Legal restrictions on alcohol availability have been used to address violence and injury in the world’s remote Indigenous communities. In Australia, alcohol management plans (AMPs) were implemented by the Queensland Government in 2002. This study reports changes in indicators of alcohol-related violence and injury in selected communities.Design and setting: A longitudinal observational study was conducted in four Aboriginal and Torres Strait Islander (Indigenous) communities in Cape York, far north Queensland. All communities are similarly-isolated from population centres where alcohol is available.Data: For 2000 to 2015 inclusive: 1019 Royal Flying Doctor Service aeromedical trauma retrievals; 5641 Queensland Police Service records of unique assault occurrences, including 2936 involving alcohol; and records for 2741 unique assault victims were examined.Data analysis:Rates (per 1000 population) of trauma retrievals, assault occurrences and assault victims (per 1000 population) were compared across three policy phases.Phase 1: 2000 to 2008. Initial restrictions on possession and consumption of alcohol in ‘restricted areas’ were implemented during 2002–2003.Phase 2: 2009 to 2012. All alcohol was prohibited in three study communities and its legal availability limited in the fourth from 2009.Phase 3: 2013 to 2015. Government reviews of AMP policies in light of legal challenges and community responses characterise this phase.Compared with Phase 1, in Phase 2 retrieval rates declined by − 29.4%, assault occurrences by − 34.1% with less than one-third involving alcohol, and assault victims by − 21.1%, reaching historically low levels in 2010–2012. These reductions did not continue consistently. Compared with Phase 1, in Phase 3 retrieval rates, assault occurrence rates and assault victim rates declined by somewhat lesser amounts, − 13.9%, − 15.0% and − 13.4%, respectively. In Phase 3, the proportion of assault occurrences involving alcohol in communities 2, 3 and 4 rose towards pre-2008 levels.Early successes of these controversial alcohol restrictions are jeopardised. Indicators of violence and injury appear to be rising once more in some AMP communities. Importantly, rates have not generally exceeded the highest levels seen in Phase 1. Fresh policy action is required with rigorous monitoring to prevent erosion of initial important successes. With 5.1% of the global burden of disease and injury attributable to alcohol . For theLiquor Act 1992 with controls on quantities and types of alcohol permitted in a restricted area designated under S173H [Liquor Regulation 2002 [In Australia, legal restrictions on alcohol’s physical availability in Indigenous communities became known as ‘Alcohol Management Plans’ (AMPs) towards the end of the twentieth century –14. AMPsion 2002 .all of the 19 AMP communities; bringing complete prohibition to seven of them [As already described in detail in a previous publication, these place-based controls were first introduced in 2002 . By 2006At the end of 2012, the Queensland Government implemented formal processes to review AMPs , 19. As Using data provided by Australia’s Royal Flying Doctor Service (RFDS), we have monitored rates of aeromedical retrievals for serious injury in four AMP communities over the 15 year period from 1995 to 2010 , 10. ThiCriminal Code 1995 with a local police presence in most remote communities, including the four in this study. This analytical approach ensures that the indicators examined reflect changing circumstances in these four communities, independent as far as practicable from external confounding factors, providing the opportunity to consider the local impacts of Queensland-wide policy initiatives and shifts.The communities, their populations and the reasons for choosing them as sentinel communities for monitoring have already been described in detail , 10. In This longitudinal observational study assesses the effects of a policy intervention which was developed and implemented with a changing focus over the past two decades. The very complex legislative and regulatory background for AMPs in all Queensland Indigenous communities has already been described and summarised . The fouSince the 1980s, each community’s elected Local Government Council (LGC) had been operating licensed premises, variously known as a ‘tavern’ or ‘canteen’ , from whFollowing Justice Fitzgerald’s 2000 inquiry , over a During 2007, legislation and regulation to control alcohol availability across the wider region saw ‘minimising harm’ conditions placed on the licences of liquor retailers in the ‘catchment’ areas of restricted area communities . RetaileLiquor Act 1992 was amended removing the option for LGCs to hold a liquor licence while also making it an offence to ‘attempt’ to bring prohibited quantities and types of liquor into a restricted area [In 2008, the ted area . For thrted area . The on-Significant legal challenges were mounted to the legitimacy of Queensland’s AMPs commencing during the previous Phase 2 , 35 and While this important period of review and legal challenge has unfolded, liquor restrictions in the four communities have not changed since 2008, with one exception. In mid-2014, in one community with prohibition, a venue formerly run by the LGC was permitted to operate once again, with limited trading hours and also with the on-premises purchase and consumption of liquor containing ≤4% alcohol by volume permitted under a ‘Restricted Liquor Permit’ .The causal mechanisms linking alcohol use and interpersonal violence are well known , 41, 42 In the general population, it is widely recognised that alcohol is involved in more than half of all occurrences of interpersonal violence with alcohol’s acute effects having the greatest impact on violent behaviour . For theIn addition, since QPS records also include information about individuals affected by violent occurrences, and since the victims of assault often require some kind of acute care medical intervention, to capture as wide a spectrum of relevant violent incidents as possible, we used the indicator of victims of ‘violence against the person’ recorded by QPS.A Government-funded clinic has provided local health care services in each study community throughout the observation period. The RFDS remains the principal provider of emergency medical transport to definitive care of all people presenting to the local clinic with serious injuries. There have been no substantive changes in RFDS aeromedical retrieval processes, logistics or management for these communities across the time frame of our previously published , 10 and As in these previous studies , 10, serThe QPS has a presence in each of the four communities in the form of a Division station. Two of the stations, i.e. those in the two largest communities, are staffed by the equivalent of eight permanent officers and, in the smaller communities, by the equivalent of two permanent officers. Some LGCs employ Community Police Officers to assist. In 2008, to support the ‘dry as possible’ phase, the Queensland Government committed funding for additional officers across all 19 AMP communities [Data were provided by QPS for all occurrences of person-to-person violent offences using Australian Standard Offence Classification Group Codes for ordering offences  includinFor harm observed by police in connection with an assault occurrence, or experienced or reported by victims, data for the same types of person-to-person violent offences  were useRates of RFDS trauma retrievals and QPS registered occurrences of assault and victims of assault (per 1000 population) were calculated for all years between 2000 and 2015 for all four communities , measures of statistical uncertainty  are unnecessary and would be factually misplaced.The Human Research Ethics Committees of James Cook University and the Cairns and Hinterland Health Services District provided relevant ethical approvals for the study to be conducted in these communities . The QPSA total of 1019 aeromedical trauma retrievals were extracted from the RFDS database and used in the analysis for the four communities for 2000 to 2015 inclusive. During the same period, data for 5641 unique assault occurrences were provided by QPS. In 2936 (52%) of these assault occurrences, alcohol was recorded as being involved. During the observation period 2741 individuals were victims of an assault, as recorded by QPS. The resulting rates for retrievals and assaults for the four communities as well as the average over all communities over the single years of the observation period are numerically detailed in Table Consistent with previous results , 10, TabFigure Table Table Communities 1 and 2 have not fared so well . This case required attention to international human rights conventions and the Australian Constitution [Liquor Act 1992 were invalid by reason of inconsistency with s 10 of Australia’s Racial Discrimination Act 1975 (Cth). Section 10 of this Commonwealth act provides that a State law cannot have the effect that persons of a particular race do not enjoy a right enjoyed by other races, or do not enjoy it to the same degree. In dismissing the appeal, the Court of Appeal held that s 10 did not apply, as it was a ‘special measure’ taken for the sole purpose of securing the adequate advancement of the Indigenous residents of Palm Island and to prevent human rights violations, such as violation of the right of vulnerable groups to live in a safe environment [In the background, during Phase 2, significant legal challenges to the legitimacy of AMPs were commenced , 36, onetitution , 38. An titution . The groThe issue of consultation for ‘special measures’ and the proportionality aspects of AMPs in Australia’s Northern Territory have recently raised concerns for the Australian Parliamentary Joint Committee on Human Rights . In its It is of great concern that rates of retrievals for serious injury, assault occurrences and victims show a generally increasing trend since 2012 in these four study communities. This is in the absence of any substantial policy change affecting alcohol’s legal availability. The underlying causes for these trends warrant urgent, refreshed policy action informed by rigorous objective monitoring to ensure the successes gained by AMPs in Queensland are not placed in further jeopardy.


Stimuli-responsive materials, also known as smart materials, can change their structure and, consequently, original behavior in response to external or internal stimuli. This is due to the change in the interactions between the various functional groups. Graphene, which is a single layer of carbon atoms with a hexagonal morphology and has excellent physiochemical properties with a high surface area, is frequently used in materials science for various applications. Numerous surface functionalizations are possible for the graphene structure with different functional groups, which can be used to alter the properties of native materials. Graphene-based hybrids exhibit significant improvements in their native properties. Since functionalized graphene contains several reactive groups, the behavior of such hybrid materials can be easily tuned by changing the external conditions, which is very useful in biomedical applications. Enhanced cell proliferation and differentiation of stem cells was reported on the surfaces of graphene-based hybrids with negligible cytotoxicity. In addition, pH or light-induced drug delivery with a controlled release rate was observed for such nanohybrids. Besides, notable improvements in antimicrobial activity were observed for nanohybrids, which demonstrated their potential for biomedical applications. This review describes the physiochemical properties of graphene and graphene-based hybrid materials for stimuli-responsive drug delivery, tissue engineering, and antimicrobial applications. N-vinylcaprolactam), poly(N-isopropylacrylamide), poly, and other copolymers are frequently used as a smart material for various biomedical applications [2-bonded carbon atoms with a honeycomb morphology. The higher surface area of graphene facilitates effective binding with several drugs through various interactions and is frequently used in targeted and controlled drug delivery applications [Nowadays, on-demand release of active materials in desired areas has drawn tremendous attention in the rapidly developing field of materials science. For this purpose, stimuli-responsive materials (SRMs), which are also known as smart materials, are frequently used. They can change their shapes or dimensions in the presence of external stimuli such as electric field , 2, magnications . For bioications . The proications –29. Accoications –32. Grapications , 34. Theications . The strications . Extraorications . Graphenications . Enhanceications . Moreoveications .  Figureications .This review describes the salient features of graphene and its biomedical applications such as stimuli-responsive drug delivery, tissue engineering, and antibacterial materials in the presence of different polymer matrices. Different techniques such as in situ polymerization, solution casting, and extrusion were used to fabricate graphene-based nanohybrids for desired applications.2-hybridized carbon atoms arranged in a honeycomb structure. It was initially described by Boehm et al. in 1986 followed by identification and isolation by Geim and Novoselov in 2004 [σ bonds with a delocalized π electron network. These delocalized π electrons provide a high electron density above and below the 2D planar structure of graphene. Because of the planar structure and delocalized π electrons, graphene undergoes various reactions such as cycloadditions, click reactions, and carbine insertions [Among various nanomaterials, graphene has a variety of advantages and gained tremendous attention from the scientific community. Graphene is a 2D single atomic layer of graphite with sp in 2004 , 43. In sertions . Pure grsertions . In addisertions . Some saSeveral techniques such as force displacement, force volume, nanoindentation atomic force microscopy (AFM) –48, and σ bonding and delocalized π electrons makes graphene a unique electrically and thermally conductive material with a low thermal expansion coefficient. The thermal conductivity of defect-free graphene is much higher than that of other carbon nanomaterials. Its thermal conductivity is approximately in the order of 4.5–5.5 × 103 W/mK, whereas it is approximately 2 × 103, 3 × 103, and 3.5 × 103 W/mK for graphene oxide, multiwalled carbon nanotubes, and single-walled carbon nanotubes, respectively [4 S/cm. The electrical mobility of GO is 10−1 S/cm [The presence of strong ectively , 57, 58.ectively –61. The 0−1 S/cm . With it0−1 S/cm , 64.In addition to exceptional mechanical strength and thermal and electrical properties, graphene has an excellent optical property. Single layer defect-free graphene has shown 97.7% incident light transmission over a wide range of wavelengths . This prπ-π stacking interactions between the drug and the polymer matrix [π-π stacking and hydrogen bonding interactions. A faster drug release from the nanohybrids was observed at pH 5.3 than at pH 7.4, which indicated its potential as a targeted and pH-mediated anticancer drug delivery vehicle [π-π stacking and hydrophobic interactions between the drug and the nanohybrids. However, drug release was observed only in an acidic medium but not in basic and neutral media, which are found in a tumor environment; this suggests the formation of a suitable site-specific drug carrier [N-isopropylacrylamide (pNIPAAm) polymer matrices. They noted that a faster drug (doxorubicin hydrochloride) release from the hydrogel occurred in the acidic medium than in the neutral and alkaline media due to the protonation of polar groups. Furthermore, the developed hydrogels exhibited temperature-mediated drug release, which was more controlled at higher temperatures due to dehydration of the hydrogel leading to a more compact structure that hinders the diffusion of the drug. On-demand or targeted drug release from biomedical devices has attracted great attention in the field of medical science. It has been noted that targeted drug release from carrier molecules exhibits high efficiency with a controllable release and minimum side effects. Several factors such as light, heat, pH of the medium, ultrasound waves, and electric or magnetic fields are responsible for the controlled release of drugs –72. For r matrix . In anot vehicle . Kurapat vehicle . Further carrier . Hydroge carrier . In anotγ (INF γ) biomolecules, a larger population of neuron cells occurred, while in the presence of a platelet-derived growth factor (PDGF), a larger population of oligodendrocytes occurred, suggesting its potential for controlling the NSC differentiation tendency for therapeutic applications [ε-caprolactone) (PCL) matrix to evaluate the cellular response toward stem cells. They observed that the composite with diamine-modified GO showed a higher proliferation and differentiation of human MSCs (hMSCs) followed by the GO composite. This was due to better interactions between the amine moiety and the cells [For tissue engineering applications, materials should be biocompatible, nontoxic, and biodegradable in nature. In addition, materials should not show any negative response in biological conditions –84. Tissications . In anotications . Chemicahe cells . Zhang ahe cells . Notablyhe cells . Furtherhe cells . Sayyar he cells . Hydroxyhe cells , 98. Thehe cells . A similhe cells . In anothe cells . Degradahe cells . In addiE. coli and S. aureus (~100% of E. coli and ~95.3% of S. aureus) by 10 μg/mL for 2.5 h. The higher antibacterial activity of GO-PEG-Ag composites was due to the damage of the bacterial structure and the production of reactive oxygen species, which led to cytoplasm leakage and decrease in metabolism [L-lysine) (PLL) composites through electrostatic interactions and covalent bonding between the graphene derivatives and PLL and evaluated their cytotoxicity and antibacterial behavior. They observed that the composites showed a strong antibacterial nature and biocompatibility toward human adipose-derived stem cells and non-small-cell lung carcinoma cells (A549), which indicated its dual functionality that can be used to inhibit bacterial growth as well as enhance human cell growth [E. coli (ATCC 25922) and Gram-positive S. aureus (ATCC 6538) by the plate count method and disk diffusion method. Significant antibacterial activity was observed for the nanocomposites, which suggested its potential use in biomedical applications [E. coli (~97.9% reduction) after 3 h of contact, indicating its multidynamics applications with strong antibacterial and separation performances. The antibacterial activity of the PDA-rGOC-modified membrane is shown in N-vinylcarbazole) (PVK) and graphene/GO. The PVK-GO-modified membrane exhibited a strong antibacterial activity toward B. subtilis and E. coli. This was due to the production of reactive oxygen species by the nanoparticles, which influenced the metabolic activity of the microorganisms [S. aureus [E. coli and S. aureus bacteria, respectively, were killed by the developed hybrids under irradiation of 660 nm light due to the formation of radical oxygen species; this indicated the strong photocatalytic activity of the hybrid toward microorganisms [S. aureus, E. coli, and C. albicans. A significant improvement in antimicrobial activity was observed for the GIO-based hydrogel film compared to chitosan-GO and chitosan-iron oxide films [2/Ag3PO4/graphene composite synthesized by an ion-exchange method and a hydrothermal approach [E. coli and S. aureus with negligible cytotoxicity was observed for silver-incorporated ZnO-chemically converted graphene nanocomposites synthesized by a low-temperature technique using zinc acetate dehydrate, silver nitrate, and GO [Nowadays, several antibiotics and antimicrobial agents have been developed for the treatment of various infectious diseases. However, lethal microorganisms remain a challenge for public health, causing several infectious diseases annually . Antibiotabolism . Some etl growth . In anotications . Microbiications . Severalications . Nanofilications –121. Zhurganisms . Liu et . aureus . It is w. aureus . A similrganisms . Konwar de films . Antibacapproach . Materiaapproach . Further, and GO .Nanomaterials with unique intrinsic physiochemical and biological properties, which are absent in their macro forms, have drawn significant attention in materials science for various applications. Several nanomaterials such as a graphene, carbon nanotubes, fullerenes, zeolite, and metals in different forms are frequently used to improve the native properties of materials for desired applications. Nowadays, graphene, an allotrope of carbon with excellent thermal, electrical, optical, mechanical, and biological properties and a higher surface area, is intensively used to enhance material properties. Moreover, properties of graphene can be tuned by surface functionalization with various groups. The higher surface area of graphene and the high charge density on the graphene surface facilitate the loading of several drug molecules, and they consequently act as a nanocarrier with tune rate in the biological medium. In addition, the excellent physical property of the graphene surface facilitates the proliferation and differentiation of cells. Moreover, its light-absorbing behavior plays an essential role in light-triggered drug delivery or cellular response. A significant improvement in antibacterial activity without cytotoxicity was observed for various graphene-based hybrids, suggesting its potential as a biomaterial for various applications. Hence, a discovery of wonder graphene nanomaterial has opened a new area of research to produce lightweight, high-performance hybrid materials for various biomedical applications.


Vcnp, which strongly influences the FM results, and developed a calibration method using electrochemical gate-voltage pulse sequences. This graphene-based multifunctional device shows great potential for use in a simple, low-cost, and ultracompact nanomaterial-based nodal architecture to enable continuous, real-time event-based monitoring in pervasive healthcare IoTs, ubiquitous security systems, and other chemical/molecular/gas monitoring applications.Modern internet of things (IoTs) and ubiquitous sensor networks could potentially take advantage of chemically sensitive nanomaterials and nanostructures. However, their heterogeneous integration with other electronic modules on a networked sensor node, such as silicon-based modulators and memories, is inherently challenging because of compatibility and integration issues. Here we report a novel paradigm for sensing modulators: a graphene field-effect transistor device that directly modulates a radio frequency (RF) electrical carrier signal when exposed to chemical agents, with a memory effect in its electrochemical history. We demonstrated the concept and implementation of this graphene-based sensing modulator through a frequency-modulation (FM) experiment conducted in a modulation cycle consisting of alternating phases of air exposure and ethanol or water treatment. In addition, we observed an analog memory effect in terms of the charge neutrality point of the graphene,  In general, the sensor, modulator, and memory that are necessary components of a sensor network are separate and independent modules11. For example, a graphene chemical sensor would need to be combined with a silicon-based modulator to make it compatible with wireless radio frequency (RF) operation for data synchronization in internet-of-things (IoTs). However, in the case of nanosensors2–6 based on nanomaterials and nanostructures, their small sizes and heterogeneous features compared with conventional solid-state electronic devices could make their integration particularly challenging13. To integrate nanomaterial sensors with commercial silicon-based modulation interfaces, complicated post-transfer processes must be performed13; this results in a high-cost and non-compatible nodal structure and therefore limits the applicability of nanomaterial sensors for the internet of nano-things (IoNT)14.Advanced nanostructures such as two-dimensional carbon nanomaterials, namely, graphene6, which lead to a nonlinear effect in the control of the gate voltage with respect to the drain current. On the basis of this unique property, special modulation functions, such as full-wave rectification, frequency mixing, amplitude modulation (AM), frequency modulation (FM), and phase modulation (PM), can be directly achieved with a single graphene device, which is not possible for conventional semiconductor devices15–19. Here we propose a different modulation mechanism utilizing chemical gating, instead of the application of an electrical signal, to directly modulate the input carrier signal by means of the chemical dopants  to which a GFET is exposed. This monolithic all-graphene paradigm can enable virtually simultaneous in situ chemical sensing and signal modulation, thereby significantly reducing the integration complexity and cost, which is of great interest for ubiquitous sensor network applications.It has recently been reported that graphene field-effect transistors (GFETs) can exhibit not only ultrahigh mobility but also interesting gapless electron-hole spectra and ambipolar transport properties20–23 because of the low-signal dimensionality. One advantage of the proposed chemically modulated sensing device, compared with other nanomaterial sensors, resides in its two-dimensional (2D) output signal, which provides information through both the amplitude and frequency of an RF signal. Very recently, Lee et al. have demonstrated that electrically gated modulation on graphene exhibits a transition from a fundamental input tone to a second-harmonic output tone19, which enables AM to FM functions using a single device. Here we show that this signal transition is not abrupt and that the fundamental and second-harmonic tones instead coexist, with gradually varying strengths depending on the potential level of the graphene, which can be modulated by means of a chemical  gating effect. In this scenario, mixed AM/FM modulation can be achieved and the amplitude responses at both frequencies constitute a 2D output signal, thereby improving the overall reliability and the tolerance to environmental noise and interference.For most graphene sensor applications, the sensor output signal, in information theory, is one-dimensional (1D), in terms of either direct current (DC) or electrical resistance. This 1D spectrum offers limited information processing, storing, and distributing capabilitiesVcnp) depends not only on the chemical agents to which it is exposed but also on the exposure time, in an integral fashion not found in other semiconductor devices. This interesting feature could be advantageous for continuous-time biomedical and environmental monitoring, considering that the calibration of the memory effect on site is regarded as an important issue for practical applications in long-period and multicycle sensing. Thus far, several methods of tuning Vcnp, on which the modulated output signal depends, have been reported, including annealing the device under high vacuum, post-heating at high temperature, and applying a specialized gas/wet treatment after the adsorption of substances onto the graphene25. However, these calibration methods require extreme conditions to initiate the memory effect in the graphene and thus are not feasible for pervasive sensing or continuous monitoring applications. A simple and robust approach that can be used to precisely calibrate the graphene device would be preferable.We also investigate for the first time the memory effect of chemically gated graphene devices, in which the shift of the charge neutrality point of the graphene , this graphene-based sensor no longer requires an FM modulator because the sensor output is already an RF signal (second-harmonic tone), which is fully compatible with wireless communication mediaVcnp. When a single-tone input at a frequency f is applied to the gate with zero DC gate bias, the modulated output shows a visible change as Vcnp is altered through chemical gating: (1) if Vcnp is zero, then the drain output has a vanishing component at f and a strong component at 2f (gray curve); (2) if Vcnp is up-shifted above the critical point, then the signal component at f is amplified, whereas the 2f tone decreases (blue curve); (3) if Vcnp is far from the critical point, then the output signal begins to monotonically decrease, exhibiting drops at both f and 2f. With a large shift in Vcnp, the second-harmonic tone is completely suppressed, as the fundamental tone continues to decrease (red curve).Vcnp is shown in 27. The best-signal linearity is observed at two critical conditions: the peaks in the fundamental-tone curve (blue shaded region in Vcnp=±1.4 V). This variation in nonlinearity is explained by the schematic diagram presented in Vcnp of graphene can be shifted from positive to negative values, over a wide range, by means of exposure to p-/n-type dopants of different concentrations. As a result, there exists an ideal operating region in which both components are monotonic functions of Vcnp, as shown in the gray shaded region in A theoretical analysis of the mixed modulation output as a function of Vcnp shift, the variation in carrier mobility also affects the output RF signal of the GFET. In fact, although the attachment of most chemical agents to graphene can affect its Vcnp14, there is no material that shows a measurable impact on graphene’s carrier mobility15. The results calculated for the two-tone output via 2D vector mapping , thereby demonstrating excellent robustness in sensing compared with conventional approaches involving the detection of only 1D outputs .In addition to the chemical-gating-induced  mapping  clearly 2O/O2 redox system, residues such as photoresist could also contribute to the formation of charge traps. The synthesized graphene was first transferred, using polymethyl methacrylate as a support, onto a SiO2/Si substrate with a 285-nm thermal oxide layer as the dielectric to form a back-gate device structure. Raman spectroscopy with a 442-nm blue laser was used to confirm the quality of the graphene after transfer (see the inset of −1 and a symmetric 2D band at 2695 cm−1 (bandwidth of ~32). Back-gated field-effect transistors were formed by means of the electron-beam evaporation of 2-nm Ti+48-nm Au metal contacts using a shadow mask to measure the electrical properties of the devices. The area of the metal contacts was 200 μm×200 μm, which defined the graphene channel width; the devices were fabricated with various channel lengths of 25, 50, 100 and 200 μm. Further details regarding the graphene preparation28–30 and device fabrication can be found in our previous work31. The intrinsic sensitivity of a graphene chemical sensor can be extremely high6. However, in this study, our focus was on demonstrating a novel multifunctional sensor/modulator module and its applicability for the sensing of time-varying chemical events rather than demonstrating the high intrinsic sensitivity of graphene-based devices.For our experiment, the GFETs were fabricated using a shadow mask without any lithography step to preserve the pristine surface of the graphene. This is because in addition to the HVcnp calibration demonstration, the DC drain current to gate voltage (IDS–VGS) curves of the GFET were recorded using the HP4145B semiconductor parameter analyzer under ambient conditions, with a drain-to-source bias of 100 mV. Droplets of 200-proof ACS/USP ethyl alcohol (100% ethanol), as the wet treatment agent used in the experiment, were directly applied to the device on the probe station in the ambient environment at room temperature. We note that the test bench setup was suitable for kHz modulation frequencies. However, the GFETs are applicable at higher frequencies, potentially up to several GHz18, which would require a different measurement setup. In practice, the frequency of the input/output signals of the modulator will depend on the transmission medium. For example, if the sensor-modulator is used in a low-frequency RFID communication system, then the operating frequencies will typically be in the kHz range. However, if it is used in a Bluetooth system, then the corresponding frequencies will be 2.4 GHz/4.8 GHz.In the chemical-gating-enabled self-modulation demonstration, the GFET was vacuum fixed on a probe station and the back-gate and drain contacts of the GFET, which had a channel length of 100 μm, were connected to bias tees to combine and separate the RF and DC signals. The input RF signal was generated by a function generator, and the DC drain bias was generated by a voltage source. The output signal at the drain was measured using an Agilent DSO-X 3034A digital oscilloscope  with the spectrum analyzing function based on the fast Fourier transform. In the 2, O2, and ambient air) or an electron donor , inducing charge transfer between the graphene and the adsorbed substance6–8. For instance, under ambient air, the IDS–VGS curve of a GFET naturally shifts rightward (p-type shift) over time, as verified by the DC characteristic measurement shown in 31 patterned directly with a shadow mask, which exhibited reliable device characteristics  and the H2O/O2 redox-coupled DOS reach equilibrium. In our test, Vcnp had undergone a marked shift from 3 to 12 V after 10 min of air exposure.The chemical gating effect typically results from the adsorption of an oxidizer  high-vacuum annealing or high-temperature heating, which causes the desorption of the substances on the graphene24, and (2) wet chemical treatment, although most studies of this method are based on photoresist-contaminated GFETs and the recovery chemicals involved are hazardous 35. In this study, ethanol treatment could serve as a cost-effective, non-toxic and ecofriendly method of resetting the Vcnp of a photoresist-free GFET. During each wet treatment phase, a naturally p-doped GFET was treated with an ethanol droplet for 20 s, followed by a drying process using a nitrogen gun. We found that the instantaneous measured Vcnp could be reduced from 16 to 3 V, as seen in 2O/O2 redox system on the graphene surface and hence shifts the Fermi level of the graphene to its original energy level prior to air exposure, as depicted in 14, which relies on the chemical reaction between HF and H2O14, here the H2O/O2 redox system is removed because it is readily soluble in ethanol; therefore, this wet treatment is a chemical-reaction-free and environmentally friendly process that is suitable for practical applications.There are several ways to restore the intrinsic electrical characteristics and tune the Vcnp of the fabricated GFET is ~3 V, and the output vector, consisting of the fundamental tone at 25 kHz and the second-harmonic tone at 50 kHz, has a signal-to-noise ratio (SNR) of <50 dB, 16 dB>. Vcnp. Typically, the shift in Vcnp is quite sensitive to different chemical events, as will be discussed below. f1, SNRf2> may provide redundant information for signal post-processing and analysis in sensing systems, which may improve their robustness and enable absolute accuracy in sensing; details of the decoding of sensing signals are provided in the Here, mixed modulation controlled by chemical gating is demonstrated in an air exposure-ethanol recovery modulation cycle using the original GFET represented in Vcnp value of a graphene device can be used as an analog state to label its level of chemical exposure over a finite period of time, having a role similar to that of an analog memory. More interestingly, this memory-like effect is adjustable with the assistance of a voltage pulse sequence applied at the gate. Under ambient air exposure, the charge traps on the graphene surface predominantly consist of the H2O/O2 redox system. Here we demonstrate that one can dynamically control the hysteresis-like behavior of the GFET by repeatedly applying a unidirectional positive (negative) voltage-pulse sequence, resulting in a faster (slower) Vcnp shift during air exposure. Vcnp shifts measured under identical air exposure conditions and time durations but with voltage-pulse sequences of different polarities applied at the back gate. IDS–VGS curve shifts at different points in time during air exposure without the application of gate-voltage pulses. IDS–VGS curve shifts with the application of a positive gate-voltage pulse sequence, where each pulse is swept from 0 to 30 V . IDS–VGS curve shifts with the application of a negative gate-voltage pulse sequence, where each pulse is swept from −20 to 0 V . When the gate-to-source voltage pulse is positive and the drain-to-source DC bias is small, electrons are temporarily pumped into the graphene channel and the inward electric field (with respect to the air regions) near the graphene layer will preferentially accelerate the electron charge transfer33 from the graphene to the H2O/O2 charge traps. When the pulse ends, the accumulated electrons transferred from the graphene to the charge traps cause a stronger p-type shift of Vcnp compared with that in the case without a gate-voltage pulse  once the ethanol has dried. This change is marked compared with the unbiased scenario represented in Vcnp of 3 V). This phenomenon has previously been investigated through hysteretic voltage sweeping, which shows that when an external electrical field is applied to a polarized ionic liquid on a graphene surface, the capacitive gating effect can lead to an increase in the carrier density of the graphene32. Similarly, in the ethanol treatment applied here, the capacitive (ion) gating enables rapid control of the carrier density through repeated unidirectional voltage pulses. Because ethanol is also a polar solvent, when a positive pulse is applied at the back gate, the inward electric field causes the molecular dipoles in the ethanol droplet to orient toward the graphene surface, thereby increasing the electron carrier density in the graphene channel and consequently enhancing the n-doping effect observed after treatment under a positive pulse sequence, as shown in the inset of 2O/O2 redox charge traps from the ambient air are triggered simultaneously.The behavior of the graphene in the wet chemical treatment phase can also be adjusted using a gate-voltage pulse sequence. From but with  no gate but with  a positibut with  a negatiVcnp shift is reversed from an n-type shift to a strong p-type shift, with a final Vcnp higher than 50 V, as shown in By contrast, with the application of a negative gate-voltage pulse sequence during ethanol treatment, the Vcnp during air exposure for the same GFET represented in Vcnp) during air exposure. 2H6O) and water (H2O). The different capabilities of ethanol and water in removing the H2O/O2 redox system from the graphene surface may lead to different profiles of the Vcnp shift over time, resulting in very different event-driven modulation cycles. It is seen from The memory effect of GFET modulators is sensitive to both electrical gate bias and chemical gate conditions. The benefit of this feature for IoT-based continuous monitoring applications can be seen from the time-domain presentation of the experimental data shown in 36. The passive sensor modulator is very suitable for integration with communication systems containing a wireless interrogating power source, for example, RFID or NFC. Considering the large numbers of sensor nodes required in IoTs or IoNTs, the power savings over a long period of time could be enormous. The use of either an active or passive modulation scheme will depend on the application type and the system-level design for sensing and communication. For example, for typical indoor sensing applications, a lithium-ion battery or any other power source can be conveniently connected to the drain terminal of the GFET to provide DC power. For other power-critical applications, such as state-of-the-art bio-implantable devices, the use of on-site batteries is not possible and a passive GFET modulator is more desirable.The proposed multifunctional graphene module, with its highly simplified integration and operation stages , arguablMycobacterium tuberculosis), as a third material to be added to the chemical gating modulation cycle. For the sake of brevity, the measurement results are presented in the Vcnp of the GFET could provide information regarding to which type of protein the graphene may have been exposed. In this context, this nanomaterial sensor node may have great potential to become part of future point-of-care platforms. Smart phones or other portable gateways could receive the sensor signal and instantly decode and synchronize the information to the cloud, providing a seamless, convenient and ultra-low-cost healthcare IoT solution in the big data era.We also considered, as another example, two different protein solutions, namely, milk and ESAT-6  frequency modulation, sensing, and an analog memory effect to record the history of various chemical events. This new frequency-modulated sensing paradigm may enable the development of practical graphene-based nanosensors with wireless connectivity for the continuous monitoring of surface chemical events. We have demonstrated in an air exposure-wet treatment cycle that the modulated chemical gating of the proposed graphene field-effect device and the resulting RF output signal may provide reliable diagnoses of different types of treatment options and solvents, providing a promising route toward infection and biometric monitoring


Xenopus embryos. CEP3 is specifically expressed in somite tissue during somite segmentation. Loss-of-function experiments showed that CEP3 is not required for the specification of paraxial mesoderm, nor the differentiation of muscle cells, but is required for the segmentation process. Live imaging analysis further revealed that CEP3 is required for cell shape changes and alignment during somitogenesis. When CEP3 was knocked down, somitic cells did not elongate efficiently along the mediolateral axis and failed to undertake the 90° rotation. As a result, cells remained in a continuous sheet without an apparent segmentation cleft. CEP3 likely interacts with Cdc42 during this process, and both increased and decreased Cdc42 activity led to defective somite segmentation. Segmentation defects caused by Cdc42 knockdown can be partially rescued by the overexpression of CEP3. Conversely, loss of CEP3 resulted in the maintenance of high levels of Cdc42 activity at the cell membrane, which is normally reduced during and after somite segmentation. These results suggest that there is a feedback regulation between Cdc42 and CEP3 during somite segmentation and the activity of Cdc42 needs to be fine-tuned to control the coordinated cell shape changes and movement required for somite segmentation.Somitogenesis is a critical process during vertebrate development that establishes the segmented body plan and gives rise to the vertebra, skeletal muscles, and dermis. While segmentation clock and wave front mechanisms have been elucidated to control the size and time of somite formation, regulation of the segmentation process that physically separates somites is not understood in detail. Here, we identified a cytoskeletal player, Cdc42 effector protein 3  that is required for somite segmentation in  Somitogenesis is an essential step during vertebrate development. During somitogenesis, the paraxial mesoderm is partitioned into bilaterally symmetric blocks called somites, which later give rise to the dermis, skeletal muscles, and axial bones including vertebrae and ribs  in a cyclic manner, which determines the speed of segmentation , which is specifically expressed in the developing somite. CEP3 belongs to a small family of Cdc42 effector proteins, also named binders of Rho GTPases (Borgs)  during early cleavage stages. standard control MO  and Cdc42 effector protein 3(CEP3)-MO (5′- GGAATGAAATACGCAGATGTCAGAT−3′) that hybridizes to −32 to −8 position relative to the translational start site of Xenopus CEP3 (GenBank Accession No. NM_1095138) were used in the study. GFP-wGBD was a gift from William Bement (Addgene plasmid # 26734). pcDNA3-EGFP-Cdc42-T17N and pcDNA3-EGFP-Cdc42-Q61L were gifts from Gary Bokoch (Addgene plasmid #12976/ #12986). RNAs of nuclear beta-galactosidase , membrane-tethered EGFP, GFP-wGBD, Cdc42(T1N), Cdc42(Q61L), and CEP3 were synthesized with linearized templates using SP6 polymerase (Ambion mMessage mMachine Kit). To target the paraxial mesoderm, mRNA or MO was injected into the lateral marginal zone at the 2-cell stage. Five to ten nanograms of MO or 0.05–0.3 ng of RNA was injected into one side of the embryo as indicated in each experiment. All experimental procedures were performed according to the USDA Animal Welfare Act Regulations and had been approved by the Institutional Animal Care and Use Committee, in compliance of the Public Health Service Policy.in situ hybridization was performed. Whole-mount in situ hybridization was performed as previously described , mef2d (NM_001096493), myf5 (NM_001101779), myf6 (NM_001088008), myod1 (NM_001087823), and myog (NM_001085857) were synthesized with T7 RNA polymerase with linearized plasmids. Immunohistochemistry was performed as previously described  until they turned red. The embryos were refixed for 2 h in MEMFA, and stored in methanol before Wilson explants were isolated from embryos at stage 13–14 as previously described  posterior to the forming somite. The expression pattern of CEP3 suggests that it may play a role in somitogenesis.Cdc42 is involved in various cell and tissue morphogenesis events, and consistently, it is broadly expressed during embryonic development  against CEP3. Ten nanograms of CEP3-MO together with a lineage tracer nuclear beta-galactosidase  was injected into one side of 2-cell stage embryos, leaving the contralateral side as an internal control. Embryos were fixed at gastrula stage (stage 13), early tailbud stage (stage 22–23), and late tailbud stage (stage 28–30), and subject to in situ hybridization against different mesodermal and myogenic genes. During gastrulation, the paraxial mesoderm is specified by gradients of morphogens such as bone morphogenetic proteins (BMPs), FGFs, Wnt, and Noggin. At this stage, CEP3-MO did not affect the expression of a pan-mesodermal gene tbxt (Xbra) (n = 9/9). The somite in Xenopus embryos primarily gives rise to myotome tissue, and some myogenic genes are already expressed in paraxial mesoderm at gastrula stages. When CEP3 was knocked down by CEP3-MO, the expression of myogenic genes myf5 and myod1 was not affected . These results indicate that CEP3 is not required for the specification of paraxial mesoderm.Since t (Xbra) ; n = 9/9Mef2d and myod1 are expressed in a segmented pattern in the developing somite and uniformly in the PSM. When CEP3 was knocked down by CEP3-MO, the segmented expression pattern of mef2d and myod1 was lost . The expression levels of both genes were relatively unchanged, but they were expressed in a diffused and continuous manner. This result indicates that CEP3 is required for somite segmentation. Later in development, the somite is further differentiated, and more muscle-specific transcription factors are expressed. Myog is expressed continuously in the central domain of each somite, and in a segmented manner in the dorsal and ventral aspect of the somite. When CEP3 was knocked down, the expression level of myog was unaffected, but the segmented pattern was lost (n = 12/17). Similarly, the segmented expression of myf6 and muscle marker 12/101 were disrupted by CEP3-MO . Unlike the chevron shaped somites seen in control embryos, somitic tissue appeared in a disorganized and continuous form. These results confirmed that somite segmentation, but not myogenesis was inhibited by the loss of CEP3. The expression pattern of myogenic genes at late developmental stages also indicated that CEP3-MO did not cause a delay in somite segmentation, but rather inhibited segmentation. When CEP3 RNA without 5′UTR (therefore cannot be blocked by CEP3-MO) was coinjected with CEP3-MO, the segmentation defect was partially rescued , suggesting that the segmentation defect is specific to the loss of CEP3. Results obtained from the above in situ hybridization experiments were pooled together and summarized in At early tailbud stages when somite segmentation is underway, the first wave of myogenesis has occurred to generate differentiated myotome , with circularity = 1 for a perfect circle and circularity = 0 for a straight line). Over 100 cells were analyzed from 3 explants, with a similar number of cells picked on control and experimental sides at each axial level . While control cells have an average circularity of 0.454 ± 0.10, CEP3-MO cells have an average circularity of 0.698 ± 0.09. This difference was statistically significant .Besides a failure to organize into distinct somitomeres, cells deprived of CEP3 also appeared different in shape compared to control cells. On the control side, cells elongated mediolaterally before segmentation took place. After segmentation, cells maintained their elongated shape and gradually rotated 90° to adopt an anterior-posterior orientation  and constitutively active (CA-) Cdc42 to alter the activation level of Cdc42 . Both DN-Cdc42 and CA-Cdc42 disrupted the segmentation expression pattern of myogenic genes , suggesting that tightly controlled Cdc42 activity is required for proper segmentation process. A rescue experiment was performed next to determine whether CEP3 mediates Cdc42 during somite segmentation. While co-expression of CA-Cdc42 with CEP3-MO could not rescue segmentation defects caused by CEP3-MO, co-expression of CEP3 RNA with DN-Cdc42 partially rescued segmentation defects caused by DN-Cdc42 . This result suggests that Cdc42 activity needs to be decreased during somite segmentation and this partly explains why CA-Cdc42 also affected somite segmentation. Moreover, CEP3 may provide a negative feedback to Cdc42 to fine-tune Cdc42 activity in regulating somite segmentation.In neural crest cells, we previously showed that CEP3 paralog CEP1 provides feedback regulation of Cdc42 by regulating the subcellular localization of active Cdc42  (Watanabe et al., In this study, we showed that CEP3 is required for somite segmentation in CEP3 in somitic cells increases after somite segmentation and that intersomitic fissures can form but are not maintained at the loss of CEP3, it is possible that CEP3 also influences matrix deposition and assembly around the somitomeres. In endothelial cells, Rac1 and Cdc42 levels influence fibronectin matrix assembly during vascular morphogenesis (Fernandez-Sauze et al., The second possible way through which CEP3 regulates somite segmentation is the regulation of matrix deposition. Both fibronectin and laminin matrix are deposited and assembled at the intersomitic boundaries, as well as boundaries between notochord and the PSM. This extracellular matrix not only protects the integrity of each somitomere but also plays essential roles in somitic cell elongation, alignment, and rotation (Kragtorp and Miller, Lastly, to maintain somite segmentation, CEP3 could also regulate cell rotation and alignment. The rotation of somitomeres has also been observed in zebrafish (Hollway et al., CEP3 is an effector protein for Cdc42; therefore, its role in somite segmentation likely involves its interaction with Cdc42. Our results suggest that there is cross-regulation between CEP3 and Cdc42. CEP3 acts downstream of Cdc42 such that segmentation defects caused by DN-Cdc42 can be partially rescued by CEP3 RNA, but segmentation defects caused by CEP3-MO cannot be rescued by CA-Cdc42 or DN-Cdc42. Conversely, CEP3 also modulates the activity of Cdc42. When CEP3 is knocked down by CEP3-MO, active Cdc42 on the plasma membrane is maintained, suggesting a feedback regulation between CEP3 and Cdc42. This is similar to our recent report that in neural crest cells, CEP3 paralog CEP1 provides feedback regulation of Cdc42 by regulating the subcellular localization of active Cdc42 (Cohen et al., This study was carried out in accordance with the recommendations of USDA Animal Welfare Act Regulations and Public Health Service Policy. The protocol was approved by the Institutional Animal Care and Use Committee.HS performed the initial characterization of loss of CEP3 phenotype, which is confirmed by MK. MK further examined the activity of CEP3 in somite segmentation with live imaging and examined the interaction between CEP3 and Cdc42. SN designed and guided the experiments, and prepared the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Simplified microbial communities, or “benchtop microbiomes,” enable us to manage the profound complexity of microbial ecosystems. Widespread activities aiming to design and control communities result in novel resources for testing ecological theories and also for realizing new biotechnologies. Simplified microbial communities, or “benchtop microbiomes,” enable us to manage the profound complexity of microbial ecosystems. Widespread activities aiming to design and control communities result in novel resources for testing ecological theories and also for realizing new biotechnologies. There is much to be gained by reconciling engineering design principles with ecological processes that shape microbiomes in nature. In this short Perspective, I will address how natural processes such as environmental filtering, the establishment of priority effects, and community “blending”  can be harnessed for engineering microbiomes from complex starting materials. I will also discuss how future microbiome architects may draw inspiration from modern practices in synthetic biology. This topic is based on an important overarching research goal, which is to understand how natural forces shape microbial communities and interspecies interactions such that new engineering design principles can be extracted to promote human health or energy and environmental sustainability. Microbes rule our soils, our oceans, many of our bodily functions, our built environments, and our food, and many of us also find them fascinating. We have had a long and rich history of harnessing microbial ecosystems for engineering purposes, which is a practice being revolutionized by innovations borrowed from synthetic biology. Modern microbiome sciences have shown us that most microbes live in ultracomplex communities that are subject to spatial and temporal heterogeneity and often influenced by stochastic processes . In shorEscherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae. But what about microbial communities? The criteria for building or adopting useful model microbial communities depend completely on the context of investigation. For example, many studies require explicit linkages to habitats such as soil, the human body, or marine euphotic zones. In these cases, it is difficult to imagine a single model microbiome ever becoming as widely adopted as the E. coli example has been for single-species investigations, because our collective efforts are spread across seemingly endless ecological categories. However, as we get better and faster at building ecosystem-relevant consortia, there may soon be an explosion in the number of model systems that can be shared and standardized to enhance collaboration. I support a vision—shared by others—in which cultivable microbial consortia will soon be archived and shared by a similar mechanism to modern day culture collections. Some challenges must be overcome for this to be realized; such as the ability to arrest or manage community-level adaptation. These efforts should, in turn, lead to new platforms, or “chassis,” for innovation and expansion in synthetic biology.Why do we need model systems in biology, and what do they give us? Organismal biology has shown us through multiple famous examples that model systems such as the mouse or zebra fish accelerate research by providing a platform for standardized and reproducible investigations. Many major advances in microbiology are the result from a century or more of collective deep-dive research into model organisms: www.addgene.org), and the Registry of Standard Biological Parts (http://parts.igem.org/).Other reasons that fundamental scientists have for building microbial communities are more system agnostic and typically seek to understand generalizable principles such as the emergence of higher-order properties or energetic drivers of metabolite exchange , 4. ArchThere are two common approaches—within the current state of the science—for developing model microbial communities: top-down selection of natural microbiomes toward representative consortia with reduced complexity  and bott8–2–Microbiome engineering can also be practiced through hybrid approaches, where different complex communities and elements of their associated environments are mixed by means that enable the engineer to capitalize on natural ecological processes as design principles . This isEnvironmental mixing itself can be a controllable property and therefore can also be used as an engineering design tool. For example, careful control over environmental mixing can be harnessed to both design and control the degree of “environmental filtering,” which is the process by which an environment selects against certain species. This can be realized by preconditioning one community as a starting material prior to blending it with donor communities that have been conditioned to different environments and/or different selection forces. A “priority effect” is the ability for a given species to influence later stages of community structure and/or function. The assignment and utilization of priority effects may also be realized as an engineering design strategy that can again be controlled by preconditioning one community—or even monocultures—prior to the addition of another. One potential application would be successive challenges of a model community with new communities in an effort to strengthen priority effects that enhance resistance or resilience against invasion. An obvious application for employing this type of microbiome engineering applies to the growing interest in developing designer human microbiomes to combat or resist invasion by pathogens.The “functional trait space” of a community can be defined as the number of measurable characteristics that affect the performance or fitness of individual members or the whole community . The abi2–As a researcher that operates at the juxtaposition of microbial ecology and bioengineering, I often struggle with defining boundaries between the fundamental and applied aspects of my research on microbial communities. There is a common, yet seemingly artificial, compartmentalization of scientific culture, language, and approach that partitions the two practices of observing nature and engineering or reengineering it. Language and accepted definitions are the obvious and most difficult places to begin reconciling our understanding of engineering design principles with ecological processes. There are so many examples, so I will give just one here. What is the definition of a community? Traditionally, “communities” have been defined as multispecies assemblages where members live together in a contiguous environment and interact. However, synthetic ecology has already challenged this definition by assembling microbial consortia from metabolically engineered and functionally differentiated members of the same species . Are the


A series of thermoelectric responsive shape memory hydro-epoxy (H-EP) composites filled with different contents of graphene were developed and characterized. Compared with traditional actuation materials, these novel shape memory composites exhibit attractive properties, such as light weight, large deformation, good processability and high response speed, making them good candidates for actuator materials. The effect of graphene content on the shape memory composites was studied in terms of mechanical, dynamic mechanical analysis (DMA), electrical properties, and thermoelectric responsive shape memory test. The results show that when graphene content was 2 wt %, the bend strength of the composite improved by about 47% with a storage modulus larger than other composites. The shape recovery ratio of the composites was about 100%, and the shape recovery speed increased with the increment of graphene content, applied voltage, and temperature. Due to the excellent actuation performance, the graphene/hydro-epoxy composite has potential applications in the actuator in the future. Actuators are mechanical devices that are used to change or control mechanisms or systems, which are operated by responding to appropriate external stimulus, such as heat, electric, light, magnetic, pH, specific ions, and pneumatic . ActuatoSince 1984, SMPs have been known for the attractive properties, such as light weight, large deformations (strain of about 300%–400%), and being easily formable into arbitrary shapes compared to SMA and SMC ,8,9. Man−8 Ω/m) and thermal conductivity (5000 Wm−1·K−1), high surface area, excellent mechanical strength , and high flexibility. Due to these properties, graphene can be used to improve the properties of the SMPs and render the shape memory polymer composites to be a possible competitive candidate for actuation materials [In this paper, graphene was used to fill the shape memory hydro-epoxy (H-EP) to prepare novel thermoelectric responsive shape memory polymer composites. Graphene is a stable two-dimensional single-carbon atom layer crystal material. It has a large number of excellent properties, such as excellent electrical  was supplied by SigmaAldrich . All of them were used without further purification.The H-EP  was produced by Yantai Aolifu Chemical Industry Co., Ltd. . The curing agent methyl tetrahydro-phthalic anhydride  was purchased from Shanghai Western Science and Technology Co., Ltd. . The accelerant agent 2,4,6-tris(dimethylaminomethyl) phenol  was provided by Guangzhou Kay Trade Co., Ltd. . High purity graphene (purity > 98 wt %) was obtained from Chengdu Organic Chemistry Co., Ltd. . The samples were fabricated by incorporating different contents of high purity graphene  into H-EP resin. Stable dispersions of graphene were created by ultrasonic dispersion (100 W) for 1 h in DMF. A certain mass ratio of the H-EP, MeTHPA, and DMP-30 (the weight ratio is H-EP:MeTHPA:DMP-30 = 100:80:0.8) were mixed at 60 °C, the mixture was stirred by magnetic stirrer until the mixture was evenly mixed. Then, the graphene/DMF solution was added to mixtures stirred by a high-speed magnetic stirrer at a speed of 2000 rpm/min for 10 min, and the mixtures were sonicated by an ultrasonic dispersion instrument for 20 min. The mixture was heated to 60 °C in an oil bath to evaporate the solvent and the magnetic stirring speed is controlled to 300 rpm/min for 2 h. After that, the mixture was placed in a vacuum oven at a temperature of 80 °C for 10 h. Finally, the mixture was poured into a glass mold for curing and the curing temperature process was 80 °C/2 h + 120 °C/2 h + 150 °C/3 h.The tensile and bending tests were carried out on a universal testing machine  according to ASTM D638-14  and ASTMThe resistance of the graphene/H-EP composites was tested using a modern digital multimeter (UT61E).ρ is the volume resistivity, R is the resistance, A is the contact area between the electrode and the composite (1.0 × 0.2 cm2), and L is the length of the sample.The volume resistivity of the samples was calculated by the following formula:The specimens were frozen in liquid nitrogen, and then brittle fractured. The broken surfaces were sputter-coated with gold and observed with a SEM .3, and the samples were sanded with 800 grit sandpaper . DMA was performed in a single cantilever clamping fixture and carried out using a Q800 . The temperature was increased to 180 °C from 20 °C with a rate of 5 °C/min, the amplitude and dynamic load were 10 um and 1 Hz, respectively.The samples were cut into 5 × 10 × 30 mm3) were cut to investigate the thermo-active shape memory effect of the graphene/H-EP composites. Shape memory performance of the composites were tested as follows: (i) in the oven, heating the samples to the glass transition temperature (Tg) and held for 5 min; (ii) bending the samples into a “U” shape and then quickly dipping the samples into ice water to maintain the external force; (iii) putting the “U”-shaped samples into hot water and the temperature of the water set to Tg, Tg + 10 °C, and Tg + 20 °C of the composites; and (iv) observing the shape recovery process of composites, when the shape of the samples did not change, recording the recovery time. i/tθ, and the shape recovery ratio is defined as (iθ – fθ)/iθ × 100% [iθ is the deformation angle (o), t is the recovery time, and fθ is the angle of recovery after deformation.Rectangular samples  were cut to test the electro-active shape memory behavior under different voltages. The first two steps in the process of the electro-active test were the same as thermo-active shape memory test. However, the third step is different. In the electro-active shape memory test, different voltages were applied to the samples to observe the shape memory process and recorded the shape recovery time.Rectangular samples . Generally, when the temperature is lower than the shape memory transition temperature, the storage modulus of a good shape memory polymer composite is 2–3 orders higher than that when the temperature is above the shape memory transition temperature . In thisgT is an important parameter of themomechanical property and shape memory effect of shape memory polymer composites. In this study, the Tgs of the SMPs are the peaks of the tanδ curves, as shown in Tgs of the bulk specimens with 1.0, 2.0, and 3.0 wt % graphene are 54.3, 56, 57 and 61.5 °C, respectively. The Tg increases slight with the increasing of the graphene content, this phenomenon is due to the doped graphene hindering the mobility of the H-EP molecular chain [ar chain .Due to the excellent electrical properties of graphene, the volume resistance of graphene/H-EP composites decrease as the graphene content increases, as shown in Tg and deformed into a temporary U shape. After cooling, the temporary shape is fixed. Then, putting the sample in the oven and raising the temperature to 55 °C, the sample takes 120 s to completely return to its original shape.The thermo-active shape memory property results show that the graphene/H-EP composites can be fully recovered within a few minutes at three different temperatures. This means that the shape recovery ratio of the graphene/H-EP composites are about 100%. This indicates that the graphene/H-EP composites have good shape memory behavior. In addition, it also shows that the addition of small amounts of graphene do not affect the shape memory behavior of graphene/H-EP composites. The shape memory process of sample with 1.0 wt % graphene is shown in Tg, Tg + 10°C, and Tg + 20 °C, it can be seen that the shape recovery time decreases with increasing graphene content. During the process of the thermo-active shape memory test, the specimen is deformed at high temperature, and then quickly cooled under the condition of keeping the external force. At this moment, the stress will be frozen as the recovery force. When the sample is heated again, the frozen stress is released, which drives the deformed sample to recover to its original shape. Therefore, the shape recovery speed and shape recovery time are related to the recovery force. The faster the recovery force is released, the faster the shape recovery speed and the lower the shape recovery time [gT more quickly, which results to the recovery time decreases with the increment of the graphene content.ery time . MeanwhiThe electric field-triggered shape recovery of sample with 2.0 wt % graphene is given in To investigate the electroactive shape memory property of the graphene/H-EP composites, rectangular samples were tested under 60–160 V voltage. The results show that all of the composite samples can recover to their initial shape under a certain voltage, and the shape recovery ratios are close to 100%. This denotes that the graphene/H-EP composites have excellent electro-active shape memory effect. The shape memory effects of the composites under different voltages are presented in The creep, plastic deformation, and residual stress will affect the shape memory properties of the composites. Therefore, we tested the shape memory effect of the composites several times to analyze the effect of cycling times on the recovery ratio of graphene/H-EP composites. As is evident from Novel thermo-electrical responsive shape memory graphene/H-EP composites was fabricated and these composites could be developed as MEMS actuation materials for large deformation, multi-stimuli response, and rapid response. Due to the homogeneous dispersion of graphene in the H-EP matrix, the mechanical and electrical conductivity of the composites are improved significantly. The graphene/H-EP composites exhibits excellent electro-active and thermo-active shape memory performance. With the increase of the content of graphene and temperature, the thermo-active shape memory effect is better, and the electro-active shape memory performance of the composites are improved by the increase of the graphene content and applied voltage. Though the cycling times have a disadvantageous effect on shape memory behavior, the shape recovery ratio of the composites are greater than 95%. In their present form, the materials developed are suitable for devices employing low cycling conditions (<5 cycles).


TERT, TERC, DKC1, TINF2, RTEL1, PARN, NAF1, ACD, NHP2 and NOP10 genes in any patient. Moreover, telomere length was normal in the two patients tested. This could suggest that although the co-occurence of IPF and MDS are suggestive of TRG mutation in patients < 65 years old, in the elderly it may occur without germline mutations and could negatively affect prognosis. Physicians should be aware for possible IPF deterioration and therapeutic options for MDS should be wisely considered.Previous studies have shown that the co-existence of bone marrow failure and pulmonary fibrosis in a single patient or in a family is suggestive of telomere related genes (TRG) germline mutations. This study presents the genetic background, clinical characteristics, and outcome of a group of five Greek patients co-affected with IPF and MDS. Four out of five patients developed an IPF acute exacerbation that was not reversible. We failed to detect any mutation in the  TERT) or telomerase RNA component (TERC)] germline mutations [Both myelodysplastic syndromes (MDS) and idiopathic pulmonary fibrosis (IPF) constitute irreversible diseases of the elderly, and both share comorbidities that adversely affect their prognosis , 2. Theiutations , 7 and sutations . Mutatioutations . Furtherutations . The stuTERT, TERC, Dyskeratosis congenita 1 (DKC1), TERF1-interacting nuclear factor 2 (TINF2), Regulator of telomere length 1 (RTEL1), poly(A)-specific ribonuclease (PARN), Nuclear Assembly Factor 1 Ribonucleoprotein (NAF1), Adrenocortical dysplasia (ACD), H/ACA ribonucleoprotein complex subunit 2 (NHP2) and H/ACA ribonucleoprotein complex subunit 3 (NOP10) by next generation sequencing. Two of the five patients had their telomere lengths measured in peripheral blood cells by the telomeric restriction fragment (TRF) assay as already described [After written informed consent, all patients diagnosed with both IPF and MDS, underwent genetic testing for escribed . IPF wasescribed . AutoimmTERT, TERC, DKC1, TINF2, RTEL1, PARN, NAF1, ACD, NHP2 and NOP10 germline mutations were not detected in any patient. The telomere length was not pathologically reduced in the two patients tested  years, 60% ex- smokers were studied. Pulmonary function and hematology parameters are reported in Table  4 weeks  and two  4 weeks –14 monthBased on this case-series four out of five patients with the co-existence of IPF and MDS developed an IPF acute exacerbation that was not reversible. An extensive work-up was performed to exclude obvious causes of the deterioration such as infection, aspiration or drug toxicity . AlthougTERT, TERC, DKC1, TINF2, RTEL1, PARN, NAF1, ACD, NHP2 and NOP10 genes in any patient. Moreover, telomere length was normal in the two patients tested. In the study of Parry and co-workers [Despite previous studies showing that the co-existence of bone marrow failure and pulmonary fibrosis in a single patient or in a family is very suggestive of TRG germline mutations , 7, we f-workers  all patiTherefore, although the co-occurrence of IPF and MDS are suggestive of TRG mutation in patients < 65 years old, in the elderly it may develop without germline mutations or may relate to mutations in other genes that are still unknown. Both IPF and MDS are diseases of the elderly and may be triggered by physiologic aging processes affecting both the lungs and the bone marrow, such as stem cell exhaustion, mitochondrial dysfunction, epigenetic alterations and disturbances of DNA methylation , 4.This case series suggests that in the elderly patients the co-occurrence of IPF and MDS may develop without germline mutations and could negatively affect prognosis. Physicians should be aware for possible IPF deterioration and therapeutic options for MDS should be wisely considered.


TCF7L2, KCNQ1, HNF1B, ADCY5 and GCK, respectively) and with additional 67 SNPs in LD with known T2D SNPs . There was enrichment of open chromatin regions near highly expressed genes in human islets. Moreover, 1,078 open chromatin peaks, annotated to 898 genes, differed in prevalence between diabetic and non-diabetic islet donors. Some of these peaks are annotated to candidate genes for T2D and islet dysfunction  and some overlap with SNPs associated with T2D (e.g. rs3821943 near WFS1 and rs508419 near ANK1). Enhancer regions and motifs specific to key TFs including BACH2, FOXO1, FOXA2, NEUROD1, MAFA and PDX1 were enriched in differential islet ATAC-seq peaks of T2D versus non-diabetic donors. Our study provides new understanding into how T2D alters the chromatin landscape, and thereby accessibility for TFs and gene expression, in human pancreatic islets.Impaired insulin secretion from pancreatic islets is a hallmark of type 2 diabetes (T2D). Altered chromatin structure may contribute to the disease. We therefore studied the impact of T2D on open chromatin in human pancreatic islets. We used assay for transposase-accessible chromatin using sequencing (ATAC-seq) to profile open chromatin in islets from T2D and non-diabetic donors. We identified 57,105 and 53,284 ATAC-seq peaks representing open chromatin regions in islets of non-diabetic and diabetic donors, respectively. The majority of ATAC-seq peaks mapped near transcription start sites. Additionally, peaks were enriched in enhancer regions and in regions where islet-specific transcription factors (TFs), e.g. FOXA2, MAFB, NKX2.2, NKX6.1 and PDX1, bind. Islet ATAC-seq peaks overlap with 13 SNPs associated with T2D (e.g. rs7903146, rs2237897, rs757209, rs11708067 and rs878521 near  Genome-wide association studies (GWAS) support islet dysfunction to be a key defect in T2D3. A large proportion of T2D-associated SNPs are located at regulatory regions and enhancer elements which control gene transcription4. Both the transcriptome and methylome are altered in T2D islets7. However, additional studies are needed to fully dissect the molecular mechanisms contributing to islet dysfunction. Importantly, a map of the open chromatin has not been generated in pancreatic islets of numerous subjects with T2D.Genetic, epigenetic and environmental factors contribute to development of type 2 diabetes (T2D)8. These open chromatin or nucleosome free regions are sensitive to nuclease enzymes. Chromatin immunoprecipitation (ChIP)-seq and DNase-seq studies from ENCODE show that open chromatin regions include all classes of cis-regulatory elements (cis-REs) e.g. promoters, enhancers and insulators10. Studies performed in human islet cells revealed that T2D-associated loci reside at open chromatin regions and enhancers15. However, these studies were mainly performed in islets/cells of non-diabetic donors and knowledge of how T2D alters the open chromatin structure in islets is needed.Transcription factors (TFs) bind to DNA in a sequence-specific manner by removing or moving the nucleosomes to form open chromatin16. We further integrated our human islet ATAC-seq data with RNA-seq and published islet ChIP-seq data of histone modifications and TFs.To map the open chromatin landscape in relation to T2D, we performed assay for transposase-accessible chromatin using sequencing (ATAC-seq) in human pancreatic islets from T2D and non-diabetic donors. ATAC-seq is a sensitive recently developed method with ability to map open chromatin in a small number of cells6. All islet preparations used in this study had a purity above 80%. Fresh islets were picked with pipette under stereo microscope and snap frozen in aliquots (~30 islets) (n = 5 donors) or immediately used for ATAC-seq (n = 5 donors). Frozen islets were obtained from our biobank and were thawed on ice (n = 5 donors). This information is presented in Supplemental Table Human islets of 9 non-diabetic donors and 6 donors diagnosed with T2D were obtained from the Nordic Network of Islet Transplantation, Uppsala University, Sweden. Donor characteristics are presented in Table 16 in our human islets .ATAC-seq was performed as previously describedSee Supplemental material and methods.The ATAC-seq peaks were annotated to genomic elements such as transcription start sites (TSS), transcription termination sites (TTS), exons and introns using GENCODE version19 for GRCh37. Each peak was annotated in relation to all these elements and may thereby have multiple gene annotations.See Supplemental material and methods.et al.11 and (ii) looking at overlap between islet ATAC-seq data and putative TF binding motifs. TF binding motif analysis of ATAC-seq data was performed using HOMER v4.7.217. Only known motifs from HOMER’s motif database were considered. We studied motifs enriched in ATAC-seq peaks of non-diabetic donors, and motifs enriched in T2D peaks relative non-diabetics. The latter analysis was performed by findMotifsGenome.pl with the peak set for non-diabetics as background.We performed two different TF analyses of the ATAC-seq data; (i) looking at overlap between islet ATAC-seq data and binding of specific TFs in human islets using ChIP-seq data generated by Pasquali See Supplemental material and methods.https://genome.ucsc.edu/cgi-bin/hgLiftOver) was used to convert the original assemblies of published datasets into GRCh37/hg19 using BED files or chromosome coordinates.UCSC lift-over online tool (6. The siRNAs used for knockdown were s144488 (siGabra2) and s132130 (siSlc16a7) and a custom negative control siRNA (5′-GAGACCCUAUCCGUGAUUAUU-3′) (siNC). Knockdown was verified with qPCR and TaqMan assays for Gabra2 (Rn01413643_m1) and Slc16a7 (Rn00568872_m1). Assays for Hprt1 (Rn01527840_m1) and Ppia (Rn00690933_m1) were used as endogenous controls and knockdown was calculated with the geometric mean method. Insulin secretion was analyzed as previously described18, except that insulin was determined with an ELISA . All siRNA and TaqMan assays were ordered from Thermo Fisher Scientific, Waltham, MA, USA.INS-1 832/13 rat beta-cells were used in functional experiments as describedU-tests were used for clinical data. Fisher’s exact tests were used to test if the number of ATAC-seq peaks were significantly associated with T2D and gene expression. Enrichments were analyzed by chi-square tests and a 5% overlap was considered by chance, i.e. 5% of the total number of peaks for each group. False discovery rate (FDR) analyses were used to correct for multiple testing.Mann-Whitney 19 were used for an affinity-based analysis (i.e. to identify islet ATAC-seq peaks where the mean number of mapped reads differ between the groups). Gender and sample treatment (frozen/non-frozen) were included as covariates in the design matrix. The whole ATAC-seq workflow was implemented in Snakemake20. qPCR and insulin secretion experiments performed in beta-cells were analyzed with the non-parametric Mann-Whitney test.Fisher’s exact test was used for an occupancy-based analysis (i.e. to identify islet ATAC-seq peaks that were found in significantly more non-diabetic compared to T2D donors or vice versa). The R Diffbind package and edgeR packageInformed consent for organ donation for medical research was obtained from pancreatic donors or their relatives in accordance with the approval by the regional ethics committee in Lund, Sweden (Dnr 173/2007). This study was performed in agreement with the Helsinki Declaration.We performed ATAC-seq to map the open chromatin accessible regions in human islets of 15 donors (6 with and 9 without T2D). The characteristics of these donors are presented in Table PDX-1, a transcription factor regulating beta-cell development and insulin gene expression21, and FOXA2, a regulator of beta-cell development, are presented in Fig. ATAC-seq peaks were only considered if they exist in at least three islet samples. Using this cut-off, 79,255 open chromatin peaks were identified when we combined ATAC-seq data from non-diabetic and diabetic islet donors  further supporting the quality of our data  with ENCODE open chromatin data generated using FAIRE-seq and DNaseI-seq as well as with ATAC-seq data generated by Thurner ata Fig. . The oveWe further examined the genomic distribution of ATAC-seq open chromatin peaks. As anticipated, a large proportion of ATAC-peaks are located close to TSS Fig. . Moreoveet al.23 and 2,207 SNPs in LD with these based on r2 = 0.8–1, 1000 genomes, phase 3 and hg19 (http://raggr.usc.edu/). SNPs with a minor allele frequency less than 0.01 were excluded. We found 13 SNPs from Scott et al. and 67 LD SNPs which were located within our islet ATAC-seq peaks  with these overlap with our islet ATAC-seq peaks  were enriched at islet ATAC-seq peaks of non-diabetic donors (q < 0.001)  Fig. . In cont01) Fig. . A similWe also studied enrichment of histone modifications at islet open chromatin regions annotated to different genomic regions i.e. TSS, exons, introns, TTS and intergenic regions Fig. . For exa26. Open chromatin regions annotated to intron and intergenic regions may also represent enhancers. We found that histone marks associated with enhancer regions are enriched at ATAC-seq peaks annotated to intron and intergenic regions in islets (q < 0.001)  functional enhancer candidates across different human cell types and tissues by using cap analysis of gene expression data (CAGE-tag)et al. where promoters, inactive enhancers, active enhancers, and CTCF bound sites were classified11. A large number of their classified promoters (C1 sites), active enhancers (C3 sites) and CTCF bound sites (C4 sites) are located at islet ATAC-seq open chromatin regions in non-diabetic donors   Fig. , support01) Fig. . The mos01) Fig. . We also01) Fig. .PDX1, GCG, FOXA2 and NEUROD1 are in the high-expressed category (data not shown). These data support that the chromatin is more open in high-expressed than in non-expressed genes. We also generated a Venn diagram showing that ATAC-seq peaks were annotated to the majority of high-expressed transcripts, while only a small proportion of non-expressed genes have open chromatin nearby . A total of 60,517 GENCODE transcripts were categorized as either not expressed  < 0.1) or as low- , medium-  and high-expressed  transcripts based on three equally sized groups of expressed transcripts (TPM > 0.1). While ATAC-seq peaks were annotated to the majority of high-expressed transcripts, only a small proportion of non-expressed genes have open chromatin nearby Fig. . There w7. Here, we found that these T2D-associated DMRs are located at 7,412 open chromatin regions in human islets, including regions annotated to PDX1 and SLC20A2  in islets of T2D versus non-diabetic donors by using whole-genome bisulfite sequencing and these DMRs were enriched in binding sites for islet-specific TFsCLEC16A, ELAVL4 , FOXO3, FST, GLIS3, MTNR1B, PARK2, WFS1 and ZMIZ1 have previously been associated with T2D by GWAS and/or found to affect islet function40. Sequencing tracks of ATAC-seq peaks that are more prevalent in either donors with T2D (e.g. MIR1178) or non-diabetics (e.g PTPN9) are presented in Fig. 42.To identify T2D-associated changes in the open chromatin landscape, we used Fisher’s exact test to examine whether islet ATAC-seq peaks were more prevalent in T2D versus non-diabetic donors. Here, 1,078 open chromatin peaks were found to differ between T2D and non-diabetic islet donors  and the functional database OMIM to identify disease-related pathways with enrichment of genes that had islet ATAC-seq peaks with a different prevalence in T2D versus non-diabetic donors. Interestingly, categories including Noninsulin-dependent Diabetes Mellitus, Familial Hypercholesterolemia, Essential Hypertension and Obesity were significantly enriched , 19.4% with inactive enhancers (C2 sites), 22.4% with active enhancers (C3 sites) and 11.7% with CTCF bound sites (C4 sites) 5.To identify TF motifs significantly enriched at the 1,078 ATAC-seq peaks with different prevalence in T2D, we performed a motif search using HOMER. Intriguingly, motifs specific to key TFs in islets, including BORIS, BACH2, FOXO1, FOXA2, NEUROD1, MAFA and PDX1 as well as the motif for CTCF were significantly enriched in the ATAC-seq peaks with different prevalence in T2D islets  was significantly enriched among the genes annotated to the 88 differential ATAC-seq peaks .We then used R Diffbind and edgeR packages178 Fig. , BACH1, SLC16A7 and GABRA2, were selected for functional follow-up experiments based on these criteria  and with differential expression in T2D versus non-diabetic islets have a functional role in beta-cells. Two genes, lls Fig. , which f2. GWAS placed the pancreatic islets in the center of attention as many of the loci were associated with impaired insulin secretion3. Although large efforts have been made to understand how these SNPs may cause disease further studies are needed. Interestingly, several risk genes identified by GWAS such as HHEX, HMGA2, GLIS3, MTNR1B and WFS152 had differential ATAC-seq peaks in T2D islets annotated to them. It is possible that T2D-associated SNPs change the chromatin structure and thereby gene regulation. In support for this, we have previously shown that SNPs linked to HHEX, HMGA2 and WFS1 associate with epigenetic changes, e.g. differential DNA methylation, in human islets38. Here, we found T2D-associated differences in ATAC-seq peaks annotated to genes which also show differential DNA methylation patterns in islets from T2D versus non-diabetic donors e.g. SOX67 (Supplemental Fig. MTNR1B expression is increased and insulin secretion decreased in islets from G-allele carriers which increases T2D risk. MTNR1B deficient mice secreted more insulin and MTNR1B overexpression affect insulin secretion in beta-cells35. Moreover, beta-cell mass and insulin secretion were reduced in rats where exon 5 of Wfs1 was deleted53. Also, GLIS3 controlled beta-cell proliferation in response to high-fat feeding and Glis3 deficiency leads to diabetes54. The potential causal consequences of differences in ATAC-seq peaks between donors with T2D and controls should be examined further and some caution should be considered regarding the gene annotations of these peaks. Here, we performed functional follow-up experiments of two identified genes with both differential ATAC-seq peaks and expression in islets from donors with T2D versus non-diabetic controls. We found that knockdown of Slc16a7, which exhibits fewer ATAC-seq peaks and lower expression in islets from non-diabetic donors, increased both insulin secretion and content in beta-cells. This supports a role for the identified chromatin differences in the secretory insufficiency that is characteristic of T2D. We cannot exclude that altered levels of Gabra2 affect other phenotypes than impaired insulin secretion, characterizing islets from donors with T2D.This study used ATAC-seq to establish T2D-associated changes in the open chromatin landscape of human islets. Interestingly, we found 1,078 differential ATAC-seq peaks in T2D versus control islets. T2D is driven by interactions between genetic and environmental factors. A major breakthrough in genetic research on T2D came with GWAS where ~100 loci were identified55. Gene expression is regulated by TFs that bind to DNA in open chromatin regions in a sequence-specific manner56. Given this, we studied TF binding and putative TF binding motifs in open chromatin regions of T2D and non-diabetic islets. As may be expected, ATAC-seq peaks were enriched in regions occupied by islet-specific TFs such as FOXA2, MAFB, NKX2.2, NKX6.1 and PDX1. These TFs are important for islet development and function as well as for proper insulin expression21. For example, PDX1 regulates development of beta-cells and insulin expression in mature beta-cells. While expression of PDX1 seems to be regulated by epigenetic mechanisms in T2D islets57, its function may be controlled by the chromatin structure and PDX1’s ability to bind to its target genes. Putative TF binding motifs were also enriched in the islet ATAC-seq peaks. Interestingly, the FRA1 motif was the most significantly enriched among islet ATAC-seq peaks. Our result is in line with data from a recent study where the motif for FRA1 was enriched in ATAC-seq peaks in alpha- and beta-cells14. FRA1 is involved in MAPK signaling and oxidative stress pathways. However, there is limited knowledge of its role in islets and future studies evaluating its binding and function would be interesting. The motif for CTCF was also significantly enriched in our islet ATAC-seq peaks as well as in the peaks that differed between T2D and non-diabetic islet donors. Varshney et al. previously did a reconstruction of the CTCF motif using ATAC-seq TF footprint allelic bias data of two islet donors15 and Ackerman et al. found this motif to be enriched in ATAC-seq peaks from sorted alpha-cells14. Moreover, alpha- and beta-cell-specific peaks e.g. ARX, GCG, DPP4 and SLC27A6 presented by Ackerman et al. were also identified in our islet ATAC-seq data14.Insufficient insulin secretion is a hallmark of T2D. Altered expression of insulin and numerous other genes seems to contribute to islet-dysfunction7. Here, we integrated ATAC-seq peaks with the methylome and found that T2D-associated DMRs are located at 7,412 open chromatin regions in human islets. Earlier, we found DMRs in and decreased expression of PARK2 in T2D versus control islets7. PARK2 regulates mitochondrial function in beta-cells and its silencing impaired insulin secretion. Here, we fund differential ATAC-seq peaks annotated to PARK2 in T2D islets, further supporting its role in diabetes. Given the importance of posttranslational modifications of histones, we related islet ATAC-seq peaks to histone marks associated with enhancers, active promoters and inactive genes. While histone marks of active enhancers and promoters were enriched, histone marks of inactive genes were underrepresented in open chromatin regions. These results were further supported by CAGE data and data generated by Pasquali et al.28 and are in line with muscle ATAC-seq data58.Epigenetic modifications are closely linked to chromatin structure. We have studied epigenetic alterations in T2D versus control islets by genome-wide DNA methylation analyses12. It should be noted that the number of islets used for ATAC-seq was optimized and 25–30 islets per donor gave the best result. Importantly, our data seem technically sound since a large proportion of our ATAC-seq open chromatin peaks overlapped with ENCODE validated peaks. Of note, islets contain several cell types. However, our previous data support that there are no differences in islet cell composition between T2D and non-diabetic donors6. Moreover, in the present study, we found no differences in expression of islet cell specific genes in T2D versus control donors further supporting no differences in islet cell composition in our cohort (Supplemental Fig. There are advantages with ATAC-seq compared with earlier methods. It requires smaller number of cells and has higher sensitivity and resolution. Indeed, we only used ~30 islets per donor5 as well as RNA-seq data from the donors with available ATAC-seq data.Additionally, since the current study included data from 15 donors, the differences in islet ATAC-seq peaks identified between donors with T2D and controls should be replicated in future studies. The donors with T2D have a higher BMI than the controls and it is possible that this affects our results. Subsequently, it will be of interest to only study the effect of BMI on the chromatin structure in human islets. Also since whole islets contain several different cell types, and these cell types may be in different proportions in different donors, future studies should perform ATAC-seq in sorted islet cells from donors with T2D and controls. To maximize our statistical power regarding analysis of RNA-seq data, we used both published expression data from a larger number of islet donors including 12 with T2D and 66 controlsTogether, we provide the first map of open chromatin regions in islets from numerous T2D donors. Dissection of open chromatin regions revealed enrichment for islet-specific TF binding motifs and enhancer regions. Numerous SNPs associated with T2D reside within islet ATAC-seq peaks. T2D candidate genes showed differential ATAC-seq peaks in diabetic versus non-diabetic islets. Our study improves current understanding of the link between altered chromatin structure and disease.Supplementary infoTable S2Table S3Table S4Table S5Table S6Table S7Table S8Table S9Table S10Table S11Table S12Table S13Table S14Table S15Table S16