for 30 min at 30 °C. The barrier-layer-opened AAO was utilized as the Au-mesh patterning templates. A 25-nm Au film was deposited on the opened barrier-layer side of AAO. Subsequently, the AAO disk was removed in a KOH solution, thereby leaving only the Au mesh with aligned hole arrays. The KOH solution was then replaced to DI water. The Au mesh as catalyst of MaCE was transferred on the p-type Si substrate.The AAO disks were prepared in an electrochemical shell containing an oxalic acid solution at 40 V for 24 hr, and the barrier layer of AAO was then selectively removed in a solution of 5% H2 and 20-sccm CH4 flowing at a pressure of 3 Torr during growth34Large-area graphene layers were grown on 70-μm-thick Cu foils in a graphite-heater-based CVD quartz tube furnace at a growth temperature of 1000 °C with 10-sccm HThe synthesized oligonucleotides with specific sequences and fluorescent labels are provided from BIONEER Corporation. The purchased oligonucleotide was dispersed in DI-water of controlled volumes for making p-ss, t-ss, and d-ss oligonucleotide solutions at a molar fraction of 1000 nM, which was further diluted to make the solutions in the range of molar fraction from 0.1 to 500 nM. 10 μL of the prepared solution was taken by using a micropipette, and dropped onto the opened surface of the device without the top graphene layer, so that the solution could be permeated into the gap of surface modified Si NW arrays .I-V measurements to monitor the electrical signals of graphene/Si NWs biosensors were conducted using a Keysight B1500A semiconductor analyzer controlled by an installed program. The probe station was employed for the precise electrical contact on the top- and bottom-electrodes. The PL spectra were measured at RT by using a 488 nm line of Ar ion laser as an excitation source. The PL microscopy was carried out at RT using an epifluorescence microscope (Nikon Instruments). The light beam was focused on the sample surface through a microscope objective with magnifications between 20x and 40x.How to cite this article: Kim, J. et al. Precise and selective sensing of DNA-DNA hybridization by graphene/Si-nanowires diode-type biosensors. Sci. Rep.6, 31984; doi: 10.1038/srep31984 (2016).