Mongolia’s riverine landscape is divided into three watersheds, differing in extent of permafrost, amount of precipitation and in hydrological connectivity between sub-drainages. In order to assess the vulnerability of macroinvertebrate communities to ongoing climate change, we consider the taxonomic and functional structures of stream communities in two major watersheds: The Central Asian Internal Watershed (CAIW) and the Arctic Ocean Watershed (AOW), together covering 86.1% of Mongolia’s surface area. We assess the consequences of the hydrological connectivity between sub-drainages on the nestedness and distinctness of the stream communities. And accordingly, we discuss the expected biotic changes to occur in each watershed as a consequence of climate change.Gamma and beta diversities were higher in the CAIW than the AOW. High community nestedness was also found in the CAIW along with a higher heterogeneity of macroinvertebrate assemblage structure. Assemblages characteristic of cold headwater streams in the CAIW, were typical of the drainages of the Altai Mountain range. Macroinvertebrate guilds of the CAIW streams exhibited traits reflecting a high stability and low resilience capacity for eutrophication. In contrast, the community of the AOW had lower nestedness and a combination of traits reflecting higher stability and a better resilience capacity to disturbances.Higher distinctness of stream communities is due to lower connectivity between the drainages. This was the case of the stream macroinvertebrate communities of the two major Mongolian watersheds, where connectivity of streams between sub-drainages is an important element structuring their communities. Considering differences in the communities’ guild structure, hydrological connectivity and different magnitudes of upcoming impacts of climate change between the two watersheds, respective stream communities will be affected differently. The hitherto different communities will witness an increasing differentiation and divergent adaptations for the upcoming changes. Accordingly, in an increasing awareness to protect Mongolia’s nature, our results encourage adapting conservation planning and management strategies specifically by watershed. Over the last two decades human-caused climate change has emerged as a leading threat to biodiversity conservation -3. The pTo predict the response of communities to climate change, we need first to understand how communities are currently structured and regulated. The structure of a stream invertebrate community is the result of several multiscale filters representing both historical factors and ecological constraints. The historical factors are considered to operate on a larger geographical scale and over geological time periods; while the ecological constraints are considered to operate mainly on the habitat scale and on shorter time scales ,13. The The impact of climate change on stream macroinvertebrate communities has been mainly studied in Europe  and Nort2). The AOW is bounded by Russia to the north, by the Hentei Mountain range to the east and by the Hangai Mountain range to the south and west. The CAIW is also bounded by Russia to the north, and is separated from China by the Gobi Desert to the south  The AOW covers only 20.6% of Mongolia’s surface area yet receives 51% of the annual precipitation of Mongolia while the CAIW covers 65.6% of Mongolia’s surface area and receives only 12% of the annual precipitation .2) The AOW has a highly connected network of sub-drainages that exit Mongolia by draining to the north, providing about 50% of the water inflow to Russia’s Lake Baikal , while the CAIW has 11 disconnected sub-drainages believed to be remnant of Tertiary and Quaternary drainages . Therefo3) The AOW is located at the boundary of the Siberian cryosphere, therefore having the highest permafrost extent in Mongolia Figure B, while http://clade.ansp.org/entomology/mongolia/] during multiple expeditions carried out in Mongolia from 2003 to 2010. Each stream site was sampled once during the 8 years timeframe. Sample sites in the AOW had a median elevation of 1432 m a.s.l.  and sample sites of the CAIW had a median elevation of 2029 m a.s.l. . This difference in altitudes of the stream-sites can be considered as an uncontrolled bias as high altitude streams from the AOW were scarce overall in the watershed and the majority were dry during the sampling expeditions. A recent sharp decrease of the glaciers in the Hangai Mountains may have contributed to the lack of summer-flow in the headwater streams of this mountain range . The frost-free period in Mongolia extends on average from late May to late August; therefore, all the sampling expeditions were carried out in July.The database for this study was obtained from 114 streams (37 from the AOW and 77 from the CAIW) sampled by the Mongolian Aquatic Insect Survey [At all sites the invertebrate communities were sampled following a kick sampling protocol using a 500 μm net mesh size and complying with the US EPA Rapid Bioassessment Protocol . Inverteet al. [Temperature, precipitation and humidity patterns, snow-cover decrease, glaciers melting and permafrost thaw are considered to be the main abiotic elements strongly affected by climate change in Mongolia. A substantial increase of the mean temperature by 1.6°C was observed over a 60-year period in Mongolia (1940-2001) . In addiet al.  found thet al. . In addiet al.  and the et al.  suggestsThese changes will be directly affecting the stream invertebrate communities. For instance, water temperature and thermal regimes regulate invertebrate population growth, population dynamics and overall niche expansion . IncreasFor these reasons, all biological traits deemed to be sensitive to or to describe affinities to temperature, disturbance, substrate structure, trophic status and eutrophication Table  were usehttp://purl.oclc.org/estimates.The total number of taxa per watershed constitutes gamma diversity. Since the numbers of sampled stream-sites per watershed were not equal, richness accumulation curves  were uset-test, (significance threshold 5%), followed by an analysis of nestedness to assess the impact of rare species on the community structure. Nestedness represents the degree to which small assemblages of taxa are subsets to successively larger assemblages and can be viewed as a region-wide outcome of a taxa pool being filtered by site-specific environmental constraints [T) of 0° shows a perfectly nested distribution (rare taxa are in diversity rich sites) and a T of 100° shows a random distribution of the taxa. Matrix temperatures were tested for statistical significance using a Monte-Carlo test (comparison to 999 randomly simulated matrices). Nestedness analysis was performed using NESTEDNESS software [Beta diversity, between stream diversity, was used to quantify the level of distinctness of the stream invertebrate communities at the watershed scale. Beta diversity values between watersheds were compared using straints ,38. To qsoftware .We excluded all rare taxa  and performed a Correspondence Analysis (CA) to describe the structure of the metacommunities. The CA was conducted on a reduced matrix of 100 taxa (59 taxa were removed) and 114 stream-sites. Prior to the CA, data were transformed to logarithmic values (ln(x + 1)) in order to reduce and normalize the variance.et al.[Taxa were assigned to particular biological trait categories Table  accordinet al.. The datBiological traits were available for 69 of the 100 taxa that occurred more than once in our data set. Therefore, the analysis was computed on a matrix of 69 taxa and 114 stream-sites. Ten biological traits described in 43 categories were included in the analysis  were computed using the ade4 package in R .p < 0.001) between the two watersheds. Values were higher at the CAIW having a median value of 124, while the median value was 68 in the AOW. The nestedness analysis showed also that communities of the CAIW had a higher nested structure than communities of the AOW . Therefore, these 3 results show that the CAIW has a higher heterogeneity of its stream communities when compared to the AOW.In total 159 taxa were identified from 114 stream-sites, including 125 different genera  and taxa adapted to intermittent stream flow . The other taxa of this assemblage were mainly trout-stream type taxa, like Suwallia sp., Suwallia teleckojensis, Isoperla sp., and Acentralla sp. While 28 stream-sites out of 77 in the CAIW (≈ 36%) were correlated to this assemblage  reported previously in glacier streams of the Himalaya and Tian-Shan Mountain ranges [The differences in the structure of communities between the two watersheds are the result of geological history and natural variation in the riverine landscape. No dams or channelization has occurred in the Mongolian rivers, so the lower biodiversities (both gamma and beta diversities) of the AOW cannot be attributed to anthropogenic homogenization across drainages [sensu, . A predin ranges . The assHigher beta diversity values and lower nestedness temperature in the CAIW confirm our initial hypothesis that lower sub-drainages connectivity would support higher distinctiveness of communities. The importance of dispersal through the riverine network was also indicated by guild structure analysis. The disconnected sub-drainages of CAIW prevent passive dispersal via the riverine network ; consequently both aquatic and aerial passive dispersals were less relevant to the structure of the communities in this watershed Figure B. In conet al.[A higher connectivity of sub-drainages also means a higher connectivity of vegetated riparian buffers that can provide sheltered habitat corridors for the dispersal of aquatic imagos. In Mongolia, such dispersal routes (i.e. following the network of riparian vegetation) can play an important role due to the structure of the Mongolian landscape. The lack of dense vegetation on the xeric Mongolian short grass steppe and a windy environment may restrict swarming activities of aquatic imagos to the riparian vegetation. This hypothesis corroborates the outcome of Petersen et al. finding The consequences of global warming on the stream communities in Mongolia will be dependent on 1) their current composition, and 2) the magnitude of the thermal and hydrological changes. Macroinvertebrates in streams of the AOW have more traits providing better resistance and resilience to disturbance, and many taxa there are typified by r-selected traits. Correspondingly, the AOW community has more taxa with r strategy, having a higher occurrence of r-selection traits . Small bBeyond the functional differences between communities of the watersheds, remarkable differences in the extent of permafrost  due to local extinction and reduced colonization by better adapted taxa. Conservation planning must incorporate the connected nature of drainages, a key factor for the conservation of freshwater biodiversity  when undThe authors declare that they have no competing interests.AM participated to the field expedition in 2010, compiled data, ran the analysis and wrote the paper. JG served as PI on the two grants supporting this work and led the expeditions of the Mongolian Aquatic Insect Survey. All authors read and approved the final manuscript.


Trilayer graphene is of particular interest to the 2D materials community because of its unique tunable electronic structure. However, to date, there is a lack of fundamental understanding of the properties of epitaxial trilayer graphene on silicon carbide. Here, following successful synthesis of large-area uniform trilayer graphene, atomic force microscopy (AFM) showed that the trilayer graphene on 6H-SiC(0001) was uniform over a large scale. Additionally, distinct defects, identified as flower-shaped domains and isolated wrinkle structures, were observed randomly on the surface using scanning tunneling microscopy and spectroscopy (STM/STS). These carbon nanostructures formed during growth, has different structural and electronic properties when compared with the adjacent flat regions of the graphene. Finally, using low temperature STM/STS at 4K, we found that the isolated wrinkles showed an irreversible rotational motion between two 60° configurations at different densities of states. The peculiar band structure of massive Dirac fermions in multilayer graphene is driving intense activity in fundamental research, as well as for applications in next generation optoelectronic devices. Most of these studies have been focused on ABA and ABC trilayer graphene, with their tunable electronic properties, which depend on the type of stacking123467910121011et al. suggested that they were caused by compressive strain212225Epitaxial graphene, as well as the other large scale graphenes, can possess a variety of structural defects that can affect the material's properties. For example, a high number of substrate steps151718Here, we investigate the effect of such corrugations on epitaxial trilayer graphene grown on 6H-SiC(0001). Scanning tunneling microscopy and spectroscopy have allowed us to study the effect of wrinkles on the structural and electronic properties of epitaxial trilayer graphene on SiC. Distinct protrusions, identified as flower-shaped domains and isolated wrinkles were observed over the sample surface and were associated with the deformation of the standard hexagonal bonding within the carbon lattice. Moreover, the results of STM/STS mapping show that these flower-shaped domains have distinctly different electronic properties when compared with their adjoining flat regions. Furthermore, the wrinkle-like structures can be rotated by 60° during STM/STS measurements, confirming that these features are flexible.The graphene used in this study was obtained by annealing 6H-SiC at 1550°C in 800 mbar argon for 10 min [see Methods]. The typical morphology of the graphene sample can be seen in the AFM image displayed in −1, corresponding to second-order Raman bands originating in the SiC substrate. Graphene contributions were also observed, identified by three main structures: i) the D band at 1355 cm−1, ii) the G band (symmetric Eg2 phonon mode) at 1595 cm−1 and iii) the 2D band at 2720 cm−1. In both spectra, the D band, which corresponds to disorder, was weak in comparison with the G and 2D bands (double resonant electron-phonon process). The low intensity of this peak showed that there was only a small number of defect/disorder in the graphene structure. This was an indication of the high quality of the epitaxial graphene produced. The G and 2D lines of the spectrum recorded on the step edge had blue shifted with respect to those of the spectrum obtained for the terrace. It is known that the position of both peaks depends on strainIn addition to acquiring AFM images, we characterized the graphene layer using Raman spectroscopy30311s XPS spectrum of the sample, collected for hν = 510 eV, is shown in 1s peak, labeled G, located at 284.5 eV in binding energy, indicates the presence of sp2 hybridized C–C bonds. The C 1s spectrum showed three components at 283.4, 284.4, and 285.3 eV in binding energy32K point of the first Brillouin zone. K-point of the Brillouin zone of graphene. The detailed dispersion of the different π-bands was well resolved, as shown in the 2D plots in panel (c). By counting the number of π-bands, three graphene layers could clearly be distinguished. In order to extract more information about the stacking geometry in our sample, we shifted the energy reference previously calculated (ab initio) band structures of both ABA and ABC stacked trilayer filmsF and ED was roughly in the range of 200 to 400 meV.We probed the electronic properties of the sample using X-ray Photoelectron Spectroscopy (XPS) and Angle Resolved Photoelectron Spectroscopy (ARPES) experiments . The C 1To obtain detailed information about the morphology of the graphene trilayer, STM images of different surface regions were also acquired. 14When the trilayer graphene was imaged, various linear defects were found over the surface. We revealed two interesting types of protrusion: isolated wrinkles and flower-shaped domains. The STM image in s = 0 mV) and another at the Dirac point (Vs = −360 mV). Interestingly, the STS spectrum obtained in the middle and on the petal-shaped domains of the flower differed from those obtained on the adjoining flat region. In addition to the expected STS of a trilayer graphene, there was a resonance peak located −264.5 mV and −344.4 mV below the Fermi level in the valence band, in the vicinity of our simple ARPES estimate of the position of the Dirac point energy. The first peak located at −264.5 mV, confirmed the presence of a strongly localized defect in the middle of the flower. Recent theoretical calculations have shown that such a peak can be induced by a highly localized impurity on graphene, causing a perturbation in the onsite potential energy at its locationIn addition to their specific structural features, the flower-shaped graphene domains exhibited a singular electronic structure, as demonstrated by the STS measurements, shown in α or β atoms of the outermost layers. To this end, theoretical LDOS derived from Density Functional Theory (DFT) calculations describing the ABA and ABC trilayers as shown in F) is shifted from the Dirac point (ED) due to substrate doping, the Dirac point in the calculated LDOS has not been aligned to the experimental position of ED in our theory spectra. Therefore, only the relative positions at the Dirac point of the peaks are relevant, suggesting a good agreement between the theoretical LDOS  could be associated with an ABC structure embedded within an ABA trilayer graphene. When compared to the usual ABA structure, ABC-stacked few-layer graphene exhibits unusual band dispersions near the Dirac level. Specifically, the low-energy bands near the Dirac level (flat band) in ABC-stacked few-layer graphene has surface states with wave functions distributed on either cal LDOS  and the cal LDOS . It is wWe spatially mapped the LDOS at three resonance peaks located at −344.4 mV, −264.5 and −83.4 mV . The gras = 0.05 V and tunneling current It = 200 pA. As can be seen, the position of the overlying wrinkle changed substantially, being rotated out of the area by 60° after occasional spectroscopy measurement instability. Recently, several theoretical studies have discussed either the rotation or the translation of graphene layers on graphite or graphene substrates432, which corresponded to a 1 nN, where A is the Hamaker constant (1 × 10−19 J), R the STM tip radius (30 nm) and D the tip - sample distance (0.7 nm). In some cases, the force generated by such interactions, seems to be sufficient to make some corrugations disappear or create new ones.The STM image in ED is consistent with that of pristine graphene. The Dirac point ED was in the same position as the Fermi level, suggesting very low charge transfer between the graphene and the substrate. This is consistent with the tunneling spectrum of quasi-free standing graphene monolayer. The curves  in D = −50 mV. The position of the Dirac point ED is slightly above the Fermi level, suggesting a charge transfer between the graphene and the substrate. It is interesting to note that the shapes of the two spectra (j and k) are identical, showing a clear correlation between the structural and the local electronic properties of the corrugations observed. The STS measurements agree with the STM morphological observations reported in Focusing now on the electronic properties of the wrinkles before and after the rotation, In the case of heteroepitaxial growth, the natural strain in the epilayers is a result of the misfit in lattice parameters and thermal expansion coefficient and of the plastic relaxation process. Epitaxial trilayer graphene on 6H-SiC(0001) can be regarded as a model system to study the interplay between epitaxy and changes in the resulting crystal structure. Trilayer epitaxial graphene on SiC is an extreme example of heteroepitaxial systems because this combined material has a large lattice misfit and a difference in crystal symmetry . This residual strain can induce a myriad of defects on the atomic-scale, on grain boundaries and on miscellaneous forms of corrugation; ripples, wrinkles, ridges and partial dislocations are encountered in all epitaxial multilayer graphenes. Partial dislocations in trilayer graphene cause direct changes in the crystal structure, in our case, for example, from Bernal stacking to rhombohedral stacking (ABA-ABC). Concerning the origin of the dislocations, our STM studies show very small holes and the spiral structure of ABC stacking indicate that the origins of the dislocations must already be present at the graphene/SiC interface. Most likely, the dislocations and the wrinkles form as a result of residual strain either during growth of the graphene on the SiC or during cooling of the samples from 1550°C to room temperature.Concerning the structural properties of wrinkles, our results suggest that these structures are flexible and the interaction with the STM tip causes an occasional rotational instability of the wrinkles during the spectroscopy measurement. Several STM experiments were performed to understand the dynamics of such features. From all the STM images taken sequentially, we noticed that a wrinkle can rotate under STM-tip effects, but its orientation is always found to be close to a high symmetry direction in the graphene latticeCareful examination of the STM images  reveals Therefore, we propose that the wrinkle could switch by rapid rotation from an initial state to a stable state in registry with the underlying graphene layer, as schematically illustrated in In summary, we have combined complementary techniques to investigate the structural and electronic proprieties of nanostructures present in epitaxial trilayer graphene. These nanostructures are interconnected flower-shaped domains and isolated wrinkles that are in fact bulging regions of the graphene layer, which appear to relieve the compressive strain induced by a mismatch in the thermal expansion coefficients of the graphene layer and its supporting SiC substrate. The flower-shaped domain is attributed a partial dislocations between ABA and ABC stacking in trilayer graphene. Our STM/STS study revealed that the wrinkles are preferentially oriented close to the high symmetry direction in the graphene lattice and they have distinctly different structural and electronic proprieties in comparison to the adjoining flat regions of trilayer graphene. Furthermore, we have found that it is possible to manipulate these corrugations and even create new ones through the tip-surface interactions during scanning, voltage pulses or spectroscopic instabilities.2) at 1550°C to produce well-ordered atomic terraces of SiC. Subsequently, the SiC sample was heated to 1000°C in a semi UHV chamber and then further heated to 1550°C in an Ar atmosphere. This graphitization process resulted in the growth of an electrically active graphene layer on top of the buffer layer, covalently bound to the substrate. The sample is cooled down to room temperature and transferred ex-situ to perform different measurements. Before STM/STS and XPS/ARPES, the sample is outgassed at 600°C for one hour at a pressure of 5 × 10−10 mbar.The graphene trilayers studied in this paper was produced via a two-step process beginning with a starting substrate of 6H-SiC(0001). Prior to graphitization, the substrate was hydrogen etched , and scanning tunneling microscopy and spectroscopy (STM/STS). The micro-Raman spectroscopy was performed at room temperature with a Renishaw spectrometer using 532 nm excitation wavelength laser light focused on the sample using a DMLM Leica microscope with a 50× objective and a power of 5 mW with spot size of about 1 μm. The AFM measurements were carried out under ambient conditions and the images were recorded in non-contact mode.The XPS/ARPES measurements were performed on the Tempo beamline at the Soleil synchrotron . The photon energy (hv = 50 eV) and sample orientation were set in order to explore the k space region around the K point of the first Brillouin zone.T = 4.2 K, the I(V) characteristics were acquired while the feedback loop was inactive, the differential conductivity dI/dV , proportional to the LDOS, was measured directly by using a lock-in technique. For this purpose a small AC modulation voltage Vmod was added to V  and the signal dI/dV detected using a lock-in amplifier in order to determine the differential conductivity dI/dVmod. Our STM tips were prepared from 0.25 mm W polycrystalline wire. They were electrochemically etched in a NaOH solution and then passivated in an HF bath. The quality of this etching procedure and the global apex shape was checked using scanning electron microscopy. The tips were then introduced into an ultrahigh vacuum preparation chamber connected to the STM chamber. There, they were flashed by Joule heating to temperatures up to white light emission. This procedure allowed the removal of contaminants and oxides from the apex.STM/STS measurements were carried out using an Omicron ultrahigh vacuum low temperature scanning tunneling microscope (UHV-LT-STM). STM/STS measurements were acquired at 4.2 K in the constant current mode for different bias voltages V applied to the sample. For the STS measurements, performed at B.L. and A.O. grow the graphene sample, E.P. characterized it by AFM and Raman spectroscopy. A.O., M.G.S. and F.S. conducted the measurements by XPS and ARPES. J.C.G. and C.D. carried out STM/STS experiments. B.L., E.P., S.L., J.C.G. and A.O. analyzed the data. B.L. and A.O. wrote the paper with all authors contributing to the final version. A.O. planned the experiments and supervised the project.


The application of graphene in sensor devices depends on the ability to appropriately functionalize the pristine graphene. Here we show the direct writing of tailored phospholipid membranes on graphene using dip-pen nanolithography. Phospholipids exhibit higher mobility on graphene compared with the commonly used silicon dioxide substrate, leading to well-spread uniform membranes. Dip-pen nanolithography allows for multiplexed assembly of phospholipid membranes of different functionalities in close proximity to each other. The membranes are stable in aqueous environments and we observe electronic doping of graphene by charged phospholipids. On the basis of these results, we propose phospholipid membranes as a route for non-covalent immobilization of various functional groups on graphene for applications in biosensing and biocatalysis. As a proof of principle, we demonstrate the specific binding of streptavidin to biotin-functionalized membranes. The combination of atomic force microscopy and binding experiments yields a consistent model for the layer organization within phospholipid stacks on graphene. The sensitivity and selectivity of graphene-based biosensors depends on attaching various functional groups to graphene. Hirtz et al. use dip-pen nanolithography to directly write phospholipid membranes on graphene, which enables multiplexed and heterogeneous non-covalent functionalization. Graphene in its pristine state and various functionalized derivatives has yielded excellent results in sensing experiments, such as single-atom gas sensitivity23111in vitro membrane model202223Dip-pen nanolithography16Here we present our results towards the fabrication of lipid membrane patches onto graphene using L-DPN, demonstrating the precise and selective spatial positioning and shaping of the pattern, enabling multiplexed and localized heterogeneous functionalization. Moreover, it is a direct writing method that does not require masks or other protective layers, therefore minimizes unwanted exposure of the graphene to additional chemicals and processing steps.N-(lissamine rhodamine B sulfonyl)  . The line features are increasing in width with increasing humidity and decreasing writing speed  and 2,691 cm−1 (2D)p-doped), the G peak broadens and the 2D to G intensity ratio increases, all of which are consistent with electron transfer from the n-doping DOPA in the lipid membrane. This can be distinguished from p-doping, where the 2D peak will further upshift rather than downshifting. In addition, no defect related D peak is observed in the graphene before and after the L-DPN writing, indicating that the lithography process does not damage the graphene.The charge transfer interaction between lipids and graphene and the integrity of the graphene during the writing process were confirmed by Raman spectroscopy. Having established that (1) lipids form uniform membranes on graphene, (2) lipid membranes can be assembled on graphene in a multiplexed fashion in close proximity, and (3) there is a charge transfer interaction between the lipid membranes and graphene, we propose that lipid membranes can be used as a non-covalent approach to present functional groups on a graphene surface for applications such as biosensors, biocatalysis and interfacing with living cells. As a demonstration for biosensing capabilities and membrane stability, we performed streptavidin-binding experiments on biotinylated lipid membranes assembled on graphene.A typical outcome of binding experiments with streptavidin is shown in The results presented above describe the controlled delivery and subsequent self-assembly of phospholipid membranes on graphene and shed light on the organization of the obtained artificial biomembranes, as will be discussed in the following. As described above, only D-layers were observed on graphene, whereas patches on silicon dioxide always consist of a base S-layer with additional D-layers on top. This agrees with previous studies of lipid patches on silicon dioxide that show additional layers on top of the thin base layer are always of same thickness—the interpretation being that the thin base layer is a phospholipid monolayer, facing headgroups towards the hydrophilic substrate, decorated by stacks of phospholipid bilayers with the hydrocarbon chains facing outwards34The situation on the hydrophilic silicon dioxide substrate is depicted in L-DPN has previously been demonstrated on a variety of substrates such as silicon dioxide, titanium and polystyreneIn summary, we have demonstrated the assembly of active lipid membranes on graphene of controlled location and size with submicron resolution. The membrane organization can be understood in terms of hydrophilicity differences between silicon dioxide and graphene. On the graphene, inverted phospholipid bilayers (hydrocarbon chains out) are formed in air that reorganize into single layers (hydrocarbon chains facing to the graphene) on immersion into buffer solution. Although the phospholipids are found to be more mobile on the graphene substrate, allowing for uniform spreading, the formed membranes are still stable enough to allow for immersion into liquid and binding of analytes from solution. More importantly, it was demonstrated that multiplexing of different membrane compositions in close proximity can be achieved on graphene by L-DPN, enabling the crucial control over spatial distribution and chemical composition lacking in self-assembled membranes yielded by vesicle fusion. On the basis of the current results and taking into account appropriate measures to control spreading in immersion, for example, by tailoring graphene devices with distinct graphene stripes or applying chemical barriers in forms of polymers or photoresist, L-DPN-generated membranes could become active elements in graphene-based sensor devices and graphene-based biointerfaces with cells.The graphene flakes used in this work were prepared by micromechanical cleavage3840−1 carrier solution of DOPC was admixed with appropriate amounts of either the fluorescently labelled Liss Rhod PE to obtain a 1-mol% concentration of Liss Rhod PE in DOPC, or the biotinylated Biotinyl Cap PE or the negatively charged DOPA to obtain a 5-mol% concentration of Biotinyl Cap PE or DOPA in DOPC, respectively.All phospholipids used in our experiments were obtained dissolved in chloroform from Avanti Polar Lipids, USA and used as received. The 20 mg ml−1.All writing processes were performed on an NLP2000 system  with M-type one-dimensional cantilever arrays (NanoInk). Matching inkwells (NanoInk) were loaded with 1.5 μl of the different phospholipid mixtures, and then dried in a vacuum desiccator for 15 min. The cantilever array was inked in the inkwells for 10 min at a humidity of 70% R.H. After inking, excess ink was wiped off by writing on a sacrificial area on the sample. Lithography on graphene was typically performed at humidities of 20–30% R.H. with a line pitch of 500 nm and a writing speed of 5–10 μm sFor the streptavidin-binding experiments, the sample was first covered with 100 μl PBS and imaged optically for characterization ). After this, PBS was replaced with 50 μl of a solution of 5% BSA in PBS and allowed to rest for 10 min. Subsequently, the BSA solution was removed and the sample was washed three times by pipetting 50 μl PBS on and off for three times each. Then the sample was incubated for 10 min with a solution of 1:100 streptavidin-cy3  in PBS. Before fluorescent imaging, the sample was again washed three times with 50 μl PBS (each time pipetting on and off for three times) and covered with 100 μl PBS in the end. All procedures were done at room temperature.−1), with a laser power well <1 mW to avoid sample damage. The peaks were fitted with a single Lorenzian line shape to determine peak position and full width at half maximum. The areas of the dot features for determining the transfer constants were measured with ImageJSurface enhanced ellipsometric contrast microscopy43M.H. and A.V. conceived the research and designed the experiments. A.O. fabricated the graphene substrates. M.H. performed the functionalization, AFM and optical characterization and binding experiments, and T.G. and A.O. performed Raman spectroscopy. M.H. and A.V. analysed and interpreted the results, in discussion with H.F. All authors contributed to writing the manuscript.How to cite this article: Hirtz, M. et al. Multiplexed biomimetic lipid membranes on graphene by dip-pen nanolithography. Nat. Commun. 4:2591 doi: 10.1038/ncomms3591 (2013).Supplementary Figures S1-S5 and Supplementary Table S1


Understanding the molecular underpinnings of chemoresistance is vital to design therapies to restore chemosensitivity. In particular, metadherin (MTDH) has been demonstrated to have a critical role in chemoresistance. Over-expression of MTDH correlates with poor clinical outcome in breast cancer, neuroblastoma, hepatocellular carcinoma and prostate cancer. MTDH is also highly expressed in advanced endometrial cancers, a disease for which new therapies are urgently needed. In this present study, we focused on the therapeutic benefit of MTDH depletion in endometrial cancer cells to restore sensitivity to cell death. Cells were treated with a combination of tumor necrosis factor-α-related apoptosis-inducing ligand (TRAIL), which promotes death of malignant cells of the human reproductive tract, and histone deacetylase (HDAC) inhibitors, which have been shown to increase the sensitivity of cancer cells to TRAIL-induced apoptosis. Our data indicate that depletion of MTDH in endometrial cancer cells resulted in sensitization of cells that were previously resistant in response to combinatorial treatment with TRAIL and the HDAC inhibitor LBH589. MTDH knockdown reduced the proportion of cells in S and increased cell arrest in G2/M in cells treated with LBH589 alone or LBH589 in combination with TRAIL, suggesting that MTDH functions at the cell cycle checkpoint to accomplish resistance. Using microarray technology, we identified 57 downstream target genes of MTDH, including calbindin 1 and galectin-1, which may contribute to MTDH-mediated therapeutic resistance. On the other hand, in MTDH depleted cells, inhibition of PDK1 and AKT phosphorylation along with increased Bim expression and XIAP degradation correlated with enhanced sensitivity to cell death in response to TRAIL and LBH589. These findings indicate that targeting or depleting MTDH is a potentially novel avenue for reversing therapeutic resistance in patients with endometrial cancer. A common problem with cancer therapy is the development of resistance, and an improved understanding of the underlying pathways involved with drug resistance could lead to the development of new strategies to overcome this resistance. A recently discovered gene, metadherin  has emerged as a potentially crucial mediator of tumor progression, metastasis, and resistance to chemotherapies In vitro and in vivo chemoresistance analyses confirmed that MTDH knockdown sensitizes various types of tumors — including breast cancer, hepatocellular carcinoma, prostate cancer, and neuroblastoma— to multiple chemotherapy agents such as 5-fluorouracil (5-FU), cisplatin, paclitaxel, and doxorubicin Initial interest in MTDH as a factor in chemoresistance arose as a consequence of NCI60 pharmacogenomic data, which found that the genomic copy number gain on 8q22 is a defining event in chemoresistance Tumor necrosis factor (TNF)-α-related apoptosis-inducing ligand (TRAIL) recently emerged as a promising targeted therapeutic strategy in various types of cancers due to its pro-apoptotic characteristics We examined the effect of depleting MTDH levels on restoring sensitivity to TRAIL-based targeted therapies. The data reported herein demonstrate that MTDH regulates cell cycle and cell survival in response to treatment with HDAC inhibitors and TRAIL, suggesting that MTDH is a promising therapeutic target to increase the efficacy of TRAIL and HDAC inhibitor combinatorial treatment.MTDH was highly expressed at the protein level in all six endometrial cancer cell lines tested  specific for MTDH. A 3.49-fold reduction in MTDH gene expression was detected by quantitative real-time PCR (qRT-PCR) in MTDH shRNA-transfected Hec50co cells compared to control scrambled shRNA-transfected cells . DepletiTo identify downstream genes mediating the tumorigenic effects of MTDH in endometrial cancer cell lines, an Affymetrix oligonucleotide microarray (Human gene array 1.0 ST) was used to detect differentially expressed genes in Hec50co cells stably transfected with either MTDH shRNA or a control shRNA . Depletion of MTDH by knockdown resulted in at least two-fold gene expression alteration of 57 genes . SeveralData from the array indicated that MTDH regulates expression of genes involved in invasion, tight junctions, as well as chemoresistance  and S2. We next sought to understand how MTDH regulates the PI3K/Akt pathway. Expression of galectin-1 is upregulated in various types of tumors and is reported to mediate tumor invasion and metastasis by increasing expression of matrix metalloproteinase MMP-9 and MMP-2 and promoting reorganization of the actin cytoskeleton To overcome resistance to therapies, HDAC inhibitors such as LBH589 have been used to restore TRAIL-induced apoptosis in various types of cancers To study the molecular mechanism of cell death in MTDH knockdown cells, expression of proteins associated with apoptosis (both pro- and anti-apoptotic factors) were tested in MTDH control and knockdown cells after single treatment (LBH589 or TRAIL) or after combined treatment (LBH589 and TRAIL). The combination treatment did not enhance expression of the TRAIL receptors DR4 and DR5. Additionally, there were no changes in expression of anti-apoptotic genes Bcl-xL, Mcl-1, or FLIP in MTDH control and knockdown Hec50co cells after the combinatorial treatment .Interestingly, the pro-apoptotic BH3-only protein Bim was up-regulated in untreated MTDH knockdown Hec50co cells compared to control cells , which iIn control cells, post-translational modification of XIAP occurred upon treatment with LBH589, as demonstrated by a higher migrating species, and reduction of both total and modified XIAP was only detected with LBH589 and TRAIL combination treatment . This efIn order to determine the precise mechanism by which MTDH confers resistance to TRAIL and LBH589 treatment, we next investigated the role of MTDH in cell cycle control. Control and MTDH knockdown Hec50co cells that were untreated or treated with TRAIL alone had similar cell cycle profiles . In contTo investigate the mechanism by which MTDH regulates the cell cycle, expression of cell cycle regulators was measured. Increase of Aurora A at the protein level was observed in MTDH knockdown cells as compared to control cells . When coAlthough MTDH has been linked to both metastasis and resistance to chemotherapy in various types of cancers st annual meeting abstract 301). Our data indicate that MTDH-mediated up-regulation of galectin-1 and CALB1 may be the mechanism by which MTDH stimulates an increase in PIP3 levels, thereby activating the PI3K/AKT/mTOR pathway. This pathway plays an important role in endometrial carcinogenesis, either by the mutation of PTEN or by other mechanisms, constitutive activation is the rule. Our identification of the over-expression of MTDH leading to increased PIP3 levels provides yet another potential explanation for the high activity of PI3K and AKT frequently found in endometrial cancers.Using a microarray approach, we identified many candidate genes that are differentially regulated in cells lacking (MTDH shRNA) or expressing (control shRNA) MTDH. Of particular interest were calbindin 1(CALB1) and galectin-1, two proteins that have previously been linked to pro-survival signaling pathways. CALB1 is a calcium binding protein with four active calcium binding domains. CALB1 was reported to interact with Myo-inositol monophosphatase (IMPA) using phage display and to induce IMPA activity up to 250-fold Inhibition of MTDH was shown to increase cell death induced by LBH589 and TRAIL combination treatment . TRAIL cIn response to LBH589 treatment alone or with TRAIL, the two principal effects of MTDH knockdown on cell cycle were a decrease in the percentage of cells in S and a concomitant increase in the percent of cells in G2/M, where the cells appear to be arrested. At the cellular level, however, knockdown MTDH alone in the absence of therapy did not affect the expression of canonical cell cycle regulatory proteins. For example, LBH589 significantly enhanced histone H3 acetylation on lysine 9  which maThis is the first report of MTDH's impact on resistance in targeted therapy for endometrial cancer. For these studies, we chose the combination of LBH589 and TRAIL because of the impact of these agents on proapoptotic pathways. We demonstrate herein that knockdown of MTDH sensitizes cells to programmed cell death though a mechanism that involves inhibition of PDK1 and Akt phosphorylation, increase in Bim, and reduction in XIAP. Therefore, we can target MTDH to enhance sensitivity to not only chemotherapy but also targeted agents that work through programmed cell death. These data provide compelling evidence that, by downregulating MTDH in endometrial cancer, we can magnify the proapoptotic pathways and response to therapy.Normal endometrial tissues and endometrial tissues from endometrial cancers were collected with the consent of patients under the University of Iowa Institutional Review Boards approved protocol. We obtained informed written consent from all participants involved in this study.2 and 95% air. To generate an MTDH knockdown model, Hec50co cells were transfected with MTDH sh RNA or control non-silencing shRNA constructs per the manufacturer's instructions . Individual cell clones resistant to puromycin were expanded and subjected to screening for MTDH expression by Western blotting.Human endometrial cancer cell lines Hec50co, RL95, AN3CA, ECC1, Ishikawa H, and KLE were maintained as described previously Normal endometrial tissues and endometrial tissues from endometrial cancers were collected from the University of Iowa Hospitals and Clinics. The endometrial disease spectrum  tissue array (UT 801) was purchased from US Biomax, Inc. .Human recombinant TRAIL (TNF-related Apoptosis inducing ligand) was purchased from R&D Systems, Inc. . LBH589 was purchased from Selleck . The following antibodies were used: anti-MTDH and anti-β-actin antibodies were from Sigma ; and anti-cleaved caspase-3, caspase-8, DR4, DR5, Bim, Bid, FLIP, XIAP, p21, PARP-1 p-AKT S473, p-AKT T308, PDK1, p-PDK1 S204, histone 3, p-histone 3 serine10 and histone 3 lysine 9 acetylation antibodies were from Cell Signaling Technology Inc. . Whole-cell protein lysates were prepared and analyzed by Western blotting as previously described mirVana miRNA Isolation Kit , and cDNA was generated using the High-Capacity cDNA Reverse Transcription Kit . A human gene array 1.0 ST  was interrogated using the biological triplicates in the DNA Core Facility at the University of Iowa, and Partek Genomics Suite  software was used to generate gene expression values. All data is MIAME compliant and the raw data has been deposited in a MIAME complaint database GEO, as detail on the website http://www.ncbi.nlm.nih.gov/projects/geo/query/acc.cgi?acc=GSE26134. The accession number for Hec50co cells is GSE26134 and for Ishikawa H cells is GSE27828. Several MTDH downstream genes were randomly chosen to validate the microarray data using TaqMan primers and probes  for quantitative RT-PCR analysis. Experiments were performed using biological triplicates.Total RNA was isolated from MTDH knockdown and control endometrial cancer cell lines  using the Cells were seeded in 96-well cell culture plates and treated the next day with the agents as indicated. The number of viable cells was determined using the WST-1 assay (Clontech Laboratories Inc.), as previously described DNA content analysis by flow cytometry has been described previously Table S1mRNAs that are more than two fold increased in control shRNA expressed cells compared with MTDH shRNA expressed Hec50co cells.(TIF)Click here for additional data file.Table S2mRNAs that are more than two fold decreased in control shRNA expressed cells compared with MTDH shRNA expressed Hec50co cells.(TIF)Click here for additional data file.Table S3mRNAs that are more than five fold increased in control shRNA expressed cells compared with MTDH shRNA expressed Ishikawa H cells.(TIF)Click here for additional data file.Table S4mRNAs that are more than five fold decreased in control shRNA expressed cells compared with MTDH shRNA expressed Ishikawa H cells.(TIF)Click here for additional data file.


While gallium scintigraphy has been recommended, This report comprises the cases of 12 Japanese patients. Two were male, and ten were female. Their age range was between 32 and 93 years. The average age of the patients was 63. We found internal involvement of sarcoidosis in eight (89%) of nine patients by positron emission tomography-computed tomography and in two (67%) of three patients by gallium scintigraphy. Myocardial sarcoidosis was detected in four (33%) of twelve patients, and specifically in three (75%) of four facial multiple plaque type sarcoidosis patients.The myocardial lesions detected by positron emission tomography-computed tomography could not be detected with conventional electrocardiogram or echogram. Positron emission tomography-computed tomography can detect sarcoid lesions of the whole body and is useful for the follow up of patients. We recommend positron emission tomography-computed tomography for those patients having cutaneous sarcoidosis, especially facial multiple plaque type sarcoidosis. Sarcoidosis is a systemic granulomatous disease with involvement of various organs . The var67Ga) scintigraphy has been recommended as a diagnostic tool, 18F-fluorodeoxyglucose positron emission tomography-computed tomography (PET-CT) imaging might be more sensitive in detecting sarcoidosis [Myocardial sarcoidosis is known as a significant complication, but Holter electrocardiographic monitoring and echocardiograms might not be sensitive enough to detect cardiac involvement . Althougcoidosis . Previoucoidosis . BecauseIn this report, we investigated the relationship of cutaneous sarcoidosis and internal involvement, including myocardial sarcoidosis, using PET-CT and Ga scintigraphy. Among 12 patients, we found that 10 patients (83%) had internal involvement . We used special stains, including, Bacille Calmette-Guérin stain, Grocott’s methenamine silver stain, periodic acid–Schiff stain or Ziehl–Neelsen stain, to visualize acid-fast bacteria, but we could not find acid-fast bacteria, fungus or bacterial cutaneous infection in any of the biopsy samples. Cutaneous sarcoidosis was diagnosed on the basis of histopathology by the pathologist.The other 12 patients were also Japanese. Two were male, and ten were female. Their ages ranged from 32 to 93 years. Their average age was 63 (Table Sarcoidosis is a multisystem granulomatous disease of unknown etiology . We perfThe various types of cutaneous sarcoidosis have been described previously, but the relation between cutaneous sarcoidosis and the severity of general sarcoidosis has been shrouded in mystery . Our patSarcoidosis is accompanied by many complications, which include various autoimmune diseases that may cause lymphadenopathy ,8. Our pMycobacterium and Propionibacterium have been identified as causative antigens of sarcoidosis [et al. suggested that the autoimmune etiology of sarcoidosis might occur through a process of molecular mimicry of infectious or other environmental antigens to host antigens [A recent report showed the relation of sarcoidosis to hepatitis C viral infection . One is coidosis . The higantigens .PET-CT can be used to detect sarcoid lesions of the whole body and is useful in patient follow-up. PET-CT has been shown to be quite useful for the detection of myocardial sarcoidosis, which was highly associated with the multiple facial plaque type of sarcoidosis. Although other associated involvements were noted in our patients, the significance in terms of the development of clinical sarcoidosis remains to be determined.Written informed consent was obtained from all of the patients for publication of this case report and any accompanying images. Copies of the written consents are available for review by the Editor-in-Chief of this journal.ACE: Angiotensin-converting enzyme; ANA: Antinuclear antibody; LN: Lymph node; PET-CT: Positron emission tomography-computed tomography; HLA: horizontal long axis; VLA: vertical long axis; SA: short axis.The authors declare that they have no competing interests.SN conceived of the study, participated in its design and coordination and drafted the manuscript. KN and KT participated in the design of the study and performed the statistical analysis. TM and YH conceived of the study. TD and HI drafted the manuscript and decided the results after discussion with SN. All authors read and approved the final manuscript.


In amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, TAR DNA binding protein 43 (TDP-43) accumulates in the cytoplasm of affected neurons and glia, where it associates with stress granules (SGs) and forms large inclusions. SGs form in response to cellular stress, including endoplasmic reticulum (ER) stress, which is induced in both familial and sporadic forms of ALS. Here we demonstrate that pharmacological induction of ER stress causes TDP-43 to accumulate in the cytoplasm, where TDP-43 also associates with SGs. Furthermore, treatment with salubrinal, an inhibitor of dephosphorylation of eukaryotic initiation factor 2-α, a key modulator of ER stress, potentiates ER stress-mediated SG formation. Inclusions of C-terminal fragment TDP-43, reminiscent of disease-pathology, form in close association with ER and Golgi compartments, further indicating the involvement of ER dysfunction in TDP-43-associated disease. Consistent with this notion, over-expression of ALS-linked mutant TDP-43, and to a lesser extent wildtype TDP-43, triggers several ER stress pathways in neuroblastoma cells. Similarly, we found an interaction between the ER chaperone protein disulphide isomerase and TDP-43 in transfected cell lysates and in the spinal cords of mutant A315T TDP-43 transgenic mice. This study provides evidence for ER stress as a pathogenic pathway in TDP-43-mediated disease. TAR DNA-binding protein 43 (TDP-43) is a protein constituent of pathologic cytoplasmic and intranuclear inclusions in neurons and glia of patients with sporadic and familial forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) Met) required to initiate protein translation in vivo remain unclear, conditions including endoplasmic reticulum (ER) stress, heat shock, oxidative stress, osmotic stress, and serum deprivation can all cause TDP-43-positive SG formation in certain cell types in cell culture systems SGs form rapidly in response to a variety of cellular insults and lead to translational repression of incorporated mRNAs ER stress and induction of the unfolded protein response (UPR) are central to ALS pathophysiology ER stress precedes the appearance of clinical features in ALS-linked mutant superoxide dismutase 1 (SOD1) transgenic rodents The chaperone protein disulphide isomerase (PDI) is induced by ER stress and is up-regulated in human sporadic ALS and in animal models of mutant SOD1-linked ALS In this study we examined whether ER stress could act as a stressor that leads to cytoplasmic accumulation of TDP-43 and subsequent incorporation of TDP-43 into SGs. Six different ALS-linked TDP-43 mutants were examined: A315T and M337V, which have been reported in multiple familial ALS pedigrees BamHI restriction enzyme site into a pcDNA3.1(+) Myc-tagged human TDP-43-encoding construct HindIII and BamHI restriction sites in pmCherry.N1 (Clontech), to allow expression of human TDP-43 with a C-terminal mCherry tag. The subsequent wildtype Myc-TDP-43-mCherry construct was used as a template to produce six ALS-linked mutant TDP-43 expressing vectors  by QuikChange site-directed mutagenesis (Stratagene) according to the manufacturer's instructions. The 218–414 TDP-43-mCherry construct was produced by cloning of a PCR product from a primer incorporating a unique HindIII restriction enzyme site immediately 5′ to the M218 codon of TDP-43 into a pCR2.1-TOPO vector (Invitrogen) with subsequent sub-cloning into pmCherry.N1 using HindIII and BamHI restriction sites. EGFP-TDP-43 constructs were as described previously Site-directed mutagenesis was performed to remove the stop codon and insert a unique g for 10 min (the SDS-soluble fraction). Protein concentrations of cell and tissue lysates were determined using the BCA protein assay (Thermo Scientific) by comparison with bovine serum albumin (BSA) standards.Mouse neuroblastoma Neuro2a (ATCC cell line CCL-131), human epithelial HeLa (ATCC cell line CCL-2) and human embryonic kidney HEK293T cell lines were maintained in high glucose DMEM (Gibco) with 10% heat-inactivated fetal calf serum (Gibco), 100 µg/mL penicillin and 100 µg/mL streptomycin. Cells were treated as indicated with thapsigargin (Sigma) to induce ER stress, MG132 (Sigma) to inhibit proteasomal function, cycloheximide (Sigma) as a general translation inhibitor, and arsenite (Sigma) as a standard oxidative stress inducer of SGs. For immunocytochemistry, cells were plated on poly-L-lysine-coated 12 mm glass coverslips. Cells were transfected with constructs using Lipofectamine2000 with PLUS reagent (Invitrogen) according to the manufacturer's protocol. Cell lysates were collected in TN buffer (50 mM Tris-HCl pH 7.5 and 150 mM NaCl) with 0.1% SDS, 1% protease inhibitor cocktail (Sigma) and 1% phosphatase inhibitor (Sigma) by incubation on ice for 10 min. The supernatant was cleared by centrifugation at 16,100 Cells were fixed with 4% paraformaldehyde at room temperature for 15 min, permeabilised with 0.1% triton X-100 in PBS for 10 min and then blocked with blocking buffer (1% BSA in 0.03% triton X-100 in PBS) for 30 min at room temperature. Primary antibodies were diluted in blocking buffer and incubated overnight at 4°C. Primary antibodies were: mouse anti-PDI Ab2792 (Abcam), rabbit anti-TDP-43 10782 (Protein Tech Group), mouse anti-HuR 39-0600 (Invitrogen), rabbit anti-ERGIC53 E1031 (Sigma), rabbit anti-XBP-1 sc-7160 (Santa Cruz), mouse anti-GADD153/CHOP sc-7351 (Santa Cruz) and mouse anti-GM130 610823 (BD Transduction Labs). Cells were washed in PBS and then incubated with secondary antibody at 1∶5000 in PBS for 1 h at room temperature. Secondary antibodies used were: goat-anti-rabbit IgG AlexaFluor® 488/594, goat anti-mouse IgG AlexaFluor® 647 or rabbit-anti-mouse IgG AlexaFluor® 488 . Nuclei were stained using TO-PRO®-3 Iodide stain (Invitrogen), Hoechst 33342 or DAPI diluted 1∶2000-1∶10000 in PBS for 10 min. Images were acquired with constant gain and offset settings where appropriate using an Olympus Fluoview 1000 inverted confocal laser-scanning microscope or an Olympus IX81 inverted fluorescence microscope with Olympus digital camera. For SG quantification, 40X images of at least five random fields of view taken at ½ radius of the coverslip were used for analysis from at least three independent experiments. Cells bearing two or more HuR-positive and/or TDP-43-positive non-nuclear foci of greater than ∼1 µm in diameter were counted as cells containing SGs using ImageJ software. For detection of ER stress, 120 cells were counted in each group. An ATF6-GFP reporter construct was co-expressed with TDP-43 constructs and cells were immunostained for GM130 to visualise the Golgi apparatus. Cells in which ATF6 fluorescence co-localised with GM130 fluorescence, indicating translocation of ATF6 to the Golgi, were scored as having ER stress A315T TDP-43 transgenic mice were obtained from Jackson Laboratories and bred on a C57BL/6 background Protein samples were electrophoresed through SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were blocked with 5% skim-milk in TBS or 3% BSA in TBS-T for 30 min then incubated with primary antibodies at 4°C for 12–16 h. Primary antibodies used were: mouse anti-FLAG M2 (Sigma), mouse anti-β-actin AC-15 (Sigma), mouse anti-TDP-43 2E2-D3 (Abnova), rabbit anti-PDI SPA-890 (Stressgen), mouse anti-V5 R960-25 (Invitrogen) and rabbit anti-red fluorescent protein . Membranes were incubated with secondary antibodies for 1 h at room temperature. Secondary antibodies used were: HRP-conjugated goat anti-rabbit AB132P or goat anti-mouse AB326P at 1∶10000 (both from Chemicon). Signals were detected using ECL reagent (Roche) with Biomax MR film (Kodak) or ChemiDoc imaging system (BioRad). Densitometry of immunoblots was performed using ImageJ software (NIH).g for 2 min, and Sepharose pellets were washed twice in TN buffer + NP40. For immunoblotting, immunoprecipitates were liberated by boiling in 4× SDS sample buffer with 20% β-mercaptoethanol.Cell lysates (1 mg protein) at 24 hr post-transfection were incubated with anti-PDI or anti-RFP antibody or irrelevant, isotype-matched control antibody  and 100 µl 50% (wt/vol) protein A-Sepharose CL-4B (Amersham Biosciences) in TN buffer with 0.5% NP40 and incubated on a rotating wheel overnight at 4°C. Samples were centrifuged at 2000 Data are presented as mean ± standard error of the mean (SEM) from at least three independent experiments and were analysed by unpaired t-test or ANOVA followed by Bonferonni's post-test unless otherwise stated. p<0.05 was considered statistically significant.Six TDP-43 mutations previously linked to ALS were chosen for investigation in this study: A315T, M337V, D169G, G294A, Q331K and N390D There was moderate cytoplasmic fluorescence in approximately 10–20% of cells expressing either wildtype or mutant TDP-43-mCherry . The subER stress was chronically induced in Neuro2a cells expressing TDP-43–mCherry by treating with 100 nM thapsigargin for 24 h. The cells were fixed 48 h after transfection, and examined by confocal fluorescence microscopy. Cytoplasmic TDP-43-mCherry was detected in approximately 50% of wildtype and mutant TDP-43 expressing cells treated with thapsigargin, compared with 10–20% of untreated cells . In compSG formation was identified by immunoreactivity to antibodies against the SG component Hu-antigen R (HuR) Both TDP-43 and HuR were located primarily in the nucleus with no evident cytoplasmic puncta in the vast majority of vehicle treated cells regardless of pre-treatment with either vehicle control, 50 µM salubrinal or 50 µg/mL cycloheximide . TreatmeNext, the role of ER stress in SG assembly was investigated by acutely treating vehicle-, salubrinal- or cycloheximide-pre-treated cells with 10 µM thapsigargin for 1 h, which is consistent with conditions reported by others previously to induce SGs These results demonstrate that ER stress is a potent inducer of TDP-43 recruitment to SGs, and that inhibiting eIF2α dephosphorylation with salubrinal specifically enhances the formation of TDP-43-positive SGs. Recruitment of TDP-43 to cytoplasmic SGs appeared to be specifically related to ER stress since treatment with arsenite to induce oxidative stress did not cause TDP-43 redistribution to SGs under the conditions used in these experiments.The sub-cellular positioning, relative to ER-Golgi proteins, of SGs formed by endogenous TDP-43 in thapsigargin-stressed was examined in HeLa cells, because their flat extended morphology lends itself to the examination of protein co-location using confocal microscopy. The TDP-43-positive SGs did not co-locate with PDI or markers of the ER or the Golgi apparatus (KDEL and ERGIC-53), implying that they form in the cytoplasm and outside of the ER-Golgi system (data not shown). We next produced a vector to allow expression of mCherry-tagged TDP-43 residues 218-414 , since fUnder basal conditions, the expression of 218–414 TDP-43-mCherry could not be detected by immunoblotting up to 72 h after transient transfection in Neuro2a cells, however diffuse cytoplasmic mCherry fluorescence was evident under confocal microscopy at very low levels in a small proportion of cells . In contNeuro2a or HeLa cells were transiently transfected with 218–414 TDP-43-mCherry, treated with MG132 and the sub-cellular location of 218–414 TDP-43-mCherry inclusions, relative to PDI and other proteins of the ER and Golgi apparatus was examined by immunocytochemistry. Small peri-nuclear inclusions accompanied by one or two larger juxta-nuclear inclusions were observed in most cells . These iIn the well-studied model of ALS induced by mutant forms of SOD1, ER stress and UPR induction precede inclusion formation Others have made the observation that CHOP, but no other UPR marker, is up-regulated following viral overexpression of wildtype TDP-43 in cell culture We showed that mutant TDP-43 induces ER stress and since abnormal TDP-43 disulphide bonding occurs in FTLD in vivo model of TDP-43 proteinopathy was then examined using spinal cord tissues from transgenic A315T mutant TDP-43 mice The involvement of PDI in an In this study, we demonstrate that ER stress leads to accumulation of both wildtype and ALS-linked TDP-43 mutants in the cytoplasm and to incorporation of TDP-43 into cytoplasmic SGs, We also demonstrate that C-terminal TDP-43 inclusions, induced by proteasome inhibition, are closely associated with PDI and the ER-Golgi compartments, suggesting potential disturbances to these organelles in TDP-43-linked disease. Indeed, over-expression of wildtype TDP-43, and to an even greater extent ALS-linked mutant TDP-43, induced ER stress via multiple ER stress signalling pathways. Also, PDI, a chaperone induced during ER stress, interacted with TDP-43 and co-located with TDP-43 in the spinal cords of transgenic mutant A315T TDP-43 mice, consistent with the increasing evidence that alterations of PDI location and function are common features in ALS. These results suggest that ER stress is involved in modulating TDP-43 sub-cellular distribution, with potential implications for triggering pathology and neuronal death in TDP-43 proteinopathies .It is well established that ER stress is an upstream event in ALS and this study confirms that ALS-associated mutant TDP-43 joins the list of disease proteins that induce ER stress. ER stress is a feature of sporadic human ALS We found that transfection with ALS-linked mutant TDP-43, and to a lesser extent wildtype TDP-43, increased ATF6 and XBP-1 activation and induced CHOP as markers of ER stress, at 18–24 h post-transfection. However at later time points the induction of ER stress was variable or not detected (data not shown). The reason for this is unclear but may be linked to accumulation of TDP-43 in the cytoplasm, which we found to vary considerably depending on time after transfection. This may explain the findings of a recent study which reported only up-regulation of CHOP, but not other markers of ER stress, in NSC-34 cells with viral over-expression of wildtype TDP-43 at 24–48 hr post-infection G93A mouse model of ALS G85R mice with hemizygous deletion of PERK C. elegans model of TDP-43 proteinopathy We also interpret our findings as showing that ER stress is a key pathway in the formation of eIF2α-mediated SGs and the incorporation of TDP-43 into these SGs. Salubrinal, which inhibits dephosphorylation of eIF2α and potentiates the PERK pathway of ER stress, markedly increased TDP-43-positive SG formation following thapsigargin treatment. This argues that ER stress induces SGs via the eIF2α-mediated pathway. Indeed, ER stress activates PERK to inhibit protein translation, which is also the primary consequence of SG formation. The PERK arm of the UPR may be a defence against ALS because salubrinal is protective in a mutant SOD1in vivo.It remains to be determined how different cellular stressors are able to differentially modulate TDP-43 redistribution into SGs. Previous studies have been conflicting in the effect of arsenite, a typical SG trigger, to form TDP-43 SGs. Some studies have indicated that arsenite does cause TDP-43 SG formation We previously provided evidence that PDI can attenuate the effects of mutant SOD1 in cell models of ALS in vivo models, in order to conclusively identify differences in dysfunction of wildtype versus mutant TDP-43 with respect to ER stress in disease. Indeed, our results also indicate that ER stress is able to drive cytoplasmic relocation of wildtype TDP-43, suggesting that upstream cellular stress responses could drive TDP-43 pathology in sporadic ALS patients. It remains important to determine the upstream triggers of TDP-43-dysfunction in ALS, and to determine if modulation of ER stress responses can be used as a target for disease modifying therapies in TDP-43 proteinopathies.In conclusion, this study demonstrates that ER stress can be triggered by over-expression of wildtype TDP-43 and to an even greater extent ALS-linked mutant TDP-43, and that ER stress modulates TDP-43 sub-cellular distribution and SG formation, reminiscent of disease features. These findings imply that TDP-43 mutation carriers may be more susceptible to ER stress induction than other ALS patients. However, further experiments will be required, possibly comparing the effects of wildytpe and mutant TDP-43 in Video S1Association of 218–414 TDP-43-mCherry inclusions with the Golgi apparatus. Neuro2a cells were transfected with the 218–414 TDP-43-mCherry construct, and treated with 10 µM MG132 for 24 h starting at 24 h post-transfection. Cells were processed for immunocytochemistry against GM130 (green) and serial z-stack images were taken at 0.50 µm intervals using confocal microscopy and post-processed using Olympus Fluoview v2.0 software. mCherry fluorescence is shown in red and nuclei (Hoechst dye) are shown in blue. Scale bar represents 10 µm.(MOV)Click here for additional data file.


An electronic nose (E-nose) has been designed and equipped with software that can detect and classify human armpit body odor. An array of metal oxide sensors was used for detecting volatile organic compounds. The measurement circuit employs a voltage divider resistor to measure the sensitivity of each sensor. This E-nose was controlled by in-house developed software through a portable USB data acquisition card with a principle component analysis (PCA) algorithm implemented for pattern recognition and classification. Because gas sensor sensitivity in the detection of armpit odor samples is affected by humidity, we propose a new method and algorithms combining hardware/software for the correction of the humidity noise. After the humidity correction, the E-nose showed the capability of detecting human body odor and distinguishing the body odors from two persons in a relative manner. The E-nose is still able to recognize people, even after application of deodorant. In conclusion, this is the first report of the application of an E-nose for armpit odor recognition. The corrected reference point [rxR(n)] is calculated by projecting the sample point onto the baseline. As a result, the baseline-corrected difference between the sample and reference resistances is calculated via the following formula:From For later data analysis and to compare data of different types of sensors, it is better to calculate the percentage change of resistance:2.2.As discussed in the introduction, most chemical gas sensors are sensitive to humidity. Therefore, if two identical samples with a different humidity are measured, the results can be different. In our work, we propose two methods as solutions to this problem. The first is a hardware-based method, where the sample was handled so as to have almost the same humidity as the background. Under such condition, the humidity signals will be equivalent for the sample and the reference, thereby only signals from the odors of interest result. To produce a constant humidity background, the carrier gas was directed to flow through a liquid water container that is immersed in a temperature-controlled heat bath see . The temThe second solution to the humidity problem is a software-based approach. A mathematical model describing the resistance of each gas sensor at different humidity level can be calibrated to subtract the humidity signal from the total signal. Although each aforementioned approach can be used independently, we have employed both schemes concurrently to achieve maximum accuracy. In addition, it should be noted that these humidity corrections could be applied in other E-nose systems or in the field conditions. Samples other than the human body odor can also be used with this algorithm. However, varying humidity (10–90%) should be tested before a measurement of a desired sample.2.3.Human body odors from armpits were collected from two male volunteers. The experiment was performed for five days with a sample collection of the armpit odors in the morning right after waking up  and in the afternoon (8 hours later). Cotton pads were used to transfer the odors from the armpits to the E-nose. A cotton pad must be in direct contact with the armpit for 10 minutes and stored in a special sample glass bottles with a screw-on closure. Once the morning samples were collected, the glass bottles were transferred to laboratory for E-nose measurement. To minimize the odor change due to bacteria, the samples, transferred via a heat-protection container, were measured within 30–50 minutes after sample collection. For the afternoon samples, E-nose measurement can be done immediately after odor sampling.During the experiment period, the volunteers were requested to go about their ordinary life and activities: for example, they took a shower twice a day (before going to bed and after waking up following the morning sample collection). To avoid fluctuation in odor samples, they were not allowed to have sex and/or consume alcohol. To study the effects from deodorant, the volunteers were requested to use deodorant, after taking shower in the morning, but only on the right arm.2.4.et al  was varied from 30% to 80%. Resistances arisen from humidity of TGS 813, TGS 825, and TGS 2602 sensors are displayed in Exponential equation of TGS 813:Exponential equation of TGS 822:Exponential equation of TGS 880:Polynomial equation of TGS 2602:Polynomial equation of TGS 825:These mathematical models were included in the data acquisition and analysis codes, thus allowing the response of sensors to humidity of the samples to be corrected on the fly. However, to achieve the maximum accuracy, hardware correction as shown in The hardware-based method helps to reduce the effect arising from the humidity reference between the reference and the sample. It can be said that the sensor signals consist in a large part of the contribution from the odors of interest. Thus, relative humidity at 75% was applied in all experiments for measuring the human body odors in this paper.3.2.The response of each sensor to the isovaleric acid prepared according the intensity levels 1 to 5 is displayed in As shown 3.3.Armpit odors of two volunteer persons were measured by an E-nose during five days using a combined hardware/software humidity correction. From M×N. The row M represents different repetition of the experiment and the column N represents the number of independent sensors. In our case, M equals to 20 and N equals to 5.Get data from matrix, XNorm(XM×N), by the mean subtraction. The mean of each N column is calculated and subtracted from the data set. Hence, the new data set has a zero value of mean.Normalize the data matrix, Cov(XM×N), and calculate eigenvectors and eigenvalues of the covariance matrix. The calculated eigenvectors must be unit eigenvectors.Calculate the covariance matrix, Rearrange the eigenvectors and eigenvalues. The eigenvectors are ordered by eigenvalues from highest to lowest, Obtain the PCA result by matrix multiplication and transpose, To allow an identification of human odors from two persons, we have adopted a pattern analysis based on principle component analysis (PCA). Only the data from the afternoon measurement was introduced into PCA. PCA is a popular statistical technique usually used to visualize in two or three uncorrelated dimensions transformed from all correlated information. In principles, PCA process contains five following steps:The PCA result is shown in 4.In this paper, the detection and classification of human body odor by E-nose measurement have been demonstrated. We have proposed a scheme to minimize the humidity effect that is usually a serious problem for the detection of human body odor. This scheme employs a humidity generator (hardware) for creating smooth background humidity that can dominate the humidity of samples, as well as mathematical models (software) for humidity response of all sensors that can be used to eliminate signals from the humidity in a real-time fashion. The E-nose in conjunction with PCA method was shown to differentiate the body odors of two persons with similar life style and activities. In addition, we have found that deodorant does not effect the relative identification of these two persons. In order to extend the discrimination of human body odors beyond two persons, a number of improvements are required such as increasing sensor types that response to a variety of volatile molecules. It is hoped that the preliminary results presented in this paper will open the door to the field of human body odor biometrics.


Capacitive sensors are the most commonly used devices for the detection of humidity because they are inexpensive and the detection mechanism is very specific for humidity. However, especially for industrial processes, there is a lack of dielectrics that are stable at high temperature (>200 °C) and under harsh conditions. We present a capacitive sensor based on mesoporous silica as the dielectric in a simple sensor design based on pressed silica pellets. Investigation of the structural stability of the porous silica under simulated operating conditions as well as the influence of the pellet production will be shown. Impedance measurements demonstrate the utility of the sensor at both low (90 °C) and high (up to 210 °C) operating temperatures. Humidity sensors are based on a wide range of working principles and technologies –4. AmongHowever, for many industrial processes a high durability and resistance to harsh environments are of particular importance. For example, industrial branches with high energy saving potential, such as wood drying or textile processing, demand sensors with high tolerance to corrosive/oxidizing species or contaminants with high molecular weight at high temperature (>200 °C). These requirements often exclude the utility of conventional sensor materials ,4 as the2.2.1.−1).Mesoporous SBA-15 silica  was syntPowder X-ray diffraction (XRD) was carried out on a “Panalytical X'Pert Pro” diffractometer . Nitrogen physisorption was conducted at −196 °C on a Quantachrome Autosorb 6; samples were degassed at 120 °C for 24 h prior to measurement. Specific surface areas were calculated from the adsorption data in the relative pressure interval from 0.05 to 0.2 using the BET method .2.2.ca. 20 kN/cm2 was found to be optimal for the stability of the pellet (see Results section). Pellets were coated with gold by vapor deposition using a Tectra Mini-Coater with quartz crystal microbalance. The gold forms porous layers to serve as electrodes, keeping silica accessible to water vapor; an optimum thickness for each gold layer was found to be 50 nm (see Results section). The gold layers were investigated by scanning electron microscopy (SEM) using a HREM EDX Leo Gemini 982 microscope. The shape of the electrodes was controlled by shadow masking. The gold-coated pellets were mounted into a specially designed sensor housing made of a high temperature stable polyimide (Vespel) as shown in A plate-type capacitor geometry was chosen for the capacitance measurements. Pellets of 23 mm diameter were made from mesoporous silica powders by using a MB 100400L hydraulic molding press . The thickness of the pellets was in the range of 0.5 to 1.5 mm. A pressure of 2.3.Humidity measurements were carried out with two different setups. For low-temperature measurements up to 95 °C an environmental chamber  was used. Up to this temperature the absolute humidity in the chamber can be regulated from 0 to 98%. For high-temperature measurements a specially designed setup was used . PressurIn both cases, low and high temperature, an impedance analyzer  was used to record the changes in capacitance of the sensor at different frequencies. For low-temperature measurements a commercially available digital humidity sensor was used as a reference  image  which sh3.2.As described above, the general principle of a capacitive humidity sensor is based on the humidity-dependent change of the dielectric constant of the porous medium which is located between the adjacent electrodes of the capacitor. This change originates from the physical adsorption of increasing amounts of water in the porous medium with increasing (relative) humidity. As a result, a change in the capacitance  becomes a measureable parameter for humidity.In order for the mesoporous silica to serve as the dielectric medium a sensor device had to be fabricated in which a defined amount of silica is placed between adjacent plate-like electrodes. The electrodes must have a defined size and distance to each other. At the same time, the silica needs to be easily accessible to water vapor. These requirements are fulfilled by forming the silica powder into a solid pellet and by applying porous gold layers at both faces of the pellet. These gold layers serve as the capacitor plates. They intrinsically exhibit porosity due to the gas-phase deposition method; their pores are wider than those in the silica pellet (see below) in order to not obstruct the accessibility of the pellet for water vapor, as will be demonstrated in the following.2 physisorption and XRD (data not shown). The results show that the pressure applied in the molding press (20 kN/cm2) does not significantly compromise the mesoporosity and periodic structural order of the sample. The specific surface area and total pore volume are slightly lower than before the molding press  The layers must be continuous and have a well-defined area for the calculation of the base capacitance. (ii) The layers must exhibit high porosity to allow diffusion of water vapor into (and out of) the dielectric layer. Both requirements can be controlled by varying the thickness of the gold layers. On the one hand only a sufficiently thick layer will be continuous and therefore exhibit defined conductivity as required. On the other hand, if the layers are too thick, then the porosity will naturally be too low. Hence, a compromise had to be found with respect to the gold layer thickness. This was accomplished by conductivity and SEM measurements. For the conductivity measurement the gold layer was contacted at opposite sides with a maximum electrode distance of 21 mm. To minimize the influence of contingent inhomogeneities at the contacts each measurement was repeated after changing the contact points.ca. 100 nm in size) which are poorly interconnected with each other. (Due to the low interconnectivity this layer could be measured by SEM only at regions close to the contact between sample and sample holder.) The thicker layer shows larger islands with good interconnectivity. At the same time large pores are visible which ensure efficient diffusion of water vapor through the gold layers. Hence, a gold layer thickness of 50 nm was chosen for the device.In the following, two gold layers with thicknesses of 10 nm and 50 nm, respectively, will be compared exemplarily. 3.3.The graph shows the change of capacitance of the sensor prepared with mesoporous silica (top) as well as the results from a SHT11 sensor (bottom) which serves as the reference ; its sigca. 8 pF from 0% to 50% rel. humidity with a signal resolution comparable to that of the commercial reference sensor.Fluctuations in both signals, especially at 25% rel. humidity, are caused by the adjustment of the climatic chamber. The signal from the silica-based sensor shows a good reliability over the entire measurement period of 24 h. The capacitance shows a strong change of To point out the above-mentioned influence of the measurement frequency on the sensing results 3.4.2O) leads to a change in capacitance of e.g., 30%. At a temperature of 210 °C a change in relative humidity from 0% to 4.5% (corresponding to 854 mbar H2O) leads to a change of 8%. The sensor shows a near-linear response behavior to relative humidity with only little influence of the temperature. The sensor response  is 1.6 at 150 °C and 1.7 at 210 °C. The data confirm that the sensor is sufficiently sensitive, e.g., for humidity control in drying processes.In the preceding section it was proven that the sensor based on mesoporous silica is suitable for the detection of 0% to 50% relative humidity at a relatively low temperature of 90 °C. For this temperature region other commercial capacitive humidity sensors are available (such as the SHT11 reference sensor), and the silica-based sensor delivers similarly reliable results. Measurements at higher temperatures  are substantially more challenging, since relative humidity is generally very low at these temperatures; in addition most organic sensor materials are not stable, therefore commercial capacitive humidity sensors are not applicable at high temperatures. The sensor presented here has a high thermal stability which makes it generally eligible for high-temperature applications. 4.A capacitive sensor design based on a mesoporous silica pellet as the dielectric with macroporous gold layers as electrodes is capable of measuring humidity at low (<90 °C) and high temperatures (up to 210 °C). Measurement frequencies of 200 kHz were found to be suitable to minimize the influence of leak currents caused by ionic surface conductivity. A linear response curve in the high-temperature region (150 °C–210 °C) and a consistent response make the sensor eligible for high-temperature applications such as industrial drying processes. The regular porosity of the mesoporous silica was proven to tolerate long-term treatment comparable to operating conditions and processing during sensor preparation.