Our work demonstrates that the removal of less than 10% of the low-complexity sequence at N- and C-termini generates amyloid fibrils with comparable macroscopic features but different local structural arrangement. It highlights that the assembly mechanism of TDP-43, in addition to the aggregation of the hydrophobic region, is also driven by complex interactions involving low-complexity aggregation-prone segments that are a potential source of structural polymorphism.Aberrant aggregation of the transactive response DNA-binding protein (TDP-43) is associated with several lethal neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. Cytoplasmic neuronal inclusions of TDP-43 are enriched in various fragments of the low-complexity C-terminal domain and are associated with different neurotoxicity. Here we dissect the structural basis of TDP-43 polymorphism using magic-angle spinning solid-state NMR spectroscopy in combination with electron microscopy and Fourier-transform infrared spectroscopy. We demonstrate that various low-complexity C-terminal fragments, namely TDP-13 (TDP-43 Misfolding and deposition of proteins into fibrillar aggregates are causative of many neurodegenerative diseases, including Alzheimer’s and Parkinson’s disease, and many other cognitive disorders . These pTARDBP gene and widely expressed in neurons. The protein binds to DNA and RNA  essential for TDP-43 function, and disruption of these interactions contributes to TDP-43 dysfunction and pathology  1–81) , a nucleathology . Recentlynuclein .postmortem brain tissue of FTLD patients  adopt multiple structurally distinct molecular conformations  to TDP-35 (TDP-4390–414) and full-length TDP-43. We used a construct lacking the GaroS2 domain ) to demonstrate that although GaroS2 is critical for the formation of amyloid fibrils in the full-length construct, its removal still results in the formation of fibrillar aggregates with cross-β properties (in vitro but using a different aggregation procedure (in vitro aggregated TDP-43 (311–360) and TDP-43 (262–414), respectively. By comparing the NMR chemical shift signature of these various TDP-43 fibrillar polymorphs, as well as to those formed after seeding with proteins from bacterial inclusion bodies , TDP-11 (TDP-43 residues 300–399) and TDP-10 (TDP-43 residues 314–414).. The polymorphic capability of the TDP-43 CTD amyloid fibrils has significant relevance in TDP-43 pathology and understanding its structure and function can provide insights into the underlying mechanisms of neurodegenerative diseases and guide the development of effective therapeutical strategies. The three fragments used in our study comprise the hydrophobic segment (318–340) and the Q/N rich segment (341–367) but varied in sequences from the GaroS1 and GaroS2 subdomains. The nomenclature of different C-terminal segments is chosen to be consistent with Mompean et al. and Shenoy et al.  low-complexity constructs studied here have been selected to be shorter than the LCD (267–414) as defined by Eisenberg and coworkers . In TDP-E. coli and purified under denaturing conditions following the procedure reported previously  is a good indicator because the spectral dispersion in this region allows for unambiguous identification of most aromatic contributions. Phenylalanine and tyrosine signals are observed in the INEPT spectra  of the TDP-11 construct (Hα-Cα correlation peaks are indicated with dotted squares in 13C–13C spectroscopy with a short proton-driven spin diffusion (PDSD) mixing time of 50 ms to probe intra-residue correlations of rigid residues. The aliphatic region of the 13C–13C correlation spectra of CTF aggregates is displayed in 13C line width of ∼100 Hz , indicative of the presence of residues associated with a relatively well-ordered structural arrangement within the amyloid core. Comparable 13C line widths have been reported for TDP-43311–360 by Lu and coworkers  techniques. CP favors the detection of rigid residues in amyloid aggregates. Hence, we employed 2Doworkers , TDP-432g et al.  and for oworkers . However primarily arises from backbone carbonyl stretching vibrations and is directly related to secondary structure (−1) corresponds to CN stretching and NH bending. ATR-FTIR analysis also evidences a composite secondary structure in TDP-43 CTF fibrils (−1), β-sheet , turns  and random coil   spectroscopy. FT-IR is a powerful method to obtain a rapid analysis of the secondary structure of peptides and proteins in their aggregated state . We perftructure , while t fibrils ; Table 146 cm−1) . The throid core .13C–13C correlation experiments with a mixing time of 150 ms to favor the detection of sequential  connectivity  of the 2Dspectrum , suggestspectrum . These ispectrum . Unfortuspectrum .et al. have pointed out that the interactions of the Phe-Gly motifs are a main driving force for the TDP-43 prion domain fibril formation from droplets by using solution and solid-state NMR analysis . This observation is in line with our previous findings that the GaroS2 domain is involved in the TDP amyloid core . Recentlanalysis . Interesanalysis . Strikinanalysis . Althouganalysis  for TDP-analysis .in vitro conditions. The CTD of TDP-43 is composed of the hydrophobic domain (318–340) and the Q/N-rich domain (341–367), surrounded by two low-complexity domains (GaroS1 and GaroS2). By shortening the primary sequence of these two low-complexity domains, we generated three CTF constructs, and demonstrated that using in vitro conditions, already employed for full-length TDP-43 aggregation , TDP-11 (300–399) and TDP-10 (314–414). The presence of specific C-terminal fragments is one of the structural hallmarks of the pathology of TDP-43 proteinopathies. Interestingly, the length and the starting amino acid of the fragments seem to be controlled during the disease process, e.g., ∼18–35-kDa CTFs are found in the brains of ALS and FTLD patients , while tregation , triggerregation  by increregation . Here wef TDP-43 , indicatus study . Such obus study  and TDP- shared the same solid-state NMR spectral fingerprint; however, it was not the case for the CTF TDP-16 (267–414). Interestingly, in the present work, we show that shorter CTFs are more prone compared to the larger constructs TDP-43 and TDP-35, to structural polymorphism and to aggregate into distinct structures despite the absence of a few residues from the CTD. For TDP-11, we truncated a part of the GaroS2 domain (residues 400–414) that is not observed by solid-state NMR as the rigid component of the so-called “second core”  by Murray et al.  or fibrils and droplets  by digestion with NdeI and XhoI, followed by ligation using T4 DNA ligase (New England Biolabs).E. coli strain BL21 (DE3) pLysS transformed with the pET24-TDP vector. The expression and purifications were either in LB or M9 minimal media supplemented 15NH4Cl and 13C-glucose. The bacterial cultures were induced with 0.75 mM IPTG at OD600 0.8 and grown for 3 h 30min at 37°C. The cells were harvested by centrifugation  and frozen (−80°C) until purification. Frozen bacterial pellets were thawed and resuspended on ice in 20 ml of lysis buffer , sonicated , and centrifuged at 15,000 g for 1 h at 4°C. The pellet was then resuspended in 20 ml of lysis buffer supplemented with 2% Triton X-100, incubated at 37°C , and centrifuged . To remove the detergent, the pellet was washed with lysis buffer  and then centrifuged . The pellet thus obtained was resuspended in 10 ml extraction buffer  supplemented with dry guanidine hydrochloride until saturation and incubated overnight . The solution was sonicated and centrifuged . The protein was purified using a 5 ml Histrap HP column with a gradient of 10–500 mM imidazole in denaturing buffer . The fraction containing pure protein was pooled and dialyzed two times against 20 mM MES buffer pH 7.5 to remove urea and imidazole and was allowed to self-assemble for 1 week at room temperature. It was then extensively washed in water, centrifuged, and transferred to the ssNMR rotor. The sample quantity per rotor was ∼10 mg. Note that the limited solubility of CTD constructs has hampered further solution NMR analysis of soluble monomers.The TDP proteins were expressed and purified following the same protocol as described in our previous study . In shor1H Larmor frequency spectrometers (Bruker Biospin) at a MAS frequency of 11 kHz and 278K on a triple resonance HCN 3.2 mm MAS probe , using 90 kHz SPINAL-64 1H decoupling. DSS was used as an internal reference. The rigid part of the fibrils was probed using two-dimensional 13C–13C correlation spectra using a PDSD mixing scheme. A cross-polarization (CP) contact time of 1 ms was used in the initial transfer. Intra-residue correlations were probed using a PDSD mixing time of 50 ms for acquisition times of 20 ms (direct) and 7.5 ms (indirect), 288 scans for TDP-13, 480 scans for TDP-10 and 384 scans for TDP-11 and a recycle delay of 3 s led to a total experimental time of ∼5 days. Sequential correlations were probed using a PDSD mixing time of 150 ms for acquisition times of 20 ms (direct) and 6.3 ms (indirect), 792 scans and a recycle delay of 3 s leading to a total experimental time of ∼7 days. The 2D 1H-13C INEPT was recorded using acquisition times of 20 and 6 ms in direct and indirect dimensions, respectively, with 128 scans, for a total experimental time of ∼18 h. All two-dimensional spectra were processed using Bruker Topspin with a sine-bell shift of pi/4 and analyzed using CCPNMR  at the copper wavelength. The concentrated hydrated samples were mounted in a MicroLoops™ from Mitegen  on a goniometer head under the cold nitrogen flow. Each diffraction pattern corresponds to a 360° rotation along the phi axis with an exposure time of 720 s after subtraction of a ‘blank’ image of the same exposure time with only the loop on the goniometer head.TDP fibrils were diluted twice in 150 mM acetic acid buffer and applied to glow-discharged 300 mesh carbon-coated copper grids for 1 min, washed with water, stained with a 2% uranyl acetate (w/v) solution for 1 min, and dried in dark condition, and monitored in an FEI CM120 transmission electron microscope at an accelerating voltage of 120 kV under TEM low-dose mode at a magnification of 200,00×. TEM images were recorded using a Gatan USC1000 2 k × 2 k camera .−1 and a one-level zero filling. The hydrated TDP fibril samples were deposited (10 μl) on a germanium ATR crystal. Excess water was removed with a nitrogen gas flow, and the spectrum was recorded with 200 scans. The spectra were analyzed to determine the secondary structure elements of each protein with an algorithm based on a second-derivative function and a self-deconvolution procedure  to examine the number and wavenumber of individual bands within the spectral range of the amide I band .The ATR-FTIR spectroscopy was performed on a Nicolet 6700 FT-IR spectrometer  equipped with a liquid nitrogen-cooled mercury–cadmium–telluride detector , with a spectral resolution of 4 cm


Using this concentration in the seed amplification assay, ACI-5891.9 was able to neutralize the transactive response DNA-binding protein of 43 kDa pathogenic seeds derived from amyotrophic lateral sclerosis patient CSF. This translational work adds to the evidence of transmission of transactive response DNA-binding protein of 43 kDa pathology via CSF that could contribute to the non-contiguous pattern of clinical manifestations observed in amyotrophic lateral sclerosis and demonstrates the ability of a therapeutic monoclonal antibody to neutralize the toxic, extracellular seeding-competent transactive response DNA-binding protein of 43 kDa species in the CSF of apparently sporadic amyotrophic lateral sclerosis patients.In amyotrophic lateral sclerosis, a disease driven by abnormal transactive response DNA-binding protein of 43 kDa aggregation, CSF may contain pathological species of transactive response DNA-binding protein of 43 kDa contributing to the propagation of pathology and neuronal toxicity. These species, released in part by degenerating neurons, would act as a template for the aggregation of physiological protein contributing to the spread of pathology in the brain and spinal cord. In this study, a robust seed amplification assay was established to assess the presence of seeding-competent transactive response DNA-binding protein of 43 kDa species in CSF of apparently sporadic amyotrophic lateral sclerosis patients. These samples resulted in a significant acceleration of substrate aggregation differentiating the kinetics from healthy controls. In parallel, a second assay was developed to determine the level of target engagement that would be necessary to neutralize such species in human CSF by a therapeutic monoclonal antibody targeting transactive response DNA-binding protein of 43 kDa. For this, evaluation of the pharmacokinetic/pharmacodynamic effect for the monoclonal antibody, ACI-5891.9,  et al. report the detection of seeding-competent transactive response DNA-binding protein of 43 kDa (TDP-43) in CSF of amyotrophic lateral sclerosis patients using seed amplification assay. These seeding species can be neutralized by an antibody targeting the C-terminal of TDP-43 providing evidence for an immunotherapy approach to target amyotrophic lateral sclerosis.Audrain   Staging of TDP-43 pathology based on post-mortem immunohistochemical analysis of ALS brain and spinal cord has revealed the spatio-temporal spread of pathology from the initially affected region to other neuroanatomically connected brain regions.7 These data advocate for the presence of specific pathogenic forms of TDP-43 that can transmit from cell to cell and transform conformation of the native protein into pathogenic form in a prion-like mechanism. The cell-to-cell spreading of TDP-43 pathology in brain has been demonstrated in transgenic mouse models injected with pathogenic TDP-43 seeds derived from brains of patients with TDP-43 proteinopathies.8 More recently, spinal cord extracts from ALS patients were demonstrated to seed and spread TDP-43 pathology in ALS cerebral organoids.9 However, this mechanism by itself does not explain the non-continuous clinical manifestations frequently seen in ALS.10 To account for this, it has been hypothesized that CSF can be a vector for local and distal propagation with subsequent disease progression occurring independently either via axonal11 transport (anterograde and/or retrograde), tunneling nanotubes,12 extracellular vesicles13 and CSF transmission.14 In fact, the neurotoxicity conferred by CSF of ALS patients in vitro suggests the presence of one or more toxic factors in ALS CSF,15 supporting an implication in disease pathogenesis.16 In ALS, a primary TDP-43 proteinopathy, it is hypothesized that disease-associated misfolded TDP-43 forms resulting from neurodegeneration in the CNS might be present in patient’s CSF and thereby contribute to disease pathogenesis.10Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder with high unmet medical need due to the lack of disease-modifying therapies.in vitro12 and in vivo.17 Recently, an ultrasensitive assay based on amplification of seeds has emerged that enabled the detection of misfolded TDP-43 forms in CSF from confirmed genetic ALS patients that ‘seed’ or ‘induce’ aggregation of native TDP-43.18 Such a seed amplification assay (SAA) was originally conceptualized for antemortem detection of prions under the name of protein misfolding cyclic amplification and was based on the tendency of misfolded prion seeds to serve as template and induce native protein to shift to pathogenic conformation.19 Subsequently, the assay was successfully adapted to detect tau,20-22 α-synuclein23-25 and TDP-4318 seeds and has been reported under the name of real-time quaking-induced conversion. In this report, based on the recent consensus, such a technique is referred to as a SAA to reflect the proposal for a unifying terminology for this concept.26Several attempts have been made to establish the impact of ALS patient CSF exposure on TDP-43 aggregation and the associated downstream features 27 The published data clearly demonstrated, using multiple in vitro and in vivo models of ALS and frontotemporal dementia (FTD), that mAbs such as ACI-5891 with epitopes in the C-terminal region result in the reduction of pathology as well as confer neuroprotection from toxic patient brain-derived TDP-43 aggregates.27 Furthermore, the sequence of ACI-5891 was humanized to a therapeutic suitable format, ACI-5891.9, which involved insightful antibody engineering, producing a candidate with optimal potency and pharmacokinetics to maximize successful disease modification in upcoming clinical trials.28To study the seeding propensity of CSF obtained from ALS patients and establish the translational potential to neutralize TDP-43 seeds with immunotherapy, the C-terminal region targeting TDP-43 monoclonal antibody (mAb), ACI-5891, was assessed. Previously, we have established that this region of TDP-43 is key for efficacy.Thus, to study patient-derived extracellular seeding-competent TDP-43 and implications for clinical research, a robust SAA was established, employing a unique reaction substrate to both confirm the presence of seeding-competent TDP-43 species and determine the effect using a therapeutic mAb, ACI-5891.9. In parallel, a novel target engagement assay complemented with pharmacokinetic (PK) data from non-human primates (NHPs) was created to obtain the pivotal data to model the dose needed for target saturation in patients. Together, the data confirmed that CSF of apparently sporadic ALS patients contains species which can induce TDP-43 aggregation. Furthermore, when using the high-affinity mAb, ACI-5891.9, full inhibition of seed amplification was observed at clinically relevant concentrations. Hence, these data add to the growing body of literature to support that spreading of pathology in ALS occurs at least in part by species found in CSF. As such, treating patients with a passive immunotherapy approach by ACI-5891.9 should result in slowing and ameliorating devasting TDP-43-mediated neurodegenerative diseases such as ALS.Human CSF was provided by the Northeast Amyotrophic Lateral Sclerosis Biofluids Repository, Boston, USA. All samples were collected and used with written informed consent. Participants were not tested for all known ALS-causative genes, however, as these participants had no family history of ALS or FTD, they were classified as ‘apparently sporadic ALS’. Due to the small size of cohorts, the healthy controls (HCs) were not perfectly age matched, but majority of them overlapped with the ALS patients in age . CSF was352NNQNQGNMQREPNQAFGSGNNSYSGSNSGAAIGWGSASNAGSGSGFNGGFGSSMDSKSSGWGM414-OH) was chemically synthesized at Pepscan and obtained with a purity >99% (UPLC/UV215). Lyophilized powder was first dissolved in pure hexafluoro-2-propanol (HFIP) for 5 days under agitation at RT and let dry under the hood overnight (200 µL of HFIP for 0.5 mg of lyophilized substrate). Films were then resuspended in ice-cold H2O, aliquoted and kept at −80°C. Concentration was assessed by measuring UV absorbance at 280 nm using NanoDrop Spectrophotometer (Thermo Fisher).The TDP-43 peptide used as assay substrate (H-Reactions were performed in 96-well black/clear bottom plates (Thermo Fisher #265301) in a total volume of 200 µL per well and in the presence of one glass bead of 3 mm diameter (Supelco #104015) in each well. The reaction master mix contained 40 mM Tris-HCl, 5 mM DTT, 0.002% SDS, 50 µM thioflavin T (ThT) and 10 µM substrate peptide  was arbitrarily compared at 12 h between the different conditions. Additionally, the difference in time to reach 3000 rfu between each sample incubated with ACI-5891.9 versus isotype control (ratio) was assessed.The morphology of assay substrate assemblies was assessed by transmission electron microscopy in a CM 100 Biotwin transmission electron microscope (Philips) following adsorption onto formvar carbon-coated 400 mesh grids and negative staining with 1% uranyl acetate. The images were recorded using a bottom-mount TVIPS F416 camera (4K × 4K).Escherichia coli as a fusion protein with N-terminal His-SUMOstar tag. Protein expression was induced with 0.1 mM IPTG at 18°C for 16 h in Luria–Bertani medium (Sigma) supplemented with kanamycin. Following cell lysis, the protein was purified from the soluble fraction using affinity chromatography. Subsequently, the His-SUMOstar tag was removed using the SUMOstar Protease I (Tebu-bio). Finally, the protein was applied to size exclusion chromatography using 16/600 Superdex 75 column to obtain high-purity soluble protein in buffer .Human TDP-43 was expressed in d-galactosidase . Plates were washed between each incubation using a magnetic microplate washer (Quanterix Corporation) and read with a SR-X SIMOA® instrument (Quanterix Corporation). Assay optimizations were accomplished by adjusting the concentrations of mAbs either conjugated to the paramagnetic beads or after biotinylation. The molar ratio of biotin and linker used for mAb–biotin conjugations and the concentrations of SBG were also adjusted. Paramagnetic beads conjugated to the capture antibody were diluted in bead diluent (Quanterix Corporation), and human CSF samples were diluted in the general detector and sample diluent (Quanterix Corporation). SBG was diluted in SBG diluent (Quanterix Corporation). The lower limit of quantification was determined as the mean blank for a readout of average enzyme per bead (AEB) + 2.5 times the standard deviation. The lower limit of detection was set based on the signal at the lowest concentration of the calibrator (0.023 AEB). For calibrator, full length recombinant TDP-43 with a polyglycine tag in N-terminal was used (described above). The raw values in AEB rather than the extrapolated concentrations for CSF were used as the levels of TDP-43 signal was between lower limit of detection and lower limit of quantification.The Single Molecule Array  technology was used for the development of high-sensitivity custom immunoassays using two proprietary murine IgG2a mAbs binding TDP-43: ACI-5891 and ACI-5965. ACI-5891 was conjugated to paramagnetic beads (Quanterix Corporation) as a capture reagent, and ACI-5965 was biotinylated (Thermo Fisher) to be used as a detection antibody. Assays were performed using a three-step protocol with all incubation steps performed at 25°C on a microplate shaker set at 800 rpm (Quanterix Corporation). The incubation times were 30 min with the antibody-conjugated capture beads, 10 min with the biotinylated detection antibody, and 10 min with streptavidin-β-The assay described above was used to quantify free (unbound) TDP-43 in human CSF. Different concentrations (from 0 to 5000 ng/mL) of ACI-5891.9 (ACI-5891 humanized variant on human IgG1 backbone) were spiked into three different human CSF samples prior to use in the assay. This resulted in concentration-dependent saturation of the epitope by ACI-5891.9, preventing capture of TDP-43 by the ACI-5891-coated magnetic beads and allowing assessment of target engagement by measuring free TDP-43. A four-parameter logistic regression model was used to plot each curve for each human CSF sample independently.28 Murine complementarity-determining regions (CDRs) of ACI-5891 VH and VL were grafted onto selected frameworks VH1-69-2/VK2-28. Back-mutations were introduced at critical framework residues known to influence CDR conformation and based on the molecular model of ACI-5891 Fv domains to generate ACI-5891.9.28 For recombinant antibody productions, CHO cells were transiently transfected with equimolar quantities of heavy- and light-chain vectors. Antibodies were purified from supernatants by protein A chromatography . The identity and purity of the purified antibodies were confirmed using native and reducing SDS-PAGE. Concentrations of antibodies were measured using the NanoDrop Spectrophotometer (Thermo Fisher).The Fv domain of ACI-5891 was discovered by hybridoma technology, from mice immunized using the SupraAntigen® vaccine technology.in vivo study was approved by the national ethical guidelines and performed in an animal facility accredited by the provincial animal management office. NHPs ≥ 2 years old, weighing ≥ 2 kg, were purchased from Hainan Jingang Laboratory Animal Co. Ltd. ACI-5891.9 was administered by intravenous bolus injection single dose at 40 mg/kg (n = 4). Serum was collected from each animal at the following time points: pre-dose (−24), 0.05, 1, 2, 4, 8, 16, 24, 48, 168, 336, 504, 672, 840, 1008, 1176 and 1344 h (Day 56).The Serum PK time points were selected to accurately capture the initial antibody concentration at the start of the study, the distribution phase during the first 48 h and the typical inflexion point of the PK profile . Following this, weekly time points were collected for 8 weeks to characterize the elimination phase. PK parameters were estimated by non-linear mixed effect modelling using Monolix  by a two-compartmental model with linear elimination from central compartment. PK parameters are reported as typical population PK parameters, and the terminal half-life was derived from it.CSF was collected at the following time points: pre-dose (−24), 48 and 1344 h (Day 56), except for the NHP#1 for which both the pre-dose and 48 h time points were not collected as per protocol. The pre-dose time point allowed baseline quantification of TDP-43. The time point at 48 h post-dose allowed assessment of antibody penetration in the CNS when serum concentration was expected to be high, and the terminal time point was used to assess antibody concentration at the end of the study when antibody serum concentration had declined. Animals were checked daily for body condition, natural behaviour and appearance. Haematology and blood chemistry were performed pre-dose and at 168 h post-dose.y2 weighting.The total concentration of human antibody in NHP serum was determined by enzyme-linked immunosorbent assay (ELISA). A biotinylated mouse polyclonal anti-human kappa chain antibody (Southern Biotech) diluted to 1.5 µg/mL in phosphate buffered saline (PBS) with 0.05% Tween 20 was captured onto a streptavidin-coated microplate (Microcoat Biotechnologie GmbH) for 1 h at RT. Plates were washed four times with PBS with 0.05% Tween 20 and blocked with 100 µL of PBS with 1% bovine serum albumin (BSA) and 0.05% Tween 20 (dilution buffer) for 1 h at RT. Standard curves prepared from a 2-fold serial dilution starting at 50 ng/mL and test samples diluted in 1:100 dilution buffer supplemented with 1% cynomolgus monkey serum (Neo-Biotech) were loaded to the plates, incubated for 90 min at RT and washed as previously described. Bound human antibodies were detected with an anti-human kappa chain antibody conjugated to horseradish peroxidase (Southern Biotech) diluted to 4.0 ng/mL in dilution buffer. Plates were incubated for 30 min at RT and washed as previously described. Finally, TMB substrate (SeraCare) was added to each well and incubated for 10–15 min. Reactions were stopped by adding 100 µL of Stop Solution (Bethyl) to each well. Absorbance was read at 450 nm with a reference wavelength at 690 nm using a BioTek microplate reader. Concentrations of all serum samples were back-calculated against a reference standard using a non-linear four-parameter regression fit including a 1/2 (Jackson), at 20 nM (3 µg/mL) in PBS with 1% BSA and 0.05% Tween 20 for 1 h was performed at 37°C. Fifty microlitres per well of the TMB substrate (SeraCare) for 15 min at RT in the dark was used for the detection, and the reaction was stopped by adding 50 µL per well of H2SO4. Absorbance was read at 450 nm using a Tecan plate reader. Concentrations of CSF samples were also back-calculated using a reference standard using a non-linear four-parameter logistic model. The sensitivity of the assay was 0.93 ng/mL.The total concentration of human antibody in NHP CSF was also determined by ELISA. Briefly, coating of 96-well plates was done with an anti-human IgG at 10 nM (=1.5 µg/mL) in carbonate–bicarbonate buffer  using 50 µL/well at 4°C overnight. The washing steps were done using a plate washer and 200 µL per well of PBS/Tween 20 (0.05%). Blocking used 5% BSA in PBS/Tween 20 (0.05%) at RT for 30 min. Fifty microlitres per well of the sample  were incubated at 37°C for 1.5 h. CSF was diluted to 1/100. Incubation with the detection antibody, anti-human IgG F(ab’)A similar assay design to that described for CSF was used to assess target engagement in the serum of NHPs previously treated with ACI-5891.9 during the PK study. However, capture and detection antibodies were inverted. The RNA recognition motif (RRM) binding TDP-43 mAb ACI-5965 was coupled to acceptor beads to capture total TDP-43, while detection was performed using a biotinylated ACI-5891. Additionally, serum samples were diluted (1/10) in a diluent  specifically formulated for optimum assay performance in serum. The concentration of free TDP-43 was extrapolated in picograms per millilitre or as a fold change compared to the pre-dose serum for each animal.n values) and statistical tests are also indicated in ﬁgure legends. For data in t-test (two-tailed) was used whereas for data in post hoc analysis was used. The normal distribution of all data has been checked. P-values are reported as asterisks based on the following values: ****P < 0.0001, ***P < 0.001 and *P < 0.05.Graphs represent the mean of all samples in each group ± SEM or SD. Sample sizes  with characteristics suitable for SAA was identified. Following an initial lag phase, the substrate alone showed a concentration-dependent aggregation as monitored by ThT fluorescence, confirming the formation of β-sheet-containing fibrils . FollowiFollowing the assay qualification, CSF from 20 apparently sporadic ALS patients and 12 HC  technology was set up. A mouse anti-human C-terminal targeting, TDP-43 mAb, ACI-5891, was coupled to acceptor beads allowing capture of total TDP-43 in human CSF. However, when ACI-5891.9, a humanized version of ACI-5891, was pre-incubated in human CSF sample, immune complexes with TDP-43 were formed impeding their capture in the assay due to the unavailability of the ACI-5891 epitope (50) of 129.2 ng/mL was calculated for ACI-5891.9 confirming its high binding affinity to TDP-43 (90 (1100 ng/mL), these data demonstrated that a mAb concentration of approximately ≍1100 ng/mL was required to saturate all forms of TDP-43 in human CSF  has been shown to represent the amyloid core of pathological TDP-43 aggregates in the brain of ALS patients,46 regions adjacent to this core have been demonstrated to adopt β-sheet conformation.47 This was further demonstrated in this study by the ThT-positive fibrillar structure adopted by the assay substrate (amino acids 350–414).To maximize the chances of capture, neutralization and clearance of all seeding-potent forms of TDP-43 including the C-terminal fragments found to be enriched in TDP-43 aggregates found in patients,in vitro and in vivo for use of an immunotherapy approach with ACI-5891.9 can be used in support of clinical rational and dosing regimens for trials in patients with apparently sporadic ALS. Furthermore, the SAA and target engagement assays can be used to help stratify and follow efficacy of disease-modifying therapies in patients.In conclusion, in addition to providing the field with an optimized SAA, this study provides evidence for extracellular seeding-competent disease-associated TDP-43 species in the CSF of patients with apparently sporadic ALS. The extensive work to determine saturating conditions both fcad306_Supplementary_DataClick here for additional data file.


Here, we reported a fast preparation method of self-propagating high-temperature synthesis to realize in situ compositing of BiCuSeO and Cu2Se to optimize the service stability. Additionally, using the interface design by introducing graphene in these composites, the carrier mobility could be obviously enhanced, and the strong phonon scatterings could lead to lower lattice thermal conductivity. Ultimately, the Cu2Se-BiCuSeO-graphene composites presented excellent thermoelectric properties with a ZTmax value of ~2.82 at 1000 K and a ZTave value of ~1.73 from 473 K to 1000 K. This work provides a facile and effective strategy to largely improve the performance of Cu2Se-based thermoelectric materials, which could be further adopted in other thermoelectric systems.Thermoelectric materials can realize direct conversion between heat and electricity, showing excellent potential for waste heat recovery. Cu 2Se-based materials. They obtain a maximum ZT value of ~2.82 at 1000 K on Cu2Se-BiCuSeO-graphene composites.Here, the authors devise a synthesis strategy to optimize the stability and thermoelectric performance of Cu A good thermoelectric performance is needed for further application, which is generally determined by the dimensionless figure of merit, ZT = S2σT/κ. The parameters S, σ, κ, and T stand for the Seebeck coefficient, electrical conductivity, thermal conductivity, and absolute temperature, respectively. Thus, achieving an outstanding ZT value relies on a large power factor (PF = S2σ) and a low κ, where the κ is mainly consisted of carrier thermal conductivity (κc) and lattice thermal conductivity (κl)6. However, the strongly coupled correlation among S, σ, and κc has always been a big challenge for improving ZT values8. To overcome this problem, various strategies have been proposed, such as band structure engineering11, mobility optimization14, lattice thermal conductivity suppression17, and so on. To date, polycrystalline SnSe recorded the highest ZT value of ~3.1 at 783 K18. The maximum ZT (ZTmax) values over 2.0 have also been reported in Cu2Se20, PbTe22, GeTe24, and single-crystal SnSe26 thermoelectric materials.Waste heat will be a potential and huge energy resource if it can be properly utilized. Based on carrier and phonon transport, thermoelectric technology with the capacity of direct energy conversion between heat and electricity could be an attractive alternative for waste heat recovery, which has been widely investigated in the past decades2Se-based thermoelectric materials with high ZT values are highly promising and notable in mass production and commercial applications29. The reason for the high thermoelectric performance of Cu2Se could mainly be the highly mobile Cu+ ions, which cause strong phonon scattering and further lead to intrinsically low κl30. Meanwhile, the superionic nature has detrimental effects on the stability of Cu2Se and then damages its thermoelectric performance under some certain application conditions31. Thus, improving the stability and remaining a high ZT value is a challenge for Cu2Se-based materials. Inhibiting the long-range migration of Cu+ ions by introducing ion-blocking interfaces has been proposed as an effective strategy to improve the stability of Cu2Se34. It is proposed that Cu2Se/BiCuSeO interfaces could stabilize Cu2Se by blocking the long-range migration of Cu+ ions and obstructing the reduction reaction of Cu+ into Cu metal precipitates34.Due to the elemental abundance, non-toxicity, and wide working temperature range, Cu2Se (lower than 20 cm2 V−1 s−1) and BiCuSeO (lower than 10 cm2 V−1 s−1) hinders the further improvement of thermoelectric performance, especially the average ZT (ZTave) values39. Thus, optimizing carrier mobility is urgent for achieving higher ZT values of Cu2Se-based materials. Recently, carbon materials have been attractive in optimizing thermoelectric performance since it has excellent electrical properties42, which can introduce extra electrically conductive paths and are expected for improving the carrier mobility of composites. In addition, previous work has shown that introducing carbon structures can also decrease lattice thermal conductivity because of the interfacial phonon scattering and strong phonon reflection at phase interfaces41.However, the relatively low carrier mobility of Cu0.88Pb0.06Ca0.06CuSeO (BPCCSO)35 than that of pristine BiCuSeO and the robust level of carrier mobility in graphene41, the Cu2Se-BPCCSO-graphene composites were designed and prepared by a fast method of combining self-propagating high-temperature synthesis (SHS) with spark plasma sintering (SPS). We found that compositing with BPCCSO and graphene synergistically optimized electrical and thermal properties. Excellent electrical performance with the highest average PF (PFave) value of 14.49 μW cm−1 K−2 from 473 K to 1000 K was obtained, which could be mainly attributed to the optimized weighted mobility. Meanwhile, the multiple interfaces brought strong phonon scatterings to achieve a relatively low lattice thermal conductivity value of 0.11 W m−1 K−1 at 1000 K. Ultimately, attractively high ZT values including a ZTmax value of ~2.82 at 1000 K and a ZTave value of ~1.73 from 473 K to 1000 K were realized, which exhibited good potential for future practical applications.Considering the higher thermoelectric properties of Bi2Se-BPCCSO composites and explored the optimum compositions for further investigation. For comparison, Cu2Se and BPCCSO were also prepared by the same method in the meantime. All the samples were highly dense with a relative density of over 95%, as shown in Supplementary Table 2Se-BPCCSO composites in the whole temperature range was much higher than that of Cu2Se . For instance, the carrier concentration increased from 2.22×1020 cm−3 of Cu2Se to 2.80×1020 cm−3 of 0.9Cu2Se-0.1BPCCSO  is shown in Supplementary Fig.ρ) can be seen in Supplementary Table Cp) was estimated according to the Neumann–Kopp rule based on the measured Cp of Cu2Se in this work . The temperature-dependent κl and Lorentz constant (L) are presented in Supplementary Fig. 2Se and BPCCSO, the Cu2Se-BPCCSO composites had lower lattice thermal conductivity due to the interfacial phonon scatterings.By using the SHS-SPS method, we first prepared Cu2Se Fig. , which w2Se Fig. , Cu2Se-Bues Fig. . FurtherZT values were calculated and shown in Fig. 2Se-0.1BPCCSO sample possessed the highest ZTmax value of ~2.14 at 973 K. To test the service stability of such composites, we re-measured the 0.9Cu2Se-0.1BPCCSO sample and conducted heating and cooling cycles test. As shown in Supplementary Fig. ZT values all presented excellent stability with only small changes under experimental uncertainty. Furthermore, it is proposed that electrical conductivity has a positive correlation with critical voltage and can be a simple indicator to rapidly predict the stability of Cu2Se-based materials, where the higher electrical conductivity is, the better stability will be43. Therefore, the Cu2Se-BPCCSO composites should have better stability than Cu2Se. In addition, with the compositing of BPCCSO, the bending strength of Cu2Se-BPCCSO composites largely improved from ~45 MPa to ~70 MPa  can be analyzed via the X-ray diffraction (XRD) patterns, as shown in Supplementary Fig. 2Se-0.1BPCCSO-x wt% graphene composites  were almost the same and mainly consisted of two phases, Cu2Se and BPCCSO, indicating the high efficiency of the facile and fast preparation method for fabricating such complex composites. Owing to the low content of graphene, it was difficult to detect its phase by XRD. Besides the main phases, two minor peaks of Bi2O3 around 27.5° and 32.7° were observed in composites as well, which formed different scales of precipitates embedded in the matrix to strengthen phonon scatterings, bringing a further decrease in lattice thermal conductivity. The existence of Bi2O3 may be ascribed to the evaporation of Bi elements, and a small amount of Bi2O3 was unreacted as impurity phases.The phase structure of 0.9Cu2Se-0.1BPCCSO-x wt% graphene composites  is presented in Supplementary Fig. 2Se, which was confirmed by energy dispersive spectroscopy (EDS)  plane of BPCCSO was parallel to the (−206) plane of Cu2Se. Such in situ formed interfaces can effectively scatter phonons without impeding carrier transport, which is beneficial to decoupling carrier-phonon interactions. In addition, graphene with different sizes has been found in composites, as shown in Supplementary Fig. The fracture microstructure of 0.9Cuses Fig. , which wses Fig. . Parts o2Se-0.1BPCCSO-x wt% graphene composites  are presented in Fig. 2Se-0.1BPCCSO composite, the addition of graphene  further increased the electrical conductivity of the Cu2Se-BPCCSO-graphene composites, where the maximum value of 1538 S cm−1 was 36% higher than that of the 0.9Cu2Se-0.1BPCCSO composite without graphene and the value was approximately twice than that of Cu2Se. The results could be attributed to that the appropriate content of graphene could introduce extra electrically conductive paths and effectively improve the mobility of composites. Here, the Hall mobility reached 39.63 cm2 V−1 s−1, which was 57% higher than that of the 0.9Cu2Se-0.1BPCCSO composite  is shown in Fig. 2Se-0.1BPCCSO-x wt% graphene composites  were significantly improved within the entire temperature range, reaching 16.97 μW cm−1 K−2 at 1000 K of 0.9Cu2Se-0.1BPCCSO-0.035 wt% graphene, which was much higher than that of the Cu2Se-BPCCSO composite and Cu2Se sample. At the same time, the average PF values in the mid-to-high temperature range significantly increased. The average PF value of 0.9Cu2Se-0.1BPCCSO-0.035 wt% graphene reached 14.49 μW cm−1 K−2 in the temperature range from 473 K to 1000 K  composites possessed much higher weighted mobility than others, which was consistent with the Hall mobility and PF values, proving their good electrical transport properties.To get a better understanding of inherent electrical transport properties, the weighted mobility of the composites was calculated based on the measured Seebeck coefficient and electrical conductivity, as shown in Fig. 2Se-based thermoelectric materials are well known for their relatively low thermal conductivity due to their superionic nature. The temperature-dependent thermal conductivity of 0.9Cu2Se-0.1BPCCSO-x wt% graphene composites  is shown in Fig. x = 0.02 to x = 0.04, the thermal conductivity first increased and then decreased. Compared to 0.9Cu2Se-0.1BPCCSO, although 0.9Cu2Se-0.1BPCCSO-0.035 wt% graphene showed higher electrical conductivity, they had close thermal conductivity, which may be due to the much lower lattice thermal conductivity. Therefore, by subtracting carrier thermal conductivity, we obtained the lattice thermal conductivity of 0.9Cu2Se-0.1BPCCSO-x wt% graphene composites . The temperature-dependent lattice thermal conductivity and Lorentz constant (L) are presented in Supplementary Fig. 2Se-0.1BPCCSO composite sample possessed a relatively low lattice thermal conductivity, it is noted that the lattice thermal conductivity of the 0.9Cu2Se-0.1BPCCSO-x wt% graphene  composite samples was even lower. With the increasing content of graphene, the lattice thermal conductivity increased, which may be ascribed to the aggregation of graphene that reduced the interfacial phonon scattering effects. As revealed by the STEM results, due to the increase of multiple interfaces among BPCCSO, Cu2Se, and graphene, more phonon scattering sources strengthened the phonon scattering and improved the interfacial thermal resistance.In addition to the excellent electrical transport properties, Cuvl) and shear sound velocity (vs) were obtained. And the average sound velocity (va), phonon mean free path (lph), and elastic properties including Young’s modulus (E), Poisson ratio (νp), and Grüneisen parameter (γ) were calculated and listed in Supplementary Table va and lph values of 0.9Cu2Se-0.1BPCCSO-x wt% graphene  were also plotted in Fig. −1 to 1428 m s−1, which meant the softening of the phonons. Meanwhile, the declined phonon mean free path from 0.34 nm to 0.20 nm also proved the increase of phonon scattering.Furthermore, by measuring sound velocity, the longitudinal sound velocity  experiments were performed to have measured the generalized phonon density of states (GDOS) for Cuδ) was introduced, which is defined as ω) is the normalized intersection height of GDOS at a frequency ω51. The δ of Cu2Se-BPCCSO-nano graphite was 0.376. As a comparison, the δ of Cu2Se-nanographite, and BPCCSO-nano graphite were also calculated and the values were 0.437 and 0.514, respectively. The poor overlap provided a good explanation for the ultralow lattice thermal conductivity of Cu2Se-BPCCSO-graphene composites. Moreover, the peaks shifted to lower energy when measured at 600 K  were calculated and demonstrated in Fig. ZT values of the Cu2Se-BPCCSO-graphene composites were significantly improved. The ZT values of 0.9Cu2Se-0.1BPCCSO-0.035 wt% graphene reached 2.82 at 1000 K. Compared to Cu2Se and 0.9Cu2Se-0.1BPCCSO, the ZTmax value increased by ~50% and ~32%. Furthermore, the average ZT (ZTave) values of 0.9Cu2Se-0.1BPCCSO-0.035 wt% graphene in the temperature range from 473 K to 1000 K was over 1.7, which was superior to most Cu2Se-based materials with good thermoelectric performance45, as shown in Fig. ZTmax and ZTave values, the Cu2Se-BPCCSO-graphene composites in this work could meet the needs of thermoelectric devices.The temperature-dependent 2Se-BPCCSO-graphene composites were successfully prepared by a fast preparation method of combining self-propagating high-temperature synthesis (SHS) with spark plasma sintering (SPS). The Cu2Se-BPCCSO-graphene composites presented excellent thermoelectric properties with a ZTmax > 2.8 at 1000 K and ZTave > 1.7 in the mid-to-high temperature range. The underlying mechanisms of carrier and phonon transport were deeply investigated. On one hand, the electrical performance was largely improved with the highest PFmax value of 16.97 μW cm−1 K−2 at 1000 K and PFave value of 14.49 μW cm−1 K−2 from 473 K to 1000 K, which could be attributed to the optimized carrier concentration and Hall mobility by compositing with BPCCSO and graphene. On the other hand, multiple scattering sources like interfaces between BPCCSO, Cu2Se, and graphene revealed by STEM strongly scattered phonons, resulting in extremely low lattice thermal conductivity. The weak interface thermal transport in such composites was further proved by inelastic neutron scattering (INS) techniques. This work gives a paradigm to improve the thermoelectric performance of Cu2Se-like superionic semiconductors, which could be further adopted in other thermoelectric systems.In this study, Cu2Se-0.1Bi0.88Pb0.06Ca0.06CuSeO (BPCCSO)-x wt% graphene  composites were fabricated by a fast preparation method of combining self-propagating high-temperature synthesis (SHS) with spark plasma sintering (SPS). Stoichiometric amounts of Bi , Bi2O3 , Cu , Se , PbO , and CaO  powders were thoroughly mixed by hand grinding, then cold pressed into pellets and underwent the SHS process. For each sample, the weight of the pellet was about 10 g. The pellet was put in an alumina crucible. The SHS process started by heating the bottom of the crucible to the ignition temperature with an alcohol lamp. Once the reaction began from the lower melting point component, we put the lid on the crucible and moved the alcohol lamp away. The combustion wave was persisted by the energy released from the initial reaction and spread to the whole pellet in few seconds. The obtained bulks were ground into fine powders and fully mixed with graphene  by hand grinding for more than 30 min, and then sintered by an SPS furnace at 973 K under a uniaxial pressure of 40 MPa for 3 min in vacuum.The 0.9Cuλ = 0.15406 nm) at the scanning rate of 1° min−1. The samples for XRD measurement were fine powders crushed and ground from the sintered bulks. The microstructure of different samples was observed by field-emission scanning electron microscopy  using backscattering electron imaging (BEI) mode at 15 kV. The crystallography, compositions, and interfacial structure were further investigated by a double CS-corrected scanning transmission electron microscopy  equipped with probe and image correctors simultaneously. The probe convergence angle and high angle annular dark-field (HAADF) acceptance angles were 25 mrad and 48-200 mrad, respectively. The TEM specimens were prepared by ion milling with an Ar gas source, and the energy was 2 kV for perforation and 0.5 kV for the removal of the amorphous layer.The identification of phase purity and crystal structure for the composites was analyzed by X-ray diffraction  with a Cu Kα radiation  as a function of energy transfer at the neutron-energy-gain side. The GDOS accounts for the temperature dependence of the scattering function, which is related to the thermal population of excitations.The INS experiments were conducted on the time-of-flight cold neutron spectrometer, Pelican, at the Australian Nuclear Science and Technology Organisation (ANSTO). The five powder samples are Cuσ) and Seebeck coefficient (S) were measured by a commercial thermoelectric measurement system  from room temperature to 1000 K under the protective atmosphere of helium gas. The sample size was about 3 × 3 × 11 mm3. The carrier concentration (n) and Hall mobility (μΗ) were determined from room temperature Hall coefficient (RH) measurements by using the van der Pauw method on a homemade system equipped with a maximum 5 T superconducting magnet . The sample size was about 10 × 10 × 0.5 mm3. Thermal conductivity (κ) was calculated from the equation, κ = DρCp, where ρ is the density of the specimen, Cp is the specific heat capacity, and D is the thermal diffusivity, respectively. The ρ was measured by the Archimedes method. The Cp was measured by differential scanning calorimetry on a DSC  under an argon atmosphere and estimated according to the Neumann–Kopp rule in this work. And the D was measured by a laser flash method  from room temperature to 1000 K under a continuous argon flow. The longitudinal and shear sound velocities were obtained at room temperature via an ultrasonic pulse-echo method using an Olympus 5072 PR pulser/receiver. The sample size used for thermal diffusivity and sound velocity measurements was about 10 × 10 × 1.5 mm3. The electrical and thermal properties of all the samples were measured in the same direction (perpendicular to the direction of pressure). The uncertainties of the measured Seebeck coefficient and electrical conductivity are approximately 3% and 5%, respectively. The uncertainty of thermal conductivity measurement is about 10%. As a result, the uncertainty of ZT is estimated to be about 20%.The values of electrical conductivity (Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


The drop in capacitance over 1000 cycles was 4%, indicating a sufficiently high FLG stability, allowing us to consider FLG as a prospective material for use in supercapacitors.Graphene nanostructures (GNSs) are among the most promising materials for producing supercapacitors. However, GNSs are still not used in creating supercapacitors due to the impossibility of obtaining large volumes of high-quality material at an acceptable cost. In our previous works, we have shown the possibility of synthesizing large volumes of few-layer graphene  from cyclic biopolymers under conditions of self-propagating high-temperature synthesis (SHS). Using the SHS process makes it possible to synthesize large volumes of FLG without Stone–Wales defects. This work is devoted to the study of the possibility of using FLG synthesized under the conditions of the SHS process in the creation of supercapacitors. It was found that the synthesized FLG makes it possible to obtain better results than using classical materials, namely activated carbon (AC). It was found that the sample based on FLG had a higher specific capacitance of 65 F × g Solving the problem of rapidly growing global energy consumption, combined with the critical issue of climate change, is one of the important challenges for scientists and engineers worldwide, requiring the development of new sustainable and renewable energy sources . Energy Supercapacitors are energy storage devices with high power characteristics, demonstrating a high charge/discharge rate, cycle life, shelf life, a wide operating temperature range, and safety. . GeneralAmong all supercapacitors, one can highlight devices based on aqueous electrolytes, which are both high-power, environmentally friendly, and attractive for mass production ,12,13,142 × g−1) [The concept of adding graphene to supercapacitors was first described in 2008 . The aut2 × g−1) , increasoperties ,32, distoperties ,22,33.The capacitance characteristics of graphene-based supercapacitors depend on key material characteristics such as the specific surface area, pore size distribution, interlayer spacing, heteroatom doping, surface functionalization, and conductivity ,35. MostAccording to the global requirements for sustainable materials, some green technologies have been used to synthesize graphene nanostructures for supercapacitor applications. These approaches aim to minimize toxic chemicals, reduce energy consumption, and utilize renewable resources, making graphene synthesis more environmentally friendly and sustainable. The methods typically use renewable and sustainable carbon sources such as biomass , plants Green-synthesized graphene can be used in supercapacitors by directly using it as an electrode material ,42,43 orHowever, despite all the promise of using GNSs in creating supercapacitors, their practical application has yet to occur. One of the critical reasons for this is the high cost of GNSs, which makes the use of GNSs unprofitable. The high price of GNSs is due to the imperfection of the GNS synthesis methods, which do not allow for the synthesis of large volumes of high-quality material at an acceptable cost. Considering the methods of the synthesis of GNS, two main approaches can be distinguished: “top–down”  and “botIn our previous works, we reported on the possibility of synthesizing large volumes of few-layer graphene (FLG) from cyclic biopolymers under the conditions of the self-propagating high-temperature synthesis (SHS) , free ofThis work is devoted to the study of the possibility of using few-graphene synthesized under the conditions of the SHS process in the creation of supercapacitors.FLG particles synthesized by the SHS method were taken as the basis for the electrode. The initial biopolymer  in a 6 to 4 ratio using a “drunk barrel” type homogenizer  for 15 min (60 rpm). Then, the resulting mixture was placed in a reactor and heated to a temperature of 220 °C . After the completion of the synthesis, the resulting graphene (in the form of a powder) was washed with isopropyl alcohol  and dried in an oven until the weight loss ceased. The yield of graphene was 40 wt. % by weight of the original starch. The procedure for obtaining FLG is described in detail in .To obtain electronic images of the graphene particles, a TESCAN Mira-3M scanning electron microscope (SEM)  and an FEI Tecnai G2 30 S-TWIN transmission electron microscope  were used. To obtain electronic images of graphene particles by the TEM method, FLG was placed in toluene (concentration 0.05 mass. %) and sonicated until a stable suspension was obtained. Then, the resulting suspension was applied to a carbon grid and dried until the complete evaporation of toluene  at 60 °C). The laser diffraction method ) was used to measure the linear dimensions of the particles. For measurement purposes, a suspension of FLG particles in water (0.05 mass %) was prepared by sonication and an ultrasonic bath “Sapphire” .X-ray phase analysis ) was used to measure the number of layers in the FLG. The scanning rate was 1 deg/min.Raman spectroscopy ) was used to measure the quality of the FLG. FLG particles deposited from a toluene suspension (concentration of 0.05 mass %) on a silicon substrate were used for the measurements.The Brunauer–Emmett–Teller (BET) method  was used to measure the specific surface area of the FLG sample. Before measurement, the FLG sample was dried for 2 h at 300 °C under vacuum.®, Merck KGaA, Germany) as a solvent were carefully mixed with ultrasound until a homogeneous mass (slurry) was obtained. Next, a commercially available MF-2012 glassy carbon electrode  was evenly coated with the above suspension using a micropipette (0.7 µL) and then dried at 60 °C for 2 h. The load of electroactive material on each received electrode was 51 µg (0.73 mg × cm−2) (without considering the mass of PVDF). As a reference sample, electrodes were made from commercial activated carbon , specific surface area 740 m2 × g−1) using a similar procedure.The electrodes were prepared according to the following procedure: graphene or carbon powder (22 mg), 2 mg of poly(vinylidene fluoride)  as a binder, and 0.3 mL of N-methyl-2-pyrrolidone  as an auxiliary electrode and a standard silver chloride reference electrode MF-2056 (Ag/AgCl (3 M KCl); BASi, USA). All potentials in the work are given relative to the Ag/AgCl reference electrode.The electrochemical properties of the prepared electrodes were studied on a VSP multichannel potentiostat-galvanostat  in a three-electrode electrochemical cell containing a glassy carbon plate  in distilled water in the range potentials from −1 to 0.8 V. CV measurements were carried out at various scan rates in the range from 5 to 200 mV × s−1.The prepared graphene electrodes were tested as electrodes for supercapacitors using cyclic voltammetry (CV), galvanostatic charge–discharge (GCD), and electrochemical impedance spectroscopy (EIS) techniques in 1 M LiClOsp, F × g−1) was calculated based on the data obtained by the CV and GCD measurements according to Equations (1) and (2), respectively [Q is the accumulated charge in coulombs ; m (g) is the mass of the active substance; ∆t (s) is the discharge time; ∆V (V) is the voltage window; I (A) is the current during discharge.The specific capacitance (Cectively .(1)Csp=−1 in the potential range (a voltage window range) from −1 to 0.8 V. EIS measurements were carried out in the frequency range (100 mHz–100 kHz) with a sinusoidal potential amplitude of 5 mV rms at open circuit potential (OCP). Before the EIS measurements, the electrodes were preliminarily soaked in the electrolyte for 5 min to ensure stability. All parameters were obtained using EC-Lab V11.02 software.GCD was carried out at specific currents from 0.2 to 5 A × gAs can be seen in As can be seen from An X-ray diffraction study was carried out to determine the number of layers in the synthesized FLG sample. The results of the experiment are shown in Based on the position of the 002 peak and its full width at half maximum (FWHM), using the Scherer formula , the cry−1 with the wavy structure of the samples, the presence of a large number of edges, and the multidirectional superposition of the graphene layers on each other. It is essential to note that the FLG samples synthesized under the conditions of the SHS process did not contain Stone–Wales defects. This was shown by us using a chemical technique described in detail in our previous work [As can be seen from ous work .2 × g−1.As can be seen from 4 solution. −1 (Samples made from FLG and activated carbon (AC) were studied by cyclic voltammetry (CV) as model electrodes of supercapacitors in a three-electrode cell in 1 M aqueous LiClOtion. −1 b in the For both scan rates, the samples showed an electrical double-layer capacitance behavior (no pronounced peaks).−1 and 5 mV × s−1, these values were 170 and 64 F × g−1, respectively. It should be noted that despite the fact that the specific surface area of the FLG was much smaller than that of AC , it demonstrated greater efficiency than AC.The specific capacity of the obtained samples was calculated using Equation (1). −1.It can be seen from the presented graphs that for both samples, the values of the current densities became larger with an increasing scan rate. At lower potential scan rates, all samples showed curve shapes close to rectangular (rectangular-like shape), which is typical for double-layer behavior. With an increase in the sweep rate, the shape of the CV curves was distorted (fusiform), which is associated with the resistance of electron transfer. Still, the general shape of the curves remained close to rectangular, which indicates the relatively high-power characteristics of the materials.p)–lg(v) for the FLG and AC. The values of Ip were used at the plateau around 0.0–0.2 V, where the contribution of ohmic polarization did not have a significant effect on the CV shape.b = 0.86), which suggests insignificant limitations of mass transfer in the electrode material. A noticeable contribution of limited mass transfer was observed for the FLG sample only at high values of scan rates .The slope of this dependence enables the estimation of the limiting stage of the process . The slo−1. All samples were characterized by deviations from the linearity of the charge–discharge curves, especially when discharging low potentials, which is probably associated with side irreversible reduction processes. Most likely, there is slow water splitting with the hydrogen evolution. Although the overpotential of the hydrogen evolution reaction at carbon electrodes reached high values, at potentials around −1.0 V vs. Ag/AgCl and at low scan rates/discharge currents, the process may have an impact [The galvanostatic charge/discharge method was used to study the capacitive properties of the obtained graphene samples. n impact ,59.−1 were 265 and 75 F × g−1, respectively. All calculated capacitance values from CV and GCD are presented in The gravimetric capacitances of the electrodes were calculated using Equation (2). The capacitances of the electrodes based on graphene made from FLG and AC at a current of 0.2 A × gThe higher FLG efficiency may be due to a more optimal pore distribution since it is well-known that starting from some value of the surface area, its further increase in surface area does not lead to an increase in capacitance due to limitations of diffusion and electrode penetration into the porous system of the material . AdditioTo better understand the electrochemical properties of the electrodes, the samples were studied by electrochemical impedance spectroscopy (EIS). Spectra in Nyquist coordinates are shown in 4 aqueous solution at a current of 2 A × g−1 for 1000 cycles. The resulting dependence is shown in The stability of electrode materials based on few-layer graphene was tested by the GCD method in 1 M LiClO−1, and after a thousand cycles, it was 138 F × g−1), which indicates a sufficiently high stability of the materials, and allows them to be considered for use in supercapacitors. The decrease in capacity may be associated with irreversible reduction processes (slow water splitting and hydrogen evolution as discussed above), which was confirmed by an increase in the values of the Coulomb efficiency with cycling. It is recommended to use a narrower range of potentials to improve stability.The drop in capacitance for 1000 cycles was 4%  was achieved by the few-layer graphene electrode in an aqueous LiClO4 electrolyte. The achieved capacitance values were 2–4 times higher than the activated carbon-based electrode with a higher surface area. Furthermore, the few-layer graphene electrode maintained its capacitive performance cycling (1000 cycles). Thus, the findings suggest that the few-layer graphene composite could be a promising electrode material for supercapacitors.As a result, few-layer graphene has the optimal structure and particle size distribution and thus achieves higher capacitance values. A high capacitance of 265 F × g


A person’s first language (L1) affects the way they acquire speech in a second language (L2). However, we know relatively little about the role different varieties of the L1 play in the acquisition of L2 speech. This study focuses on German (L1) learners of English (L2) and asks whether the degree to which German speakers distinguish between the two vowels /eː/ and /ɛː/ in their L1 has an impact on how well these individuals identify /æ/ and discriminate between the two English vowels /ɛ/ and /æ/. These two English vowels differ in both vowel quality and duration (/æ/ is longer than /ɛ/). We report on an identification and a discrimination experiment. In the first study, participants heard a sound file and were asked to indicate whether they heard “pen” or “pan” . The stimuli differed from each other in terms of both vowel quality  and duration . In the second study, participants had to signal whether two sound files they were exposed to differed from each other. We modeled the percentage of /æ/  selection , accuracy , and reaction time  by implementing the German Pillai score as a measure of vowel overlap in our analysis. Each participant has an individual Pillai score, which ranges from 0 (= merger of L1 German /eː/ and /ɛː/) to 1 (=maintenance of the contrast between L1 German /eː/ and /ɛː/) and had been established, prior to the perception experiments reported here, in a production study. Although the findings from the discrimination study remain inconclusive, the results from the identification test support the hypothesis that maintaining the vowel contrast in the L1 German leads to a more native-like identification of /æ/ in L2 English. We conclude that sociolinguistic variation in someone’s L1 can affect L2 acquisition. This idea is central to several well-established models of L2 speech, such as the Speech Learning Model SLM, , the Perpen, and /æ/ (TRAP vowel), as in pan. Remember that well-known varieties of English, such as Standard American or Southern British English, distinguish between the two front, unrounded vowels /ɛ/ and /æ/ , as in  /æ/ see . The two /æ/ see .1pen and pan, respectively , pose a possible challenge to L1 German (or Dutch) speakers. The objective of the present paper is to expand previous research by investigating whether some L1 German speakers show fewer difficulties in identifying /æ/ and discriminating /æ/ and /ɛ/ than others. That is, we examine whether the properties of one’s L1 vowel inventory affect the way one deals with the two English vowels /ɛ/ and /æ/. More precisely, we ask whether the presence/absence of the distinction between the two German vowels /eː/-/ɛː/ in a person’s L1 vowel space facilitates the identification of /æ/ and the discrimination of the English contrast between /ɛ/ and /æ/. We do so by examining both binary (identification rate of English /æ/ in the identification and reaction accuracy in the discrimination task) and continuous data (reaction times) in order to get a comprehensive picture and detect potential burdens in the processing of L2 sounds. Before going into detail, however, we consider, more generally, which factors might affect how a person acquires speech in an L2.Since /æ/ is not part of the Standard German vowel inventory, it frequently poses a challenge to learners of English, who may not distinguish between English /ɛ/ and /æ/ and/or rely on a close category they are familiar with from German, such as /ɛ/ or /ɛː/ for vely see .2 Note tvely see . Weber a1.1.In this section, we discuss several factors that can affect the acquisition of speech in an L2. The factors are age and amount of experience (section 1.1.1) and similarities between a person’s L1 and the L2 (section 1.1.2).1.1.1.A first factor is the age at which a person acquires the L2. If the acquisition of the L2 starts later, it is often more difficult to hide a foreign accent, that is, the L1 shapes L2 speech more than if the L2 acquisition begins at an earlier age see . For insA second factor is the amount of experience a person has in the L2. Usually, individuals with more experience in the L2 sound more native-like see . For exaduration difference between the two English vowels and was, so to speak, native-like with respect to the duration contrast. A further finding from Although the two factors age and amount of experience play a crucial role in the acquisition of an L2 in general and with regard to how much the L1 shapes the L2, we see that even highly advanced language users still differ from native speakers. For instance, 1.1.2.A third factor is the degree of similarity between specific phenomena in the L1 and L2. It has been stated that a higher degree of similarity between phenomena in the L1 and L2 typically results in greater L1 influence on the L2 and more difficulties in correctly acquiring the L2 sound see . Bohn an1.1.3.dehnen, [ˈdeːnən], “to stretch”) and /ɛː/  in their L1 has an impact on how well these individuals identify /æ/ and discriminate between the two English front, unrounded vowels /ɛ/  and /æ/ . We therefore intend to expand previous research on how a high degree of similarity between a phenomenon in the L1 and one in the L2 affects L2 speech acquisition in detail, by considering sociophonetic variation across different native speakers of German.To reach an intermediate summary, we can say that whether and to what extent the L1 affects the acquisition of the L2 has been widely examined in the literature, and several factors have been taken into consideration. However, we feel that another factor has been mostly neglected, namely the role of sociolinguistic aspects of an individual’s L1 in the context of L2 speech acquisition. It is the aim of the present paper to investigate this issue. Precisely, we aim at investigating whether the degree to which German native speakers distinguish between the two front, unrounded vowels /eː/  and /ɛː/  are distinct phonemes  and speakers who merge the two vowels (henceforth: /eː/-/ɛː/ mergers). We ask whether these speaker groups identify /æ/ and discriminate the English vowels /ɛ/ and /æ/ differently in speech perception. Assuming that there are two groups, /eː/-/ɛː/ maintainers and /eː/-/ɛː/ mergers, we test the following hypotheses.The identification of English /æ/ and the discrimination of the English /ɛ/−/æ/ contrast is:H0: The same in both groups.H1: More native-like in the group of /eː/-/ɛː/ maintainers.These hypotheses derive from the patterns summarized in 2.2.1.In Experiment I, participants saw two pictures, heard a word, and had to decide which of the two concepts represented in the pictures they had heard.2.1.1.Fifty-one native speakers of German from northern Germany participated in the experiment .All participants were well-educated and had taken English as one of the central school subjects for at least 8 years. That is, all of them had a solid knowledge and command of English. Despite this similarity between our participants, they varied to some extent with regard to their level of English. We grouped our participants into three proficiency groups . Categorizing participants into the three groups was done in the following way. The first author of the paper and a native speaker of English listened to sound files of items, which were recorded in a short production experiment prior to the perception experiments. They examined the pronunciation in terms of nativelikeness, and assigned the participants to the groups “low,” “mid,” and “high.” If the first author and the native speaker of English did not agree, the case was discussed until agreement was reached. Our evaluation of the participants’ level of English is thus based on their pronunciation only. The participants’ level of English was later entered as a control variable into the statistical models.dehnen, [ˈdeːnən], “to stretch”) and 29 items containing /ɛː/  in Standard German. A key element of our work is whether and to what extent our participants distinguish between the German vowels /eː/ and /ɛː/. In order to obtain an objective reflection of this, we relied on the data from the production experiment reported in 2.1.2.pan/pen and paddle/pedal. The four words were spoken and recorded by a 24-year-old male US American from California in a silent room. The four sound files were normalized (70 dB) and subsequently used as the basis for the following manipulations. We created a spectral continuum with 11 steps from an extreme /æ/ (Step 1) to an extreme /ɛ/ (Step 11) based on a Praat script . Hence, the identification task consisted of six distinct blocks . The three blocks relating to one minimal pair always appeared one after the other in the same order . All the pan/pen blocks appeared first, all the paddle/pedal blocks afterwards. Each person was tested on 198 trials . Hence, each of the six blocks contained 33 cases (11 steps of the continuum × 3 times each sound file).The two minimal pairs 2.1.4.The two response variables were the percentage of the selection of the picture representing the /æ/ word (Selection of /æ/) and ReactionTime. We used a binomial logistic regression to examine the first and linear mixed effects models to analyze the second one in R , using tModel fitting proceeded in the following way , Step  were conducted using the emmeans package  in R to 2.1.5.We start presenting the results for Selection of /æ/. The most important results are the two significant interactions GermanPillai × Step and GermanPillai × Duration see , 2. The We see two significant interactions, GermanPillai × Step and GermanPillai × Duration. Let us start with the first one. In In We now turn to the analysis of ReactionTime, where we found a significant interaction of GermanPillai × Duration see , 7.post hoc test revealed further that participants responded faster to Step 2 than to Step 6 , to Step 2 than to Step 7 , and to Step 2 than to Step 8 .In the analysis of ReactionTime, there is a main effect of Step. Participants reacted more quickly to Step 1 than to Step 6. A Tukey Furthermore, we also detected a significant interaction, namely GermanPillai × Duration. In 2.2.In Experiment II, participants heard two sound files and were asked to indicate whether the two were acoustically identical or different.2.2.1.The participants were those from Experiment I.2.2.2.The materials/sound files were those from Experiment I.2.2.3.The experiment was conducted in a silent room. Participants sat about 60 cm in front of a computer screen and wore KOSS R-80 over-ear headphones. We used E-Prime 3 for stimulus presentation. In each trial, participants heard two sound files and indicated via button press, using the left and right arrow on a regular keyboard, whether they believed they were identical or different. We counterbalanced the association of arrow and identical/different across participants. As in Experiment I, both the two minimal pairs and the three vowel duration types appeared in different blocks. The spectral distance between the two sound files of a trial was 3, 2, 1, or 0 steps. That is, for instance, if the first sound file contained Step 3 and the second sound file Step 4, the distance between the two was 1 step. The interstimulus interval (ISI) was 1 s.2.2.4.The two response variables were Accuracy and ReactionTime. We used binomial logistic regression to examine the first and linear mixed effects models to analyze the second one in R, using the lme4 and lmerTest packages. In the analysis of ReactionTime, only correct answers were considered (40% of the answers), following common practice , Duration , LevelOfEnglish , and the three interactions GermanPillai × StepDistance, GermanPillai × Duration, and GermanPillai × LevelOfEnglish in the initial model. The model was reduced step by step and significances were checked using the technique outlined in section 2.1.4. Planned pairwise comparisons were used as outlined in section 2.1.4.2.2.5.We start looking at accuracy. Here, the interaction GermanPillai × StepDistance reached significance see . The stapost hoc test further showed that middle vowels were responded to more accurately than short vowels .In the analysis of Accuracy, we found a main effect of Duration, indicating that short vowels were responded to less accurately than long ones. A Tukey Further, we detected a significant interaction GermanPillai × StepDistance. We see in post hoc test indicated that the response time was longer for middle than for short vowel durations . Second, participants needed more time to respond when the StepDistance between the two sound files of a trial was 1, 2, or 3, in comparison to when it was 0. A Tukey post hoc test showed that the response times were longer for a StepDistance of 1, compared to StepDistances of 2 and 3 . Also, participants responded more slowly for StepDistance 2 than for StepDistance 3 .We now turn to ReactionTime see , 11. The3.The present paper reported on an identification and a discrimination task and aimed at investigating whether the degree of overlap between the vowels /eː/ and /ɛː/ in L1 German affects the way individuals identify the new vowel /æ/ and discriminate between the vowels /ɛ/ and /æ/ in L2 English. We distinguished broadly between /eː/-/ɛː/ maintainers, that is, L1 German speakers who articulate the vowel contrast in their L1, and /eː/-/ɛː/ mergers, that is, L1 German speakers who do not articulate the vowel contrast in their L1.In the identification task, we found that Step 1 was reacted to faster than Step 6 and Step 2 was responded to more quickly than Steps 6, 7, and 8. That is, some of the more extreme steps were easier to process. The significant interaction GermanPillai × Duration for the reaction time data signaled that /eː/-/ɛː/ mergers needed more time to process the difficult intermediate category (middle duration), in comparison to the categories of short and long duration, than /eː/-/ɛː/ maintainers.Moreover, and crucially, we detected two significant interactions, namely GermanPillai × Duration and GermanPillai × Step in the data for the Selection of /æ/. Considering the first interaction, we saw that while /eː/-/ɛː/ maintainers selected English /æ/ slightly more often than /eː/-/ɛː/ mergers for long vowels, /eː/-/ɛː/ mergers picked English /æ/ slightly more frequently than /eː/-/ɛː/ maintainers for short vowels. Importantly, while /eː/-/ɛː/ mergers chose /æ/ for short and long vowels roughly equally often, /eː/-/ɛː/ maintainers selected this vowel more frequently for long than for short vowel durations. This supports H1: /eː/-/ɛː/ maintainers are more native-like in the identification of the new vowel /æ/ than /eː/-/ɛː/ mergers. While both speaker groups assimilate English /ɛ/ to German /ɛ/, the two groups seem to differ with regard to how they treat English /æ/. /eː/-/ɛː/ mergers, who only rely on /ɛ/ in L1 German and do not distinguish between /ɛ/ and /ɛː/, seem to assimilate English /æ/ to German /ɛ/, they fail to make adequate use of the duration parameter. /eː/-/ɛː/ maintainers perform more successfully for the parameter of vowel duration, which might derive from the fact that they are familiar with the distinction between /ɛ/ and /ɛː/ from the L1 German. /eː/-/ɛː/ maintainers either could have assimilated English /æ/ to German /ɛː/ or could have developed the new category /æ/.Interestingly, the second interaction supports H1, too, although /eː/-/ɛː/ maintainers should not necessarily have an advantage when it comes to vowel quality . We observed that /eː/-/ɛː/ maintainers selected English /æ/ more frequently than /eː/-/ɛː/ mergers for Steps 1 to 4. Step 5 shows a slight tendency for this, too. Acoustically speaking, these steps clearly represent English /æ/ and we have thus evidence that /eː/-/ɛː/ maintainers identify this vowel more accurately than /eː/-/ɛː/ mergers. This is further supported by the fact that /eː/-/ɛː/ mergers chose English /æ/ more frequently than /eː/-/ɛː/ maintainers for Steps 7 to 11, which carry the acoustic properties of English /ɛ/. Step 6, which is exactly in the middle of the continuum and can be considered to be neutral, joins for some reason the pattern we found for Steps 7 to 11. Overall, the significant interaction GermanPillai × Step we saw in the analysis of the response variable Selection of /æ/ in the identification task also supports H1: /eː/-/ɛː/ maintainers perform more native-like in the identification of /æ/ than /eː/-/ɛː/ mergers. Remember that we stated above that the results of the significant interaction GermanPillai × Duration could mean that /eː/-/ɛː/ maintainers either assimilated English /æ/ to German /ɛː/ or have developed the new category /æ/. The results of the second interaction, GermanPillai × Step, seem to be more compatible with the interpretation that /eː/-/ɛː/ maintainers have developed the new category /æ/. They outperform /eː/-/ɛː/ mergers not only on the level of vowel duration but also on the spectral level.It is an interesting finding that vowel quality makes such a difference although both /eː/-/ɛː/ maintainers and /eː/-/ɛː/ mergers are familiar with the vowel quality of /ɛ/ but unfamiliar with the vowel quality of /æ/ from their L1 German. A potential explanation might be related to the fact that all of our participants were advanced speakers of English. Possibly, advanced speakers are generally aware of the differences between English /æ/ and German /ɛː/ / /ɛ/ see also . NeverthIn the discrimination study, we found a lower accuracy for the short in comparison to both middle and long vowel durations. That is, more information led to higher response accuracy. The reaction time data revealed that short vowels were responded to more quickly than middle or long vowels. In addition, middle vowels triggered longer reaction times than long vowels. It seems thus that the intermediate category is the hardest one to deal with. Looking at the response times, we further found that participants gave the fastest answers for a StepDistance of 0, followed by StepDistances of 3, 2, and 1. Hence, identical cases posed the smallest challenge to the individuals; beyond that, a greater distance between two sound files of a trial accelerated the reaction.Further, the interaction GermanPillai × StepDistance reached significance in the accuracy data. Despite the significance, the interaction is neglectable. On the one hand, we saw that /eː/-/ɛː/ maintainers responded slightly more accurately than /eː/-/ɛː/ mergers for a StepDistance of 0; on the other hand, /eː/-/ɛː/ mergers answered slightly more correctly for the StepDistances 1 and 2. StepDistance 3 falls, roughly speaking, in the middle. Overall, the discrimination study can only be interpreted to support H0: /eː/-/ɛː/ maintainers and /eː/-/ɛː/ mergers do not differ with regard to the discrimination of the English vowels /ɛ/ and /æ/.We realize that sociophonetic aspects of the L1, in our case the degree of overlap of the two German vowels /eː/ and /ɛː/, affect how language users deal with certain phenomena in the L2. Our results provide a piece of evidence that the concept of the L1 and its function in L2 acquisition is heterogenous. Although there is by now a good amount of research targeting different types of sociolinguistic variation in the L2 itself see , that isFinally, we would like to point out another avenue for future research. As we noted in section 1, varieties of English differ with regard to how the phonological contrast between /æ/ and /ɛ/ is articulatorily and acoustically realized. It goes without saying that it is hard to assign an L2 speaker of English to a specific variety since everyone is exposed to several sources of input, each having its distinct nuances. For instance, one might have had a teacher speaking a variety of US American English at school but later lecturers from the United Kingdom at university. To complicate matters even further, learners can be confronted with yet other varieties of English through the media, in international communities, or during extended stays in English-speaking countries. That being said, and although it would not be a trivial task to find L2 speakers who have been exposed to and used a single variety of English only or at least primarily, disentangling the influence of various Englishes would provide us with even more pieces of the puzzle of how individuals acquire speech in an L2.4.Our findings show that sociophonetic variation within a person’s L1 plays a role in L2 acquisition. Individual variation adds an essential piece to the puzzle of how the L1 determines how language users deal with phenomena in the L2. Here, we showed that the degree to which two vowels are merged in someone’s L1, such as /eː/ and /ɛː/ in German, seems to affect the identification of a new vowel (/æ/) in the L2. We feel that there are promising avenues for future research in this field and hope that this work inspires others to enrich our knowledge on the role of individual variation in the interplay between L1 and L2 speech.The R scripts and the datasets are included in the The studies involving human participants were reviewed and approved by Research Ethics Committee, University of Oldenburg, Germany. The participants provided their written informed consent to participate in this study.MS and JP developed together the research questions against the background of the relevant literature in the field. MS and MF compiled the materials, designed the experiments, collected the data, conducted the statistical analyses, and wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.