Methods: Sixteen patients  with non–small cell lung cancer were recruited. Primary tumor PD-L1 expression was measured by immunohistochemistry. NM-01 was radiolabeled with [99mTc(OH2)3(CO)3]+ complex binding to its C-terminal hexahistidine tag. Administered activity was 3.8–10.4 MBq/kg, corresponding to 100 μg or 400 μg of NM-01. Whole-body planar and thoracic SPECT/CT scans were obtained at 1 and 2 h after injection in all patients, and 5 patients underwent additional imaging at 10 min, 3 h, and 24 h for radiation dosimetry calculations. All patients were monitored for adverse events. Results: No drug-related adverse events occurred in this study. The mean effective dose was 8.84 × 10−3 ± 9.33 × 10−4 mSv/MBq (3.59 ± 0.74 mSv per patient). Tracer uptake was observed in the kidneys, spleen, liver, and bone marrow. SPECT primary tumor–to–blood-pool ratios (T:BP) varied from 1.24 to 2.3  at 1 h and 1.24 to 3.53  at 2 h (P = 0.005). Two-hour primary T:BP ratios correlated with PD-L1 immunohistochemistry results . Two-hour T:BP was lower in tumors with ≤1% PD-L1 expression . Nodal and bone metastases showed tracer uptake. Heterogeneity (>20%) between primary tumor and nodal T:BP was present in 4 of 13 patients. Conclusion: This first-in-human study demonstrates that 99mTc-labeled anti–PD-L1-single-domain antibody SPECT/CT imaging is safe and associated with acceptable dosimetry. Tumor uptake is readily visible against background tissues, particularly at 2 h when the T:BP ratio correlates with PD-L1 immunohistochemistry results.Immunotherapy with checkpoint inhibitor programmed cell death 1 (PD-1)/programmed death ligand-1 (PD-L1) antibodies demonstrates improvements in treatment of advanced non–small cell lung cancer. Treatment stratification depends on immunohistochemical PD-L1 measurement of biopsy material, an invasive method that does not account for spatiotemporal heterogeneity. Using a single-domain antibody, NM-01, against PD-L1, radiolabeled site-specifically with  Non–small cell lung cancer (NSCLC) is the leading cause of cancer mortality, with nearly 1.6 million cancer-related deaths each year worldwide ,3. The rPD-1 receptor, expressed on the surface of activated T cells, is a negative costimulatory receptor, and its ligands, PD-L1 or PD-L2, are normally expressed on the surface of dendritic cells or macrophages ,13. CertDiscrepancies in treatment response have highlighted potential deficiencies in the current methods to evaluate PD-L1 expression in a clinical setting . ImmunohSingle-domain antibodies (sdAbs) are the smallest naturally derived antigen-binding fragments from camelid antibodies and demonstrate great potential for molecular imaging in both preclinical and clinical models –21. Beca99mTc-labeled sdAb (99mTc-NM-01) that specifically binds to human PD-L1 is feasible, is safe, and correlates with PD-L1 immunohistochemistry results. In this first-in-human study, we aimed to assess safety, radiation dosimetry, and imaging characteristics of 99mTc-NM-01 and sought to compare imaging and PD-L1 immunohistochemistry results in patients with advanced NSCLC.Our hypothesis was that SPECT imaging of NSCLC with a n = 16) between March and November 2018  and obtained approval from Shanghai General Hospital Ethics Committee . All patients enrolled into this study gave written informed consent to participate.This was an open-label, nonrandomized early phase I diagnostic study in patients with untreated NSCLC (99mTc(OH2)3(CO)3]+ complex (pH 7.0–7.5) was added to a sealed vial containing 200 μg of NM-01 in 100 μL of phosphate-buffered saline (pH 7.4), and the mixture was incubated at 37°C for 1 h . PD-L1 expression was determined in primary tumor tissue obtained by core needle biopsy.Patients aged between 18 and 75 y with histopathologically confirmed NSCLC and an Eastern Cooperative Oncology Group Performance Score of 1 or less were eligible to participate in this study  was also processed in parallel. After collection, formalin-fixed paraffin-embedded blocks were prepared according to instructions set in the PD-L1 IHC 22C3 pharmDx immunohistochemical assay kit manual . Briefly, each specimen was fixed in 10% neutral buffered formalin. After being rinsed, samples were dehydrated by sequential immersion in ascending concentrations of ethanol in water for 2 h at each concentration, starting at 80% and reaching 100% in 5% increments. Dehydrated samples were cleared in xylene and subsequently infiltrated with melted paraffin at 55°C.Formalin-fixed paraffin-embedded blocks were cut into 4-µm sections in an RM2235 Rotary Microtome (Leica Biosystems). Sections were mounted onto DAKO Flex IHC microscope slides , stored in the dark at 2°C–8°C and used within 30 d of preparation.Further processing and immunostaining of primary tumor samples and controls were performed in a DAKO Autostainer Link 48 SK006 immunohistochemistry stainer  using the pre-programmed PD-L1 IHC 22C3 pharmDx staining protocol and reagents, including the mouse anti–PD-L1 (clone 22C3), provided in the assay kit. Briefly, an automated 3-in-1 process of deparaffinisation, sample rehydration, and target retrieval was followed by an automated staining procedure with mouse anti–PD-L1 (clone 22C3) antibody, followed by hematoxylin (Baso Diagnostic Inc.) counterstaining. Microscopic and histopathologic evaluations of PD-L1 immunostaining were performed on a DAKO Autostainer Link 48  and results validated by a pathologist. Representative images were recorded using a Leica DM6000B microscope (Leica Biosystems) and Leica DFC 550 digital camera (Leica Biosystems).99mTc-NM-01 in an intravenous bolus (3.8–10.4 MBq/kg), corresponding to 100 µg  or 400 µg  of NM-01. Patients were asked to drink 300–500 mL of water after injection then void their bladder before imaging on a GE Discovery NM07 SPECT/CT scanner . Planar scans were acquired with the patient supine at 1 and 2 h after injection at 10 cm/slice/min. A CT scan was acquired at 60 min followed by local tomographic SPECT imaging focused around primary and suspected secondary as follows. All scans were acquired using low-energy high-resolution collimators in a 20% energy window centered around 140 keV, in a 256 × 1,024 matrix for planar images and 64 × 64 matrix for tomographic images. A 10% energy window centered around 120 keV was also collected during tomographic acquisitions for attenuation and scatter correction. SPECT images were acquired over 360° in 60 frames per full rotation with 20-s acquisition per frame.Patients were administered All scans were assessed by a single observer with more than 25 y of nuclear medicine and radiology experience. Maximum counts were recorded from regions of interest drawn manually on SPECT scans, using the CT for guidance, around the primary tumor; any lymph node metastases within the thoracic field of view; a 3-cm normal-lung ROI in the right upper lobe (or left upper lobe if pathology existed in the right upper lobe) for tumor-to-lung ratios (T:L); and a ROI, within the aortic arch avoiding the walls of the aorta, for calculation of tumor–to–blood-pool ratios (T:BP).Five patients underwent extra whole-body planar imaging at additional time points  to obtain radiation dosimetry data. A calibration source of 37 MBq at injection time was placed above the head of each patient to provide quantitative calibration of counts to activity. Visible organs were drawn on the initial anterior and posterior whole-body images for each patient at 10 min using OsiriX software (Pixmeo) by an experienced operator and transferred to all remaining time points. The remainder of body was assigned activity not accounted for by organ delineation or excretion. All organs for each patient were assumed to be of the same volume over the imaging time frame. Counts from the anterior and posterior images were geometrically averaged. Time–activity curves were converted to percentage injected dose and used as an input to the OLINDA/EXM dose calculation software (version 1.1) . Organ nU test, and Spearman correlation using IBM SPSS Statistics (version 24) software. A P value of less than 0.05 was taken for statistical significance.Differences and correlations between parameters were tested using the Wilcoxon signed-rank test, Mann–Whitney Average radiochemical purity of radiolabeled NM-01 was 96.9% ± 0.9% (95.4%–98.5%); purification after radiolabeling was not necessary . Endotoxin levels remained below 0.4 EU/mL.99mTc-NM-01 was 372 ± 62 MBq  in group 1 and 659 ± 25 MBq  in group 2  with histopathologically proven NSCLC  were included. Clinical stage and other characteristics are summarized in  group 2 .99mTc-NM-01 was administered in 16 patients; no drug-related adverse reactions were reported during the 7-d follow-up period. Vital signs were measured during the screening period, before injection and then every 60 min up to 5 h after injection and first follow up period (48 h after SPECT/CT scan). Clinical laboratory testing, including standard hematologic and comprehensive metabolic panels , was performed during the screening period and 48-h follow-up period. In an additional step to ensure the safety of patients, a phone interview was conducted 7 d after the SPECT/CT scan to follow-up on patients, and a questionnaire was filled out by the investigator as presented in the case report form. Symptoms related to advanced-stage lung cancer, that is, frequent coughing, dyspnea, and fatigue, reported by some patients were not attributed to the radiopharmaceutical by the supervising physician. Blood and urine clinical laboratory tests performed in the follow-up period were in the reference range of National Cancer Institute–Common Toxicology Criteria for Adverse Events .,29.28,29No noticeable differences in image quality and no statistically significant differences in tumor-to-background ratio were observed between group 1 and group 2 .n = 5). All patients had normal liver and kidney functions. The kidneys showed the highest organ dose (0.036 ± 0.018 mSv/MBq), followed by the bladder (0.026 ± 0.011 mSv/MBq), spleen (0.022 ± 0.011 mSv/MBq), and liver (0.011 ± 0.0025 mSv/MBq) and in line with the uptake and excretory pathway of the imaging agent. Time–activity curves for organs with highest radiotracer uptake are shown in P = 0.034), allowing reasonably good tumor conspicuity against normal background lung activity in patients with high PD-L1 expression (P = 0.005). Intratumoral heterogeneity of tracer uptake was noted in some primary tumors (P = 0.048). When receiver operating characteristic analysis is used, this gives an optimal threshold for the 2-h T:BP of 2.32 .The primary tumor T:L ratio was greater at 2 h than 1 h . At 2 h, primary tumor and maximum lymph node metastasis T:BP ratios (n = 13) showed a correlation . Intrapatient heterogeneity of T:BP ratios was noted between the primary tumor and lymph node metastases in several patients with a mean difference of 19% (±17%), with 4 of the 13 patients with lymph node metastases showing a greater than 20% difference . Statistically significant correlations were not found for T:L ratios or 1-h T:BP ratios.Thoracic lymph node metastases T:BP ratios were also greater at 2 h, although this difference did not reach statistical significance  and similar to other SPECT agents in clinical use. The kidneys received the highest organ dose (0.036 ± 0.018 mSv/MBq), being the major route of excretion, and although it remained tolerable, urinary tract dose could be further minimized by good hydration or coinjection of amino acids or peptides .Our initial results show promise that the simple measurement of T:BP ratios at 2 h correlates with PD-L1 expression and if confirmed in subsequent studies could potentially replace or complement immunohistochemistry in some circumstances. We noted heterogeneity of activity within primary tumors in some patients, and between the primary tumor and metastases within patients, suggesting that intra- and intertumoral heterogeneity of PD-L1 expression is relatively common and is another factor that could be measured noninvasively with imaging to allow more relevant information to be available for patient treatment decisions. Intratumoral PD-L1 expression heterogeneity may also explain the imperfect correlation with T:BP  could be improved, further scanning beyond 2 h could be considered in subsequent studies. The relatively low spatial resolution of SPECT does not allow more than gross visual intratumoral heterogeneity observations. Although a PET tracer, in theory, might allow more accurate and detailed heterogeneity analysis similar to what has been reported with in NSCLC , recent in NSCLC . In addi99mTc-labeled anti-PD-L1-sdAb SPECT/CT using 99mTc-NM-01 is a safe diagnostic procedure, delivering a tolerable radiation dose and presenting favorable biodistribution and image characteristics correlating with PD-L1 immunohistochemistry results in patients with NSCLC. As PD-1/PD-L1 immune checkpoint inhibitors are more frequently used in the treatment of NSCLC, a companion imaging methodology is becoming increasingly sought after for the staging and monitoring of patients. Anti–PD-L1-sdAb SPECT/CT using 99mTc-NM-01 has the potential to be used for the close monitoring of changes in PD-L1 expression levels during PD-L1 immunotherapy. In addition, the heterogeneous and dynamic nature of PD-L1 expression in most malignancies could make imaging a preferred method, compared with more invasive core needle biopsies, in diagnosing and staging patients with PD-L1–positive malignancies.This study demonstrates that This research is supported by Nanomab Technology Limited and Shanghai Shenkang Development Center for Clinical Skills Innovation (16CR3052A). The authors acknowledge financial support from the King’s College London/University College London Comprehensive Cancer Imaging Centres funded by Cancer Research U.K. and Engineering and Physical Sciences Research Council in association with the Medical Research Council and the Department of Health (C1519/A16463) and the Wellcome Trust EPSRC Centre for Medical Engineering at King’s College London (WT203148/Z/16/Z). No other potential conflict of interest relevant to this article was reported.Click here for additional data file.


Finally, decay rates for a dipole emitter are significantly enhanced in the proximity of finite-area biaxially crumpled graphene flakes. Our findings indicate that crumpled graphene provides a platform to engineer graphene-based plasmonics through broadband manipulation of strong plasmonic resonances.Graphene nanostructures with complex geometries have been widely explored for plasmonic applications, as their plasmonic resonances exhibit high spatial confinement and gate tunability. However, edge effects in graphene and the narrow range over which plasmonic resonances can be tuned have limited the use of graphene in optical and optoelectronic applications. Here we present a novel approach to achieve mechanically reconfigurable and strongly resonant plasmonic structures based on crumpled graphene. Our calculations show that mechanical reconfiguration of crumpled graphene structures enables broad spectral tunability for plasmonic resonances from mid- to near-infrared, acting as a new tuning knob combined with conventional electrostatic gating. Furthermore, a continuous sheet of crumpled graphene shows strong confinement of plasmons, with a high near-field intensity enhancement of ~1 × 10 An innovative approach for making tunable plasmonic nanoparticles using crumpled graphene opens the door to new optical and optoelectronic devices. Graphene is a promising plasmonic material due to its low optical loss, high spatial confinement, and tunable plasmonic resonances. However, its structural patterns fix the resonance wavelengths, making broadband tunability across near- to mid-infrared wavelengths very challenging, limiting its range of applications. This led Pilgyu Kang from the University of Illinois at Urbana-Champaign in America, in collaboration with fellow American and South Korean colleagues, to turn to crumpled graphene, a form of graphene with unique optical and physical properties. By modelling the plasmonic resonances of crumpled graphene, the researchers demonstrated mechanically reconfigurable structures that enable a much broader range of spectral tunability, leading to new applications from optical sensors to photovoltaic devices. Graphene-based plasmonic devices have shown potential in broad applications, including optical biosensing3, optical communications4, photodetectors5, and plasmonic metamaterials6. In particular, desired plasmonic resonances are easily excited in lithographically patterned graphene structures14 using a far-field coupling method. However, such structural patterns in graphene are not reconfigurable, thus fixing their resonance wavelengths. Although one can tune the resonance wavelength of graphene through electrical gating15, it is challenging to achieve broadband tunability across the near- to mid-infrared wavelength ranges. While graphene grating structures have been proposed for on–off switching and plasmonic modulation19, post-fabrication reconfiguration of plasmonic structures has not yet been realized.Graphene is a promising plasmonic material with superior properties that distinguish it from conventional noble metals22. However, the potential of crumpled graphene structures for reconfigurable plasmonic materials has not been explored. In this work, we report on the use of mechanically reconfigurable crumpled graphene structures to realize strong plasmonic resonances with broadband tunability. We introduce a resonant electrical inductor–capacitor (LC)-circuit model to describe the characteristics of plasmonic resonances in the crumpled graphene. By varying the geometry of the crumpled graphene structures, we show a tunable spectral range for plasmonic resonances that is fivefold broader than that achieved by conventional electrical gating. Our results show that mechanical reconfiguration of crumpled graphene serves as a new way to manipulate strong plasmonic resonances over a broad spectral range.It has recently been demonstrated that mechanical crumpling of graphene can be used to create different surface structures while maintaining reversibilityJs = σEt, where Js is the surface current, σ is the optical conductivity of graphene, and Et is the tangential component of the electric field to the graphene surface24. We used a mobility value of 10,000 cm2/(V·s), which is achievable for crumpled graphene based on the reported range of mobilities (μ) for flat graphene15 from ~1000 cm2/(V·s) for chemical vapor deposition grown graphene to 230,000 cm2/(V·s) for suspended exfoliated graphene. Moreover, the mobility of wrinkled graphene was shown to be similar to that of flat graphene26. For the optical absorption/extinction spectra of uniaxially and biaxially crumpled graphene in Figs. T/T0, where T and T0 are the transmittance values with and without crumpled graphene, respectively. To investigate substrate-induced effects on plasmonic resonances in Fig. 2) substrate27 was used as a model material. In Fig. We performed full-wave simulations using the finite element method  to investigate the plasmonic resonances of crumpled graphene structures. We modeled graphene as a two-dimensional surface current tangential to the crumpled structure: σ) for varying Fermi energy (EF) and carrier mobility (μ) values of graphene at a constant temperature of 300 K using a random phase approximation method29. The optical conductivity is expressed by σ(ω) = σintra(ω) + σinter(ω), where σintra(ω) and σinter(ω) are the intraband and interband conductivities, respectively23. They are expressed as below, where ω is the frequency of the incident light, e is the charge of an electron, τ is the Drude relaxation rate, T is the temperature, kB is the Boltzmann constant, and ℏ is the reduced Planck constant. EF is the Fermi energy, determined by EF = ℏvf(πn)1/2, where vf is the Fermi velocity of electrons (~106 m/s) and n is the carrier density of graphene. The Drude relaxation rate τ is determined by τ = µEF/evf2, where µ is the carrier mobility of graphene.ωF = EF/ℏ and ωT = kBT/ℏ.We calculated the optical conductivity of graphene (λc) and crumple height (h)  at a constant λc of 250 nm  redshifted from ~4.5 to ~11.3 µm with increasing h/λc from 0.25 to 2.0. The optical absorption and extinction (σabs and σext) also increased with higher h/λc from 0.25 to 1.0 but showed no significant change from h/λc = 1.0 to 2.0  and observed redshifts for λres from ~3.2 to ~13.8 µm and an increase in σabs and σext from ~0.12 to ~0.41 and from ~0.14 to ~0.50, respectively  is the curvature factor for the crumpled structures. By using Leff and Ceff, we derived the plasmonic resonance wavelength (λres) of the crumpled graphene expressed as λres = 2πc(LeffCeff)1/2 (see SI for detailed derivation).To describe the plasmonic resonance in crumpled graphene structures, we introduced a resonant ons Fig. . An effeLC-circuit model, we compared the value for λres predicted by the LC-circuit model with that predicted by numerical simulations. With varying λc from 50 nm to 1 μm and h/λc from 0.5 to 2, the λres obtained by the full-wave simulations (dots) agreed with that of our LC-circuit model (solid curves) , leading to a shift in λres. This implies that controlling the charge interactions on confronting graphene surfaces by varying h/λc enables the tunability of λres across the near- to mid-infrared wavelength ranges. The mechanically reconfigurable crumpled graphene structures are thus an excellent platform for broadband tunability of plasmonic resonances.To validate our es) Fig. . In contEF and carrier mobility µ  for varying EF was ~3.7 µm, while ∆λres for varying λc was ~20.1 µm . The redshift of λres is attributed to confinement of resonant plasmons exclusively in the apex of the crumpled graphene on the undulating substrate  were enhanced approximately twofold for the undulating substrate compared to the free-standing crumpled graphene (14.0 vs. 7.6% and 63.8 vs. 26.1)   Fig. . However6%) Fig. . These rλxc, = λyc,)  2) compared to uniaxially crumpled graphene  for the uniaxially crumpled graphene (∆λres ~ 6.1 µm)   Fig.  and an eµm) Fig. . Notablyµm) Fig.  and Q-faene Fig. . The abs·s) Fig. . The Q-fity Fig. . The biax and y directions. Plasmonic resonance peaks were observed for various values of h/λc. The near-field distributions at the plasmonic resonance peaks show that resonant plasmons were strongly confined to the center of the crumpled graphene flake  showed the same plasmonic resonance peak wavelengths as those with lower mobilities, including at 1000 and 4000 cm2/(V·s)  Fig. . Crumple39 of crumpled graphene flakes, we introduce a dipole emitter placed 40 nm above the center of a free-standing biaxially crumpled graphene flake. The orientation of the dipole moment for the emitter is parallel to the x crumpling direction  and total decay rates (Γ), which were normalized by those of the emitter in free space without the crumpled graphene flake (Γ0). Figure h/λc. We observed significant enhancements in the radiative decay rates, up to three orders of magnitude, at the plasmonic resonance peaks  of ~1.6 × 105  showed the same plasmonic resonance peak wavelengths as those with a lower mobility of 1000 cm2/(V·s)  Fig. . We also·s) Fig.  and tota·s) Fig.  for vari4 for biaxially crumpled graphene flakes. Furthermore, we showed strong light–matter interactions for the crumpled graphene flakes, which enabled large decay rate enhancements to yield a maximum Purcell factor of ~1.6 × 105. We believe that our crumpled graphene structures, with strong and broadly tunable plasmonic resonances in the near- to mid-infrared, can find broad applications43, including photonic and optoelectronic devices, ultrasensitive chemical and biological sensing, photovoltaic devices, and spectroscopy.In summary, we have demonstrated, for the first time, mechanically reconfigurable crumpled graphene exhibiting broadband tunability of strong plasmonic resonances from the mid- to near-infrared. The reconfigured crumpled graphene structures, with varying crumple wavelengths and heights, enabled the manipulation of plasmonic resonances over a tunable wavelength range five times broader than that afforded by conventional electrical tuning of graphene. We have also demonstrated efficient excitement of graphene plasmons in crumpled graphene structures by far-field coupling of light as well as strong confinement of the excited plasmons in crumpled graphene. The strong plasmonic resonances enabled optical absorption enhancements of up to ~0.46 as well as high near-field enhancements on the order of 1 × 10Supplementary InformationSupplementary Movie


Gases, such as nitrogen dioxide, formaldehyde and benzene, are toxic even at very low concentrations. However, so far there are no low-cost sensors available with sufficiently low detection limits and desired response times, which are able to detect them in the ranges relevant for air quality control. In this work, we address both, detection of small gas amounts and fast response times, using epitaxially grown graphene decorated with iron oxide nanoparticles. This hybrid surface is used as a sensing layer to detect formaldehyde and benzene at concentrations of relevance (low parts per billion). The performance enhancement was additionally validated using density functional theory calculations to see the effect of decoration on binding energies between the gas molecules and the sensor surface. Moreover, the time constants can be drastically reduced using a derivative sensor signal readout, allowing the sensor to work at detection limits and sampling rates desired for air quality monitoring applications. Several toxic air pollutants in more than 80% of the urban areas where air pollution is monitored exceed the World Health Organization’s (WHO) recommended safe exposure levels. Poor air quality has been associated with several negative health aspects ranging from less severe conditions, such as skin and eye irritation, to more acute respiratory problems, cancer, or death. Air pollution has been estimated to cause 8 million annual deaths and to financially burden the European region by about 1.6 trillion US dollars per year .2O) and benzene (C6H6), at levels of relevance to human health. Benzene is a genotoxic aromatic compound, especially associated with leukemia. There are no safe exposure limits for C6H6 according to the last published WHO recommendations [Air quality (AQ) monitoring and control using extremely sensitive sensors are crucial from the viewpoint of preventing further deaths and diseases correlated with toxic air substances. However, commercial sensors/instruments available today are either large, expensive, and complex or small but limited by poor selectivity, sensitivity, and a slow sampling rate . In addindations , howeverndations  recommenbenzene CH6, at leIn the future, AQ assessment should ideally be based on real-time monitoring of air pollutants with high spatial resolution , allowin6H6 at the concentrations of relevance for AQ monitoring [6H6 detection limit down to 0.5 ppb. Unfortunately, none of the sensors are selective to a particular VOC, making it impossible to distinguish, e.g., the C6H6 concentration. Some of the reviewed sensors also include a selective absorbing cartridge for benzene. Unfortunately, the best limit of detection for commercially available sensors selective to C6H6 was found to be 10 ppb for the Ion Science Tiger Select .In a report by Spinelle et al. from 2017, commercially available portable low-cost sensors for VOCs were reviewed, and it was found that few sensors can detect Cnitoring , with mo6H6 [Among state-of-the-art research studies on portable AQ sensors , there a6H6 . Both Si6H6  and meta6H6  operated6H6 , in whic6H6 . HoweverA low cost, portable sensor capable of detecting benzene at 1 ppb or lower concentrations would thus constitute a breakthrough in the field of air quality monitoring.2), C6H6 and CH2O [3O4) decorated multiwall carbon nanotubes [6H6. However, poor reproducibility and insufficient detection limits make the state-of-the-art sensors ill-suited for human safety applications. So far, these sensors were able to detect the mentioned gases down to tens of ppb, but not reliably lower. In this paper, which is an extension of a conference contribution [Using the unique properties of graphene as a transducer allows fabrication of sensor devices that can be used for gas detection where low concentrations can be detected, including air quality control for human health. Besides a high sensitivity, also interaction with specific target analytes and a good selectivity must be addressed to get a useful sensor device. It has already been shown that decoration of the graphene surface with metal/oxide nanoparticles can lead to a higher sensitivity and selectivity towards certain gases, e.g., nitrogen dioxide  through a sublimation process were an on-axis, semi-insulating (0001) 4H-SiC substrate (7 mm × 7 mm) is used for the formation of graphene in argon (Ar) at a temperature of 2000 °C and a pressure of 1 bar  was used3O4 was present on the surface.Before and after the deposition of nanoparticles, a series of characterization measurements was conducted to determine the graphene uniformity and quality, and to see if any damage of the graphene occurred during the deposition. Atomic Force Microscopy (AFM) (Quadrexed Dimension 3100 with a Nanoscope IVa controller) was used in tapping mode to obtain topography images of the sensing layers. The measurements were performed using silicon (Si) tips (PPP-NCHR-50 from Nanosensors) with a tip radius of curvature below 7 nm. X-ray photoelectron spectroscopy (XPS) studies using a Microlab 310-F spectrometer were performed to investigate possible alterations made to the sample after deposition of NPs and to establish if Fe2 and 20% O2 was used as a purging and carrier gas. A more detailed description of the measurement system can be found in a previous work [As a first step, titanium on gold  contact pads were thermally evaporated onto the epitaxially grown graphene on SiC (EG/SiC) before functionalization. The contacts have a size of 1 mm × 1 mm with a distance of 1 mm between them. The sensor chip and a Pt-100 resistance thermometer were glued (Aremco Ceramabond 571) onto a ceramic heater (Heraeus GmbH) to enable a controlled temperature loop, and welded to a 16 pin TO8 header to establish the electrical contacts. This device was mounted on top of a TO8-socket and connected to its pins using gold-wire bonding and silver glue (Epotek E3081). The final sensor was placed in a flow chamber connected to a gas mixing setup. A Keithley 2601B SourceMeter was used in a two-wire mode to measure the resistance between the contacts during gas exposure. The total gas flow was kept constant at 100 mL/min, and a dry mixture of 80% Nous work . 6H6 and CH2O) on pristine EG (PEG) and Fe3O4-decorated epitaxial graphene (DEG) on Si-face 4H-SiC was investigated based on hybrid gas-phase density functional theory (DFT) calculations performed by using Gaussian 16 Rev. B.01 program package [3O4 located on PEG. All dangling bonds are passivated by the hydrogen atoms. The calculations were carried out using M06-2X level of theory with consideration of split basis set [The adsorption of gas molecules (C package . As a moasis set . It is iasis set . A 6-31Gasis set . All atoEads) was calculated by using the following Equation:6H6 or CH2O), respectively, whereas The adsorption energy of gas molecules (r (BSSE)  was appl3O4 NPs. Neglecting the characteristic steps corresponding to the SiC step bunching , the as-grown graphene surface in 3O4 NPs onto graphene was investigated by means of XPS. The elemental compositions of as-grown EG/SiC prior to and after deposition of Fe3O4 NPs (Fe3O4/EG/SiC) were obtained to demonstrate the presence of Fe3O4 NPs on the latter sample. The XPS survey spectra for EG/SiC sample and Fe3O4/EG/SiC sample are shown in 3O4/EG/SiC showed the presence of iron and gold (Au) with XPS peaks Fe2p, Au4d, and Au4f found at binding energy position about 711 eV, 340 eV and 84 eV [3O4/EG/SiC are shown in 3O4/EG/SiC, based on the XPS O1s and C1s core level spectra has been performed. An increase of O, estimated to be a fivefold increase, and a decrease of C, reduced to about half for Fe3O4/EG/SiC, were observed. The strong increase in oxygen signal is shown for Fe3O4/EG/SiC sample, with an additional peak at binding energy position 530.0 eV, in good agreement with the presence of the chemical structure Fe-O in Fe3O4 NPs. XPS Fe2p core level spectra before and after deposition of Fe3O4 NPs are shown in 3O4/EG/SiC. The corresponding XPS C1s core level spectra for EG/SiC and Fe3O4/EG/SiC are presented in The effect of deposition of Fend 84 eV . The gol3O4 NPs, gas measurements were performed. 2O. The sensor was exposed to CH2O and C6H6 in a dry background of synthetic air using concentrations ranging from 5 parts per million (ppm) to 1 ppb, and a pulse duration of 30 min. Tests to identify a good operating temperature have been conducted and 150 °C, which is a good compromise between stability and high response, was chosen for all measurements shown here. Similar measurements with pure graphene did not show any response to both test gases. In an earlier work of ours [3O4 nanoparticle-decorated graphene sensors can be used for formaldehyde and benzene detection, but not over such a large range and down to a single ppb. The same measurement was repeated twice in the same environment and was repeated once more approximately one year later with the same sensor. The measured responses show some small deviations between the measurements without a clear trend of decreasing response over time. The sensor response shown here is from the first measurement. The response is defined as R is the saturated resistance signal and R0 corresponds to the baseline resistance before the gas exposure. The raw sensor signal in 6H6 at the sensor surface at 150 °C  is easily reached with a relative response above 0.1%.After the successful decoration of the graphene surface with Fe of ours , it has 0 °C see . The reldR/dt (after smoothening the data with a 500-point moving average filter), where the time derivative signal reaches its maximum value after about 50 s. It is evident from the insert that dR/dt exhibits distinct peaks in response to CH2O and the peak values are concentration dependent. Because of the smaller time constants needed to reach maximum dR/dt, the actual sensor signal does not have to be saturated when using the rate of change instead of the absolute value of resistance. It is therefore possible to significantly improve the speed of the sensor by utilizing dR/dt as a sensor parameter. Besides a very good sensitivity, also time constants of these measurements need to be addressed. For an application in indoor air monitoring, the time constants for the sensor should be in the range of half a minute to several minutes, depending on the application. However, as shown in 3O4-decorated epitaxial graphene was performed. Energetically favored adsorption configurations of considered gas molecules on PEG and DEG are illustrated in 6H6 on DEG, Eads = 1.795 eV, is significantly higher than on PEG with a value of Eads = 0.284 eV. In the absence of the Fe3O4 nanoparticle, C6H6 molecule adsorbs in the flat geometry, which is parallel to the surface. In this case, the center of C6H6 ring lays on top of a carbon atom belonging to graphene. On the other hand, a functionalization of graphene with Fe3O4 causes the formation of a strong chemical bond between C6H6 ring and one of the Fe atoms, facilitating the C6H6 adsorption in the tilted geometry regarding the graphene surface. Like the situation for C6H6, we found that CH2O molecule prefers to accommodate in a parallel geometry to graphene surface with weak adsorption energy of 0.149 eV, while the energetically preferred adsorption mode for CH2O on DEG is related to the formation of strong Fe-O covalent bond with a bond length of the 1.98 Å. The calculated adsorption energy of 1.870 eV is much higher than the value predicted for adsorption on pristine graphene sample, implying the strong chemisorption case. To summarize, one can conclude that weak physisorption of gas molecules on non-decorated graphene is immediately changed to strong chemisorption when iron oxide NPs are involved in adsorption phenomena. From an experimental point of view, the observed chemical similarity between adsorption configurations of benzene and formaldehyde enables us to anticipate comparable responses of Fe3O4-decorated epitaxial graphene to exposure of both gases, with slightly higher sensitivity towards formaldehyde.An efficiency of the decoration approach towards gas sensing can be directly demonstrated and validated by the DFT results. For this aim, a comparative analysis of the adsorption of benzene and formaldehyde onto non-decorated and Fe3O4 NPs. We could verify the decoration and the integrity of the graphene surface using AFM and XPS. With this sensor platform, concentrations down to a single ppb of toxic VOCs could be quantitatively measured, which makes it very promising for air quality monitoring. DFT calculations show that the gas molecules are more likely to bind to the decorated sensor surface. Moreover, by evaluating the first-order time derivative of the sensor signal, it was possible to significantly enhance the speed of the sensor, yielding response times of less than one minute, in turn allowing sampling rates desired in air quality monitoring.Benzene and Formaldehyde sensing properties were investigated using epitaxially grown graphene on silicon carbide decorated with Fe


Transactive response DNA-binding protein 43 kDa (TDP-43) was identified as a major disease-associated component in the brain of patients with amyotrophic lateral sclerosis (ALS), as well as the largest subset of patients with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U), which characteristically exhibits cytoplasmic inclusions that are positive for ubiquitin but negative for tau and α-synuclein. TDP-43 pathology occurs in distinct brain regions, involves disparate brain networks, and features accumulation of misfolded proteins in various cell types and in different neuroanatomical regions. The clinical phenotypes of ALS and FTLD-TDP  correlate with characteristic distribution patterns of the underlying pathology across specific brain regions with disease progression. Recent studies support the idea that pathological protein spreads from neuron to neuron via axonal transport in a hierarchical manner. However, little is known to date about the basis of the selective cellular and regional vulnerability, although the information would have important implications for the development of targeted and personalized therapies. Here, we aim to summarize recent advances in the neuropathology, genetics and animal models of TDP-43 proteinopathy, and their relationship to clinical phenotypes for the underlying selective neuronal and regional susceptibilities. Finally, we attempt to integrate these findings into the emerging picture of TDP-43 proteinopathy, and to highlight key issues for future therapy and research. The pathology of proteinopathies appears in distinct brain regions, involves disparate brain networks, and features accumulation of misfolded proteins in various cell types and in different neuroanatomical regions. The clinical phenotypes of these diseases correlate with the characteristic distribution patterns of the underlying pathology across specific brain regions with disease progression. Individuals may exhibit rapid or slow neurodegeneration even within the same syndrome. However, little is known to date about the reasons for this, although an understanding of the basis of the selective cellular and regional vulnerability would be helpful for the development of targeted and personalized therapies. Notably, there is growing evidence that these proteinopathies are modulated by genetic risk factors, differences in the conformation of pathological proteins, and various environmental factors. These factors may play a role in differential cellular susceptibility or resilience, and hence result in phenotypic diversity.Transactive response DNA-binding protein 43 kDa (TDP-43) was identified as a major disease-associated component in the brain of patients with amyotrophic lateral sclerosis (ALS) and the largest subset of patients with frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) , 116. FTIn this review, we summarize recent advances in the neuropathology, genetics and animal models of TDP-43 proteinopathy, and discuss their relationship to clinical phenotypes, their place in the emerging picture of TDP-43 proteinopathy, and their relevance to the development of therapeutic interventions.TDP-43 was originally discovered in a screen for protein factors that bind the long terminal repeat transactive response element of HIV-1 . SeveralTDP-43 shuttles between the nucleus and cytoplasm under physiological conditions, and a nuclear localization signal enables the active import of the protein into the nucleus , 159. ThThe neurotoxicity of pathological TDP-43 protein has not been fully clarified. Although the redistribution of TDP-43 from the nucleus to the cytoplasm is generally considered to cause loss of TDP-43 nuclear function , some stBiochemical, immunochemical and protein-chemical analyses of materials isolated from the brains of patients with TDP-43 proteinopathies have uncovered the characteristic features of pathological TDP-43. Immunoelectron-microscopic studies demonstrated that the protein accumulates as abnormal fibrils or filaments with cellular organelles, irregularly shaped structures and lipofuscins. The fibrils appear to be amyloid-like, even though they are intracellular proteins, whereas the classical histopathological “amyloid” exists as extracellular proteinaceous deposits exhibiting beta sheet structure , 68, 116As in other neurodegenerative diseases, hyperphosphorylation seems to occur in the aggregated TDP-43 proteins. Many phosphorylation sites, including serine, threonine, or tyrosine residues, have been reported within TDP-43 in cellular experiments, and these residues may have some functional role in TDP-43. However, intensive immunochemical studies using more than 20 phosphorylation-site-specific antibodies for the human TDP-43 sequence revealed that several of the antibodies  stained the pathological TDP-43 inclusions. These antibodies specifically recognize hyperphosphorylated full-length and cleaved forms of abnormal TDP-43, and thus are frequently used for immunostaining and immunoblotting studies , 68. TheNotably, patients with PGRN gene (GRN) mutations have high frequency of neuronal intranuclear inclusions (NIIs) containing TDP-43. The formation mechanism of NIIs is still unknown, but one possibility is that the production of NIIs might be associated with the deficiency of PGRN, which is involved in the regulation of lysosomal function since this would lead to the increased lysosomal gene expression and protein levels , 145 Fia.Previous research on physiological microenvironments associated with neurodegeneration in TDP-43 proteinopathy has focused on stress granules (SGs). These are membrane-less discrete cytoplasmic structures, containing mRNA and associated proteins that form in cells as a protective response to stress . SGs havIn addition to neurons, TDP-43 is abundantly expressed in glia, as well as many other cell types. Oligodendrocytes with accumulations of pTDP-43 frequently appear in FTLD/ALS brains , 26, andRecent studies of mice with TDP-43 pathology induced with TDP-43 from patients with TDP-43 proteinopathy established that white matter oligodendrocytes and astrocytes are capable of developing phosphorylated TDP-43 aggregates , and abuMany neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS), are classified according to their clinical symptoms and neuropathological features. Indeed, major clinical symptoms correspond well to the anatomical brain regions showing neuronal loss with reactive gliosis. These pathological changes are associated with the accumulation of abnormal proteins with conformational changes and modifications, which occur intracellularly in neurons or glial cells, or extracellularly .ALS and FTLD-TDP are sporadic and familial neurodegenerative diseases characterized neuropathologically by cellular aggregates of TDP-43. The differences in TDP-43 pathology are associated with differences in clinical manifestations, and ALS and FTLD are generally diagnosed on the basis of clinical symptoms. However, some patients may show symptoms common to both diseases. For example, clinical overlap between ALS and behavioral variant frontotemporal dementia (bvFTD) has been recognized, with up to 50% of ALS patients showing some executive function deficits , 127. AlThere are three major clinical phenotypes of frontotemporal lobar degeneration (FTD): bvFTD, semantic variant primary progressive aphasia (svPPA) and nonfluent/agrammatic primary progressive aphasia (naPPA) , 44, 48.Loss of empathy, apathy, and selfishness are more frequent features of bvFTD, but may be found commonly in all subtypes . These sIn FTLD-TDP and ALS, neuronal loss, reactive gliosis, microglial activation and superficial laminar spongiosis are seen in the neocortex and hippocampus . TDP-43 The deposited protein is hyperphosphorylated, ubiquitinated and abnormally cleaved to generate C-terminal fragments , 68, 116TDP-43-positive NCIs are frequently found in the frontotemporal neocortex and dentate granule cells of the hippocampus in ALS, FTLD and FTLD–MND , 157. ThTDP-43 pathology is typically classified into four subtypes (types A–D) in FTLD-TDP, based on the predominant type of TDP-43-positive structures and their distribution . Type A TDP pathology should be evaluated from multiple points of view. Neuropathological comorbidity has recently attracted attention. TDP-43 pathology frequently appears in other neurodegenerative diseases, including AD, argyrophilic grain disease (AGD), dementia with Lewy bodies (DLB), chronic traumatic encephalopathy (CTE), hippocampal sclerosis (HS), and Guam ALS , 162. CoTDP-43 pathology is present in a high proportion of cognitively normal elderly persons . In caseGRN mutation [As we described above, TDP-43 pathology frequently occurs in AD and DLB cases. In 20–30% of these cases, the morphological pattern of cortical TDP-43 immunoreactivity and the band pattern of C-terminal fragments of insoluble TDP-43 on immunoblots are similar to those of type A FTLD-TDP, which is seen in cases with mutation .TMEM106B protective (GG) and risk (CC) haplotypes (SNP rs3173615 encoding p.T185S) is high in type-α compared to type-β in non-FTLD-TDP cases [Another study classified TDP-43 pathology in non-FTLD brains into two subtypes, TDP type-α and TDP type-β. The former shows typical TDP-43 immunoreactive inclusions, while the latter is characterized by the presence of TDP-43 immunoreactivity adjacent to/associated with NFTs in the same neuron . The ageDP cases  Fig. 6)TMEM106B Recently, the new term limbic-predominant age-related TDP-43 encephalopathy (LATE) was proposed to describe the presence of TDP-positive lesions in AD, as well as in older adults . LATE negranulin precursor (GRN) [valosin-containing protein (VCP) [TAR DNA-binding protein (TARDBP) [chromosome 9 open reading frame 72 gene (C9orf72) [sequestosome 1 (SQSTM1) [ubiquilin 2 (UBQLN2) [TANK-binding kinase 1(TBK1) [T cell-restricted intracellular antigen-1 gene (TIA1) [, cyclin F (CCNF) [, coiled-coil-helix-coiled-coil-helix domain-containing protein 10 (CHCHD10) [OPTN) [SOD1) has also been linked with ALS [C9orf72 repeat expansion is the most frequent genetic factor in sporadic and familial cases [C9orf72 expansion repeat mutation have additional p62-positive and TDP-43-negative NCIs, which are composed of dipeptide repeat proteins (translated from the C9orf72 expansion repeats) [C9orf72 repeat expansion carriers [C9orf72 repeat expansion might have high incidences of psychiatric illness [GRN is mostly associated with type A, and C9orf72 with type B, or less commonly with type A [The pathological classification of TDP-43 proteinopathy is supported by clinical and genetic correlations. svPPA is associated with type C, and FTLD-MND or clinical signs of MND with type B, and naPPA with type A, although bvFTD is not as strongly correlated with any special pathological type , 85, 100or (GRN) , 42, valin (VCP) , TAR DNA(TARDBP) , chromosC9orf72) , sequest(SQSTM1) , ubiquil(UBQLN2) , TANK-bi 1(TBK1) , T cell-e (TIA1) , cyclin F (CCNF) , coiled-CHCHD10)  and opti) [OPTN) . Superoxwith ALS . C9orf72al cases , 66, butcarriers , 138, alcarriers . Also, f illness . GRN is h type A . Associah type A . Type D h type A . As a reh type A .APOE), granulin (GRN), transmembrane protein 106B (TMEM106B), ATP-binding cassette sub-family member 9 (ABCC9) and potassium channel subfamily M regulatory beta subunit 2 (KCNMB2), have been reported to be risk alleles associated with pathological manifestations in FTLD-TDP and LATE [Various related genes, apolipoprotein E  since it is considered to reflect the TDP pathology in human brain. However, the results are conflicting. One report found that ALS and FTLD patients had higher TDP-43 levels in CSF, compared to control patients , but anocontrols . These rcontrols . Furthercontrols , 54. HowSeveral neuroimaging studies of FTLD-TDP and ALS have been reported. Volumetric structural MRI scans, cortical thickness analyses, diffusor tensor imaging, and to a lesser extent functional techniques, including functional MRI and FDG-PET have been utilized to aid diagnosis in clinical practice , 33. GenImaging studies indicate that neurodegenerative diseases are caused by degeneration within specific intrinsic functional connectivity networks vulnerable to their pathologies , 165. fMC9orf72 in 2011, the role of the repeat expansion in TDP-43 proteinopathy has been investigated by means of imaging techniques. Recent neuroimaging analyses demonstrate that patients with C9orf72-positive bvFTD show symmetric atrophy most prominently in the anterior insula, anterior cingulate, and frontotemporal cortex, in keeping with the sporadic bvFTD-associated pattern [C9orf72 carriers exhibit atrophy of the parietal lobe [C9orf72 expansion show convergent large-scale network breakdowns despite having distinctive atrophy patterns [C9orf72 genotype, compared to the relatively limited extramotor pathology in patients with C9orf72-negative ALS [C9orf72 genotype [C9orf72-positive ALS had a more widespread central nervous system involvement than C9orf72-negative ALS, with or without FTD comorbidity [Since the discovery of  pattern , 155. Sotal lobe , 155 andtal lobe , 155, whpatterns . In ALS,tive ALS . The chagenotype . As for orbidity .Understanding the underlying causes is crucial for building effective treatment strategies. As described, neurodegeneration in ALS selectively involves the upper and lower motor neurons within the pyramidal motor system . Motor nTDP-43 accumulations are detected in unaffected cells in ALS, though some Betz cells, which mainly degenerate in ALS, are known to lack TDP-43 accumulation , 59. ThiMany physiological and pathological studies indicate system degeneration in ALS and FTLD. ALS patients present focal clinical symptoms showing upper or lower motor neuron predominance and clear alterations in motor function in one body region at a specific time, typically within a timescale of weeks to months . The neuThe vulnerable circuits and cell types in FTD have recently been investigated. Especially in bvFTD, von Economo neurons and fork cells within the anterior cingulate and ventral anterior insular cortices are affected in early degeneration , 111. ThThe different banding patterns of abnormal TDP-43 fragments in ALS and FTLD may represent different TDP-43 strains with different conformations. Biochemical analysis of different brain regions and spinal cord in individual patients with TDP-43 proteinopathy revealed three distinct C-terminal banding patterns , 68, 147In prion diseases such as CJD and bovine spongiform encephalopathy, the different patterns of protease-resistant bands can be used to identify the etiology of the diseases , 120. FoA hypothesis that has received much attention recently is that neurodegeneration might spread from cell to cell by a mechanism that is seen in prion diseases . AccordiDrosophila models of ALS indicated that repetitive trauma promotes the accumulation of TDP-43, with SGs in the brain, and also exacerbates neurodegenerative phenotypes as indicated by mortality and locomotor dysfunction [Clinical studies suggest that traumatic brain injury (TBI) might be associated with an elevated risk of ALS, as well as other devastating neurodegenerative diseases , 126. Chfunction . A singlfunction . The preIn terms of selective vulnerability, exposures outside the CNS may influence motor neurons themselves. Especially in the spinal cord, motor neurons lack the protection of the blood–brain barrier at the axon termini. The neuromuscular junction seems vulnerable and the retrograde transport of aberrant factors may be able to occur at this site in patients .The development of multiple experimental models of TDP-43 proteinopathy is essential to clarify the pathogenic mechanism underlying TDP-43 propagation. Protease-resistant banding patterns of pathological insoluble TDP-43 are useful for biochemical classification of the subtypes . Cell-toE. coli, and so it is difficult to form synthetic amyloid-like TDP-43 fibrils [TARDBP gene, associated with downregulation of two known modifiers of neurodegeneration, Atxn2 and Arid4a, and upregulation of myelination and translation genes. Identifying the environmental factors that influence the delicate balance in the transcriptome of the brain might be a promising topic for future studies [As in the cases of tau and α-synuclein, the prion-like propagation of TDP-43 protein in vivo has been investigated by injecting brain extracts from aged transgenic mice showing pathology, or synthetic protein fibrils, or extracts from brains of patients into mouse brains. Animal models of TDP-43 propagation are not easy to prepare, compared to models of tau and α-synuclein propagation. It is very difficult to prepare substantial amounts of natively folded recombinant proteins or fragments in  fibrils . In addi fibrils , which m fibrils . The pro studies . Other p studies , 154. FuIncreasing evidence indicates that ALS/FTLD-TDP disease progression is induced by cell-to-cell propagation of intracellular aggregated proteins. Extensive biochemical and biophysical studies needed to clarify the pathomechanisms leading to the presence of distinct pathogenic TDP-43 strains in patients’ brains. Using genome editing technology, it should be possible to generate large knock-in animal models or human iPSCs to overcome overexpression, off-target, and mosaic effects, which could offer promising approaches for therapy . In addiGenome-wide association studies have uncovered over 100 loci that are linked with ALS/FTLD . Some ALRecent years have seen a rapid development of fluid biomarkers for FTLD. In C9orf72 mutation carriers, the concentration of poly GP, which is produced as an abnormal dipeptide repeat protein, could be a target biomarker, in addition to TDP-43 . The devIdentification of imaging biomarkers, such as amyloid-imaging probes, that predict future clinical syndrome will be critical for the identification of suitable candidates for clinical trials. In addition, clinicopathological studies of TDP-43 are expected to provide deeper insights into disease mechanisms, although we should pay attention to the limitations involved in investigating patients with end-stage disease. Such studies should provide further support for the idea that clinical heterogeneity in patients is associated with the heterogeneity of TDP-43 neuropathology resulting from different TDP-43 strains.Small molecular compounds that can pass through the blood–brain barrier, enter the cells, and bind to intracellular aggregates of abnormal proteins would be promising candidates for pharmacotherapy of neurodegenerative diseases by promoting clearance of pathological TDP-43 proteins, e.g., by activating degradation or protein quality-control systems. Antibody therapy may also be feasible.In conclusion, combinations of behavioral, functional, and physiological approaches to study the changes in the neuroanatomical system will be crucial for the development of future therapy. In addition, the clinically stereotyped nature of the disease progression, and neuronal brain imaging studies, suggests that a system level vulnerability is a significant aspect of the biological underpinning of TDP-43 proteinopathy.


Supplemental Digital Content is available in the text The aim of present study is to quantitatively evaluate the association between different interventions and abstinence rates based on network meta-analysis.P values (SUCRA). Funnel plot symmetry was used to detect publication bias. Moreover, pair-wised comparison was also conducted to determine the statistical difference and forest plots were generated to calculate the differences between the groups. The Grades of Recommendations Assessment, Development and Evaluation (GRADE) criteria were utilized for the recommendations of evidence from pairwise direct comparisons.Following PRISMA guidelines, randomized clinical trials that compared different alcoholic interventions for alcohol use disorders associated with abstinence rates in treatment sessions or/and follow-up sessions were recruited. Main data synthesis was performed by Bayesian random-effects network meta-analyses, and the relative ranking of interventions was estimated by cumulative probability A total of 137 RCTs containing 27,282 participants and 8 variations of psychotherapy, pharmacotherapy, contingency management, and brief intervention used as treatment interventions were included. In summary, contingency management plus psychotherapy was demonstrated to be effective and possessed the best rank of achieving the highest abstinence rate in treatment sessions . Pharmacotherapy plus psychotherapy also revealed its efficacy and was associated with the highest abstinence rate in follow-up sessions . More importantly, psychotherapy alone was demonstrated not to be associated with higher abstinence rates in both treatment  and follow-up sessions , yet pharmacotherapy seemed to be the only intervention associated with higher abstinence rates compared to controls in both sessions  .Contingency management plus psychotherapy and pharmacotherapy plus psychotherapy were demonstrated to be associated with the highest abstinence rates in treatment sessions and follow-up sessions, respectively. However, contingency management and pharmacotherapy seemed to be the substantial crucial factors allowing for the maintenance of the highest abstinence rates in respective sessions, although we need more evidence for further validation. AUDs remain widespread in developed countries, and they are the key factor in liver cirrhosis. Alcohol affects the health of not only the drinking individual but also the fetus in pregnant women. The neurotoxic effects of alcohol may cause a range of congenital defects, including fetal alcohol spectrum disorders and fetal death, stillbirth, and infant and child mortality.The AUD is also a psychiatric diagnosis–8 which is similar to other chronic illnesses, AUDs have physiological and behavioral components as well as relapse rates. Therefore, AUDs may cause a wide range of medical, psychological, social, personal, and economic problems.Alcohol use disorders (AUDs), encompassing various serious forms of consumption, is the leading preventable cause of morbidity and a major contributor to health care costs,–12 It comprises systematic and stepped continuous therapy but requires costly support from experienced psychologists. Pharmacotherapy was also considered physiologically effective, and some medications were approved by the US Food and Drug Administration (FDA).–14 Additionally, some brief interventions were applied in the community population, such as simple group counseling, brief mobile electronic program monitoring, and even brief educational interventions derived from psychotherapy.–16 All these approaches are now utilized for treating AUDs, but at the same time, the effectiveness of these interventions compared with one another is controversial. More importantly, the association of different alcoholic interventions and abstinence rates has not been fully analyzed.Currently, various alcoholic interventions are reportedly aiming to reduce alcohol abuse. For example, psychotherapy is described as psychologically based interventions that exclude any pharmacological treatments and are aimed at reducing consumption behavior or alcohol-related problems. Therefore, a comprehensive systematic review and Bayesian network meta-analysis were conducted to summarize the evidence from RCTs comparing various alcoholic interventions for the elucidation of the association between alcoholic abstinence rates and interventions.Since the emergence of the concept of AUDs, hundreds of randomized controlled trials (RCTs) of various alcoholic interventions were reported. However, the availability of alcoholic management options poses a challenge when making evidence-based management decisions due to superior interventions not yet having been determined, although there was an initiative proposed years ago.2 Moreover, this review was registered online at the Research Registry Center with obtained UIN number review registry 532.This study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement extension for network meta-analysis.2.1Randomized clinical trials (RCTs) that compared any alcoholic interventions with no treatment control or with each other were considered eligible if they reported ≥ 2 weeks of treatment or/and follow-up sessions.Non-RCTs or observational trials, trials without sufficient parametric data, and trials without interesting  were excluded. In addition, papers of reviews, comments and basic science were also excluded.2.2Three globally recognized databases  were searched from inception to December 2017 without publication status restriction. The search strings were based on MeSH terms (example search strategy in MEDLINE is presented in Table S1 of supporting information). Full English texts had to be addressed if the trial was considered for inclusion. Study retrieval and identification were conducted by all members of our group under the guidance of the same eligibility criteria and search strategy.2.3General information and intervention-related characteristics were abstracted into a standardized form (Table S2 in the supporting information). For the outcome of interest, we preferred abstinence rates rather than other subjective data after unanimous discussion. We deemed abstinence the terminal purpose of alcoholic treatment, and the measurement of abstinence rates may be more accurate than continuous variables (such as the amount of drinking). Furthermore, even some objective parametric data  were considered inhomogeneous in different trials ; thus, these data were not appropriate for data synthesis. Nevertheless, data on abstinence rates, which was defined as no alcohol use, were essentially homogeneous for pooled estimation.–20 Data extraction was performed by 2 independent investigators, and any debates were resolved by group discussion.For extraction of outcomes, the raw data of abstinence rates in treatment sessions and in follow-up sessions were individually extracted for separate comparisons. Treatment sessions were defined as the whole therapeutic period under respective alcoholic intervention until the end of the treatment. Follow-up sessions were considered from the endpoint of treatment to the end of follow-up without any interventions in each group. Self-reports of no alcohol use, continuous negative detection of monitoring devices, or/and rates of continuous negative bio-sample testing were considered available abstinence data for comparison. Data of intention-to-treat patients were preferred for extraction. In addition, for those papers presenting only survival curves, Engauge Digitizer (version 4.1) was used to extract raw data of abstinence proportions.2.4 to address the bias risk of individual studies with following requirements:Included trials were assessed by the Cochrane Risk of Bias assessment tool1.Free of selection bias,2.Free of performance bias,3.Free of detection bias,4.Free of attrition bias,5.Free of reporting bias, and6.Free of other bias.A graphic summary of the overall and study-level risk of bias was conducted using Review Manager Software (version 5.3). Five factors that may reduce the quality of evidence were considered . Additionally, 3 factors that can enhance the quality of evidence were reviewed . Furthermore, in this approach, the rating of indirect estimate starts at the lowest rating of the 2 pairwise estimates that contribute as 1st-order loops to the indirect estimate but can be downgraded further for imprecision. All investigators assessed the quality of the examined studies through discussion until reaching agreement. Explanations for the Cochrane Summary of Findings Table of the GRADE system were made by the software GRADE profiler (version 3.6).To confirm the reliability and quality of the present study, the Grades of Recommendations Assessment, Development and Evaluation (GRADE) criteria were selected to assess the methodological quality of evidence.2.5–25 The surface under the cumulative ranking (SUCRA) probabilities of the P values were presented to clarify the pros and cons of different alcoholic interventions. The highest P value represented the possibility of achieving the highest abstinence rate, and these methods were described previously.–27 Odds ratios (ORs) derived from network meta-analysis were calculated to exhibit the comparison of different interventions. Publication bias was assessed by examining funnel-plot symmetry. Inconsistency model approach was used to test the consistency of main results based on the node-splitting analysis. No statistical inconsistency was shown at P > .05.Indirect pooled estimation of alcoholic interventions was conducted to make comprehensive network comparisons based on the Bayesian theorem. This approach can be considered an extension of the traditional pairwise meta-analysis because it incorporates both direct and indirect information through a common comparator to obtain estimates of the relative interventional effects on multiple intervention comparisons.Moreover, sensitivity analyses were performed to assess the robustness of the main outcomes. Random effects Bayesian network meta-analyses were also performed among the following: only USA trials; treatment and follow-up sessions ≥ 12 weeks; only continuous abstinence rates. All relative ORs and credible intervals (CIs) were estimated for sensitivity analysis.I2 index statistic) in the study design was used to estimate a data mode for using fixed- (I2 < 50%) or random- (I2 > 50%) effects models. The associated 95% CIs were calculated, the level of statistical significance was set at P < .05, and all statistical tests were 2-sided.Direct meta-analysis was conducted to complete direct comparisons for further investigation. In this condition, heterogeneity  .Data manipulation and statistical analyses of network meta-analysis and pairwise analysis were conducted using the Stata software package (version 12.0).3The searches identified 92,149 records, of which 2307 were considered relevant clinical studies after titles and abstracts were reviewed. Eventually, based on the review of full texts, 137 trials containing 27,282 participants were included for quantitative analysis. Most of them  were 2-arm studies, and the rest of them were 3-  or 4-arm  studies. Moreover, given the intensive review of studies, all relative interventions were classified into 5 categories: psychotherapy ; pharmacotherapy ; CM ; BI ; and control . We categorized studies according to these 5 interventions and summed 4 additional combinations  for the final estimation. The available direct comparisons and network plot of included trials are shown in Figure 3.1In general, all included 137 RCTs were published from 1979 to 2017, containing 27,282 cases. For 8 included interventions and controls, the sample sizes ranged between 79 and 8129  were conducted with random sequence generation, but only 57 trials (42%) applied blinding methods. Overall, included studies were considered to be at high risk of bias. The details of the overall and study-level risk of bias assessments were summarized .3.2P values, CM plus psychotherapy was ranked 1st in its association with the highest alcohol abstinence rate in treatment sessions (cumulative P = .61), followed by CM (P = .37), psychotherapy plus BI (P = .01), and pharmacotherapy plus BI (P = .01) . Network odds ratios (ORs) for each possible comparison of all 8 interventions and the control were estimated and presented in Figure 01) Fig. .P = .40), followed by pharmacotherapy (P = .31), pharmacotherapy plus BI (P = .17), CM (P = .08) and CM plus psychotherapy (P = .03) . After quantitative comparison, the network meta-analysis suggested that the control was associated with lower odds of abstinence than 4 interventions: pharmacotherapy, with an OR of 0.68 ; pharmacotherapy plus BI, with an OR of 0.70 ; pharmacotherapy plus psychotherapy, with an OR of 0.63 ; and psychotherapy, with an OR of 0.99  ; sessions ≥ 12 weeks ; and continuous abstinence . The relative ORs were estimated and presented in detail (Table S3 in supporting information). Overall, the results of sensitivity analysis were similar to the main outcomes.3.4I2 = 67.3%) (Table S4 in supporting information). Moreover, 16 reports from 12 trials covering 5 alcohol interventions compared with controls in follow-up session were pooled-analyzed . Overall, the application of alcohol interventions could significantly enhance abstinence rates compared with controls  with low heterogenicity (I2 = 12.2%) (Table S4 in supporting information) in follow-up sessions. Next, we further separated each intervention to make a comparison with controls in the treatment session. The results suggested that pharmacotherapy plus psychotherapy , pharmacotherapy , CM , BI , and psychotherapy plus BI  revealed significant differences for abstinence rates compared to controls in treatment sessions, yet psychotherapy did not . Meanwhile, we also observed that the application of pharmacotherapy exhibited significant differences with regard to abstinence rates in follow-up sessions , yet BI , rather than the application of psychotherapy  (Table S4 in supporting information). Therefore, we may understand that the application of some certain interventions may not reveal any benefits in both treatment and follow-up sessions.To further validate our main results, we conducted a direct meta-analysis to verify the significant differences. We 1st estimated 60 reports from 43 trials , which covered 6 interventions directly compared with controls, and the results indicated that the application of the intervention could significantly increase the alcoholic abstinence rate with significant differences in treatment sessions  with high heterogenicity (I2 = 70.7%). On the other hand, based on 9 reports from 6 trials, a direct meta-analysis of pharmacotherapy plus psychotherapy versus other interventions in follow-up sessions was conducted . The results illustrated that pharmacotherapy plus psychotherapy showed significant efficacy in increasing alcohol abstinence rates compared to other interventions  with low heterogenicity (I2 = 0%) (Table S5 in supporting information). For further exploration, we next separately compared CM plus psychotherapy with each specific intervention. However, only the direct comparison between CM plus psychotherapy and psychotherapy was available, and the results suggested that CM plus psychotherapy revealed a statistically higher rate of alcohol abstinence  in treatment sessions (Table S5 in supporting information). In follow-up sessions, pharmacotherapy plus psychotherapy was directly compared with 2 other interventions: pharmacotherapy plus BI and pharmacotherapy. With direct comparison, we observed that pharmacotherapy plus psychotherapy showed significant differences in alcoholic abstinence rates compared to psychotherapy alone  but not pharmacotherapy plus BI  (Table S5 in supporting information).As mentioned above, we addressed the finding that CM plus psychotherapy and pharmacotherapy plus psychotherapy were associated with the highest alcohol abstinence rates in the treatment session and follow-up session, respectively. To statistically determine their validation of practical benefit, we performed direct comparisons between CM plus psychotherapy and other interventions in treatment sessions and comparisons between pharmacotherapy plus psychotherapy and other interventions in follow-up sessions. A total of 11 reports from 8 trials reported the findings of CM plus psychotherapy versus other interventions . Pooled estimation demonstrated CM plus psychotherapy to have significant benefits in enhancing alcohol abstinence rates compared with other interventions in the treatment session  with high heterogenicity  (Table S6 in the supporting information). Based on all these results, we may conclude that our results did not reveal obvious publication bias and exhibited good consistency.We did not observe any evidence of publication bias in either treatment sessions or follow-up sessions according to funnel-plot asymmetry after quantitative calculations . Additionally, to test network coherence, the differences between the direct and indirect effects in the closed loops were estimated by a node-splitting model. This approach was used to access the network inconsistency and coherence. No significant difference was detected in either the treatment session or in the follow-up session after assessment , which was classified as high or moderate. In addition, for follow-up sessions, only 6 (17%) comparisons were rated as high or moderate (Table S7 in the supporting information).4In the present study, direct and indirect evidence from 137 RCTs containing 27,282 participants with AUDs was analyzed to elucidate the association of various alcoholic interventions with abstinence rates in treatment sessions and follow-up sessions based on Bayesian network meta-analysis. We demonstrated that alcoholic interventions were effective in both treatment sessions and follow-up sessions. In treatment sessions, the application of CM plus psychotherapy was associated with the highest probability of achieving the highest abstinence rate with a good quality of evidence. On the other hand, pharmacotherapy plus psychotherapy was associated with the highest abstinence rate in follow-up sessions, yet the recommendation of evidence was limited. Additionally, the applications of CM plus psychotherapy and pharmacotherapy plus psychotherapy were proven to have higher abstinence rates than other interventions at the statistically significant level. For the 1st time, although we quantitatively analyzed the association of various alcoholic interventions with abstinence rates, the objective results still need to be further explained and discussed. Petry et al 1st demonstrated that CM was associated with higher alcoholic abstinence rates, and this approach was demonstrated to be effective in the treatment of AUDs in the following years.–34 The CM was developed based on psychological therapy, and some psychiatrists refer to CM as part of psychotherapy. However, performing CM is directly aiming at abstinence, which is triggered by goods or money. This approach does not need complicated systematic psychotherapy, and the participants do not even need to meet the conductors, who may not even need to be professional psychologists either. We deemed CM an independent approach, which was the only “yes or no” alcoholic treatment procedure directly focusing on abstinence, and CM plus psychotherapy was proven to be associated with the highest probability of achieving abstinence. Here, speaking of psychotherapy, which focuses on motivational and behavioral switches, the results of the direct meta-analysis showed that psychotherapy alone did not confer any benefit to alcoholic abstinence compared to controls (Table S4 in the supporting information). Meanwhile, previous RCTs have illustrated that psychotherapy may ameliorate alcoholism regarding some subjective parametric data but exhibited no significant benefit in achieving or maintaining higher abstinence rates.–41 In addition, evidence-based medicine also proved that there was no substantial enhancement of abstinence using psychotherapy.–43 Thereby, we may conclude that CM was the key factor in achieving or maintaining the highest abstinence rates in treatment sessions.In treatment sessions, CM plus psychotherapy was verified to be the best intervention, associated with the highest alcohol abstinence rate, followed by CM. The CM is an intervention in which reinforcers, such as vouchers or prizes, are provided, typically multiple times per week, when individuals demonstrate substance abstinence.–14,44–47 Additionally, the direct meta-analysis in the present study concluded that pharmacotherapy was the only intervention that may increase the abstinence rate in follow-up sessions (Table S4 in the supporting information). Although pharmacotherapy plus psychotherapy possessed the best rank based on network comparison, direct meta-analysis exhibited that pharmacotherapy plus psychotherapy revealed no significant difference compared to pharmacotherapy plus BI (Table S5 in the supporting information). As mentioned above, psychotherapy conferred no benefit to abstinence, and a large sample RCT reported by Anton et al also concluded that pharmacotherapy may show similar efficacy on abstinence with or without psychotherapy. Moreover, BI was confirmed to validate the achievement of higher abstinence in treatment sessions (Table S4 in the supporting information),–51 yet it was also determined to have no efficacy on post-treatment session.–53 Thus, pharmacotherapy plus psychotherapy revealed similar benefits compared with pharmacotherapy plus BI. Taken together, these findings indicate that we regard pharmacotherapy as the crucial and maybe the only factor of achieving the highest alcoholic abstinence rates in follow-up sessions.On the other hand, for the outcomes of follow-up sessions, we determined that pharmacotherapy plus psychotherapy seemed to have the best chance of achieving the highest abstinence rate. Pharmacotherapy, which aims to lessen the physiological dependence for AUDs, was approved by the FDA and had been demonstrated to bring significant benefits to reduce alcoholism to maintain abstinence and prevent relapse.In summary, by network quantitative analysis, we concluded that CM plus psychotherapy possessed the best rank of achieving the highest abstinence rate in treatment sessions, and pharmacotherapy plus psychotherapy possessed the best rank in follow-up sessions. Despite the fact that psychotherapy was involved in the best rank in both sessions, we confirmed that it may not have relative efficacy. Meanwhile, it should be noticed that pharmacotherapy was the only intervention that was statistically confirmed as effective in both sessions (Table S4 and Table S5 in the supporting information). The CM revealed great efficacy in treatment sessions, but we do not have enough direct statistical evidence to estimate CM in follow-up sessions, although CM-related interventions were similar to pharmacotherapy-related sessions in maintaining the highest abstinence rates in follow-up sessions , so some conclusions could not be further confirmed, and the validation of direct comparisons could not be fully completed. Finally, ranking probabilities may be influenced by unequal numbers of trials per comparison and network configuration. For these reasons, these results require further statistical validation and should be interpreted with caution.In summary, the application of alcoholic interventions was effective to achieve and maintain abstinence rates. In addition, among these interventions, contingency management plus psychotherapy and pharmacotherapy plus psychotherapy were demonstrated to be associated with highest abstinence rates in treatment sessions and follow-up sessions, respectively. In addition, contingency management and pharmacotherapy seemed to be the key factors affecting alcoholic abstinence in treatment sessions and follow-up sessions, respectively. However, this conclusion still requires more evidence for further validation.The authors report no financial relationships with commercial interests.Conceptualization: Jiamin Gao.Data curation: Jiamin Gao.Formal analysis: Jiamin Gao.Funding acquisition: Jiamin Gao.Investigation: Jiamin Gao, Jun Cao.Methodology: Jiamin Gao.Project administration: Jiamin Gao, Yunyue Xiao.Resources: Jiamin Gao, Jun Cao.Software: Jiamin Gao, Jun Cao.Supervision: Jun Cao, Tao Guo, Yunyue Xiao.Validation: Jun Cao, Tao Guo.Visualization: Jun Cao, Tao Guo.Writing – original draft: Jun Cao, Tao Guo.Writing – review & editing: Tao Guo, Yunyue Xiao.