Integrating knowledge from across the natural and social sciences is necessary to effectively address societal tradeoffs between human use of biological diversity and its preservation. Collaborative processes can change the ways decision makers think about scientific evidence, enhance levels of mutual trust and credibility, and advance the conservation policy discourse. Canada has responsibility for a large fraction of some major ecosystems, such as boreal forests, Arctic tundra, wetlands, and temperate and Arctic oceans. Stressors to biological diversity within these ecosystems arise from activities of the country's resource-based economy, as well as external drivers of environmental change. Effective management is complicated by incongruence between ecological and political boundaries and conflicting perspectives on social and economic goals. Many knowledge gaps about stressors and their management might be reduced through targeted, timely research. We identify 40 questions that, if addressed or answered, would advance research that has a high probability of supporting development of effective policies and management strategies for species, ecosystems, and ecological processes in Canada. A total of 396 candidate questions drawn from natural and social science disciplines were contributed by individuals with diverse organizational affiliations. These were collaboratively winnowed to 40 by our team of collaborators. The questions emphasize understanding ecosystems, the effects and mitigation of climate change, coordinating governance and management efforts across multiple jurisdictions, and examining relations between conservation policy and the social and economic well-being of Aboriginal peoples. The questions we identified provide potential links between evidence from the conservation sciences and formulation of policies for conservation and resource management. Our collaborative process of communication and engagement between scientists and decision makers for generating and prioritizing research questions at a national level could be a model for similar efforts beyond Canada.Generación de Preguntas de Investigación Prioritarias para Informar a las Políticas y Gestión de la Conservación a Nivel Nacional Although we recognize the benefits of ongoing curiosity-driven research, we believe there is clear value in aligning research in the natural and social sciences more closely with the information needs of decision makers. Scientific research can inform decisions regarding domestic environmental protection and enhance a country's ability to contribute to international solutions to global environmental challenges . In CanaTargeted research can have instrumental effects , which dRecent projects have been undertaken at the global  and natibiodiversity, defined here as “the variety of genes, species and ecosystems and the ecological processes that allow them to evolve and adapt to a changing world” , environmental nongovernmental organizations (n= 3), industry associations (n= 2), policy analysis or advisory organizations (n= 6), and academia (n= 7). The core team of eight organizers and four student assistants brought the total number of participants to 40. Twenty-eight of the participants attended a workshop in Ottawa, the national capitol, in April 2010. Although provincial and municipal governments also have substantive, often paramount, responsibilities for ecosystem management, involving participants from a full range of regional government organizations was not feasible logistically. Academic participants were drawn from across the country and many team members had extensive regional policy experience.A core team of eight organizers (the eight lead authors) designed and implemented the project. A larger group—who participated in solicitation and synthesis of questions—included policy advisors and decision makers in governments and nongovernmental organizations  and academics with expertise in conservation science. Of 57 invitees to that group, 43 responded and 28 accepted. The final group of participants was drawn from federal government agencies with national responsibilities . Questions were sought from policymakers and their advisors in government, policy specialists in the nongovernmental and private sectors, Aboriginal leaders and science advisors, researchers in the natural and social sciences, and the philanthropic community. We used a bilingual (English and French) website established for this exercise to collect questions over 5 weeks.yes, no, or it depends response, (5) contain a subject of the intervention, an intervention, and a measurable effect if related to interventions and effects, and (6) increase the effectiveness of policy about, and management of, resource use and biodiversity in the face of environmental stressors if answered.Instructions for individuals who wished to submit questions were similar to those for other priority-setting exercises , although policy applications were stressed. Individuals were asked, “What research question, if answered, would substantially advance the development of effective policies and management strategies for species, ecosystems, and ecological processes in Canada?” Aspirational criteria for questions were that they (1) be answerable through a realistic research design, (2) be answerable on the basis of facts rather than value judgments, (3) be of a spatial and temporal scale that could be addressed realistically by a research team or program, (4) not be answerable simply with a A total of 396 questions were received in 271 submissions. Questions were submitted by individuals with diverse organizational affiliations . Over 62% (159 of 255) of individuals who voluntarily provided background professional information had more than 10 years experience in natural resource management. Core team members culled candidate questions that failed to meet the aspirational criteria and combined and refined questions to avoid substantial redundancies. This process reduced the list to 242 questions for participants to consider in the workshop. All questions are included in the Supporting Information.At the workshop, participants selected the top 40 questions from those submitted through a process developed for similar priority-setting exercises in the United Kingdom and United States . The workshop opened with a plenary session that included participant introductions, an overview of how participants were selected, highlights from recent exercises, an explanation of the policy emphasis of this exercise, and a review of workshop processes and ground rules. The balance of the day was spent in three breakout sessions (75–90 minutes each) composed of three concurrent groups per session. Each breakout group discussed one loosely themed set of 25–30 candidate questions, narrowing the set to four candidates for the top 40 and two alternates. Participants in breakout groups sometimes combined candidate questions, developed alternate questions that captured key ideas from candidate questions, and referred to the original list of 396 questions. The 54 questions that resulted from the first day  were discussed during a plenary session on the second day, during which the top 40 were selected by consensus.After the workshop, the core team and participants, including those individuals unable to attend the workshop in person, refined the questions by consensus-based email dialogue. During the refinement phase, it became apparent that some questions were quite similar. Combining three pairs of questions allowed us to include other candidates that were not on the initial list of 40. Participation of scientists helped ensure that questions were sufficiently narrow to form the basis of a research program. Participation of policymakers helped ensure that the final questions were relevant.We grouped the top 40 questions into themes: ecosystem structure and function, land cover and habitats, populations and species, resource-based industries, parks and protected areas, environmental change, environmental values, economic benefits and costs, individual and community well-being, adaptive management, and policy and governance. The organization of these themes was drawn from the adaptive-management logic framework outlined in Ecosystem attributes that provide benefits to humans  are increasingly a focus of biodiversity policy . The alt1. To what extent can ecological function and the supply of ecosystem services be predicted on the basis of ecosystem composition and structure?2. How does managing ecosystems for particular ecological functions or services affect other elements of biodiversity?3. How can transition points or thresholds among ecosystem states best be identified and predicted?Current approaches to ecosystem management seek to link land-cover types, species’ habitats, and ecosystem stability and services in part to understand how natural and human-induced changes in land cover might affect ecological, social, and economic well-being e.g., .4. How are populations of animals and plants affected by different configurations of land cover?5. At what spatial and temporal scales are management or policy interventions most likely to achieve various objectives for conservation of land cover?6. What ecological principles and information standards employed in the identification of critical habitat under the Species at Risk Act are most likely to contribute to species recovery?7. To what extent can habitat restoration or rehabilitation compensate for loss of quantity or quality of existing species’ habitat?Many ecosystem functions and services depend on the maintenance of particular populations or species e.g., . Populat8. What investments and management interventions will be required to maintain or increase the abundance of harvested populations when harvesting is one of multiple stressors acting on those populations?9. What are the cumulative demographic and genetic effects of harvest on target and nontarget populations and species?10. How do transboundary migrations of terrestrial and aquatic animals affect efforts to manage populations of those species?Natural-resource industries account for a substantial portion of Canada's economic activity . Where past policies have failed to effectively manage biodiversity there have sometimes been enormous economic and social impacts. For example, collapse of the Newfoundland cod fishery led, in 1992, to the largest single layoff of workers in Canadian history . Policy 11. What are the potential effects of large-scale energy exploration, development, and extraction projects on ecosystems and species?12. How does management of terrestrial and aquatic food-production systems affect species, ecological functions and services, and economic benefits?13. What are the magnitude and extent of effects of terrestrial resource-development activities on aquatic ecosystems?14. How do extractive and nonextractive industrial activities affect ecological functions and biodiversity in aquatic ecosystems?Canada has an extensive system of terrestrial parks and other protected areas  and a growing system of freshwater and marine parks for which federal, provincial, territorial, and regional governments all have responsibilities. Parks are managed to meet different objectives and mandates to protect species and ecosystems, and provide recreational opportunities that sometimes conflict. There are gaps in knowledge of how networks of parks can and should be designed, connected, and managed to meet ecological, social, cultural, and economic objectives.15. To what extent can species be maintained over the long term within existing parks and protected areas in the absence of proactive, external management?16. What are the ecological consequences of existing human activities in parks and their implications for future management?17. What are the impacts of alternative configurations of, and management strategies for, aquatic reserves on ecological function, species, and social well-being?18. What are the effects of different configurations of, and management strategies for, protected areas on biological diversity across landscapes and beyond the boundaries of terrestrial protected areas?Canada is already affected by climate change, especially in northern regions . Underst19. How is climate change likely to affect the abundance, rates of growth, and distribution of and interactions among populations?20. How can undesirable ecological, social, and economic effects of future changes in freshwater availability be reduced or mitigated?21. How will northern coastal ecosystems respond to changes in climate and industrial activity if reduction in ice cover increases human access to those ecosystems?22. What policies and management strategies most effectively mitigate the undesirable effects of non-native invasive species, potential disease vectors, and emerging pathogens?23. What are the effects of different policies and management strategies on the capacities of humans and other species to adapt to environmental change?Environmental values are influenced by demographic, cultural, experiential, and educational variables . They ca24. How does the integration of conservation science into public education influence environmental values, ecological literacy, and behaviors that affect the environment?25. How does urbanization and immigration affect environmental values, behavior, and conservation outcomes?26. How do individual and societal values about collective responsibility for common- pool resources affect private conservation initiatives and stewardship efforts?International agencies, governments, and researchers have highlighted the economic value of biodiversity. Accounting for the full costs and benefits of ecological change is central for credible economic analyses . The way27. What are the true direct and indirect contributions of ecosystems to economic well-being and how are these benefits likely to change in the future?28. What are the comparative ecological, economic, and social impacts of economic instruments versus input or output controls for ecosystem management?29. What are the benefits and costs of horizontally and vertically integrating policies and regulations within and across different policy domains such as environment, health, and trade?Policymakers are concerned with aspects of individual, community, and societal well-being beyond economics. Environmental change affects human health e.g., , communi30. How do terrestrial and aquatic conservation policies directly or indirectly affect human health?31. What are the effects of conservation initiatives on the health, livelihoods, traditional cultures, and identities of Aboriginal peoples?32. What are the effects of different strategies for building community capacity on levels of citizen engagement in environmental stewardship, restoration, and conservation?Canadian governments are committed to working together with citizens using “an ecosystem and adaptive management approach to achieve shared outcomes” . Ecosyst33. How can local and traditional knowledge be most effectively communicated and synthesized with scientific knowledge to inform conservation science and management?34. What forms of scientific evidence, risk assessment, and knowledge transfer most effectively and increase the probability of achieving ecosystem-management objectives?35. What monitoring methods can effectively and efficiently assess long-term, incremental, and cumulative environmental changes?36. What are appropriate indicators to evaluate the effects of public, private, and nongovernmental-sector policies, programs, and initiatives on biodiversity?Canada is a parliamentary democracy with a complex federal structure and diverse citizens who expect to participate in collective decisions. This situation complicates the formation and implementation of national evidence-based and results-oriented policy. It is difficult to develop consistent policies across jurisdictions and to determine the most-appropriate level of governance to address a given environmental problem. It is also difficult to balance the legitimacy of governance (a function of structure and inclusive processes) with explicit action to conserve biodiversity as required by laws, international commitments, and moral values.37. What factors influence the likelihood of compliance with environmental legislation and regulations?38. How do policy, legal, or institutional arrangements shape the effectiveness of integrated management for terrestrial watersheds and adjacent coastal environments?39. How do the actions of Aboriginal governments and organizations influence conservation strategies and the assignment of responsibilities for environmental governance in Canada?40. How can environmental laws, policies, and regulations be integrated and harmonized across multiple jurisdictions and sectors to achieve conservation objectives?Answers to questions about conservation policy can be informed by scientific evidence about the ecological, social, and economic effects of governance interventions. The Some of the themes identified in this Canadian exercise coincide closely with those identified in the United States . Relative to the United States, however, there was less emphasis on environmental stressors (27% vs. 55% of the 40 questions) and more emphasis on governance and management issues (67% vs. 32% of the 40 questions). In Canada there was strong emphasis on the effect of aboriginal governance on federal and provincial conservation policy and, conversely, on the effect of governmental conservation policy on individual and community well-being for Canada's Aboriginal population.We suspect that the contrasting priorities within North America arise from cultural and political differences . In other fields of study, political context has been identified as an important factor influencing the effect of research , so it wThe questions we identified provide potential links between evidence from the conservation sciences and the formulation of environmental policies for conservation and resource management. Integrating knowledge from across the natural and social sciences is necessary to effectively address societal tradeoffs between use and conservation of biodiversity. The collaborative process of communication and engagement between scientists and decision makers can change the ways that decision makers think about evidence, enhance levels of mutual trust and credibility, and potentially help advance the conservation policy discourse in Canada and internationally.


In this work, we synthesized graphene oxide from silk cocoon embarking its new dimension as a magnetic fluorophore when compared with its present technical status, which at best is for extracting silk as a biomaterial for tissue engineering applications. We produced graphene oxide by pyrolysing the silk cocoon in an inert atmosphere. The collected raw carbon is oxidized by nitric acid that readily produces multilayer graphene oxide with nano carbon particulates. Structural properties of the graphene oxide were analyzed using scanning electron microscopy, transmission electron microscopy, Fourier transform infra-red spectroscopy, and Raman spectroscopy. The oxidized sample shows remarkable fluorescence, multi-photon imaging and magnetic properties. On increasing the excitation wavelength, the fluorescence emission intensity of the graphene oxide also increases and found maximum emission at 380 nm excitation wavelength. On studying the two photon absorption (TPA) property of aqueous graphene oxide using Z-scan technique, we found significant TPA activity at near infrared wavelength. In addition, the graphene oxide shows ferromagnetic behavior at room temperature. The observed fluorescence and magnetic property were attributed to the defects caused in the graphene oxide structure by introducing oxygen containing hydrophilic groups during the oxidation process. Previously silk cocoon has been used extensively in deriving silk-based tissue engineering materials and as gas filter. Here we show a novel application of silk cocoon by synthesizing graphene oxide based magnetic-fluorophore for bio-imaging applications.The online version of this article (doi:10.1007/s13205-013-0128-2) contains supplementary material, which is available to authorized users. Carbon, the sixth most abundant element, exhibits the unique property of forming a wide variety of structures and allotropes. Because pre-historic times, only two allotropes of carbon are known widely: graphite and diamond. However, provoked by the discovery of zero-dimensional allotrope (0D) fullerenes by Kroto et al. , there isp2 bonded carbon atoms. It is the “thinnest material” that is stable in free form  and potassium chlorate (KClO3) resulting in brilliantly transparent yellow pallets (Brodie 4) and potassium chlorate (KClO3) by dissolving them in concentrated sulfuric acid (H2SO4) , transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy. The synthesized graphene oxide also exhibit excellent fluorescence property and two-photon activity at near-IR (NIR) excitations (800 nm), thus making it a very strong candidate for two-photon microscopy applications. Magnetic property measurements of graphene oxide showed ferromagnetic behavior at room temperature indicating its excellent potential in magneto-optic studies also.All chemical reagents were of analytical grade, and used without further purification and Tasar silk cocoon were collected from the state of Chhattisgarh in India as previously described  for 24 h. Then, the acid was decanted off and the brown mass was thoroughly washed with distilled water till it was free from acid and becomes neutral. The brown residue was finally dried in vacuum and subjected for further analysis.The collected cocoons were grounded in mortar and pestle. Three grams of grounded Tasar silk cocoon was pyrolysed in a muffle furnace at 400 °C for 2 h in argon (Ar) atmosphere. This resulted in the formation of black solid which was collected at room temperature. The powdered raw carbon was taken in a thimble made of Whatman filter paper. Then, this raw carbonized cocoon was subjected to soxhlet purification. Continuous washing of raw carbon powder were done by petroleum ether followed by acetone. The raw carbonized cocoon was oxidized by treating with concentrated nitric acid (HNOMorphology of both the raw carbonized cocoon (containing graphene) and oxidized carbonized cocoon (containing graphene oxide) were characterized using SUPRA 40VP field emission scanning electron microscope (FESEM) equipped with energy dispersive X-ray (EDX) facility (Carl Zeiss NTS GmbH). To analyze the soxhlet purified raw carbonized cocoon (containing graphene), 3 mg of sample was taken in 4 ml isopropanol solution and sonicated for 30 min. 50 μl of the sonicated solution was placed on brass stubs and vacuum dried. For analyzing graphene oxide, 3 mg sample was taken in 4 ml isopropanol and sonicated for 3 min. 50 μl of the sonicated solution was placed on brass stubs and vacuum dried and subjected to SEM analysis.TEM was performed using Tecnai 20 G2 300 kV, STWIN model. An acceleration voltage of 200 kV was used for TEM analysis. Samples for TEM analysis were prepared by dropping 5 μl of the re-dispersed raw and oxidized carbon on to a carbon-coated copper TEM grid with a mess size of 400.FT-IR spectra were recorded using Bruker Vertex 70 FT-IR spectrophotometer using KBr disc.After Raman spectra were recorded using a Raman spectrometer (WITEC model) with 514 nm excitation.Fluorescence microscopy was performed using Leica Microsystems DM 2500 microscope.Fluorescence spectra of graphene oxide were obtained using Perkin Elmer LS 55 fluorescence spectrometer with peltier temperature programmer (PTP-1). The fluorescence excitation spectrum was recorded at the highest emission wave length of 460 nm.−1 energy and 150 fs pulse width was focused on to the sample with a 20 cm focal lens. Across the focal plane of this lens, a 1-mm path length cuvette containing the clear aqueous sample solution was scanned in the laser propagation direction [so is the name Z scan  shows the SEM images of purified raw carbon and oxidized carbon. The raw carbon contains layers of sheets with carbon particulates and amorphous carbon. These amorphous carbons were removed during oxidation process and layered graphene oxide is separated out.TEM image of oxidized carbon shown in Fig. FT-IR spectroscopy was performed to study the presence of different functional groups. FT-IR spectra of graphene oxide in Fig. sp2 and sp3 hybridized forms of carbon in the graphene oxide . The D-band occurs due to the number of defects and broken symmetry of basal plane of graphitized carbon atom. The G-band corresponds to the Eg vibrational mode of sp2 hybridized carbon atoms in both rings and chain structures. The Raman spectrum of the oxidized soot is shown in Fig. −1. The first one is associated with the stretching vibrations of sp2 hybridized carbon atoms and the second one is attributed to the disorder-induced stretching modes of graphite. The D-line of oxidized soot has almost the same intensity as that of the G-line when compared with the insoluble raw soot residue  at the zigzag sites was one of the possible explanations for the fluorescent emission . Radiative recombination of this e–h pair in these localized sp2 clusters give rise to fluorescence property  of fluorescent excitation study of graphene oxide are ~0.78 eV (4.42–3.64 eV), ~1.24 eV (4.42–3.18 eV), and ~0.45 eV (3.64–3.19 eV). Hoffman demonstrated that for a triplet ground state carbene, the difference in energy (δE) between σ and π orbital must be less than 1.5 eV  of the system. The schematic of the experimental set-up for the measurement of the optical properties is shown in Fig. 2 of supplementary material. Figure T(z) is the normalized energy transmittance as a function of z position for a sample thickness L; I0 is the incident laser intensity (1.09 nJ pulse−1) at the focus of the lens; and λ (780 nm) with a minimum beam radius ω0 (30 μm) at the focus. Because all the parameters in Eq.  data at different z positions easily gives the value of β as 0.55 × 10−9 cm W−1.The efficiency of any nonlinear two-photon material is measured in terms of the TPA coefficient (Hc) of graphene oxide is 143.75 and saturation magnetization (Ms) is 0.005 emu g−1. Low coercivity values indicating the graphene oxide is a soft magnetic material. To understand the origin ferromagnetic behavior of graphene oxide several theoretical works has been done, but it is not yet fully understood. Theoretical study has been suggested that graphene oxide show ferromagnetism due to the presence of various defects and long-range ordered coupling among these defects. In graphene oxide, the presence of C–OH, –COOH, and C=O groups which creates various defects may be responsible for ferromagnetic behavior of graphene oxide. Introduction of this hydrophilic group on the topological structures of raw carbon obtained from pyrolysis of silk cocoon may leads to increase defects due to intra or inter molecular hydrogen bonding among them.Room temperature ferromagnetic behavior of graphene oxide is shown in Fig. In summary, we have demonstrated the synthesis of graphene oxide from Tasar silk cocoon by pyrolyzing at 400 °C in argon atmosphere followed by its oxidation with nitric acid. Different characterization techniques were utilized to characterize the raw carbon and oxidized carbon. Synthesized water soluble graphene oxide shows remarkable fluorescence property. On increasing the excitation wave length to 380 nm, emission peak was observed at 460 nm. Its tunable fluorescence property with different wavelength can be used in applications like bio-imaging. Soft ferromagnetic behavior of graphene oxide which also exhibits single and two-photon fluorescence characteristics can open the new door for wide potential application, such as, magneto-optics, carbon-based magneto resistance, spintronics, magnetic memory devices, and so on.Supplementary material 1 (DOC 1402 kb)Below is the link to the electronic supplementary material.


We investigate the effect of water adsorption on the electrical properties of graphene oxide (GO) films using the direct current (DC) measurement and alternating current (AC) complex impedance spectroscopy. GO suspension synthesized by a modified Hummer's method is deposited on Au interdigitated electrodes. The strong electrical interaction of water molecules with GO films was observed through electrical characterizations. The DC measurement results show that the electrical properties of GO films are humidity- and applied voltage amplitude-dependent. The AC complex impedance spectroscopy method is used to analyze the mechanism of electrical interaction between water molecules and GO films in detail. At low humidity, GO films exhibit poor conductivity and can be seen as an insulator. However, at high humidity, the conductivity of GO films increases due to the enhancement of ion conduction. Our systematic research on this effect provides the fundamental supports for the development of graphene devices originating from solution-processed graphene oxide. Many oxygen-containing groups, including hydroxyl, epoxy and carboxylic acid, were bonded to the two-dimension network. The presence of sp3-bonded hybridized carbon atoms weakens the conductivity and enhances the hydrophilic property of GO. Recently, exploring the feasibility of integrating GO into graphene-based electronic devices has motivated immense studies on the intrinsic electrical [Graphene oxide (GO), a single thin sheet of graphite oxide that conventionally serves as a precursor material for preparing graphene -3, has rectrical ,8 and meectrical ,10 propeectrical , electriectrical -14, tempectrical ,16, lighectrical , etc. Dusp3-bonded hybridized carbon atoms, it is worth noting that GO can capture water vapor from external environment easily, owing to its notable hydrophilicity [In earlier works ,8, severhilicity -20. Henchilicity ,19. The Graphite oxide was synthesized via the oxidative treatment of natural graphite using the modified Hummer's method . Then, g2 (300 nm) formed by thermal oxidization. Ti/Au layers with the thickness of 100:400 nm were deposited on SiO2 layer using magnetron sputtering. The Au electrodes with a 20-μm-wide gap were formed through photolithography followed by wet etching. Before being functionalized by GO films, the IDE was rinsed with distilled water and ethanol and dried in vacuum overnight. The device was fabricated by dispersing the GO suspension onto Au IDE. A few drops (4 μl) of the GO suspension were cast onto Au IDE by a micro-syringe. After drying at room temperature for 6 h, a discrete network of GO sheets was left on the Au IDE.Interdigitated electrodes (IDEs) were fabricated on an n-type silicon wafer with a top layer of SiOThe schematic diagram of the experimental setup is shown in Figure −1) is shown in Figure −1. The peaks between 1,800 and 1,050 cm−1 are due to C = O, C–H, C–OH, C–O–C and C–O stretching at 1,730, 1,629, 1,396, 1,240 and 1,060 cm−1, respectively.The photograph of the stable GO dispersion (1 mg mlI-V) characteristics of the GO-film-functionalized IDE, is measured with a voltage sweeping mode at various humidity points. In this configuration, one electrode of the IDE is loaded with the sweeping voltage bias, and the other electrode is grounded. Recent works have noted that the DC electrical property of GO films can be influenced by the amplitude of sweeping voltage [I-V characteristics of GO films with low (−1 to 1 V) and high (−4 to 4 V) sweeping voltages at various humidity levels, respectively.The DC electrical property, i.e., current–voltage  provides a powerful tool to analyze the electrical interaction of the investigated material with water molecules -24. To k+) arising from hydroxyl of GO is bonded to excess adsorbed water molecules to form hydronium (H3O+) ion. However, the hydronium ions formed by charged carriers were alone and not enough to yield the continuous conduction path due to insufficient adsorption of water molecules. Thus, this process does not bring out conductance obviously. However, the increasing formation of hydronium ions can be seen as an accumulate layer. The mechanism is illustrated by a cartoon image shown in Figure RGO and geometric capacitance CGO of the GO films, as shown in Figure At lower RH (<54% RH), only semicircle-type impedance is observed in Figure + afresh, leading to a formation of a liquid layer around the interlayer of GO sheets by two-dimensional capillary or swelling effect. This process increases the interlayer distance of GO sheets largely, which can be sufficient to accommodate a monolayer of water. The formation of a liquid layer provides a conduction path across, between GO films and electrodes as illustrated in Figure +). As a result, ion conduction, i.e., Warburg impedance Zw, appears. Therefore, Warburg impedance Zw is added in the equivalent circuit shown in Figure As the humidity increased stepwise above 54% RH, straight line-type impedance appears at the low-frequency region, and semicircle-type impedance appears at the high-frequency region shown in Figure Figure sp3-bonded hybridized carbon atoms. As RH increased stepwise above 54%, the conductivity of GO films increased sharply due to strong water-adsorption-induced ion conduction. The results are beneficial to the development of graphene-based electronics, especially graphene device arising from solution-processed GO. Finally, the exciting-frequency-dependent impedance of the GO films versus humidity was discussed, and this result suggested a potential application of GO films in impedance-type humidity sensor.We used the DC measurement and AC complex impedance spectroscopy methods to investigate the effect of ambient humidity on the electrical properties of GO films. The strong interaction of water molecules with GO films was observed through electrical characterizations. The DC measurement results show that the electrical properties of GO films were affected by ambient humidity and the amplitude of applied voltage. The electrical sensing mechanism of GO films was discussed by analyzing the characteristics of AC complex impedance spectroscopy. At low RH (<54%), GO films exhibited poor conduction property due to the presence of The authors declare that they have no competing interests.The experiment and initial manuscript were prepared by YY and XDC. XYL helped in the AFM scan. ZQW helped in the FT-IR analysis. BQZ and JFZ contributed valuable idea and useful discussion for this manuscript. All authors read and approved the final manuscript.


Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid expression system in silkworm larvae. Two constructs, full-length L1 (500 a.a) and C-terminal-deleted short L1 (479 a.a), and three PCR-manipulated antigenic loops at amino acids 55–56, 174–175, and 348–349 regions were incorporated with whole enhanced green fluorescent protein (EGFP). Expressed in full, short L1 proteins and variants were purified in heparin affinity column chromatography and confirmed by SDS-PAGE and western blot. The presence of self-assembled virus-like particles (VLPs) and EGFP incorporation on the surface of VLPs were confirmed by the observation of transmission electron and immunoelectron microscopies, respectively. HPV 6b L1 major capsid protein was successfully expressed in silkworm, and effective manipulation on the antigenic regions showed the path to versatile vaccine development based on HPV L1-VLPs.Human papillomavirus (HPV) 6b L1 capsid protein was expressed using the  Virus-like particles (VLPs) are empty virus particles, which lack virus-derived genome DNA or RNA, and composed of virus-capsid or matrix proteins. A non-enveloped virus, VLPs are mainly composed of virus-capsid proteins, which can be self-assembled both in vitro and in vivo. VLPs have been utilized as a vaccine because of its high immunogenicity and capability of inducing cellular and humoral immune responses . AlternBorrelia burgdorferi were inserted into the immunodominant c/e1 epitope region of hepatitis B virus capsid protein without the disruption of VLP’s shape . An enhz et al. ; Nassal z et al. ). In genIn this study, human papillomavirus (HPV) 6b L1 protein was expressed in silkworm larvae, and HPV 6b L1-VLPs were purified from the fat body of silkworms. HPV is a non-enveloped virus that has approximately 50–60 nm in diameter and ~8 kbp of double-stranded circular DNA genome. HPV L1 protein can be self-assembled as a VLP when this protein is expressed in various expression systems . To disHPV 6b L1 protein has a basic domain at its C-terminus, which can bind to genomic DNA and pack into virus particles  . ChimerThree L1-EGFP fusion proteins did not have green fluorescence, but EGFP was displayed on the surface of its capsomeres. To date, a genetic whole protein display system on the VLP has not been established, except for hepatitis B virus nucleocapsid protein VLP . HPV 6b L1 full-length gene . Table EcoRI-KpnI site in pFastBac1. The recombinant BmNPV CP- bacmid containing each L1-EGFP fusion gene was constructed according to the protocol described above.Each L1-EGFP fusion gene was obtained by two-step PCR. To obtain L1-EGFP55, two truncated L1 genes coding a.a.1–55 and 56–500 were amplified by PCR using primers Eco-HPV 6b L1-F and HPV 6b L1-55R-LINKER and HPV 6b L1 LINKER-56F and Kpn-HPV 6b L1-R . Five micrograms of extracted BmNPV CP- bacmid DNA, together with a helper plasmid, was mixed with 1/10 volume of DMRIE-C reagent (Invitrogen) and incubated at room temperature for an hour. This mixture was injected into silkworm larvae. The DNA-injected silkworm larvae were reared using Silkmate 2S  as a diet for 6–7 days and followed by the collection of fat body from silkworm larvae. Collected fat body was stored at −80°C before use.A recombinant BmNPV CPThe fat body was suspended in a homogenate buffer (50 mM Tris–HCl buffer [pH 7.5] containing 150 mM NaCl) and sonicated to extract expressed proteins. The homogenate was centrifuged at 30000 × g for 15 min. A supernatant was applied to the HiTrap heparin affinity column chromatography . After loading the sample, the column was washed with 20-column volumes of homogenate buffer, followed by an elution by NaCl concentration gradient to 2 M. Fractions containing L1 protein were collected and dialyzed against homogenate buffer overnight. The dialyzed sample was applied to Mono S 5/50GL column chromatography . The column was washed with 10-column volumes of homogenate buffer, and proteins were eluted by NaCl concentration gradient to 1 M.The samples were subjected to SDS-PAGE on a 10 or 12% polyacrylamide gel with the Mini-protean II system . The total number of proteins on the SDS-PAGE gel was detected with Coomassie Brilliant Blue (CBB) R-250. In the case of western blot, the proteins found in the gels were blotted onto a polyvinylidene fluoride (PVDF) membrane using the Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). After being blocked in 5% skim milk in a Tris-buffered saline containing 0.1% Tween 20 (TBST), the membrane was incubated in a 1:10000 diluted mouse anti-HPV 16 L1 antibody  for an hour. This antibody can also recognize HPV 6b L1 protein. The membrane was washed with TBST and then incubated in a 1:20000 diluted either anti-mouse or anti-rabbit labeled with horseradish peroxidase (HRP)  for an hour. Detection was performed using ECL Plus Western Blotting Reagent . Specific bands were detected using a Fluor-S/MAX Multi-Imager (Bio-Rad). Protein band intensity was analyzed by Quantity One software (Bio-Rad).The homogenate of the fat body was laid on a 25–60% sucrose density gradient. This suspension was centrifuged at 96000 × g for 3 h at 4°C. Each 0.5 ml of the fraction was collected at the top of the tube. Each fraction was analyzed by SDS-PAGE and western blot.Purified VLPs were immobilized on the grid and blocked with 4% BSA in PBS (pH 7.4). This grid was soaked in either mouse anti-HPV 16 L1 antibody diluted by 30-fold with 1% BSA in PBS for 2 h. After washing with PBS, the grid was soaked in either 10 nm gold-conjugated goat polyclonal anti-mouse IgG+IgM (H+L)  diluted by 25-fold with 1% BSA in PBS for 1 h. After washing with PBS, the grid was stained with 2% phosphotungstic acid. VLPs were observed by JEM-2100F  at 200 kV.


We demonstrate that near-edge X-ray-absorption fine-structure spectra combined with full-field transmission X-ray microscopy can be used to study the electronic structure of graphite flakes consisting of a few graphene layers. The flake was produced by exfoliation using sodium cholate and then isolated by means of density-gradient ultracentrifugation. An image sequence around the carbon K-edge, analyzed by using reference spectra for the in-plane and out-of-plane regions of the sample, is used to map and spectrally characterize the flat and folded regions of the flake. Additional spectral features in both π and σ regions are observed, which may be related to the presence of topological defects. Doping by metal impurities that were present in the original exfoliated graphite is indicated by the presence of a pre-edge signal at 284.2 eV. These occupied bands have counterparts on the unoccupied side of the Fermi edge (σ* and π* bands). In graphene sheets, the pz orbital lies perpendicular to the basal plane of the planar lattice, while the sp2 orbitals are set in the basal plane. The high directionality of these electronic orbitals is strongly reflected in the electronic properties of graphene-based materials [The demonstration of the remarkable transport properties of graphene in 2004 by Geim and Novoselov triggered intense interest in its electronic structure –11. A keaterials .−1, π*) final states being selectively excited when the E-vector lies perpendicular to the basal plane, and  final states being selectively excited when the photon E-vector lies in the basal plane. The presence of defects, nonplanarity and edges relax these symmetry selection rules. The peak energy positions and line shapes of the measured NEXAFS resonances reflect the unoccupied density of states, although the energies and intensities are modified by core–hole interactions [E-vector. Deviation from the expected signal intensity can be associated with nonplanarity, structural defects, etc. [The near-edge X-ray-absorption fine-structure technique (NEXAFS) is ideally suited for the study of graphene-based materials, because the carbon K-edge is very sensitive to the bonding environment, providing diagnostic information about the structure, defects and doping . Photoabractions . When liractions . When coractions –18. Linets, etc. .24H39O5Na) is used to promote graphite exfoliation, resulting in graphene–surfactant complexes having buoyant densities, which vary with the graphene thickness. Raman studies on samples produced by the same technique found no impurities, such as amorphous carbon, in the sample [Here we use NEXAFS spectromicroscopy, performed with the Helmholtz Zentrum Berlin (HZB) full-field transmission X-ray microscope (TXM) installed at the electron storage ring BESSY II , to image sample –22.The setup of the HZB full-field X-ray microscope  is analoWe examined several different flakes to ensure that we had selected a typical specimen; electron microscopy images are given in E parallel to the basal plane). However, we see a residual intensity due to the folding of a few graphene layers, implying that there is a nonzero angle between the polarization vector and the basal plane in places. This is most notable in the central spectrum, confirming that this region corresponds to a fold in the flake.The peak near 292 eV corresponds to the σ* threshold. This peak is composed of two distinct features at 291.5 eV and 292.6 eV. The sharp feature at 291.5 eV is an exciton , its shaE-vector of the light is always in the horizontal plane, which is thus in the plane of the graphene sheets for the unfolded area, but approximately perpendicular to the folds. Thus the blue spectrum , which could appear, for example, due to residual intercalation in the flake.We also scanned over the O 1s spectral range 528–556 eV, and the absence of signal confirms that the sample is essentially oxygen free .In summary, the distinctly different X-ray spectra of the flat and folded regions of the sample, which are related to the strong dichroism of the C 1s spectrum of graphene, have been used in a fitting procedure to generate maps of the flat and folded regions of the flake. A low-lying peak at 284.2 eV is associated with doping states in the electronic structure of the flake induced by metallic impurities present in the graphite that was exfoliated to make the multilayer graphene sample. We tentatively associate a peak at 287.4eV with defects created during the exfoliation using sodium cholate  and acquisition time.The data analysis was accomplished by using aXis2000 . The NEXThe sample was obtained from NanoIntegris  in the form of “PureSheets” (MONO). The aqueous solution (0.5 μg per 10 μL) with an ionic surfactant (2% w/v) contains pristine multistacked graphene flakes with different numbers of sheets, which have not been oxidised, reduced, or chemically modified in any way.Schematic of the morphology of the studied sample, electron microscopy images of typical samples and oxygen K-edge spectrum.File 1Additional figures.


Integration of electronics and photonics for future applications requires an efficient conversion of electrical to optical signals. The excellent electronic and photonic properties of graphene make it a suitable material for integrated systems with extremely wide operational bandwidth. In this paper, we analyze the novel geometry of modulator based on the rib photonic waveguide configuration with a double-layer graphene placed between a slab and ridge. The theoretical analysis of graphene-based electro-absorption modulator was performed showing that a 3 dB modulation with ~ 600 nm-long waveguide is possible resulting in energy per bit below 1 fJ/bit. The optical bandwidth of such modulators exceeds 12 THz with an operation speed ranging from 160 GHz to 850 GHz and limited only by graphene resistance. The performances of modulators were evaluated based on the figure of merit defined as the ratio between extinction ratio and insertion losses where it was found to exceed 220. In recent years it has become evident that bandwidth-limited electrical interconnects can no longer meet the growing demand on data processing and telecommunication traffic. Besides the limited capacity, electrical wires suffer from large energy consumption, signal attenuation and significant operational costs as interconnects densities rise. As opposed to it, photonics enables huge amounts of data to be moved at very high speeds with extremely low power over very small optical waveguides. One of the key components of the future telecommunication networks are optical modulators which serve as the gateway from the electrical to the optical domain. These are particularly attractive for low energy transmitters because they do not have a threshold that could limit the minimum operating energy and they may be easier to integrate with the available silicon platform1211Recently, the broadband electro-absorption modulator based on interband absorption of graphene was demonstrated with an overall length of 40 μm and with a modulation depth of 0.16 dB/μm. This was achieved by placing a double-layer graphene on top of a silicon waveguide0), and accordingly shifts a Fermi level: 0 is the offset voltage caused by natural doping, α is estimated from a simple capacitor model (α = ε0εd/de), ℏ is the Planck constant, vF is the Fermi velocity, and n is the electron/hole doping concentration.The proposed double-layer graphene optical modulator is based on the rib waveguide configuration with the Si rib waveguide deposited on the buried-oxide layer. To maximize the influence of the graphene on the modulator performances, the double-layer graphene was placed between Si layer (slab) and Si ridge . The dou1(ω) + iσ2(ω) of graphene, consisting of interband and intraband contributions , was obtained from the complex optical conductivity σ(ω) = σibutions .The dielectric constant of graphene varies very fast from ε(0.4 eV) = 13.66 + i6.71 to ε(0.7 eV) = −4.21 + i0.69 with a dip in the magnitude curve corresponding to μ = 0.51 where “dielectric graphene” is transforming to “metallic graphene” with ε(0.51 eV) = −0.50 + i0.50.i.e., between a buried-oxide and the Si ridge  and high-κ dielectric respectively .eff = 2.353 for the hBN spacer to neff = 2.450 for the high-κ dielectric spacer. Secondly, for the modulator with the low-κ dielectric spacer (hBN), the minimum mode effective index occurs at μ = 0.495 eV whereas for the high-κ dielectric spacer it shifts to μ = 0.500 eV. At the same time, the maximum mode effective index is not affected by the spacer and remains at μ = 0.530 eV. Apart from it, the difference between maximum and minimum mode effective index increases from Δn = 0.118 for the low-κ dielectric spacer (hBN) to Δn = 0.144 for the high-κ dielectric spacer. In terms of a chemical potential, the change between minimum and maximum mode effective index is Δμ = 0.035 eV for the hBN spacer and Δμ = 0.030 eV for the high-κ dielectric spacer. However, to achieve the same charge carrier mobility in graphene, a lower voltage change is required for the high-κ dielectric spacers. The required voltage change was calculated to be ΔV = 0.505 V for the hBN spacer while for the high-κ dielectric spacer it drops more than four times to ΔV = 0.141 V. Additionally, to introduce a π-phase shift between both arms of the graphene-based Mach-Zehnder interferometer, an active arm length of 6.57 μm is needed for structure with the hBN spacer and only 5.4 μm with the high-κ dielectric spacer.As shown in bit = 0.23 fJ/bit with calculated capacitance of 46 fF and ΔV = 0.141 V.For a 6.57 μm-long and 0.4 μm-wide modulator with the hBN spacer, the capacitance of the device was calculated to be 18 fF, which in tandem with the voltage change of ΔV = 0.505 V needed to ensure a full modulation results in an energy per bit on the order of 1.16 fJ/bit. Conversely, for a 5.4 μm-long and 0.4 μm-wide modulator with a high-κ dielectric it was calculated to be only Ebit = 0.26 fJ/bit for modulator with the hBN spacer and Ebit = 0.96 fJ/bit with the high-κ dielectric spacer respectively.Since the optical absorption in graphene can be controlled through electrical gating, the graphene can be used as the active medium in an optical electro-absorption modulator. Therefore, conductivity change induced by an applied gate voltage has a strong effect on the propagating mode . RegardlOne of the ways to increase the modulator attenuation is to push mode down into a slab such that graphene resides closer to the mode field maximum. Thus, the interaction between mode field and a graphene is maximized. It can be achieved either by increasing the slab thickness or by decreasing the ridge dimensions. In our calculations the slab thickness and ridge width were kept constant while the ridge height was decreased from 260 nm to 200 nm . The anaApart from high modulation speed, small footprint and high modulation strength (efficiency), the novel integrated modulators require large optical bandwidth for applications in on-chip optical interconnects. However, due to the poor electro-optic properties of regular materials the conventional electro-optic modulators suffer either from very large footprint or narrow bandwidth. In comparison to compound semiconductors, the ultrahigh carrier mobility and optical absorption of graphene which is independent of wavelength, enables a new optical modulators with ultra-broad optical bandwidth.To evaluate the bandwidth of the presented rib waveguide photonic modulator, the dependence of chemical potential on graphene's dielectric constant and, in consequence, on the mode effective index and MPA was studied for different wavelengths covering the entire telecommunications bandwidth. As shown in Thus, for a chemical potential of 0.512 eV, the dip in the magnitude of dielectric constant is observed at a wavelength of 1550 nm, what fulfill the requirements of maximum losses of the modulator. Away from the central wavelength, the MPA decreases. Consequently, the wavelength spanning from 1520 nm to 1580 nm only results in a decrease in modulation depth of ~1.5 dB/μm.Consequently, for a lower potential, μ = 0.46 eV, the dip in the real part of the dielectric constant moves towards longer wavelengths with a maximum MPA corresponding to λ ≈ 1710 nm. Thus, for a 1 μm-long modulator, a 3 dB optical bandwidth was calculated to be 14.1 THz near 1550 nm, 12.3 THz near 1480 nm and 13.6 THz near 1710 nm for a chemical potential of 0.512 eV, 0.54 eV and 0.46 eV respectively. For a lower chemical potential, μ = 0.42 eV, a 3 dB modulation requires at least 1.5 μm-long waveguide with the optical bandwidth calculated for a 2 μm-long modulator exceeding 15.1 THz.Apart from the chemical potential, the wavelength dependence of the MPA and mode effective index was studied for different spacer materials and for both supported modes – TE and TM . For a hFor the TE supported mode  with a mTherefore, this modulator shows broadband operation so hundreds of channels from different systems can be processed in the same device because of weak wavelength dependence.Due to the exceptionally high carrier mobility and high saturation velocity, the operation bandwidth is not likely limited by the carrier transit time. The relaxation time is inversely proportional to the degree of crystalline disorder in the graphene so with a high quality of graphene it can operates on the timescale of picoseconds, which implies that graphene-based electronics may operate at 500 GHz. In practice, the maximum operating bandwidth is limited by the RC constant of the device.i.e., small losses for OFF voltage state, to obtain a 3 dB modulation it requires to introduce a modulator to a high loss regime, which is achieved by increasing the chemical potential. For a 0.72 μm-long modulator with a low-κ dielectric spacer (ε = 3.9), a 3 dB modulation is achieved with an operation speed of 0.51 THz while keeping an energy per bit at a low level of 0.96 fJ/bit. Conversely, for modulator with a high-κ dielectric spacer, the active length of around 0.595 μm is sufficient to achieve a 3 dB modulation with an operation speed of 0.16 THz and Ebit = 0.26 fJ/bit. Thus, it can be confirmed there is fundamental energy-speed tradeoff. Additionally, it should be noted that adaptation of longer modulators will not affect the modulation speed but will improve the modulation depth which will be however, at the cost of Ebit. Furthermore, it can be summarized that low-κ dielectric spacers offer a higher operation speed but at the cost of energy and footprint  simulations at the telecom wavelengths using commercial software COMSOL. The FEM is a well know technique for numerical solution of partial differential equations or integral equations, where the region of interest is subdivided into small segments and the partial differential equation is replaced with a corresponding functional. In the calculations, the refractive indexes of the Si rib, the SiO0 = e2/(4ℏ) = 60.8 μS is the universal optical conductance, by using: g = 0.7 nm is a thickness of the graphene layer.To evaluate the mode effective index and mode power attenuation of the considered structure the gate-dependent complex dielectric constant of graphene has to be calculated. The complex dielectric function ε(ω) can be obtained from the complex optical conductivity of graphene, consisting of interband and intraband contributions, 1 = 1.2 ps for interband conductivity and τ2 = 10 fs for intraband conductivity.As the complex conductivity σ depends on the angular frequency ω, the charge particle scattering rate Γ = 1/τ with τ being the relaxation time, the chemical potential μ and temperature TJ.G. conceived the idea, designed the structures, performed theoretical calculations and FEM simulations and wrote the manuscript. D.T. supervised the project.


Interleukin-8 (IL-8) is a potent chemo-attractant cytokine responsible for neutrophil infiltration in lungs with idiopathic pulmonary fibrosis (IPF). The IL-8 protein and mRNA expression are increased in the lung with IPF. We evaluated the effect of single nucleotide polymorphisms (SNPs) of the IL-8 gene on the risk of IPF.One promoter (rs4073T>A) and two intronic SNPs (rs2227307T>G and rs2227306C>T) of the IL-8 genes were genotyped in 237 subjects with IPF and 456 normal controls. Logistic regression analysis was applied to evaluate the association of these SNPs with IPF. IL-8 in BAL fluids was measured using a quantitative sandwich enzyme immunoassay, and promoter activity was assessed using the luciferase reporter assay.p = 0.006 and 0.007, respectively). The IL-8 protein concentration in BAL fluids significantly increased in 24 subjects with IPF compared with 14 controls (p = 0.009). Nine IPF subjects homozygous for the rs4073 T>A common allele exhibited higher levels of the IL-8 protein compared with six subjects homozygous for the minor allele (p = 0.024). The luciferase activity of the rs4073T>A common allele was significantly higher than that of the rs4073T>A minor allele (p = 0.002).The minor allele frequencies of rs4073T>A and rs2227307T>G were significantly lower in the 162 subjects with surgical biopsy-proven IPF and 75 subjects with clinical IPF compared with normal controls in the recessive model (OR = 0.46 and 0.48, The common allele of a promoter SNP, rs4073T>A, may increase susceptibility to the development of IPF via up-regulation of IL-8. Idiopathic pulmonary fibrosis (IPF) is a devastating disease of the idiopathic interstitial pneumonia family. It predominantly affects the lung parenchyma and is characterized by progressive dyspnea and worsening lung function . AlthougCharacteristic of IPF is neutrophilia of the bronchoalveolar lavage fluid. The recruitment and activation of neutrophils plays a fundamental role in the development of lung injury, which precedes aberrant wound repair in the pathogenesis of IPF. InterleIn humans, the gene encoding IL-8 is located on chromosome 4q12-q21 and consists of four exons and three introns . Polymorn = 162) or using radio-clinical criteria (n = 75), i.e., the presence of clinical, functional, and high-resolution computed tomography patterns strongly consistent with IPF. None of the patients with IPF had any evidence of the underlying collagen vascular diseases clinically or by laboratory diagnosis. The institutional review board by Soonchunhyang University hospital for human studies approved the protocol, and informed written consent was obtained from all subjects.Subjects with IPF were recruited from the Korean Cohort of Interstitial Lung Disease. The study population comprised 237 patients with IPF recruited from January 1984 to November 2004 from eight university hospitals. Normal (control) subjects (n = 456) were the spouses of the patients or volunteers from the general population. Control subjects were at least 50 years old, had no respiratory symptoms, exhibited normal FVC and FEV1 , and normal findings on a simple chest posterior-anterior view x-ray. The diagnosis of IPF was based on an international consensus statement by ATS/ERS with compatible findings via surgical lung biopsy  was used to design both the PCR primers and the MGB TaqMan probes. One allelic probe was labeled with the FAM dye and the other was labeled with fluorescent VIC dye. The PCRs were run on the TaqMan Universal Master mix without UNG (Applied Biosystems), with a PCR primer concentration of 900 nM and a TaqMan MGB-probe concentration of 200 nM. The reactions were carried out in a 384-well format in a total reaction volume of 50 ul using 20 ng of the genomic DNA. The plates then were placed in a thermal cycler  and heated to 50°C for 2 min and 95°C for 10 min followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The TaqMan assay plates were then transferred to a Prism 7900HT instrument (Applied Biosystems), which measured the fluorescence intensity in each well of the plate. The fluorescence data files from each plate were analyzed using automated software (SDS 2.1). Detailed information concerning the primers is presented in additional file To genotype polymorphic sites, primers and probes were designed for TaqMang for 5 minutes. IL-8 in BAL fluids was measured using a quantitative sandwich enzyme immunoassay kit . The lower limit of detection for IL-8 was 15.6 pg/mL. Values below this limit were assumed to be 0 pg/mL for the statistical analysis. The inter- and intra-assay coefficients of variance were below 10%. Protein concentration of BAL samples was measured for standardization using a micro BCA protein assay kit .BAL had been performed in the most affected lobe by computed tomography in the 24 subjects without any immunosuppressive therapy and in the right middle lobe of 14 normal controls, as described previously. The supGGTACC(KpnI)ACATTACTCAGAAA-3', reverse; 5'-CCTACGCGT(MluI)GTCTCTGAAAGTTTG-3') for construction of the IL8 reporter plasmid. The amplified fragment of the promoter region of the IL8 gene (-79 to -743 bp from the transcription start site) was cloned using the pGEM-T easy vector system , was ligated with pGL-3 basic Luc+ reporter vector (Promega). Cloned DNA sequences were determined by a DNA direct-sequencing service . One day before transfection, 293 T cells were seeded at 5 × 105 cells per well (6-well plate) in 2 ml with 10% FBS. A 2-μg aliquot of the IL8-pGL3 basic constructor plasmid and 50 ng of PSV-galactosidase reporter vector  were diluted in 250 μl OptMEM  without serum. The 4 μl of lipofectamine 2000  was diluted in 250 ul OptMEM (GIBCO BRL) per well. The diluted DNA was combined with the diluted lipid . Then, 500 μl of transfection complex was added, and the cells were incubated at 37°C with 5% CO2 in humidified air for 48 h. β-galactosidase activity was measured by ortho-nitrophenyl-D-galactopyranoside (ONPG) hydrolysis using β-Gal Assay kit (Promega). The cells were solubilized by scraping with 400 μl of cell lysis buffer of Luciferase Assay System kit (Promega). Luciferase activity was measured using the Luciferase Assay System and luminometer . And the relative luciferase activity was normalized to the protein concentration and β-galactosidase activity.The promoter region of IL-8 was amplified using PCR. The genomic DNA fragment was isolated from B cell lines of the IPF subjects using a genomic DNA preparation kit . The first PCR product was amplified using the following primers: forward; 5'-TGCCTTTGGAAGATTCTGCT-3', reverse; 5'-GCCAGCTTGGAAGTCATGTT-3'. The primary PCR reaction mixture was diluted and used as a template for a nested PCR reaction using the nested primers containing restriction enzyme sequences  developed by Stephens et al. [We applied widely used measures of linkage disequilibrium to all pairs of biallelic loci: Lewontin's D' (|D'|)  and r2. s et al. . The gens et al. . Mantel-s et al. .p < 0.01). The subjects with biopsy-proven IPF and those with clinical IPF had the comparable impairment of FVC and DLCO.Clinical profiles of the study subjects are summarized in Table IL8 gene were genotyped in IPF patients and normal subjects . The LDs were calculated, and haplotypes of IL8 polymorphisms were constructed  and two intronic SNPs (rs2227307T>G and rs2227306C>T) within the T>A and tp = 0.009, Figure p = 0.024). The IL-8 concentration in BAL fluids did not differ among individuals with the rs2227307T>G genotype .The amount of IL-8 protein was measured in BAL fluids from 24 subjects with IPF and 14 NC. IL-8 concentrations were significantly increased in IPF patients compared with NC  vector and ONPG activity. The luciferase activity of the rs4073T>A TT allele was significantly higher than that of the rs4073T>A AA allele  were evaluated in patients with systemic sclerosis with (n = 78) or without fibrosing alveolitis (n = 50), those with cryptogenic fibrosing alveolitis (n = 71), and normal healthy subjects in the UK [IL8 with the risk of pulmonary fibrosis. The discrepancy between ours and the previously reported results may be due to the small study population in the previous study[Our logistic regression analysis of a case-control study determined that the diseases . Two SNPn the UK . These sous study or to etous study and bronous study. In addious study. This daous study. Further>A compared to those with the minor allele. This result indicates that the rs4073T>A allele within the promoter may result in increased IL-8 production when compared with the minor allele.We did not validate the association between the SNPs of the IL-8 gene in an independent replication population. We evaluate the effect of the SNP on IL-8 gene or protein expression instead. We measured IL-8 protein concentrations in the lung. IL-8 protein was increased in the BAL fluids of patients with IPF compared with normal controls. The IL-8 protein level in BAL fluid was significantly increased in the subjects with IPF having the common allele of rs4073TT>A TT allele was significantly stronger than that of the rs4073T>A AA allele. This is in accordance with a previous study, which reported that the rs4073T>A TT allele exhibited 2- to 5-fold stronger transcriptional activity than did the rs4073T>A AA counterpart [T allele on the promoter may enhance the IL-8 transcription compared with the rs4073 A allele. Putative transcription factor binding sites in the promoter of the IL8 gene were searched using the TFSEARCH and TESS websites. The candidate binding protein for the transcription of IL8 at rs4073 was eEF1A1 : Idiopathic pulmonary fibrosis; (IL-8): Interleukin-8; (ONPG): ortho-nitrophenyl-D-galactopyranoside; (|D'|): Lewontin's D'; (MHC): Mantel-Haenszel chi-square;The authors declare that they have no competing interests.MHA performed all experimental steps; BLP, SHL, and HDS analyzed statistics and wrote the manuscript; SWP, JSP, DJK and ASJ provided experimental assistance; JSP, HKS, SU, YK, YWK, SKH, KSJ, KYL, SHJ, JWP, BWC, IWP, MPC, JWS, DSK and YSS supervised this project; CSP conceptualized of the study and wrote the first draft of the manuscript. All authors read and approved the final manuscript.http://www.textcheck.com/certificate/ox3Vhg.The authors thank the editors from textcheck.com, both native speakers of English, for their proofreading for grammar and typographic errors. For a certificate, see The fluorescence labeled allelic probe for amplification of IL8, IL8RA and IL8RB genes. The data provided represent the probe for amplification of IL8, IL8RA and IL8RB genes.Click here for fileSNPs on the map of the IL8 gene, linkage disequilibrium, and haplotypes of IL8 genes. The figure provided represent the map of the IL8 gene, linkage disequilibrium, and haplotypes of IL8 genes.Click here for fileThe Minor allele frequency (MAF), Heterozygosity, Hardy-Weinberg equilibrium (HWE) of IL8 gene polymorphisms. The data provided represent the MAF, HWE of IL8 gene polymorphisms.Click here for fileThe candidate binding protein for the transcription of IL8 at rs4073. The figure provided represent the putative transcription factor binding sites in the promoter of the IL8 gene.Click here for file


Trans-activation Response DNA-binding Protein-43 (TDP-43) lesions are observed in Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration with ubiquitin inclusions (FTLD-TDP) and 25–50% of Alzheimer's Disease (AD) cases. These abnormal protein inclusions are composed of either amorphous TDP-43 aggregates or highly ordered filaments. The filamentous TDP-43 accumulations typically contain clean 10–12 nm filaments though wider 18–20 nm coated filaments may be observed. The TDP-43 present within these lesions is phosphorylated, truncated and ubiquitinated, and these modifications appear to be abnormal as they are linked to both a cellular heat shock response and microglial activation. The mechanisms associated with this abnormal TDP-43 accumulation are believed to result in a loss of TDP-43 function, perhaps due to the post-translational modifications or resulting from physical sequestration of the TDP-43. The formation of TDP-43 inclusions involves cellular translocation and conversion of TDP-43 into fibrillogenic forms, but the ability of these accumulations to sequester normal TDP-43 and propagate this behavior between neurons pathologically is mostly inferred. The lack of methodology to produce soluble full length TDP-43 and recapitulate this polymerization into filaments as observed in disease has limited our understanding of these pathogenic cascades.The protocols described here generate soluble, full-length and untagged TDP-43 allowing for a direct assessment of the impact of various posttranslational modifications on TDP-43 function. We demonstrate that Casein Kinase II (CKII) promotes the polymerization of this soluble TDP-43 into 10 nm diameter filaments that resemble the most common TDP-43 structures observed in disease. Furthermore, these filaments are recognized as abnormal by Heat Shock Proteins (HSPs) which can inhibit TDP-43 polymerization or directly promote TDP-43 filament depolymerization.These findings demonstrate CKII induces polymerization of soluble TDP-43 into filaments and Hsp90 promotes TDP-43 filament depolymerization. These findings provide rational for potential therapeutic intervention at these points in TDP-43 proteinopathies. Trans-activation response DNA-binding protein (TDP-43) is highly conserved among species and ubiquitously expressed in humans Since the identification of TDP-43 in neuronal cytoplasmic inclusions (NCI) and dystrophic neurites (DN) in FTLD and neuronal intranuclear inclusions (NII), skein-like inclusions and glial inclusions in ALS, it has become apparent that abnormal TDP-43 localization and accumulation into amorphous aggregates and filamentous structures must be related to numerous pathological stressors in vitro and ubiquitin-positive, sarksoyl-insoluble TDP-43 aggregation in vivoThe observation abnormal nuclear and cytoplasmic TDP-43 aggregation can correlate with disease progression in ALS and FTLD indicates that if this accumulation can be inhibited the resulting phenotypic changes may be alleviated magnifying the importance of understanding the mechanisms leading to these TDP-43 lesions TDP-43 forms amorphous aggregates when isolated but can assemble into filamentous structures when prompted by an activating or nucleating event indicating that endogenous TDP-43 solubility is likely regulated by its binding partners and that when not sequestered it is vulnerable to either aggregation or polymerization in vitro assembly of tagged, full-length, wild-type TDP-43 demonstrated that the protein is extremely aggregation-prone when purified to homogeneity in2 as reported, but this led to negligible Thioflavin S fluorescence characteristic of cross-β amyloid structures as shown in Previous reports on the in vivo, reactions were set up using CKI or CKII, for which TDP-43 is a known substrate. These results demonstrated this preparation of rTDP-43 is recognized by these kinases and is in a conformation that permits phosphorylation. As shown in Non-specific aggregation provides little detail as to the possible cellular activity of these preparations so to characterize the potential functionality of this soluble TDP-43 its ability to interact with known binding partners was examined. First, the ability to bind a physiologically relevant substrate was confirmed. TDP-43 is reported to specifically bind TG-repeated single-stranded DNA sequences, but not AC-repeated DNA sequences, and this assay has been used to demonstrate functionality using other tagged rTDP-43 preparations Previous reports demonstrated that phosphorylation of enriched rTDP-43 fractions with CKI would promote the polymerization of 10 nm wide filaments that could be immuno-gold labeled using anti-TDP-43 antibodies Aggregation of rTDP-43 preparations is well documented, but the presence of pore-like structures has not been reported. As shown in It is difficult to assess the true filament assembly properties of TDP-43 using traditional high-throughput techniques as the filaments are reported to be predominantly ThT- negative and TDP-43 aggregates into amorphous precipitates so readily it precludes the use of light scattering or pelleting assays that rely on ultracentrifugation in vitro while also promoting Tau function in vivoGiven that the same kinases are implicated in both the polymerization of TDP-43 and Tau filaments, and HSPs can inhibit the polymerization of Tau oligomers and filaments TDP-43 expressed using recombinant methods has been shown to be highly prone to aggregation, and this has limited research efforts directed at understanding how full-length wild-type TDP-43 can assemble into ordered filaments. Here, the data demonstrate the TDP-43 Figure 1The development of biochemical techniques to produce soluble wild-type recombinant TDP-43 without protein tags or the use of chemical stabilizers and denaturants provides the cleanest system to study the effects of post-translational protein modification on TDP-43 function. This should permit the effects of modifications such as phosphorylation, acetylation and proteolysis on TDP-43 function, protein folding, stability, oligomerization and polymerization to be studied effectively without the concerns that arise from the use of artificial constructs or the chemical modifiers used in other studies. Furthermore the assembly of this soluble rTDP-43 into 9.9 +/− 0.9 nm diameter filaments that characterize the predominant 10–12 nm forms of filamentous TDP-43 inclusions observed in ALS, FTLD and AD provides the best resource to date for studying the effects of TDP-43 filament assembly on TDP-43 function and the ability of these filaments to sequester wild-type TDP-43 or other cellular components. These filaments should more accurately represent the structures found in disease because they are formed from purified TDP-43 which does not contain potential bacterial proteins that may have contaminated enriched TDP-43 fractions used in the only other study reporting 10 nm wide filaments with TDP-43 phosphorylation The results reported here using CKII most closely recapitulate what might be expected when soluble and mislocalized, but not yet fibrillogenic, TDP-43 finds its way to the cytoplasm where it might be further modified and converted to polymerization competent forms. CKII levels are found to be up regulated in AD cases The recognition and disassembly of these CKII induced rTDP-43 filaments by Hsp90 suggest the phosphorylated TDP-43 is abnormal and contains exposed Hsp90 binding sites further strengthening the argument that these filaments are pathologically relevant. The generation of filamentous TDP-43 should facilitate the determination of the role particular TDP-43 filaments play in sequestering normal TDP-43 intraneuronally or through cell-to-cell propagation interneuronally, and the functional consequences and cellular responses of this conversion of normal TDP-43 into filamentous aggregates can be examined. Polymerized rTDP-43 filaments could be introduced into cells using protein transfection reagents, and the ability of filamentous TDP-43 to recruit normal endogenous TDP-43 from the nucleus could be determined using immunofluorescence, while the functional effects of seeding could be assessed through the examination of TDP-43 RNA client transcripts Filamentous TDP-43 inclusions form pathological lesions in some of the most prominent and debilitating neurodegenerative diseases including the motor neuron disease ALS where TDP-43 mutations are causative The polymerization of soluble wild-type TDP-43 into 10 nm filaments characteristic of ALS, FTLD and AD upon CKII phosphorylation suggests this signaling pathway is involved in disease pathogenesis. The novel techniques described here for untagged TDP-43 purification and the unprecedented demonstration that CKII can drive TDP-43 filament assembly that outpaces its generic aggregation indicates that free TDP-43 deposition may be controlled in a disease specific manner resulting from the confluence of multiple independent mechanisms. The identification of CKII as a kinase that can promote TDP-43 assembly without any further modification means this pathway may be susceptible to therapeutic modification in the treatments of these diseases, and the binding of Hsp90 to TDP-43 following phosphorylation by CKII demonstrates this protein is recognized as abnormal. The potential of targeting this CKII phosphorylated TDP-43 though pharmacological manipulation of the Hsp90 chaperones system is enhanced dramatically by the demonstration Hsp70 and Hsp90 can both inhibit CKII mediated TDP-43 filament assembly and promote the depolymerization of TDP-43 filaments once formed. Indeed CKII phosphorylation of aggregates composed of only carboxy-terminal fragments of TDP-43 has been shown to target these for disassembly in cell culture, but it was not determined whether the HSPs then promote any beneficial TDP-43 degradation also http://www.mayo.edu/research/departments-divisions/department-neuroscience-florida/brain-bank). Written informed consent from the donor or the next of kin was obtained for brain autopsy and the use of tissues for research purposes. All studies involving autopsy materials were approved by the Mayo Foundation Institutional Review Board. All animal procedures were approved by the Mayo Institutional Animal Care and Use Committee (Protocol # A30911) and are in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication 80–23).Human postmortem brain tissue was provided by the Mayo Clinic Brain Bank  was purchased from Fisher Scientific Incorporated in Pittsburg PA, and Phosphatase Inhibitor Cocktail II (#5726) and III (#P0004) were from Sigma Corporation in Cream Ridge NJ. DNAase (#5025), ADP-agarose (#A2810) and dithiothreitol (#D0632) were from Sigma-Aldrich Corporation in St. Louis MO. Bicinchoninic acid reagents  and bovine serum albumin  were from Pierce Thermo Fisher Incorporated in Rockford IL. Electron microscopy carbon/formvar coated 400 mesh copper grids (#FCF400-Cu) were purchased from Electron Microscopy Sciences in Hatfield PA. The kinases used were obtained as follows: Casein Kinase I (#P6030S) and Casein Kinase II (#P6010L) were from New England Biolabs in Ipswich MA. Streptavidin labeled horse radish peroxidase (#016-030-084) was from Jackson ImmunoResearch Laboratories in West Grove PA.2, 1 mM dithiotreitol, 1 mM PMSF, 1x PIC and 21 units/ml DNAase, pH 7.4) and lysed by probe sonication for 30–90 seconds on ice at 50% power in a Bronson sonicator. Cell debris and inclusion bodies were removed by centrifugation at 12,000×g for 20 minutes, and saturated ammonium sulfate solution was added to 15% saturation while on ice for 10 minutes. The precipitated proteins were collected by centrifugation at 12,000×g for 10 minutes, and the pellets were resuspended in 8 ml of assembly buffer  by rocking at 4°C for 30 minutes. The samples were clarified at 12,000×g for 10 minutes and dialyzed 3 times against 200 volumes of assembly buffer. The rTDP-43 was then clarified again at 12,000×g for 10 minutes, and then aliquotted and snap frozen in liquid nitrogen before storage at −80°C. Before use, the soluble rTDP-43 was collected after clarification at 100,000×g for 37 minutes, and the protein concentration was determined by BCA analysis with BSA standards. Purity was analyzed by SDS-PAGE and Coomassie brilliant blue staining.Human TDP-43 cDNA was cloned into pET30b for expression under the T7 promoter. Overnight 3 ml culture were prepared in Luria broth with 20 µg/ml kanamycin using frozen glycerol stocks and grown overnight with shaking at 37°C. These were used to inoculate 300 ml of LB media again with 20 µg/ml kanamycin, and these were grown with shaking at 37°C until the OD600 reached 0.5 absorbance. These cultures were then chilled at 4°C for 30 minutes without shaking before induction and then transferred to a 16°C shaker for overnight induction with 0.3 µM Isopropyl β-D-1-thiogalactopyranoside. Cells were then collected by centrifugation, washed 1x in Tris-buffered saline , collected and frozen at −80°C. These cells were then resuspended in lysis buffer  12 or (AC)12 DNA probes were incubated with soluble 0.1–1.0 µM TDP-43 proteins in TDP assembly buffer for an hour at 37°C. The incubation reaction was then filtered through a pretreated nitrocellulose membrane overlaid on an assembly buffer equilibrated nylon membrane in a 96-well slot blot apparatus. After washing with 3 ml of assembly buffer per well, the bound biotin-labeled (TG)12 or (AC)12 DNA probes were cross-linked to the membranes by shortwave ultraviolet radiation for 2 minutes on each side. The membranes were then blocked with 5% milk in TBS with 0.1% Triton X-100 for 60 minutes and then incubated with streptavidin-HRP diluted at 1∶5000 for 60 minutes at room temperature. The biotin-labeled DNAs on the membranes were developed with standard ECL reagents. To reduce nonspecific single-stranded DNA binding to the nitrocellulose membranes that was not TDP-43 dependent, the membranes were presoaked for 60 min in 0.4 M KOH, rinsed in water and then equilibrated in assembly buffer for 30 minutes before slot blotting. The nylon membranes were not treated, but they were also incubated in assembly buffer for 30 minutes prior to the filtration 2, pH 7.5) as recommended by the kinase provider. These reactions contained 2.5 µM rTDP-43 supplemented with 500 µM ATP and the kinase indicated. Control reactions used heat denatured kinases or 1 x KB in substitution of the kinase volume. Typically 1 µl of kinase was used in a 30 µl reaction. All reactions were incubated at 37°C without shaking for the time indicated, and then these were immediately prepared for western blotting or electron microscopy as described. Experiments to examine the effects of Hsp90 on rTDP-43 assembly were carried out as described except Hsp90 was added at 0.0–2.5 µM for the duration of the phosphorylation assembly reactions. Experiments to examine the effects of Hsp90 on pre-assembled rTDP43 filaments were carried out as described except the concentration of rTDP-43 and kinases and ATP were proportionally scaled up by a third to 3.25 µM rTDP-43, 1.3 µl CKII and 667 µM ATP and incubated as described for 2 hours to reach maximal filament assembly before adding 4× Hsp90 so the final rTDP-43 concentration was again 2.5 µM and the Hsp90 was 0.0–5.0 µM. These disassembly reactions were then incubated as described for 0–4 hours, and grids for EM were taken during this time as indicated.Recombinant TDP-43 filament phosphorylation assembly reactions were set up in 1 x AB buffer supplemented with 1x kinase buffer  for 60 minutes. Blots were then incubated overnight with either total TDP-43 antibody or phosphorylation specific S409/410 TDP-43 antibodies. Blots were developed using ECL Plus reagents and traditional film.Tissue was collected from the parahippocampal gyrus of formalin-fixed brains from pathologically confirmed cases of FTLD-TDP. All tissue was dehydrated in ethanol and embedded in LR White Reaction samples were diluted 4x in 1XTBS, and 10 µl was adsorbed onto carbon/formvar-coated 400 mesh copper grids (EM Sciences) for 45 seconds. These were then stained with 2% uranyl acetate for 45 seconds, and the grids were examined with a Philips 208S electron microscope . For quantification, twelve images were collected randomly at three predetermined coordinate locations that remained fixed throughout these studies.These 12 images were collected at 5,000× magnification, and the average filament number, average filament length and total filament length per field was measured manually using Image J freeware TDP-43 filament assembly was monitored by fluorescence of thioflavin S binding using a Cary Varian Eclipse Spectrofluorimeter  with an excitation wavelength of 440 nm and a slit width 10 nm, and emission spectrum collected from 460–600 nm and a 10 nm slit width. Measurements were performed at room temperature after incubating 30 µl of TDP-43 phosphorylation reaction with 90 µl of 0.006 mg/ml thioflavin S for 30 minutes. Negative controls included buffer alone, kinase and buffer, and reactions containing TDP-43 alone, but each with thioflavin S as above. Thioflavin S binding intensity was measured by integrating the curve between the ranges of 460–600 nm using the Cary Eclipse Scan software.


The right temporo-parietal junction (TPJ) is widely considered as part of a network that reorients attention to task-relevant, but currently unattended stimuli . Despite This viUnderstanding TPJ is important not only because attention is a core cognitive function that contributes to many other domains of information processing, but also because TPJ has been identified as a key structure in seemingly unrelated domains . In all of these domains, the attentional account of TPJ has played a critical role to either explain function or fuel debate over functional specialization . In bothThe purpose of this review is to critically evaluate two core assumptions about the role of TPJ in attentional control in order to highlight the need to reconsider theories of TPJ function. As an alternative account, we then propose the “contextual updating” hypothesis in which the function of TPJ is to update internal models of the current behavioral context for the purpose of generating appropriate actions; contextual updating is therefore particularly important when unexpected stimuli occur. A similar idea has long existed within the event-related potentials (ERP) literature with respect to the P300 component, which is thought to have a number of neural sources including TPJ .While the contextual updating hypothesis implies an unitary account for TPJ function, we readily acknowledge that it is likely that there are multiple specialized sub-regions in “TPJ” . HoweverThe review is structured in five sections: the first reviews the evidence for the role of TPJ in attention based on human functional magnetic resonance imaging (fMRI) and neuropsychology; the second reconsiders the evidence for two commonly accepted characteristics of TPJ in attention; the third discusses the contextual updating hypothesis for TPJ with respect to studies of attention; the fourth section evaluates (necessarily in brief) the generalizability of the contextual updating hypothesis to other psychological domains; finally, in the fifth section, we discuss recent anatomical work that contributes importantly to the anatomical definitions for the region of cortex broadly referred to as TPJ.22.1Perhaps the most prevalent theory of TPJ function, particularly in the right hemisphere, comes from Maurizio Corbetta and colleagues . In thisIn addition to spatially “invalid” targets, TPJ is also sensitive to relevance defined by other stimulus dimensions such as: target-similarity , cues th2.2Support for the notion that right TPJ plays a crucial role in reorienting attention to unexpected sensory stimuli comes from spatial neglect. Spatial neglect is a neuropsychological phenomenon that is defined by behavioral symptoms of a deficit in spontaneously reorienting attention to stimulus information in the contralesional visual field. Spatial neglect cannot be attributed to either sensory or motor defects alone  and is a3While the neuroimaging and neuropsychological data converge in support of the 3.1If TPJ is to send a fast-latency “interrupt” signal to the dorsal network that reorients attention, it logically follows that TPJ must generate an earlier output signal in response to sensory inputs than the dorsal system regions. However, the evidence from electrophysiology and TMS suggests that FEF actually responds earlier, not later, to sensory information than TPJ. For example, the latency of stimulus-evoked responses in FEF range from 50 to 138 ms  and whennot stimulus onset) and lasted 200 ms with an approximate peak at 200 ms  following selective damage to each hemisphere . Those fA more recent model for neglect by There appear to be clear hemispheric differences in attentional control, but there is also increasing evidence that the left TPJ plays an important role in control processes related to attention. For example, many studies in which right TPJ is found in response to task-relevant stimuli also report left lateralized activation in similar conditions, although the left hemisphere activation is rarely investigated in detail . A notabThe left TPJ is also frequently activated in studies of memory . This ha4In the previous section we reviewed evidence against the idea that TPJ provides an early stimulus-driven signal that triggers attentional reorienting. However, the existing data also clearly indicate that TPJ is engaged in processing task-relevant stimuli, particularly when they are unexpected. Here we suggest that the observed effects in TPJ can be understood in terms of “contextual updating” (see above) . AlthougThe contextual updating hypothesis is consistent with extant findings of TPJ activation in standard attentional tasks. For example, invalid targets in the Posner paradigm are infrequent and violate expectations and therefore produce an update to stimulus-response mappings of what action to take and what to expect in the future. Similarly, a valid target would also be predicted to activate TPJ, albeit to a lesser degree, because it signals the need to make a context-appropriate response based on the new occurrence of a target stimulus, but there is no violation of expectations .The contextual updating hypothesis provides similar explanations for why target-colored distracters produce TPJ activation . The appThe contextual updating hypothesis also fits in well with the overall goal of organisms to establish predictive expectancies that facilitate behavioral actions . We do nThe flexible integration between sensory, motor, and decisional regions can also be illustrated between domains. Although there are still many details that need to be specified with regard to the contextual updating hypothesis, the framework is a significant deviation from the current theories and therefore stands as a reasonable alternative hypothesis: instead of initiating “bottom-up” attentional reorienting, we hypothesize that TPJ has a more general post-perceptual function in evaluating and integrating stimulus information with internal models of task performance and expectations. We note that the contextual updating hypothesis does not leave attentional reorienting without a mechanism. It is reasonable that unattended, but relevant stimuli be assigned attentional priority through “dorsal attentional” network regions in a manner similar to other salient or relevant stimuli . More st5Any theory of TPJ function must go beyond any one sub-discipline in order to account for its involvement in different cognitive processes. The contextual updating hypothesis is appealing because similar ideas have been suggested in many domains. For example, it has been proposed that right TPJ encodes an internal model of the body and engages in a “test-for-fit” process to determine whether sensory events belong to one's own body . This hySimilarly, the contextual updating hypothesis helps to reconcile the debate between TPJ as a cognitive module for theory-of-mind (ToM) and attention tasks by suggesting a common computation occurs in both situations. The contextual updating hypothesis specifically refers to the TPJ activation and does not suggest that attentional reorienting itself does not occur during ToM . While there may be anatomically separate functional modules in the posterior parietal cortex that map onto existing cognitive domains , we arguAlthough a comprehensive review of other cognitive domains is beyond the scope of this current review, we note that the idea of contextual updating would be compatible with a number of other studies showing TPJ involvement in altruism  and empaThe broader characterization of TPJ in contextual updating is consistent with its role in attentional and social-cognitive domains. Although these situations are superficially very different, they all involve a stimulus-triggered event update of internal mental models of the external environment (or person), which are then used to predict future outcomes . A challenge for future studies of TPJ will be to provide insight into how the general function of contextual updating is made specific for a highly variable range of tasks and cognitive domains .6A critical aspect of the discussion about TPJ relates to its anatomical location. Thus far, we have used the term TPJ loosely to refer to a cluster of regions in the inferior parietal lobule extending into the superior temporal gyrus see . One posNevertheless, there are general conventions and TPJ is typically described as being near “the posterior sector of the superior temporal sulcus (STS) and gyrus (STG) and the ventral part of the supramarginal gyrus (SMG)” p. 307  cf. Fig. More prOne exciting recent development has been creation of a new cytoarchitectonic maps based on postmortem human brains . WhereasInterestingly, studies that have used the cytoarchtectonic parcelation scheme to identify fMRI activations in TPJ, have been more mixed. For example, in a cued attention task, invalid targets activated both PGa and PFm, regions in the anterior portion of the angular gyrus and posterior supramarginal gyrus . This is7Regions of the inferior parietal lobe within the supramarginal and angular gyri have commonly been referred to collectively as the temporoparietal junction. In the extant literature on attention, the exact location of TPJ has varied between studies considerably since the definition is frequently based on functional contrasts across a group rather than individual functional or anatomical locations. It is possible that different studies are actually referring to different subregions that are near anatomical neighbors. Although it may well be the case that the results we reviewed will someday be discovered to be anatomically distinct, the review was aimed at addressing the question of what common computation the results attributed to TPJ might perform; even if the specific nature of representations between subregions are separable, neighbors are likely to be computationally similar.Here we have proposed that a likely framework for TPJ function can be found in the idea of contextual updating: that the purpose of TPJ is to update internal models of the environment (including other people) for the purpose of constructing appropriate expectations and responses. These may occur for attention to simple stimuli as well as for understanding social cognition and our bodies. The anatomical connectivity between TPJ and the medial temporal lobe and frontal regions situates it well to integrate internal (or memory) representations of the current context with context-appropriate sensory-to-motor transformations. Such a framework describes well the myriad deficits that are involved with brain damage to this region and the many different tasks that produce activation in this region, but there is still much to be done to characterize the functional profile of TPJ as well its anatomical demarcations. Nevertheless, we hypothesize that the context of current events and behavioral goals will be critical to the function of this region. Much is still to be discovered about the function of TPJ, but the evidence indicates that we need to reevaluate the idea that it is involved in sending a fast-latency interrupt signal to dorsal attentional control regions and that its function is right-lateralized.