Ubiquilin-2 (UBQLN2) is a member of the ubiquilin family, actively implicated in the degradation of misfolded and redundant proteins through the ubiquitin-proteasome system and macroautophagy. UBQLN2 received much attention after the discovery of gene mutations in amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). The abnormal presence of positive UBQLN2 inclusion in the cytosol of degenerating motor neurons of familial and sporadic forms of ALS patients has been newly related to neurodegeneration. Only recently, data have emerged on its role in liquid-liquid phase separation, in stress granule development and in the formation of secondary amyloid structures. Furthermore, several animal models are available to investigate its involvement in TDP-43 pathology and neuroinflammation in ALS. This review addresses the molecular pathogenetic pathways involving UBQLN2 abnormalities which are converging toward defects in clearance mechanisms. UBQLN2. C9ORF72 (Chromosome 9 open reading frame 72), SOD1 (Superoxide dismutase 1), TDP-43 (TAR DNA-binding protein 43) and FUS (Fused in Sarcoma) [Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disorder associated with degeneration of upper and lower motor neurons . The disSarcoma) , 53, 61.UBQLN2 gene were found to be associated with dysfunction of autophagy [UBQLN2 is a shuttle protein implicated in the ubiquitin-proteasome system (UPS). One of the most actively studied mechanism in UBQLN2-related pathology is the mutant-related dysfunction of the UPS. However, its implication in the cytoplasmic mislocalization of TDP-43 into insoluble aggregates is also well described in ALS. More recently, ALS-linked mutations in utophagy , neuroinutophagy , 50 and utophagy . These rIn this review we will describe the structure and the function of UBQLN2, and we will propose an integrative mechanism for the pathogenesis of UBQLN2 in ALS and FTD.UBQLN2 gene, also called Chap1/Dsk2 or PLIC, is located on the Xp11.21 chromosome. UBQLN2 is a single-exon coded, 66 kDa protein member of the ubiquilin family [UBQLN2 gene are located in the PXX domain. Yet, it is unclear how the mutations impact the secondary structure and functions of the PXX domain. The 3-dimensional structure of UBQLN2 is formed by five β-strands, an α-helix of 3.5 turns and an additional 310-helix [The n family , 29. UBQn family . UBQLN2 n family  Fig. 1)UBQLN2 ge10-helix .Fig. 1ScUBQLN2 is located in the cytosol and has been reported to be mainly expressed in the brain, spleen, heart, liver, pancreas and other tissues . The oth2+ homeostasis and maintaining mitochondrial function  proteins. UBQLN2 can interact with the ubiquitin regulatory X domain-containing protein 8 protein (Ubxd8), which is localized in the ER membrane and it facilitates the transport of endoplasmic-reticulum-associated protein degradation (ERAD) substrates to the cytosol . UBQLN2 P497H measured the autophagic flux with an LC3 turnover assay [P497H compromised autophagy-lysosomal pathways in an age-related manner.The implication of UBQLN2 in autophagy is now well established. Autophagy, specifically macroautophagy, is crucial for the bulk turnover of redundant cellular components, including protein aggregates and damaged organelles. A specific region of the cytosol is isolated in an intracellular double membrane stretch called autophagosome, secondarily fusing with lysosomes for degradation , 43, 68.UBQLN2 mutations were first identified in five unrelated families, suffering of ALS/FTD [n = 23), fALS without mutations in SOD1, TDP-43 and FUS (n = 5), ALS with dementia (n = 5) and fALS with G298S mutation in TDP-43 (n = 21) [P506T mutation were analysed. All fALS cases exhibited co-localization of UBQLN2 and TDP-43 cytoplasmic inclusions in the brain and the spinal cord neurons [ ALS/FTD . From th ALS/FTD , 60, 64  ALS/FTD . The aut(n = 21) . Also, b neurons . Interes neurons , 17.TablG76V_GFP was detected in Neuro2a cells expressing mutant UBQLN2P497H [G76V_GFP degradation were significantly slower in UBQLN2 mutations (P497H and P506T) when compared to the WT, suggesting the impairment of the protein degradation pathway by UBQLN2 mutations. Another group took advantage of the endogenous Myc as a proteasome efficiency reporter, as it is quickly degraded by the proteasome when its synthesis is blocked by cycloheximide [P497H in Neuro2A cells sequestered lys48_ bound ubiquitin and resulted in a reduction in the efficiency of the proteasome [The degradation of misfolded or redundant proteins is critical for the maintenance of cellular health, specifically in neurons which are not proliferating cells. There is evidence that UBQLN2 mutations can provoke proteasome as well as autophagy impairments. First, an accumulation of the proteasome efficacy reporter UbiquitinLN2P497H . They fuheximide . Indeed,heximide . Those rheximide . RecentlP506T mouse, that UBQLN2 works with the HSP70 system for proteasomal degradation of insoluble ubiquitylated protein aggregates [Another study reported, with a knocked-in hUBQLN2gregates . Under ngregates . Defect gregates . They algregates . Thus, aLastly, optineurin (OPTN) is a ubiquitin-binding multifunctional adaptor protein and is mainly implicated in the autophagy processes. It has also been proposed to be an important factor implicated in regulation of inflammation . UBQLN2 WT or mutated hUBQLN2P497H in Neuro2A cells can promote the formation of TDP-43 cytoplasmic inclusions [It has been reported that cytoplasmic inclusions of mutant C-terminal TDP-43 colocalized with either WT or mutant UBQLN2 in co-transfected Neuro2a cells . UBQLN2 clusions , 27, 34.clusions , 49.P497H and hTDP-43G348C vectors led to the accumulation of cytoplasmic TDP-43. However, ubiquitin overexpression reduced the cytoplasmic aggregation of TDP-43 [P497H and hTDP-43G348C transfected cells. This assay revealed a significant reduction in proteasome efficacy in all transfected cells as compared to control cells. The presence of up-regulated ubiquitin levels corrected the proteasome dysfunction in cells transfected with hUBQLN2P497H and in cells co-transfected with both hUBQLN2P497H and hTDP-43G348C. These results suggested that the proteasome impairment caused by hUBQLN2P497H up-regulation may participate to cytoplasmic TDP-43 accumulation. In summary, UBQLN2 overexpression or mutation exacerbated TDP-43 pathology in vitro , a potent inflammatory mediator [P497H;TDP-43G348C mutations also exhibited increased levels of NF-κB in spinal cord as compared to TDP-43G348C mice [P497H transgene was only expressed in neurons. The increased inflammation markers could suggest that microglia were hyperactivated in reaction to UBQLN2/TDP-43 neuronal pathology. These results also suggested that UBQLN2 pathology may favorize neuroinflammation and the reduction of UBQLN2 cytoplasmic accumulation may be a potential treatment approach to reduce glial cells activation observed in ALS.The implication of UBQLN2 in inflammation has emerged in the last two to 3 years. The overexpression of hUBQLN2mediator . Neverthmediator . TDP-43 mediator , 48, 58.48C mice . The douStress granules (SGs) are cytoplasmic inclusions of proteins and maintenance mRNAs with the aim of promoting stress response proteins translation in stressful situations, such as oxidative stress, osmotic stress, infection, ER stress and inhibition of the proteasome . Under sStress granules contain various proteins, including fALS related proteins, TDP-43, FUS, hnRNPA1 and 2. A disturbance in stress granules homeostasis can lead to disruption of RNA processing  L619A in neurons could prevent UBQLN2 self-assembly and could inhibit LLPS of UBQLN2 [L619A induced a 50% increase in neuronal death compared to WT. Collectively, those results suggest that the UBA domain promotes self-assembly of the protein leading to an increase in UBQLN2 aggregation with toxic consequences for neurons.Ubiquitin is also a common component of SGs , 33. Thef UBQLN2 . Also, eThe LLPS capacity of a protein is based on its multivalent interactions in “sticker” regions which are separated by “spacers” . RecentlP497H under the control of the hUBQLN2 promoter [P497H was comparable to the levels of endogenous UBQLN2WT. Another group used a transgenic hUBQLN2P497H rat model with TRE-doxycycline system [P506T mice were generated and showed cognitive impairment but no motor deficits [Animal models are essential to study in vivo pathological mechanisms as well as to test experimental therapeutic avenues, and numerous animal models were generated for ALS research (recently reviewed in ). Severapromoter . The anie system . Their mdeficits . The knoP497S or hUBQLN2P506T mutations exhibited motor neurons loss and cognitive impairments [P497S mice and 40% of hUBQLN2P506T mice developed hindlimb paralysis. Interestingly, TDP-43 mislocalization could be noted in neurons. Transgenic mice overexpressing hUBQLN2WT also showed loss of upper motor neurons as well as hippocampal neurons. Nevertheless, the effects were less important than those observed in the mutant transgenic mice. Knockout models were also generated, but phenotypes were highly divergent between species (Table Drosophila melanogaster (DM) created a severe motor phenotype with TDP-43/Ubiquitin/UBQLN2 aggregation [Transgenic mice bearing Thy1.2-driven hUBQLN2airments . The levregation . The cauregation . ConsequP497H and hTDP-43G348C mutations using a NFH promoter with low levels of expression [P497animal models are summarized in Table Lastly, our team generated the first double transgenic mouse model harboring both hUBQLN2pression . The simCollectively, these multiple reports suggest that UBQLN2 is acting on various ALS pathological mechanisms and is not only causing an UPS dysfunction as previously recognized. It is now documented that UBQLN2 is acting on stress granules formation, on TDP-43 mislocalization into cytoplasmic inclusions, on cellular clearance pathways and neuroinflammation. Thus far, the pathological role of mutated UBQLN2 is more defined, but many questions remain on the exact role of the WT UBQLN2 in sporadic cases of ALS. Nevertheless, the tight control in the levels of UBQLN2 appears to be a primary factor for its role in the clearance pathway and stress granules formation. A small disturbance in its levels could promote oligomerization and trigger dysfunction in LLPS formation, inducing aggregates formation and causing toxicity in neurons. To our knowledge, the levels of UBQLN2 in tissues from sporadic ALS cases without UBQLN2 mutation have never been evaluated nor neuropathological studied and this question should be addressed. Indeed, the WT UBQLN2 form also appears to have a toxic role when overexpressed , 49. It


By cooperatively tuning the input power on the IDTs, we can manipulate the propagation direction of Iae such that the measured Iae can be deliberately controlled to be positive, negative, or even zero. We define the zero-crossing Iae as Iae can be switched with a ratio We implement a logic switch by using a graphene acoustoelectric transducer at room temperature. We operate two pairs of inter-digital transducers (IDTs) to launch surface acoustic waves (SAWs) on a LiNbO Recent developments in graphene field-effect transistors (GFETs) suggest that graphene holds great promise in radio frequency (RF) applications5. For digital electronics, adopting new materials as a successor to Si must perform excellent switching capabilities with a low off-state dissipation power and a high on/off current ratio7. Nevertheless, graphene shows a serious hurdle for its applications in logic circuits2, because the pristine graphene does not possess an energy bandgap1. As a result, GFET cannot be turned off efficiently, leading to a low on/off current ratio typically less than 102. Subsequently, the research efforts are geared toward two different directions: engineering graphene material to open a bandgap9, or exploiting layered 2D semiconductors with a naturally occurring bandgap, e.g. transition metal dichalcogenides (TMDs) and black phosphorus (BP)7.Graphene –a two-dimensional (2D) sheet of carbon atoms arranged in a honeycomb lattice– exhibits various unique properties beneficial for post-silicon electronicsIae. We will show that Iae induced by dual SAWs can be modulated by discretizing RF signals. In this regard, a graphene acoustoelectric transducer(GAET) can function as a logic switch. The switching performance is demonstrated by the successful generation and detection of the digital text carried by Iae with a switching rate up to few tens kHz.In this work, we report a different approach to implementing graphene for logic devices by utilizing acoustoelectric effects. Here graphene is used as a channel material to convert surface acoustic wave (SAW) into acoustoelectric current 10. SAW can be induced by distributed comb-like metallic structures, such as interdigital transducers (IDTs), deposited on the surface of the piezoelectric substrate. Triggered by the piezoelectric effect, the RF input signal at the transmitting IDT stimulates the SAW. For a typical SAW device, a second IDT is employed, served as a signal processing unit and a transducer, to convert the acoustic waves back into a RF signal. Nowadays, the SAW devices have been widely used in various RF signal processing techniques for telecommunications and sensors12.A surface acoustic wave is an acoustic wave traveling along the surface of the piezoelectric materials, with its displacement amplitude exponentially decaying into the material so that it is roughly confined within one wavelength beneath the surfacevs and the SAW attenuation factor Γ. For example, SAWs can interact with two-dimensional electron gas (2DEG) placed nearby and the corresponding changes in both vs and Γ have been used to probe the distinct electronic states of 2DEG16. In addition, the interaction between the SAW and the charge carriers of 2DEG can also induce a macroscopic direct current, acoustic current Iae, which is known as the acoustoelectric effect.The propagation of SAW is sensitively influenced by the local changes of the host medium, which causes the variations of the SAW velocity 25. Owning to the linear energy dispersion and gapless nature of graphene, electrons in graphene can absorb sound waves over a wide frequency range26 and in theory Γ is strikingly diminished as the Fermi level EF is tuned across the charge neutral point (CNP)20. However, graphene does not possess piezoelectricity because of its central symmetric lattice structure, unlike to GaAs-2DEG. The major obstacles in studying and utilizing the acoustoelectric effects of graphene lie on how to generate SAW and maintain its propagation under the control of EF. Early experiments reveal that SAW of graphene can be excited by placing graphene either on or in close contact to a substrate with high piezoelectricity, e.g. LiNbO3 substrate18. By incorporating ion liquid gate and IDTs, EF of graphene can be tuned across CNP, and Iae exhibits an ambipolar effect- the sign of Iae is reversed associated with the change of charge carriers from n- to p-type23. Furthermore, Γ of grapehene is extremely weak, approximately ~0.4 to 6.8 m−1 depending on the carrier density ns, which is three orders of magnitude smaller than that of GaAs 2DEG systems23. These fascinating properties make graphene an ideal material for various acoustoelectric devices, ranging from acoustic tweezers, branch switch, flip-chip devices etc.29.The acoustoelectric properties of graphene have been extensively studied30. The acoustic current in a closed-circuit measurement (or voltage in an open-circuit measurement) is induced by a loss of wave energy associated with a proportional loss of SAW momentum, which is analogous to a force applying on the absorber (the charge carriers of graphene in this study). For a 2D system, we can assume that the acoustic current density jae is proportional to Γ with the coefficient Λ and flows along the direction of SAW propagation31:i(=x or y) is the spatial index, x(y)-direction. It has been known that Λ can be described by16σ is the DC conductivity of graphene. We assume that both Γ and Λ are spatially uniform because graphene is an isotropic material. Note that one may need to treat Γ and Λ in a tensor form when the SAW propagates on an anisotropic substrate or the carriers are in the presence of an external magnetic field31.A theoretical model to describe acoustodynamic effects in semiconductors was developed by G. Weinreich23. Consequently, one can define a true zero-current state or an “off”-state at CNP although the channel is not completely closed. On the other hand, a fair on/off ratio ~20 has been reported by defining an on/off state away from CNP in our earlier study23. In principle, if the off-state is set exactly at CNP, one can get a much higher ratio (>107). There are competitive advantages to utilize GAET for logic devices. For commercial SAW filters used in the RF front-end, the device requires sufficient high-power durability. In general the SAW device can withstand power levels ≥30 dBm, which is high enough to generate Iae with a decent S/N ratio. Moreover, no quiescence power source is needed because GAET is activated by the energy of the RF input signals received by the IDT transceiver. Nevertheless, if one operated GAET like GFET that the RF signal is sent through the gate electrode4, the modulation speed of GAET would be too slow for practical applications23. It is mainly because the ionic liquid is adopted for the gate electrode in the present GAET design23. Note that the gate electrode made of conducting materials will severely damp the propagation of SAWs. This is the key bottleneck for GAET to be used for the logic devices.Because of the ambipolar effect of graphene, 3 piezoelectric substrate in a nearly orthogonal arrangement. Each IDT comprises two sets of interleaved fingers and the acoustic current density induced by IDT1 and IDT2 is indicated as jIDT1 and jIDT2, respectively. Two current sensing leads are placed along the positive x-direction , IDT1 functions as the source contact to inject the channel current and IDT2 serves as a gate electrode to turn “on/off” the device. We will demonstrate below that by digitizing RF signal applied on IDT2 or IDT1, the GAET can perform as a logic switch.Our design concept is illustrated in Fig. 3 substrate, graphene, four electrodes on graphene labeled as leads 1 to 4, and a micro-bead of an ion-gel coated on graphene33, a gate electrode of the polymer electrolyte for applying gate voltage Vg. The two sets of opposite ITDs, IDT1-IDT3 and IDT2-IDT4, are separated with a distance LT = 1.4 mm and backed by metallic strips to damp reflected waves. In this study, we only operate IDT1 and IDT2, and conduct their counterpart IDT3 and IDT4 as a passive receiver for checking the SAW properties. Each IDT comprises two sets of interleaved fingers with NIDT = 25 finger pairs made by 5 μm wide electrodes with 8/70 nm of Cr/Au. The acoustic aperture WT ~ 600 μm, the overlap between electrodes, is aligned between two opposite IDTs along the [011] direction of the z-cut single-crystal LiNbO3 substrate. The optical micrograph of device can be found in the Fig. λSAW determined by the pitch of the IDT electrode is 20 μm and the SAW velocity vs is approximately 3795 m/s34. The central resonance frequency f = vs/λSAW is estimated to be 190 MHz. Figure S21 as a function of frequency measured by a network analyzer (RS ZVA24). It exhibits a peak at approximately central frequency fc = 191 and 187 MHz for IDT1 → IDT3 and IDT2 → IDT4 respectively, which fairly agrees with the designed value.Figure 37. Graphene is gently placed between two IDTs, and tailored to a rectangular shape of length LG = 600 μm and of width WG = 400 μm. Caution must be taken to ensure that graphene residues will not short the Au electrodes of IDTs. Four electrodes deposited along the side border of graphene are used for resistance and acoustoelectric current measurements. They are made of an Au/Cr bilayer with 8/70 nm in thickness and 20 μm in width, among which 450 μm in length for leads 1 and 4, and 250 μm in length for leads 2 and 3  of graphene as a function of Vg, where V24 is the voltage measured across the lead 2 and 4, and I13 is the current passing through the lead 1 to 3. The R versus Vg trace of graphene reaches a maximum with resistance ~2.5 kΩ at CNP, where Vg ≡ VCNP = −0.549 V. We have measured five devices with the same structures and got consistent results. Data presented below are mainly obtained from one of the devices.Graphene is prepared by the chemical vapor deposition. We refer the readers to our previous publications for the details of graphene growth, characterization, and transfer proceduressee Fig. . FinallyIae measurement. Here we use IDT1 and IDT2 to generate the SAWs and take leads 4 and 1 to sense Iae, while keep the rest IDTs and electrodes inactive and open. We modulate RF signal at a frequency of 10 kHz and employ standard lock-in technique to measure Iae. Note that the propagation direction of the Iae measured by leads 4 and 1 aligns with that of the SAWs induced by IDT1. Figure Iae as a function of bias voltage Vg with various RF powers PIDT1 applied on IDT1 at the central frequency of 191 MHz, while keeping IDT2 inactive. With the present arrangement of the current leads displayed in Fig. Iae is positive, negative, or zero as graphene is biased in the hole-rich regime, electron-rich regime, or at Vg ~ VCNP. The gate bias dependence of Iae manifests the unique Dirac dispersion relation of graphene23. We note that an on/off ratio of Iae up to 107 can be achieved, for example, if one defines the on-state at Vg − VCNP = 0.5 V and the off-state at Vg = VCNP for PIDT1 = 10 dBm. Extracted from Fig. 18, as indicated in Eq.  and a duty cycle D(=0.2). Oscilloscope traces of the applied RF-signal on IDT2 is shown in Fig. Iae taken from the oscilloscope at various PIDT2. As PIDT2 is within active time of the pulse, Iae exhibits a dip feature due to the cancellation by negative Iae. As PIDT2 increases up to 6 dBm, Iae is nearly vanished. Here the GAET functions as an active-High logic switch that processes information as either a “1” or a “0”, depending on whether the switch is off − getting finite acoustoelectric current Iae− or on − Iae measured zero. We estimate the on/off ratio of Iae is approximately 104 based on the noise level of the on and off states.Next we will show that by dynamically controlling the on/off state of Iae, we note that the maximum switching rate - the key parameter to limit sampling rate in digital communications - is determined by the transition time of Iae in response to the modulated RF pulse. We can switch Iae simply by modulating RF signal applied on a single IDT. Unlike the dual-SAW scheme discussed above, one can view such operation as an active-Low logic switch. In terms of GFET, graphene provides a nature 2D conducting channel such that a digital on/off state can be simply achieved by modulating the source-drain bias without applying a gate voltage, if the signal gain is not a concern. On the other hand, a RF signal can be directly converted to an electric signal in GAET. In this regard, GAET has an advantage over GFET as a logic switch. Figure Iae by operating IDT1 alone. Figure Iae pulse waveform generated by a square wave-modulated PIDT1(=17 dBm). The on-time ton is set about 20 μs. Based on the 90% and 10% threshold levels of the pulse amplitude, we determine the Rise tR and Fall tF time to be about ~6 μs. Figure Iae with different modulation frequencies. For comparison, we normalize Iae to its quiescence value I0(=1.6 μA), and time to the modulated period Tm. As displayed in Fig. Tm up to 20 μs, corresponding to dynamic switch rate of 50 kHz. That is to say, the peak value of the Iae waveform with a pulse width ~4 μs is within 90% of the full amplitude. The propagation delay time tp - a parameter to evaluate the jitter effects- is about 0.6 μs. We find To characterize the response time of switching CIDT ~ 6.25 pF and the circuit input impedance around 448 Ω, giving rise to the RC time constant around 2.8 ns, which is much shorter than the tR and tF measured. Therefore, we argue that the intrinsic RC delay is likely due to the impedance mismatch. We conceive the switching rate can be immediately raised up by shrinking the channel width of graphene. To optimize impedance-matching one may employ tapered IDTs, functioning as an impedance-transformer40, which impedance match to 50 Ohm transmission line at one end and to the characteristic impedance of the tailored graphene and the leads at other end. To this end, one also needs to characterize the output impedance of GAET and match it with that of the transmission line extended to the measurement ports.Data shown in Figs 41 and would be interesting subjects for future studies.The dynamics of acoustoelectric effects of emerging post-graphene 2D materials – e.g. transition metal dichalcogenides (TMDs) and black phosphorus (BP) are much less exploredvs and the channel width. There are several approaches to increasing the switching rate. One may try to fabricate the device on substrates with a relatively large electromechanical coupling coefficient, e.g. 42° Y-X LiTaO3 or 64° Y-X LiTaO3 substrate, which have been widely applied to the SAW devices for mobile communications. However, the tradeoff is that larger leaky wave may yield a lower Iae. In principle, a high-slew rate of Iae can be obtained from a wide-band SAW device, which can be implemented by an apodized IDT design or simply reducing the number of fingers in the IDT. A narrower channel width may give a shorter response time, but it in turn reduces Iae or requires a larger PIDT. This drawback makes GAET unsuitable for latch operation. Regarding the operation scheme, a single IDT is sufficient for the active-Low switch. Using collinear dual IDTs such as IDT1 and IDT3 (or IDT2 and IDT4), one can apply lower and balanced PIDT for the active-High switch. Nevertheless, evident interference due to reflected waves should be taken into account. For the dual-SAW operation, two SAWs can be excited at different frequencies. However, it makes SAW attenuation become more pronounced at a higher frequency. In addition, by properly utilizing four leads and IDTs, we can directly measure Iae to make GAET act as an acoustoelectric branch switch28. We note that recent studies reveal several intriguing interface elastic properties of van der Waals materials44 and may offer a mean to speed up the switch rate of GAET by engineering the interfacial acoustoelectric properties. Maybe the switch rate is slow (audio frequencies) and the design is a little complex, we think that the GAET opens a route for developing graphene-based logic switch. In this work, we only demonstrate the feasibility of GAET as a logic switch and leave aforementioned issues for future studies.Finally, we wish to make few comments on future development of GAET logic devices. The ultimate response time of the GAET switch is limited by the SAW velocity 4 at room temperature. By manipulating the propagation direction of Iae, the measured values of Iae can be fine tuned to zero - an ideal off state for a logic switch. We demonstrate the dynamic switch rate of Iae can be up to 50 kHz by modulating the amplitude of the input RF-signal applied on IDTs. By deliberately controlling the digitized RF-power applied on a pair of crossed-IDT or a single IDT, the output Iae can be either active-High or active-Low, respectively. The digital modulation rate can achieve ~10 KB/s. The performance of the GAET is suitable for processing digital audio signals. Even the switch rate is slow, our work provides a means to integrate the SAW device and the acoustoelectric effects for future development of graphene-based logic devices.In conclusion, we present an accessible operation scheme of GAET for a logic switch with a moderate on/off rate of ~10


When calibrated, the weather station results were well within ±3% RH. During the winter 2015/2016 weather stations were placed in the living room in eight wooden buildings. A period of significantly increased fire risk was identified in January 2016. The results from the outdoor sensors compared favorably with the readings from a local meteorological station, and showed some interesting details, such as higher ambient relative humidity for a home close to a large and comparably warmer sea surface. It was also revealed that a forecast predicting low humidity content gave results close to the observed outdoor weather station data, at least for the first 48 h forecast.During January 2014, Norway experienced unusually cold and dry weather conditions leading to very low indoor relative humidity (RH) in inhabited (heated) wooden homes. The resulting dry wood played an important role in the two most severe accidental fires in Norway recorded since 1923. The present work describes testing of low cost consumer grade weather stations for recording temperature and relative humidity as a proxy for dry wood structural fire risk assessment. Calibration of the weather stations relative humidity (RH) sensors was done in an atmosphere stabilized by water saturated LiCl, MgCl Fire is a major cause of accidental injury and results in over 300,000 deaths annually ,2,3. TheIn these areas, as well as in the majority of the country, wooden homes dominate the building style due to the abundance of wood as a construction material. One of the precursors for the severe fires was ambient subzero temperature air of low relative humidity for a few weeks before these fires. This resulted in dry outdoor combustible materials and extremely dry indoor wood in inhabited (heated) wooden structures ,6. A cor2 capacitive high speed sensor with 35 s response time [However, such an application reveals the need for low cost sensors for recording ambient and indoor temperature and RH. Recently, a fast TiOnse time  and an ense time  have beeThe objective of the present work is to report on the experience of testing consumer grade weather stations for relative humidity measurements as a proxy for the winter fire risk in wooden constructions . Section−1) is the molecular mass of water and −1 mol−1) is the molar gas constant.The relative humidity in air is dependent on the absolute water vapor content as well as the air temperature. The saturation vapor pressure of water is a near exponential function of temperature and may be described by :(1)Psat=Cooling this air so that it theoretically contains more than 100% relative humidity, the surplus water condenses at small dust particles, etc. to make fog. It is a quite normal phenomenon to observe fog in a cold night and morning dew on the grass after a cold night. It should be noted that cooling the air results in a volume reduction, which also increases the water vapor concentration. The focus of the present paper is, however, the indoor heating of the air in cold climates. Heating the cold ambient air (at constant pressure) to the higher indoor temperature, www.eklima.no. In the Haugesund area, the meteorological station at the Haugesund airport is well equipped and professionally maintained for recording data relevant for the present study. Based on retrieved temperature and relative humidity data, the historic values for the ambient water vapor concentration may be calculated by using Equations (1) and (2).In Norway, historic weather data at 1 h frequency may be retrieved for free from a number of meteorological stations by the application provided at 3, was indeed very low compared to the rest of the year. If such dips are short, i.e., less than a day, a wooden home does not have time to adjust to the new dry conditions. This is partly due to the finite air exchange rate as well as slow diffusion processes of water in wood and humidity being released from the drying wood to the indoor air. However, if the dry conditions persist for some days or weeks, the wood dries out and the fire risk related to the drier wood increases [The calculated outdoor water concentration based on data from the meteorological station at the Haugesund airport for the period July 2015 through June 2016 is shown in Indoors, there are usually also sources of moisture supply present, e.g., humans, pets and pot plants. Hygroscopic materials, e.g., wood and other cellulose based materials such as upholstery, clothes, carpets, etc. may also release humidity when the indoor climate gets drier. If the indoor air gets more humid, these materials absorb humidity from the air. These materials may also show hysteresis when changing from the mode of adsorption to desorption and vice versa . Since sThe best way to measure the water content of indoor combustible hygroscopic materials would probably be to record the mass change of selected objects. This is not easy to do with a good precision. Measuring the indoor relative humidity does, however, provide a good alternative for obtaining information about the indoor climate contribution to structural wood fire risk. Recording the humidity levels, and keeping track of especially low indoor humidity levels, gives a good indication about the structural wood fire risk development. Warnings may be considered based on weather forecasts so that focus can be shifted to monitoring indoor relative humidity development and fire risk evaluations when needed.The first idea was to record the mass of representative indoor wooden objects in order to consider their combustibility. This could be e.g., wooden plates of different thicknesses placed on separate balances, and then record the mass at a convenient frequency. The recorded data could then be transferred via the internet to researchers analyzing the data. It was, however, realized that it would be quite challenging to e.g., have sufficiently stable balances. A number of precision balances would also have been very costly. During discussions with an automation engineer , it was A very low cost, and not web based, weather station was therefore purchased and evaluated against a precision psychrometer . The psychrometer calibration data are presented in The weather station and the precision psychrometer were placed in a 17 L transparent plastic box with a lid. Operation of the precision psychrometer without disturbing the internal atmosphere was provided for by a 2.5 mm plastic rod through a sealed hole. The internal relative humidity was adjusted to get varying relative humidity (RH) in the plastic box. To get a low RH, the box was taken outdoor and filled with low temperature air which became dry when adjusted to indoor conditions at 22 °C. To get high RH, droplets of water were allowed to evaporate in the plastic box, inspired by a previous study . Compari2-concentration. The outdoor unit sends the recorded data to the indoor unit, which automatically forwards the recorded indoor and outdoor data to the Netatmo servers for instant or later user access and data retrieval. The complete weather station, including indoor and outdoor units, is shown in Eight Netatmo weather stations , as sho® SP10302D Pt100 Temperature Reference Instrument, Ahlborn Mess- und Regelungstechnik GmbH, Holzkirchen, Germany) for the temperature interval relevant for the indoor and outdoor sensors. Only minor adjustments were needed to achieve temperature recordings well within ±0.3 °C for the temperature region of interest in the present work.The producer of the Netatmo units states that the equipment can read temperatures within ±0.5 °C and relative humidity (RH) within ±3% RH. The PC interface allows for a temperature calibration by the user. In the present work it was decided to calibrate the temperature output of the units against a precision temperature instrument , the sensors to be calibrated were placed, four at a time, in a transparent plastic box of dimensions 50 cm by 40 cm by 30 cm height. A lid was applied to the box and the USB power cables for the indoor units were let out through the back wall. The hole was sealed to prevent air exchange with the surroundings.2 and NaCl, all salts pro analysis quality, were used to respectively achieve RH values of 11.3 ± 0.31% RH, 33.07 ± 0.18% RH and 75.47 ± 0.14% RH [Water saturated inorganic salt solutions, i.e., LiCl, MgClSaturated water salt solutions were placed at four 8 cm diameter plastic plates. Two of these plates were placed on the plastic box floor level and two plates were placed at about 10 cm elevation. This was done in order to quickly obtain equilibrium RH conditions after any handling of salts or weather station units in the plastic box. Air circulation within the box was achieved using a USB fan, which also speeded up the process of establishing a constant RH level. When changing to NaCl solutions, water droplets were added to an aluminum plate (for good heat transfer to the droplets ) placed The previously mentioned battery powered precision psychrometer was also placed inside the plastic container for instant recordings of temperature and relative humidity. As with the preliminary testing, a plastic rod was arranged for operating the psychrometer. The psychrometer readings were observed through the transparent plastic box lid. The box was loosely covered by sheets of aluminum foil to reflect light radiation to prevent any plastic box “greenhouse effects”. The system was then left to achieve equilibrium conditions governed by the applied saturated salt solution. The psychrometer readings were used to confirm that a constant RH level was achieved inside the plastic box. Another two hours were allowed for ensuring constant weather station readings. The temperature and RH for each weather station unit were then obtained by the web interface together with the psychrometer readings. The procedure was repeated for all the relative humidity sensors (four at a time) and for all the saturated inorganic salt solutions. Separate testing was done to establish the sensor response times when exposed to a sudden change in relative humidity.The sensors generally gave very erroneous results compared to the equilibrium conditions ensured by the water saturated salt solutions. Several of the detectors were up to, and even more than, 10% RH wrong either at low (11.3% RH) or high relative humidity (75.5% RH). The sensor outputs were, however, very linear with respect to the real relative humidity. This indicated that a linear equation could be used for converting the recorded result to correct relative humidity values. A linear correction curve, similar to the one shown in Equation (4), was therefore fit to the RH data recorded for the indoor units (at 22 °C) and outdoor units (at 2–5 °C). The results are presented in It should be noted that when extrapolating the linear curves based on the correction coefficients presented in A number of tests were also done in the range 40% to 60% RH, where the precision psychrometer, corrected by its calibration curve, was used as the reference. No deviation from linearity was detected for any of the weather station units. To the surprise of the author, when testing an outdoor unit at indoor temperatures, the results were still within 2% RH. The calibration of the outdoor sensors at 2 °C to 5 °C was therefore taken as valid also for temperatures below 0 °C.The sensor response time was also tested. This was done by setting the weather station number 1 indoor and outdoor units in an atmosphere stabilized by water saturated NaCl solution for 6 h. Then, the sensors were suddenly taken out and placed in the measurement container where the atmosphere was in equilibrium with water saturated LiCl solution. It should, however be mentioned that this does not represent a clean cut step function from humid (75% RH) atmosphere to dry atmosphere (11% RH) since during the sensor handling, the air in the plastic box chamber was partly diluted by indoor air of about 35% RH. Nevertheless, compared by the recording time of several hours, given the experimental setup this was as close to a step function as practicably possible. The results are shown in The most interesting period during the winter 2015/2016 was in January 2016. Early in January, the wind direction changed and the wind came from the mountain plains east of Haugesund, Norway. This led to adiabatically heated ambient air and lower than normal outdoor relative humidity. The recorded temperature and relative humidity from the local meteorological station at Haugesund airport for January 2016 is presented in The adiabatically heated dry subzero temperature air is easily identified in the first days of January. However, as both the temperature and the relative humidity vary, it is easier to interpret the data when studying the actual ambient air water concentration.The water vapor concentration calculated by Equations (1) and (2), based on the Netatmo weather stations outdoor temperature and relative humidity at two homes, as well as the Haugesund airport, is shown in The indoor relative humidity recorded at the homes presented in Especially during winter time, with large outdoor to indoor temperature differences, there will be, and should be, sufficient air changes in a building to prevent rot formation in colder parts of the thermal insulation, etc. Due to the chimney effect, the indoor air will gradually be exchanged with ambient air. The ambient air entrained into the home is then heated to the indoor temperature. Wind pressure also increases the air change rate. Indoors, there will usually be some moisture gain in inhabited buildings as a result of persons and pets breathing or perspiring, dishwashing, pot plants and wood sorption processes. The wood sorption processes may go both ways, i.e., represent a positive or negative humidity gain. This also holds for other hygroscopic materials, such as furniture upholstery, pot plant soil, etc. However, assuming that there is no moisture gain, and that the ambient air is heated to the indoor conditions, a theoretical indoor relative humidity may be calculated based on the ambient conditions. The indoor weather station unit supplies the indoor temperature needed for the calculations while the ambient temperature and RH may be taken from the outdoor unit or from the local meteorological station. The best way is, however, to record the indoor RH directly, as seen in It has recently been demonstrated in ¼ scale wooden compartments that the time for reaching flashover, i.e., the sudden transition from a gradually increasing fire to a fully engulfing compartment fire, was very dependent on the wood fuel moisture content (FMC). At equilibrium conditions at 20% RH, the wood equilibrium moisture content (EMC) is about 4.5% . This dr31 December 2015, at 23:00, a weather forecast API from the Norwegian Meteorological Institute was run to retrieve the forecasts for the following 10 days period. For the first 48 h, the data are detailed to each hour. Then, the data is gradually presented with less frequency. Five days later, the API was run again supplying data for the following 10 days period. The calculated ambient air water concentration based on the forecasted data is presented in The only outdoor weather station sensor results that differed significantly from those obtained by the other sensors were from the home in Skudeneshavn, see While the other homes studied were close to the town of Haugesund, the one standing out was in Skudeneshavn, i.e., on the southern tip of the Karmøy island. As can be seen in middle left part of The major fjord Boknafjorden, between Haugesund and Stavanger, has open water the whole year while the inner parts of the attached smaller fjords are covered by ice in January. In wind from east, the air passing over the open sea has a long fetch and therefore a long contact time for picking up humidity from the open water surface before arriving at Skudeneshavn. This is especially the case during winter time when the sea has a temperature well above the ambient air temperature, and when the ambient air from the east is dry due to adiabatically heating. The reason for the unit in Skudeneshavn showing the highest RH values is therefore most likely due to the dry wind from the east picking up humidity on this 25+ km long fetch during the first 12 days of January 2016.The smaller fjord arms and most upwind lakes east of Haugesund were covered by ice during January 2016. The other weather stations close to the town of Haugesund, as seen in the upper left part of The purpose of this study was to test whether consumer grade weather stations for recording temperature and relative humidity could be used for indicating structural wooden home fire risk during winter time. The present work demonstrated that calibration was needed for the relative humidity sensor results to be within ±3% RH. The correction curves were simple linear equations. Without calibration, the readings were quite erroneous, i.e., the errors in relative humidity were up to more than 10% RH.2 and NaCl pa salt saturated water solutions. Internal air circulation was ensured by an USB powered fan. This represented a low cost setup for the relative humidity calibration. An available high precision temperature unit was used to calibrate the weather station by the built-in user based temperature calibration app. The recorded temperatures were well within 0.3 °C when tested after this user calibration. Using the high precision temperature unit may, however, be seen as an overkill since a normal type K thermocouples would probably have been sufficiently accurate for the temperatures of interest in present work.The calibration was done in a transparent plastic box where the air was conditioned by LiCl, MgClThe precision psychrometer was used to check that the inorganic salts did indeed produce the correct relative humidity. This was also in principle not necessary, as the saturated salt solutions would ensure equilibrium conditions at a known relative humidity level . The psyThe selected weather stations had a built-in web application which allowed for remote retrieval of the data recorded at 5 min intervals. The recorded data was downloaded in the form of spread sheets. It was therefore easy to apply the proper relative humidity correction equation for each indoor and outdoor detector unit.The tested weather stations had a resolution of 1% RH. This made it difficult to detect any weather station hysteresis regarding the relative humidity recordings. The weather station response was slow, i.e., a time period of about 2 h was therefore allowed to ensure correct results. The sensors can therefore not be used in situations where a fast response time is required. When the ambient water vapor concentration decreased considerably in January 2016, it was seen that a typical home needed a few days to equilibrate to the new and drier condition, as seen in −1 allows for detecting quite rapid changes in the indoor relative humidity, e.g., as a result of airing. However, given the slow response of the units, i.e., close to two hours, any such potential sudden changes in indoor relative humidity could not be discovered. Peaks due to cooking activities were, however, discovered. Due to the slow response, the peak values were probably not reliable as the sensor recordings were probably lagging behind such rather rapid changes in indoor relative humidity.In principle, the measurement frequency of 12 hThe weather station response time must be compared to the building response time, i.e., a few days. The conservative two hours weather station response time was an order of magnitude faster than the building indoor response time. This was taken as a proof of the sensor response time being sufficiently fast for the purpose of the present work, i.e., demonstrating that consumer grade weather stations can be used to monitor the dry indoor climate fire risk levels of wooden buildings.It was clearly seen that the indoor sensor had a longer response time compared to the outdoor sensor when tested under the same conditions, i.e., 2 h versus 1 h to 95% of a step change in relative humidity. This may simply be due to the larger internal air volume of the indoor sensors. For future measurement campaigns it is recommended to try to optimize the response time by e.g., allowing for better air convection and thereby faster response.During the calibration, the outdoor relative humidity sensors were, as always, powered by internal batteries. The indoor relative humidity sensors were, however, supplied USB power from transformers outside the box, i.e., the current wires had to be led through the wall. This was also the case for the USB fan power cable. Instead of relying on external power, using USB power banks for supplying these units would take away the clutter of wires through the wall. Utilizing USB power banks as the power supply source are therefore recommended for future calibration and characterization studies.The accuracy of the recordings, after the calibration curves were established and applied to the retrieved data, was found to be within ±3% RH. This was also the case for three weather stations that were tested after four months of operation during December 2015 through March 2016. This accuracy was still well within the goal for the present study. Advanced relative humidity sensors may display results far better than this, and with response times of 35 s  or even 2 may be available at hardware stores as an ice melting powder, at least in cold climate areas. MgCl2, which in contrast to LiCl, is not poisonous, may therefore represent a safe second calibration point.An interesting research possibility regarding web based weather stations may be related to the geography teaching for primary and secondary schools. The influence of the sea and the mountains on the local relative humidity, as clearly observed in the present work, may be demonstrated in the class room. The changes in relative humidity when the temperature is changing may also be worthwhile presenting to school children. The calibration procedure for teaching purposes may be done without using pro analysis quality salts. Commercial grade NaCl is available in any grocery store, though maybe producing equilibrium conditions 1% RH off due to a minor content of other salts. Consumer grade MgClAnother research possibility may be to use private weather stations for suppling data for increased wildfire risk awareness, especially during summer time , but alsThe main conclusion of the present study is that consumer grade weather stations, at least the type tested in the present study, have been shown to quite accurately measure the indoor and outdoor relative humidity when properly calibrated. This indicates that such weather stations could become central in a future attempt to develop a cold climate structural fire risk warning system.


Graphene-based materials are highly interesting in virtue of their excellent chemical, physical and mechanical properties that make them extremely useful as privileged materials in different industrial applications. Sonochemical methods allow the production of low-defect graphene materials, which are preferred for certain uses. Graphene nanosheets (GNS) have been prepared by exfoliation of a commercial micrographite (MG) using an ultrasound probe. Both materials were characterized by common techniques such as X-ray diffraction (XRD), Transmission Electronic Microscopy (TEM), Raman spectroscopy and X-ray photoelectron spectroscopy (XPS). All of them revealed the formation of exfoliated graphene nanosheets with similar surface characteristics to the pristine graphite but with a decreased crystallite size and number of layers. An exhaustive study of the particle size distribution was carried out by different analytical techniques such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA) and asymmetric flow field flow fractionation (AF4). The results provided by these techniques have been compared. NTA and AF4 gave higher resolution than DLS. AF4 has shown to be a precise analytical technique for the separation of GNS of different sizes. Particle size analysis is a key element because many properties of nanomaterials are size dependent . This paIt is well known that graphene has excellent properties that provide a multitude of technological applications in different fields. It is formed by a pattern of hexagonal rings of carbon atoms constituting a huge flat molecule . Some ofo-dichlorobenzene (ODBC) [Many methods for obtaining graphene have been reported. Graphene layers can be obtained by liquid-phase exfoliation of commercial graphite in different organic solvents using the ultrasonic technique. This method requires special experimental conditions at which very high pressures and temperatures are reached in very short periods of time . The cole (ODBC) ,12. By me (ODBC) . By carre (ODBC) . On the e (ODBC) . For inse (ODBC) . For thie (ODBC) .o-Dichlorobenzene is an organic solvent that has excellent properties to be used as liquid phase for the process of graphite exfoliation. It is commonly applied as a reaction medium in fullerene chemistry and it is well known that it forms very stable dispersions due to its very efficient interactions with graphene via π-π stacking [stacking . On the stacking . Graphene-based materials have been characterized by a variety of different techniques, such as X-ray diffraction, transmission electron microscopy and Raman spectroscopy, among others ,20. Alth6 to 109 particles/mL, aggregation and fluorescence [NTA is a relatively novel technique for the study of particle size distribution. Like DLS, NTA uses light scattering and the Brownian motion of liquid suspensions of particles to obtain the size distribution in a sample. This technique allows real-time visualization and recording of nanoparticles during the measurement by combining a charge-coupled device with laser light scattering microscopy . In thisrescence . The conrescence . Based orescence .2 nanoparticles [AF4 is one of the most promising techniques for the characterization of nanoparticles due to the high precision and wide separation range it provides, as well as the great variety of nanomaterials that can be analysed. Currently, this technique is in continuous progress and shows high versatility, because it has been used to characterize many types of nanomaterials such as nanodrugs, silica nanoparticles, nanocellulose and nanoplastics, among others ,29,30,31articles  and in carticles , renderiHerein, we report the synthesis of unfunctionalized, non-oxidized and isolated graphene nanosheets (GNS) applying the ultrasound-assisted method by liquid-phase exfoliation of a micrographite. The nanosheets obtained have been characterized using different techniques, particularly related to the study of particle size. Thus, besides XRD, TEM and Raman spectroscopy, three techniques, specifically oriented to the determination of particle size, i.e., DLS, NTA and AF4, were investigated. The proper choice of the parent micrographite has been essential because it has provided GNS particles measurable by all these techniques. A comparative study of the results obtained with the different characterization techniques is reported. Microcrystalline graphite powder (98%) was purchased from Nanostructured & Amorphous Materials Inc. . 1,2-dichlorobenzene , dichloromethane  and lanthanum hexaboride  were obtained from Sigma Aldrich .o-dichlorobenzene (ODCB) for 30 min. Subsequently, the resulting dispersion was subjected to treatment with a pulsed ultrasound probe for 2 h using an Ultrasonic Homogenizer 4710 Series from Cole Parmer Instrument Co.  . The power supplied by the equipment was 9.96 W, according to a calorimetric calibration method [g (4000 rpm) for 30 min to remove the non-exfoliated micrographite. The remaining suspension was again centrifuged at 12,096× g  to isolate the graphene nanosheets. They were washed three times with dichloromethane by successive redispersions and centrifugations to remove the ODCB. A Sorvall Super T-21 Tabletop Superspeed Centrifuge  was used to carry out all centrifugation processes. Dichloromethane was evaporated from the resulting graphene nanosheets dispersion at room temperature for 12 h and, finally, the powdered exfoliated GNS were dried under vacuum at 80 °C overnight.250 mg of microcrystalline graphite powder (MG) were stirred in 50 mL of n method   with a monochromatic CuKα radiation source. The scanning conditions for structural analysis were an angular range of 15°–80° (2θ), a 0.040° step size and 1.05 s per step. Additional measurements of X-ray diffraction were carried out using a Bruker D8 Advance. The scanning condition for this analysis were an angular range of 5°–40° (2θ), a 0.002° step size and 9 s per step.Transmission electron microscopy (TEM) images were recorded on a Jeol JEM-1400 transmission electron microscope  operated at an accelerating voltage of 120 kV. The instrument has a high-contrast objective-lens polepiece, enabling high-contrast TEM image observation at all magnifications and good image quality, as well as easy handling. In addition, it has an advanced vacuum system. The digital-camera configuration includes a CCD camera that enables focusing and image verification on the operation screen. The measurement CCD camera (MC), orthogonal to the alignment camera, has a resolution of 1344 × 1024 pixel (12 bits/pixel). The measurements have been made in a magnification range between 8000× and 150,000× with an instrument resolution of 0.38 nm between points.−1). Spectra were obtained by excitation with green laser light (532 nm) from 150 to 3500 cm−1. A total of 32 scans per spectrum were acquired to improve the signal-to-noise ratio.Raman spectra were acquired with a Renishaw Raman instrument  equipped with a Leica microscope furnished with various lenses, monochromators and filters in addition to a CCD camera. A silicon standard sample was used as reference for calibration (520 cmX-ray photoelectron spectroscopy (XPS) was performed through a SPECS mod. PHOIBOS 150 MCD spectrometer  using non-monochromatic Mg Kα radiation and a multichannel detector. All spectra were fitted to Gauss–Lorentz curves to better identify the different chemical environment of the elements in each material. The binding energy values were calibrated with the adventitious carbon C 1s signal (284.8 eV).Particle sizes were measured in a Zetasizer ZSP  at 25 °C based on laser Doppler velocimetry and dynamic light scattering (DLS) techniques. Previously, the suspension was homogenized using an ultrasonication probe for a period of 5 min. h) of the GNS particles, the hydrodynamic diameter (Dh) was calculated by using the Stokes–Einstein Equation (1):B is the Boltzmann constant, T the temperature in K degrees, t the translational diffusion coefficient.The refraction index values were set at 1.33 and 2.38 for the dispersant (deionized water) and the material (carbon), respectively. The analysis was carried out by triplicate and medium and standard deviation were calculated. To obtain the hydrodynamic radius , equipped with a sample chamber and a 488 nm laser.B is the Boltzmann constant and h in a medium of defined viscosity and temperature. All measurements were performed at room temperature.For the NTA measurements, 1 mg of GNS was suspended in 1 mL of water. Before the analysis, the solution was homogenized for 3 min in an ultrasonic bath. The samples were injected into the chamber with sterile syringes. Many individual particles are tracked during their Brownian motion by the software, which uses the Stokes–Einstein Equation (2) to provide sample information:An AF4 system  coupled online to a 21-angle MALS detector  and UV-Vis diode array detector  was used. The MALS detectors were calibrated using bovine serum albumin monomer and detectors at different angles were normalized with respect to a 90° detector measuring a sodium poly(styrenesulfonate) (PSS) standard. The AF4 channel was trapezoidal-shaped, 350 μm thick (defined by a spacer), 29.8 cm long (inlet to outlet) and had a maximum width of 2 cm. Particle recoveries over AF4 were tested with several commonly used membranes. The analytical parameters and characteristics of our AF4 system are presented in 6 was used as a pattern to determine the instrumental broadening). FWHM for GNS was higher than for MG  and graphene nanosheets (GNS) samples were examined by XRD. r MG see . The cryinstrumental is referred to LaB6 and equal to 0.130.The instrumental broadening effect on FWHM was subtracted out using Warren’s method on the assumption of a Gaussian peak :β2 = β2s002) due to the exfoliation of the MG [Liquid-phase exfoliation by ultrasound-assisted synthesis for obtaining graphene nanosheets (GNS) produced smaller crystallite sizes. Moreover, the thickness of the crystallites along c axis decreased because the full width at half-maximum (FWHM) of the peak (002) was higher than that of the starting micrographite (MG) ,43. Thusf the MG . In thisThe surface morphology of the original graphite and the synthesized graphene was examined by TEM images. Nanometric particles of graphite flakes are shown in 2g in the Brillouin zone. The intensity of the D band indicates the degree of disorder that the material possesses, which is related to several factors such as functionalization in the graphene macromolecular sp2 ring or, in our case, the ultrasonic exfoliation in ODCB. In many studies, the ratio of ID/IG intensities is used to verify that the material has undergone a structural change during the synthesis process [D/IG ratio indicates a smaller sheet size because the ratio of edge defects is greater for smaller layers [D/IG = 0.10 while this ratio increased to 0.38 for the exfoliated GNS. These values confirmed the exfoliation of the graphite in ODCB using the ultrasound probe, which caused a certain disorder in the material. Raman spectroscopy was performed to confirm the exfoliation of the micrographite by ultrasound irradiation. The typical bands of graphene materials were observed in the spectra . The G p process . In addir layers . The ref−1 and so, applying the equation proposed by Wang et al. [−1. It has been shown that in the case of multilayers graphene, the 2D peak shifts toward higher Raman shift and becomes broader [−1, similar to MG, although there was a clear difference in peak intensity between them. It is common to calculate the ratio of peak intensity I2D/IG. An approximate value of 2 identifies a single layer of graphene [The calculation of the number of graphene layers is very relevant in this type of study. Many investigations relate the position of the G peak with the number of graphene layers. The G peak of the GNS was at a Raman shift of 1582 cmg et al. , it can g et al. . Normall broader . In the graphene . The oriN-methyl-2-pyrrolidone [In order to study the composition of MG and GNS, XPS spectra of these samples were measured. rolidone .h), which is defined as the radius of an equivalent hard sphere diffusing at the same rate as the particle under observation, and so it is indicative of the apparent size adopted by the GNS in the aqueous dispersion. GNS exhibited a Gaussian peak located at 450 ± 14 nm. The measurement carried out for the MG material revealed a particle size outside the quantification limits of this technique (>1 μm).The size distribution and intensity correlation function normalized between one and zero of the GNS using dynamic light scattering (DLS) are shown in 2. A value of PDI < 0.1 indicates that the sample is monodisperse. Instead, if PDI is between 0.1 < PDI < 0.2, the sample would have a narrow particle size distribution. In the case of GNS, PDI was 0.36, value between 0.2 < PDI <0.5, which indicates that the sample has a wide particle size distribution according to the results reported by J. Lohrke et al. [DLS also provides the polydispersity index (PDI), which indicates the width of the particle size distribution, being calculated as (peak width/peak height)e et al. .9 ± 6.48 × 107 particles/g. However, most particles had sizes between ca. 30–200 nm, although the concentration of particles around 300 nm was also significant. The number of larger particles (>350 nm) was much lower. A comparison between DLS and NTA reveals that DLS could overestimate the particle sizes of the sample. Both techniques provide a hydrodynamic radius (Rh) value but larger particles scatter stronger and are preferably detected by DLS than the smaller particles since the scattering intensity effect depends on size rather than on the number of particles with similar diameter. DLS is a fast, simple and non-destructive technique, but it presents this inherent feature when analysing samples that present some polydispersity [In order to further define the size of the graphene nanosheets, nanoparticle tracking analysis (NTA) was used . This tespersity . In thesspersity .Firstly, the influence of the membrane composition on retention properties and recovery from the AF4 channel was studied. Polyvinylidene fluoride (PVDF), polyacrylonitrile (PAN) and regenerated cellulose (RC) were used. 0 are the peak areas without void peak of the detector signal , obtained with and without cross-flow, respectively. The MALS detector allows the direct measurement of the radius of gyration in the 1–1000 nm particle size range [The recovery from AF4 runs was determined as follows:ze range .The fractograms (90° light scattering detector) showed a low recovery using PAN and PVDF (less than 30%) due to the retention of graphene nanosheets in these membranes. It was easily visualized because they became black. This had to be due to the interaction of the graphene sheets with these polymeric membranes. However, the fractogram exhibited a broad peak centred at around 33 min using a RC with a recovery of 30%, thus suggesting a weaker interaction with this membrane. To increase the recovery, pure water was essayed as carrier. g), which is defined as the mass weighted average distance from the core of a particle to each mass element in the particle. Rg is more dependent on the structure of the particle than the value of Rh. Rg increases with increasing the elution volume. The size distribution derived from the MALS detector showed the presence of different populations of particles . The mosf). Df gives a detailed information of the shape of the materials, thus allowing to understand the distribution of particles in aqueous media. A sphere has a Df of 3, whereas Df is 2 for a planar structure and 1 for a rigid rod [MALS detector coupled to AF4 can provide relevant information about the mechanism of aggregation and nanoparticle structure. This information is included in the fractal dimension structural parameter  and Rh is the Z-average hydrodynamic radius provided by DLS (450 nm). The theoretical value of p for a hard sphere is 0.778 and for a stiff rod of approximately 2 [Also, additional information on the particle shape can be obtained by calculating the shape factor, p = R by AF4 3 nm and Rmately 2 ,62. The In summary, AF4 and NTA give much richer information on particle size than DLS when dealing with a polydisperse material as graphene nanosheets. AF4 can be considered a promising technique for the separation and characterization of a variety of graphene structures, including not only graphene nanosheets, as described in this work, but also graphene oxide, graphene quantum dots and other carbon nanostructures. Unlike DLS and NTA, AF4 is a separation technique and can be coupled to a great variety of detectors, thus gathering rich information from the sample. It separates particles in the range from 1 nm to 1–2 μm, although other field flow fractionation techniques could expand this range up to particle sizes higher than 100 μm. Interestingly, the existence of semipreparative channels would allow the separation and collection of milligram quantities of these graphene structures. In addition, NTA can provide the concentration of GNS nanoparticles. Nevertheless, stability concerns of aqueous dispersions of large graphene particles, a more limited range of particle size determination and the current limited availability in many laboratories are among the disadvantages of NTA and AF4 relative to DLS.The ultrasound-assisted exfoliation of micrographite (MG) to graphene nanosheets (GNS) was confirmed by different techniques, such as XRD, TEM (sheets size between 200–500 nm) and Raman spectroscopy. The particle size distribution of GNS was firstly determined by DLS (ca. 450 nm), which provided the hydrodynamic radius and polydispersity of GNS. Subsequently, two different techniques, i.e., NTA and AF4, were applied here for the first time to graphene. NTA provided an average size of ca. 150 nm and higher resolution than DLS as well as approximate concentrations. In addition, it proved that DLS gave an average particle size whose intensity was biased towards the larger graphene particles. AF4 was able to separate various populations of GNS with different radius of gyration (centered at approx. 360 nm) and in addition it provided information concerning the structure and shape of the particles. Clearly, the application of NTA and AF4 besides DLS allows gathering much rich information on graphene particles. The above-mentioned advantages for NTA and AF4 reveal their usefulness for the determination of the actual polydispersity in graphene preparations, evaluation of their synthetic procedures and size determination and fractionation in aqueous media for studies related to biological applications, sensing and toxicity.


Long interspersed nuclear element 1 (LINE-1 or L1) is a non-long terminal repeat (LTR) retrotransposon that constitutes approximately 17% of the human genome. Since approximately 100 copies are still competent for retrotransposition to other genomic loci, dysregulated retrotransposition of L1 is considered to be a major risk factor of endogenous mutagenesis in humans. Thus, it is important to find drugs to regulate this process. Although various chemicals are reportedly capable of affecting L1 retrotransposition, it is poorly understood whether phytochemicals modulate L1 retrotransposition. Here, we screened a library of compounds that were derived from phytochemicals for reverse transcriptase (RT) inhibition with an in vitro RT assay. We identified capsaicin as a novel RT inhibitor that also suppressed L1 retrotransposition. The inhibitory effect of capsaicin on L1 retrotransposition was mediated neither through its receptor, nor through its modulation of the L1 promoter and/or antisense promoter activity, excluding the possibility that capsaicin indirectly affected L1 retrotransposition. Collectively, capsaicin suppressed L1 retrotransposition most likely by inhibiting the RT activity of L1 ORF2p, which is the L1-encoded RT responsible for L1 retrotransposition. Given that L1-mediated mutagenesis can cause tumorigenesis, our findings suggest the potential of capsaicin for suppressing cancer development. The eukaryotic genome contains retrotransposable elements, also known as retrotransposons, with or without long terminal repeats (LTRs). Long interspersed nuclear element 1 (LINE-1 or L1) is a non-LTR retrotransposon and constitutes approximately 17% of the human genome . ORF2p iThe active retrotransposition of L1 is considered to be a major source of endogenous mutagenesis in humans ,6. In adAsiasari Radix and Capsicum annuum [Sho-seiryu-to. The compound is a homovanillic acid derivative and is known as a potent analgesic that is used for treating pain and inflammation [Capsaicin is a major chemical constituent of m annuum  and alsoammation . The anaammation . HoweverHere, we screened a library of compounds that were derived from natural medicines for RT inhibition by an in vitro RT assay with Moloney murine leukemia virus (M-MLV) RT. Among the 96 compounds we examined, we found that capsaicin inhibited the activity of M-MLV RT. Capsaicin also suppressed L1 retrotransposition in a dose-dependent manner, while it did not affect the L1 promoter and antisense promoter activities. These results suggest the potential of capsaicin as a novel RT inhibitor that can inhibit L1 retrotransposition. To identify novel RT inhibitors that can affect L1 retrotransposition, we sought to screen compounds that inhibit RT activity. For efficient screening, we used M-MLV RT as a model RT, because M-MLV RT shares several features with L1 ORF2p  and was 50 was calculated as 34.6 µM . To reveal the mechanism of how capsaicin inhibits L1 retrotransposition, we next evaluated its effect on L1 expression. Since the L1 5′ UTR promoter activity is responsible for L1 RNA expression, we conducted an L1 5′ UTR promoter assay. We detected a slight reduction in the L1 5′ UTR promoter activity by capsaicin only at a concentration of 10 µM, but did not detect any significant dose-dependent suppression of promoter activity in the range of its L1 inhibitory effect A. Recentposition . TherefoPhytochemicals are beneficial for therapeutic use because they are active in many biological processes and have less toxicity than pharmaceutical agents. In this report, we tested the effects of such chemicals on L1 retrotransposition. We identified capsaicin as a novel RT inhibitor that suppresses L1 retrotransposition. Since capsaicin suppressed the activity of M-MLV RT, did not show dose-dependent suppression of the L1 promoter and antisense promoter activities, and was still active in the TRPV1-knocked down condition, it is likely that capsaicin suppressed L1 by inhibiting the RT activity of L1 ORF2p. A main concern of this study is the use of an in vitro RT assay with M-MLV RT to screen compounds that regulate L1 retrotransposition. Indeed, L1 ORF2p, the RT of L1, was more suitable for our purpose, but the use of M-MLV RT provided several advantages over the use of L1 ORF2p. First, quality-certified M-MLV RT was commercially available from Thermo Fisher Scientific and ready to use for screening. Second, the reaction condition of the in vitro M-MLV RT assay has been already established in the kit, making it easy to set up screening. Third, some nucleoside RT inhibitors of retroviral RT are also known to inhibit L1 RT, which suggests that M-MLV RT and L1 ORF2p share several properties . AdditioEmerging evidence indicates that capsaicin exhibits an anti-cancer and/or growth-inhibitory effect in various cancers ,24. AlthIn conclusion, we demonstrated the potential of capsaicin for inhibiting retroviral RT activity and suppressing L1 retrotransposition, possibly via inhibition of RT activity of ORF2p. Our findings suggest the potential of capsaicin for suppressing cancer development and they may facilitate the studies of capsaicin or related compounds, capsaicinoids, for cancer prevention and treatment. 293T cells (a human embryonic kidney cell line) were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 5% fetal bovine serum (FBS). Sho-seiryu-to extract was included in the INM deposited WAKANYAKU library. The compound library that was derived from natural medicines, the INM deposited WAKANYAKU library, was kindly supplied from the Institute of Natural Medicine, University of Toyama. Capsaicin was purchased from Sigma-Aldrich . The PstI and NheI sites of the 5′ UTR of L1, as previously reported [BbsI sites of pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro (Addgene #28289).The reporter plasmid for L1 retrotransposition, pYX014, was kindly provided by Dr. Wenfeng An  . The repreported , in antiThe activity of the M-MLV RT (400 U/mL), SuperScript II Reverse Transcriptase , in the presence of a test compound (300 µM) was determined using the EnzChek Reverse Transcriptase Assay Kit  according to the manufacturer’s instruction. The values of treated wells were normalized with those of the control wells. Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System  according to the manufacturer’s instruction in a single-well luminometer . Since Renilla luciferase was constitutively expressed from the reporter construct, Firefly luciferase activity was normalized to the corresponding Renilla luciferase activity. For knockdown experiments, 293T cells were co-transfected with an L1 reporter plasmid and a plasmid expressing sh-TRPV1. The assay was conducted in duplicate.The 293T cells were transfected with pYX014 using Lipofectamine 2000 reagent . At 24 h after transfection, cells were treated with the indicated concentrations of test compounds. After 3 days of treatment, The 293T cells were treated with the indicated concentrations of test compounds for 3 days. The cell viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instruction. Values of the treated wells were normalized to those of the control wells. The assay was conducted in duplicate.Gaussia and Cypridina luciferase activities were measured using the Gaussia and Cypridina Luciferase Assay Kit (New England BioLabs) according to the manufacturer’s instructions. Gaussia luciferase activity was normalized to the corresponding Cypridina luciferase activity.The 293T cells were co-transfected with pGLuc-5′-UTR or pGLuc-ASP, together with pCMV-CLuc  using Lipofectamine 2000 reagent. At 24 h after transfection, cells were treated with the indicated concentrations of test compounds. After 3 days of treatment, the Total RNA was extracted from the indicated cells and reverse transcribed using a Verso cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction. Quantitative real-time RT-PCR assays were carried out using Fast SYBR Green Master Mix (Thermo Fisher Scientific) and gene-specific primers with a QuantStudio 6 (Thermo Fisher Scientific). The assay was conducted in duplicate.The sequences of the primers used in this study are as follows:TRPV1-forward primer, 5′-ATG ACA GTG TGA TGG AGA GTC-3′TRPV1-reverse primer, 5′-CCT TGC TGG ATC CTC TGT GG-3′GAPDH-forward primer, 5′-AGC GAG ATC CCT CCA AAA TC-3′GAPDH-reverse primer, 5′-AAA TGA GCC CCA GCC TTC TC-3′t-test with a threshold of p < 0.05. Statistical significance was assessed using a two-tailed Student’s


Graphene, the first true two-dimensional material, still reveals the most remarkable transport properties among the growing class of two-dimensional materials. Although many studies have investigated fundamental scattering processes, the surprisingly large variation in the experimentally determined resistances is still an open issue. Here, we quantitatively investigate local transport properties of graphene prepared by polymer assisted sublimation growth using scanning tunneling potentiometry. These samples exhibit a spatially homogeneous current density, which allows to analyze variations in the local electrochemical potential with high precision. We utilize this possibility by examining the local sheet resistance finding a significant variation of up to 270% at low temperatures. We identify a correlation of the sheet resistance with the stacking sequence of the 6H silicon carbide substrate and with the distance between the graphene and the substrate. Our results experimentally quantify the impact of the graphene-substrate interaction on the local transport properties of graphene. Measurement of charge transport in epitaxial graphene is challenging. Here, the authors quantitatively investigate local transport properties of graphene prepared by polymer assisted sublimation growth using scanning tunneling potentiometry and report local sheet resistances with a variation of up to 270% at low temperatures. The high quality and its 2D nature make epitaxial graphene the perfect system to study fundamental transport properties on the nanometer scale. Consequently, in a series of experiments—based on scanning tunneling potentiometry (STP)2 or four-point-probe microscopy3—several groups have focused on local properties like the sheet resistance and the impact of scattering centers like single substrate steps5 or the transition from monolayer to bilayer graphene on transport7. From these results, it is qualitatively well understood that the transport properties of epitaxial graphene are not homogeneous on the nanometer scale. Substrate steps or monolayer-bilayer junctions act as local scattering centers. In addition, for epitaxial graphene on SiC(0001) it is well known that interaction with the substrate drastically affects graphene’s transport properties. In order to reduce this inherent proximity effect, i.e., to improve the transport properties of the graphene sheet, different strategies were pursued such as the refinement of the growth process10, the use of suitable dielectric substrates like boron nitride11, the decoupling of the substrate by intercalation12, or the preparation of suspended graphene13. Moreover, the proximity effect can be deliberately exploited to specifically tune the properties of a graphene sheet18. For example, the almost negligible spin-orbit coupling can be significantly increased by bringing the graphene layer into contact with transition metal dichalcogenides15 and proximity superconductivity can be observed in graphene in the vicinity of superconducting materials19.Charge transport in epitaxial graphene has been subject of theoretical and experimental investigation since its first electronic characterization25. The strong variation in the experimental values of sheet or defect resistances determined by local probe measurements is likely due to the lack of information about the actual local current density. Replacing the probe by a single-electron transistor allows simultaneous measurement of local voltage drop and current distribution in 2D materials26 with a lateral resolution in the range of 350 nm26. In comparison, STP has an angstrom resolution27 and measures the local electrochemical potential with high accuracy, but local variations in the current density are experimentally not accessible and are indistinguishable from spatial variations of the sheet resistance. For conventionally grown graphene on SiC(0001), typically monolayers as well as bilayers are present. Monolayer-bilayer transitions represent strong scattering centers and cause a significant variation of the local current density. Having no better approach, so far local transport properties have been determined using an averaged (sometimes even macroscopic) current density for the whole sheet.In the context of charge transport in epitaxial graphene, a locally varying potential landscape and a spatially inhomogeneous current density are induced by local defects like substrate steps and local variations of the coupling between the graphene layer and the substrate. Analyzing the published results for resistances assigned to specific defects in epitaxial graphene, one finds a large spread28 on SiC substrates with ultra-low step heights. This allows for local transport investigations of monolayer graphene on terraces with different SiC terminations free from bilayer and step edge effects.In this study, we show that the high quality of epitaxial monolayer graphene samples grown by polymer assisted sublimation growth (PASG) opens a promising way to quantify also delicate local transport properties with high precision. Applying the PASG method, it is possible to grow large-scale monolayer graphene sheets without bilayer formationThe aim of this work is to test for local variations in the sheet resistance of epitaxial graphene and to unravel possible intrinsic proximity effects induced by the presence of the substrate.28, drastically reduce lateral current density variations29. In our STP setup, the current flow was deliberately driven parallel to the miscut of the SiC sample, resulting in an overall voltage drop perpendicular to the substrate steps  is visible. It is induced by a lattice mismatch of the graphene sheet and the substrate and originates from actual height corrugation as well as from electronic contrast38. However, this 6 × 6-quasi corrugation is structurally not perfect  at 8K provide insight into the local electronic structure. dI/dV spectra it can be seen that the electronic properties on the two terraces are not identical and even on a given terrace we find local deviations  exhibits six different  terminations , the buffer layer is partially covalently bonded and thus strongly coupled to the SiC substrate60 and electron–electron scattering61. In addition, potential scattering, screening, and their interplay have to be considered in the discussion. While all these processes depend on the charge carrier density, the electron–electron scattering has been found to be most dominant at high temperatures and low doping62. ARPES measurements reveal a high charge carrier concentration of Within the proposed model, we now discuss the temperature-dependence of the sheet resistance, i.e., an increasing conductivity with decreasing temperature. In the semi-classical Boltzmann transport, an increase in the conductivity with decreasing temperature is attributed to freezing out of electron–phononSince the Fermi wavelength of the electrons roughly corresponds to the wavelength of the potential modulations, we have additionally considered phase-coherent transport phenomena. We predominantly observed classical Lorentz magnetoresistance in macroscopic magnetotransport measurements  spectra acquired on S2 and S3 that show slight differences in the spectra at e.g., −300 mV.Having identified the distance between the graphene and the substrate as an important parameter that controls the sheet resistance in general, the question arises whether this parameter also explains the different sheet resistances of the graphene sheet on terraces S2 compared with terraces S3. Figure In summary, the spatial homogeneity of PASG graphene allows for a quantitative analysis of electronic transport on a local scale. We have shown a direct correlation of the structural as well as the electronic transport properties with the substrate. In particular, PASG graphene shows a locally inhomogeneous sheet resistance, which is governed by both the substrate termination of the SiC and the distance between the graphene layer and the substrate. A locally varying distance to the substrate is accompanied by a variation of the impact of the interface states such that a larger distance leads to a reduced resistance. By analyzing the temperature-dependence of the sheet resistance we have disentangled different scattering mechanisms and have thereby revealed a large inhomogeneity in the sheet resistance at low temperatures associated with the imperfections of the 6 × 6-quasi corrugation and localized defects.66. In this context, the question arises as to the limitations of the reported effect, i.e., a maximum variation in the sheet resistance of 270% at low temperatures. Such a strong local inhomogeneity of the electronic transport is an important quality in the field of epitaxial graphene. It e.g., implies that nanometer-sized devices could exhibit a local variation in the mobility of up to 270%. We are convinced that the findings of this study can be generalized and should be considered for other 2D materials grown on and in proximity with a substrate.Besides the investigation of fundamental processes in the interaction between substrate and graphene, the reported anisotropy could be exploited in further transport experiments. For example, it is interesting to simply rotate the sample by 90° such that the current is applied parallel to the steps instead of perpendicular. Generally, terraces S2 with 28. The idea of this method is to support the growth process with an external carbon source. A polymer is deposited on the substrate using liquid phase deposition before high-temperature sublimation growth is initialized28. The samples were initially annealed in vacuum (p = 900 mbar) at 1200 °C and 1400 °C for 10 and 2 min, respectively. Afterward, the samples were heated directly up to 1750 °C and annealed (6 min) while argon flux was kept at 0 sccm10. Samples prepared with this method are almost defect- and bilayer-free and exhibit shallow step heights, as verified in Raman mapping and AFM topographies28.Graphene samples investigated in this study were grown on the (0001) Si-terminated face of semi-insulating 6H-SiC wafers with small nominal miscut angle of 0.06° toward −10 mbar at 300K, 77K, and 8K) using electrochemically etched tungsten tips. STS spectra were acquired using standard lock-in technique and a modulation amplitude of 10 mV. The concept of the STP setup is depicted in Supplementary Fig. Vcross is applied across the sample via two gold contacts. The voltage IT vanishes and is additionally recorded as a function of position. The resulting potential map (Supplementary Fig. j as follows4The experiments were performed in a custom-built low-temperature STM and in a custom-built room temperature STM under UHV conditions (base pressure < 10® using the AC/DC module. As input parameters, we enter the macroscopic ohmic resistance and the global geometry of the sample. Additional topographic information like substrate steps, bilayer regions, and corresponding monolayer-bilayer transitions are included according to the structural information from constant current topographies. Step resistivities used in this study are set to 6 Ωμm, 12 Ωμm, 18 Ωμm for single, double, and triple substrate steps, respectively.The local current density Supplementary Information