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 .


There have been relatively few studies on sign language interaction carried out within the framework of conversation analysis (CA). Therefore, questions remain open about how the basic building blocks of social interaction such as turn, turn construction unit (TCU) and turn transition relevance place (TRP) can be understood and analyzed in sign language interaction. Recent studies have shown that signers regularly fine-tune their turn-beginnings to potential completion points of turns Groeber, . Moreove Subsequent research in Conversation Analysis (henceforth: CA) has further demonstrated the robustness of Sacks et al.'s  signers actively accomplish smooth transitions between the current and the next signer, collaborating thereby in a situated and collaborative construction of turns. The findings add to recent research in spoken and in signed interaction that proposes to conceive turn boundaries as flexible and interactionally achieved.With this paper I intend to add to this topic by a detailed investigation of those instances in signed interaction where participants actually overlap each other's turns, by focusing specifically on the sequential environment of these overlaps . This type of investigation sheds light on two issues. First, the analysis of the sequential moment of overlaps aims at revealing whether they present an orderly organization, as it has been shown for overlap in spoken language interaction (cf. Section Overlaps in Spoken and Signed Interaction). Second, the analysis of overlapping signing provides insight into how four participants of a signed language conversation accomplish the actual transitions from one signer to the other. On the basis of a 33-min video-recording of a multi-party interaction with 4 female signers in Swiss German Sign Language (DSGS), the paper shows that (1) signers overlap each other's turns regularly I start with providing some details with regard to turn-taking and overlap in signed languages (Section Research on Turn-Taking and Overlap in Sign Language), presenting my conception of turn and further detailing the issue of this study. Then I present the methodology and procedure I followed for the current study (Section Method), specifying the annotation practice and the established categories for analysis. In Section Sequential Environments of Overlapping Signing, I present the results on different types of overlaps before I discuss these findings in Section Discussion.sign, which consists of a distinct combination of four sublexical manual parameters, namely handshape, location (in the signing space), orientation  and movement , the stroke or independent hold  and the retraction  . Put simply, a participant can design a complete syntactic construction but prosodic resources may indicate that there is more to come . Within such a perspective, the delimitation of TCUs becomes less important an issue, because the focus of analysis is not on linguistic constructions but on the practices for the organization of activities  are traditionally conceived of as linguistic units, several researchers put into question a logocentric definition of turns-at-talk, where the turn and the TCU have to be conceived of in signed interaction are still scarce . This would be an argument to consider such non-lexical resources as proper parts of signed turns.A first fundamental issue in research on signed interaction is how to define the beginning and the end of a turn. Lackner  considerturn includes the preparation phase of the turn-initial sign. The preparation phase of a turn-initial sign is comparable to a hearable inbreath in spoken interaction, described as a pre-beginning element  and post-completions  as not being part of the core TCU, but as a part of the possible turn. Against the background of this, I maintain a differentiation between turns as participant's overall contributions including resources beyond grammar, and the grammatical units that such turns (can) host.The beginning of the stroke of a potentially last sign builds a first TRP, constituting also the end of the core grammatical unit . Consequently, holds after the stroke of the potentially last sign as well as additional gestural elements such as palm-up gestures are considered as being part of the turn. These additional elements extend the turn and finally propose a new TRP , providing a first visual index that the participant launches a turn. In case of absence of mutual gaze between participants, the incipient signer uses an attention-getting device  in order to establish recipiency. Baker . This recalls the difference that is made in spoken interaction between overlapping conducts that are troublesome and others that are not troublesome. For example, in spoken interaction an overlap between the end of a lexical unit by speaker A with the inbreath of the incipient speaker B is of a different quality than the overlap between two lexical units . An issue on this behalf is that all types of overlaps are indicated with the same transcription symbol —as mentioned in Groeber and Pochon-Berger  between four young deaf women: Denja, Nathalie, Isaline and Melinda. All participants have deaf and signing parents and learned DSGS as their L1. The data comes from a larger corpus of DSGS narratives and interactions that has been gathered within the project “Gaze and Productive Signing in a Corpus of Interactions of Deaf and Hard-of-Hearing Signers of Swiss German Sign Language (DSGS),” conducted at the University of Applied Sciences of Special Needs Education, and funded by Swiss National Science Foundation. Within this project, participants were invited to attend to a whole day of recording at a film studio producing programs in sign language. While there are some semi-experimental data, the film under study here comes from the lunch break that was recorded with the aim of having a maximally natural interaction. All data was filmed with three cameras. Two cameras were positioned to record two participants frontally, and the third camera captured all participants together.The current analysis is based on a 33-min four-party interaction in Swiss German Sign Language  that roughly describe the meaning of a sign. These glosses function as labels for a sign and are not a precise translation. In the transcript they are always written in capitals. As the project team is working with iLex, the glosses in the transcripts are automatically linked to the sign databank for Swiss German Sign Language. When a sign is not available in the lexicon, the annotators create a new gloss, which is automatically added to the lexicon.In the current state, the basic transcript consists of a content translation (done by two interpreters), an annotation of manual signs and participants' gaze conduct. Manual signs have been annotated with is the beginning of sign 2, and that there are no gaps between signs  Each overlap was tagged in ELAN on a separate analysis tier. On the basis of participants' gaze conduct I then chose to distinguish between two types of overlaps. Most frequently, overlaps occur between participants who are gazing at each other, either at the beginning, the end or over all their turns. This implies that at some point there is mutual orientation and participants can perceive their simultaneous production. Another type of overlap consists of simultaneous signing between two participants while they are addressing a third person through their action and gaze conduct. This type of overlap can go totally unnoticed by the participants  Description of the sequential environment of the overlapping turn's onset with respect to the overlapped turn. The analysis of all relevant overlaps resulted in a categorization of the overlaps in three main sequential environments:The overlap occurs at a first possible completion place reached by participant A (cf. cases A and B in the table below).The overlap occurs near the end of a unit or the potential end of a unit (but not exactly at the transition place) (cf. cases C and D in the table below).The overlap occurs in the midst of a unit (cf. case E).(3)turn-level, i.e., whether the overlap occurs at the beginning, in the midst or the end of a possible turn. The overlap onset at the lexical level  is visible also in the transcripts, but it is not the focus of analysis in this paper , another participant B , or it can be syntactically dependent (Section Overlapping a Current Signer's Dependent Unit at Places of Possible Completion).The first excerpt illustrates an overlap occurring at a place of possible completion between a current signer, Nathalie, and a new signer, Melinda. The segment stems from a repair sequence initiated by Melinda concerning an exchange that she missed between Nathalie and Isaline. Isaline said that she really dislikes her eyes without make-up because they appear so big. Nathalie responds to this by expressing her surprise. During this exchange, Melinda averts her gaze from the participants while she is drinking . After Nathalie's display of surprise, Melinda initiates repair by asking Nathalie what Isaline dislikes. When Nathalie reaches a potential completion of her repair, Melinda starts a turn  while Nathalie is continuing with a “new” unit, resulting in simultaneous signing.The excerpt is presented as follows: a rough gloss annotation of the larger sequence is provided first, followed by a content translation. The part of these transcripts that are in bold are then represented again in the format of the ELAN annotation, so as to provide some more details on the temporal unfolding of the overlap under discussion Figure . In the IX(isa) on the tier LH_Gloss_1_Nat; l.07) which is doubling the same pointing at the beginning of the unit. This type of reduplication of a pronoun is frequent in several signed languages and has been described as a prosodic marker , reporting her response to Isaline (highlighted in blue).Nathalie's repair turn (l.07-08) consists of two parts. She starts with reporting that Isaline told her that without make-up Isaline's eyes look so big (red rectangle). This part finishes with the referential pointing toward Isaline . Interestingly, Melinda's response to Nathalie's repair is finely tuned to the moment when the element she addressed as repairable has been mentioned, namely what does Isaline dislike (“her big eyes”). At this point, she therefore orients to pragmatic completion, as her repair initiation did not ask for anything more than “what does she/Isaline hate.” Note also that Melinda launches a response to the repair after the reduplication of the sign IX(isa), where the first part is markedly finished on a syntactic and prosodic level. What we see here is thus precisely what Jefferson  the current speaker, perfectly within his rights, keeps going.”  that is overlapped with a stroke by Melinda (RH_phas_Mel). The other overlaps concern strokes and preparation phases. Interestingly, the preparation phase of Melinda's I (RH_phas_Mel) is rather long (350 ms)I in response to the fact that Nathalie is extending her turn . This second part of the turn is overlapped with Nathalie's acknowledging response that can be translated as “you see” with a PALM-UP gesture directed toward Melinda (l.06).Excerpt 2 presents a simple case of a short overlap between Melinda and Nathalie within a discussion on the different possible origins of pimples Figure . MelindaPALM-UP gesture directed to Melinda , in precise overlap with the last item of Melinda's turn, the gesture PALM-DOWN . Considering the movement phases of the overlapping gestures, it becomes clear that Nathalie launches her responsive PALM-UP near the end of the stroke of Melinda's sign WORK, the final sign of her first unit. Thus, Nathalie orients to this moment as reaching potential completion where she can acknowledge Melinda's telling and display her understanding. Melinda's turn however continues with a sign PALM-DOWN, which she adds without any manual prosodic disruption . Despite the absence of a manual prosodic break after WORK, Melinda deploys a resource that indicates a possible completion after WORK—she averts her gaze from her co-participant (to the right side) during the deployment of WORK (cf. DD on the tier Gaze_3_Mel). Baker . In fact, the excerpt represents a recurrent way of how signers in the present data end their turns. We will take this issue up again with excerpts 3 and 5.Nathalie launches her acknowledging response to Melinda's telling, a ). Baker  describe Rossano  describeExcerpts 1 and 2 have shown the fine coordination of incipient signers to possible completion, which implies that the overlapping signing is not the result of mistiming. Signers therefore precisely time their turn beginnings to such places within the temporal unfolding of turns, where syntactic boundaries occur. The overlaps are the result of the fact that the current speaker is not ending her turn at this point, but extends her turn.A further environment where simultaneous signing occurs is just after a possible completion of a signer's unit, i.e., when some pause has occurred after the first possible completion place . This pause can consist of a full or partial retraction, or it can be filled with a holdWhen participant A continues with his turn after some pause after a possible completion, he can do this either by starting a new unit or by adding elements that are somehow dependent on the first part of the turn (as in Section Simultaneous Signing at Places of Possible Completion). In this section I present an example of the first case in excerpt 3 Figure . Denja fBUT LEGISLATION D-HAND BAD STOP -H, on the tiers RH_Gloss_Den/BH_Gloss_Den; l.01-02, with headshake that starts in parallel to the sign BAD). At the end of the sign BAD, Denja's turn reaches a first possible syntactic and pragmatic completion. She continues however with the sign STOP, after which again speaker change may occur. This sign STOP is then held for 0.6 s, and she expands her headshake that she began in parallel to the sign BAD). Nathalie provides an acknowledging response by means of a head nod that is precisely timed to the end of the sign BAD; this head nod extends simultaneously to the sign STOP and the further -H (hold). With this non-manual acknowledgment the course of action has reached some completeness and there is no constraint regarding who of the participants takes a turn. Nathalie might take a full turn during Denja's -H, i.e., she could launch a turn going beyond her non-manual acknowledgment, but her hands remain in rest position. By contrast to Nathalie's turn-ending in excerpt 2, where her gaze aversion from the co-participant was interpreted as a possible sequence closing resource, Denja is gazing at Nathalie during her -H. I suggest that by means of these resources she invites Nathalie to take a turn and elaborate on the topic . In overlap with the preparation phase of I, also Nathalie finally takes a turn by launching the preparation phase of her turn-initial sign SIMILAR , orienting thereby to Denja's previously deployed resources (-H and gaze) as yielding the floor to her. This simultaneous beginning after the completion point results in overlap (highlighted in blue).The segment starts with Denja affirming that Switzerland is a very safe country, but that there is poor legislation resulting in too mild sentences, as for example for the punishment of rapists or murderers  where participants deal with the determination of a next signer in a subtle and situated manner. This is a most relevant interactional task especially in moments where no projection is pending, which means that there is neither constraint with respect to the next action to be accomplished, nor with respect to whom of the participants will get the floor .I suggest that from the end of SCHOOL I, l.04) as soon as the simultaneous signing quits.It is interesting to note that when Nathalie has finally taken over the turn, Denja does not drop out of the overlap immediately, but she brings her new unit to an end (l.03). However, Nathalie clearly orients to the simultaneous signing as a potentially troublesome overlap, as she restarts the overlapped turn-beginning  ALREADY prod-MAN-needle TRY YOU, tier RH_Gloss_3_Mel; l.03; red rectangle]. Denja launches an answer  to this question while Melinda is still producing the stroke of her last sign of her question, the sign YOU . Such examples show that signers can project incipient turn completions and launch into the transition even before the current signer has actually finished. Surprisingly, in the data under study here at least, cases where a signer provides minimal responses or launches a new turn in overlap with a sign “under way” (during the stroke deployment) that will actually be the last sign of the turn are very rare . In the next section I will focus on those instances in more detail and propose a discussion on the possible reasons for the frequent accomplishment of this type of turn transition.Denja is reporting that she was not able to squeeze the pimple despite the fact that it was all yellow. In overlap with this , Melinda is addressing a question to Denja, namely whether she has already tried to pick it with a needle [Current signers are recurrently overlapped during a potentially last item of a unit, after which they continue their turn . The sign HOW-LONG (produced on the right hand) is accompanied with a head tilt back and raised brows  she deploys an acknowledging response that consists of a lifting of a PALM-DOWN gesture with a prolonged mouthing of the letter—e-PALM-DOWN, also Nathalie lifts her left hand (while on the right hand she still signs HOW-LONG) and produces a PALM-UP , which she holds for a moment.Nathalie says that she wonders for how long a rapist who is freed from prison will go before relapse (HOW-LONG). However, Nathalie still prepares and holds her PALM-UP on the left hand until Isaline has again retracted her hands to rest position. Different hypotheses can be drawn up for explaining Nathalie's -H: first, Nathalie may thereby indicate that she does not want to relinquish the turn, i.e., that she is holding the floor. However, -H as a floor-holding device has been described as being accompanied with gaze aversion from the participants . A second possibility is that, by means of her -H, Nathalie embodies her expectation of Isaline's response  during which Nathalie averts her gaze from the co-participant (cf. excerpt 2 for a similar case). It is only after the simultaneous and sequential combination of all these resources that Nathalie retracts her hands to rest position. However, even after having retracted her hands to rest position (on the table), she then further extends the turn with a short PALM-UP accompanied with a shrug of the shoulders . This is again accompanied with a glance toward Isaline, who still does not take a turn.Interestingly, when Nathalie sees Isaline's preparation phase of her acknowledgment (raising the hands out of rest position), Nathalie could relinquish the floor by retracting her hands into rest position .within units. Jefferson . While the sequential moment of these overlaps does not manifest any coordination between the participants with respect to turn management, an interesting question to ask is what actions participants accomplish by means of these overlapping turns. In what follows I provide a brief description of the principal actional environments in which these overlaps occurred. Note however that each case is very specific with respect to the involvement of the participants and the coordination processes .A first environment where participants overlap each other with interjacent onset is in courses of actions involving repair (7/35 cases). The overlapping party is either self-initiating other-repair, providing a self-initiated other-repair, or displaying his understanding after having launched a repair sequence . In order to understand how members of a community accomplish courses of action, and establish and negotiate meaning in a situated and dynamic way, we have to first understand how turns and turn-taking are organized. There is still little research on social interaction in signed languages that adopts a conversation-analytic perspective. To date, we have been provided with some descriptions of how signers indicate their incipient speakership, or their willingness to keep a turn or assign a turn to a next participant. These descriptions do not necessarily follow the analytical mentality of conversation analysis. As a consequence of this, the notions of turn, TCU and TRP are still rarely discussed in detail in this field. Moreover, a most basic issue that has given rise to contradictory assumptions among researchers is the question of whether Sacks et al.'s  turn-taksequential environments of overlaps in signed interaction. Using the conversation analytic tools for studying the organization of signed interaction data allowed us to see how participants themselves treat ongoing turns (as being complete or not) and overlaps (as being troublesome or not). The preceding analyses have shown that the vast majority of overlaps produced among four young female signers frequently occur within the sequential environment of possible completion , i.e., they rarely occur in the midst of syntactic units . This shows that participants finely tune their turn beginnings to those places in the dynamics of interaction, where turns are possibly complete. Hence, the majority of overlaps results from the fact that incipient signers anticipate a turn-end and overlap it, and/or that current signers continue beyond a first possible completion. The findings therefore underpin the observed orderliness of overlapping talk in spoken language interaction , or by accomplishing actions that can reasonably overlap ongoing turns because of their urgency (repair initiations) or because of the interactional effects it thereby creates . It seems therefore that the observations made on overlaps in spoken interaction are also applicable to the data under study in this article. Consequently, prolonged simultaneous signing and the existence of one-at-a-time principle are not necessarily mutually exclusive.The data showed that signed overlap is not messy but organized. Admittedly, however, this does not disprove the assumption that signed interaction presents n-Spence  or Lacknn-Spence . It mighPALM-UP, PALM-DOWN, STOP, -H). Moreover, after these add-ons, signers often finish their turns. This provides evidence, from a participants' perspective, that there is in fact no attempt to hold the floor. By contrast, on the basis of a fine-grained investigation of the collaborative work accomplished by the signer and his recipient's during possible turn transition, the present analyses suggest that participants deploy such short add-ons as an interactional resource for the management of turn transitions. Concretely, participants step out of their turns in a gradual and smooth manner.An important finding of the present study is that ‘last item overlaps’ are scarce (cf. Section Overlapping a Current Signer's Last Item of a Unit). By contrast, participant B often overlaps a potentially last item of participant A who however continues past a possible completion . This may raise the question of whether participants erroneously predict turn-endings. Another hypothesis would be that current signers who are confronted with overlapping signing by their co-participants continue their turns as a means of holding the floor. While the methodological tools of CA do not allow answering the first question, the sequential analyses of overlapping signing provide some evidence for answering the second one. The analyses have shown that at first places of possible completion, current signers' continuations regularly consist of one or more items, short add-ons. These added items often consist of non-lexical elements or signs that do not contribute substantially new information (These add-ons can occur after a pause (Section Simultaneous Signing after Places of Possible Completion) or latched to a first possible completion . In the first case, the turn continuation can serve as a resource for treating a problem of recipiency, similarly to turn extensions in spoken language  and a limited range of courses of action . Whether the same type of turn transition can be found with other participants  is an open question. Moreover, as the excerpts in Sections Simultaneous Signing after Places of Possible Completion and Simultaneous Signing before Places of Possible Completion suggest, a systematic analysis of turn-endings that set a strong actional projection as compared to those without strong projection  will be necessary for a more detailed understanding of signers' management of turn-taking.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The intestinal microbiome represents a complex network of microbes that are important for human health and preventing pathogen invasion. Studies that examine differences in intestinal microbial communities across individuals with and without enteric infections are useful for identifying microbes that support or impede intestinal health.n = 200) and 75 healthy family members to identify differences in intestinal community composition. Stools from 13 patients were also examined post-infection to better understand how intestinal communities recover. Patient communities had lower species richness, evenness, and diversity versus uninfected communities, while principle coordinate analysis demonstrated close clustering of uninfected communities, but not the patient communities, irrespective of age, gender, and race. Differences in community composition between patients and family members were mostly due to variation in the abundance of phyla Proteobacteria, Bacteroidetes, and Firmicutes. Patient communities had significantly more Proteobacteria representing genus Escherichia relative to uninfected communities, which were dominated by Bacteroides. Intestinal communities from patients with bloody diarrhea clustered together in the neighbor-joining phylogeny, while communities from 13 patients’ post-infection had a significant increase in Bacteroidetes and Firmicutes and clustered together with uninfected communities.16S rRNA gene sequencing was conducted on stool DNA from patients with enteric infections  contains supplementary material, which is available to authorized users. Salmonella spp. predominated (11 %) followed by Clostridium perfringens (10 %) and Campylobacter spp. (9 %). Shiga toxin-producing Escherichia coli (STEC) O157 and non-O157 also caused 0.7 and 0.6 % of infections, respectively, while Shigella spp. caused 1.4 % during this same time frame [C. jejuni can cause neuropathies  and paralysis [In the US, diarrheal diseases are mostly caused by 31 different foodborne pathogens and were reported to contribute to 9.4 million infections, 55,961 hospitalizations, and 1351 deaths each year [me frame , though 12 microbes/ml [Bacteroidetes and Firmicutes, have been found to dominate in the intestines of healthy individuals, though considerable variation has been observed [Bacteroidetes and Lachnospiraceae has been observed [Bacteroidetes and Firmicutes [Bacteroidetes and Firmicutes was suggested to result in enhanced susceptibility to enteric infections as it shifts the microbial balance away from a healthy state. A prior study in mice demonstrated that intestinal communities with a high abundance of species closely related to pathogens were more likely to be affected by those pathogens [E. coli, for example, were more susceptible to inflammation induced by Salmonella enterica infections, whereas diverse intestinal communities were more resistant to infection. Commensal microbes were also suggested to alter pathogen virulence gene expression profiles by modifying environmental conditions [Human intestinal communities were previously suggested to comprise >10robes/ml  with keyrobes/ml , 7. Membobserved . Prior sobserved  and thatrmicutes . Decreasathogens . Communinditions . These dCitrobacter rodentium, a model for human enterohemorrhagic and enteropathogenic E. coli infections, alterations in the composition and abundance of the intestinal microbiota could be restored to a pre-infection state upon elimination of the pathogen [Several studies have also demonstrated that intestinal communities are capable of recovering following perturbations. In mice infected with pathogen . Similarpathogen , 18.Despite the recent interest and research on the intestinal microbiome, few population-based human studies have been conducted to better understand how the microbiota is impacted by enteric pathogens with different lifestyles. Here, we have compared the composition and abundance of intestinal microbes between patients with acute enteric infections and their uninfected family members by 16S rRNA sequencing. We have also examined how the intestinal microbial communities from a subset of patients change up to 14 weeks post-recovery, and have identified specific microbial populations that are correlated with clinical symptoms.n = 615) identified between January 1, 2011 and May 7, 2014 to all cases (n = 7435) identified throughout the State of Michigan during the same time period. For each pathogen, the frequency of cases was similar even after stratifying by gender, though a difference was observed after stratifying by age group (data not shown). Because the ERIN network targeted hospitals with the largest pediatric populations, we have captured more pediatric cases, particularly those in the youngest age group.As part of the Enteric Research Investigative Network (ERIN) Cooperative Research Center (CRC) at Michigan State University, we developed an active surveillance system to identify patients with enteric infections at four participating hospitals and the Michigan Department of Health and Human Services (MDHHS). To ensure that the ERIN surveillance network is representative of statewide enteric infection frequencies and that our results are generalizable, we compared the frequency of all ERIN cases (Campylobacter (n = 71), Salmonella (n = 66), Shigella (n = 34), and STEC (n = 28); the pathogen was not known for one patient. A total of 75 stools from otherwise healthy family members were also included for comparison, with 64 (85 %) representing 22 families ranging in size between two and six participants.A total of 310 stool samples were included for 16S rRNA sequencing analysis. These stools originated from 200 patients infected with P < 0.0001) and Bray-Curtis dissimilarity index (P < 0.0001). Moreover, principal coordinates analysis demonstrated tighter clustering of intestinal communities from uninfected family members relative to patient communities based on both the number and abundance of OTUs  and abundance , respectively. Significant differences were also observed using UniFrac weighted and unweighted distances calculated by the analysis of similarities (ANOSIM) and multivariate ANOVA based on dissimilarities (ADONIS) methods (data not shown).Rarefaction analysis revealed that communities were sampled completely with between 60 to 120 OTUs per sample and at 1000 sequences per sample  cases; two patients infected with Salmonella and STEC had communities comprising 99 % Proteobacteria. A total of 28 OTUs representing phylum Proteobacteria were classified in the uninfected communities, though variation in OTUs was observed in the communities infected with the four different pathogens . Variation in the abundance of Proteobacteria was also observed as 37 % of patients with Campylobacter, 29 % with Salmonella, 18 % with STEC, and 38 % with Shigella infections had over 50 % Proteobacteria. Among these infected communities, Gammaproteobacteria represented more than 92 % of the Proteobacteria detected  across the infected communities relative to the uninfected communities. The mean abundance of Escherichia in communities infected with Campylobacter, Salmonella, Shigella and STEC was 0.21, 0.14, 0.24, and 0.21, respectively, relative to uninfected communities (0.01). More specifically, the population of Gammaproteobacteria from Campylobacter cases represented 29 genera with Escherichia comprising 60 % followed by Trabulsiella (7 %) and unclassified Pseudomonadaceae (7 %). Genus Campylobacter was only detected on average in 0.01 % of 41 of the 71 Campylobacter cases, which represented 3.7 % of the total Proteobacteria levels. Salmonella cases had 27 genera represented from Gammaproteobacteria with Escherichia and Trabulsiella predominating at 45 and 21 %, respectively, followed by Citrobacter (12 %) and unclassified Enterobacteriaceae (10 %). Only 22 of the 66 Salmonella cases had detectable levels (average 0.002 %) of genus Salmonella, which represented 0.52 % of the total Proteobacteria levels. For STEC cases, 21 genera were identified with Escherichia comprising 77 % of the Gammaproteobacteria population identified, which represented the total level of Proteobacteria detected in these cases. A total of 23 genera were detected in Shigella cases with most belonging to Escherichia (69 %), unclassified Pseudomonadaceae (8 %), and Klebsiella (6 %). Genus Shigella was not detected in any of the cases, though unclassified Enterobacteriaceae was found at an average of 0.007 %. None of the cases had levels of unclassified Enterobacteriaceae exceeding 10 %. Together, these data demonstrate that the distribution of genera differed among the intestinal communities from patients, which was also indicated in the NPMANOVA analysis; however, Escherichia predominated in most infected communities regardless of the pathogen causing the infection.To further demonstrate that the differences observed across patients infected with the different pathogens were not attributable solely to an increase in the abundance of the actual pathogen, we also examined genera abundance and composition across patients. Notably, genus Enterobacteriaceae followed by Pateurellaceae, Lactobacillales, Streptococcaceae, and Lachnospiraceae. At the genus level, Bacteroides dominated in 86.7 % of the samples from healthy family members with a range between 6 and 75 %, while Prevotella were found in 13.3 % with a range of 16 to 65 %. Among the intestinal communities from healthy individuals, a total of 38 microbial features were differentially abundant. At the family level, most of these features were classified as Rikenellaceae, Bacteroidaceae, Ruminococcaceae, Porphyromonadaceae, Bifidobacteriaceae, Alcaligenaceae, Odoribacteraceae, Barnesiellaceae, and Lachnospiraceae. The genera Roseburia, Lachnospira, and Blautia of the Lachnospiraceae family were most abundant in healthy family members compared to patients. Overall, no microbial features were differentially abundant in the communities of patients infected with different pathogens.Similar findings were observed utilizing the linear discriminant analysis (LDA) effect size (LEfSe) method , which in = 153) of the infected communities belonged to clusters I or II, whereas 91 % (n = 68) of the uninfected communities belonged to clusters III and IV. Based on the placement within the dendrogram, communities of cluster V, which originated from one uninfected individual and five patients, were grouped together with communities belonging to clusters III and IV for subsequent analyses. Overall, intestinal communities from patients were significantly more likely to group within clusters I or II, while uninfected communities grouped within clusters III–V (likelihood ratio chi-square (χ2) = 109.2; P = <0.0001).Microbial community composition was examined based on the Bray-Curtis dissimilarity index to compare microbiota profiles between patients (infected communities) and healthy family members (uninfected communities). Five distinct clusters were identified with the infected and uninfected communities separating into different sections of the neighbor-joining tree Fig. . SpecifiP < 0.0001) when compared to clusters III–V. A significantly higher average percentage of phylum Proteobacteria (42 %) was observed in clusters I/II relative to clusters III–V (6 %), while Bacteroidetes (38 %) and Firmicutes (17 %) were more common in the latter three clusters. Among the uninfected communities belonging to clusters III–V, the abundance of Bacteroidetes (59 %) and Firmicutes (33 %) was even higher. Significant differences were also observed at the genus level. Intestinal communities representing clusters I and II (n = 160) were dominated by Escherichia, Trabulsiella, Citrobacter, unclassified Pseudomonadaceae, or Bacteroides, whereas communities of clusters III–V (n = 115) were dominated by Bacteroides or Prevotella. Notably, Escherichia was significantly more abundant in communities belonging to clusters I and II compared to communities of clusters III, IV, and V as were Trabulsiella and unclassified Enterobacteriaceae (P < 0.0001). Bacteroides, Parabacteroides, Faecalibacterium, Roseburia, Ruminococcus, Suturella, and unclassified Lachnospiraceae, however, were significantly more abundant in clusters III–V . Indeed, only 14 of the 85 patients (17 %) with bloody diarrhea had intestinal communities belonging to clusters III–V. No association was observed between specific intestinal microbiota profiles and length of hospitalization, patient age group, or history of nausea, headache, fatigue, and chills.To determine whether differences in the intestinal microbiota profiles were associated with disease severity and other patient characteristics, we evaluated epidemiological data by the cluster groups defined in the Neighbor joining phylogeny Fig. . No signSalmonella, Shigella, and Campylobacter . Nonetheless, these data suggest that individuals with more severe disease, as measured by the presence of bloody diarrhea, were more likely to have intestinal communities with similar profiles regardless of age and infection by three of the four pathogens.While adjusting for age, gender, and intestinal microbiota profile, logistic regression analyses demonstrated that patients with intestinal communities belonging to clusters I/II were significantly more likely to have bloody diarrhea (odds ratio (OR), 2.3; 95 % CI, 1.12, 4.82). Male gender, however, resulted in a protective effect , and no association was observed for length of hospitalization. Addition of pathogen type to the logistic regression model resulted in similar associations for gender and cluster, but also showed that patients with STEC infections were significantly more likely to have bloody diarrhea when compared to patients with Campylobacter (n = 3), Salmonella (n = 6), and Shigella (n = 4) infections were examined to assess how intestinal communities changed following an enteric infection. In each case, a shift in the intestinal community was observed following recovery. Collectively, the recovered intestinal communities had greater richness, evenness, and diversity relative to the communities that were present during the acute infection , while the level of Proteobacteria was significantly greater (P < 0.0001) . Such differences are likely attributable to variation in the time that each recovered patient was evaluated as patients submitted a follow-up sample between 1 and 14 weeks post-infection. Indeed, Proteobacteria levels decreased over time across the 14 week follow-up period in all patients regardless of the pathogen causing each infection. At the genus level, this decrease in Proteobacteria was mostly attributable to a decrease in Escherichia, which coincided with an increase in Bacteroides, Prevotella, Parabacteroides, and Rikenellaceae in phylum Bacteroidetes and Faecalibacterium belonging to phylum Firmicutes  intestinal communities when compared to the uninfected communities of healthy family members. Both Bacteroidetes and Firmicutes have previously been associated with intestinal health [Proteobacteria compared to an average of only 4.3 % of the healthy individuals (n = 75). Although the total number and relative abundance of OTUs uncovered in the intestinal communities from individuals were lower than in prior studies, all stool samples were preserved in the same conditions with Cary-Blair media, which had little effect on the presence and abundance of specific community members following experimental inoculation of cattle feces  that are characterized by an increase in Proteobacteria and not Bacteroidetes and Firmicutes, which represent dominant commensals and the most common phyla in human intestinal communities. Patients with similar microbiota profiles were more likely to have severe disease outcomes, suggesting that increases in specific microbe populations, which were primarily attributable to Escherichia, occur during an infection. This finding is supported by the result that patient intestinal communities recovered over time to mimic those communities of healthy family members, which were classified by an increase in phyla Bacteroidetes and Firmicutes and decrease in Proteobacteria belonging to genus Escherichia. Future enteric disease prevention strategies could therefore aim to decrease intestinal Escherichia levels at the time of an acute infection while simultaneously boosting microbes including Bacteroidetes that are most important for restoring intestinal health.Enhancing our understanding of how intestinal microbial communities change during an enteric infection and identifying factors that influence these changes are important for the development of novel prevention and treatment strategies. In our analysis of 200 intestinal communities from patients and 75 healthy family members, we found that specific intestinal microbiota are more common among patients during an acute enteric infection regardless of the type of enteric pathogen. Specifically, the intestinal microbial communities from patients were more frequently dominated by Campylobacter, Salmonella, Shigella, and STEC at four participating Michigan hospitals. Surveillance began on January 1, 2011. Stools from each patient were added to Cary-Blair transport media and submitted to the MDHHS for de-identification, culture, and transport to MSU for community DNA isolation and 16S rRNA sequencing. Family members of patients (n = 75) were enrolled by phone and asked to submit a stool specimen preserved in Cary-Blair transport media and to complete a questionnaire containing demographic information, exposures , and signs of illness including duration, type, and severity of clinical symptoms. Such information was also available for each patient through the Michigan Disease Surveillance Network (MDSS) maintained by the MDHHS Bureau of Epidemiology. A subset of 13 patients was also re-contacted post-infection for stool submission and questionnaire completion. Each participant was required to provide informed consent prior to enrollment and was given a monetary incentive after samples were received at MDHHS. All protocols were approved by the Institutional Review Boards at MSU (IRB# 10-736SM), the MDHHS (842-PHALAB), and each of the four participating hospitals .An active surveillance system was created through the MDHHS Bureau of Laboratories to identify 200 patients infected with n = 741) received via the ERIN surveillance system through May 7, 2011. Stool DNA recovery (260/280 nm) and yield (ng/μl) were initially compared using the QIAamp DNA Stool Mini Kit  and the MoBio Fecal Isolation Kit  and standardized using the NanoDrop (Thermo Fischer Scientific). Four stools were compared using the manufacturer’s instructions, except for the addition of a denaturation step (95 °C) followed by bead beating for the QIAamp kit to enhance DNA isolation of gram-positive bacteria as described [Stool samples were processed soon after receipt by both MDHHS and MSU and time period from collection to processing was noted. Following homogenization and centrifugation, aliquots of stool were stored in triplicate at −80 °C until further use. A total of 310 stools were examined in this analysis, which represent 41.8 % of the total number of stools  in triplicate with initial denaturation at 95 °C for 2 min followed by 30 cycles of 95 °C for 20 s, 50 °C for 30 s, and 72 °C for 5 min. An amplicon library of 48 communities were prepared for each sequencing run on 454 GS Junior Titanium chemistry, by individually purifying the product using the Agencourt AMPure XP  beads, quanitification by pico green assays  followed by normalization and pooling.Isolated community DNA was amplified for the 16S rRNA gene spanning the V5–V3 regions using universal primers 357F and 926R tagged with the 454 universal primers A and B at either end with barcodes for multiplexing samples. PCR amplification was performed using AccuPrimeOver 1 million reads were processed in the Quantitative Insights Into Microbial Ecology (QIIME) software  and aligThe phylogenetic data from QIIME was further analyzed using Paleontological Statistics Software Package For Education and Data Analysis (PAST) based on the NPMANOVA and to calculate genera important for dissimilarities between groups using SIMPER. Additional 2D principal coordinate analysis plots based on both the Bray-Curtis and Jaccard similarity coefficients were also constructed for a weighted and unweighted analysis (28). Box plots were constructed using SigmaPlot 10.0.http://tree.bio.ed.ac.uk/software/figtree/). We tested for differences in the frequency of clinical characteristics including HUS, diarrhea, bloody diarrhea as well as for various factors including age and gender, across the different microbial community clusters using the likelihood chi-square test or Mantel-Haenszel chi-square (for variables with more than two outcomes) distributions using odds ratios with 95 % confidence intervals performed with Statistical Analysis Software (SAS) (version 9.3).Links to epidemiological data were made after clustering relationships were identified between the stool communities based on abundance profile calculated by Bray-Curtis dissimilarity index in PAST for generation of neighbor-joining tree followed by tree construction in FigTree (Raw sequence data is available at (http://dx.doi.org/10.6084/m9.figshare.1447256), while the metadata and taxonomic classifications have been included as Additional files


The crosslinking mechanism under different temperatures indicated two crosslinking pathways: (a) crosslinking on the sulfonic acid groups; and (b) crosslinking on the backbone. It was observed that membranes crosslinked at a temperature of 150 °C lead to low proton conductive membranes, whereas an increase in crosslinking temperature and time would lead to high proton conductive membranes. High temperature crosslinking also resulted in an increase in anisotropy and water diffusion. Furthermore, the membranes were investigated for a Vanadium/Air Redox Flow Battery application. Membranes crosslinked at 200 °C for 30 min with a molar ratio between 2:1 (mol repeat unit:mol benzenedimethanol) showed a proton conductivity of 27.9 mS/cm and a 100 times lower VO2+ crossover compared to Nafion.Highly conductive and low vanadium permeable crosslinked sulfonated poly(ether ether ketone) (cSPEEK) membranes were prepared by electrophilic aromatic substitution for a Vanadium/Air Redox Flow Battery (Vanadium/Air-RFB) application. Membranes were synthesized from ethanol solution and crosslinked under different temperatures with 1,4-benzenedimethanol and ZnCl These applications require highly conductive and stable membranes. The vanadium/air redox flow battery (Vanadium/Air-RFB) is one of the most promising energy storage technologies based on such membranes.2+/V3+, where the second half-cell contains either water vapor or air, depending on the charge or discharge modes of the system. The Vanadium/Air-RFB has been chosen as a special type of redox flow system, since it employs only vanadium in its 2+/3+ oxidation state, but not in the 4+/5+ oxidation state. Due to that, stability issues caused by the 4+/5+ oxidation state  on the square of the wave vector (q2) is shown in q2 range, where a “fast” diffusion occurs and from which apparent diffusion coefficients, namely Dfast, can be extracted.The authors of a recent paper  measuredmposites . The watη = (D⊥ − D∥)/D⊥, where D⊥ corresponds to the through-plane diffusion coefficient and D∥ the in-plane diffusion coefficient, respectively. The anisotropy of water diffusion, as shown in slowD, is more affected by the experimental errors and, therefore, is not shown in this work.The “fast” diffusion is characterized by a higher diffusion coefficient for the perpendicular orientation (through-plane) of THE gradient relative to the membrane plane than for the parallel one (in-plane). The SPEEK membrane crosslinked at 200 °C for 60 min shows the highest in-plane and through-plane diffusion among the studied membranes. The anisotropy of water diffusion in the cSPEEK membranes is defined as fastD in the crosslinked SPEEK membranes at elevated temperatures (200 °C) would indicate an improvement of proton conductivity through the membrane, which is beneficial for applications, such a vanadium redox flow batteries or fuel cells.The increase of 3.3.i.e., From eat unit . To provi.e., after exposition at 200 °C for 60 min. The resulting conductivities support the previous hypothesis of the sulfonate ester appearance at lower crosslinking temperature and the change of the crosslinking position of the 1,4-benzenedimethanol at higher temperatures and longer crosslinking duration.As illustrated in i.e., IEC for the non-crosslinked SPEEK  data. Indeed, IEC is directly related to the number of the ed SPEEK . Therefo3.4.4 compartments over time, where 2+ through these membranes.The vanadium permeability of the synthesized and commercial membranes were studied. This was done in a diffusion cell, which consists of two half-cells separated by the membrane under investigation. i.e., increasing the crosslinking). However, the vanadium permeability remains still higher than through a Nafion 117 membrane. This may be a result of the highly sulfonated SPEEK, which has a high water uptake. To overcome this issue, the ratios of crosslinked It can be seen that the vanadium permeability decreased with decreasing the amount of SPEEK (fion 117 . HoweverThe reason for the low vanadium cross over in the case of the crosslinked SPEEK lies in its glassy properties and rigid backbone. Furthermore, the dead end hydrophilic channels of SPEEK also decrease the vanadium permeability, since these are not as well interconnected as in the case of Nafion.4.Highly sulfonated crosslinked SPEEK membranes for a Vanadium/Air-RFB application were prepared as described elsewhere ,23. For