Here, we develop a graphene FET with a regenerative sensing interface based on dynamic covalent chemistry (DCvC). The LoD down to 5.0 × 10−20 mol L−1 remains even after 10 regenerative cycles, around 4–5 orders of magnitude lower than existing transistor sensors. Owing to its ultra-sensitivity, regeneration ability, and advantages such as simplicity, low cost, label-free and real-time response, the FET sensor based on DCvC is valuable in applications such as medical diagnosis, environment monitoring, etc.Field-effect transistor (FET) sensors require not only high sensitivity but also excellent regeneration ability before widespread applications are possible. Although some regenerative FETs have been reported, their lowest limit of detection (LoD) barely achieves 10 Dueive FETs . Consideive FETs .2+) is employed as a representative example [2+ efficiently hydrolyzes PyCDH to pyrene-1-carboxaldehyde (PyCHO), leading to an n-doping effect upon graphene  is generally used in designing self-healing materials ,23,24. T example ,28. Aftegraphene c. The n-−1) was prepared in DMSO/H2O (4:1 v/v). The ethanol solution of hydrazine hydrate (10 mmol L−1) was prepared by dissolving hydrazine hydrate  and HCl  in absolute ethanol. Mono-layer graphene was grown by thermal chemical vapor deposition on high-purity copper foils. After growth, graphene was transferred by a standard procedure using a PMMA temporary substrate by electrochemical delamination (bubbling transfer) method. The samples were measured by Raman , NMR  Co., Ltd., Shanghai, China), and laser scanning confocal microscope  Co., Ltd.).Copper foil (LOT: I26Z009) was purchased from Alfa Aesar a Johnson Matthey Co., Ltd. . 1-Pyrenecarboxaldehyde was purchased from Aladdin Chemistry Co., Ltd. . Hydrazine hydrate was purchased from Sinopharm Chemical Reagent Co., Ltd. . Analytical grade metal salts were procured from Energy Chemical Co., Ltd. . The solution of PyCHO (5 mmol L2 (285 nm)/Si wafer via a bilayer lift-off photolithography process, and a polydimethylsiloxane (PDMS) well containing the graphene FET chemical sensor was formed on the substrate to hold the analytical solutions [2/Si substrate using standard photolithography and metal deposition techniques. Then, the device was kept in Remover PG overnight at room temperature to remove the residual photoresist. The FET had a channel length of 100 μm and a width of 2 mm (W/L = 200). Next, the CVD graphene was transferred onto the substrate by the electrochemical delamination method. Finally, PyCDH was immobilized onto the graphene surface by π-π stacking to fabricate the PyCDH/graphene FET.The solution-gated graphene FET chemical sensor was fabricated on a SiOolutions . First, Vg) was set lower than ±400 mV (versus Ag/AgCl), and the Vds was set to 25 mV to avoid any electrochemical reaction on the electrodes.The electrical signal was measured by a semiconductor parameter analyzer  Co., Ltd., Beijing, China) and a probe station  at room temperature. All the aqueous solutions were prepared using deionized water with a resistivity of 18.2 MΩ cm. The Ag/AgCl reference electrode was used as the liquid gate electrode. The gate voltage  was added to PDMS well to incubate the probe PyCDH at room temperature for 12 h. Finally, the FET was washed with deionized water three times and sealed with deionized water (100 μL) until the next use. The above-mentioned series of processes were regarded as one cycle.The graphene FET, which detected 5.0 × 102/Si wafer with photo-lithographically patterned gold electrodes. The Raman spectrum of the graphene  of 25 mV (Vg) at minimum drain-source current (Ids) corresponds to the Dirac point (VDirac). The charge transfer between graphene and H2O leads to hole doping [p-type behavior in the deionized water with a VDirac at 54 mV . With increasing Cu2+ concentrations from 5.0 × 10−18 to 5.0 × 10−8 mol L−1, the VDriac negatively shifted from 92 to 76 mV. Furthermore, the Ids at VDriac gradually increased from 55.97 to 57.18 μA /Ids,0| vs. time, where Ids,0 is the drain current at Vg = 0 V  were prepared. Moreover, the influence of ions on the transfer characteristics of the device was investigated with a normalized response . The mean fluorescence intensity of graphene surface functionalized by PyCHO was about 635 arb.unit (a.u.), reaching 1023 a.u. after condensation reaction  after 10 regeneration cycles. This technology achieves inspiring sensitivity, selectivity, and regeneration ability because reversible dynamic covalent bonds utilized in DCvC can be easily broken and reassembled to reform bonds. It might be the missing piece of the puzzle that enables ion sensors to be a comprehensive tool for not only sensitive testing but also regenerative applications in environmental monitoring and medical diagnoses.In conclusion, we report a regenerative testing methodology based on PyCDH/graphene FET sensors for the first time and address an urgent issue of ion testing that faces a trade-off between sensitivity and regeneration ability. Compared with other methods ,34,36, t


Spermatogenesis is a cyclical process in which different generations of spermatids undergo a series of developmental steps at a fixed time and finally produce spermatids. Here, we report that overexpression of PD‐L1 (B7 homolog1) in the testis causes sperm developmental disorders and infertility in male mice, with severe malformation and sloughing during spermatid development, characterized by disorganized and collapsed seminiferous epithelium structure. PD‐L1 needs to be simultaneously expressed on Sertoli cells and spermatogonia to cause spermatogenesis failure. After that, we excluded the influence of factors such as the PD‐L1 receptor and humoral regulation, confirming that PD‐L1 has an intrinsic function to interact with PD‐L1. Studies have shown that PD‐L1 not only serves as a ligand but also plays a receptor‐like role in signal transduction. PD‐L1 interacts with PD‐L1 to affect the adhesive function of germ cells, causing malformation and spermatid sloughing. Taken together, these results indicate that PD‐L1 can interact with PD‐L1 to cause germ cell detachment and male infertility. We found that the overexpression of PD‐L1 can cause complete infertility in male mice. Spermatids show maturation defects during the stages of spermiogenesis, characterized by premature degeneration of spermatids, apoptosis, sloughing into the lumen, and few spermatids produced in the functional lumen that exhibit malformed heads with disorganized alignment. Most importantly, PD‐L1 overexpression in the seminiferous and Sertoli cells of the seminiferous tubules only caused spermatid release disorder. Therefore, the results obtained from the study of PD‐L1 in Sertoli and germ cells may provide us with an investigational basis regarding the potential regulatory mechanisms of PD‐L1 in affecting cell adhesion function.22.1Experimental animal materials included male and female C57BL/6 (B6) mice . All experimental procedures were approved by the Institutional Animal Care and Use Committee of South China University of Technology. Transgenic mice and wild‐type mice were fed in an SPF‐grade animal laboratory and sacrificed at 22, 40, 60, 70, 90, 100, 120, 140, 160 and 190 days. At each time point, wild‐type mice and transgenic mice were divided into WT and PD‐L1 groups, with five mice in each group. Testis and epididymis were immediately taken, photographed and weighed, and the average weight of testicular and epididymal tissues of mice was calculated.2.2Xho I and EcoR I sites of pCAGGS (a gift from Dr. Miyazaki J) to construct the pCAG‐PD‐L1 plasmid. The pCAG‐PD‐L1 plasmid was digested with Sal I and Avr II, and linearized CAG‐PD‐L1 transgenic fragments were collected and prepared for pronuclear microinjection of fertilized eggs  and EcoR I  restriction enzymes to liberate PD‐L1‐IRES2‐EGFP fragments, which were then subcloned into the pCAGGS vector to construct the pCAG‐PD‐L1‐IRES2‐EGFP vector. We linearized this vector using Spe I  and Ssp I  prior to pronuclear microinjection of fertilized eggs  to construct the pPD‐L1‐IRES2‐EGFP plasmid. The pPD‐L1‐IRES2‐EGFP plasmid was digested with s Figure . Similars Figure .2.32 and covered with mineral oil . Two‐cell embryos were picked up and transferred to pseudopregnant ICR foster mothers. Positive PD‐L1 transgenic mice were detected by PCR using specific primers .Superovulation procedures were performed following the previously described procedures.2.4Periodic acid‐Schiff staining was performed as previously reported.2.5Haematoxylin‐Eosin staining was performed as previously reported.2.6For IF, place paraffin slices in 75°C for half an hour to prevent slices from falling off, dewaxing and hydration. Then, tissue sections were placed vertically in Tris‐EDTA antigen repair solution  and heated for 10 min at 65°C to repair the antigens, and removed to room temperature. After that, blocked it with 10% goat serum  for 1 h at room temperature, and then incubated with DAPI/PI/anti‐PD‐L1 antibodies  overnight at 4°C. After washing with 1× TBST  for 5 min, the sections were incubated with the appropriate secondary antibody  for 30 min at room temperature. For the negative control, sections were incubated with the secondary antibody only. Further detection was done by following the manufacture protocol. Photographs were taken under the same conditions with a fluorescence microscope . The experiment was repeated three times.2.72 , while the wild‐type mice in the negative control group and the transgenic mice in the experimental group were injected with the same volume of normal saline. Wild‐type mice and transgenic mice were divided into CdCl2, WT and TgPD‐L1 groups, with five mice in each group. Ten milligrams of FITC‐lectin  was injected into the tail vein after 24 h. After 20 min, the mice were euthanized and the testis were removed via abdominal incision. Tissues were fixed overnight with 4% paraformaldehyde, and then dehydrated with 25% sucrose solution. Finally, 12‐μm sections were mounted onto glass slides for observation with a fluorescence microscope .Both wild‐type mice and transgenic mice were adult mice of the same age. The wild‐type mice in the positive control group were given 3 mg/kg CdCl2.8We used TgGFP and TgPD‐L1 transgenic male mice for our parabiosis test using the following procedure that was adopted from published methods.2.92 and 37°C.The human colorectal cancer cell line SW480 and the human embryonic kidney 293T cell line were purchased from ATCC . The HEK293T cell line was cultured in DMEM  supplemented with 10% FBS  and 1% penicillin‐streptomycin solution . The SW480 cell line was cultured in RPMI 1640 medium  supplemented with 10% FBS and 1% penicillin‐streptomycin solution. All cell lines were cultured at 5% CO2.10Full‐length human PD‐L1‐coding cDNA with HA and Flag labels was PCR amplified from CS‐U0767‐pIRES and CS‐U0767‐M83 . Primers for PCR are in Table 2.11Total RNA was extracted with TRIzol reagent  followed by RaPure Cell RNA Mini Kit  treatment using the manufacturer's protocol and transcribed into cDNA by using PrimeScript™ RT Master Mix  according to the manufacturer's instructions .https://www.ncbi.nlm.nih.gov/tools/primer‐blast/). Primer sequences are listed in Table For reverse transcription‐PCR (RT‐PCR), cDNA was then amplified using gene‐specific primers designed using NCBI primer design tool  according to the manufacturer's protocols. Reactions were run on an LightCycler® 96 System  and samples were analysed in triplicates. The qRT‐PCR cycling conditions were 95°C for 10 s, 58°C for 20 s and 72°C for 15 s. Melt curve data were obtained to confirm amplification of the correct product in each well. Gapdh was used as an internal reference gene, and all data analyses were performed using the comparative Ct method. Primers for PCR are shown in Table For quantitative reverse transcriptase‐PCR (qRT‐PCR), the cDNA was diluted 1:50 and qRT‐PCR assays were performed by using the SYBR2.12The cells were collected by a cell scraper  with cool lysate  for protein extraction. The lysate was incubated with anti‐Flag antibodies  or an equal amount of mouse IgG  on a rotating wheel overnight at 4°C and then incubated with protein A agarose beads  at 4°C for 10 h. Beads were collected by centrifugation, washed, boiled in 2× PAGE loading buffers  and analysed by Western blotting. The experiments were repeated three times.2.13The protein was separated by 10% SDS‐PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes . The membranes were blotted with anti‐HA antibodies , PD‐L1 , anti‐GAPDH antibodies  and secondary antibodies  to detect the proteins. GAPDH protein was used as a loading control. The experiments were repeated three times.2.14p < 0.05 (two‐tailed) was considered statistically significant.All experiments were repeated independently at least three times. Statistical analysis was performed using GraphPad Prism Software. Quantitative data are displayed as the mean ± SEM in each experiment. Comparisons among multiple groups were performed through one‐way ANOVA with Newman–Keuls post‐test. 33.1Pdcdl1 gene under the CAGGS (CAG) promoter to generate TgPD‐L1 mice by fertilized egg microinjectionPdcdl1 gene was inserted at 93,439,972 bp on mouse chromosome 17 without inserting into known genes in micePD‐L1 (programmed cell death ligand‐1), also known as B7‐H1, was initially reported to be involved in the negative regulation of cell‐mediated immune responses as a B7 family member and was later found to be one of the ligands acting as PD‐1 (programmed cell death‐1).3.2The testis size of TgPD‐L1 was much smaller than that of WT mice Figure . Compare3.3The stages in the cycle of seminiferous epithelium and developmental steps of germ cells were evaluated via cross sections of testes in WT and TgPD‐L1 mice. At 22 days of age, there were no distinguished differences in histological observation by PAS staining between WT and TgPD‐L1 mice Figure . At appr3.4Egfp reporter genes under the CAG promoter (TgPD‐L1‐EGFP)  Figure . We obtaHowever, we also found that Line11 mice with high expression of EGFP in testes could normally be fertile , which was shown to be translated at spermatid stages during development3.5−/−) mice to produce TgPD‐L1/PD‐1−/− mice. Unfortunately, TgPD‐L1/PD‐1−/− mice were still infertile, with smaller testes, germ cells sloughing in the seminiferous epithelium and no mature sperm in the epididymis ; Methodology ; Writing – original draft . Rui Feng: Conceptualization ; Methodology ; Writing – original draft . Weiye Liang: Conceptualization ; Methodology ; Writing – review & editing . Fangfang Liu: Conceptualization ; Methodology ; Writing – original draft . Ganlan Bian: Formal analysis (lead); Visualization . Caiyong Yu: Investigation ; Validation . Hongmin Guo: Validation ; Visualization . Yihui Cao: Investigation ; Visualization . Mingkai Liu: Formal analysis ; Visualization . Jia Zuo: Formal analysis ; Visualization . Yinglong Peng: Formal analysis ; Visualization . Jie Zhao: Investigation ; Validation . Rui‐Xia Sun: Resources ; Supervision ; Writing – original draft . Jiajie Shan: Resources ; Supervision ; Writing – original draft . Jian Wang: Funding acquisition (lead); Resources (lead); Writing – review & editing .Fig S1Click here for additional data file.Fig S2Click here for additional data file.Fig S3Click here for additional data file.Table S1Click here for additional data file.


TMEM106B risk allele. Pathologically, LATE-NC was associated with comorbid hippocampal sclerosis (HS), myelin loss, and vascular disease in white matter (WM). Unbiased hierarchical clustering of TDP-43 inclusion morphologies revealed discernable subtypes of LATE-NC with distinct clinical, genetic, and pathologic associations. The most common patterns were: Pattern 1, with lamina II TDP-43 + processes and preinclusion pathology in cortices of the amygdala region, and frequent LATE-NC Stage 3 with HS; Pattern 2, previously described as type-β, with neurofibrillary tangle-like TDP-43 neuronal cytoplasmic inclusions (NCIs), high Alzheimer’s disease neuropathologic change (ADNC), frequent APOE ε4, and usually LATE-NC Stage 2; Pattern 3, with round NCIs and thick neurites in amygdala, younger age at death, and often comorbid Lewy body disease; and Pattern 4 (the most common pattern), with tortuous TDP-43 processes in subpial and WM regions, low ADNC, rare HS, and lower dementia probability. TDP-43 pathology with features of patterns 1 and 2 were often comorbid in the same brains. Early and mild TDP-43 pathology was often best described to be localized in the “amygdala region” rather than the amygdala proper. There were also important shared attributes across patterns. For example, all four patterns were associated with the TMEM106B risk allele. Each pattern also demonstrated the potential to progress to higher LATE-NC stages with confluent anatomical and pathological patterns, and to contribute to dementia. Although LATE-NC showed distinct patterns of initiation in amygdala region, there was also apparent shared genetic risk and convergent pathways of clinico-pathological evolution.Transactive response (TAR) DNA-binding protein 43 kDa (TDP-43) pathology is a hallmark of limbic-predominant age-related TDP-43 encephalopathy (LATE). The amygdala is affected early in the evolution of LATE neuropathologic change (LATE-NC), and heterogeneity of LATE-NC in amygdala has previously been observed. However, much remains to be learned about how LATE-NC originates and progresses in the brain. To address this, we assessed TDP-43 and other pathologies in the amygdala region of 184 autopsied subjects (median age = 85 years), blinded to clinical diagnoses, other neuropathologic diagnoses, and risk genotype information. As previously described, LATE-NC was associated with older age at death, cognitive impairment, and the The online version contains supplementary material available at 10.1007/s00401-022-02416-5. The amygdala region comprises an anatomically heterogeneous set of brain structures that normally play a role in cognitive function, emotional processing, integration of olfactory input, and autonomic responses , 33, 54.The amygdala region appears particularly susceptible to neurodegenerative disease pathology . This brThe amygdala has been reported to be the earliest anatomic location of TDP-43 pathology in aging (LATE-NC Stage 1) , 39, 42.The aim of the current study was to enhance our understanding of LATE-NC heterogeneity, and to elucidate how TDP-43 pathologic patterns are associated with genetics, clinical disease severity, and comorbid pathologies. Pathologic features in the amygdala region were graded blind with respect to clinical diagnoses, genetic risk factors, and comorbid pathologies, and all included brains were assigned to TDP-43 pathologic pattern subtypes that were generated using an unbiased clustering algorithm. These patterns were then correlated with other parameters. Our findings build on prior studies about how TDP-43 pathology in LATE-NC originates and evolves in the aging brain.Samples and data evaluated were from autopsy study participants from two different institutions: Houston Methodist Hospital  and the University of Kentucky Alzheimer’s Disease Research Center at Sanders-Brown Center on Aging , or other rare neurological diseases . Samples were also excluded if review of anatomic sections showed poor orientation, incomplete sampling of the region, or acute hypoxic/ischemic injury.For evaluation of pathologic features in the amygdala region, formalin-fixed paraffin-embedded (FFPE) tissue blocks were sectioned at 6 µm, mounted on charged slides, and dried overnight at 60 °C. Serial sections were stained for hematoxylin and eosin (H&E) and cresyl violet/ luxol fast blue (LFB), using a previously described modified Klüver–Barrera protocol . All incAll samples were stained for TDP-43 using phosphorylation-dependent  and non-phosphorylation-dependent antibodies , and were also stained for phospho-tau (AT8). The immunostaining procedure used has previously been described in detail , 22 and OTCtx), transentorhinal and entorhinal cortex (TECtx/ECtx), corticomedial amygdala (CM), basolateral amygdala (BL), lateral subdivision of the lateral nucleus (La-L), and white matter (WM). Structures that were not consistently present across samples included the centromedial nuclear group—a dorsally located nuclear group in amygdala, the basal forebrain with magnocellular neurons of the nucleus basalis, the ventral extension of claustrum and putamen, and anterior hippocampus. However, pTDP and TDP-43 IHC pathologic observations were recorded when these structures were present.The regions-of-interest (ROIs) examined here are shown in Fig. OTCtx was defined as neocortex of fusiform gyrus lateral to the collateral sulcus [TECtx and ECtx were defined here as used previously [TECtx and ECtx into two ROIs since large multipolar neurons in the pre-α layer of ECtx intercalate with laminae III and IV of TECtx [CM amygdala ROI included several fields in and around the uncus, including the subpial zone and molecular layer overlying amygdala, the underlying ventral cortical nucleus of amygdala containing several layers of pyramidal neurons [BL amygdala represented the largest study ROI , comprising large neurons of lateral, basal and accessory basal subnuclei, and scattered islands of small neurons  [La-L amygdala was the lateral-most ROI, very distinct as stripes of gray matter with medium-sized and large neurons and intervening white matter. The WM ROI focused on the white matter positioned between amygdala and entorhinal cortex. This WM ROI includes perivascular and periventricular white matter, enriched in corpora amylacea, veins with thickened and fibrotic walls (“venous collagenosis”), and rarefied white matter on LFB stain . This area, also referred to as “subamygdaloid white matter” [The ROIs were defined as follows using H&E and LFB/Nissl-stained sections . l sulcus , 27, 35.eviously . No atte neurons , and thenuclei”) . La-L am matter” , containTDP-43 IHC and pTDP IHC pathologies were scored blinded to clinical and pathologic data. Cases designated as “Positive” had inclusions in the same ROI by both TDP-43 and pTDP IHC. In addition, 6 morphologic subtypes of TDP-43 and pTDP IHC pathology were recorded for each participant as being present or absent. The 6 recorded morphologies were as follows:(1) neurofibrillary tangle (NFT)-like neuronal cytoplasmic inclusions, consistent with type-β NCIs as previously described ; 2) rou rou2.Fig (online resource). Briefly, each amygdala sample was scored for pTDP-43 pathology as very mild pathology in 1 ROI (Score 1), multifocal and mild pathology in ≥ 2 ROIs (Score 2), multifocal and moderate pTDP-43 pathology (Score 3), and severe and extensive pathology (Score 4). For each ROI, pTDP-43 inclusion density was recorded in a representative 400 × field, recording 0–30 inclusions per 400 × field . We have previously demonstrated that TDP-43 IHC inclusion density assessed this way is strongly correlated with image-based analyses of pTDP IHC [For each case, semiquantitative measures were applied to determine the severity of pTDP-43 pathology across the entire amygdala region and pTDP-43 pathologic burden within each ROI. The four-tiered semiquantitative scoring approach using pTDP-43 IHC is shown in Supplemental Fig. 4pTDP IHC .Phospho-tau AT8) immunostaining was used to assess argyrophilic grain pathology  immunost and agin (online resource). A four-tiered scale was also used to rate small vessel arteriolosclerosis in the section as being absent, mild, moderate, or severe. Vascular pathology with rarefaction in subamygdaloid WM was rated as present  or absent.Blinded to pTDP and TDP IHC data, H&E and LFB/Nissl stains were used to assess neuronal loss, vascular pathology, reactive astrocytosis, and myelin loss in the amygdala region, using FFPE sections obtained serially adjacent to those used for Tau, TDP-43, and pTDP IHC studies. The grading scheme for each of these pathologies is shown in Supplemental Fig. 5The immunofluorescence protocol used has been described in detail previously , 22 and APOE rs429358/ rs7412, ABCC9 rs704180, GRN rs5848, KCNMB2 rs12496790, and TMEM106B rs1990622  on a Bio-Rad CFX 96 Real-Time System . Interpretation was performed using the Allelic Discrimination function in CFX Maestro Software . Further details of SNP genotyping are provided in the Supplemental Methods file (online resource).For SNP genotyping, DNA was isolated from FFPE samples using the QIAamp DSP DNA FFPE Tissue kit  following manufacturer instructions. SNP genotyping was performed using TaqMan™ probes for After pathologic data and genotyping were completed, additional clinical and pathologic data were added to the study database. Demographic and clinical data included participant age at death, sex, and cognitive status  proximal to death. Pathologic measures included brain weight, ADNC level (NIA/AA 2012)  , presencχ2) testing, as implemented in the software program R [https://software.broadinstitute.org/morpheus) was used to perform unbiased hierarchical clustering of LATE-NC + samples. Post hoc evaluation of group differences between clusters was performed using a non-parametric Kruskal–Wallis rank sum test, as implemented in R.Two-sided Wilcoxon ranked-sum tests and Chi-square  and had a greater level of cognitive impairment (P < 0.02) (Table P < 0.0001), fibrillary astrocytosis (P < 0.001), WM vascular pathology (P < 0.05), and myelin and neuron loss (both P < 0.01) (Table Participants with LATE-NC (LATE-NC +) were older at death   in As shown in Fig. Pattern 1 was distinguished by prominent lamina II TDP-43 pathology in cortical sites of the amygdala region , granular preinclusions, and processes in subpial and WM sites. Double labeling with Tau demonstrated these Lamina II inclusions did not co-localize with Tau . In any given microscopic field, the granular preinclusions in Pattern 1 histologically resembled those often seen in ALS and FTLD-TDP patients.Brains designated as Pattern 2 samples had fibrillary, NFT-like TDP-43 NCIs, corresponding to the previous description of type-β by Josephs and colleagues . These wA common combination of pathologies was seen with features of both Pattern 1 and Pattern 2 in the same brains. These participants had type-β NCIs (NFT-like), as well as small lamina II NCIs and neurites in cortical regions  (FTLD-like), and NCIs in amygdala. Granular preinclusions were variably seen, as were subpial and WM pTDP-43 immunoreactive processes.Pattern 3 was characterized by round amygdala pTDP-43 NCIs and thick neurites in amygdala. Brains with this pattern mostly lacked other features of Patterns 1 and 2. pTDP-43 immunoreactive NCIs in Pattern 3 were seen in both CM and BL ROIs and thick neurites were more apparent on pTDP IHC than TDP-43 IHC staining.Pattern 4 was the most frequent pattern in this study and was represented by subpial and/or WM TDP-43 + processes, often with no or rare NCIs. In these brains there were tortuous subpial TDP-43 + processes often wrapped around corpora amylacea. In WM, they could also be identified adjacent to thick-walled blood vessels, or in rarefied white matter. Double labeling studies demonstrated the TDP-43 processes were bounded by CD44-positive membranes . CD44 highlights astrocytic membranes , and the TDP-43 processes were in areas enriched for GFAP labeling (not shown), suggesting some component of this pathology is within astrocytes. The TDP-43 + processes did not co-localize exactly with tau pathology when ARTAG was also present , although both pathologies could affect the same anatomic region. In addition, these TDP-43 + processes did not co-localize with the axonal markers MAP-2, Tubulin, or pan-neuronal marker .APOE ε4 allele, and frequency of normal cognition.Table . [APOE ε4 allele was not enriched in this group.Pattern 1 cases, which may be referred to as “type-α”, had the highest frequency of Stage 3 LATE. . In distinction from Pattern 1, this group had more frequent LATE-NC Stage 1, and especially LATE-NC Stage 2 cases.Pattern 2, which we designated “type-β” for consistency with Josephs et al. , had theAPOE ε4 (like Pattern 2 samples). Features of Pattern 1 were also present, including lamina II pTDP-43 NCIs. The rates of cognitive impairment (100%) and comorbid HS (82%) were greatest in this group.The combination Pattern 1 + 2 group had a similar profile to Pattern 2, with higher ADNC and higher NFT stage, and was similarly enriched for Stage 2 LATE-NC and Pattern 3 generally lacked features of Patterns 1 and 2, and instead had rounded amygdala NCIs. This pattern was most notable for the high frequency of transitional and diffuse LBD (60%) as compared to other pTDP-43 pathologic patterns , ventral extension of claustrum , the centromedian nuclear complex of the amygdala , and the magnocellular neurons of basal forebrain.TMEM106B risk genotype. Among amygdala region pathologies, LATE-NC was associated with myelin loss, entorhinal cortex cell dropout, astrocytosis, and white matter vascular pathology, as well as amygdala region ARTAG. Using a clustering algorithm, we found four different patterns of LATE-NC pathology, sometimes apparently initiated outside the “amygdala proper”, but within the amygdala region. Although TDP-43 pathologies converged on common anatomic foci at higher stages, the patterns identified were clinically, pathologically, and genetically differentiable. One of these patterns  was commonly seen in persons dying without documented cognitive impairment, and in isolation may represent a subclinical pathology.This study builds on prior work to generate insights into the heterogeneity and evolution of LATE-NC in aged brains. As in earlier studies, LATE-NC was associated with HS, older age, cognitive impairment, and the Methodological strengths of the study included that LATE-NC pathology was determined blinded to other pathologic, clinical, and genetic data. Participants included both TDP-43-negative samples and many brains in early stages of LATE-NC. The TDP-43 pathologic patterns were also determined using relatively unbiased clustering methods, and we correlated the pathologic findings with a large variety of additional clinical and neuropathologic data. Both phosphorylated and non-phosphorylated TDP-43 antibodies were used on serially sectioned brain portions, with the determination that either staining approach is suitable for the neuropathological evaluation of LATE-NC.Earlier studies of TDP-43 pathology have identified stage 1 pathology within amygdala , 39, 42,A primary finding in the present study was that there were four patterns of LATE-NC, based on the clustering of 6 common types of TDP-43 pathologies. These patterns broadly replicate and extend an earlier landmark study, which identified two distinct patterns of LATE-NC, termed type-α and type-β . A new pAnalogous to the prior study by Josephs et al. , PatternAll TDP-43 pathologies studied here were identified using both phospho-TDP-43 and non-phospho-TDP-43 antibodies. The use of both antibodies is not required, although pTDP IHC was more sensitive to certain inclusion types . Conversely, the non-phospho-TDP-43 antibody demonstrates both loss of nuclear labeling and early granular cytoplasmic inclusion pathology, and was more sensitive to granular preinclusions, as previously reported in ALS/FTLD-TDP spectrum brains , 13, 17.TMEM106B risk allele. This finding replicates earlier studies reporting an association between TMEM106B and LATE/HS-Aging [TMEM106B in this study is notable provided the modest sample size, the widely varying severity and patterns of TDP-43 proteinopathy, and the inclusion of cognitively normal participants; thus, the TMEM106B risk allele is a key risk factor for LATE-NC across all patterns of pathology. Although the APOE risk allele did not necessarily associate with LATE-NC in Patterns 1 and 4, APOE ε4 was enriched in samples with prominent type-β NCI pathology (Pattern 2), and in samples with Pattern 3 (type γ).Brains with LATE-NC significantly differed from those without LATE-NC in several ways that were previously described, including increased frequencies of cognitive impairment  and comoHS-Aging , 27, 43.Two novel patterns described here are proposed as Pattern 3 (type γ) and Pattern 4 (type δ). Pattern 3 samples had round NCIs and thick neurites in amygdala, usually without other forms of LATE-NC pathology. Pattern 3 was always associated with cognitive impairment and had the highest rate of transitional and diffuse LBD. Others have found that LATE-NC may have a unique phenotype in patients with LBD , 5, 52. Pattern 4 (type δ) was the most subtle subtype of LATE-NC, featuring TDP-43 + processes in subpial amygdala and/or subamygdaloid WM, often in the absence of NCI pathology. This corresponds to a morphology of TDP-43 first reported in detail by Arnold and colleagues , who alsAPOE ε4. Most Pattern 4 cases were LATE-NC Stage 1, and nearly half of the group was cognitively normal. Since some Pattern 4 cases were cognitively impaired, the pathological diagnosis is likely still warranted with the expectation that LATE-NC stage 1 may often be subclinical. However, our cross-sectional data, including many Pattern 4 cases with LATE-NC stage 1, raise the question as to whether some brains with Pattern 4 TDP-43 pathology in isolation will not progress further in terms of pathologic severity, perhaps analogous to how PART-may not progress to neocortical Braak NFT stages even in very advanced age [TMEM106B risk allele (90.5%), compared to samples without LATE-NC (74%). This does suggest a common pathogenetic pathway in the different LATE-NC subtypes, even with very mild pathology, and suggests that there may be compensatory mechanisms in some Pattern 4 cases. To test these hypotheses will require future studies.Participants with Pattern 4 pathology in isolation had the lowest rates of ADNC and HS, and the lowest frequency of nced age . An alteAPOE risk allele rate (38.2% of study samples) indicated that there was a recruitment bias with a tendency for study samples to have higher-than-normal risk for developing ADNC. Other aspects of potential bias include the general tendency for brain banks to be enriched for high-socioeconomic status patients who may die at older ages than the general population and thus have elevated dementia risk. For definitive establishment of associations between LATE-NC patterns and clinical features, more work will be required. Future studies in larger cohorts, including more diverse samples from additional centers, are needed. An additional area not examined here and requiring further study is whether specific clinical syndromes were associated with LATE-NC patterns, or specific anatomic sites of involvement e.g., selective memory impairment, anomia, other language impairments, and/or prosopagnosia.There are limitations to the current work. The sample size was modest with a total of 184 cases studied. In addition, among study participants, the APOE risk alleles, HS, higher NFT stages, Lewy body pathology, and clinical impairment severity were associated with given patterns of LATE-NC. However, the TMEM106B risk allele was elevated in all LATE-NC patterns, which is remarkable given the heterogeneity of the TDP-43 pathology identified. The different TDP-43 patterns also tended to converge in the more severely affected brains, and each pattern could be seen at any given LATE-NC stage. Thus, our findings support the idea that there are meaningful commonalities under the umbrella diagnosis of LATE-NC.In summary, early foci of LATE-NC TDP-43 pathology occurred at several anatomic locations in the “amygdala region” and not only within the amygdala proper. Certain parameters and features, e.g., Supplementary file1 (DOCX 25 KB)Supplementary file2 (DOCX 97 KB)Supplementary file3 Supplemental Figure 1. Scanned LFB/Nissl sections of two study samples, without (A) and with (B) atrophy in the amygdala region. The major study ROIs are shown, as also depicted in the schematic of Figure 1. (TIF 8410 KB)Supplementary file4 Supplemental Figure 2. Microscopic heterogeneity within the amygdala region. LFB/Nissl images of representative fields as shown in the schematic at top center . Note the heterogeneity of cell density, cell orientation and cell size, even with components of “amygdala”, including ventral cortical nucleus (labeled “CM”), basal (BL) and accessory basal (AB) nuclei, lateral nucleus (Lat) and its lateral division (La-L). Because of the variability across cases, BL, AB, and Lat were grouped into a single ROI (“BL ROI”), and VCo and the parahippocampal-amygdaloid transition area (PHA) was grouped into a single ROI (“CM”). The central and medial nuclei were not incorporated into the CM nucleus because they were not consistent present across all samples; when present, these were never a significant site of early pTDP-43 pathology. (TIF 11119 KB)Supplementary file5 Supplemental Figure 3. Subamygdaloid white matter and surrounding structures. H&E  and LFB/Nissl  stains show subamygdaloid white matter (“WM”), which is positioned between the basolateral division of amygdala  and entorhinal cortex (“ECtx”). This is a common site for conspicuous vascular pathologies, including venous collagenosis (panel C) and perivascular space widening . Corpora amylacea may also be abundant in this region (panel C). One of the morphologic measures in the amygdala region of this study was the combination of rarefied white matter (H&E) with loss of myelin (LFB), venous collagenosis, perivascular space widening and abundant corpora amylacea (scored as present or absent). The sample shown here demonstrates an example of the pattern being “present”. (TIF 11156 KB)Supplementary file6 Supplemental Figure 4. Scoring of pTDP-43 inclusion density in amygdala region. pTDP-43 staining in four representative ROIs are shown in four different amygdalae with LATE-NC (one subject is shown per row). ROIs shown include basolateral nuclear group of amygdala (“BL”), the parahippocampal–amygdaloid transition zone of the CM ROI, layer 2 of the occipitotemporal cortex (“OTCtx”), and the subamygdaloid white matter (“WM”) that is positioned between amygdala and entorhinal cortex. The definitions of scores 0–4 are further defined in the Methods and considered all study ROIs, not just those depicted here. (TIF 8320 KB)Supplementary file7 Supplemental Figure 5. Additional pathologic measures in the amygdala region. Pathologies assessed in the amygdala region, blinded to pTDP-43 status, included fibrillary astrocytosis (H&E) (top row), assessed as present/absent, as well as three-tiered scales to assess neuronal loss in entorhinal cortex (Nissl/LFB) (middle row) and myelin loss (bottom row), particularly in white matter between amygdala and entorhinal cortex. Top row images are from the deep laminae of entorhinal cortex and conspicuous reactive astrocytes are indicated by white arrows. Middle row images are centered on the layer V (pri-α) of entorhinal cortex, and this layer as well as the overlying lamina dissecans (“Lam. Diss”) are labeled. Scale bars for right-most images apply to all images on that row. (TIF 10386 KB)Supplementary file8 Supplemental Figure 6. Overlapping and non-overlapping TDP-43 and Tau pathologies in LATE-NC. (A) The short non-tapering neurites in lamina II that characterized Pattern 1 (type-α) did not co-localize with tau-positive pathology. (B) In contrast, TDP-43 and Tau co-localized in the same neurons and even cytoplasmic foci in this example of a Pattern 2 (type-β). (C) LATE-NC was significantly associated with amygdala region ARTAG, although the TDP-43 processes of Pattern 4 and tau-positive ARTAG did not co-localize. (TIF 6085 KB)Supplementary file9 Supplemental Figure 7. CD44 and TDP-43 labeling in WM and subpial amygdala. CD44 is a non-specific marker, that can be used to highlight astrocytic membranes, as shown in the control CD44-GFAP image in panel A. In WM , CD44 staining and TDP-43 stains show the proximity of TDP-43+ processes and CD44+ membranes, suggesting some of this TDP-43 pathology may occur within astrocytes. Note that in panel D , TDP-43+ process wrap around corpora amylacea, appearing as a negative space in the image surrounded by CD44+ processes. Panel C is enlarged from the region-of-interest in panel B. Panels D and E are enlarged, and a scale bar is provided for both. (TIF 10262 KB)Supplementary file10 Supplemental Figure 8. TDP-43-positive processes and axonal markers. Select samples with Pattern 4 (δ), characterized by TDP-43+ processes, were also investigated using double labeling for TDP-43 and three neuronal and axonal markers. As shown in WM (A) and subpial amygdala (B), no overlap was seen. The possibility that some of this pathology is neuritic/axonal cannot entirely be excluded, but better co-localization was seen in foci of dense CD44 staining  and GFAP labeling (not shown). Scale bar applies to both images. (TIF 8378 KB)Supplementary file11 Supplemental Figure 9. Regions resistant to pTDP-43 pathology in LATE-NC. Paired LFB/Nissl and pTDP-43 stains in ROIs in a single study subject with mild LATE-NC in the amygdala region. Three ROIs frequently positive in LATE-NC are highlighted with a red outline at the bottom right . In contrast, negative ROIs dorsally and laterally are shown, including ventrolateral extent of basal forebrain, ventral extension of claustrum (near La-L), the lateral subdivision of the lateral nucleus of amygdala (La-L), the inferior longitudinal fasciculus (ILF) , and centromedian nuclear complex. This subject is representative of many cases with this medial to ventrolateral and lateral gradient of pTDP-43 pathology in LATE-NC. (TIF 8882 KB)Below is the link to the electronic supplementary material.


Inter-digitate electrodes (IDEs) were printed for humidity monitoring  whilst a meander-based pattern was printed for the temperature measurement  on each side of the PET substrate using silver ink. Moreover, active layers with different concentration ratios were coated on the electrodes using a spray coating technique. The as-developed sensor showed an excellent performance, with a humidity measurement range of (10–90% RH) and temperature measurement range of (25–90 °C) with a fast response  and recovery time . The reliability of the sensor during mechanical bending of up to 5.5 mm was validated with a reliable performance. The sensor was also used in real-world applications to measure human respiration. For this, a suggested sensor-based autonomous wireless node was included in a 3D-printed mask. The manufactured sensor was an excellent contender for wearable and environmental applications because of its exceptional performance, which allowed for the simultaneous measurement of both quantities by a single sensing device.In this paper, we report a fast, linear wide-range hybrid flexible sensor based on a novel composite of strontium titanate (SrTiO Humidity and temperature are co-related environmental conditions with great importance in many industrial and domestic fields such as medical care , agricul2 and LiCl-doped TiO2 [2 [The characterization of hybrid sensors is highly dependent on the properties of the selected materials, which have been chosen by researchers to achieve better stability and reproducibility, a high sensitivity, and a rapid response and recovery time with certain economical fabrication methods ,23. The ped TiO2 , WS2 [28 TiO2 [2 , and two TiO2 [2 . However TiO2 [2 . Therefo TiO2 [2 ; collage TiO2 [2 ; and a p TiO2 [2 . However3) and poly 3,4 ethylenedioxythiophene polystyrene sulfonate (PEDOT:PSS) was used. Inter-digitated-type sensor electrodes (IDEs) for humidity-sensing and meander-type electrodes for temperature-sensing were screen-printed on a flexible PET substrate. The hybrid sensor showed stable results in response to RH for the range (10–100% RH) and temperature for the range (25–90 ℃), with fast transient times of 3 s and 5 s and recovery times of 6 s and 9 s for humidity and temperature, respectively. The performance of the fabricated sensor was assessed in a custom-made electronically controlled chamber and a human-wearable mask was prepared to evaluate the practical human breathing application of the device. Furthermore, real-time and online patient breathing activity and body temperature-monitoring were analyzed with the advanced IoT-based functionality ThingSpeak API.In this paper, we present a hybrid flexible sensor containing a combination of a wide-range flexible humidity sensor and a wide-range temperature sensor for wearable human health-monitoring applications. As the active layer, a composite of strontium titanate (SrTiO3) and poly 3,4 ethylenedioxythiophene polystyrene sulfonate (PEDOT:PSS) were purchased from the Shanghai Macklin Biochemical Co. Ltd.  and Shanghai D&B Biological Science and Technology Co. Ltd. , respectively. Furthermore, a silver epoxy paste (Sigma Aldrich) was used to make the connections to the sensor electrodes. Each of the solvent materials was dissolved separately into acetone and stirred for an hour at room temperature (32 ℃). By taking a specific ratio of 25 mg of SrTiO3 and 1 mL of PEDOT:PSS, a smooth solution was developed. Using a gravity spray gun, the resultant novel solution was sprayed as active layers of the hybrid sensor.For the fabrication of the hybrid sensor electrodes, a polyethylene terephthalate (PET) substrate with 200 Fabrication of the electrodes for the hybrid flexible sensor. For the proposed hybrid sensor, inter-digitated electrodes were printed on one side of the flexible PET substrate for humidity-sensing and a meander-type pattern was printed on the other side for temperature-sensing. Each of the electrode-specific dimensions was designed on the 2D designing software Inkscape. The dimensions for each of the 7 fingers of the designed humidity sensor were 8 mm in length, with a 40 µm space between each electrode pair and 50 µm in thickness. The dimensions for the temperature sensor were 2000 µm in length, a 400 µm space, and 180 µm in thickness, as shown in Spray coating of the conductive slurry. For the spray coating of the specified electrodes of the hybrid sensor, an active layer was created using SrTiO3 and poly 3,4 ethylenedioxythiophene polystyrene sulfonate (PEDOT:PSS) at a ratio of 25 mg and 1 mL, respectively. As an organic component with a high binding force, 1 mL of N, N-Dimethylformamide (DMF) was introduced into the prepared conductive slurry. As an active sensing layer, a properly mixed composite chemical layer was achieved using a hot-plate stirrer. The sensors coated with chemical layers were placed in a laboratory dry oven (DHA-9030A) at 70 °C for 2 h to ensure the optimum film treatment.Sensor characterization. The composition of the material used as the active layer was created using a LabRAM HR Raman spectrometer. A solid state laser of 514 nm was used as an excitation wavelength to acquire the Raman spectra. As shown in With the exception of the dry gas compressor, which was manually operated, the in-house setup used loop-controlled inlets controlled by electronic mass flow controllers (MFCs). The dry air compressor was used to reduce the humidity to its lowest possible level of zero, and, by favoring it, the desired levels of humidity were attained. A smart air cooler was employed to lower the temperature. In contrast, a humidifier and an electronic heater were utilized, respectively, as humidity and temperature sensors to increase both quantities. The temperature of the chamber was maintained at 30 °C throughout the experiment because the ambient temperature would have had an impact on the observations. To receive the real-time and remote graphs of the impedance versus time results on a smartphone, an android application was designed with the help of an online source MIT app inventor.3/PEDOT:PSS is shown in −1 represented the first-order band and the second-order band was from 500 to 1150 cm−1. Herein, various modes of O-Sr-O (TiO2) and O-Sr-O (TiO3) were associated with those peaks. Moreover, a significant change was observed in the first-order band compared with the addition of PEDOT:PSS for the preparation of the composite material. However, the second-order showed the attributes for the prepared active layer. The relatively higher peak of 1095 cm−1 could have been related to the C-O out-of-plane bending vibration for PEDOT:PSS.The Raman spectra of the composite SrTiOThe chemical properties of the composite materials that were sprayed determined how our hybrid sensor functioned. Due to the hydrophilic behavior of the materials, changes in humidity and temperature tended to alter the impedance of the sensor. Both sensors displayed a capacitive and resistive shift in their response, but the impedance change had the main effect. The capture of the airborne water vapors of the detecting layer led to an increase in the conductivity of the sensor. The impedance was seen to decrease as a result. Additionally, in the case of temperature-sensing, the rise in impedance was compared with the rise in temperature. The chemisorption and physisorption phenomena discussed here could each be used to describe the response of the proposed hybrid sensor. In the process of chemisorption, humidity rises and more water vapor is produced. The sensing layer traps such vapors, which enhances the ionic conductivity and triggers the protonic conduction, which initiates the current flow between electrodes. The use of physisorption of the sensing devices is a natural behavior ,34.The reaction of the proposed hybrid sensor was based on the chemical characteristics of the sensing layers, including the structure of the sensor. The layers formed from the composite material drew heat and water vapor from the environment, comparatively enhancing the conductivity by lowering the impedance of the sensor. The decrease in the impedance of the sensor was inversely proportional to the rise in relative humidity and temperature. As conductive electrodes with a dielectric space in between, humidity-sensing IDEs have a structure that is both capacitive and resistive in nature. As a result, the impedance value was calculated in relation to changing humidity values, and temperature-resistive behavior was noted. The constructed sensors were cured for two hours at 70 °C prior to the electrical investigation. The electrical data were measured at a variety of frequencies, although the responses at 1 kHz and 10 kHz were more appropriate for this article. For the electrical characterization, the fabricated sensor was analyzed in a custom-developed electronically controlled chamber. The results showed that the change in the impedance was inversely proportional to the positive change in humidity and directly proportional to the change in temperature. The electrical response of the device is shown in It was apparent from the analysis that the general behavior of the sensor was not the same throughout the range. There was a fast change in the impedance against the humidity; for large values of humidity, there was a small change in the impedance due to adsorption. The device showed an average change of 77.5 kΩ with each percent change in relative humidity. For the sensor at both frequencies (1 kHz and 10 kHz), the sensing pattern was significantly matched. The results shown in The outcomes shown in The response and recovery times of a sensor are crucial, especially for any potential real-time applications. A repeated cycle of instantaneous humidification and dehumidification was applied to the e-controlled chamber in order to measure the transient response of the sensor with the response and recovery time. For that purpose, controlled quick-responding valves of a humidifier and a nitrogen gas cylinder were used ,36,37. FThe sensor, as created, was eventually utilized to track the breathing activity of a human. A wearable mask was 3D-manufactured for this purpose, as illustrated in The human body, which is made up of joints wrapped in stretchy skin, necessitates flexible devices that are not impacted by the flexibility or stretchability of the skin. As a result, preferable gadgets in human health-monitoring systems are those whose results are unaffected by bending. Herein, the silver conductive electrodes for both humidity and temperature were fabricated on a flexible PET substrate. The performance of the sensor was stable, linear, sensitive, and reliable with a bending radium of 5.5 mm, which showed the mechanically analyzed flexibility of the sensor. This mechanical evaluation supported the application of the sensor in a wearable invasive and non-invasive medical aspect. The mechanical bending process and unaffected observations are shown in Altogether, a flexible biocompatible hybrid sensor with a fast response and recovery time that was highly sensitive to large ranges in humidity and temperature is reported here. The results defend its performance in many fields of work, particularly related to health-monitoring applications.In this paper, we designed, built, and tested a hybrid flexible sensor with high linearity and stability, and a wide sensing range for humidity and temperature. The electrodes of the hybrid sensor were created using a screen-printing process. As the sensing layers for the hybrid sensor, thin films of a new combination of SrTiO3 and PEDOT:PSS were used with a spray coating on the electrodes. The electrical, chemical, and mechanical characteristics of the sensor were examined to evaluate the properties. The response and recovery times of the sensor in respect to humidity were 5 s and 8 s, respectively; these were 4.2 s and 4.4 s in the case of the temperature sensor. The fabricated sensor was mechanically stable with a consistent response to bending at a radius of 5.5 mm, showing an excellent sensitivity with 77.5 kΩ/% RH at a wide range of 10–90% RH. Moreover, the sensitivity of 0.3 ohms/°C was recorded from 30–80 °C. For the practical application of the as-developed sensor, a mask was 3D-printed, which carried all the necessary components to be a wireless breath-monitoring apparatus in addition to the sensor. The sensor could easily distinguish between different breathing styles, enabling it to be a strong commercial candidate for wearable health-monitoring applications.


Our results revealed that advanced EFL learners had not yet attained native-equivalent implicit grammar knowledge in English questions, despite their English proficiency level. The results also indicated that the learners’ different L1 languages do not impact advanced learners’ overall implicit knowledge acquisition but constitute influential factors for particular morphosyntactic inflections in English question formation.This study aims to explore whether advanced EFL learners can acquire implicit knowledge of basic sentence structures, such as English questions. We ran a reaction-time experiment, a word monitoring test experiment to test learners’ implicit knowledge by checking advanced EFL learners’ grammatical sensitivity to English questions with five types of grammatical errors. The study recruited three groups of participants: native English speakers ( One important topic in second language acquisition (SLA) concerns L2 learners’ acquisition of implicit knowledge and, specifically, to what extent L2 learners can acquire implicit language knowledge. A high level of implicit knowledge correlates with learners’ high linguistic competence ,2,3. TheTo address the aforementioned research concerns, we raised the following two research questions for the present study.RQ1: Did advanced EFL learners acquire implicit knowledge that was equivalent to the level of native speakers?RQ2: What might potentially impact Spanish and Chinese EFL learners’ implicit knowledge acquisition, in terms of English question formation?wh in situ” language [wh-movement, nor any subject-verb inversion or auxiliary insertion in questions. Chinese yes/no-questions are formed by adding particles, such as ma or ba, at the end of a declarative sentence (as shown in (1c)), with no morphological inflection on verbs [wh-questions do show wh-movement, as in English, but without auxiliary do-support or subject–auxiliary inversion, basic Spanish yes/no questions are built by just changing intonation in sentences, as exemplified in (1b) and (2b). Although Spanish does have auxiliaries, such as haber, “have”, or ser, “be”, for perfective and passive tenses, respectively, their syntactic behavior differs from those in English [The acquisition of morphosyntactic inflections in question formation is difficult for L2 English learners since the syntactic manipulation of interrogatives is far more complex than the syntax of statements . Diversilanguage . That ison verbs . Meanwhi English .(i)English: John has gone to Tarragona.—Has John gone to Tarragona? vs. *John has gone to Tarragona?(ii)Spanish: Juan ha ido a Tarragona.—*Ha Juan ido a Tarragona? vs. Juan ha ido a Tarragona? To illustrate the syntactic differences between English and Spanish auxiliaries, see the contrast in (i)–(ii), where examples can be said to be word-by-word translations.Yes-no Questions(1) a. English:Doyou get permission to enter the office?b. Spanish:¿Tienespermisoparaentrara laoficina?2sg.PrsIndget.permissiontoenterto theoffice?c. Chinese:Nidedaoxukequjinbangongshi ma?YougetpermissiontoenterofficeqWh-Questions(2) a. English:Wheredotheywant tobuildtheirnewfactory?b. Spanish:¿Dóndequierenconstruirsunuevafábrica?wherepl.PrsIndwant.3buildtheirnewfactory?c. Chinese:Tamenxiangzainalixiujiantamen dexingongchang?Theywant.toatwherebuildtheirnewfactory?The following example questions in English, Chinese, and Spanish illustrate basic morphosyntactic differences in question structures in these three languages. We use the following abbreviations for glosses: 2: second person; 3: third person; sg: singular; pl: Plural; PrsInd: Present Indicative; q: Question marker.wh-questions and embedded questions [wh-questions [wh-questions by Cantonese learners of English. More importantly, those learners who seem to have gained a high communicative ability still produce basic English grammatical errors, such as dropping plural markings or creating third-person present-tense morphemes. Acquisition difficulty in morphology is also reflected in morphosyntactic inflections, in the context of forming English questions. For example, Hong Kong ESL learners had difficulty in acquiring wh-questions, and exhibited grammatical errors when using be and do , or failing to invert verb structures [yes/no questions in their L1 language affected their choice of linguistic forms [Morphological inflection is problematic for FL/L2 learners from various L1  languageuestions . In a couestions  reporteduestions ,26. Pozzuestions  found thuestions . Ma (201uestions  found a ructures . Moreoveic forms .*Is your son like doing extreme sports as much as you?), GAUXT errors , GAUXA errors , GAUXO errors , and GAUXM errors . Among all five categories of errors, grammatical errors made by Chinese preliminary learners were mainly distributed between GAUXC and GAUXT errors, while those from preliminary Spanish learners were evenly located among GAUXA, GAUXC, and GAUXT errors [Although a series of studies have investigated the difficulties found in acquiring morphosyntactic features in English questions from different perspectives, morphosyntactic errors in English question formation have not been systematically reviewed and categorized. To find out whether cross-linguistic transfer from L1 hindered the formation of English questions in preliminary L2 learners, He and Oltra-Massuet  classifiT errors . HoweverTherefore, a study targeting advanced EFL learners’ grammatical sensitivity to the fine-grained types of morphosyntactic errors displayed by preliminary learners was conducted to uncover the learners’ degrees of implicit knowledge acquisition. Moreover, focusing on a specific sentence structure with a group of advanced EFL learners from two different L1 backgrounds should reinforce the results regarding the potential influence of L1 on the learners’ acquisition of implicit knowledge.In the field of second-language acquisition (SLA), the concept of implicit knowledge has been defined in comparison with explicit knowledge, as its characteristics are elucidated, to a large extent, by the contrasting properties between the dyads of implicit and explicit knowledge. Ellis (2005)  differenImplicit knowledge is acquired in a natural and simple process that involves no conscious operations . AlthougHowever, previous studies have shown differing views on whether L2 learners can acquire implicit L2 knowledge and on how implicit knowledge is acquired. In reflecting on frequency effects in language processing, N. Ellis (2002)  evaluateBy comparing cross-linguistic experiments, Hopp (2010)  showed tExisting studies ,40,41,42As for whether EFL learners’ L1 influences their L2 morphosyntactic acquisition, previous studies ,45,46,47In the study of the acquisition of plural noun phrases by Spanish and Korean learners of English, Ionin and Montrul  found thOther research has suggested that the L1’s influence constantly affects L2 learners’ acquisition of target structures. These studies revealed that learners’ L1 had an influence on L2 morphosyntactic acquisition, and L1–L2 morphosyntactic congruity was one of the most prominent factors. Jarvis  found thThe present study intends to explore this issue from the perspective of the morphological congruency hypothesis of Jiang et al. . This wamorphological congruency hypothesis is as follows:Jiang et al.’s “When L2 learners reach an advanced or near-native level of L2 proficiency, only congruent learners  are able to reach native-like proficiency in acquiring an L2 morpheme. Incongruent L2 learners will find it extremely difficult, if not impossible, to develop native-like competence with respect to the same L2 morpheme.”  (i)Advanced L2 learners encountered great difficulty in acquiring native-like knowledge in grammatical morphemes when the morpheme in their L1 was not congruent with the target morpheme in an L2.(ii)For L2 learners with different L1s, learners with congruent L1 morphemes performed better than those without congruent L1 morphemes.(iii)For L2 learners from the same L1 background who were learning different morphemes, the learners performed better concerning grammatical morphemes that were congruent than on those that were incongruent with their L1.As illustrated by Jiang et al. (pp. 943–944) , their hWhen exploring the potential challenges in the acquisition of English questions for the EFL learners under investigation, we formed three predictions, based on the morphological congruency hypothesis.Prediction 1: Advanced EFL learners are confronted by great difficulty in the acquisition of native-like implicit knowledge of morphosyntactic features in English question formation that are not instantiated in their L1.Prediction 2: Spanish EFL learners outperform Chinese EFL learners in acquiring morphosyntactic features in English question formation because there are morphological inflections on verbs in Spanish, but not in Chinese.Prediction 3: For Spanish EFL learners, there are congruent morphosyntactic features and incongruent morphosyntactic features in question formation. Spanish EFL learners are expected to perform better on English morphosyntactic features that are congruent with morphological inflections in Spanish.The goal of the present study is to find out to what extent advanced EFL learners can attain implicit language knowledge and investigate whether EFL learners from two different L1s displayed a difference in their acquisition of implicit knowledge. This study further explores what might constitute difficulties for advanced EFL learners in acquiring implicit knowledge of morphosyntactic features in English question formation. Based on previous studies on the acquisition of implicit knowledge and the theoretical framework of morphological congruency, we formulated the following two hypotheses:Hypothesis 1 (H1).Advanced EFL learners cannot acquire an implicit knowledge level equivalent to native speakers’ level.Hypothesis 2 (H2).Particular types of morphosyntactic inflections in English question formation constitute an influencing factor for EFL learners’ implicit knowledge acquisition of English questions.n = 32) and advanced Spanish EFL learners (n = 37). We collected data from 75 participants, and obtained valid data from 69 participants, with an effective rate of 92%. The study recruited 81 participants online, comprising 12 monolingual native English speakers and 69 advanced EFL learners, ranging from 20 to 40 years old. All advanced English learners were reported to possess a C1 English proficiency level (common European framework of reference for language (CEFR)), which was further confirmed via a short online C1-level test (Cambridge English test) immediately prior to completing the experimental tasks. The EFL learners consisted of two groups of participants: advanced Chinese EFL learners (https://app.gorilla.sc/ (accessed on 25 July2021). All participants were recruited via the experiment participants’ recruitment platform, https://www.prolific.co/ (accessed between 20 June 2021 and 22 December 2021), which routes participants directly to the experiment platform. Every participant was required to give informed consent and read the experiment instruction before entering the experiment page. Participants started the experiment after filling out the background information questionnaire and passing the English proficiency test by presenting at least three correct answers out of five questions. The whole experiment lasted for about 30 min.The experimental procedures in the present study were reviewed and approved by the Research–Innovation Ethics Committee from the Universitat Rovira i Virgili, Spain. The experiment was conducted online on the behavioral experiment platform, There is a battery of tests that were designed by R. Ellis ,30 to meThe experimental procedure is displayed in An example of an experimental item is shown in the following:*Did she enjoyed Jean’s birthday party last evening?Sentence audio: Target word: JeanConceptual sentence for judging the meaning: Jean had her birthday party last evening .While the participant was listening to the experimental sentence *There were 5 practice items, 60 critical experimental sentences, and 30 filler items in the experimental design. Participants had to complete all 90 items in the experiment. Among the 60 critical experimental sentences, 30 ungrammatical sentences were experimental items, and 30 grammatical sentences were used as control items to calculate the RT. The grammatical sentences were used as “a baseline for determining if the phenomenon of lags in response, driven by implicit knowledge, is present” . Thirty a. GAUXC:*Are you get the permission to enter the office?Do you get the permission to enter the office?b. GAUXT:* Before their final decision, what do they say in the meeting?Before their final decision, what did they say in the meeting?c. GAUXA:*Do your uncle live in the new neighborhood nearby?Does your uncle live in the new neighborhood nearby?d. GAUXO:*Will be he giving a presentation at the conference?Will he be giving a presentation at the conference?e. GAUXM:*Have you get a new job offer after the interviews?Have you got a new job offer after the interviews?Experimental items :*When did they went to the fun park yesterday?/When did they go to the fun park yesterday?, by comparing the participants’ Reaction Time (RT) on the target words the fun park, we can determine whether participants are sensitive to grammatical violations. The preposition “to” was not included as part of the target words, because of its status as an unstressed functional word, which is easily missed in audio recordings. As pointed out by Jiang (2013: p. 196), “One can be quite flexible in choosing targets. It can be any word that happens to be at a particular location in a sentence.” The concept of the grammatical sensitivity index (GSI) developed by Suzuki [This study is designed to find out whether EFL learners can acquire implicit knowledge of typical English questions, as measured by participants’ reaction times in response to the target word in both grammatical and ungrammatical sentences. The reaction time reflects the participants’ sensitivity to grammar violations in morphosyntactic errors in English questions. For example, in the paired test sentences, y Suzuki  was intrEach learner’s language background is an important variable that may influence their acquisition of implicit knowledge. Therefore, language background was manipulated as an independent variable in the study, which included Spanish and Chinese EFL learners as experimental groups and native speakers as the referring group. In addition, in order to detect what constitutes an influential factor in EFL learners’ implicit knowledge acquisition, we also introduced five types of morphosyntactic errors in question formation as an independent variable to measure their degree of acquisition, compared to native speakers’ data.t-test, with the number of years of English classes as an independent variable and the group as the dependent variable. The Kolmogorov–Smirnov test suggested that the GSI was not normally distributed; however, we selected parametric tests for data analysis for the following two reasons: (1) the Kolmogorov–Smirnov test results flux and are not always reliable as the sample size varies, especially with a large sample size [p-value of 0.05 was considered to be the statistical significance threshold.After the experiments were completed, we pre-processed the data, and then input them into the Microsoft Excel database. The IBM Statistical Package for Social Sciences (SPSS) version 22.0  was used for statistical description and statistical inference. The participants’ demographic characteristics were described using descriptive statistics, including the frequency and constituent ratio. The difference in categorical variables in the demographics between groups was tested using the chi-square test, while the difference in the number of years of taking English classes between Chinese speakers (CS) and Spanish speakers (SS) was tested using an independent two-sample ple size ; (2) a nple size . TherefoThe participants’ grammatical sensitivity index (GSI) values were computed based on the RT difference derived from the mean RT of ungrammatical items, minus the mean RT of the grammatical items. Based on the pre-designed criterion for eliminating outliers, we excluded 105 items from the NS group, 132 items from the CS group, and 158 from the SS group. The mean GSI of the native-speaker group was 125.8 ± 25.8; for the Chinese EFL learner group, it was 40.4 ± 11.4, and for the Spanish EFL learner group, it was 58.9 ± 11.7.= 5.630, p = 0.004). The post hoc tests using the LSD test further suggested that the NS group had a mean GSI of 125.8, which was significantly higher compared to that in the CS group  and SS group  indicating that both the Spanish and Chinese advanced EFL learners did not acquire a general implicit knowledge level that was equivalent to the native speakers’ level.The results of the ANOVA indicated a significant difference among the three groups in terms of mean GSI . The results are depicted in However, the difference between the CS and SS groups in terms of the mean GSI was not statistically significant , GAUXC , GAUXM , GAUXT , and GAUXO , suggesting a significant difference among the three groups in terms of GAUXC, that is, the choice of auxiliary. However, the mean GSI scores of the NS group are numerically higher than those of the SS group, followed by the CS group in all types of errors, except for the GAUXT items (see p = 0.004) and between the Spanish group and the native group (p = 0.021). Meanwhile, the Chinese group showed significantly lower sensitivity compared to the native group (p = 0.028) in terms of GAUXT when detecting the morphological inflection related to verb tense in forming questions, while the Spanish group did not show significant differences compared to the native group (p = 0.163).For the five types of error, the results of the ANOVA for each type of error among the three groups are as follows: GAUXA  the GSI difference between the native-speaking group (NS) and two EFL groups , exploring the degree of implicit knowledge of English questions acquired by advanced EFL learners; (2) the statistical results between advanced EFL learners and the NS group with respect to the morphosyntactic inflections involved in English question formation, revealing whether morphosyntactic inflections may impede EFL learners’ implicit acquisition regarding English questions.wh-questions [As we hypothesized, we found a significant difference in GSI between the NS group and the two EFL groups, revealing that advanced EFL learners did not attain a general level of implicit knowledge in English questions that is equivalent to that of native speakers. As summarized in uestions , Indian uestions , Cantoneuestions , and Spauestions ,48. The The present study also revealed that the two advanced EFL groups of Chinese and Spanish learners showed a similar overall level of implicit knowledge in terms of English questions. Similar conclusions were drawn on the issue in previous studies. Hopp’s  study onSince we found that the two advanced EFL learner groups did not acquire a native-like level of implicit knowledge of English questions, we further explored the experimental data on five different types of errors, in order to ascertain what might constitute the specific difficulties impeding EFL learners from progressing to a native-like level of implicit knowledge. The statistical results in do-support auxiliary is required to form questions, even if the language does have auxiliaries in other contexts. Advanced EFL learners may be able to use auxiliaries on specific occasions, when they are consciously paying attention to the language forms, but the participants did not detect the grammatical violations when their focus had been directed toward key-pressing and sentence meaning in the experiment, which indicates that no implicit knowledge was available for them to recognize the error subconsciously.We can relate these specific morphosyntactic problems to Jiang et al.’s  morpholoAs for the other four types of morphosyntactic errors, GAUXA, GAUXO, GAUXT, and GAUXM, which involve morphological inflections on number, person, and tense, Spanish EFL learners showed a high degree of implicit knowledge, as this first language displays a complex morphosyntactic system for person, number, tense, mood, and aspect features in verbs . This suWith respect to Chinese, this language has neither auxiliaries nor morphological verb inflections of person, number, or tense (as seen in examples (1c) and (2c), above); thus, no morphological congruency exists at all between Chinese and English questions. Therefore, most Chinese EFL learners rely heavily on the grammatical knowledge they have learned at different stages, progressing to high proficiency by means of practice and communication, which facilitate their language acquisition. Although advanced Chinese EFL learners showed similar levels of implicit knowledge to their Spanish counterparts, they were confronted with more challenges in recognizing GAUXC and GAUXT errors, which means that they did not acquire implicit knowledge in either the choice of auxiliary or in verbal tense morphology in English questions. This finding is in line with the prediction that Spanish EFL learners would outperform Chinese EFL learners in acquiring morphosyntactic features in English question formation because of Spanish’s congruent morphological inflections in verbal phrases.Our findings showed similarities and differences between advanced Chinese and Spanish EFL groups in the acquisition of the implicit knowledge of morphosyntactic features in English questions. The experimental results revealed that the degree of implicit knowledge acquisition of English questions is similar for EFL learners from two different L1 backgrounds when they are at an equivalent proficiency level. However, the two groups encountered different challenges in the acquisition of implicit knowledge regarding different types of morphosyntactic features, due to the differences in L1–L2 morphosyntactic congruity. As shown in previous studies ,30,60,61The present study was conducted within an online environment. There are many advantages of using the online experimental mode. For example, recruited participants can be drawn from different areas of the same L1 background, which may be more representative than targeting participants from a specific language class. However, the drawbacks of the present study cannot be ignored. Although we explicitly stated that participants should have a quiet and undisturbed experimental environment, quality equipment in terms of audio and recorder toolsets, a stable internet connection, and be in a peaceful state, we could not control for other potential factors that may have disturbed them, such as noise, interactions with co-inhabitants, and other distractions affecting their concentration. A pure experimental environment, such as that in a laboratory, would be optimal for the stricter control of disrupting factors. Moreover, we need to acknowledge that L2 learners are more tolerant of errors; however, the present study did not introduce acceptability judgment tasks to the model to investigate our participants’ degree of tolerance of the different types of morphosyntactic errors, which is worthy of exploration in future studies.The hypotheses and experiments were well-grounded in previous studies; however, the sample size of this study is relatively small. In order to magnify the significance and explanatory power of the study, future studies may collect a larger sample from different proficiency levels and L1 backgrounds for more robust hypothesis support.In the current study, we adopted the word monitoring task, to measure the degree of implicit knowledge acquisition of English questions for advanced EFL learners from two different L1 backgrounds and explore the potential effect of L1 morphological influence on their language acquisition process. The grammatical sensitivity index comparing participants’ RTs to (un)grammatical sentences was analyzed, and an in-depth exploration was performed with a fine-grained classification of the five different types of grammatical problems, based on the different aspects of the morphosyntactic inflection of English questions. The L1 morphosyntactic features of these questions were discussed to reveal the inhibiting factors for implicit knowledge acquisition for different advanced EFL learners. To sum up, this study revealed that: (1) it is difficult for advanced EFL learners to acquire implicit language knowledge that is equivalent to that of native speakers, despite their having acquired a high level of language knowledge and achieved high English proficiency; (2) EFL learners’ different L1 backgrounds do not affect their overall implicit knowledge acquisition; (3) EFL learners encounter distinct difficulties in acquiring implicit knowledge of morphosyntactic inflection in the context of questions, due to L1–L2 incongruency.Future studies involving EFL learners at different proficiency levels, including beginner, medium, and advanced levels, who are drawn from different L1 backgrounds, may ideally uncover the trajectory of how the influence of the L1 evolves in the L2 acquisition process. In addition, studies investigating the language learning context within the framework of social-cultural theory may be of great significance to improving our understanding of EFL learners’ acquisition of implicit knowledge in their unique language contexts.


A composite was prepared using two suspensions: nano-zirconia obtained by sol-gel synthesis and oxygen-free graphene obtained sonochemically. The morphology of oxygen-free graphene sheets, phase composition and the morphology of a zirconia powder, and the morphology of the synthesized composite were studied. The effect of the graphene sheets on the rheological and sintering properties of a nanostructured zirconia-based composite powder has been studied. It has been found that graphene sheets in a hybrid nanostructure make it difficult to press at the elastic deformation stage, and the composite passes into the plastic region at a lower pressure than a single nano-zirconia. A sintering mechanism was proposed for a composite with a graphene content of 0.635 wt%, in which graphene is an important factor affecting the process mechanism. It has been determined that the activation energy of the composite sintering is more than two times higher than for a single nano-zirconia. Apparently, due to the van der Waals interaction, the graphene sheets partially stabilize the zirconia and prevent the disordering of the surface monolayers of its nanocrystals and premelting prior to the sintering. This leads to an increase in the activation energy of the composite sintering, and its sintering occurs, according to a mixed mechanism, in which the grain boundary diffusion predominates, in contrast to the single nano-zirconia sintering, which occurs through a viscous flow.The use of a nanostructured graphene-zirconia composite will allow the development of new materials with improved performance properties and a high functionality. This work covers a stepwise study related to the creation of a nanostructured composite based on ZrO In the last decade, the growing interest of the developers of new materials has been turned to graphene-ceramic hybrids in particular, based on zirconia, in whose structure the graphene particles or its derivatives (reduced oxidized graphene or oxidized graphene) are incorporated, since they have unique physicochemical properties . The spe2-electron system that provides unique electronic properties. Compared to the oxygen-free graphene, the oxidized graphene and the reduced oxidized graphene showed only a slight increase in the fracture toughness through their reduced mechanical strength and structural defects  were constructed graphs, the tangent of the angle of which S2 makes it possible to determine the apparent activation energy of the sintering by the Equation (3):The same heating rate graphs in the coordinates 1/T—ln [T:2 nanopowder (ii), the obtaining of the hybrid nanostructures using the graphene suspension and the nanocrystalline ZrO2 powder (iii). The synthesis of the graphene-zirconia nanostructured powders consisted of three stages: exfoliation of the oxygen-free graphene sheets using the sonochemical method and the stabilization of its suspension (i), preparation of the ZrO−1, the main spectral characteristic is observed in the form of a narrow band G, which arises as a result of the stretching vibrations in the plane of the carbon atoms bound by the sp2 bonds. The small width of the G band indicates the predominance of a low-layer, low-defect oxygen-free graphene. The latter is also confirmed by the EELS analysis data (inset): the absence of the 532 eV peak, characteristic of the oxidized graphene indicates the absence of the O-containing functional groups on the surface of the graphene sheets. At the same time, a broad peak centered at 284 eV, indicates the transition from 1s to π* and confirms the presence of the sp2 carbon atoms in the system. The D band in the region of 1347 cm−1, characterizes the degree of disorder in the electronic structure of the carbon layers, which occurs when the lattice moves from the middle of the Brillouin zone. A large value of intensity ratio IG/ID indicates a low degree of defectiveness of the graphene. The 2D mode in the region of 2711 cm−1, is an overtone that characterizes the internal properties of the graphene and appears as a result of the double resonant Raman scattering. The overtone G* in the region of 2441 cm−1, is also characteristic of the graphene and is due to the presence of a few defects since the peak intensity is low [According to the Raman shown in y is low ,40.According to the TEM data, the graphene in the suspension, consisted of submicron blocks sheets a. The el2 adsorption–desorption methods. The residual carbon content in the prepared single zirconia powder was 0.07 (±0.01) wt%.The phase composition and morphology of the zirconia nanopowder obtained by the sol-gel synthesis, were studied by XRD, TEM, electron diffraction, and N2 tetragonal modification tP6 (JCPDS card no. 24-1164) and 54 wt% from the ZrO2 monoclinic mP12 (JCPDS card no. 05-0543). The average size of the zirconia crystallites, calculated using the Rietveld method, was 9 nm for the monoclinic modification and 10 nm—for the tetragonal modification. According to TEM data, the zirconia powder consisted of agglomerates with sizes of 100–300 nm formed by 7–13 nm crystallites  of the powder, c of the nanostructured hybrid is several times higher than that of the single nano-zirconia. We have seen a similar pattern earlier [A comparative study of the effect of adding graphene on the compaction dynamics, during the pressing of nano-zirconia was carried out, and  earlier , and thiIt is probable that the reason for the observed phenomenon, is the elasticity of the graphene sheets, which prevents the system from being compressed at the stage of the elastic deformation. As a result, the composite passes into the plastic deformation region at a lower pressure than the single nano-zirconia. It can also be seen that a higher pressure is required to achieve the same degree of deformation for the single nano-zirconia. It can also be assumed that, at the stage of the elastic deformation, due to the van der Waals interaction of the hybrid nanoparticles, the particles of a more regular shape, close to isometric, are formed because by optimizing their arrangement in the volume.Next, the parabolic sections of the curves correspond to the transition of the systems to the stage of accommodation (adaptation), where the particles begin to move relative to each other to more effectively fill the voids between the larger particles with smaller ones. At this stage, only an insignificant contribution of the particle deformation to the process of the compaction of powders was observed .The final, exponential sections of the shrinkage curves correspond to an intense increase in the stress with small changes in the deformation, apparently because there is practically no free space left in the compact, and only the particles themselves can be deformed. A comparison of the experimental data for the graphene-containing composite and the initial single nano-zirconia, shows that the compressibility modulus of the latter is much lower, while it should be noted that a higher pressure is required to achieve the same degree of deformation for single nano-zirconia.A comparative study of the shrinkage of the nanostructured graphene-zirconia and the nano-zirconia powders, was carried out. 0 curve with a sharp increase in the shrinkage rate (dL/dt) from 0.42%/min (1210 °C) to the maximum value, 0.85%/min (1395 °C), in the region of 1400 °C. The observed break in the curve coincides with the temperature of the ZrO2 phase transition from the monoclinic to the tetragonal modification (1170 °C). The total shrinkage of the sample over the entire temperature range studied (from room temperature to 1600 °C), was more than 24%.In the temperature range of 1170–1178 °C, a break is observed on the dL/L2 nanopowder, which was used to obtain a composite with graphene. The initial thermal expansion of the sample was 2.06%. The start of the sintering was recorded at the temperature of 595.8 °C, and it corresponded to a sharp increase in the shrinkage rate of the sample, which exceeded 0.26%/min at 681 °C. The observed temperature of the phase transition from the monoclinic to the tetragonal modification was 1165–1166 °C, when the sample shrinkage (dL/L0) did not exceed 0.5%. Then, the accelerated growth of the shrinkage was observed at the final stage of the experiment, in the temperature range 1439–1600 °C. The maximum speed, in this case, was more than 0.38%/min at 1561 °C, and the total shrinkage of the sample in the entire experiment was more than 9%.For comparison, For comparison, the results of a dilatometric study of the synthesized graphene-containing composite and a single nano-zirconia, are presented in the summary The effects found can only be explained by the presence of the graphene sheets, which, firstly, restrain the expansion of the zirconia agglomerates and, secondly, partially prevent the disordering of the surface monolayers of the zirconia crystallites, prior to the sintering. Apparently, the graphene sheets “enveloping” the zirconia crystallites partially reduce the surface diffusion rate of the atoms forming these crystallites at the initial and middle stages of the sintering in the temperature range up to 1000 °C. At the same time, in the region of 650–700 °C, the shrinkage is approximately 8 and 7% of the initial sample sizes for the composite and the single nano-zirconia, respectively.2-electron system of the graphene (δ−). 2 nanocrystals. The increased defectiveness of the crystal structure is known to be typical of nano-objects, which is confirmed by the XRD data for the single nano-zirconia synthesized, which was subsequently used to obtain a composite with graphene.The effect of the graphene sheets on the rate and magnitude of the shrinkage is most pronounced at temperatures above 1000 °C. An increase in the shrinkage rate and the total shrinkage of the composite, compared to a single nano-zirconia, may be due to an increase in the surface energy of the zirconia crystallites, as a result of the van der Waals interaction with the spAs shown above, based on the analysis of the dilatometric data, as well as the results of the kinetic calculations, the effect of the addition of 0.635 wt% graphene on the sintering dynamics of the nanostructured zirconia powder and the mass transfer mechanism during the sintering was shown. According to the results of the calculation presented in With an increase in temperature, the disordering occurs in the surface monolayers of the zirconia particles, especially on the edges and tops of the crystallites, as well as on the protrusions of the agglomerates, i.e., in the regions of higher surface energy. At the initial stage, at a relatively low activation energy and a large surface area of the particles, the viscous flow dominates, due to the high shear stresses in the contact area.The beginning of the sintering is accompanied by an increase in the contact area between the particles and their initial association with each other; in this case, the particles retain their structural individuality, and their contours are practically preserved. Subsequently, because of the mass transfer, an exchange occurs between the vacancies and the highly mobile atoms, and in the interparticle spaces, the latter are concentrated and have become shared by several particles. This leads to the gradual settling of vacancies, the filling of pores and compaction. With an increase in temperature, the bulk diffusion becomes more intense, and with the strengthening of the interparticle bonds, the diffusion at the grain boundaries becomes more active.2 monolayers parallel to the graphene sheets, resulting in a change in volume and shape and the compaction of the ceramic particles [In the case of a composite powder, a change in the sintering mechanism is observed: instead of the viscous flow process, a mixed mechanism is realized in the contact areas of the zirconia particles, in which the grain boundary diffusion prevails. In this case, the second component of the mechanism is the volume diffusion in those areas where the clean surfaces of the zirconia nanoparticles interact with the subsequent coalescence and the formation of the uniform grain boundaries. Where the graphene sheets are between the zirconia particles, most likely, the grain-boundary diffusion occurs with the movement of the surface ZrOarticles . The comThe effect of the graphene sheets on the rheological and sintering properties of a nanostructured composite powder, based on the zirconia, has been studied. The work covers the research from the synthesis of the nano-zirconia powder to a graphene-containing nanostructured composite, based on it. The paper reports on a method for synthesizing a nanostructured composite, using two suspensions: the nano-zirconia obtained by the sol-gel synthesis and the oxygen-free graphene obtained by a sonochemical method. The morphology of the oxygen-free graphene sheets, the phase composition and the morphology of the zirconia powder, as well as the morphology of the synthesized composite were studied. A comparative study of the rheology, the dynamics of the sintering, and the activation energy of the sintering of the pure nano-zirconia and a graphene-containing composite based on it, has been carried out.It has been found that the graphene sheets in a hybrid nanostructure make it difficult to press at the stage of the elastic deformation, and the compressibility modulus of the nanostructured hybrid is several times higher than that of the pure nano-zirconia. As a result, the composite goes into the region of the plastic deformation at a lower pressure than the single nano-zirconia. A mechanism for sintering a composite with a graphene content of 0.635 wt% is proposed; it explains the effect of the graphene sheets on the process and its result. It has been determined that the activation energy of the composite sintering is more than two times higher than for the single nano-zirconia. Apparently, due to the van der Waals interaction, the graphene sheets partially stabilize the zirconia structure. They inhibit the disordering of the surface zirconia monolayers and their sub-melting, the two processes preceding the sintering. The determined effects lead to an increase in the activation energy of the sintering for the composite and a complication in its mechanism. As the analysis of the obtained data shows, in the presence of the graphene sheets in the hybrid structure, its sintering occurs according to a mixed mechanism, in which the grain boundary diffusion predominates, in contrast to the sintering of the pure nano-zirconia, which proceeds through a viscous flow stage. The study of the physicochemical properties of the graphene-containing composites shows that the oxygen-free graphene sheets impart the rheological and morphological features to the composite powders based on the nano-zirconia, and also increases its sintering activation energy. The determined patterns open up new capabilities for the modeling of the compacting and sintering processes when creating new types of fine-grained ceramics for the electrodes of lithium-ion batteries, sensors, details of small-sized devices, and lightweight protective coatings.