Local communities, particularly the poorest households, benefit from work and trade opportunities offered by gold mining and agricultural companies but continue to depend on forests for house construction materials, food, and income from the sale of forest resources. However, gold mining and agricultural concessions reduce tree cover, potentially reducing access to forest resources and further marginalizing these households. Our analyses do not provide evidence that long-term resident communities contributed to forest cover loss between 2000 and 2010. We argue that landscape management, which recognizes local community rights to customary community use areas, and appropriate zoning for commercial land uses and protected areas could contribute to both local livelihoods and protect biodiversity throughout Myanmar during economic growth.Myanmar offers unique opportunities for both biodiversity conservation and foreign direct investment due to projected economic growth linked to natural resource exploitation. Industrial-scale development introduces new land uses into the landscape, with unknown repercussions for local communities and biodiversity conservation. We use participatory mapping of 31 communities, focus groups in 28 communities, and analyses of forest cover change during 2000–2010 using MODIS vegetation continuous fields images, to understand the social and environmental impacts of gold mining and agricultural concessions in Myanmar’s Hukaung Valley (~21,800 km Following the lifting of trade embargos since 2011, international economic interests can investigate opportunities offered by Myanmar’s relatively abundant natural resources and human capital14678910One example of an industrial-scale commercial land use which may expand in Myanmar is mining. Myanmar has diverse mineral resources, including gold57662326Like gold mining, the environmental and social impacts of biofuel concessions are hotly debated, even as biofuels are heralded as a sustainable alternative to fossil fuels28293438392 and has already experienced growth in industrial-scale natural resource extraction due to increases in gold mining and the granting of land for agricultural concessions. The interaction between the development of these concessions, gold mining and natural resource use by communities represents a unique landscape that grants the opportunity to understand potential environmental and social impacts of industrial-scale land use and draw lessons for future landscape management in Myanmar. Understanding the complex interactions and feedbacks between socio-economic and ecological systems that emerge through the growth of gold mines and creation of agricultural concessions can be used to propose policies that reconcile economic development and biodiversity conservation in Myanmar.This study focuses on the social and environmental impacts of gold mining and agricultural concessions in the Hukaung Valley in northern Myanmar. With Myanmar’s increased market access and a growing economy based on natural resource exploitation—industrial-scale extractive industries such as mining, logging and agricultural concessionsth century, but gold mining in the Hukaung Valley landscape has substantially increased since the mid-1990s after a ceasefire between the government and Kachin ethnic forces, and by 2001 the nearby Hpakant area was flooded by an estimated half a million migrants from other areas of Myanmar2, were granted in Hukaung Valley in 2006. Although the Jadeland concession remains largely undeveloped, the main crops grown in Yuzana have been jatropha (Jatropha spp.) and cassava (Manihot esculenta), intended for use as biofuels.Mining in Myanmar is nominally the responsibility of the Ministry of Minesth century), where the main livelihood activities are agriculture  and huntingGold mining and agricultural concessions have expanded in Hukaung Valley, but the environmental and social impacts of this expansion for local communities have not been quantified. There are 64 communities in the Hukaung Valley  Which areas of the local environment are used by communities, which natural resources do they value, and how is availability of these resources perceived to have changed as a result of expanding gold mining and agricultural concessions?(2) Do competing land uses  and landscape features  correlate with tree cover change between 2000 and 2010?2, but this ranged from 13.4 km2 to 443.2 km2. The agricultural concessions overlapped with eight mapped CUAs, completely encompassing the CUAs of two communities, which housed around 1100 people. This meant that 15% of the total mapped CUA was inside agricultural concessions. Gold mines were found inside the CUAs of three communities, and two of these communities also overlapped with the Yazana concession. 230 local natural resources were considered important by at least one of the 1061 participants in 56 focus groups. Over 20% of the 1061 participants in the focus groups valued the bamboo wanet , which is used mostly for walls and floors in house construction (Rusa unicolor), was eaten by participants, but also sold to workers at the gold mines and in the concessions. Most of the ten most important resources  were reported by all groups which discussed their population trends (21 and 26 groups respectively). Discussions of population changes in the other eight most important natural resources were more varied, with some groups reporting little change, while others reported increases or decreases. Two communities reported the bamboo waboe  to be locally extinct. During focus groups it was stated that decreases of natural resources were due to increased demand for charcoal, meat, and house construction materials on migrant workers who came to work in the gold mines and concessions.The community use areas (CUAs) around 31 communities adjacent to Hukaung Valley Wildlife Sanctuary (notified in 2004) were mapped, representing 49% of the 63 communities in the Hukaung Valley landscape. The 31 communities had been founded between seven and more than 200 years before the start of the study, with a mean community age of 50 years. In total, these CUAs supported an estimated 24,650 people, with a mean community size of 747 individuals . These 31 communities had an average CUA of 141.7 ± 102.9 (SD) kmtruction . Anotheresources  are usedQuantitative information on income was not available at the household level, but there was evidence that communities adopted new economic opportunities; ten communities identified gold mining, panning and selling as important income sources, and residents of 11 communities were employed in agricultural concessions. However, income generating activities connected to natural resource extraction and the agricultural concessions were mostly conducted by households perceived by participants as lower income. For example, gold panning and selling of minor forest products was only carried out by lower income households. These households were also more often associated with hunting, rattan and bamboo collection and wage labour for agricultural concessions. In contrast, growing opium and gold dealing were only conducted by households perceived by participants as higher income.st and 3rd quantile of altitude respectively) increased by 2.12% . Although this relationship was statistically significant in 84.5% of models between 2005–2010, it was only statistically significant for 16.5% of models between 2000–2005. Between 2005 and 2010, the two agricultural concessions granted in 2006 were strongly associated with changes in percentage tree cover, with 6.49% greater decreases in tree cover  within agricultural concessions when compared to outside  in 2000 and an annual estimate of 0.094% forest cover loss between 2000 and 2005 for Hukaung Valley Wildlife Sanctuary based on MODIS VCF and Landsat images56575859Dendrocalamus longispathus may reduce the availability of grazing fodder for wild species. Similarly, declines in Sambar deer (Rusa unicolor) population may impact threatened predator species such as the Indochinese tiger (Panthera tigris corbetti), which are found within the Hukaung Valley Wildlife Sanctuary65One drawback of using satellite data of percentage tree cover as an indicator of environmental impacts of land uses is that it does not consider other impacts such as mercury poisoning through the gold extraction process, overhunting and fishing or overuse of specific species. For example, multiple pathways link mining activities and the degradation of ecosystems that support livelihoods in Western Ghana6163Managed populations are consistent with existing governance structures in Myanmar. Governmental recognition of community managed forests in Myanmar is formalized through certified Forest User Groups that manage their own community area and activities such as reforestation. Collection of natural resources is permitted, but private agricultural use is not. The efficiency and inclusiveness of Forest User Groups variesRapid changes in social and environmental conditions will likely occur across Myanmar in the near future as annual rates for economic growth are expected to be as high as 8% and tightly linked to natural resource exploitation242, these protected areas are smaller than the proposed area due to removal of a large central area  includes tropical and temperate broadleaf forests, conifer forests and some areas cleared for human activities including shifting cultivationral area , but remCUAs of 31 communities within the Hukaung Valley landscape were mapped between 2005 and 2010 through collaborations between community inhabitants, the Nature and Wildlife Conservation Division (NWCD) of the Forestry Department of the Ministry of Environmental Conservation and Forests and the Wildlife Conservation Society (WCS) Myanmar field team. Communities adjacent to the Hukaung Valley Wildlife Sanctuary (notified in 2004) were selected for participation. The CUAs were mapped as part of participatory rapid rural appraisals conducted by NCWD and WCS Myanmar to initiate long-term relationships with communities and develop sustainable natural resource management plans. The NCWD and WCS field team met with the head of each community and explained their intention to map the areas used for crops, timber and non-timber forest products. Community elders and those considered knowledgeable were consulted about the traditional CUA boundaries and constructed a sketch map. Knowledgeable individuals in each community accompanied the NCWD and WCS team to map the sketched boundaries with a GPS. Boundaries between neighboring communities were established through joint meetings with both communities. Once the CUA was mapped and approved by community members, a shapefile of all CUAs was produced by WCS Myanmar shown in .ca. 20,000) participated and provided information on the uses and management of key resources, and details of conflicts and issues associated with natural resource use. As part of the participatory rapid rural appraisals, the NCWD and WCS also collected information on the date communities were founded and number of residents.In 28 communities , male and female focus groups were formed  and participants asked to list anything eaten, sold or extracted from forested areas surrounding the community. Focus groups are routinely used in the social sciences as a cost-effective method of gathering data on specific themes. They can be used to generate a broad range of data, including highlighting consistent and contrasting attitudes, perceptions and beliefs2 grid cells consistent with the MODIS VCF products. The VCF 2000 product was cross-validated using a ground-truthed supervised land cover classification of Landsat 7ETM+ imagery from February 2000–20012 = 0.9708) with percent tree cover calculated from a Landsat 5 image811155We conducted separate analyses for the periods 2000–2005 and 2005–2010 to examine tree cover changes after two agricultural concessions were granted in 2006. We used the Moderate Resolution Imaging Spectroradiometer (MODIS) vegetation continuous fields (VCF) product for the years 2000, 2005, and 20102 raster grid squares which were in forested areas of the Hukaung Valley landscape in 2000 (including inside CUAs). For each grid square, dependent and independent variables were extracted from the rasters using the package ‘raster’ in R 3.2.1To investigate the impact of different human activities on forest across the whole landscape, analyses were conducted on the 230 mTo access the impact of different drivers of forest change within community use areas, we generated 40 random points inside each mapped CUA within the Hukaung Valley landscape. Additional independent variables (population density in the CUA and number of years before 2010 that the community was founded) were extracted from the participatory rapid rural appraisals at each community. For consistency in the analysis, two communities were excluded as the majority of their CUA lay outside the Hukaung Valley landscape, leaving a total of 29 communities. For each community, the mean distance to gold mines, accessibility and water bodies were calculated from the 40 random locations, and each community was classified as either inside (8 communities) or outside (21 communities) an agricultural concession. For the eight communities classified as inside an agricultural concession, between 2% and 100% of the CUA lay inside the concession (median = 54%). As there were only 29 communities, altitude and slope could not be considered in this analysis, due to insufficient degrees of freedom for robust analyses. Altitude and slope were excluded as an exploratory analysis did not suggest any relationship between mean altitude or slope and changes in tree cover within CUAs.How to cite this article: Papworth, S. et al. The impact of gold mining and agricultural concessions on the tree cover and local communities in northern Myanmar. Sci. Rep.7, 46594; doi: 10.1038/srep46594 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Numerical calculations show good qualitative agreement with the experimentally obtained results for the photoresponse of G-FET.Graphene/semiconductor Schottky junctions are an emerging field for high-performance optoelectronic devices. This study investigates not only the steady state but also the transient photoresponse of graphene field-effect transistor (G-FET) of which gate bias is applied through the Schottky barrier formed at an n-type Si/graphene interface with a thin oxide layer, where the oxide thickness is sufficiently thin for tunneling of the charge carrier. To analyze the photoresponse, we formulate the charge accumulation process at the n-Si/graphene interface, where the tunneling process through the SiO A graphene/semiconductor heterojunction is an emerging field of recent optoelectronic devices because of their low light absorption of graphene of 2.3%2, which is appropriate for high-performance transparent electrodes of optoelectronic devices such as photosensors and photovoltaic applications13. In such applications, the charge carrier transport through the graphene/semiconductor heterojunction is crucially important to assess the device performance. Generally, a graphene contact to the doped semiconductor substrates is described as a Schottky contact, which is useful in high-performance optoelectronics and photovoltaic devices21. In addition, the presence of the thin tunnel barrier at graphene/semiconductor interface modifies the current-voltage characteristics of the junction. In the case of a metal/Si Schottky junction with a 2–10-nm-thick oxide layer at the interface, which is sufficiently thin for tunneling of charge carriers, the open circuit voltage and short circuit current under light irradiation are strongly suppressed because the photogenerated carriers recombine at the interface states in the thin oxide followed by tunneling23. Photoresponse similar to the ideal Schottky diode is observed only in a reverse bias regime. Similar characteristics were observed for a graphene-Si Schottky contact20. In this case, both the trap state at the interface and the unique density of states of graphene should be considered for detailed analyses, where the energy position of Fermi level of graphene can be modulated by the field effect through the oxide layer.Graphene is anticipated for use in high-performance electronic devices because of its extraordinary carrier transport property with single-atomic-layer thickness11. Although detailed analysis of current–voltage characteristics of the graphene/Si Schottky junction with thin oxide layer has been conducted14, detailed analysis of the G-FET with the Si Schottky junction with the thin oxide gate remains an open subject.The transfer characteristics of graphene field-effect transistor (G-FET) are governed by the induced charge carrier density by application of the gate voltage. For a graphene/doped Si junction with thin oxide layer at reverse bias, the induced charge carrier in graphene should be balanced to that of Si depletion layer induced by the reverse bias corresponding to the gate bias. Under light irradiation, the excess charge carrier generated in the depletion layer by light irradiation is accumulated at the Si/oxide interface because of the presence of the thin oxide layer, which increases the charge carrier density in the graphene and which consequently increases the drain current of the G-FET. This effect can be used to amplify the photoresponse of the graphene/Si Schottky junction, where the photoresponse appears on the drain current of G-FETx layer is considered in addition to the recombination of the accumulated holes and the electrons in the graphene at the surface states on SiOx layer.This study investigates not only the steady state but also the transient photoresponse of G-FET of which gate bias is applied through the Schottky barrier formed at the n-type Si/graphene interface with a thin oxide layer, where the oxide thickness is sufficiently thin to tunnel the charge carrier. To analyze the photoresponse, we formulate the charge accumulation process at n-Si/graphene Schottky interface, where the tunneling process through the SiOx layer acts as the insulating layer of the gate. All processes were performed in air, so that the channel part of the graphene is contacted to the Si surface through a 2–5 nm thick native oxide layer, which is sufficiently thin to tunnel the charge carrier23. As depicted schematically in Fig. VGS) corresponding to the reverse bias of the Si/graphene Schottky junction.Figure VGS > 0, the photoinduced hole–electron pair at the depletion layer is separated by the build-in potential, as presented schematically in Fig. −1, so that the penetration depth of the 623 nm light is ~3 μm, which is much greater than the depletion layer thickness. Consequently, the hole–electron pair is generated throughout the depletion layer. The photo-excited electrons at the depletion region drift to the back gate electrode that is positively biased. The holes at the depletion region also drift toward the graphene channel. Thus, the either direct or trap controlled recombination of photogenerated hole and electron in the depletion region is suppressed. For an ideal Schottky junction, the holes are swept out to the graphene without restriction. However, under the presence of the thin SiOx, the holes are accumulated at the interface, followed by tunneling to the graphene, which closely resembles the inversion layer at MOS interface for n-type semiconductor. In this case, the electrons are induced in the graphene as the counter charge of the accumulated holes at the interface, which engenders the Fermi level shift of graphene. Additionally, one must consider recombination of the photogenerated holes and electrons in the graphene through the recombination center at SiOx. Consequently, the output current (IDS) of the G-FET is increased by light irradiation depending on the accumulated holes at the Si/SiO2 interface. Similar concept of the trap controlled band modification for the enhancement of photosensitivity has been reported in organic photodiodes28.At IDS, which is determined by the carrier density of graphene nG. For simplicity, we assume that the recombination life-times of both of electrons and holes are the same to match τR of the Shockley–Read–Hall (SRH) recombination model29. Furthermore, we assume that the intrinsic carrier density is negligibly small. Therefore, the resultant rate equation of accumulated holes, pSi, at the Si/SiO2 interface can be expressed asx and the SRH recombination of the accumulated holes and the electrons in graphene at SiOx. Also, τT−1 stands for the tunneling probability of the accumulated holes, τR−1 expresses the recombination rate of the accumulated holes and the electrons in graphene at SiOx, and g denotes the photo-generation rate of holes in the depletion layer. The density of electrons in graphene at Fermi level EFG is known to be32.EDP is the energy position of the charge neutral point, the Dirac point, vF represents the Fermi velocity of charge carriers in graphene (~108 cm/s). The tunneling probability τT−1 of holes into graphene is proportional to the graphene density DG(ETG) as final states for tunneling, where ETG is the energy position for hole tunneling. The graphene density at energy E isEFG − EDP . At VGS higher than that at the gate voltage corresponding to the Dirac point (VDirac) under the dark condition, the excess VGS (=VGS − VDirac) is applied for development of the depletion layer, so that one can assume EFG ~ EDP . At this condition, the energy state for tunneling of accumulated holes into graphene, which corresponds to the energy position of valence band top of Si at the SiO2/Si interface, is defined as Ev0. To clarify the tunneling process under light irradiation, we investigate the relation between ETG and Ev0, where we consider the Fermi level shift of graphene ΔEFG = EFG − EDP and the additional potential drop at the thin SiOx, ΔVOX, because of nG as the counter charge of accumulated holes induced by photoexcitation, pSi. The relation between ETG and Ev0 under the light irradiation is given asBased on the model described above, we will formulate the light-intensity dependence of nG at dark is negligible. In this case, one can expect nG ~ pSi. As described above, tunneling probability τΤ−1 is proportional to the final density of states asα is the constant for proportional factor including the transmittance of tunneling electrons through the thin SiO2. Inserting this equation to Eq.  dependence of pSi should be expressed as gθ with θ = 1 at the steady state, i.e., dpSi/dt = 0. For the case in which β2 originating from the voltage across the thin oxide layer is dominant at stronger light intensity, θ is approx. 0.5. As presented in Fig. Supplementary Information, the numerically calculated light-intensity dependence of pSi shows power law dependence with two slopes. It is noteworthy that nG ~ pSi. Thereby, one can expect that the light-intensity dependence of IDS shows power law dependence of gθ with θ < 1.Using the combination of Eqs  and 4),,4), one mentclass2pt{minimCG–VGS) characteristics of the Schottky junction were investigated with or without light irradiation, where the measured capacitance CG is a series capacitance consisting of a quantum capacitance of graphene CQ, the thin native oxide layer COX, and the depletion layer at the Si/SiOx interface, CD. As presented in Fig. VGS < −0.5 V corresponding to the accumulation condition for MOS junction, the gate capacitance CG decreases with increasing VGS because of the decrease of CQ34. The maximum capacitance of CG−1 = Cox−1 + CQ−1 at VGS < −1 V is greater than 0.65 μF/cm2. The SiOx layer thickness is estimated as 5 nm or less. The corresponding band diagram is portrayed in Fig. Supplementary Information. To clarify the light irradiation effect on CG, the CG difference between the dark and the light conditions, ΔCG, is shown against VGS in Fig. VGS < −0.5 V, ΔCG shows no marked light-intensity dependence.To investigate the depletion layer at the junction, the capacitance–voltage  by the Schottky barrier. Under the light irradiation, IG increases concomitantly with increasing light intensity to 63 nA at 641 μW/cm2. In this region, the photo-excited electrons are swept out through the depletion layer of n-Si, whereas the photo-excited holes are tunneled to the graphene through SiOx layer or recombined at the graphene-Si interface at the thin SiOx layer, as presented in Fig. IG at −0.5 V < VGS < 0.2 V under light illumination is suppressed, unlike the common Schottky junction behavior. This behavior is commonly observed at the Schottky junction of lightly doped n-Si with thin SiOx layer23. It originated mainly from the presence of thin SiOx layer at the interface, resulting in the increase of the direct carrier recombination of the photogenerated carriers at the depletion layer of n-Si14.Figure 1 at VG < VDirac. The drain current IDS at VG > VDirac in a dark condition remains almost constant, which implies that EFG ~ EDP, as assumed in the numerical calculations. Under light irradiation, IDS increases concomitantly with increasing light intensity of 8–11 μA with saturation behavior at higher VGS, where VDirac (VGS ~ 0.15 V) is the bias voltage for the charge neutrality point of G-FET determined at minimum IDS. The IDS difference between the dark-current and photo-current, ΔIDS, is 3 μA at 641 μW/cm2, which is 2–3 orders of magnitude larger than that of ΔIG depicted in Fig. IDS is negligible.The transfer characteristics of the G-FET under an illumination condition are presented in Fig. IDS is governed by the accumulated holes at SiOx/Si interface, even under light irradiation, as described earlier. The ionized donor (positively charged) in the depletion layer gives limited impact to IDS at VGS > VDirac under light irradiation because of the slight increase of IDS in dark conditions. The light-intensity dependence of the saturated IDS under light irradiation at VG > VDirac indicates that the charge carrier density in graphene is determined by the accumulated photo-excited excess hole at the interface, where the density of the accumulated photo-excited excess hole is regulated by the recombination rate of excess holes through the thin SiOx barrier, as described above. The field effect mobility of hole in graphene was estimated as 800 cm2V−1s−1 from CG of around VGS = −0.5 V.Based on the band model depicted in Fig. IG and ΔIDS, both showing power law dependence of the light intensity. The light-intensity dependence of ΔIG can be fitted by one slope of 0.73. By contrast, the light-intensity dependence of ΔIDS comprises two slopes as predicted theoretically earlier. The experimental data are well fitted to the numerically calculated result shown in Fig. vF = 0.8 × 106 m/s, τR = 18 ms, Evo = 0.05 eV, α = 2 × 10−34, SiOx thickness = 5 nm are used. The actual psi, which is proportional to ΔIDS, is unknown. For that reason, the vertical axis was adjusted with an arbitrary ratio of ΔIDS/psi. From a practical perspective for the photosensor, the ΔIDS is 2–3 orders of magnitude larger than ΔIG. This photocurrent amplification of G-FET with the Si Schottky gate gives rise to easier measurement of the light intensity than the simple graphene/Si Schottky diode, where the noise equivalent power (NEP) of the G-FET obtained at 8.9 μW/cm2 is 7 × 10−14 W/Hz1/2 (corresponding specific detectivity: 1 × 1011 Jones).Figure CD, internal series resistance RT, and load resistance RL, as portrayed schematically in Fig. IL is a current source determined by the light intensity. In our device structure, RL and RT consist of contact resistance, tunnel resistance, and resistance of the Si substrate. Based on the simplified model presented here, the time constant for rise time, τ0, is defined simply by CDRT, expressed as 2 interface, which induces the time dependence of IL determined by Eq. pSi corresponding to IL under various light intensities, where the used parameters are the same for those of light-intensity dependence in a steady state. The calculated data are well fitted by τrise, which depends on the light intensity. Consequently, one can expect that the transient photoresponse at light-on is given as the product of Transient response of G-FET is important for practical applications. The photoresponse time of the Schottky diode is usually explained by the simplified model with junction capacitance IDS on the G-FET, where the light intensity was varied from 0.91 to 257 μW/cm2. As might be readily apparent, the response time at light-on τrise decreases concomitantly with increasing light intensity, which is consistent with the calculated results based on the proposed model shown in Fig. τ0 determined from CDRT was fixed. Note that the lowest photoresponse obtained at 0.91 μW/cm2 is quite noisy and close to the dark signal, so that we could not obtain the reliable fitting curve. The experimentally obtained results were not fitted well by the simple exponential function. The time constant τrise obtained from the fitting clearly depends on the light intensity: it decreases from 12.5 to 1.6 ms with increasing light intensity, as portrayed in Fig. τrise obtained from the numerically calculated results presented in Fig. 2 could not be well reproduced based on our simplified model. This is most likely that the contribution of recombination process becomes more dominant at low carrier density. This is a subject for further study.Figure t > 3 ms, the decay can be expressed by τf = 17.8 ms for all light intensities, mainly because of the recombination time constant τR  of the G-FET obtained at 8.9 μW/cm2 is 7 × 10−14 W/Hz1/2. This amplification function is useful for practical applications. To analyze the photoresponse, we formulated the charge accumulation process at the n-Si/graphene interface, where the tunneling process through the SiOx layer was considered in addition to the recombination of the accumulated holes and the electrons in the graphene at the surface states on SiOx layer. The accumulated holes induce the Fermi level shift of graphene, which produces variation of the tunneling probability. The analytical framework proposed here is well described by the experimentally obtained results for both steady states and transient states. Moreover, it provides an effective means of analyzing charge carrier transport through the grapheme-based heterostructure through a thin tunneling barrier, which represents one path to achieving ultrathin opto-electronic devices in the future.We demonstrated a photosensor consisting of G-FET with gate bias applied through the Schottky barrier formed at an n-Si/graphene interface, where a thin SiO2 layer using conventional photolithography processing. The resistivity of the n-type Si substrate used in this experiment is 8 Ωcm. Unlike common back-gate G-FETs, we removed the SiO2 layer (300 nm thick) between the electrodes using buffered HF. All processes were performed in air, so that the channel part of the graphene is contacted to the Si surface through a 2–5 nm thick native oxide layer. Subsequently, after a monolayer graphene was transferred onto the substrate using polymethyl methacrylate, it was trimmed using oxygen plasma etching to form the G-FET channel, where the graphene was synthesized using low-pressure chemical vapor deposition at 1000 °C using Cu foil as catalyst36. The channel length and width are, respectively, 70 and 100 μm.The device fabrication process was the following. First, metal electrodes consisting of Cr/Au (5 nm/30 nm) as a source and drain electrodes were fabricated on a Si substrate with a 300 nm-thick SiOSupplementary Information


PD-L1/PD-1 interaction leads to a down-regulated immune response and it is one of the most promising targets for gastric cancer immunotherapy. PD-L1/PD-1 and CD8 expression were immunohistochemically investigated in a series of 169 FFPE GCs, including 33 EBV+, 59 MSI and 77 MSS/EBV− cases. PD-L1 membrane immunoreactivity in more than 5% of tumor cells was present in 31/169 GCs and was associated with high levels of CD8 intraepithelial lymphocytes . PD-L1+ cases were mainly poorly differentiated (71%), intestinal type (85%) and high lymphoid response  tumors. PD-L1 expression was only present in EBV⁺ (46%), MSI (24%) and rare MSS/EBV− (3%) GCs with high CD8+ TILs (p < 0.001). Despite being associated with a better prognosis both in the whole series (p < 0.05) and in the MSI subset, PD-L1 is not an independent prognostic factor. PD-L1 gene amplification was detected in 3/17 cases, including 2/7 EBV+ and 1/8 MSI GC. PD-1⁺ TILs were significantly higher in EBV⁺ than MSI and MSS/EBV− cases. PD-L1/PD-1 pathway is selectively activated in HLR GCs and could be considered an emerging therapeutic target, particularly for EBV and MSI GCs.Both EBV Gastric cancer (GC) is a heterogeneous disease characterized by different molecular and histological profiles ,2, whichAs in other malignancies such as melanoma and non-small cell lung cancer, GC tumor cells can also activate immune checkpoints to inhibit and escape an immune system response. One of these checkpoints is programmed cell death-ligand 1/programmed cell death protein-1 (PD-L1/PD-1) interaction. PD-L1 (B7-H1/CD274) is a type I transmembrane glycoprotein constitutively expressed on macrophages, T, B and dendritic cells, and its expression is enhanced by pro-inflammatory cytokines. In normal tissue, PD-L1 protein is less synthesized compared to its mRNA which is present in various human tissues . PD-1 is+ T cells [In cancers, up-regulation of PD-L1 expression could depend on intrinsic cellular control directed by genomic alterations, or it could be the consequence of an adaptive extrinsic control induced by CD8 T cells . In addi T cells ,6,7,8,9.The role of PD-L1 expression as a prognostic factor remains unclear because of the absence of uniform evaluating criteria for its analysis and the different tumor characteristics analyzed and correlated to survival in the various studies ,11,12,13+ tumor infiltrating lymphocytes (TILs) in whole formalin-fixed, paraffin-embedded tissue sections of a large series of 169 GCs which were well characterized histologically and enriched for EBV+ and MSI cases. The role of PD-L1 in predicting survival was also investigated. In this work we evaluated PD-L1/PD-1 expression and CD8PD-L1 immunoreactivity was observed in tumor cells and in immune cells, lymphocytes and macrophages infiltrating and surrounding tumor nests. + tumor cells, varying from 1% to 90%, showed weak to strong immunoreactivity prevalently along the cellular membrane or both along the membrane and in the cytoplasm. Five cases displayed only cytoplasm immunoreactivity and were considered negative for PD-L1.PD-L1+, showing 5% or more PD-L1+ tumor cells. They were mainly poorly differentiated GCs, intestinal in type according to the Lauren Classification, with the extent of the primary lesion (pT) being low and a prevalence of TNM stage I and II (Thirty-one out of 169 (18.3%) GCs were PD-L1I and II . They oc+ cases were high lymphoid response (HLR) GCs, 2 were ordinary cohesive  and one was an anaplastic MSI GC with a moderate number of TILs. Noteworthy, no ordinary or invasive mucinous carcinomas, including MSI cases, showed PD-L1 expression.According to the histotype-based prognostic classification , 28/31  and MSI  than in MSS/EBV−  GCs (p < 0.001). When we considered the cut-off of 1%, instead of 5% of immunoreactive tumor cells, the percentage of PD-L1+ cases increased to 61% for EBV+ and 37% for MSI GCs, whereas it remained unvaried in MSS/EBV− GCs. PD-L1+ cases, PD-L1+ tumor cells were spread out along the tumor and varied from 1% to 90%  of EBV+ and 37% (22/59 cases) of MSI GCs. Regarding the pattern of infiltration, in addition to the invasive margin the PD-L1+ immune cells infiltrated tumor in 58% and 54% of EBV+ and MSI GCs, respectively  of the cases. They were few and prevalently localized along the invasive margin (IM). TI pattern, in fact, was present in only 1/19 .In addition to PD-L1vely TI, . On the + lymphocytes were present both within tumor-cell nests and in intratumoral and peritumoral stroma. The mean number of CD8+ TILs varied from 0 to 167 and was significantly (p < 0.001) higher in EBV+  and MSI  GCs than in MSS/EBV− tumors  GCs with low CD8+ TILs, p < 0.001; Considering the previously defined cut-off value of 9.5 CD8 for HPF , PD-L1+ + TILs was observed in 137/169 GCs (81%), more frequently in EBV+  and MSI  than MSS/EBV−  GCs  compared to MSI (7 cells/HPF) and MSS/EBV⁻  GCs (The presence of PD-1.05) GCs . Overall.01) GCs .+, 8 MSI and 2 MSS/EBV⁻ GCs), including 12 high PD-L1 expression and 5 PD-L1 negative GCs with only cytoplasm immunoreactivity. The probe used for this study identified CD274,PDCD1LG2 gene cluster amplifications; high levels of amplification of this region were observed in three out of 12 PD-L1+ cases  and HLR (p < 0.0001) histotypes, low TNM stage (p < 0.0001), low pT (p < 0.0001), absence of lymph node metastases (p = 0.002) and high CD8 TILs (p = 0.01) were significantly correlated with a better outcome  gastric cancers, correlations with survival were analyzed only for advanced  GCs (AGCs). Follow up data were available for 146 AGCs. According to the literature, at univariate analysis intestinal  and high number of CD8+ TILs (p < 0.05) or HLR histotype (p < 0.05) were identified as independent prognostic factors . EBVnstrated . The hosnstrated ,17. To onstrated  and in tne cells ,12,19,20+ cases belong to the HLR group [+ T-cells infiltrating tumoral epithelium and stroma and that includes EBV+, MSI and a small number of GCs with high lymphoid infiltration but absence of virus infection and a defective MMR system. In our series about 50% of EBV+ and 25% of MSI GCs expressed PD-L1 in more than 5% of tumor cells. Outside these two distinct subgroups PD-L1 was observed in only two (3%) other cases, both MSS/EBV− GCs characterized by a high lymphoid infiltration that affects the whole tumor area or only a part of it. In addition, EBV+ cases showed a higher number of PD-L1+ cells, CD8+ TILs and PD-1+ cells than MSI and both compared to MSS/EBV− GCs. By confirming previous observations on smaller series of GCs [+ IC were also significantly more frequent in EBV+ and MSI than MSS/EBV− cases. In the last subset of carcinomas, positive IC, if present, were confined at the invasive front, whereas they infiltrate tumor in EBV+ and MSI GCs. The majority of PD-L1LR group , which is of GCs , PD-L1+ + GCs were prevalently EBV+ or MSI, their clinic-pathological profile is a mixture of both EBV and MSI characteristics. Furthermore, our GCs do not belong to a consecutive series of cases, but rather are enriched in both EBV and MSI carcinomas to compare them to MSS/EBV− subgroup. Therefore, the frequency of some features may be altered by such enrichment. The high frequency of low TNM stages in PD-L1+ cases, for instance, could be more likely related to the presence of early GCs (pT1) in EBV, but not in MSS/EBV− group, rather than to PD-1/PD-L1 pathway activation. Anyway, PD-L1+ carcinomas were prevalently poorly differentiated intestinal GCs, with larger diameter, lower pT and stage, with a preference for male and older patients. As previously observed by Ma and colleagues [+ GCs remains the presence of high CD8+ lymphocytes. Since PD-L1lleagues  all muci+ T-cells infiltration [+ lymphocytes via γ interferon secretion or by an intrinsic resistance due to oncogenic signaling. Derks et al. [− GCs. In a recent paper, Mimura and colleagues [+ T cells and tumor IFN-γ. All these data seem to suggest that an adaptive immune resistance contributes to PD-L1 expression rather than an innate immune resistance.Previous papers reported the association between PD-L1 expression and CD8ltration ,9,20, bolleagues  demonstr+ cases. Also, Clavè and colleagues [+ GCs showed a very strong immunoreaction involving a high number of tumor cells. FISH analysis demonstrated PD-L1 gene amplification in 3 out of these cases, which were two EBV+ and one MSI GCs. The induction of PD-L1 expression by inflammatory cytokines may be more efficient in tumors with gene amplification and the knowledge of the presence of an oncogenic upregulation of PD-L1, in addition to an adaptive immune response, could be useful for choosing the appropriate immunotherapy.On the other hand, the TCGA research network  reportedlleagues  describe+ GCs and is associated with high levels of CD8+ TILs, all parameters related to a more favorable prognosis. Interestingly, subgroup analyses demonstrated that cancer-specific survival was better for PD-L1+ cases in the MSI but not in the EBV+ subgroup. It could be due to the low number of EBV+ advanced GCs or the significantly higher presence of PD-1+ lymphocytes in EBV+ than MSI GCs. A more favorable prognosis was also observed when PD-L1 expression was combined with high CD8+ TILs. This result might be explained again by the CD8+ TILs positive influence on prognosis. Multivariate analysis, however, showed that PD-L1 expression is not an independent prognostic factor. Tumor stage and HLR histological type or high levels of CD8+ lymphocytes are the only independent prognostic parameters, stronger than MSI and EBV status.Controversial data are reported in the literature about the impact of PD-L1 expression on survival in GCs ,13,19,20+, MSI and rare MSS/EBV− gastric cancers characterized by abundant CD8+ lymphocyte infiltration. In these tumors the use of immune checkpoint inhibition can restore the efficiency of immune system. The presence of PD-L1 gene amplification can contribute to PD-L1 upregulation. In conclusion, PD-L1/PD-1 expression is a distinctive biological feature of EBV+ TILs, but not PD-L1 expression, are independent positive prognostic factors.Low TNM stage and high CD8The study was performed on 169 cases of advanced and early gastric carcinomas (GCs) operated on at the Ospedale di Circolo Fondazione Macchi in Varese and Fondazione Policlinico San Matteo in Pavia  between 1980 and 2012. Patients who had undergone preoperative chemotherapy or radiotherapy were excluded from the study.+) and MSI cases had to be excluded because there was no remaining tumor tissue; thus, to make the groups comparable, new cases were added from the Pavia series. Altogether, 33 cases where EBV+, 59 were MSI and 77 were microsatellite stable-EBV negative (MSS/EBV−) carcinomas.Most of the cases belong to the Varese series of GCs, previously well characterized both histologically and for the presence of EBV infection, microsatellite instability (MSI) and MMR protein expression . Some EBThe tumors were classified according to the Lauren criteria  and histPatients were followed-up either until death or for a mean period of 164 months (range 3–340).The clinico-pathological data of the GCs are summarized in The study was performed in accord with the ethical standards established by the Local Institution Review Board, and in accord with the Helsinki Declaration of 1975.2O2 in water for 15 min. Slides were treated for antigen-retrieval with pH8 EDTA buffer in a domestic microwave oven for 20 min. Immunohistochemical reactions for PD-L1, PD-1 and CD8 were performed on whole tissue sections obtained from formalin-fixed, paraffin-embedded tumor blocks. Three μm thick sections were deparaffinized and rehydrated. Endogenous peroxidase activity was inhibited in 3% HPrimary antibodies were applied overnight at −4 °C. In particular, PD-L1 rabbit monoclonal antibody  was used at a working dilution of 1/100; PD-1 mouse monoclonal antibody,  was undiluted, while CD8 monoclonal antibody  was used at a dilution of 1/20. UltraVision Quanto Detection System HRP  was used as amplification system according to the manufacturer’s instructions and with 3-3′-Diaminobenzidine as the chromogen. + immune cells were recorded as tumor infiltrating (TI) or located only along the invasive margin (IM), as reported previously [+ and CD8 TILs were counted at 400×  in ten consecutive fields and the mean value of immunoreactive TILs per high power fields was reported. The count area was selected according to the maximal number of neoplastic cells with minimal desmoplastic stroma and necrosis and only the immunoreactive lymphocytes in direct contact with tumor cells were included in the count.PD-L1 immunoreactivity was evaluated separately in tumor and immune cells. For tumor cells, only membrane staining was considered, and the percentage of positive cells was recorded, independently of the intensity. A case was considered positive if PD-L1 membrane immunostaining was present in at least 5% of tumor cells. For immune cells, both the pattern of infiltration and the intensity of immunoreaction were evaluated. The intensity was scored as absent/weak, moderate or strong, whereas PD-L1eviously . PD-1+ aAll the cases showing intense and abundant (more than 30% of tumor cells) membrane or cytoplasmic immunoreactivity for PD-L1 were tested for the presence of gene amplification. Interphasic FISH analysis was performed on 3–4 μm-thick sections used for conventional histologic examination as reported in the guidelines of the European Cytogeneticists Association  and the p value of <0.05 was considered significant. The GraphPad v.5.0  and MedCalc 11.2.0.0  software were used for statistical analyses.Statistical analysis was performed by using Fisher’s exact test or Chi-square test and Wilcoxon rank-sum test for unpaired data. Survival curves were calculated using the Kaplan-Meier estimator test. Statistical differences were tested using the log-rank test. Multivariate analysis was performed using Cox regression analysis. A + and MSI gastric carcinomas are characterized by high PD-L1/PD-1 expression and abundant CD8+ tumor infiltrating lymphocytes. PD-L1 expression is not an independent prognostic factor.EBV


Sei-1 is an oncogene capable of inducing double minute chromosomes (DMs) formation. DMs are hallmarks of amplification and contribute to oncogenesis. However, the mechanism of Sei-1 inducing DMs formation remains unelucidated.in vivo and gradually decreased following culture in vitro. micro nuclei (MN) was found to be responsible for the reduction. Of the DMs-carrying genes, Met was found to be markedly amplified, overexpressed and highly correlated with DMs formation. Inhibition of Met signaling decreased the number of DMs and reduced the amplification of the DMs-carrying genes. We identified a 3.57Mb DMs representing the majority population, which consists of the 1.21 Mb AMP1 from locus 6qA2 and the 2.36 Mb AMP2 from locus 6qA2-3.DMs formation significantly increased during serial passage Sei-1 overexpression to monitor and characterize DMs in vivo and in vitro. Array comparative genome hybridization (aCGH) and fluorescence in situ hybridization (FISH) were performed to reveal amplification regions and DMs-carrying genes. Metaphase spread was prepared to count the DMs. Western blot and Met inhibition rescue experiments were performed to examine for involvement of altered Met signaling in Sei-1 induced DMs. Genomic walking and PCR were adopted to reveal DMs structure.We employed NIH-3T3 cell line with Met is an important promotor of DMs formation. Sei-1 is a cell cycle promotor and an identified oncogene [Sei-1 is capable of promoting tumorigenesis by enhancing cell proliferation and prohibiting cell apoptosis [Sei-1 in chromosome instability has also been reported [Sei-1's oncogenic ability and potential tumor promoting pathways. Mechanisms that involve chromosome alterations that are inflicted by Sei-1 were not fully elucidated.oncogene . Sei-1 ipoptosis , 6. The reported , 7. ReceMYC and DHFR [Double minute chromosomes (DMs) are round-circle, acentric double-strand extra-chromosome DNA that usually exist in pairs. The formation of DMs is usually regarded as an important sign of genome instability –11. DMs and DHFR –16, are and DHFR –20. DMs and DHFR –24. As tin vitro and in vivo [Although DMs may be naturally lost during cell growth , 26, DMs in vivo –30 while in vivo –33, and  in vivo , 26. Rec in vivo .Sei-1 overexpressing NIH-3T3 cells not only gain a growth advantage but also produce DMs in vivo [Sei-1 overexpression in vitro and in vivo. In this study, we innovatively investigated the mechanism of Sei-1 inducing the formation of DMs in nude mouse. We found the number of DMs induced by Sei-1 was significantly increased during in vivo tissue passage but reduced during in vitro cell passage. Among the DMs-carrying genes, Met was the most prominently amplified and overexpressed. Met signaling pathway was shown to increase the Sei-1-induced DMs population, which indicated a novel function of the well-known oncogene Met to induce the formation of DMs. Additionally, a 3.57 Mb DMs structure was identified to represent the majority population of DMs with two amplicon fragments that were jointed with blunt ends.NIH-3T3 serves as a suitable model for the study of DMs in cancer biology. –38. A pr in vivo . This fiSei-1 and null-vector-transfected NIH-3T3 cell clone Sei-1/NIH-3T3 and Vec  has been well elucidated to directly bind and motivate Met and its downward pathways. An alternative method of Met accumulation that causes self-phosphorylation and activation was also previously reported [Sei-1 overexpressing the Sei-1/NIH-3T3 clone and primary cell pool CPX3, CPX6 with and without mouse recombinant Hgf treatment  were distinctly decreased 24 hours after Met inhibition as a result of the loss of DMs  with nearly identical copy changes were identified in this study: four breakpoints, namely, BP1, BP2 on the edge of AMP1, and BP3, BP4 on the edge of AMP2, which suggests a relatively simple DMs structure. Based on the aCGH results, a set of primers  were desSei-1 is a newly discovered oncogene that is particularly found to be able to induce the formation of DMs. The biological connection between its oncogenic and DMs inducing abilities was barely explored. In this study, we innovatively observed the gain/loss of Sei-1-induced DMs to investigate oncogenic mechanism of Sei-1. Our study discovered Met as a dynamic responder to enhanced Sei-1 oncogenic function. We uniquely found that Met was not only amplified in the form of DMs and overexpressed but also significantly involved in the formation of DMs itself, suggesting a novel function of Met to generate the DMs.in vivo, but progressively DMs lost during in vitro culture. A Darwinian selection pattern was previously implied to explain the increased number of DMs that harbor MYCN in neuroblastoma cells [in vitro passages. They hypothesized that the number of DMs could change dynamically as a response to significant environmental changes [Sei-1-induced DMs probably facilitate the survival of cells under in vivo pressure. In vitro, endogenous microenvironment pressure was retreated, and previous growth advantage conferred by DMs was lost. Therefore, DMs production was restricted, which caused a reduction in DMs populations. Interestingly, we observed the Sei- 1 overexpression level significantly higher in CPX6 compared to Sei-1/NIH-3T3, CPX1, CPX3 both in cells and tumor tissues .Sei-1 CDS were cloned into pcDNA3.1(+) plasmid  and transfected into cells with Lipofectamine 2000 (Invitrogen).NIH-3T3, a mouse embryo originated fibroblastic cell line , was purs.c. xenograft was conducted as previous [Five-week-old Balb/c female nude mice were obtained from SLAC laboratory  and maintained within individual ventilated cages in SPF-degree conditions. The previous , with eaColcemid  was added to the medium of fast growing cells for 2 hours, which were trypsinized, washed with PBS, and subsequently centrifuged. Suspend pellets in pre-warmed 0.075 M KCl and incubate in 37 °C water bath for exact 14 min. Fixate the cells by fresh fixative solution (mixture of 3 methanol: 1 acetic acid) three times. Drop the cell suspension on a clean slide. The slides were dyed with Giemsa and photographed using Olympus BX41 microscope  equipped with JVC TK-C75U color video camera .A micronuclei spread was prepared without colcemid treatment. The protocol, including Giemsa dying and image capturing, was identical to metaphase spread.BAC clones purchased from the BACPAC Resources Center  were labeled with fluorescence dUTPs and hybridized to metaphase/micronuclei spread as described . The sliGenomic DNA samples were extracted from fresh xenograft tissue with the DNeasy Blood & Tissue Kit . Sample quality was guaranteed by Nanodrop . The samples were properly predisposed and applied to a SurePrint G3 Mouse CGH Microarray . The procedures were conducted by Shanghai Biotechnology Co., Ltd. .The DMs were micro-dissected from NIH-3T3 metaphase karyotype that were prepared as previously described . The sam® DNA Mini and Blood Mini Kit (Qiagen) and Trizol . cDNA samples were obtained using Transcriptor First Strand cDNA Synthesis Kit (Roche). For quantitative real-time PCR, the samples were tested using the LightCycler® 480 instrument (Roche) and corresponding SYBR Green I master (Roche). Semi-quantitative PCR was performed as described [Genomic DNA and RNA samples were extracted by QIAampescribed .The Universal Genomic Walker Kit  was employed according to the manufacture instructions. The PCR products were sequenced and aligned with the mouse genome using the BLAT tool online from UCSC. The gene-specific primers (GSP) for BP1 were GSP1, 5′-TTGGTATTTATTCACGACTTTGATAC-3′, GSP2, 5′-GTCATGTACTCTGTTAAGACTGCTTT-3′. The GSP primers for BP2 were GSP1, 5′-AACAGATGAA AAAAAGTTGAGAGGCT-3′, GSP2, 5′- AACAGATGA AAAAAAGTTGAGAGGCT-3′Cells were lysed by RIPA buffer and sonication, and centrifuged at 12000 g 4°C for 30 minutes. Supernatant protein was separated by SDS-polyacrylamide gel, and transferred to PVDF membrane, which was scanned by Odyssey Imaging System  after incubation of corresponding antibodies. Image J Software was used for quantification.Antibodies against Gab1—phospho-Gab1, Met—Phospho-Met, pan-AKT—Phospho-Akt, PI3K, and Phospho-PI3K were purchased from Cell Signaling . Antibodies against Hgf were purchased from Thermo Fisher . Anti-mouse and anti-rabbit antibodies were obtained from Rockland Immunochemicals . Mouse recombinant Hgf and c-Met Kinase Inhibitor III were purchased from Millipore .t-test as well as one-way ANOVA with SNK test were employed as the statistics.Data of the DMs counts and Western blot coming evenly from three parallel studies were presented as the mean ± S.D. All experiments were independently repeated three times. Two-tailed Student's


FOXC2 gene cause a form of lymphedema primarily of the limbs that usually develops at or after puberty. In 90-95% of patients, lymphedema is accompanied by distichiasis. FOXC2 is a member of the forkhead/winged-helix family of transcription factors and plays essential roles in different developmental pathways and physiological processes. We previously described six unrelated families with primary lymphedema-distichiasis in which patients showed different FOXC2 mutations located outside of the forkhead domain. Of those, four were missense mutations, one a frameshift mutation, and the last a stop mutation. To assess their pathogenic potential, we have now examined the subcellular localization and the transactivation activity of the mutated FOXC2 proteins. All six FOXC2 mutant proteins were able to localize into the nucleus; however, the frameshift truncated protein appeared to be sequestered into nuclear aggregates. A reduction in the ability to activate FOXC1/FOXC2 response elements was detected in 50% of mutations, while the remaining ones caused an increase of protein transactivation activity. Our data reveal that either a complete loss or a significant gain of FOXC2 function can cause a perturbation of lymphatic vessel formation leading to lymphedema.Dominant mutations in the  More than 10 Mendelian forms are known, of which three are considered the major forms: Milroy disease (MD) [MIM 153100], lymphedema-distichiasis syndrome (LDS) [MIM 153400] and Meige disease (MGD) [MIM 153200] [Lymphedema is a dysfunction of the lymphatic system, a disorder characterized by abnormal swelling of one or more extremities due to impaired transport of the lymph –3. This ) MIM 15300 and MeFOXC2 gene (MIM 602402), coding for the forkhead transcription factor FOXC2, cause a form of lymphedema with variable age of onset (range: 7-40 years), often associated with distichiasis [FOXC2 plays a key role in regulation of lymphatic endothelial cells differentiation, in formation of smooth muscle cell layers and in morphogenesis of lymphatic valves. FOXC2 and VEGFR-3 act through a common genetic pathway to establish distinct properties of the lymphatic vascular architecture [Dominant mutations in the gene MIM 02402, coichiasis . FOXC2 pitecture .FOXC2 maps to 16q34.3 and it produces a 2.2 kb transcript with a 1.5 kb single exon coding region The FOXC2 protein contains 501 amino acids . The majority of FOXC2 mutations are small insertions or deletions and nonsense mutations causing truncated proteins, which have been hypothesized to be responsible for a haplo-insufficiency condition [FOXC2 haplo-insufficient state is associated to hyperplasia and distichiasis in mice [FOXC2−/− mice have been found to exhibit abnormal pericyte recruitment to the lymphatic capillaries and valve agenesis in the collecting vessels, indicating that FOXC2 is essential for the formation of a pericyte-free lymphatic network and lymphatic valve development [FOXC2-null mice [In LD patients, almost 70 different ondition . The FOX in mice . FOXC2−/elopment . Moreoveull mice , 16.FOXC2 haplo-insufficiency also in humans, until some FOXC2 missense mutations, identified in LD patients, were found to cause a gain of protein function [FOXC2 variations are missense mutations of which very few have been functionally investigated. Two mutations located inside the forkhead domain impaired the DNA-binding and transcriptional activation ability of FOXC2 protein [FOXC2 variations. We therefore performed functional characterization of FOXC2 mutations located in different protein domains to provide novel information on structure-function relationships of the FOXC2 transcription factor and to get further insight into the disease-causing mechanism of lymphedema.The pathogenesis of LD was assumed to be associated with function . Approxi protein ; in cont protein . MoreoveGenetic evaluation of LD patients has been previously reported . MoleculFOXC2 cDNA into the pcDNA3.1/NT-GFP-TOPO TA expression vector and then performed site-direct mutagenesis to generate the FOXC2-GFP plasmids with the disease causing mutations.The mutations analysed in this study were all localized outside the forkhead domain, in different regions of FOXC2 protein Figure . The evaFOXC2 mRNA levels  mutation-containing plasmid as a control in order to allow an accurate comparison of intracellular distribution, since the FOXC2(R121H) protein has been previously demonstrated to display a defect in nuclear localization, with partial cytoplasmic signal [FOXC2(M276DfsX186) recombinant plasmid caused the production of nuclear aggregates  plasmid, in our system, induced intranuclear protein aggregation  which leads to the formation of a truncated protein lacking of 235 amino acids in the C-terminal region, and with the addition of 186 nonsense amino acids due to the frameshift  and one nonsense (p.Q420X) mutations. All three AD-2 mutations significantly increased FOXC2 transcriptional ability. Surprisingly, the p.Q420X displayed the highest level of activation, despite the truncated protein lacked more than half of AD-2 sequence and the entire ID-2 region. Consistent with these findings, Van Steensel et al. also reported a gain of function for FOXC2 missense variations localized inside AD-2 domain. We did not detect any evidence of mRNA or protein stability alterations resulting from FOXC2 AD-2 mutations. Thus, it is possible that AD-2 mutations exert their influence by modifying the tridimensional structure of the C-terminal domain of the FOXC2 protein and/or changing the ability of FOXC2 to interact with regulatory proteins. In the central region of Finally, the S370T missense mutation, located in the terminal sequence of central region Figure , resulteTo date, it is not possible to extrapolate general conclusions about the relationship between FOXC2 activity and the protein variation position, because of the very limited number of different types of mutations  that have been functionally tested. However, data obtained so far indicate that missense mutations in FHD and AD-2 domain might represent an exception, causing always a loss and a gain of FOXC2 function, respectively. More data are needed to highlight possible correlation between mutation position and function in other FOXC2 protein domains.FOXC2, that are responsible for regulating the expression of a broad range of genes both during development and in adult tissues [Nowadays, it is possible to estimate the theoretical pathogenic effects of missense mutations utilizing bioinformatics tools. However, the usefulness of these programs in predicting the real contribution of a mutation becomes limited when they provide conflicting data  with the others carrying a gain of function mutations , we noted some differences in the uptake of the radiopharmacon by inguinal lymph glands and its reflux back down into the lower leg. In particular, P1 and P2 patients showed a lymphoscintigram profile/images compatible with a hyperplasia of lymphatic vessels , while P4, P5 and P6 patients reports resembled more a hypoplastic condition . While, Based upon clinical and molecular data, therefore, we can hypothesize that an increased activity of FOXC2 results in disregulated expression of proteins essential for normal development of lymphatic system, causing hypoplasia. In contrast, decreased FOXC2 transcriptional activity appears to be associated with a hyperplastic condition. Our hypothesis on hypo or hyperplasia and activating versus inactivating mutations is consistent with previously reported results , 18 and FOXC2 different mutations and the morphologic changes of lymphatic system, and with distichiasis.The association of FOXC2 mutation function and distichiasis is less clear however. Our patients with activating mutations presented with distichiasis, in contrast, 2 out of 3 patients with inactivating mutation did not. From these clinical findings, it might be tempting to speculate that distichiasis is associated with hypoplasia rather than hyperplasia. However, additional larger clinical studies are required to definitely elucidate correlations between FOXC2 mutations identified in six Italian families with lymphedema-distichiasis, providing evidences of patients with inactivating versus activating mutations of the FOXC2 transcription factor. During development, transcriptional programs of lymphatic endothelial cells are established by networks of transcriptional factors, among which FOXC2 is one of the most important. FOXC2 acts downstream of VEGFR-3 signaling pathway and is essential for the formation of pericyte-free lymphatic network. Moreover FOXC2 regulates the expression of genes essential for all steps of lymphatic valve morphogenesis and maintenance, such as connexins CX43 and CX47 [In conclusion, we have analyzed the molecular consequence of and CX47 , 29, 30.Finally, recent studies show that FOXC2 overexpression is involved in cancer progression, inducing epithelial mensenchymal transition (EMT) , 32. LDSFOXC2 cDNA was amplified by PCR using DNA clone ID 32938 (Thermo Scientific) as template and the following primers: FOXC2-F 5′-ATGCAGGCGCGCTACTCC-3′ and FOXC2-R 5′-TCAGTATTTCGTGCAGTCGTAGGAG-3′. The PCR product was subcloned into pcDNA3.1/NT-GFP-TOPO from Invitrogen to produce NT-GFP-FOXC2, expressing FOXC2 with GFP at the N-terminus. Point mutations were performed using the Phusion Site-Directed Mutagenesis Kit (Thermo Scientific). Mutations in FOXC2 cDNA were introduced using the following primers: A3G forward 5′-ATGCAGGGGCGCTACTCCGTGT-3′ and reverse 5′-ACACGGAGTAGCGCCCCTGCAT-3′; R121H forward 5′- GCAGAACAGCATCCACCACAACCTCTCGC -3′ and reverse 5′- GCGAGAGGTTGTGGTGGATGCTGTTCTGC -3′; S370T forward 5′-GTCGCCCCTGACCGCTCTCAACC-3′ and reverse 5′-GGTTGAGAGCGGTCAGGGGCGAC -3′; Q420X forward 5′- GCCGCGGCGTAGGCGGCCT-3′ and reverse 5′- AGGCCGCCTACGCCGCGGC -3′; L487P forward 5′-ACGCCGCCTCCCTATCGCCAC-3′ and reverse 5′-GTGGCGATAGGGAGGCGGCGT-3′; A492V forward 5′-TCGCCACGCAGTCCCCTACTCCT-3′ and reverse 5′-AGGAGTAGGGGACTGCGTGGCGA-3′.In order to obtain the c.826-827delAT FOXC2 mutation (M276DfsX186) were used the following two couple of primers and two rounds of mutagenesis : forward(1) 5′- CAGCGTGGAGAACATCTGACCCTGCGAACGTC-3′ and reverse(1) 5′- GACGTTCGCAGGGTCAGATGTTCTCCACGCTG-3′ and forward(2) 5′- CAGCGTGGAGAACATCGACCCTGCGAACGTC-3′ and reverse(2) 5′- GACGTTCGCAGGGTCGATGTTCTCCACGCTG-3′.FOXC2 (wild type) and NT-GFP-FOXC2 mutant plasmids were used as template to amplify FOXC2 control and mutants cDNA sequences with FOXC2-F and FOXC2-R primers (reported above). The PCR products were subcloned into pcDNA3.3-TOPO (Life Technologies) to produce control or mutant FOXC2 proteins without any tag. All final expression constructs were sequenced to verify that no additional mutations were introduced.NT-GFP-FOXC2 and GAPDH gene, in order to avoid saturation: regression curves assaying different amounts of cDNAs (corresponding to different mRNA concentrations) and different number of cycles of amplification were performed . After 48 h, cells were carefully washed with PBS, total RNA was isolated with TRIzol (Invitrogen) and 1 μg was converted to cDNA by RT-PCR using random hexamers (0.5 mg), 400 units of MMLV-RT, 1.6 mM total dNTPs, 20 units of Rnasin and 0.4 mM dithiothreitol, in a 50 μl reaction solution containing 10× RT Buffer. Before reverse transcription, RNA was treated with DNase in order to eliminate DNA contamination . RT-PCR erformed . Thirty For transient transfections, HeLa and COS cells were cultured on glass coverslips in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) and allowed to adhere overnight. The next day, the cells were transiently transfected with recombinant NT-GFP-FOXC2 plasmids using the TurboFect transfection reagent. After 24 h, cells were fixed stained with DAPI, and examined with a Leica MB5000B microscope equipped with 40X and 100X Fluorart oil immersion objectives.HeLa cells were transiently transfected with recombinant NT-GFP-FOXC2 plasmids as described above. Four flasks for each type of recombinant plasmids was transfected. After 48 h, all flasks were evaluated for transfection efficiency. Only samples reaching 80% of transfection efficiency were used to prepare protein cell extracts. Cell extracts were prepared from confluent cultures grown in serum-containing medium. After extensive washing with Dulbecco's phosphate-buffered saline (DPBS), cells were recovered by scraping with a rubber policeman in 300 μl of 0.05% (wt/vol) SDS. The total protein concentration of cell extracts was quantified using a Coomassie (Bradford) Protein Assay Kit (Pierce). Proteins (10 μg/well) were separated by electrophoresis on 8% SDS-polyacrylamide gel (Bio-Rad), transferred to a nitrocellulose membrane (Pierce) and then immunoblotted using a rabbit monoclonal antibody (dil. 1:5000) raised against green fluorescent protein (Invitrogen) and a mouse monoclonal antibody (dil: 1:5000) to GAPDH (Abnova). Specifically bound immunoglobulins were detected using the SuperSignal West Pico Complete Detection Kit (Pierce) containing ImmunoPure Peroxidase Conjugated Goat anti-Mouse and anti-Rabbit IgG (dil 1:20.000). Experiments were repeated three times.HeLa and COS cells were co-transfected with 20 ng FOXC1 luciferase reporter  and 100 The same experimental conditions were used for transient transfection of wild type or FOXC2 mutant plasmids subcloned into pcDNA3.3, a mammalian expression without tag. The dual-luciferase assays were used to obtain sequential quantification of both Photinus pyralis luciferase (reporter vector) and Renilla reniformis luciferase (control vector), as previously described (35). Luminescence detection for all transfected plasmids was performed using the Glomax luminometer (Promega). Reactions were replicated three times, using the Promega Dual Luciferase Assay kit (Promega).The statistical analysis of quantitative data of RT-PCR and of Luciferase assays was made using SPSS v.19 package . The values were compared using Student's t-test. A P-value of ≤0.05 was considered to be statistically significant.The effect of amino acid substitutions on protein function was predicted using ClustalW, SIFT and PolyPhen software. A multiple sequence alignment of mammalian FOXC2 proteins was used as imput for ClustalW. The NCBI reference sequence (FOXC2 protein NCBI accession number: NP_005242.1.) of the human FOXC2 protein was used as the input for SIFT and PolyPhen, with default query options.


Acute exacerbation of idiopathic pulmonary fibrosis (AE-IPF) has high short-term mortality with unknown causes. To predict this malignant condition in clinics is challenging. In this study, we aim to demonstrate whether there are miRNAs that differ between AE-IPF and stable IPF, which may be served as reliable biomarker for AE-IPF prediction.Human fibrotic-associated miRNAs arrays were designed to detect miRNAs expression in plasma of 3 AE-IPF patients, 3 Stable-IPF (S-IPF) patients and 3 normal controls (NC). Differentially expressed miRNAs between AE-IPF and S-IPF patients were selected for further analyses. The validation studies were carried out in plasma of 12 AE-IPF patients, 45 S-IPF patients and 51 healthy control subjects. Signaling pathways and cellular processes interacted with validated miRNAs were predicted by DIANA-miRPath.P < 0.05). In the validation studies, let-7d-5p was decreased in S-IPF and further decreased in AE-IPF, when compared to NC . While miR-25-3p was obviously decreased in S-IPF  but significantly increased in AE-IP . In receiver-operator characteristic (ROC) curve analysis, the areas under the curve (AUCs) of miR-25-3p and let-7d-5p were 0.83 and 0.75, respectively. The sensitivity at fixed specificity of 90% was improved from 50% to 66.7% when the two miRNAs were combined. The functional prediction of miRNAs suggested that the loss of anti-fibrotic capacity and the gain of uncontrolled cell growth may be required in AE-IPF pathogenesis.According to the array analysis, 6 miRNAs showed differentiated expression between AE-IPF and S-IPF patients (In conclusion, miR-25-3p and let-7d-5p in plasma were differentially expressed between AE-IPF and S-IPF. A combination of these two miRNAs may be a potential biomarker for AE-IPF from IPF. Idiopathic pulmonary fibrosis (IPF) is a progressive disease with steady worsening of symptoms and gas exchange. Most patients die from respiratory failure within 3 years –3. AlthoMature miRNAs are endogenous 18–24 nucleotide non-coding RNA molecules which act as posttranscriptional regulators of gene expression and are stable in circulation. The alterations of miRNAs in plasma or serum have been served as improved and noninvasive biomarkers for numerous pathological conditions, including cancers, autoimmune diseases and tissue injury –9. Over In this retrospective study, we initially compared the human fibrosis-related miRNAs between S-IPF and AE-IPF. Moreover, through a validation study in an expanded cohort, we determined the predictive role of the miRNAs in distinguishing between S-IPF and AE-IPF.The diagnosis of IPF was recommended by the American Thoracic Society and the European Respiratory Society (ATS\ERS) and was The diagnosis of AE-IPF was made according to the proposed diagnostic criteria that mainly focus on a previous or concurrent diagnosis of IPF with a worsening of its clinical manifestations over 30 days or less, and the absence of infection or another identifiable etiology such as left heart failure, or pulmonary embolism .A total of 63 IPF patients were admitted to Drum Tower Hospital from January 1, 2013 to June 30, 2014. Among them, 15 patients were diagnosed as AE-IPF and died within 1 month after the diagnosis. And 48 IPF patients with the stable radiographic findings and without a worsening of their clinical manifestations over a period of 30 days were defined as S-IPF. These S-IPF patients had no acute exacerbation during the follow-up period of 1–2.5 years after enrollment. In addition, 54 unrelated age-matched healthy individuals were enrolled without current or prior history of lung diseases in this study at the Center of Physical Examination of Drum Tower Hospital.This study was approved by the Ethical Committee of the Affiliated Drum Tower Hospital of Nanjing University Medical School.Chest HRCT was performed using various CT scanners with the patients in suspended inspiration. The criteria for inclusion of IPF in the study described previously . Chest HBlood samples were obtained once the patients were diagnosed as S-IPF or AE-IPF. About 5 ml venous blood samples were collected in EDTA-containing tubes. Blood samples were centrifuged within 1 h in two steps. Firstly, all samples were centrifuged at 800 g for 10 min at 4 °C, and the supernatants were collected into new RNase-free tubes for a second centrifugation at 12,000 g for 10 min at 4 °C. The supernatants were then stored for RNA extraction.Plasma total RNA including miRNAs were extracted with miRNeasy Serum/Plasma Kit (Qiagen) from 400ul plasma samples according to the manufacturer’s instructions. 100pmol/L synthesized cel-miR-39 was added into plasma as an internal calibrator to monitor extraction efficiency. The concentration and purity of the total RNA were measured by Nanodrop 2000.96-well miRNA expression profiles were used for 3 AE-IPF, 3 Stable-IPF(S-IPF) and 3 normal controls (NC). The array was designed according to miScript miRNA PCR Array  including 84 human fibrosis-associated miRNAs, an external reference cel-miR-39 and an internal reference U6. First, reverse transcription (RT) of the total RNA samples was performed with TaqMan miRNA Multiplex Reverse Transcription Kit (Applied Biosystems) according to its protocol. Next, quantitative real time PCR was performed according to the protocol of TaqMan Universal PCR master mix (Applied Biosystems) on Step One plus PCR system (Applied Biosystems).Total miRNAs were extracted from 400 μl plasma of additional 12 AE-IPF patients, 45 S-IPF patients, as well as 51 healthy controls with PARIS Kit (Ambion), mixed with 100pmol/L synthesized cel-miR-39 as an internal calibrator to monitor extraction efficiency, and were dissolved in 50 μl elution buffer. Total miRNAs were reverse transcribed into a final volume of 30 μL cDNA with specific primers (Roche) using Prime Script RT-PCR Kit (Takara) according to the instructions. Finally, quantitative PCR was performed with UPL probe kit (Roche) using Step One plus PCR system (Applied Biosystems). The reaction conditions were: 95 °C for 10 min, and 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min. Each sample was detected three times.DIANA miRPath v.2.0 was used to predict target genes by one or more miRNAs into known KEGG pathways . The gra-∆∆CT with miR-16 as a reference miRNA. Relative Concentrations of miRNAs in AE-IPF, S-IPF and control plasma samples were calculated as 2-∆CT and compared by the T- test. Receiver-operator characteristic (ROC) curve was generated under the nonparametric distribution assumption for expression levels of plasma miRNAs of AE-IPF and S-IPF patients by plotting sensitivity% vs (100%-specificity %). Areas under the curves (AUCs) were calculated. The biomarkers were considered to be useful when the value of AUCs were >0.7. Logistic regression was used in the combined markers analysis. All statistical analysis was performed with either SPSS17.0 or GraphPad Prism5.0 Software. 95% confidence intervals were used to quantify uncertainty, and P values < 0.05 were considered to be statistically significant.For the miRNA array and validation data analyses, the raw expression levels were determined by the cycle number at a predetermined threshold cycle (CT) <35. The fold-change for each miRNA from the control group to each patient group was calculated as 2Characteristics of patients and controls including age, gender, results of lung function tests were detailed in Table p < 0.05). To identify the miRNAs that were deferentially expressed miRNAs between AE-IPF and S-IPF patients, we further compared the expressions of the miRNAs in these two fibrotic groups to the control group. We found that miR-31-5p and miR-338-5p were up-regulated in S-IPF patients (p < 0.05), but down-regulated in AE-IPF patients (p < 0.05), while miR-25-3p and miR-92-3p were up-regulated in AE-IPF group, but down-regulated in S-IPF group. All the above miRNAs were selected for further validation.We initially profiled the expression of 84 fibrosis-related miRNAs in the plasma of 3 AE-IPF patients, 3 S-IPF patients and 3 healthy controls by the fibrosis-specific miRNAs arrays. With setting a threshold of CT < 35, a total of 34 miRNAs were further analyzed. As shown in the heat map Fig. , the levp < 0.05) and controls (p < 0.01). Interestingly, in the same comparison, miR-25-3p was found to be significantly decreased in S-IPF group (p < 0.01), but increased greatly in AE-IPF group (p < 0.01).Concentrations of the 6 miRNAs were detected in additional 12 AE-IPF and 45 S-IPF patients, as well as 51 healthy controls by using RT-qPCR. We found that 4 of them, miR-132-3p, miR-338-5p, miR-92-3p and miR-31-5p, were not differentially expressed between AE-IPF and S-IPF groups (data not shown). As been shown in Fig. To evaluate the effectiveness of the two miRNAs for predicting AE-IPF patients among IPF patients, ROC curves were constructed by comparing plasma measurements of let-7d-5p and miR-25-3p in the AE-IPF patients with that in the S-IPF patients. The diagnostic accuracy of the two miRNAs for AE-IPF prediction was calculated (Fig. DIANA-mirPath analysis was applied to predict the biologic targets and pathways as well as cellular processes that miR-25-3p and let-7d-5p affected. We found that the two miRNAs shared no common direct targets at transcriptional levels. We then investigated the cellular processes interacted with miR-25-3p and let-7d-5p. We found the intersections between the two miRNAs in cell cycle related processes in an opposite way, while let-7d-5p showed the suppressive effects on these processes, miR-25-3p promoted (Table During the last decade, several studies have explored biomarkers for the prediction of rapid progressive IPF , 19–21. In line with previous studies , 14, we The various functional studies on the two miRNAs, miR-25-3p and let-7d-5p, should not be neglected. First, a reduction of let-7d in epithelial cells causes epithelial-mesenchymal transition and over-expression of let-7d in fibroblasts can reduce their mesenchymal properties , 23, sugInterestingly, two genes, ATP11A and OBFC1 found to be associated with IPF susceptibility in a GWAS study  were preIn addition to the patients enrolled from a single medical center with a relatively small size in our study, one weakness of this investigation is that the current study was based on a scheme of one-time-point detection of the miRNAs without longitudinal measurements due to the clinical limitations, which may mask the relationship between the dynamic expressions of the miRNAs and the disease progression. Since the variability of the clinical courses of IPF are widely ranged and might be heterogeneous , the ideIn this study, we reported that the human fibrosis-related miRNAs, miR-25-3p and let-7d-5p, were differentially expressed between S-IPF and AE-IPF. The combination of these two miRNAs was significant in predicting an acute exacerbation of IPF. The result indicates that both loss of tissue anti-fibrotic capacity and gaining of uncontrolled cell growth may be required in AE-IPF pathogenesis.