The processing of the powders has been made using a colloidal route, and after that, a spark plasma sintering process was performed in order to densify the samples. Creep tests were performed at temperatures between 1200–1250 °C in an argon atmosphere. The microstructure obtained by SEM of the sintered and tested specimens was characterized quantitatively to elucidate the deformation mechanism. Raman spectroscopy was carried out to check the integrity of the graphene. The average grain size was in the order of 1 µm and the shape factor was 0.7 for all the studied materials. The integrity of the graphene was checked before and after the creep experiments. The careful analysis of the creep tests shows that graphene oxide or its reduced version are not efficient phases for creep resistance improvement in general, contrary to what is reported elsewhere. However, the results permit the suggestion of a creep improvement in nanocomposites at a very high temperature regime due to an enhanced reactivity of oxygen between carbon and alumina interfaces. In the case of zirconia, the results give us the conclusion that the oxidation of graphene is a highly detrimental issue regarding the improvement of high-temperature plasticity. This paper aims to give an answer to the following question: is the oxidation of graphene a critical issue for high-temperature plasticity in graphene-reinforced ceramics? To give a convincing reply, we will focus on two very different graphene-based ceramic composites: reduced graphene oxide (rGO)-reinforced alumina (α-Al Traditionally, a secondary phase is used in the world of materials to reinforce them and to try and change its mechanical properties. The result is a composite with enhanced properties ,2. In th2O3) there are a lot of studies trying to improve the creep behavior by adding a secondary phase. For example, Lessing et al. [2O3 doped with a 10 vol.% of carbon nanotubes had a better creep behavior than the pure material with a similar microstructure. In another study, Zapata-Solvas et al. [2O3/single-wall carbon nanotube composites. An extensive variety of alumina exists, with a large scatter in the diffusion kinetics processes which can also affect the mechanical properties [2O3/ carbon nanofiber composite decreased with respect to pure alumina, but at 1250 °C they found a similar behavior of the composite and the pure material. The same authors reported that the Al2O3/graphene oxide composite has a better creep resistance than the pure material. Zapata-Solvas et al. [Concerning alumina,  is the most important mechanism of deformation at high temperatures of yttria-stabilized-tetragonal zirconia (3YTZP) ,22. Somemposites . Cano-Crmposites  found thThe scientific problem is the comprehension of the importance of the reactivity of oxygen between the graphene and the alumina or zirconia grains, and if this reaction is detrimental in the mechanical properties at high temperatures. In order to give an answer, several creep experiments at very elevated temperatures using graphene as the secondary phase were performed for the first time of our knowledge. Consequently, the main goal of this study is to prepare a homogeneous dispersion of reduced graphene oxide (rGO) into alumina and zirconia matrices and to obtain nearly full density compacts by spark plasma sintering with enhanced creep resistance at high temperatures compared to pure materials. To achieve this aim, several experimental techniques were employed: scanning electron microscope, Raman spectroscopy, and creep at high temperatures. The creep experiments were analyzed according to the Equations (1)–(3) presented in the 2O3  has an average particle size of 0.35 µm and a specific surface area of 9.5 m2/g. The powder of tetragonal-ZrO2 stabilized with 3% mol of Y2O3  has an average particle size of 90 nm and a specific surface area of 6.7 m2/g. Both kinds of powders have a high purity and higher than 99.9%.The starting powders of α-alumina and tetragonal zirconia used were the same as in ,29. The 2/g was used as the secondary phase [Reduced graphene oxide  with a length of 1–4 μm, a thickness of 0.7–1.2 nm and a surface area of 103 mry phase ,31. PartN-methyl-2-pyrrolidone (NMP) with magnetic stirring, as this is considered elsewhere an excellent non-aqueous vehicle to achieve the highly efficient dispersion of reduced graphene oxide. Alumina was dispersed in water using an ammonium salt of poly(acrylic) acid  in a concentration of 0.5 wt.% on a dry solids basis and sonicated with different sonication times. Concentrated suspensions of alumina-reduced graphene oxide were prepared to solid loadings of 30 vol.% and homogenized with 1 min of ultrasonication.First of all, reduced graphene oxide was dispersed in To obtain a homogeneous dispersion of reduced graphene oxide in zirconia, both materials were dispersed independently and furtherly mixed. Zirconia was dispersed in water to a concentration of 30 vol.% using an ammonium salt of poly(acrylic) acid  in a concentration of 0.5 wt.% on a dry solid basis. Reduced graphene oxide was dispersed in 1-methyl-2-pyrrolidone at the same solids content (30 vol.% reduced graphene oxide) and both slurries were subjected to mechanical homogenization and sonication for 1 min . Once dispersed, proper fractions of both suspensions were mixed altogether to obtain two relative contents of reduced graphene oxide in the zirconia matrix of 2 and 6.7 vol.%, applying 1 min of additional sonication. The rheological behavior of the different suspensions was analyzed using a rotational rheometer.The optimized suspensions of the mixture were frozen in a rotatory chamber using liquid nitrogen as refrigerant (−196 °C). The frozen suspensions were introduced in a freeze-dryer for 24 h. The condensator temperature was −50 °C, and the conditions of the storage camera were 20 °C and 0.050 mbar .A2rGO: 98 vol.% α-alumina and 2 vol.% reduced graphene oxide.A6.7rGO: 93.3 vol.% α-alumina and 6.7 vol.% reduced graphene oxide.Z2rGO: 98 vol.% t-zirconia and 2 vol.% reduced graphene oxide.Z6.7rGO: 93.3 vol.% t-zirconia and 6.7 vol.% reduced graphene oxide.The following resultant mixtures of powders were obtained:As reference materials, pure alumina and pure zirconia commercial powders were used for the sake of comparison. These powders are named A and Z, respectively.The sintering process employed was the same as in ,29. The Raman spectroscopy was performed in order to note the integrity of the reduced graphene oxide. Measurements were performed in the same way as described in ,29.The microstructural characterization was performed by scanning electron microscopy. The surfaces of the samples were polished with diamond paste. The microstructure was described by measuring the grain size and the shape factor. Fracture surfaces were also observed to check the presence and distribution of reduced graphene oxide; to this end a high resolution scanning electron microscope  was employed in the mode secondary electrons.A is a dimensionless constant, G is the shear modulus, b is the magnitude of the Burgers vector, k is the Boltzmann constant, T is the absolute temperature, d is the grain size, p is the inverse of the exponent of the grain, σ is the stress, n is the stress exponent and D a diffusion coefficient [The creep at high temperatures of these new materials was investigated in the same way as explained in ,29. It mfficient .n) and the activation energy (Q). A value of stress exponent was obtained by changing the applied load as it can be seen in the Equation (2) and a value of the activation energy was obtained by modifying the temperature (3).The characteristics parameters of the creep experiments were the stress exponent  demonstrating their good dispersibility. Zirconia was dispersed in water, whereas reduced graphene oxide was dispersed in a non-aqueous medium (NMP) in order to improve the dispersion. The final suspensions had a solid loading of 30 vol.%. −1 observed in 2O3 stabilized ZrO2 and in Al2O3 ceramics in which there is grain boundary sliding with diffusion as an accommodation mechanism [T and σ data. Therefore, additional values of the stress exponents and activation energies were obtained. These facts and the same microstructure before and after creep permit us to affirm that the most important deformation mechanism is grain boundary sliding accommodated by diffusion [All these experimental facts are in good concordance with a general model of the creep deformation in Yechanism ,39. The iffusion ,39. The iffusion . The strOne aspect which that is very important is the analysis of the creep resistance due to the introduction of this carbon phase. To this end, and assuming that the grain size exponent is three because the accommodation process is the diffusion along the grain boundary, we are using this value to normalize strain rate versus stress plots in log–log scales in The reactivity of oxygen, present in the lattice of graphene, could be a crucial issue for the prediction of high-temperature creep resistance. Further work must be conducted to elucidate the local chemistry bonds involved in the ceramic composite at high temperatures.The microstructure is constant during the creep at high temperatures and the deformation mechanism is grain boundary sliding.The analysis of the high temperature mechanical properties allows for the conclusion that reduced graphene oxide does not provide an improvement of more than an order of magnitude in comparison to pure alumina, but it is potentially attractive in the range of nanocomposites or very high temperatures (from 1250 °C).The reduced graphene oxide reinforced zirconia composites are, inside the experimental uncertainty, similar to pure zirconia regarding high-temperature creep properties. The ability of the carbon phase to “wet” the grain boundaries and distribute homogeneously  during deformation is a critical element to predict the high-temperature plasticity.


User-interaction event logs provide rich and large data sets that can provide valuable insights into how people engage with technology. Approaches such as ecological momentary assessment (EMA) can be used to gather accurate real-time data in an individual’s natural environment by asking questions at any given instant.The purpose of this study was to evaluate user engagement and responses to EMA questions using InspireD, an app used for reminiscence by persons with dementia and their caregivers. Research findings can be used to inform EMA use within digital health interventions.A feasibility trial was conducted in which participants (n=56) used the InspireD app over a 12-week period. Participants were a mean age of 73 (SD 13) and were either persons with dementia (n=28) or their caregivers (n=28). Questions, which they could either answer or choose to dismiss, were presented to participants at various instants after reminiscence with personal or generic photos, videos, and music. Presentation and dismissal rates for questions were compared by hour of the day and by trial week to investigate user engagement.21=21.4, P<.001). Questions asked following reminiscence with personal media, especially those asked after personal photos, were less likely to be answered compared to those asked after other media. In contrast, questions asked after the user had listened to generic media, in particular those asked after generic music, were much more likely to be answered.Overall engagement was high, with 69.1% of questions answered when presented. Questions that were presented in the evening had the lowest dismissal rate; the dismissal rate for questions presented at 9 PM was significantly lower than the dismissal rate for questions presented at 11 AM (9 PM: 10%; 11 AM: 50%; χThe main limitation of our study was the lack of generalizability of results to a larger population given the quasi-experimental design and older demographic where half of participants were persons with dementia; however, this study shows that older people are willing to participate and engage in EMA. Based on this study, we propose a series of recommendations for app design to increase user engagement with EMA. These include presenting questions no more than once per day, after 8 PM in the evening, and only if the user is not trying to complete a task within the app. Digital health and well-being products such as health apps are becoming increasingly popular given that mobile technology is ubiquitous in daily life. In addition to the user data that are recorded during use of these platforms, all user interactions and events can be logged to represent usage. Such user interaction and event logs provide rich and large data sets that can reveal valuable insights into how people engage with technology. This case study explored user engagement with ecological momentary assessment (EMA) in an app designed for persons with dementia and their caregivers.Dementia includes a group of symptoms associated with ongoing cognitive decline and is highly prevalent with approximately 50 million cases worldwide . Within Many different techniques (such as EMA or experience sampling methodology) can be used to gather accurate data on daily living. These methodologies are used to capture real-time data in an individual’s natural environment through repeated sampling . This caIn our previous work, we developed an app for reminiscence which incorporated EMA for the Individual Specific Reminiscence in Dementia (InspireD) feasibility study. The study used a quasi-experimental design and investigated the use of an iPad app to allow people living with mild to moderate dementia and their family caregivers to reminisce . In phasReminiscing made up the largest proportion of total app interactions for persons with dementia (71%) and their caregivers (47%) . Across Participants living with dementia attained statistically significant increases in mutuality, quality of caregiver and patient relationship, and subjective well-being from the beginning to end of the study . AdditioIn this study, we sought to address four research questions: (1) What is the temporal engagement with EMA questions over hours of the day and across the trial period? (2) How differently do persons with dementia engage in responding to questions compared to how caregivers engage? (3) How do persons with dementia and their caregivers engage in responding to questions after reminiscing with video, audio, and photos? (4) How do persons with dementia and their caregivers engage in responding to questions after reminiscing with personal media compared to how they respond after reminiscing with generic media? The aim of this study was to evaluate engagement with EMA questions while using an app with the overall goal to inform EMA use within digital health interventions.A user engagement study was conducted in which participants used a digital health intervention, an app for reminiscence, at home for a trial period of 12 weeks. EMA questions on mutuality were presented to users at various points during the trial period while they were reminiscing using the app.The study received ethical approval in March 2016 (16/NI/0035) in line with regional and national Health Service Trust research governance. Ethical considerations were ensuring voluntary participation by supporting separate informed consent for the persons with dementia and their caregivers, secure handling and storage of data in line with University policy, and reminding participants of their right to withdraw from the study at any time. In this study, for each person with dementia who participated, a family caregiver also participated. Each dyad (person with dementia and their caregiver) was given an iPad and asked to use the app at home for a 12-week period. Participants were encouraged verbally and in writing to use the app at least 3 times per week during the trial period. Information technology training was provided on 3 occasions; 2 sessions were provided before the trial period began, and 1 session was provided in week 6 of the trial period. The purpose of the training was to provide guidance on how to use the app, how to upload media, and to provide general app support. The app recorded user event logs locally on each iPad using an SQLite database (a public domain structured query language database). User event log data were later collected in person from the iPads using a portable storage device. Specific activities and activity types were logged: entry (logging in), administrative activities , reminiscing , queries at a given instant (EMA questions), and exiting (logging out). The app allowed individuals to upload their own photos, videos, and music or to access media online. For the purposes of this study, generic media was defined as photos, videos, and music that were accessed online through the app, for example, a photograph of street where the individual lived as a child. Personal media was defined as media that was uploaded, such as an old family photograph.The EMA questions were a subset of items derived from the Mutuality Scale . Over thR studio (version 3.6.0) and the R programming language were used for data wrangling and statistical programming. For the first part of the analysis, the user log data were filtered to only contain data related to queries at a given instant in order to look at overall engagement with questions. Over the course of the 12-week trial period, a total of 832 questions were presented. Of these questions, 77 were asked during training days and so were excluded from this analysis, leaving 755 questions asked during the trial period.Data were aggregated by time of day (hourly), and by week (weeks 1 to 12) to obtain question presentation counts and dismissal rates for each user type (persons with dementia or caregivers). Data were aggregated over the 12-week trial period to obtain total question presentation counts and dismissal rates for each user type , media format , media source , and the last logged activity . Some media could not be categorized into generic or personal media and were, therefore, only included in the aggregate media category . For last logged activity, 124 out of 628 (16%) EMA questions were filtered out of the data since the previous recorded event had been an EMA question and not an action that could trigger a question .P<.05 was considered statistically significant. Chi-square tests were used to compare the proportion of questions answered versus dismissed across hours of the day, across trial weeks and for other logged activities—log-in, add or delete photo, exit app. Pairwise chi-square tests (n=36) adjusted for multiple testing using Bonferroni correction were used to compare dismissal rates for each question following reminiscence with all photos, videos and music, and then segregated into personal and generic photos, videos and music .Spearman rank correlation coefficients were used for association analysis between the number of questions presented and the number of interactions with the app, number of app interactions and the number of questions presented across trial weeks, dismissal rate and time of day, dismissal rate and trial week, number of questions presented and the number of questions dismissed per hour, where A total of 30 dyads (person with dementia and their caregiver) were recruited to the study; however, usage data was found to be corrupted in the iPad software used by 2 participants with dementia and their caregivers. Therefore, only 28 sets of tracking data (n=56 participants) were analyzed in this study. The characteristics of the participants are shown in P<.001), with roughly 1 question asked for every 10 app interactions. The overall dismissal rate for questions asked during the trial period excluding training days was 30.9%. Hence, 522 out of 755 (69.1%) questions were answered. Persons with dementia used the app more in the trial period than caregivers did [Five different EMA questions from the Mutuality Scale  were prevers did , but desA breakdown of presentation and dismissal for each question is shown in P=.001).The number of questions presented per hour and per week are shown in 21=21.4, P<.001); questions asked at 9 PM  were 5 times more likely to be answered than questions asked at 11 AM . The dismissal rate for questions was high at the start and end of the trial and was lowest in the middle of the trial .The hourly and weekly dismissal rates are shown in he trial . For exaP<.001, ρ=0.81). Weekly dismissal rates differed between user types .Dismissal rates by time of day and by week for each user type are shown in er types . There wP<.001, ρ=0.84) and persons with dementia . From investigating the residuals, there were significantly less questions dismissed at the hours of 2 PM , 9 PM, and 10 PM for both caregivers and persons with dementia. For both user types, questions in the evening were more likely to be answered than dismissed.There was a strong correlation between the number of questions presented and the number of questions dismissed per hour for caregivers  and generic music versus personal music . There was no significant difference between proportion of questions of dismissed and answered between generic videos and personal videos .As shown in 21=10.9, P<.001). The dismissal rate for questions asked after viewing personal photos  was significantly higher than the rates of those asked after generic music , personal music , and generic photos . The dismissal rate for questions asked after interacting with generic music  was significantly lower than those of personal music , personal videos , or personal photos . These results show that questions asked following reminiscence with personal media, especially those asked after personal photos, were less likely to be answered compared to those asked after other media. In contrast, questions asked after the user had listened to generic media, in particular those asked after generic music, were much more likely to be answered.Significantly more questions were dismissed after viewing any type of photo than after listening to any type of music . There was no significant difference between questions answered and questions dismissed when caregivers logged in , when caregivers added or deleted photos , or when caregivers or persons with dementia exited the app .Questions were also presented to users as they were completing other logged activities. There were significantly more questions answered than were dismissed when a person with dementia logged into the app  was deemed sufficient to meet the objectives of a feasibility study.Another limitation was the recording of log data. User events were logged in real time and were later collected in person from the iPads using portable storage devices. As a result, we could not control for lost local data due to operating system failures, app crashes, bugs, and updates. This was the case with some of the log data which was recorded. For 16% of all EMA questions asked, the event which was logged previously was also an EMA question instead of one of the actions  that shoThis study explored engagement with EMA questions using an app for reminiscence where the users were caregivers and persons with dementia. Our results show that older people are willing to participate and engage in EMA; however, more can be done to increase engagement. Notwithstanding the limitations, based on this study we propose a set of recommendations for the use of EMA to optimize user engagement within a digital intervention. EMA questions should add value and help to validate the use of the digital health app in line with the study objectives. It is important that EMA questions do not distract or interfere with the overall purpose of the app which was to allow persons with dementia and their caregivers to reminisce. To avoid overprompting the user, no more than one question should be presented per day and ideally in the evening after 8 PM as this is when people were most likely to engage. If the user is trying to complete a task within the app such as adding content then a question should not be presented. These recommendations can be broadly applied to EMA use in similar settings. Future work will be carried out to study the engagement on a larger scale with more participants, which will further support these recommendations. Future work will also involve studying app usage patterns, for example, if a user engages in an EMA question, when will they next engage, and can we predict engagement based on user log analysis.


Model organisms like C. elegans and Drosophila are powerful systems to help dissect molecular genetic aspects of complex biological processes. Here we will present a number of biological questions and approaches that have been used to study questions of neural integrity and healthful aging using these systems.


Dendropanax morbifera, which is endemic to southern regions of Asia, have long been used as a folk medicine to treat variety of diseases. However, the sap of this plant has not been widely studied and its bioactive properties have yet to be clearly elucidated. Here, we isolated extracellular vesicles from D. morbifera sap with the goal of improving the intracellular delivery efficiency and clinical effectiveness of bioactive compounds in D. morbifera sap. We further investigated the anti-metastatic effects of D. morbifera sap-derived extracellular vesicles (DMS-EVs) using a cancer metastasis model based on 3D microfluidic system that closely mimics the in vivo tumor environment. We found that DMS-EVs exerted a concentration-dependent suppressive effect on cancer-associated fibroblasts (CAFs), which are important mediators of cancer metastasis. DMS-EVs also altered expression level of genes, especially growth factor and extracellular matrix (ECM)-related genes, including integrin and collagen. Our findings suggest that DMS-EVs can act as anti-CAF agents to reduce CAFs in the tumor microenvironment. They further indicate the utility of our 3D microfluidic model for various drug-screening assays as a potential alternative to animal testing for use in validating therapeutic effects on cancer metastasis.Natural medicinal plants have attracted considerable research attention for their potential as effective drugs. The roots, leaves and stems of the plant,  Extracellular vesicles (EVs), which contain signal proteins, lipids, micro RNAs (miRNAs) and functional mRNAs, play an important role in intercellular communication in the extracellular space ,2. In adDendropanax morbifera is a plant endemic to southern parts of Asia, a region with a rich folk medicine tradition and a history of commercial production of D. morbifera golden varnish. Recent studies have reported that extracts from roots, leaves and stems of D. morbifera exhibit antioxidant, anticancer and anti-diabetic effects, as well as anti-complement, anti-inflammatory and antiplasmodial activities  fetal bovine serum  and 1% [v/v] penicillin-streptomycin . Before detachment of cells from the culture dish, the culture medium was replaced with Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% exosome-depleted FBS and 1% [v/v] penicillin-streptomycin to remove interference from FBS exosomes in media. The culture medium was then collected and concentrated by tangential flow filtration using a membrane with a 100-kDa molecular weight cutoff . The resulting exosomes were filtered with a 0.22-μm filter and stored in a −80 °C freezer until use.B16BL6 murine melanoma cells  were cultured in minimum essential media alpha , supplemented with 10% [5.0 mg/mL solution of collagen gel (pH 7.4) was prepared by add 10× phosphate buffered saline , 0.5N NaOH, distilled deionized water and collagen solution  in the given order. The hydrogel solution was then injected into the gel-filling region of microfluidic devices. Hydrogel-filled devices were place in a humidified, 37 °C incubator for 30 min. After incubation, all channels of the devices were filled with endothelial basal medium-2 , and the devices were again placed in the incubator.2 environment and used at fewer than 5 passages in all experiments. Approximately 50–60 μL of HUVEC suspensions (5 × 106 cells/mL) was seeded into the central channel of microfluidic devices and allowed to attach. After attachment of HUVECs, all reservoirs of devices were filled with EGM-2 medium, and HUVECs were incubated for 2 days to allow formation of an endothelial monolayer.HUVECs  were cultured in endothelial growth medium  at 37 °C in a 5% COHUVEC monolayers were reconstituted within 3D microfluidic devices supported by the collagen gel, which recapitulates a 3D tumor microenvironment. Cancer (melanoma)-derived exosomes (C-EVs) were delivered at a concentration of 50 µg/mL, after which interstitial fluid flow was provided through channels on one side through the collagen by virtue of height differences between side reservoirs. Our previous studies showed that the predicted interstitial flow rates in the device were range from 0.1–0.7 µm/s, which was pathophysiological level of interstitial flow ,16. CAFsCellular uptake of DMS-EVs was analyzed by fluorescence microscopy. DMS-EVs extracted as described above were tagged by incubating with red Di-I stain (1 µg/mL), a lipophilic dye. After DMS-EV treatment and uptake by HUVECs and CAFs in the 3D microfluidic device, the medium was removed and cells were fixed by incubating with 4% paraformaldehyde. The cells were prepared for staining by adding 0.15% Triton X-100 and incubating for 30 min at room temperature, after which all solutions were removed and wells were filled with 3% bovine serum albumin (BSA). After incubating for 1 h at room temperature, the solutions were again removed and Actin green 488 was added to stain the cytoplasm of the cells. Cells were then incubated for 30 min at room temperature, washed three times with 1% BSA, and incubated at room temperature for 15 min with Hoechst33342, which stains nuclei. The wells were then washed with 1% BSA and filled with PBS, after which cells were imaged under a fluorescence microscope .The viability of mammalian cells was tested using a LIVE/DEAD viability/cytotoxicity kit  as described by the manufacturer. Briefly, after culturing as described in the text, the medium was removed and cells were washed three times with PBS. Staining solution, prepared by mixing calcein AM and ethidium homodimer-1, was then added and cells were incubated for about 30 min at room temperature. The percentage of live cells stained by calcein with green fluorescence converted from calcein-AM by intracellular esterase activity and dead cells stained with orange/red by ethidium homodimer-1 (EthD-1) was assessed by imaging cells under a fluorescence microscope .v/v) for measurement of zeta potential. Diluted samples were inserted into the folded capillary cell  and detected using the same equipment indicated above.Hydrodynamic size and zeta potential of DMS-EVs were determined using dynamic light scattering (DLS) and a Zetasizer . Isolated EVs were placed in a thermostatic cell at 20 °C. The size distribution was determined by measuring hydrodynamic size. Thereafter, samples were mixed with distilled water at a ratio of 95:3 , which assesses the combined properties of light scattering and Brownian motion. Samples suspended in liquid were diluted and visualized with a Nanosight instrument  at a temperature of 25 °C using a 488 nm laser.Nanostring nCounter assays  were conducted according to the procedure outlined by the manufacturer. All tags were removed except for the DNA tags ligated onto the 3′-end of each miRNA in the samples. After each miRNA was uniquely represented, the samples were hybridized with tag-specific nCounter capture and barcoded reporter probes by reacting with 5 mL of 5×-diluted sample preparation reaction solution. Total RNA samples (100 ng) were incubated at 64 °C for at least 18 h. All excess capture and reported probes were removed. Target RNA molecules in the sample were quantified and classified by counting each fluorescent barcode with the nCounter digital analyzer.p-value < 0.05 was considered statistically significant.All data were obtained through three replicate experiments. Statistical analyses were performed using two-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test with GraphPad Prism . 9 particles/mL, and particles displayed a negative zeta potential value of −25 mV. Zeta potential provides a measure of dispersion stability, a characteristic property of the population of exosomes that reflects interactions among charged particles. A higher zeta potential results in greater electrostatic mobility between particles and a greater tendency to resist aggregation. Vesicles with minimum zeta potentials of ±20 mV are moderately stable [DMS-EVs were isolated using a sequential centrifugation step and filtration approach based on mammalian exosome isolation techniques, and their size distribution was evaluated by nanoparticle tracking analysis (NTA) and dynamic light scattering (DLS) analysis a,b. BothFluorescent DMS-EVs, labeled with red Di-I and incubated with CAFs as described in Materials and Methods, were widely distributed within cells, including in the cytoplasm and around nuclei e, indicaA 3D microfluidic model was used to monitor differentiation of CAFs from HUVECs a,b. To tAs a control, normal fibroblasts (NFs) were seeded into the medium channel, and inhibition rates for CAFs and NFs were determined in real time by counting the number of CAFs or NFs per field of view after single treatment with 1 or 10 µg/mL of DMS-EVs c,d. As sReprograming of fibroblasts into CAFs by cancer cells is mediated by miRNA. However, CAFs derived from tumor stroma have been reported to differ from normal stromal fibroblasts in terms of the expression of ECM proteins, pro-tumor factors, and cytokines ,21. ThusTo further assess the effect of concentration and treatment procedure on the suppression of CAFs by DMS-EVs, we investigated three different conditions: repetitive treatment, pre-treatment before adding cancer-derived EVs (C-EVs) and co-treatment with C-EVs and DMS-EVs together .For repetitive treatment, CAFs were treated with DMS-EVs every 24 h. With this experimental paradigm, after the initial DM-EV treatment, the inhibition rate and number of CAFs were similar regardless of the concentration of DM-EVs, and 4 days were required to completely suppress CAFs a. Thus, For pre-treatment, CAFs were treated with 1 or 10 µg/mL of DMS-EVs followed by treatment with C-EVs. In these experiments pre-treatment with 1 or 10 µg/mL of DMS-EVs decreased the survival of CAFs by 37% and 47%, respectively, on day 4 compared with DMS-EV–untreated CAFs. To date, there is limited evidence of the effectiveness of natural products in preventing cancer or cancer recurrence . Thus, oIn the co-treatment paradigm, DMS-EVs and C-EVs were applied together on a human endothelial monolayer our in vitro model. Co-treatment with 1 or 10 µg/mL of DMS-EVs together with C-EVs decreased CAF survival by 55% and 76%, respectively, on day 4 compared with DMS-EV–untreated CAFs. There is increasing evidence that natural products from dietary sources can make an important contribution to cancer development, and that diet may affect outcomes after cancer diagnosis ,27. Our To identify the major CAF-associated genes that are affected by DMS-EV treatment, we profiled mRNA expression using the Nanostring nCounter platform, which includes a panel of 770 genes known to be involved in metastasis. To this end, CAFs were treated with 1 or 10 μg/mL of DMS-EVs and differences in gene expression compared with untreated CAFs were assessed. Of the 770 genes in the Nanostring nCounter analysis system panel, approximately 20% are CAF-related genes. Heat maps used to compare differentially expressed gene levels between untreated CAFs and CAFs treated with 1 or 10 µg/mL of DMS-EVs showed clusters based on similarities and dissimilarities among groups a. IntereWe then performed a Gene Ontology (GO) enrichment analysis of differently expressed mRNAs with critical functions in CAFs, comparing untreated CAFs with CAFs treated with 1 or 10 µg/mL of DMS-EVs, as shown in To further investigate genes whose expression was changed in CAFs by DMS-EVs in a concentration-dependent manner, we focused on 10 genes that showed different degrees of differential expression between treatment with 1 and 10 µg/mL of DMS-EVs a: CD44, TFG-β2, which was significantly upregulated in CAFs in the absence of DMS-EVs treatment, was decreased by 5.8 and 6.2-fold at 1 and 10 µg/mL of DMS-EVs, respectively, indicating a dominant effect of DMS-EVs on CAFs b. The TFDuring cancer progression, CAFs are directly involved in dysregulated collagen turnover, which leads to increased stiffening of the tissue ,39. The Although these findings reveal changes in CAF-associated genes with DMS-EV treatment, whether DMS-EVs contribute directly to the inhibition of genes associated with the reduction in the number CAFs will require further investigation, because these genes exhibit interactions among the various ECM-related pathways. The traditional therapeutic modalities against cancer are chemotherapy, radiation therapy, and antiangiogenic therapy . HoweverWe investigated the inhibitory effects of DMS-EVs against CAFs in a 3D microfluidic model. We quantitatively analyzed the concentration-dependent effects of DMS-EVs on the phenotypic properties and viability of CAFs in real time, and performed a genotypic analysis to identify and determine the function of the dominant genes associated with CAFs that were affected by DMS-EVs treatment. Results obtained using our 3D in vitro model suggest that naturally derived nano-vesicles from plants have the potential to exert concentration- and time-dependent preventive and suppressive effects on the tumor microenvironment. Genes that showed differential expression in response to treatment with DMS-EVs preferentially included growth factors and ECM-related proteins, including integrins and collagens. On the basis of our results, we propose that an integrated nano-medicine approach using natural plant-based nanotechnology may provide a new direction for improving targeted stromal therapy. Further in vivo pre-clinical research and clinical studies are needed to develop safer and more effective anticancer metastasis treatments in the future.


Despite the availability of multi-“omics” strategies, insights into the etiology and pathogenesis of sarcoidosis have been elusive. This is partly due to the lack of reliable preclinical models and a paucity of validated biomarkers. As granulomas are a key feature of sarcoidosis, we speculate that direct genomic interrogation of sarcoid tissues, may lead to identification of dysregulated gene pathways or biomarker signatures.To facilitate the development sarcoidosis genomic biomarkers by gene expression profiling of sarcoidosis granulomas in lung and lymph node tissues (most commonly affected organs) and comparison to infectious granulomas (coccidiodomycosis and tuberculosis).Transcriptomic profiles of immune-related gene from micro-dissected sarcoidosis granulomas within lung and mediastinal lymph node tissues and compared to infectious granulomas from paraffin-embedded blocks. Differentially-expressed genes (DEGs) were profiled, compared among the three granulomatous diseases and analyzed for functional enrichment pathways.STAB1, HBEGF, and NOTCH4), excluded others and identified new genomic markers present in lung and lymph nodes, ADAMTS1, NPR1 and CXCL2. Comparisons between sarcoidosis and pathogen granulomas identified pathway divergences and commonalities at gene expression level.Despite histologic similarities, DEGs and pathway enrichment markedly differed in sarcoidosis granulomas from lymph nodes and lung. Lymph nodes showed a clear immunological response, whereas a structural regenerative response was observed in lung. Sarcoidosis granuloma gene expression data corroborated previously reported genomic biomarkers (These findings suggest the importance of tissue and disease-specificity evaluation when exploring sarcoidosis genomic markers. This relevant translational information in sarcoidosis and other two histopathological similar infections provides meaningful specific genomic-derived biomarkers for sarcoidosis diagnosis and prognosis. Sarcoidosis is a multisystemic disease of unknown etiology characterized by the formation of granulomatous lesions, especially in lung tissues and thoracic lymph nodes , 2. The Coccidioides immitis and C. posadasii, a soil-dwelling fungi disease, are endemic in the arid southwestern USA and northern Mexico.The etiology of sarcoidosis is unknown, and the diagnosis remains one of exclusion . SpecifiGranulomas are conglomerates of epithelioid and multinucleated giant cells surrounded by CD4+ and CD8+ T lymphocytes that result from the complex immunopathogenesis between host genetic factors, environmental or infectious triggers . SarcoidAs expression profiling of sarcoidosis granuloma using next generation sequencing expression has not been reported, one goal of the present study was to compare gene expression profiles in two sarcoidosis granulomatous tissues: lung and lymph node. The second goal of this study was to assess the ability of granuloma gene expression profiles to discriminate sarcoidosis from TB and CM. Our bioinformatic analyses included determination of unique pathways associated with sarcoidosis and overlapping pathways with CM and TB. Our results indicate that despite histologic similarities, sarcoidosis lung and lymph node granulomas exhibit distinct expression profiles. In addition, sarcoidosis pathway expression was significantly divergent compared to TB and CM. These findings suggest the importance of tissue-specific pathobiology considerations to be employed when exploring potential sarcoidosis genomic markers for potential diagnostic use and discriminate from other granulomatous diseases.We used microdissected granulomatous tissue from lung  and medi2 thickness of a 5 μm FFPE tissue were obtained, 5–10 sections per subject were lysed followed by RNA extraction-free chemistry method using the HTG®’s assay kit. Library preparation for the HTG Edge processor for nuclease protection steps was used, followed by PCR tagging, library amplification, quantitation and normalization. Library was then sequenced on an Illumina MiSeq using 150-cycle V3 kits [Formalin-fixed, paraffin-embedded (FFPE) microdissections from lung and lymph node granulomatous tissue and healthy tissue (controls) were assayed using Next Generation Sequencing-based gene expression by HTG EdgeSeq Oncology-biomarker . This system uses targeted capture sequencing to quantitate RNA expression levels of gene targets in FFPE tissues. The panel included 2535 probes and 15 housekeeper genes for quantitative analysis of targeted mRNAs. A list of the complete biomarker panel genes and the biological pathways assayed are in Additional file  V3 kits .We generated the transcriptome index using the salmon package  and a faWe validated our transcriptome results in three independent cohorts of microarray datasets publicly available in the Gene Expression Omnibus (GEO), GSE16538 and GSE63548  and an uMicrodissected lung and lymph node granulomas from 18 sarcoidosis subjects, four TB subjects, three CM subjects and six healthy controls were assessed. There were no significant differences in age (p < 0.1) in the three granulomatous disease groups  was obtained as the ratio between the averages. We identified 250 significantly dysregulated genes between all granulomatous-diseased tissues and healthy control tissues. Figure NR1H3 and CXCL9 were upregulated whereas the remainder  showed downregulation in both lung and lymph nodes. Interrogation of the expression levels of these 10 sarcoidosis genes to TB and CM DEGs revealed CCL14, CXCL9, FABP4, NR1H3 were also significantly dysregulated in TB. Only six genes  were exclusively dysregulated in both lung and lymph node in sarcoidosis granulomas but not in CM or TB . Only three DEGs, SLAMF7, DC27, and CXCL13, were dysregulated in all three diseases . Expression of CXCL13, a chemokine and B-lymphocyte chemoattractant associated to calcium influx was downregulated in both sarcoidosis (lymph node) and TB granulomas but upregulated CM granulomas. A list of the top dysregulated genes by granuloma tissue origin is available in Additional file Gene expression profiles from CM lung granulomas exhibited the fewest number of dysregulated genes, 34 DEGs with a balanced pattern of dysregulation, 18 DEGs downregulated, 16 DEGs upregulated. A total of 15 DEGs were exclusively dysregulated in CM granulomas. Eight upregulated transcripts were common to sarcoidosis lung granulomas , cytokine-cytokine receptor interaction, chemokine signaling, VEGFA-VEGFR2 and focal adhesion accounted for the most significant pathways with the most number of proportionally dysregulated genes involved, we then mapped the pathways associated to the genes that were exclusively present in sarcoidosis. Sarcoidosis pathways showed similar altered pathways to the rest of the granulomatous diseases with the exception of nuclear receptors meta-pathway . However, CXCL9 was also upregulated in the TB granulomas. Downregulated genes included FABP4, a fatty acid-binding protein associated with activation of proinflammatory macrophages in breast cancer, obesity and leukemia progression [CCL14, a chemokine ligand associated with changes in intracellular calcium concentration in monocytes. Hypercalcemia associated to granulomatous diseases is commonly reported [CXCL13 and CXCL14, both DEG in sarcoidosis and TB needs further investigation.Only ten DEGs were common to both sarcoidosis lung granulomas and lymph nodes granulomas. gression , 40. FABgression , 41. We reported –44, the CXCL13, CD27 and SLAMF7, a finding that points to diverse pathogenic mechanisms in granulomatous diseases that result in a similar histopathological conglomeration of CD4+ T cells surrounded by CD8+ T cells, fibroblasts and B cells. CXCL13 is a chemokine with key role in B cell migration by regulation of Ca++ influx [SLAMF7 DE but only in sarcoidosis lymph node granulomas.Despite similarities at the histological level, our analysis revealed sarcoidosis granulomas to be significantly divergent at the genomic level when compared to CM granulomas. TB and sarcoidosis granuloma profiles share stronger similarity at the transcriptional and pathway level. Interestingly, the number of DE genes in each comparison using the same thresholds varied significantly, indicating that the number of dysregulated genes does not correlate with the number of samples per comparison, or the size of the microdissection, suggesting that the granulomas are qualitatively distinct depending on the disease and tissue of origin. We identified only three shared dysregulated transcripts in CM, TB and sarcoidosis (lymph nodes); + influx , while S+ influx . FurtherThe activation of Th1 cells capable of producing interferon (IFN)-γ is important in the immunological pathogenesis of the granuloma formation . InterfeCXCL9, a chemoattractant for lymphocytes previously proposed as a marker of sarcoidosis severity [NOTCH4 expression to be exclusively present in lung sarcoidosis. NOTCH signaling is initiated after activation of toll-like receptors in macrophages and it has been previously reported that Notch receptor ligand Dll4 is expressed in cutaneous sarcoidosis granulomas [NOTCH4 have been reported as a sarcoidosis-associated locus in genome-wide associated study in African Americans [This study also served to demonstrate the lack of specificity of prior suggested potential sarcoidosis genomic markers due to common dysregulation in fungal or mycobacterium granulomas. For example, severity , was alsseverity . On the anulomas . Supportmericans .SERTAD1, HBEGF, KLRB1) as DEGs by oncopanel profiling. KLRB1 expression was also downregulated in TB and SERTAD1 was significantly downregulated in CM. Therefore, these two genes appear to be part of common process in granulomatous diseases. In contrast, HBEGF, a heparin-binding epidermal growth factor, was down-regulated in sarcoidosis lung granulomas  and was not dysregulated in CM or TB. HBEGF is involved in epithelialization, wound contraction and angiogenesis with an active pro-inflammatory role in skin and lung alveolar regeneration [HBEGF expression was up-regulated in contrast to the observed down-regulation in granuloma tissues. This variation in gene expression across tissues may be explained by either heritability at cis loci, by trans regulation across tissues [STAB1 as part of a miRNA-derived gene signature for complicated sarcoidosis [IL6ST was significantly dysregulated only in TB granulomas (FC − 1.42 and p 0.0008), while STAB1 gene was significantly downregulated in lymph nodes in sarcoidosis . STAB1 codes for a transmembrane receptor that is expressed in endothelial cells and lymph nodes, with functions in angiogenesis, lymphocyte homing and cell adhesion which are key aspects in the chronicity of the granuloma.We have previously generated a 20-sarcoidosis gene signature by microarray in peripheral blood from PBMCs . We now neration , pneumonneration . Importa tissues , 54 or bADAMST1, CXCL2 and FABP4, that were identified DEGs by the oncopanel. These three genomic markers were present exclusively in sarcoidosis in both lung and lymph node. NPR1, Natriuretic Peptide Receptor 1, is associated with cardiac hypertrophy and fibrosis [ADAMTS1, gene encodes a disintegrin and metalloproteinase with thrombospondin motif associated with various inflammatory processes and a potentially important regulator of pathogenic granuloma formation in sarcoidosis in both compartments.Leveraging the power derived from publicly available microarray data sets from independent cohorts, we successfully validated fibrosis  and has There are some limitations of the current study. One is related to RNA from FFPE tissues, despite the use of quantitative nuclease protection chemistry that enabled extraction-free quantitation of mRNA, RNA quantity and quality are limited in FFPE compared to cryopreserved tissues. Other limitations include the use of a sequencing panel and not whole genome sequencing, that limited number of gene probes and the expression levels of some genes was undetected. Our comparisons were against tissues and not immune cells, that and the difference in platforms (RNAseq vs microarrays) can explain the limited overlap to already published data sets. Last, the only gene expression data available in CM was microarray data in mice strains limiting the validation of our results in CM. Despite the observed differences at the pathway level in sarcoidosis granulomas at lung and lymph node that indicate disease progression, further investigations are needed to corroborate the expression of these genes in sarcoidosis heterogeneous subphenotypes since limited clinical data was available to establish these correlations.Sarcoidosis is a complex systemic disease of unknown cause with clinical care hampered by the paucity of available tools for diagnosis and prognosis. Our study is the first to identify RNAseq-derived transcriptome differences in granulomas from lung and lymph nodes of sarcoidosis subjects compared to CM and TB. Our data suggest that immunological signaling related-pathways distinguishes sarcoidosis from other granulomatous diseases and fills a crucial gap in knowledge of gene expression within granulomas. This study corroborates previous genomic markers suggested for sarcoidosis such as STAB1, HBEGF, FABP4 and NOTCH4, with HBEGF and NOTCH4 only expressed in lung granulomas. We have identified new genomic markers for sarcoidosis, ADAMTS1, CXCL2, and NPR1. This relevant translational information in sarcoidosis and other two histopathological similar infections provides important information for the future development of meaningful specific genomic-derived biomarkers for sarcoidosis diagnosis and prognosis.Additional file 1: Table S1. Oncopanel gene list. List of the complete biomarker panel genes and the biological pathways assayed.Additional file 2: Table S2. Sarcoidosis clinical phenotype. Scadding stage based on radiographic imaging which consist of Stage I: hilar enlargement only; Stage II: hilar enlargement plus interstitial lung disease; Stage III: interstitial lung disease and Stage IV: lung fibrosis. Lung involvement and other organs affected based on clinical data. All subjects have at least one reported clinical symptom related to lung involvement . NA. Information not available.Additional file 3: Table S3. Microarrays data sets used for validation of the DEG identified by the oncopanel.Additional file 4: Table S4. Sarcoidosis, TB and CM top common dysregulated genes. Common DEGs in granulomas derived this oncopanel in lung sarcoidosis and CM and in granulomas derived from lymph nodes from TB and Sarcoidosis.Additional file 5: Fig. S1. Microarray validation. Comparison of granuloma DEGs identified by the oncopanel to microarray datasets. Heat maps representing expression profiles of validated gene sets in A) Sarcoidosis Lung 14 transcripts validated, B) Sarcoidosis Lymph nodes, 30 transcripts validated and C). TB lymph nodes, 46 transcripts validated. Transcripts differentially regulated showed a fold change ±2 & FDR<0.01.


Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response.2O2 treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage.We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants , in which DNA damage was induced by etoposide or HWe demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology; cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm.This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS. Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder associated with atrophy of both upper and/or lower motor neurons, leading to muscle wasting due to denervation . ALS oveTDP-43 is a RNA- and DNA-binding protein normally located in the nucleus, where it is involved in transcription, splicing, RNA metabolism and miRNA biogenesis . Whilst The genome is under constant attack from both environmental agents and normal metabolic processes. Thus, maintaining genomic integrity is essential because damage to DNA can induce mutations and seriously compromise cellular viability. DNA damage induces the ‘DNA damage response (DDR)’, signalling pathways that aim to detect and repair the damage. However, persistent DNA damage and defects in DNA repair lead to apoptosis. The most cytotoxic lesions are double-stranded DNA breaks (DSBs) which are primarily repaired by error-prone non-homologous end joining (NHEJ) in neurons , unlike SETX, VCP, NEK1, C21orf2, and CCNF [C9ORF72, which cause a major proportion of familial ALS cases, induce DNA damage in neuronal cells and motor neurons of ALS patients [Fused in Sarcoma (FUS) has striking functional and pathological similarities to TDP-43 in ALS, and it normally performs important functions in DNA repair . Interesand CCNF –27. Recepatients . This wapatients . TDP-43 patients  and two patients , 32. It patients , and expIn this study we demonstrate that TDP-43 is a DNA repair protein that functions in classical, but not alternative, NHEJ in neuronal cells. However, ALS TDP-43 mutants A315T and Q331K lack this normally protective function, leading to the accumulation of DNA damage. Furthermore, in mouse models displaying pathological TDP-43 that develop features of ALS , DNA damage is present before the onset of disease. These mice express cytoplasmic, wildtype TDP-43 and thus bear semblance to sporadic ALS. We also show that DNA damage induces features typical of TDP-43 pathology: mis-localisation to the cytoplasm and SG formation. Moreover, inhibition of classical NHEJ repair also leads to TDP-43 mis-localisation to the cytoplasm. This study therefore places aberrant DNA damage responses onto the pathophysiolology of TDP-43, which is associated with almost all ALS cases. It therefore highlights the DDR as a novel therapeutic target for ameliorating TDP-43 pathology in ALS.Details of the patients used in this study are summarised in Table Monogenic B6;C3-Tg (NEFH-tTA)8Vle/J  mice and monogenic B6;C3-Tg (tetO-TARDBP*)4Vle/J  mice were obtained from the Jackson Laboratory (USA) and maintained on a mixed B6/C3H background  and were crossed to obtain bigenic rNLS and littermate control mice. All the mice were genotyped according to the Jackson Laboratory protocols. Expression of the hTDP-43–rNLS transgene was confirmed by cytoplasmic TDP-43 staining and increased levels of human TDP-43 by immunoblotting. Mice were provided with feed containing 200 mg/kg doxycycline  until 6–8 weeks of age.Fibroblasts from both ALS patients and pre-symptomatic carriers bearing the M337V mutation were obtained and cultured as previously described [2. Neurons were transfected with Lipofectamine 2000 at 3 or 5 DIV and fixed with 4% paraformaldehyde after 24 h.Primary cortical neuronal cultures were prepared from mouse C57BL/6 E16 brains. After isolation, cells were plated on glass coverslips covered with poly-L-lysine (Sigma-Aldrich), in B27 neurobasal medium supplemented with 1% glutamate and 1% penicillin/streptomycin and incubated at 37 °C and 5% CO2.NSC-34/CHO-9/XR-C1 or Neuro2A cells were cultured in DMEM medium with 10% FBS at 37 °C and 5% CO2O2 for 1 h. For inhibition of DNA-PK activity, a specific inhibitor was used, NU 7441 .DNA damage was induced by treatment with 13.5 μM etoposide or 100 μM HAfter washing with 1 x PBS, cells were permeabilised in 0.1% Triton X-100 in PBS for 5 min and blocked in 1% BSA in PBS for 30 min. Staining was performed overnight with the following primary antibodies at a concentration of 1:500: rabbit anti-γH2AX ; rabbit anti-53BP1 , mouse anti-DNA-PKcs (Abcam), rabbit anti-TDP-43 C-terminus (405–414) , and rabbit anti-phospho-TDP-43 . The following secondary antibodies were used: anti-rabbit Alexa Fluor 488, (Life technologies), anti-mouse Alexa Fluor 594 (Life technologies) or anti-rabbit Alexa Fluor 647 antibodies  for 2 h. The nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich) or DAPI (Sigma Aldrich).The Comet assay was adapted from a previous method . BrieflyTwo small interfering RNAs (siRNAs) described previously , targetiAfter deparaffinisation, antigen retrieval in 10 mM citrate buffer with 0.1% Tween for 30 min at 80 °C was performed. Sections were blocked with normal donkey serum for 30 min and then stained with anti-γH2AX  or anti-human TDP-43 antibodies  for 24 h at 4 °C, and after three washes with PBS, the tissues were incubated with AlexaFluor 488/564  conjugated secondary antibodies for 2 h at 4 °C. The nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich).Fluorescently labelled cells were acquired using a Zeiss AxioImager epi-fluorescence microscope (Zeiss) or a Zeiss LSM 880 confocal microscope.The following mammalian expression plasmids were used as described previously: pimEJ5GFP (Addgene # 44026) and EJ2GFP-puro (Addgene # 44025) were a gift from Jeremy Stark, TDP-43 wildtype-EGFP, TDP-43 A315T-EGFP, TDP-43 Q331K-EGFP, pEGFP-N1 , TDP-43 Mouse brain tissues were thawed on ice and sonicated in 5x volumes of BICARB buffer . Samples were centrifuged at 100,000 x g at 4 °C for 30 min. Supernatants were collected as BICARB soluble fractions. The human fibroblasts or NSC-34 cells were lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% (wt/v) SDS, 1% protease inhibitor cocktail (Sigma) and 1% phosphatase inhibitor cocktail (Sigma). Protein concentrations were determined using a BCA protein assay (Pierce). Twenty micrograms of soluble fractions were analysed by 4–15% gradient SDS-PAGE and blotted onto nitrocellulose membranes (Bio-Rad). Blots were blocked with 5% skim milk and incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. The next day, unbound primary antibodies were removed by washing and blots were incubated with HRP-conjugated secondary antibodies for 1–2 h at room temperature. Images were developed with ECL blotting substrates (Bio-Rad) and a ChemiDoc Gel imaging system (Bio-Rad). Antibodies used for immunoblotting were: rabbit anti-γH2AX , mouse anti-GAPDH , rabbit anti-GAPDH , rabbit anti-TDP-43 , goat anti-mouse IgG and IgM HRP conjugate , and goat anti-rabbit IgG antibodies, peroxidase conjugated .Immunoprecipitation was performed using GFP-Trap MA (Chromotek), which utilizes small recombinant alpaca antibody fragments covalently coupled to the surface of agarose magnetic beads. Briefly, NSC-34 transfected cells were lysed in lysis buffer  with a cocktail of protease inhibitors (Roche) and 1 m PMSF for 30 min. on ice. Then, cell lysates were centrifuged at 20,000 x g for 10 min at 4 °C. After centrifugation, the lysates were transferred to a pre-cooled new tube. The protein concentration was quantified using a BCA kit (Thermo Fisher Scientific). Five hundred microgram of the lysate was taken for further processing. Fifty microliter of diluted lysates were retained for immunoblotting analysis for the input samples. Twenty five microliter of equilibrated GFP-Trap MA beads were added to diluted lysates and rotated end-over-end overnight at 4 °C. The following day, the beads were separated magnetically from the supernatant and washed three times. Magnetically separated beads were resuspended in 100 ul 2x SDS-sample buffer and boiled for 10 min at 95 °C to dissociate immunocomplexes from GFP-Trap MA beads. The beads were separated from the supernatant magnetically and the supernatant was subjected to immunoblotting.NSC-34 cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturers’ protocol.NHEJ reporters, pimEJ5GFP or EJ2GFP-puro, were pre-digested with I-SceI restriction enzyme and the digestion products following purification were run on a 1% agarose gel to confirm that each reporter had been digested. Cells were co-transfected with mCherry-tagged TDP-43 wildtype, A315T, or Q331K, and pre-digested NHEJ reporter by nucleofection using Cell Line Nucleofector Kit V or Human Dermal Fibroblast Nucleofector™ Kit, according to the manufacturers protocol (Lonza). The efficiency of GFP restoration through NHEJ was estimated as the percentage of cells with both mCherry and GFP signals, in proportion to the total number of cells with mCherry expression , 38.NSC-34 cells were cultured in DMEM media (Gibco) on 13 mm coverslips (Menzel-Glasser) in 24 well plates and transiently transfected with EGFP-tagged TDP-43 using Lipofectamine 2000 (Invitrogen). To induce DNA damage, cells were treated with 13.5 μM etoposide  or DMSO as a control. After 48 h, cells were fixed with 4% PFA and mounted with Dako fluorescence mounting media. Images were taken with an epi-fluorescent microscope (Zeiss). For identification of SGs containing TDP-43, immunocytochemistry using a mouse anti-HuR (Invitrogen) antibody was performed. Primary cortical neurons were cultured in B27 neurobasal medium supplemented with 1% glutamate and 1% penicillin/streptomycin. Primary neurons were transfected with EGFP-tagged TDP-43 with Lipofectamine 2000 at 3 or 5 DIV. After 24 h neurons were treated with 13.5 μM  or DMSO as a control for 1 h and fixed with 4% paraformaldehyde. Slides were mounted with Dako fluorescence mounting media. Images were taken with an epi-fluorescent microscope (Zeiss).NSC-34 cells and primary neurons were selected for imaging only using the fluorescence/confocal microscopy channel displaying expression of GFP or mCherry-tagged TDP-43. Human fibroblasts were selected for imaging using the confocal microscopy channel displaying Hoechst staining of the nuclei. Hence, the channel displaying the presence of DNA damage foci was invisible to the researcher when the cells were selected for analysis. This strategy allowed us to identify transfected cells for analysis, but also it ensured that the researcher was blinded to the presence of DNA damage foci. Similarly, after imaging, cells were selected for analysis based only on the fluorescence microscopy channel displaying TDP-43 expression. Analysis of DNA damage foci was performed by three people. DNA damage images on immunofluorescently labelled NSC-34 cells or human fibroblasts were collected using a Zeiss LSM 880 confocal microscope with objective Plan-Apochromat 63x/1.4 Oil DIC M27 and z-stacks with intervals of 0.25 μm. Imaris software was used for three-dimensional reconstructions and quantification of the total area and volume of foci in NSC-34 cells. DNA damage analysis in primary cortical neurons or human fibroblasts was evaluated as the average number of γH2AX/53BP1 foci using ImageJ software. To quantify DNA damage in the TDP-43 rNLS mouse model, neurons specifically expressing the transgene were identified by double immunohistochemistry using an γH2AX antibody and an antibody specific for human, rather than mouse, TDP-43, because this model expresses human rNLS TDP-43 via a neuronal-specific promoter. Neurons were chosen at random within the cortex at different depths of coronal cross sections, and at similar levels in TDP-43 rNLS and control mice. Similar to the primary neurons, DNA damage was evaluated as the average number of γH2AX foci using ImageJ software.Fixed NSC-34 cells prepared as described above on coverglass were imaged using a custom home-built SR microscope as previously described , 40. ThiOutput TIFF stacks were processed using the ImageJ plugin ThunderSTORM with default settings and then refined further as detailed by the plugin authors . Specifip value < 0.05 was considered statistically significant.Data represent mean values and the standard error of the mean. Results were analysed using one-way or two-way analysis of variance (ANOVA) followed by Sidak, Tukey or Dunnett C post hoc tests, t-Student test or Mann Whitney test. The statistical analyses were performed using OriginPro 8.0, IBM SPSS or GraphPad Prism 7 software, and 2O2, which induces DSBs by oxidative stress [2O2 for 1 h, 24 h after transfection. The concentration of H2O2 was used as previously [2O2 treatment, foci of significantly smaller area and volume were detected in cells expressing wildtype TDP-43 compared to untransfected cells  or those expressing EGFP only , Fig. 202), or mutants (detailed below)). This result demonstrates that over-expression of wildtype TDP-43 is protective against persistent DNA damage induced by two different mechanisms. The DNA damage foci observed were all present in the nucleus, indicating that TDP-43 was protective against DNA damage in the nucleus, rather than in mitochondria.When DNA damage is induced and DSBs form, phosphorylation of the histone variant H2AX follows . The rese stress , a proceeviously . Followip < 0.05, Q331K, **p < 0.01; volume: A315T, *p < 0.05, Q331K, **p < 0.01) or H2O2 treatment  or those expressing mCherry only  and Q331K cells ) and immunoblotting ). Similar findings have been reported for FUS, which was shown to be involved in the recognition of DSBs by inhibiting H2AX phosphorylation [Next, we examined if TDP-43 knock down has the opposite effect to TDP-43 overexpression. We used two previously described specific siRNAs , targetirylation . Our datp < 0.001, Fig. p < 0.05) or Q331K (***p < 0.001) mutants compared to those expressing wildtype TDP-43 after etoposide treatment  and Q331K (****p < 0.0001). This indicates that fewer DSBs were present in wildtype TDP-43 cells, but not in mutant expressing cells, compared to control cells  of each comet is defined as the product of the comet tail length and the fraction of total DNA within the tail. Increased values indicate that more prominent DNA damage is present. This analysis revealed significantly lower OTM values for wildtype TDP-43 expressing cells after etoposide treatment compared to TDP-43 mutants A315T (*dSTORM SR imaging of immunolabelled 53BP1 [a-e). In contrast, mCherry consistently displayed a low Interaction Factor with 53BP1, indicative of low or random colocalization . This confirmed that minimal colocalization artefacts were present due to crosstalk, bleed-through, or photoconversion, which we minimized using previously described methods [p < 0.001, Fig. 5a-e).These results taken together suggest that TDP-43 functions in DNA repair. However, it is unclear whether TDP-43 is recruited to DNA damage foci, where it could perform a specific DNA repair role at the DSB. Alternatively, it could perform a more indirect function in DNA repair, and therefore may not be recruited to sites of DNA damage. Hence, next we investigated the recruitment of TDP-43 to DNA damage 53BP1) foci in more detail by super-resolution microscopy (SR), using an assay previously developed for probing individual DSB sites . In celled 53BP1 . We analed 53BP1 . Wildtyp3BP1 foci methods . InteresTo confirm that TDP-43 is recruited to sites of DNA damage, we performed immunoprecipitation of lysates from NSC-34 cells expressing TDP-43 or controls expressing GFP only. We immunoprecipitated GFP-tagged TDP-43 or GFP lysates using ChemoTek GFP-Trap magnetic beads and performed immunoblotting for 53BP1. 53BP1 precipitated together with wildtype, A315T and Q331K TDP-43, but not with GFP only  or Q331K mutants (*p < 0.05) were undergoing classical NHEJ compared to those expressing wildtype TDP-43, demonstrated by fewer GFP-positive cells   Fig. c. These p < 0.001, Fig. p < 0.001), or Q331K (***p < 0.001), compared to wildtype TDP-43 , consisting of Ku and DNA-PK catalytic subunits (DNA-PKcs), is a critical component that is recruited early in classical NHEJ . To furt-43 Fig. a. Hence,p < 0.001, Fig. To confirm these findings, we next examined the recruitment of endogenous TDP-43 to DNA-PKcs foci in cells after etoposide treatment using SR microscopy as above. The Interaction Factor was calculated between TDP-43 and DNA-PKcs. Little colocalization was detected in DMSO treated cells, as expected (Interaction Factor < 0.2). However, this increased significantly upon induction of DNA damage by etoposide, indicating that TDP-43 is recruited to DNA-PKcs specifically when DNA damage is induced, and hence to regions of DSBs (Interaction Factor > 0.4). These data confirm that TDP-43 is recruited to sites of NHEJ repair  compared to controls, and elevated expression of γH2AX was also present in tissue lysates from these mice , revealing the accumulation of DNA damage. These data therefore indicate that DNA damage is already present, together with the onset of TDP-43 accumulation in the cytoplasm, in this mouse model of ALS. In addition, they reveal that DNA damage is triggered at early disease stages, implying it is relevant to pathophysiology and not simply a consequence of neurodegeneration. Furthermore, these findings suggest that DNA damage is associated with pathological localisation of TDP-43 in the cytoplasm, as well as familial ALS-associated mutant TDP-43, thus placing DNA damage onto the broad pathophysiology of ALS, not just cases caused by TDP-43 mutations.Next, we examined the relationship between DNA damage and TDP-43 in a transgenic TDP-43 mouse model , which ep < 0.01, Fig. . However, mutants TDP-43 Q331K and A315T were recruited to a lesser extent than wildtype TDP-43 . These results indicate that TDP-43 is recruited to SGs, which are implicated as precursors of inclusions in ALS [p < 0.05)  formation. We examined this possibility in NSC-34 cells and primary mouse cortical neurons expressing wildtype or mutants Q331K or A315T TDP-43, treated with 13.5 μM etoposide for 48 h. In NSC-34 cells, induction of DNA damage by etoposide triggered the formation of SGs, confirmed by immunocytochemistry for HuR  Fig. b. Similap < 0.05, Fig. p < 0.05, Fig. As our results implicate TDP-43 in NHEJ DNA repair and DNA damage in the formation of TDP-43 pathology, we next examined whether inhibition of classical NHEJ DNA repair can directly induce TDP-43 pathology. Therefore, we inhibited DNA-PK activity pharmacologically using selective DNA-PK inhibitor NU 7441  in cellsTo confirm that our findings are related to human ALS, and are not an artefact of protein overexpression systems, we next investigated DNA damage, NHEJ repair and TDP-43 pathology in previously described fibroblasts obtained from subjects bearing the TDP-43 M337V mutation [p < 0.05, Fig. (i)). Moreover, the number of 53BP1 foci/cell was significantly increased further in the affected ALS patient fibroblasts compared to those from control individuals ) and the pre-symptomatic patient (*p < 0.05). These data therefore imply that DNA damage is present in mutant TDP-43-associated ALS and may increase during disease course. The fibroblasts obtained from the symptomatic M337V patients were difficult to culture because they were prone to undergo cell death. Hence the next experiments were performed on fibroblasts from pre-symptomatic cases only, which could be readily cultured. DNA damage was examined further by immunoblotting for DNA damage markers 53BP1 and γH2AX in the pre-symptomatic TDP-43 M337V fibroblasts, compared to control individuals  and (iii), respectively). More expression of 53BP1 and γH2AX was present in TDP-43 M337V fibroblasts, compared to controls. These results therefore reveal the presence of DNA damage in human ALS fibroblasts with the TDP-43 M337V mutation.DNA damage was first examined by immunocytochemistry for 53BP1. The average number of 53BP1 foci per cell was significantly elevated in fibroblasts derived from the pre-symptomatic cases compared to the control lines . These results therefore imply that there is an association between TDP-43 pathology and DNA damage in cells without artificial protein overexpression. These data together confirm the relationship between impaired DNA repair and human ALS associated with TDP-43 mutations.Next, we investigated NHEJ repair in fibroblasts from pre-symptomatic TDP-43 M337V patients using the EJ5GFP reporter, to quantitate total NHEJ as above. TDP-43 M337V or control fibroblasts were co-transfected with pre-digested EJ5GFP reporter and empty mCherry vector as a control for transfection efficiency. NHEJ repair was found to be impaired in TDP-43 M337V fibroblasts compared to controls, demonstrated by fewer fibroblasts with GFP expression, indicating restoration of the EJ5GFP reporter  are most relevant to neurodegeneration in ALS. Our findings point to a loss of nuclear function of TDP-43 as a pathogenic process in ALS. Whilst the RNA-related functions of TDP-43 are well described, this study also demonstrates that an important DNA-specific function is perturbed in ALS. In addition, induction of DNA damage leads to mis-localisation of TDP-43 to the cytoplasm, and the formation and recruitment of TDP-43 to SGs, revealing that DNA damage can induce features characteristic of TDP-43 pathology. SGs positive for mutant TDP-43, Q331K and A315T, were also formed upon induction of DNA damage. Interestingly, however fewer SGs were formed in these cells compared to those expressing wildtype TDP-43. The results are consistent with the notion that functional TDP-43 is required for efficient SG dynamics .Similarly, inhibition of NHEJ leads to TDP-43 mis-localisation to the cytoplasm. Therefore, this raises the possibility that persistent DNA damage and defective DNA repair could further perturb the nuclear functions of TDP-43 in DNA repair, thus exacerbating nuclear DNA damage. Surprisingly, TDP-43 knockdown prevented H2AX phosphorylation, an important event in the detection of DSBs, suggesting that TDP-43 functions in the recognition of DSBs. Similar findings were reported for FUS, implying that it functions in the detection of DSBs .The formation of 53BP1 foci in cells expressing TDP-43 implicates NHEJ, the only DSB repair mechanism available to neurons, as the mechanism of DNA repair mediated by TDP-43, and that which is lost from mutant TDP-43 in ALS. NHEJ is already more error-prone than HR , and whe, Fig. To obtain more detailed insights into TDP-43 function in NHEJ, we examined the formation of DNA-PKcs foci in NSC-34 cells following DNA damage. Classical NHEJ is initiated when a Ku70/80 heterodimer binds to DSBs, which recruits DNA-PKcs to create the DNA-PK complex, forming DNA damage foci. This event is therefore early in classical NHEJ and it facilitates binding of the other DNA repair proteins involved in this process , 64. We 52O2, a source of oxidative DNA damage. Moreover, we also demonstrate that impaired NHEJ was present in fibroblasts bearing the M337V TDP-43 mutation.In this study we also demonstrate that TDP-43 is involved in the phosphorylation of γH2AX, further implying that TDP-43 functions upstream of 53BP1. In addition, DNA damage was detected in mutant TDP-43 Q331K patient tissues, and in vitro, TDP-43 Q331K mis-localisation to the cytoplasm prevented nuclear translocation of the XRCC4-DNA Ligase 4 complex . In thisFurther SR imaging demonstrated that the TDP-43 mutants are recruited less efficiently to DNA damage foci than wildtype TDP-43, implying that this is one mechanism triggering impairment of NHEJ in ALS. However, it is important to note that the TDP-43 mutants were recruited more than mCherry only to DNA damage foci, revealing that at least a proportion of mutant TDP-43 does reach the sites of DNA damage. It is therefore possible that additional deficits also exist for mutant TDP-43, such as functional deficiencies in the repair of DSBs, which combined with impaired recruitment of TDP-43 to these sites, enhances the lack of DNA repair and thus the accumulation of DNA damage. Together, these results therefore reveal that TDP-43 is involved in DNA-PKcs-dependent DDR, confirming its role in NHEJ. Importantly, the TDP-43 mutants (Q331K and A315T) were defective in this process, revealing that they lose this important and specific cellular function, resulting in accumulation of DNA damage in ALS.Our study also describes a compelling link between DNA damage, defects in DNA repair and TDP-43 pathology in ALS. The presence of pathological forms of TDP-43 in affected neurons is a characteristic feature of 97% of ALS , 66. A pConsistent with this notion, DNA damage was present at early stages of disease in TDP-43 rNLS mice, coincident with TDP-43 accumulation in the cytoplasm and prior to the presence of neurodegeneration in this mouse model. Importantly, TDP-43 rNLS mice express mis-localised wildtype TDP-43 rather than mutant TDP-43, and therefore arguably this model is closer to sporadic ALS than TDP-43-associated familial ALS. Since these mice show an early decrease in levels of normal endogenous TDP-43 in the nucleus of neurons due to auto-regulation of expression , the incIncreasingly DNA damage is becoming linked to neurodegenerative diseases . ApoptosFinally, we investigated the relevance of our findings to human ALS using fibroblasts from an ALS patient bearing the TDP-43 M337V mutation and two pre-symptomatic TDP-43 M337V carriers. Enhanced DNA damage was present in affected and pre-symptomatic fibroblasts. Importantly, significantly more DNA damage was present in pre-symptomatic affected fibroblasts compared to presymptomatic fibroblasts, implying an active role for DNA damage in the development of ALS. However, it is possible that the presence of the TDP-43 M337V mutation renders pre-symptomatic fibroblasts more vulnerable to DNA damage in vitro. Similarly, total NHEJ was impaired in TDP-43 M337V pre-symptomatic fibroblasts compared to control fibroblasts. The upregulation of 53BP1, which promotes classical NHEJ , is alsoThe findings of our study raise the possibility that failure of DNA repair processes by mutant TDP-43 may trigger a vicious cycle, in which the resulting accumulation of DNA damage triggers TDP-43 mis-localisation in the cytoplasm, inducing more loss of nuclear TDP-43. This would enhance existing DNA damage and perturb DNA repair even further, leading to significant accumulation of DNA damage during the normal human lifespan, triggering neurodegeneration. Figure In this study we demonstrate that TDP-43 mediates DNA repair via the NHEJ pathway. In contrast, TDP-43 ALS mutants Q331K and A315T lose this function, contributing to the accumulation of DNA damage. Our findings also reveal a link between DNA damage and the formation of pathological forms of TDP-43 in ALS. Whilst the RNA-related functions of TDP-43 are well described, this study identifies an important DNA-specific function that is perturbed in ALS.Additional file 1: Supplementary Figure 1. A315T and Q331K mutants do not prevent the formation of 53BP1 foci (EGFP-tagged constructs). Top panels; Confocal microscopy of NSC34-cells expressing wildtype TDP-43 display less DNA damage compared to controls; untransfected (Un) or EGFP only cells (EGFP), determined by (i) total area, (ii) and total volume of 53BP1 foci after treatment with 13.5 μM topoisomerase II inhibitor etoposide. In contrast, cells expressing ALS-associated mutants A315T and Q331K are not protected from damage compared to wildtype TDP-43. Scale bar 10 μm. Middle panels: Quantification was performed on 3D reconstructions of z-stack images using Imaris software. 2-way ANOVA with Sidak correction for multiple comparison. Mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. At least 20 cells/group were analyzed. Bottom panels; Representative 3D reconstruction of confocal images of cells illustrating 53BP1 foci, Scale bar 5 μm.Additional file 2: Supplementary Table 1. Q331K and A315T TDP-43 mutants cause cell death in cells with DNA-PK deficiency. Expression of Q331K and A315T TDP-43 mutants in XR-C1 cells lacking DNA PK was lethal in all three replicates (crosses). Wildtype CHO-1 cells with Q331K and A315T expression survived in 2 out of 3 replicates (check marks).


Fast-response humidity sensors using nanomaterials are attractive and have been intensively studied. Among the various nanomaterials, nonporous inorganic nanoparticles are suitable for use in humidity sensitive films for sensors. Here, we focus on a nonporous inorganic nanoparticle film and investigate a humidity sensor using the film. Hysteresis error and a dynamic response to a change of humidity are fundamental specifications of humidity sensors. A humidity sensor using a 50 nm silica nanoparticle film shows a hysteresis error of 2% at 85% RH and a response/recovery time of 2.8/2.3 s in 30% RH to 70% RH. We also summarize response/recovery times and hysteresis errors of state-of-the-art humidity sensors. As compared to those of commercial sensors and porous nanoparticle-based sensors evaluated using saturated salt solutions, the fabricated sensor shows a comparative hysteresis error and shorter response time. Humidity sensing has been of great importance in environmental monitoring, planting in factories, and human care ,2. HumidRecently, there have been intensive studies to improve the dynamic response of humidity sensors. Borini et al. reported a pioneering work demonstrating the ultrafast response (≈30 ms) of humidity sensors using a graphene oxide film with a 15-nm thickness . RecentlAmong the several kinds of nanomaterials, we focus on inorganic nanoparticles as a component of humidity sensitive films. Size-regulated inorganic nanoparticles are chemically stable in air and a thin film can be formed by solution processes using colloidal nanoparticles. In previous studies, porous inorganic nanoparticles were used for humidity sensors to increase sensitivity because the nanopores inside nanoparticles easily condense water molecules ,10,11,12In this study, we investigate the humidity hysteresis and response time of a nonporous inorganic nanoparticle-based humidity sensor. Nonporous silica nanoparticles with 50 nm and 200 nm diameters were used to form a humidity-sensitive film. For humidity sensing, we utilized surface conduction via water molecular layers on the nanoparticles. We evaluated the humidity hysteresis in ascending/descending humidity levels and response times relative to dynamic changes in humidity. A remote detection of respiratory air by the sensors was shown. We summarized the response/recovery time and hysteresis error of state-of-the-art humidity sensors.We prepared gold interdigitated electrodes (line and spacing: 20 μm) on substrates as follows: gold with a titanium adhesive layer (100 and 10 nm) was sputtered  on a silicon wafer . The gold/titanium layer was patterned into interdigitated structures with photolithography and dry-etching processes. The finished electrodes were electrically isolated under high humidity.Colloidal silica nanoparticles were purchased from Sigma-Aldrich (product no.: “803073-1ML” and “803847-1ML”). The densities of the 50 and 200 nm nanoparticle colloids were diluted by adding ethanol 10 and 100 times, respectively. The colloids were spin-coated on bare interdigitated electrodes in ambient air. After the coating process, the substrate was heated to evaporate the solvent from the film.Static humidity dependence of impedance was obtained in an environment-controlled chamber . The chamber was controlled at 34% to 90% relative humidity (RH) with 3% accuracy at 20 °C. The test pressure was 1 atm and the temperature variation was ±0.3 °C. The impedance was measured by an LCR meter , which was controlled using LabVIEW . A voltage amplitude for impedance measurements was 1.0 V and a voltage frequency was 0.5 Hz to 1 kHz.2CO3, Mg(NO3)2, NaBr, NaCl, KCl, and KNO3 salts were used in this study. Abrupt changes in humidity were given by the sensor which was placed in and removed from the vessel. The zero condition of the change is in ambient air without airflow. The temperature, relative humidity, and gas pressure are 20 °C, 30% RH, and 1 atm, respectively. The actual humidity in the vessels was simultaneously monitored by a commercial humidity sensor . The measured humidity levels above the salt solutions, using K2CO3, Mg(NO3)2, NaBr, NaCl, KCl, and KNO3, were 36, 46, 52, 61, 66, 70% RH, respectively. It should be noted that we only investigated the static response time without gas flow. The sampling period of the sensor impedance was 1 s and the applied voltage was 1 V at 1 Hz.A dynamic response of impedance to a change of humidity was observed by using vessels with salt solutions. The volume of the vessels was 50 mL and the detailed dimensions are shown in the We also tested the availability of the nanoparticle-based sensor for the detection of respiration. Exhaled air has high moisture and temperature, is close to body temperature, and therefore, cyclically changed both the moisture and temperature of the air in front of the face. A mockup with a portable data logger  was packaged in a plastic test case  and a tablet with a custom-made Android API (an intrinsic source code is available from MbientLab) was used. Interdigitated electrodes on a glass (line and spacing: 100 μm) were used for the substrate of the sensor. A 50 nm nanoparticle coating was carried out by using the same method as above. A change of resistive impedance was converted to a voltage by using a voltage divider comprised with 10 MΩ. Collected data were transmitted to the tablet via Bluetooth. The measurement was approved by the internal review board.2 surface.A morphology and thickness of nanoparticle films were observed by a field-emission scanning electron microscope . Top-view and cross-section SEM images are shown in On the contrary, water molecules physically form multilayers on surface hydroxyls via hydrogen bonds. This physisorption is reversible and the thickness of the multilayers is dependent on humidity in air. Using attenuated total reflection infrared (ATR-IR) spectroscopy, Chen et al. investigated the thickness of physically adsorbed water layers over hydroxyls on a silicon wafer . They fof) dependence of impedance in a 50 nm nanoparticle film as a function of humidity (a): absolute value and (b) phase angle of impedance). Solid and dashed lines represent the measurements in the ascending and descending humidity levels, respectively. As a humidity increases from 40% RH to 90% RH, the impedance at a lower frequency decreases and shows a plateau region. This plateau region is attributed to a resistive impedance of proton conduction . A linear decrease in impedance (Z), which is independent on humidity, is due to a parasitic capacitance (pC) of the measurement system: i.e., |Z|∝(pωC)−1 = (2πfCp)−1 where |Z| is an absolute value of impedance and ω is an angular frequency (ω = 2πf) of an AC voltage.H is a relative humidity in percentage. In other words, we express this relation as This exponential relation can be attributed to the formation of water layers. According to Asay et al., a structural evolution of water molecular formation has three steps on a flat silicon-oxide surface . First, The hysteresis error is calculated by the difference between the experimental results and the empirical fitting line. We evaluated maximum humidity hysteresis at 6% at 40% RH and 2% at 85% RH in the ascending/descending humidity levels. At a lower humidity, the impedance becomes larger and its accuracy is reduced. This nanoparticle humidity sensor based on proton conduction is suitable for detecting higher humidities. It should be noted that a humidity sensor using 200 nm silica nanoparticles shows 10 times larger impedance although an exponential dependence appears a,b. BecaH1 and H2 represent as relative humidity in and out of the vessel, respectively. τ1) and recovery time (τ2) in 30% RH to 70% RH are evaluated to be 2.8 and 2.3 s, which are defined as t90 and t10  are shown in t90 and t10 were evaluated using a graphical readout. We assumed that the uncertainty of the measured response times was up to 1 s because the sampling period was approximately 1 s.A schematic setup for measuring a dynamic response to a humidity change is shown in Vout). Expiratory air is successively detected in 2 s with sharp voltage signals, as shown in Vout to actual relative humidity because temperature sensing is required for the conversion. In this proof-of-concept, the sensor does not measure relative humidity of exhaled air but only detects the existence of water vapor qualitatively. The measurement of relative humidity in breath air is a cutting-edge topic and further developments are required in our system [Using this fast response/recovery time, we show a proof-of-concept test for practical applications of a nanoparticle humidity sensor. Because a common respiratory rate ranges from 12 bpm to 20 bpm (0.05 Hz to 0.083 Hz), a humidity sensor with a 2.8/2.3 s response/recovery time can be used for sensing respiratory rate. A setup of remote sensing of respiration is shown in r system .τ) is equivalent to the total of the mixing time to obtain certain humidity in the chamber (mτ), the adsorption time of water molecules onto the sensor (aτ), and the time required for the change of the sensor property (cτ): τ = mτ + aτ + cτ. The value of mτ is related to extrinsic response and aτ and cτ are attributed to intrinsic response.It should be noted that there are two types of response times regarding diffusion of water molecules: dominated by intrinsic (sensor related) and extrinsic (environment related) processes . Sensor mτ is reduced and the evaluated recovery time is dominated by the intrinsic aτ and cτ. On the other hand, the evaluated response time may include the effect of mτ because the response time (2.8 s) is longer than the recovery time (2.3 s), as shown in In the case of the recovery process, because the volume out of the vessel (ambient air) is much larger than that of the moist air in the vessel (50 mL), mτ to obtain certain humidity is included in the evaluation of response/recovery time. In case of using a saturated salt solution, the effect of extrinsic mτ can be reduced because the humidity is stable at certain values in the vessel [We finally discuss sensor specifications, a response/recovery time, and a humidity hysteresis with state-of-the-art humidity sensors using nanomaterials. e vessel . TherefoIt should be noted that the specifications in In order to guarantee practical usability, it is essential to achieve repeatability of sensors. Therefore, we show the humidity dependence of impedance on other two sensor chips in the In summary, we investigated a humidity hysteresis and a response time of a nonporous inorganic nanoparticle-based humidity sensor. A nonporous nanoparticle film was potential to enhance fast humidity sensing with small hysteresis. A humidity hysteresis and a response/recovery time of a 50 nm nanoparticle film were evaluated to be as 2% to 6% (at 85% RH to 40% RH) and 2.8/2.3 s (a change of humidity between 30% RH to 70% RH), respectively. We demonstrated a remote sensing of respiration. According to the specifications with state-of-the-art sensors, the nonporous nanoparticle sensor showed a faster response than commercial humidity sensors and porous nanoparticle-based sensors evaluated by using saturated salt solutions. Repeatability of the sensors should be improved in future works towards practical usage.