TDP-43 accumulates in nerve cells of nearly all cases of amyotrophic lateral sclerosis  and in the majority of Tau-negative frontotemporal lobar degeneration (FTLD). There is currently no biochemical test or marker of disease activity for ALS or FTLD, and the clinical diagnosis depends on the opinion of an experienced neurologist. TDP-43 has a key role in the pathogenesis of ALS/FTLD. Measuring TDP-43 in easily accessible biofluids, such as blood or cerebrospinal fluid, might reduce diagnostic delay and offer a readout for use in future drug trials. However, attempts at measuring disease-specific forms of TDP-43 in peripheral biofluids of ALS and FTLD patients have not yielded consistent results, and only some of the pathological biochemical features of TDP-43 found in human brain tissue have been detected in clinical biofluids to date. Reflecting on the molecular pathology of TDP-43, this review provides a critical overview on biofluid studies and future directions to develop a TDP-43-based clinical biomarker for ALS and FTLD. TDP-43 has been identified as binding to the transactivation-responsive region of the HIV-1 gene, acting as transcriptional regulator . AmyotroCytoplasmic aggregation of ubiquitinated, phosphorylated, and truncated TDP-43 is a unifying pathologic observation across the clinical spectrum of ALS and FTLD , 7. TDP-Under physiological conditions, TDP-43 is a mainly nuclear protein. In most cases of ALS and FTLD, it is found translocated to the cytoplasm where it changes formation and forms aggregates Fig. . WhetherTARDBP) gene in hereditary forms of ALS, clinically and pathologically identical to sporadic cases [TARDBP mutations linked to FTD without ALS have been described [Support for a direct mechanistic link between TDP-43 and neurodegeneration came from the identification of mutations in the TAR-DNA binding protein  and additional C-terminal fragments at 18–24 kDa, which were not detected in control brains. By extraction of those phosphorylation-dependent bands, subsequent mass spectrometric analysis revealed several intrinsically cleaved peptides, N- and C-terminal peptides, and modifications of sarkosyl-insoluble TDP-43. Almost all modifications were localized in the Gly-rich C-terminal half. Importantly, several novel phosphorylation, deamidation, and cleavage sites were identified by this approach. Common modifications focused on the 180–330 residue region, suggesting that this region of TDP-43 is affected in disease [Cell-specific differences in TDP-43 distribution and in the propensity to form inclusions within regions affected by early TDP-43 pathology have been reported. In α-motoneurons of the spinal cord, loss of normal nuclear TDP-43 is always accompanied by the formation of cytoplasmic TDP-43 deposits, either granular or compact which form phosphorylated skein-like TDP-43 inclusion bodies in advanced stages. While in large Betz cells of the motor cortex, only sparse cytoplasmic deposits can be found, which are not phosphorylated . This fiThe distribution of the pathological isoforms of TDP-43 has been well established from human postmortem tissue. All TDP-43 antibodies applied in biofluid studies so far bind to aa1–260, 205–222, or 256–296 of TDP-43 . While iDisease-specific biomarkers have been highly successful in other neurodegenerative diseases such as Alzheimer’s disease, where measurement of neuron-specific proteins such as protein tau and amyloid-ß have provided a high sensitivity and specificity of diagnostic tests . HoweverThe pathological signature of TDP-43 seen in immunoblots of postmortem tissue has been only partially reproduced in CSF from patients with ALS and FTLD . A polycSOD1 mutation confirming that this subgroup of ALS is associated with a different molecular pathway.Given that it is a ubiquitously expressed protein, other studies have pursued the possibility of TDP-43 as a biomarker in blood lymphocytes . Blood fThe TDP-43 biochemical profile detected in both blood and CSF was investigated to see whether it reflected brain-derived TDP-43, or predominantly represents the physiological full-length form derived from blood . The stuGiven the indications of altered TDP-43 in biofluids, subsequent studies have attempted to quantify the degree of TDP-43 and the pathological altered TDP-43 using either a monoclonal or polyclonal TDP-43 antibody against amino acid regions 1–260, 205–222, and 256–296 or phosphorylation-dependent antibodies to aa409/410 of TDP-43. Nearly all studies used enzyme-linked immunosorbent assay (ELISA) to measure TDP-43 in CSF and plasma of different patient groups. An overview of all ELISA studies is given in Table C9orf72 repeat expansion carriers of both subgroups [n = 10) relative to the FTD patients (n = 50). In contrast to previous studies, a positive correlation with male gender was observed. However, it should also be noted that the study also reported a high inter-assay coefficient of variation.The first quantification of TDP-43 in the CSF of ALS patients used an ELISA technique against full-length TDP-43 with a commercially available chemiluminescent substrate to enhance the TDP-43 signal . With a ubgroups . TDP-43 GRN, C9orf72, and MAPT) or clinically. Remarkably, this is the first study reporting lower TDP-43 levels in patients with neurodegenerative diseases of FTLD-TDP and AD compared to controls. Thus, in lumbar CSF, full-length TDP-43 was significantly lower in FTLD-TDP compared to controls, but with high group variability, while FTLD-Tau was not different from controls or FTLD-TDP. In lumbar CSF, phosphorylated TDP-43 was not detectable. In autopsy-obtained ventricular CSF samples, lower levels of full-length TDP-43 were detected in FTLD-TDP and AD compared to controls, and non-significantly lower levels in FTLD-Tau were observed. Phosphorylated TDP-43 levels were lower in FTLD-Tau and AD, and a trend towards lower levels was observed in FTLD-TDP. This study might suggest that lower levels of TDP-43 are a feature of neurodegeneration in general, as lower levels were not restricted to TDP-43 proteinopathies. Of note, a possible breakdown of the CSF barrier postmortem is likely to have an influence on the quantification of TDP-43 in the ventricular CSF samples such as impaired antibody binding through higher IgG and albumin contamination from blood [Another study group used an ELISA with a full-length TDP-43 coating antibody for ventricular CSF obtained postmortem at the time of autopsy and lumbar CSF samples, applying a biotin-streptavidin detection method for the latter . For eacom blood .The first study to quantify the full-length TDP-43 isoform in plasma included an initial patient cohort clinically diagnosed with FTD and Alzheimer’s disease (AD) . In thisTARDBP mutations, were increased compared to healthy control subjects. A positive correlation with age was shown. A subgroup of patients had longitudinal samples at different time points but no conclusion could be drawn due to the heterogeneity of TDP-43 levels.In a separate study, plasma levels of TDP-43 in a large cohort of 219 ALS patients were measured by ELISA using a chemiluminescent substrate . By thisC9orf72 repeat expansion, GRN mutation carriers, and healthy controls [C9orf72 and GRN mutation carriers compared to FTD non-mutation carriers and healthy controls. Interestingly, full-length TDP-43 levels were found to be negatively correlated with phosphorylated TDP-43, meaning that it was lower in mutation carriers compared to non-mutation carriers and controls. Both TDP-43 forms in plasma correlated negatively with age, which was taken into account in the statistical analysis. No difference was found when investigating a smaller CSF sample group of 20 FTD patients and 5 FTD mutation carriers compared to controls.Another study measured full-length and phosphorylated TDP-43 in plasma in FTD non-mutation carriers compared to a small subgroup of FTD patients with a controls . This stA different methodology aimed to detect TDP-43 with an atomic force microscopy-based biopanning protocol, utilizing phage-displayed, single-chain antibody fragments directed against TDP-43 from an established library , 81. PhaThe pathologic specimens of TDP-43 have been well established from human postmortem studies; however, the quantification of TDP-43 isoforms in the CSF and plasma of ALS/FTLD patients has been contradicting. Although a range of antibodies can be used to detect TDP-43 in biofluids , an unacA limitation of all reviewed ELISA studies on plasma and CSF TDP-43 levels is the use of the common commercially available antibodies for TDP-43. These are restricted to a certain peptide region or phosphorylation sites of TDP-43. Immunoblotting shows that the identification of TDP-43 in CSF and serum by the commonly applied antibody combinations used for quantification represent a 45-kDa full-length form of TDP-43, rather than disease-specific truncated forms of TDP-43 while even more concerning commonly applied phosphorylation-dependent antibodies have not yet revealed a convincing immunoreaction in blood or CSF , 69. ProMonoclonal antibodies which selectively recognize the pathological forms of TDP-43 will be of critical importance for the monitoring of disease status and to assess future treatment options (such as immunization studies). An extensive study generated new monoclonal antibodies by immunization of recombinant full-length, N-terminal, and C-terminal TDP-43 peptides recognizing epitopes distributed over the entire length of TDP-43. Although many of these antibodies are suitable for common applications , there was only one antibody reported “mab138” against aa182–261 , which appeared to detect cytoplasmic, pathological TDP-43, independent of its phosphorylation status and not endogenous TDP-43 [postmortem tissue such as phosphorylation and ubiquitination are potential candidates to enrich pathological TDP-43 forms. Serial fractionation of postmortem tissue enables to isolate and enrich insoluble TDP-43 with the identification of a disease-specific pattern [To apply disease-specific TDP-43 antibodies in future studies will be essential to detect brain-derived TDP-43 and to limit the detection of normal TDP-43, which may derive from blood . These s pattern , 8, 47.  pattern , 82, and pattern . Only th pattern –89. As s pattern , 91. The pattern . Recombi pattern . The cha pattern , 93, 94. pattern , 96. New pattern . TDP-43  pattern , 95.TDP-43 is central to ALS and FTLD pathology, and the frontrunner for a suitably specific biomarker that may also reflect individual disease activity. While the simple measurement of TDP-43 from blood and CSF has failed so far, only continued advances in understanding the localization, protein interactions, and immunology of its various modified forms will deliver this full potential.


SP263 and SP142 showed strong concordance (k = 0.788) with combined+ rates of 33.3% (42/126) and 34.9% (44/126), respectively. In DLBCL‐NOS, TC+ by SP263 preferred to non‐GCB and immunoblastic variant DLBCL‐NOS (P = 0.029 and P = 0.004). Combined+ (SP263 and SP142) were associated with more than one extranodal site involved , higher ECOG PS scores , high IPI risk , and poor treatment response . IC+ by SP263 and SP142 were both independent risk factors . 9p24.1 locus amplification and gain were identified in 4.3% and 7.6% DLBCL‐NOS and indicated shorter overall survival (P = 0.004). Positive rate of PD‐L1 by RNAscope was 36.5%, while no clinical significance shown. PD‐L1 positive rates were all higher in PMBCL and DHL than in DLBCL‐NOS by SP263, SP142, RNAscope, and FISH . In conclusion, combined PD‐L1 expression by IHC was potentially reliable and convenient as a predicting biomarker. SP263 staining was easier to evaluate and recognized more PD‐L1‐stained cells, but SP142 presented a better prognostic indicator. FISH and RNAscope could be used as supplementary assays. PMBCL itself was a sensitive cohort for immunotherapy.The expression of programmed cell death ligand 1 (PD‐L1) is a biomarker for immunotherapy, but approved detection method is absent in diffuse large B‐cell lymphoma (DLBCL). Here, we performed three methods including immunohistochemistry (IHC) (clone SP263 and SP142), RNAscope, and fluorescence in situ hybridization (FISH) to evaluate PD‐L1 status on a cohort of DLBCL including 94 of DLBCL‐NOS, 25 of primary mediastinal large B‐cell lymphoma (PMBCL) and 7 of double‐hit lymphoma (DHL). SP263 with 25% for immune cell (IC) or combined cell and SP142 with 10% for tumor cell (TC), 20% for both of IC and combined cell were proved to have corresponding survival prognostic. Combined Currently, the main biomarkers for the selection of patient cohorts include PD‐L1 overexpression, microsatellite instability (MSI), and high tumor mutational burden (TMB).In the described study, the protein expression of PD‐L1 was detected by IHC with the antibodies SP263 and SP142, the mRNA level was measured by RNAscope, and the PD‐L1 genetic locus alteration of 9p24.1 was analyzed by fluorescence in situ hybridization (FISH) in a cohort of DLBCL, including DLBCL‐NOS, PMBCL, and double‐hit lymphoma (DHL). The status of PD‐L1 from different molecular levels was determined by the three methods and was compared. At the same time, the corresponding prognostic roles of PD‐L1 detected by each of the methods were analyzed. The investigation aimed to identify a more practical and reliable method for PD‐L1 detection for potentially predicting the response of DLBCL to PD‐1/PD‐L1 inhibitors in clinical practice.22.1One hundred and twenty‐six cases of large B‐cell lymphomas were selected from the files of the Department of Pathology, Peking University First Hospital. Among the 126 cases, 101 cases of DLBCL‐NOS were obtained from 2011 to 2015, while 25 cases with PMBCL were obtained from 2008 to 2017. All the 126 cases were reclassified according to the 2017 World Health Organization (WHO) classification of tumors of hematopoietic and lymphoid tissues. DLBCL‐NOS was defined as cases with diffuse proliferation of neoplastic large B cells but that could not be classified as any other specific subtype of DLBCL, and EBV‐positive cases were also excluded. As a new entity, cases with high‐grade B‐cell lymphoma, with rearrangements of MYC and BCL2 and/or BCL6, which was also called “double‐hit” or “triple‐hit” lymphoma (THL), were selected out from cases initially diagnosed as DLBCL‐NOS. PMBCL was confirmed based on the site of involvement (mediastinum), morphology (diffuse infiltration of large cells and fibrotic background), and distinctive immunophenotype . Six cases of reactive hyperplasia lymph nodes were selected as the control group. All 126 cases had adequate specimens of formalin‐fixed paraffin‐embedded (FFPE) samples, of which 30 DLBCL‐NOS, 9 PMBCL, and 3 DHL were embedded into six TMA blocks with a manual tissue arrayer . The clinical information of the patients was obtained by reviewing the medical digital database; 117 of the 126 cases had available clinical data, and 110 of the 126 cases had complete follow‐up data.2.2To assay the PD‐L1 protein expression level, two antibodies  were used for the 126 cases. IHC with SP263 was performed on freshly cut 4‐μm‐thick FFPE tissue sections using the BenchMark ULTRA automated staining platform  with an Optiview DAB IHC Detection Kit according to the manufacturer's instructions. Every section stained with the PD‐L1 rabbit monoclonal primary antibody  was set up with a matched rabbit immunoglobulin G negative control and a positive control from the placenta. IHC of SP142 was carried out with PD‐L1/PAX5 immunohistochemical double staining according to a previous procedure.+, and TPS  was estimated; for ICs, predominantly macrophages and lymphocytes  was approved by FDA in the utilized of 22C3 antibody in gastric and gastroesophageal junction adenocarcinoma.+  in DLBCL. The percentage of PD‐L1 staining cells in our study was mainly scored by two experienced hematopathologists, and the mean percentage of positive cells was used for analysis. The disputed cases were confirmed by a third hematopathologist. The determination of positive cutoff value for SP263, SP142, and the detailed reasons is described in the Results section.The scoring algorithm of PD‐L1 expression in our study was performed as reported.The antibodies used for classified cell‐of‐origin (COO) and double‐expressor lymphoma (DEL) were anti‐CD10 , anti‐BCL6 , anti‐MUM1 , anti‐BCL2 , anti‐c‐Myc . Immunohistochemical staining was performed on 10% formalin‐fixed, deparaffinized 4‐um sections using routine methods. Color development was carried out using 3,3′‐diaminobenzidine tetrahydrochloride. We use a cutoff of 50% with BCL‐2 expression and 40% with c‐Myc expression to define DEL.2.3FISH was performed on 126 DLBCL FFPE samples according to the manufacturer's instructions. PD‐L1 9p24.1/CD274) Dual‐Color probe  was performed as previously reported..1/CD274 MYC (8q24) break apart rearrangement probe ; BCL2/IGH fusion translocation t  probe  and BCL6 (3q27) break apart rearrangement probe  were performed and evaluated according to the manufacturer's instructions, and the threshold all was 20%.2.4PD‐L1 mRNA levels by ISH with RNAscope were detected in 58 samples, including 30 cases of DLBCL‐NOS, 25 cases of PMBCL, and 3 cases of DHL (six TMA samples and the remaining 16 samples were PMBCL FFPE puncture cases). An RNAscope assay  with the probes Hs‐PAX5‐C1  and Hs‐CD274‐C2 , positive control probes Hs‐PPIB‐C1/POLR2A‐C2 , negative control probe DapB , and RNAscope® 2.5 HD Duplex Reagent kit  was performed following the manufacturer's instructions. In brief, 5‐μm‐thick paraffin‐embedded TMA sections were baked for 60 minutes, deparaffinized, and then boiled with the pretreatment reagent for 15 minutes. Protease digestion was carried out at 40°C for 30 minutes, followed by probe hybridization for 2 hours at 40°C with target probes against PD‐L1 and PAX5. Hybridized signals were amplified and visualized with the RNAscope 2.5 HD Duplex Reagent kit. PD‐L1‐positive signals showed as red punctate dots in the cytoplasm or nucleus, and green punctate dots (PAX‐5) distinguished tumor cells from immune cells. PD‐L1 mRNA expression levels were categorized into 5 grades according to the manufacturer's scoring guideline: score 0, no staining or < 1 dot per 10 cells; score 1, 1‐3 dots per cell; score 2, 4‐9 dots per cell and no dot clusters; score 3, 10‐15 dots per cell and < 10% of positive cells contain dot clusters; score 4, >15 dots per cell and > 10% of positive cells contain dot clusters. Scores of 0, 1, and 2 were classified as negative, and 3 and 4 were positive.2.5P‐values involved in this study are 2 sided, and a P‐value < 0.05 was considered significant. All statistical analyses were performed with the SPSS 20.0 software.The interobserver concordance and concordance between the results of PD‐L1 presence detected by IHC, RNAscope, and FISH was evaluated by the Kappa value. A Kappa value ≤ 0.40 was poor concordance; 0.41‐0.75 was moderate concordance; >0.75 was strong concordance. The relationship between PD‐L1 alterations and clinicopathological characteristics of the patients was compared using the chi‐squared test or Fisher's exact test. The treatment outcomes were measured by overall survival (OS), which was defined as the time from the day of diagnosis to the day of death or the day of the last follow‐up. Estimates of OS were calculated by the Kaplan‐Meier method, and the log‐rank test was used to compare the difference between the two groups. Cox regression models were used to estimate the prognostic risk factors. All 33.1The 126 patients included 66 males and 60 females with ages ranging from 15 to 89 years (median 59 years). According to the 2017 WHO classification, 94 of cases were reclassified into DLBCL‐NOS, 25 were PMBCL and 7 were DHL. All the 126 patients had available samples and presented optimal results for IHC, with SP263 100% (126/126) and SP142 100% (126/126).PD‐L1 expression assessed by SP263 showed generally stronger and more complete membrane staining  cases, including 37 cases with positive expression and 77 cases with negative expression  cases were diagnosed as GCB type DLBCL, and 63 (67.0%) cases were non‐GCB type DLBCL. DEL was diagnosed in 27 (28.7%) cases. As to histologic classification, 12 (12.8%) cases were grouped as immunoblastic variant subtype, and 82 (87.2%) cases were centroblastic variant subtype. TC3.3P = 0.006, P = 0.042, respectively), higher ECOG PS scores , high IPI risk , and poor treatment response . Besides, patients with positive SP263 expression were more likely to relapse (P = 0.029). Owing to the small size of the cohort, patients with PMBCL and DHL showed no relationship with clinical features (data not shown).A total of 117 patients had available and complete clinical information, of which 91 patients were DLBCL‐NOS, 19 cases were PMBCL and 7 cases were DHL. The clinical features of 91 DLBCL‐NOS patients and the association with PD‐L1 alterations are summarized Table . Among 9+ DLBL‐NOS by IHC had inferior OS  patients died due to DLBCL‐NOS progression; 18.8% (3/16) of patients died due to PMBCL; 66.7% (4/6) of patients died due to DHL; and 5 patients died for other reasons. About 18.2% (16/88) patients with DLBCL‐NOS experienced recurrence. The five‐year survival rate of patients with DLBCL‐NOS was 71.6%. In DLBCL‐NOS, the Kaplan‐Meier analysis showed that patients with combined/88 patie3.4P < 0.001, Table P < 0.001, P < 0.001, P < 0.001, respectively; Figure FISH was performed on all 126 samples, and 119 samples yielded usable results. Among them, 15 cases were identified with 9p24.1 locus amplifications (12.6%) and 17 cases with gains 14.3%)  of the results were available, including 29 cases of DLBCL‐NOS, 21 cases of PMBCL, and 2 cases of DHL. High PD‐L1 expression score = 4) was presented in 5 (9.6%) cases; score 3 was presented in 14 (26.9%) cases; score 2 was presented in 7 (13.5%) cases; score 1 was presented in 16 (30.8%) cases; and score 0 was presented in 10 (19.2%) cases. Positive PD‐L1 mRNA overexpression (score = 3‐ 4) was detected in 36.5% (19/52) cases  can all lead to upregulation of PD‐L1.PD‐L1 mRNA expression level has been measured in solid tumors, including gastric cancer and non‐small‐cell lung cancer by using RNA‐seq or ISH with RNAscope, and it was found to be highly consistent with their IHC detection results.Previous study showed that in tumors with high PD‐L1 amplification occurrence and/or PD‐L1 overexpression, patients seemed to be more susceptible to PD‐1/PD‐L1 blockade,In conclusion, the detection of PD‐L1 by IHC could be a reliable and easy method in predicting the response to PD‐1/PD‐L1 blockade for DLBCL‐NOS, especially the combined PD‐L1 staining recording could be used in practice. The cutoff for SP142 and SP263 should be different. The staining of SP263 was easier to evaluate and was more sensitive, while SP142 was a more accurate biomarker for assessing the prognosis of patients with DLBCL‐NOS. FISH and RNAscope could also be considered as a supplementary diagnostic assay. In PMBCL, the high frequency of PD‐L1 locus amplification and PD‐L1 overexpression probably indicated their good response to immunotherapy. Nevertheless, a concrete cutoff of PD‐L1 expression and detected method chosen should be determined by their relation to the outcome or response to immunotherapy in clinical practice; hence, our study was more a suggestion than a principle in PD1/PD‐L1 blockade prediction, and treatment response and larger cohort were needed in the future study.No conflict of interest disclaimed. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


The companion diagnostic test for checkpoint inhibitor immune therapy is an immunohistochemical test for PD-L1. The test has been shown to be reproducible for expression in tumor cells, but not in immune cells. Immune cells were used in the IMpassion130 trial which showed PD-L1 expression was associated with a better outcome. Two large studies have been done assessing immune cell PD-L1 expression in lung cancer. Here, we reanalyze one of those studies, to show that, even with an easier scoring method, there is still only poor agreement between assays and pathologist for immune cell PD-L1 expression. Companion diagnostic testing has gained increased importance of the last few years. The earliest companion tests were immunohistochemistry (IHC) based (estrogen receptor and HER2). These have recently been followed by a series of molecular, mutation-based tests (EGFR and BRAF) and most recently, another IHC test for PD-L1. When the FDA clears or approves companion diagnostic tests, it is widely assumed that these tests are accurate, reproducible, and robust. In fact, the SSED (Summary of Safety and Effectiveness Documents) released by the FDA provide the evidence to justify the assumption that the tests are worthy of consumer, payer, and physician confidence. Examination of the SSEDs for the PD-L1 tests shows that the FDA clears assays after review by only 2 or 3 pathologists, often showing high overall percent agreement (OPA) that may not reflect real-world outcomes. In fact, when PD-L1 assays were assessed by multiple observers, some FDA-approved categories were found to be unreproducible, specifically including immune cell expression of PD-L1 [In October of 2018, Schmid and colleagues from Genentech reported the results of the IMpassion 130 trial in first-line metastatic setting in breast cancer , 5. As sHere, we reanalyzed the data from NCCN study  using thThe low agreement between the assays is likely attributable to previously demonstrated lower SP142 sensitivity compared to other FDA-approved and laboratory-developed test (LDT) assays [In summary, this analysis raises a significant concern for pathologists who need to provide accurate and reproducible companion diagnostic results for PD-L1. While the NCCN study data presented here are from lung cancer, not breast cancer tissue, there is no evidence that the biochemistry of the interaction has any difference between the tumor sites. While the lung cancer pathologists in the NCCN study were not trained to read IC scores, the Blueprint 2 study included 1.5 days of training for 15 pathologists and found very low concordance, suggesting that training will not solve this problem. We look forward to Genentech’s help in solving this problem. A potential solution would be a reanalysis using the SP263 assay (produced by the same vendor as the SP142 assay) or a bridging study between the SP142 assay and the SP263 assay using the IMpassion 130 tissues.


According to RM, truthful memories of actual events originate in perceptual experience and are embedded in the context of time and space. As such, they are expected to include more spatial and temporal contextual attributes  and perceptual attributes  than do false memories, which originate in self-generated thought or imagination. Derived from this prediction, the traditional use of richness in detail as an indicator of deception is based on the number of perceptual and contextual details in the interviewee's accounts. However, as a memory source-monitoring theory, RM does not take into consideration the intention of liars to deceive and consequently cannot explain the full scope of richness in detail in the field of deception  theory , is richeception . In contmories”; ]. Liars mories”; –5, for e details , 7. Affeach VA; , 8] appl applrichquality of perceptual and contextual details than it does from consideration of their quantity alone.The VA  for lie liars' dilemma hypothesis. Specifically, liars perceive richness in detail as an indicator of truthfulness . However, their strategy leaves traces in the quality of their accounts, in terms of verifiability. By assessing the quality  rather than the quantity of details provided, it is possible to reveal the liars' strategy, and thereby indicate their lies.In the last years, the validity of the VA, which was originally developed and tested in police interview setting e.g., , 12, 13], 1312, 1The first approach was recently proposed by the authors of the current paper Nisin and Nahari (in preparation), who suggest that the qualitative differences between perceptual and contextual details can serve as a potential generator of deception strategy. According to this approach, while perceptual information is actually experienced, and acquired directly by the senses, contextual information is virtual in its nature, and based on semantic knowledge and relative conceptualizations. For instance, we experience the perceptual aspects of an interaction with a friend through our senses: we see the friend and the clothes he is wearing, hear his speech, and feel his touch. Meanwhile, the contextual aspects of this interaction, such as its length and specific location, are based on conceptualization and knowledge. In fact, the contextual attributes are imposed on the perceptual details and frame them in time and space. Accordingly, the perceptual details  can be regarded as primary data, and the contextual details —as meta-data. Obviously, the truthfulness of perceptual details can be checked only when they are given by the interviewee within the framework of contextual information regarding time and space. Thus, the contextual details are those that confer the status of verifiability upon perceptual details. Considering the differences between the two types of details in light of the VA , liars wresolution of the verifiable details provided: names increase the resolution of the details. The mention of an identifiable person without a name leaves the interviewees with degrees of freedom, at least temporarily, such that the verifiability of the details is neither immediate nor easy (relatively speaking) to check. Importantly, these degrees of freedom also range, as mentioning an unnamed uncle, for example, leaves less degrees of freedom than mentioning an unnamed acquaintance. These assumptions lead to the expectation that liars, when they do provide verifiable details, will strategically prefer low-resolution over high-resolution details, while truth-tellers will prefer high-resolution details. Again, as with the contextual details, the number of low-resolution verifiable details in an account can serve as a verbal lie indicator.The second approach involves the resolution of the verifiable details provided, as determined by the immediacy in which the information they incorporate can be verify. A good example of such resolution involves the use of names, which already found significance for lie detection . Accordidetail quantity while sharpening the differences in detail quality between truthful and fabricated accounts, we stress that research should go beyond the surface of content, to look for strategies that activate verbal behaviors among liars, define the qualitative manifestations of their strategies, and then—to exploit these manifestations in indicating their lies. The VA, a new and promising paradigm for deception detection is an outcome of this research approach. Having presented two new, un-studied, strategy-based approaches to further demonstrate and extend this line of research, we propose that following this path will benefit the field both theoretically and practically.The current paper is a call for more strategy-based research in the field of verbal lie detection. By demonstrating how strategies blur the differences in GN: conceptualization and writing the first draft, revising and editing. ZN: conceptualization and review and editing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer BK declared a past collaboration with one of the authors GN to the handling editor.


Fear extinction depends on N-methyl-D-aspartate glutamate receptors (NMDARs) and brain-derived neurotrophic factor (BDNF) activation in the limbic system. However, postsynaptic density-95 (PSD-95) and neuronal nitric oxide synthase (nNOS) coupling, the downstream signaling of NMDARs activation, obstructs the BDNF signaling transduction. Thus, we wondered distinct roles of NMDAR activation and PSD-95-nNOS coupling on fear extinction. To explore the mechanisms, we detected protein-protein interaction using coimmunoprecipitation and measured protein expression by western blot. Contextual fear extinction induced a shift from PSD-95-nNOS to PSD-95-TrkB association in the dorsal hippocampus and c-Fos expression in the dorsal CA3. Disrupting PSD-95-nNOS coupling in the dorsal CA3 up-regulated phosphorylation of extracellular signal-regulates kinase (ERK) and BDNF, enhanced the association of BDNF-TrkB signaling with PSD-95, and promoted contextual fear extinction. Conversely, blocking NMDARs in the dorsal CA3 down-regulated BDNF expression and hindered contextual fear extinction. NMDARs activation and PSD-95-nNOS coupling play different roles in modulating contextual fear extinction in the hippocampus. Because inhibitors of PSD-95-nNOS interaction produce antidepressant and anxiolytic effect without NMDAR-induced side effects, PSD-95-nNOS could be a valuable target for PTSD treatment. PTSD is extraordinarily robust and difficult to treat, because of enhanced fear learning, impaired extinction or inability to modulate fear expression using contextual information2. Extinction, the learned inhibition of retrieval, is widely used in the treatment of PTSD, often under the term “exposure therapy”3. The extinction learning involves new learning of an inhibitory signal that competes with the previously learned fear memory4. Contexts, a set of circumstances around an event, are essential for abstracting situationally informed meaning from the world. Contextual processing deficits are at the core of PTSD pathophysiology2. Hippocampus has a crucial role in tasks involving learning and remembering contexts1. Therefore, understanding molecular pathways mediating contextual extinction learning in the hippocampus is particularly important to treat the disorder.Learning about potential dangers in the environment is critical for adaptive function, but the fear learning for emotional disorders, post-traumatic stress disorders (PTSD) in particular, can be maladaptive, resulting in excessive fear and anxiety7. Each NMDAR is a calcium-permeable tetrameric ionotropic receptor complex consisting of two obligatory GluN1 subunits and two GluN2 (A-D) or GluN3  subunits8. In the adult forebrain regions, GluN2A and GluN2B subunits are the main subunits available in excitatory synapses for receptor complex formation9. GluN2B-containing receptor has a preferential role in the induction of synaptic plasticity critical for the extinction of fear memories10. The carboxyl terminus of each subunit binds important intracellular signaling complexes, allowing for their efficient and selective activation by calcium influx through the opening of NMDAR channels11. One of the well-characterized intracellular signaling complexes of GluN2B is the PSD-95-nNOS complex, in which, the protein postsynaptic density-95 (PSD-95) is a scaffolding protein that links GluN2B carboxyl terminus to neuronal nitric oxide synthase (nNOS) at excitatory synapses12. Activation of nNOS depends on its association with PSD-95 and on NMDAR-mediated calcium influx13. We recently found that the PSD-95-nNOS signaling complex impairs neuroplasticity, including neurogenesis, spine growth and dendrite development14, which is clearly different from the role of NMDAR activation. Given that neuroplasticity is crucial for memory extinction15, we hypothesized that NMDAR activation and PSD-95-nNOS coupling may play different role in the modulation of contextual fear extinction in the hippocampus.Convergent evidence from animal and human studies suggests that extinction of recently and remotely acquired fear depends on N-methyl-D-aspartate glutamate receptor (NMDAR) activation in the hippocampus, basolateral amygdala and ventromedial prefrontal cortex16. The functions of BDNF are mediated by the receptor tyrosine kinase TrkB, which is present in the fraction of postsynaptic density in the adult rat brain17. Upon NMDARs activation, PSD-95 not only interacts with nNOS to form PSD-95-nNOS complex13, but also with TrkB to form PSD-95-TrkB complex18 at excitatory synapses. Based on previous reports, we speculated that nNOS and TrkB may compete with each other to form complexes with PSD-95, thus playing an important role in fear extinction.Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family identified as a critical factor that mediates synaptic plasticity associated with learning and memory, specifically in fear learning and extinction19. NO produced from nNOS in the presence of L-arginine is a potent inhibitor of Ca2+-mediated ERK activation20. Therefore, ERK activation may contribute to the role of PSD-95-nNOS in regulating BDNF expression.Extracellular regulated protein kinase (ERK) regulates hippocampal histone following contextual fear conditioningIn general, we hypothesized that disassociating PSD-95-nNOS coupling in the hippocampus may up-regulate BDNF expression via inhibiting ERK activation, enhanced the association of BDNF-TrkB signaling with PSD-95, and promoted contextual fear extinction. Conversely, blocking NMDARs down-regulated BDNF expression and hindered contextual fear extinction. NMDARs activation and PSD-95-nNOS coupling play distinct roles in modulating contextual fear extinction.21. All animals acquired contextual conditioned fear to the same extent as indicated by high freezing levels in response to the context at the end of the conditioning session and in the fear recall session performed 24 h later (data not shown). Next day after recall, the mice were randomly divided into extinction and no-extinction groups. Mice in extinction group were subjected to a daily extinction trial for 10 consecutive days, while no-extinction animals stayed in the home cages. Each extinction trial consisted of a 3 min re-exposure to the conditioned context without presenting the foot shock again. Extinction memory test was performed 24 h after the end of extinction trial, and 1 h later, protein-protein interaction in the dorsal hippocampus was detected   and increased PSD-95-TrkB level  = 23.00, p = 0.001)  Fig. , substan01) Fig.  in the dp = 0.001)  and increased PSD-95-TrkB complex level  = 6.26, p = 0.036), compared to the mice with failed extinction   Fig. . The micion Fig. . However10) Fig. . These d13, or vehicle for 4 consecutive days. Twenty-four hours later, the mice were subjected to daily extinction for 5 consecutive days in the conditioning context , compared to vehicle, suggesting increased contextual fear extinction  , a small molecular compound blocking PSD-95-nNOS bindingext Fig. . Mice trion Fig. . Moreove14) Fig. .Figure 222. Accordingly, we measured c-Fos expression in the dorsal hippocampus 90 min after contextual fear recall and extinction. The number of c-Fos positive cells was increased in the CA3 and CA1, but not in the DG region among homcage, fear recall and fear extinction groups. Moreover, c-Fos induction was higher in the CA3 than in the CA1 region with extinction exposure (% increase of c-Fos-positive cells = (number in extinction group - number in homecage group)/number in homecage group × 100%, CA3 = 304.4%, CA1 = 209%) and CA1 receives the projection of CA3, suggesting dorsal CA3 may be more important in the contextual fear extinction   = 5.268, p = 0.030)  = 4.444, p = 0.047)  Fig. , but sigion Fig. . Similar30) Fig. . Additioion Fig. . The recion Fig. , produceion Fig. . As ZL0047) Fig. .p = 0.001; post hoc comparisons, WT Vehicle vs WT ZL006, p = 0.022; WT Vehicle vs nNOS KO Vehicle, p = 0.001; nNOS KO Vehicle vs nNOS KO ZL006, p = 0.997)  or WT mice to contextual fear conditioning. Two hours after fear recall, we infused ZL006 or vehicle through implanted microcannula into the dorsal CA3 for 4 consecutive days. From next day, the mice were subjected to a spaced extinction. Although ZL006 significantly facilitated contextual fear extinction in WT mice, it had no effect on nNOS KO mice  = 7.004, 97) Fig. , suggest1–133 into the dorsal CA1 or DG through the microcannula for 4 consecutive days  = 0.009, p = 0.923)  = 0.002, p = 0.969) . And there is no changes in locomotor activity after treatment with MK801 in the open-field test  state. We used same contextual fear conditioning procedure as above, and 2 h after fear recall, we infused MK801 into the dorsal CA3 of conscious mice through the implanted microcannula for 4 consecutive days Fig. . As expep = 0.049)  Fig. .10, we specifically investigated the role of GluN2B-containing receptors in the CA3 in contextual fear extinction. Two hours after fear recall, we daily infused Ro25-6981, a selective GluN2B antagonist, into the dorsal CA3 of conscious mice through the implanted microcannula for 4 consecutive days   Fig. . The tre08) Fig. . Thus, N24, a neurotrophic factor is crucial for fear extinction16. To investigate whether BDNF is responsible for the different role of NMDARs activation and it-mediated PSD-95-nNOS binding in contextual fear extinction, we examined BDNF expression in the dorsal CA3. After fear recall, mice were subjected to spaced extinction trial or not  showed significantly increased BDNF level  = 8.29, p = 0.028)  or LV-nNOS1–133-GFP  = 5.369, p = 0.043) significantly increased BDNF expression    Fig. . We nextups Fig. . Disrupt30) Fig. , whereas01) Fig. . Thus, P25 and supports extinction learning26, which raises a possibility that uncoupling PSD-95-nNOS leads to an increase in association of TrkB with PSD-95 and thereby enhances BDNF-mediated extinction. To address this notion, we infused Tat-nNOS1–133 or vehicle through microcannula into the dorsal CA3 of mice for 4 consecutive days beginning 2 h after fear recall, and detected PSD-95-TrkB complex levels in the dorsal CA3 when extinctions were significantly different between groups  = 6.65, p = 0.017)   = 44.62, p < 0.001; Fig. p = 0.012)  and increased PSD-95-nNOS complex  = 8.47, p = 0.016)  Fig. . Disrupt15) Fig. . To furt12) Fig. . Next, w16) Fig. . Collectvs ZL006, p = 0.006; ZL006 vs ZL006 + U0126, p = 0.048. pERK/ERK: n = 6, F = 34.97, vehicle vs ZL006, p = 0.005; vehicle vs U0126, p = 0.001; ZL006 vs ZL006 + U0126, p < 0.001)  or combination of two drugs for 24 h, and measured BDNF and phosporylated ERK (pERK) levels. Indeed, blocking PSD-95-nNOS by ZL006 significantly increased pERK level and BDNF expression, ERK inhibitor reversed the effects of ZL006  = 6.09, vehicle 01) Fig. . NMDARs 1–133-GFP or its control LV-GFP into the dorsal CA3 of mice through the implanted microcannula after fear recall. Seven days later, the mice were subjected to contextual fear extinction for 7 consecutive days (E1-E7), and we infused TrkB-FC, a BDNF scavenger, into the dorsal CA3 after E1 and E2 to remove local BDNF  = 6.15, p = 0.008, p = 0.018) and up-regulated BDNF expression in the dorsal CA3, compared to LV-GFP, and TrkB-FC completely reversed the effects of LV-nNOS1–133-GFP  = 5.933, p = 0.006; post hoc comparisons, vehicle + LV-GFP vs vehicle + LV-nNOS1–133-GFP, p = 0.027; vehicle + LV-nNOS1–133-GFP vs TrkB-FC + LV-nNOS1–133-GFP, p = 0.009)  = 8.580, p = 0.004; post hoc comparisons, vehicle + LV-GFP vs vehicle + LV-nNOS1–133-GFP, p = 0.001; vehicle + LV-nNOS1–133-GFP vs ANA-12 + LV-nNOS1–133-GFP, p = 0.017)  Fig. . Next, w09) Fig. . With si17) Fig. . Togethe7, especially, the role of GluN2B-containing NMDARs10, offers an inviting strategy to treat PTSD by activating NMDARs. However, NMDARs activation has a completely opposite effect on other affective processes, such as depression- and anxiety-related behaviors30. Because PTSD is often complicated by general anxiety and depressed mood31, the treatment targeting on NMDARs for PTSD may pose a challenge, render their use problematic. Activation of GluN2B-containing NMDARs induces the interaction of nNOS with PSD-9513. Using different behavioral protocols, we demonstrated that dissociation of nNOS from PSD-95 in the CA3 promoted contextual fear extinction, while NMDARs antagonists impaired contextual fear extinction. To our knowledge, this is the first report revealing the significance of PSD-95-nNOS coupling for fear extinction, and showing opposite roles of NMDARs activation and NMDARs-mediated PSD-95-nNOS coupling in regulating extinction. Because inhibitors of PSD-95-nNOS interaction produce antidepressant and anxiolytic effect without unwanted side effects associated with NMDARs33, PSD-95-nNOS could be a valuable target for the treatment of PTSD.Impairments in fear extinction are thought to be central to the psychopathology of PTSD. Crucial role of NMDARs in the limbic system for fear extinction34. DG was reported to be involved in fear extinction learning and adult-born neurons in the DG are integral for the maintenance of remote contextual fear memory36. Moreover, inhibition of Rac1 activity in the CA1 has been shown to impair massed extinction of contextual fear37. Here, we provide strong evidence that CA3 is an important hippocampal subregion playing crucial roles in fear extinction.The hippocampus is a crucial part of the neuronal circuit mediating fear extinction. Pharmacological inhibition of hippocampal Cdk5 activity facilitates extinction in the contextual fear-conditioning procedure40. Within the hippocampus, the NMDARs presumably participate in plastic synaptic events in the CA1 and CA3, the principal pyramidal cell fields39. However, the hippocampal subfields CA1 and CA3 are functionally segregated41. Indeed, after contextual fear extinction, immediate early gene c-Fos is abundantly expressed in the CA3 but not in the CA1. Our findings that uncoupling PSD-95-nNOS in the CA3 but not in the CA1 facilitates contextual fear extinction could be a presentation of function difference between CA1 and CA3. Although the number of c-Fos-positive cells in the CA1 was rarely increased after contextual fear recall and contextual fear extinction, this change may be due to the projection from CA3 c-Fos positive cells. However, CA3 and CA1 have different efferent pathway. CA3 projects to the septofimbrial nucleus, throughout the rostral lateral septum, the dorsal medial septum, and the dorsal-most portions of the vertical limb of the diagonal band of Broca. CA1 projects to the septofimbrial nucleus, the ventral portions of the medial septum, as well as the horizontal limb of the diagonal band of Broca43. These differential targets of CA3 and CA1 subcortical efferents suggest that there may be functional heterogeneity in the classical fear conditioning44.Because of the extensive recurrent collateral system connecting pyramidal cells of the CA3 area of hippocampus, CA3 is thought to promote the flexible formation and reorganization of information-coding ensembles and serve as an associative memory network45, whereas DG NMDARs mediate rapid pattern separation in the hippocampal network46, which may explain our results that uncoupling PSD-95-nNOS in the CA3 but not in the DG promoted contextual fear extinction.For the contextual memory, CA3 NMDARs are required for the rapid formation of a salient contextual representation47. NMDARs activation not only induces BDNF gene expression in hippocampal neurons24, but also facilitates BDNF-TrkB signaling to recruit PSD-95 to synapses and promotes extinction learning26. In contrast, we found that disrupting the association of nNOS with PSD-95, downstream of NMDARs activation, up-regulated BDNF expression and association of BDNF-TrkB signaling with PSD-95. More importantly, BDNF scavenger and TrkB receptor antagonist abolished the effect of LV-nNOS1–133-GFP on fear extinction. Thus, PSD-95-nNOS interaction regulates contextual fear extinction via BDNF-TrkB signaling. Furthermore, we showed that contextual fear extinction induced a shift from PSD-95-nNOS to PSD-95-TrkB coupling. Therefore, different BDNF-TrkB signaling in the CA3 may explain different roles of NMDARs activation and NMDARs-mediated PSD-95-nNOS coupling in regulating extinction.BDNF in the limbic system are crucial for fear extinction27. Thus, ERK-BDNF signaling pathway may account for the role of NMDARs activation in fear extinction. PSD-95-nNOS interaction facilitates NO production13. nNOS-derived NO is a potent inhibitor of Ca2+-mediated ERK activation20. Here, we found that uncoupling PSD-95-nNOS increased ERK phosphorylation and ERK inhibitor reversed the effect of ZL006 and Tat-nNOS1–133 on BDNF expression. Thus, PSD-95-nNOS interaction may down-regulate BDNF expression via inhibiting ERK activation. However, we can not exclude the possibility that PSD-95-nNOS interaction may have other way to regulate BDNF, such as histone deacetylases (HDACs)-mediated epigenetic modification. HDACs play a negative role in histone H3 and H4 acetylation of BDNF promoters, thereby influence BDNF expression48. It has been reported that nitrosylation of HDAC2 is implicated in the regulation of fear memory15. Moreover, our previous study showed that NO up-regulates HDAC2 level and activity14. Thus, further investigation will be required to test whether HDACs are implicated in the regulation of BDNF expression and consequent fear extinction by PSD-95-nNOS coupling.It is generally believed that synaptic NMDAR conveys the synaptic activity-driven activation of the survival-signaling protein ERK leading to the activation of the transcription factor CREB and the production of the survival-promoting protein BDNFp = 0.010)  = 9.88, 2+-mediated ERK phosphorylation and enhances the association of BDNF-TrkB signaling with PSD-95, subsequently enhances synapse activity, thereby promotes contextual fear extinction. NMDARs-mediated PSD-95-nNOS interaction weakens the role of NMDARs activation via reducing ERK phosphorylation and BDNF-TrkB signaling with PSD-95  and their wild-type controls of similar genetic background  , and young adult (6–8 weeks) male C57BL/6 mice were used in this study. All procedures were approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. And all experiments were performed in accordance with the approved guidelines and regulations. Every effort was made to minimize the number of animals used and their suffering.Young adult (6- to 7-week-old) male homozygous nNOS-deficient mice  were unilaterally implanted 1.5 mm above the dorsal hippocampus using coordinates for the CA3: −1.7 mm AP; +1.9 mm ML; −1.9 mm DV; for the CA1: −1.7 mm AP; +1.3 mm ML; −1.8 mm DV; for the DG: −1.7 mm AP; +1.0 mm ML; −2.1 mm DV. Drug infusions at a rate of 0.06 μl/min  included ZL006, Tat-nNOS34. In brief, contextual fear conditioning was performed with a computerized fear conditioning system (CSI Systems). The freezing level was quantified from digitized video images using commercially available software . Animals were allowed to explore the training cage for 3 min followed by a mild electric shock . Context-dependent freezing, defined as the absence of movements other than those required for breathing, was assessed 24 h later by re-exposing the mice for 3 min into the conditioning context. Spaced extinction of contextual fear was performed on consecutive days, consisting of re-exposure to the training context in a non-reinforced manner for 3 min. An extinction protocol is composed by time of each extinction trial and the number of extinction trial. A complete extinction is indicated by an appearance of extinction bottom. However, the dynamics of fear extinction can vary among experiments. In other words, the number of extinction trials to get extinction bottom could be different. In our experiments, the number of extinction trials varied from 5 to 10 days, although time of each extinction trial was same. In the experiment about extinction failure and extinction success, “Extinction Failure” and “Extinction Success” were established based on the freezing values in the last extinction trial. Freezing level higher than 65% of the acquisition level was defined “Extinction Failure” and lower than 45% of the acquisition level was defined “Extinction Success”.Extinction and reminder shock procedures were carried out as described previously1–133-GFP or its control LV-GFP, and the preparations of fusion peptide, Tat-nNOS-N1–133, were performed as we previously reported49 using the procedures detailed in the Supplement.Culture of hippocampal neurons, the production of recombinant lentivirus, LV-nNOS50: the brains were removed and blocked rapidly over ice into coronal sections. Serial hippocampal sections (200 μm) were made on a vibratome (Leica) in a bath of pre-cold PBS. Then the CA3 of cannulas-implanted side was obtained in the first six pieces of hippocampal sections under a dissecting microscope and placed in the pre-cooling RIPA lysate. Samples from cultured neurons were prepared as described by our previous studies49. Western blot analysis and coimmunoprecipitation used the procedures detailed in the Supplement.The samples of CA3 in the dorsal hippocampus were obtained as followspost hoc test47. In some cases, with only one factor  followed by Tukey’s supplementary information


The laser was also used to resect epiglottitis, false focal cords, and aryepiglottic fold completely due to supraglottic swelling with a thick oedematous epiglottis. A 1.5‐ms pulse‐duration CO2 laser attached to a microscope is an effective technique of treating laryngeal sarcoidosis and preventing its hazardous complication. Beneficial effects of this method are not only an immediate improvement of the symptoms, but also this method decreases the need for long‐term medical therapy with its side effects or avoid tracheostomy due to upper airway obstruction.To improve diagnosis and treatment of laryngeal sarcoidosis, we present a rare case of upper airway obstruction of unclear aetiology, with life‐threatening complication.The patient was a 19‐year boy who presented with progressive severe dyspnoea for eight months. After extensive diagnostic evaluations with no conclusive diagnosis, biopsy showed non‐caseating granulomatous inflammation consistent with laryngeal sarcoidosis that was treated with a CO To improve diagnosis and treatment of laryngeal sarcoidosis, we present a rare case of upper airway obstruction of unclear aetiology, with life‐threatening complication in a 19‐year‐old boy. Sarcoidosis is a chronic systemic granulomatosis disease that affects usually lungs, eyes, or skin. Upper respiratory tract involvement is less common as laryngeal sarcoidosis accounts for less than 5% of all patients 2 laser attached to a microscope was used . The laser was also used to resect epiglottitis, false focal cords and aryepiglottic fold completely due to supraglottic swelling with a thick oedematous epiglottis. Patient was discharged on oral medical therapy after surgery with cefixime and omeprazole and was followed up to 4 weeks. After his final treatment there was resolution of the symptoms and the laryngeal oedema had diminished (Fig. A 19‐year‐old with no past medical history, presented with eight months of progressively worsening snoring and dyspnoea. At initial evaluation, spirometry showed upper airway obstructive pattern Fig. . Finallyhed Fig. . He is cSarcoidosis is a multisystem disease with unknown aetiology. Laryngeal sarcoidosis is a less common that usually was appeared in third decades of patients' life Timely diagnosis and treatment of laryngeal sarcoidosis due to obstruction of upper airway that is its hazardous complication is an important issue.Appropriate written informed consent was obtained for publication of this case report and accompanying images.


Glioblastoma (GBM) is the most popular primary central nervous system cancer and has an extremely expansive course. Aggressive tumor growth correlates with short median overall survival (OS) oscillating between 14 and 17 months. The survival rate of patients in a three-year follow up oscillates around 10%. The interaction of the proteins programmed death-1 (PD-1) and programmed cell death ligand (PD-L1) creates an immunoregulatory axis promoting invasion of glioblastoma multiforme cells in the brain tissue. The PD-1 pathway maintains immunological homeostasis and protects against autoimmunity. PD-L1 expression on glioblastoma surface promotes PD-1 receptor activation in microglia, resulting in the negative regulation of T cell responses. Glioblastoma multiforme cells induce PD-L1 secretion by activation of various receptors such as toll like receptor (TLR), epidermal growth factor receptor (EGFR), interferon alpha receptor (IFNAR), interferon-gamma receptor (IFNGR). Binding of the PD-1 ligand to the PD-1 receptor activates the protein tyrosine phosphatase SHP-2, which dephosphorylates Zap 70, and this inhibits T cell proliferation and downregulates lymphocyte cytotoxic activity. Relevant studies demonstrated that the expression of PD-L1 in glioma correlates with WHO grading and could be considered as a tumor biomarker. Studies in preclinical GBM mouse models confirmed the safety and efficiency of monoclonal antibodies targeting the PD-1/PD-L1 axis. Satisfactory results such as significant regression of tumor mass and longer animal survival time were observed. Monoclonal antibodies inhibiting PD-1 and PD-L1 are being tested in clinical trials concerning patients with recurrent glioblastoma multiforme. Glioblastoma (GBM) is the most common primary cancer in the central nervous system and has an extremely expansive course . The staThe proteins programmed death-1 (PD-1) and programmed cell death ligand (PD-L1) interplay, creating an immunoregulatory axis promoting invasion of glioblastoma multiforme cells in the brain tissue . PhysiolThe central nervous system (CNS) was believed an “immune privileged” organ. This idea was based on anatomical features limiting the immune response, such as the presence of the blood–brain barrier (BBB) and the lack of a typical lymphatic system ,10. SeveThe dysfunction of the BBB commonly noted in patient with glioblastoma multiforme and the concomitant inflammation enhances the CNS interplay with the lymphatic system .Glioma cells excrete suppressive cytokines to stop regional responses. Prostaglandins, especially prostaglandin E2 (PGE-2), and interleukin-10 act directly on nearby intercellular matrix; TGF-β has a similar restrictive effect on regional immune responses. Glioblastoma multiforme upregulates the superficial expression of immune checkpoint molecules such as PD-L1/PD1 which promotes immune inhibition .According to evidence, GBM has the ability to eliminate CD4+ T cell and Th1-related cytotoxic response from the neoplastic tissue. GBM may turn the immune response into chronic inflammation mediated by Th17 cells, creating convenient conditions for tumor expansion ,16.PDCDL1, localized on the 9th chromosome in p24.1 position. PD-L1 is also described as CD274 and B7-H1. This ligand was discovered and described in 1999 by Dong et al. as a member of the B7 protein family [PD-L1 is encoded by the gene n family . Seven en family .PDCD1 gene. It is information transcribed from the second chromosome and consists of 288 amino acids (50–55 kDa). It contains an IgV domain in the extracellular domain and transmembrane region [PD-L1 activity depends on binding to the PD-1 receptor encoded by the e region .The intracellular region forms a tail composed of a tyrosine-based switch motif (ITSM)–inhibitory motif. This receptor was described by Ishida et al., who used subtractive hybridization to identify genes regulating programmed cell death . PD-L1 iThe binding of PD-1 to the corresponding PD-1 receptor activates the protein tyrosine phosphatase SHP-2, which dephosphorylates Zap 70 . This prStudies revealed that glioma cells are the principal expressors of PD-1 ligands . The preToll-like receptors (TLRs) and agonists of receptors induce the immune response, activating many pathways, and cooperate with various antigens. TLRs are a conserved family of 10 receptors (TLR1–10) taking part in pattern recognition . AgonistTLR agonists as microbial antigens, activating TLR receptors to initiate precise immunological activities. Agonists of TLR that have been studied include lipopeptides , lipopolysaccharides (TLR4 agonist), LPS, flagellin (agonists of TLR5), single-stranded DNA (TLR8 agonist and TLR7), double-stranded (ds)DNA (agonist of TLR3), and the DNA CpG motif (agonist of TLR9). Later studies showed that autocrine molecules released from dead and stressed cells such as heat shock proteins  and high-mobility group box 1 proteins  are also significant agonists. Many of them appear in glioma environment, causing tumor induced-activity of TLRs ,32.In GBM cells, constitutive elevated expression of αB-crystallin, HSP27, HSP73, HSP72, and HSP90 was reported in vivo and in vitro as a result of endogenous induction. HSP–peptide complexes (HSPPCs) are able to interfere with various superficial receptors such as CD36, CD91, CD40, CD14, TLR2, TLR4 ,34,35.TLR activation in glioma cell results in signaling through two main pathways, one of which is myeloid differentiation factor 88-independent (MyD88-independent), and the other is myeloid differentiation factor 88-dependent (MyD88-dependent) . The MyDEGFR  was firstly discovered during the search for tyrosine kinases . PreviouEGFR activation requires ligand binding. Binding of TGFα or EGF to EGFR promotes the activation of selected pathways: Ras/RAF/MAPK, PI3K/Akt-1, and signal transducer and transcription-activating (STAT) pathways ,43,44,45Incidental mutation of EGFR results in the recruitment of this downstream signaling pathway which normally promotes immune escape after typical EGF binding. EGFRvIII is a modified type of EGFR receptor, transmitting immunological signals constantly in the absence of EGF. EGFRvIII expression rate is about 11% in GBM.Recent studies revealed PD-1 activation and downstream signaling of EGFR were interdependent and both present in pulmonary tumors ,47. The Features of glioblastomas are a high propensity to invade the surrounding tissues and extensive angiogenesis . They arAngiogenesis caused by the presence of a tumor is defined as the formation of new blood vessels based on existing ones, which can be visualized using ultra-high-field magnetic resonance angiography . The mecAngiogenesis occurs under the influence of various mechanisms, which we divide into hypoxia-dependent and hypoxia-independent. Hypoxia has been proven to inactivate prolyl hydroxylases. This increases the level of hypoxia-inducible factor-1α (HIF-1α), causing the release of vascular endothelial growth factor (VEGF) by glioma cells affected by hypoxia. The task of VEGF is to counteract oxygen deficiency by inducing vascular network growth ,62,63.In spite of this, hypoxia may not be the only trigger factor for angiogenesis. This applies particularly to the initial stages of carcinogenesis, when cell hypoxia is not yet severe. Regulation of VEGF secretion also takes place by a mechanism independent of hypoxia, involving dysregulated activation of mitogenic and survival pathways. This applies to phosphatidylinositol 3-kinase and Ras/mitogen-activated protein kinase ,64.The PD-L1/PD-1 axis may also have roles in regulating the level of VEGF mediating angiogenesis. In studies conducted by Shin et al., a positive correlation between PDL1 levels and VEGF expression was shown in 197 patients with renal cell carcinoma ,66. SimiSerine ProteasesMatrix Metalloproteases (MMPs)Cysteine Proteases .EGF is another molecule with similar effects. It promotes tumor angiogenesis as well as an increase in cell motility and invasiveness. This is done by phosphorylating the EGFR receptor and activating further signaling pathways ,68. It hSerine proteases are a large group of proteases which include tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) with its receptor (uPAR). Their activation affects the degradation of the extracellular space by tumors, promoting their invasiveness. It has been shown that, the PAI-1 molecule mediates the regulation of the uPA/uPAR pathway in astrocytoma cells. Its expression is promoted by an increase in EGF and inflammatory cytokines. This results in greater uPA/uPAR activity, thus increasing tumor invasiveness .Upregulation of uPA and uPAR is also achieved through the expression of the EGFR ligand transforming growth factor (TGFα) . StudiesEGF also has a potential impact on another group of proteases, i.e., MMPs. They are a large group of enzymes whose effect is based on the regulation of metabolism and cell morphogenesis. Disruption of their function also affects the loss of cell integrity. It has been shown that the activity of certain matrix metalloproteases, such as MMP-2 (gelatinase A), MMP-9 (gelatinase B), MMP-14  is related to the pathophysiology of high-grade gliomas. It correlates with the severity of tumor invasion and its degree of malignancy ,77,78,79Another group of proteases are cysteine proteases, examples of which are cathepsins. These include, among others, cathepsin B, involved in the degradation of the tumor microenvironment. Its expression (mRNA level) has been shown to correlate with EGFR expression level . It was In addition to VEGF and EGF, factors that contribute to angiogenesis also include other molecules: platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), hepatocyte growth factor, ephrins, angiopoietins, integrins, and interleukin-8 ,84.Interferons were first described in the 1950s as molecules interfering with viral replication. Signaling pathways from their receptors through Janus kinase (JAK) and STAT have been clearly described. This main signaling pathway mediates interferon-induced gene expression ,86,87,88Another consequence of activating the interferon signaling pathway is an increase in the activity of cytoplasmic GTPases, resulting in an antiviral effect. It can also lead to increased expression of myxovirus protein A (MxA) ,94.It has been shown that an increase in MxA mRNA accompanies a greater degree of tumor malignancy. The highest values were observed in the case of glioblastoma, but a good correlation was also observed in vivo in patients diagnosed with other gliomas. In this case, there was also a correlation with PD-L1 mRNA levels. MxA values can also be associated with the presence of nonclassical MHC molecules, including HLA-E and HLA-G, which promote glioma immune escape.The existence of an autocrine signaling loop in which interferon participates has been proven. In this loop, INF-a and INF-b (IFN I types) trigger intracellular signaling through IFNAR1/2.IFNAR1/2 gene silencing resulted in reduced expression of PD-L1 and MHC (class I and II). As a result, the immune cells were more susceptible to lysis. On the basis of this, it can be concluded that intracellular signals induced by the presence of interferon negatively regulate the anti-tumor immune response in gliomas [ gliomas .IRF-1 gene [The binding of type II interferon (IFN-γ) to its receptor (IFNGR) causes phosphorylation of JAK1 and JAK2 as well as the phosphorylation of STAT1. Activated dimers act after accumulation in the nucleus as transcription factors and connect to GAS elements that are located on most IFN-γ-induced genes. One of them is the F-1 gene ,97,98. IF-1 gene ,100.Evidence indicates that the presence of PD-L1 on the cell surface is regulated by downstream transcription interferon regulatory factor 1 (IRF-1). STAT1 and STAT3 also play a role .To sum up interferon pathways, researchers suggest that stimulation of the IFN-y pathway results in the upregulation of JAK2/STAT1/IRF1 and, as a consequence, increase the expression of PD-L1 as a characteristic signaling transmission element for type II interferon. Some studies noticed that IFN-y stimulated the STAT3, STAT2, and IRF9 axis which is more typical of type I interferon stimulation. This confirms a strong relation between the two molecules .Monoclonal antibodies (mAbs) have produced a turning point in the immunotherapy of cancer. They block receptors on immune effector cells or their ligands on neoplastic cells and accessory cells . Understanding the role of PD-L1 and PD-1 in the pathogenesis of neoplasms has enabled the blocking of these mechanisms in oncological therapy. Currently, there are several drugs in clinical trials that could be used to treat, among others, lung cancer, ovarian and stomach cancer, renal cell carcinoma, melanoma, lymphoma, and leukemia ,106,107.In some countries, several mAbs have been approved by the relevant authorities for clinical use . One of Preclinical GBM mouse model studies confirmed the safety and efficiency of monoclonal antibodies directed against PD-1 and PD-L1. The data obtained so far, such as significant regression of tumor mass and longer animal survival time, suggest their high anti-cancer potential ,109,110.Monoclonal antibodies as inhibitors of PD1 and PD-L1 are being tested in trials concerning patients with recurrent GBM  111,112,119,120.A study performed by Reardon et al. in 2017, called CheckMate 143 trial, included 369 patients with glioblastoma who had their first relapse after radiation and TMZ therapy. Observations compared nivolumab efficiency administered every 2 weeks at a dose of 3 mg/kg with bevacizumab administered every 2 weeks at a dose of 10 mg/kg. The median overall survival in the group of patients taking nivolumab was 9.8 months and that in in the corresponding bevacizumab group was 10 months. The median duration of response in the nivolumab cohort was 11 months versus 5.3 months in the bevacizumab cohort. Overall response rates in the nivolumab group were 8%, while in the bevacizumab group, they were 23% .n = 30). Four patients were progression-free at 12 months [Another drug from this group, Durvalumab, was tested in an open label, phase 2 trial where patients were grouped into five GBM subgroups, performed by Reardon et al. in 2017. The study design involved a monotherapy with Durvalumab at a dose of 10 mg/kg. Partial results revealed a partial response of 13,3% in bevacizumab-naïve recurrent GBM patients and a stable response in 46,7% of patients in this cohort  .Similar findings were obtained in other studies which examined the correlation between the level of PD-L1 in tumor cells, lymphocytes PD1, and clinical response. In the case of PD-1 level, the degree of this relationship was poor .However, accurate knowledge of this correlation requires further research. Many patients in the PD-L1-expressing group are unreactive to blockade of relevant checkpoints. On the other hand, it has been shown that in the case of lung cancer, a part of the study group, negative for PD-L1, showed a good clinical response ,130.GBM is highly resistant to standard therapies and thus is a challenging disease. Short OS and aggressive intercourse remain uncontrollable when using the typical therapeutic approach. The PD-1L/PD-1 immune check point axis is considered an interesting target for immunotherapy. Many pathways activating PD-L1/PD-1-dependent glioma cell immunoescape were clearly described. Blockade of the interaction between PD-1 and PD-L1 may re-establish proper immunity against GBM. Recent clinical studies on PD-1L/PD-1 inhibitors have presented effective responses. Although trials do not confirm critical extension of OS, these inhibitors may confer different benefits if combined with standard therapies. Further clinical observations are required to create the best therapeutic protocols that can bring a new hope for patients.


Features of 2D materials such as high surface area-to-volume ratios and various reaction sites are ideal characteristics for fabricating state-of-the-art high-performed chemical sensors. This review particularly focuses on the detection of pH, metal ions, and biomolecules in liquid media. The final goal of the ion/molecule sensors is a development of the electronic tongue or taste sensors that can be used in medical, food, biotechnology, and health applications. Herein, we introduce recent advances in the field of ion/molecule sensors in liquid media based on 2D materials, especially concentrating in graphene and MoS A recent development in sensor technology and increasing demands in high-quality life have led to the era of the Internet of Things (IoT). The interconnected sensor devices and numerous data acquisition from various sources including the human body, indoor air, foods, infrastructures, and so on can provide real-time information of harmful substances or human body status with adequate actions to take upon those signals  mimicking the human taste sense. Unlike electronic nose or skin aiming in various gaseous substances and external stimuli, respectively, e-tongue targets distinctive ions, or molecules  representing each of the five tastes . Although some companies have already commercialized equipment analyzing the above five taste substances, they have a relatively large scale to be adopted into the IoT platform, where small size, low power consumption, low price, and compatibility to existing circuits are essential requirements.2 hybridized carbon atoms by a long-range π-conjugation. As a result, the non-covalent interaction between the surface of graphene and ions/molecules involving π bonds is important since the electronic characteristics of π systems can modify the structure or properties , or ultrasensitive sensors that can detect a sequence of DNA. Chemical functionalization of 2D materials can also enhance selectivity toward target substances.The two-dimensional (2D) materials are emerging candidates to be used in sensing devices and, further, can be applied to the IoT platform due to their promising physical and chemical properties. Their high surface area-to-volume ratio leads to high sensitivity to target substances. 2D material-based sensors can also be fabricated in a miniaturized size due to their flexibility, great mechanical strength, and optical transparency  sensor . Hydrogen ion sensing is a vital measurement in many liquid chemical procedures such as industrial, manufacturing, pharmaceutical, and food production. The acidity of a substance may act as an indicator in many cases; food spoilage indication, monitoring clean/wastewater, chemical laboratory analysis, etc. pH meters commonly used in scientific laboratories are also called potentiometric pH meters. The basic fundamental of pH meters is measuring the electrical potential difference and monitoring the hydrogen ion concentration of a typical solution. This method is very precise in sensing the concentration of hydrogen ions, but there is a critical disadvantage that the device is not portable due to its size and the necessity of external power source. Explained above, IoT devices must be miniaturized and portable for everyday use. 2D materials can be used as pH sensors because of their high sensitivity from their chemical and physical properties. Additionally, hydrogen ion sensors based on 2D materials can be minimized in size, making them emerging candidates of hydrogen ion sensors.The most fundamental ion/molecule sensor is the hydrogen ion (HI–Vg), which has a conductance minimum point called Dirac point (VDir) or charge neutrality point (VCNP). In the case of Vg having a lower value than VCNP, the majority of charge carriers are holes and increasing Vg leads to the decline of channel current and, reversely, the majority charge carriers become electrons, which increases in quantity with increasing Vg. Consequently, GFETs have the advantage of noticing the shift of charge neutrality point by adsorption or desorption of electrons or charged ions. Sensitivity can be estimated by evaluating the degree of horizontal shift of the charge neutrality point. In other words, as chemical alteration such as ion concentration or pH increase or decrease results in the surface of GFET, the VCNP will change  by embedding a solid gate, suggesting the possibility of a practical analytic device using graphene  is used as a gate dielectric layer. The HfO2 layer has higher specific capacitance compared to conventional SiO2 solid-gated sensors, leading to higher transconductance and operation at lower Vg. Also, the HfO2 layer prevents the gate electrode to be exposed from the liquid, eliminating any errors . This device does not require any separate components such as external reference rods, which simplifies the fabrication process. Using a low Vg (1.5 V), the device can measure pH in a span of 5.3 to 9.3 with a sensitivity of ~57 mV/pH.Ohno et al. successfully demonstrated the transport behaviors of GFETs in pH solutions and protein molecules  with nitric acid (HNO3). On the other hand, Hummers treated graphite with a mixture of potassium permanganate (KMnO4) and sulfuric acid (H2SO4) , the resistance of both materials declines  and reduced graphene oxide (RGO) are also candidates for pH sensing. GO is a 2D material that is originated from natural graphene by chemical conversion  conditions or pH  containing 1-pyrenebuthanoic acid succinimidyl ester at room temperature (RT) for 2 h. Buffer solutions were used by mixing 0.1 M phosphate buffer and 0.1 M NaCl solution. + binding to the surface, eventually inducing the surface charge density to change on the gate. Precisely, the processes are different. In the case of pristine graphene, when exposed to acid electrolytes, hydronium ions (H3O+) attach to the inner Helmholtz plane (interface formed when an electrolyte and electronic conductor meets), inducing the graphene to become electron doped. On the other hand, when exposed to alkali electrolyte, hydroxyl (OH−) attaches to the inner Helmholtz plane and the graphene becomes hole doped , so these charges are impermeable across the graphene/electrolyte surface , composed of Mo  and S , which are covalently bonded. These 2D stacked layers are arranged in a row and they are held together by weak van der Waals force. This weak force makes exfoliation rather easier, and single-layered film extraction is possible  in high pH resulting in negative charges. In 2) for low-frequency noise field effect transistors for pH sensing  solution, respectively. The thickness of HfO2 was 20 nm, which was deposited by atomic layer deposition (ALD) on highly doped Si. 2 gate. IDS vs. time) on various pH values. The source–drain and the gate voltage are fixed to VDS = 0.1 V and VLG = −0.5 V, and current changes drastically and stabilizes quickly in each pH value. Shadman et al. used MoS2 and WSe2 as a double gate field effect transistor for pH sensing  is higher than the gate voltage (Vg), graphene is in the circumstance of p-type, and in contrast to this, graphene behaves as an n-type material. VCNP is in between 0.1 V and 1.0 V. In other words, in the case of Vg = 0.1 V, it is p-type, and at Vg = 1.0 V, it is n-type. In both cases, VDS = −0.2 V and an identical amount of glucose was injected with higher concentration each step. Kang et al., Shan et al., and Wu et al. all manufactured the sensor by fabricating a nanocomposite with GOx (enzyme)–graphene–chitosan. Wu et al. additionally added platinum nanoparticles for higher sensitivity while Shan et al. added Au nanoparticles. Kang et al. dispersed graphene in chitosan solution using ultrasonication. Then, GOx was coated on the graphene/chitosan film . After forming MoS2-PAH nanosheets, metal nanoparticles were hydrothermally reduced to the nanosheets. Two different metal precursors were prepared for Pt and Ag, respectively. Experimental results were confirmed by color change (blue) by each concentration of H2O2.As graphene, TMDs can also detect glucose with decorated nanoparticles. In terms of electrochemical viewpoint, nanoparticle decoration on the electrode surface not only helps electrocatalytic reactions but also presents electrochemical properties. These properties are faradic to capacitive current ratios, electron mobility, current density, and mass transport -SnS2. MWCNTs were initially mixed into nitric acid, and then after the centrifuging process, it was neutralized. These carbon nanotubes were then dispersed with yellow nanoflake SnS2 in water and vigorously mixed using ultrasonication. Then, GOx was dropped to form composites with MWCNT–SnS2. After fabrication, the reproducibility and stability of the MWCNT–SnS2 sensor are demonstrated in Not only MoSDue to population growth, increase in industrial and agricultural activities led to pollution in the environment and ecosystem. Recently, pollutant portable sensors have been studied in diverse ways to detect and eliminate the pollutants. The most representative pollutant from human activity is heavy metals leaking to the environment and threatening the ecosystem by inducing various diseases Ali, . 2D mate2 substrate. As the molecule/ion becomes absorbed by graphene, the local charge characteristics change and conductance is altered. Sudibya et al. fabricated reduced GO for the reasons of cost-effectiveness, solution process availability, and scalable production  can be bonded onto RGO by surface modification. However, K+ and Na+ had no response, indicating that the functional group (CaM) has no selectivity to K+ and Na+. Also, without functionalization (CaM), the RGO-FET had no response to Ca2+ and Mg2+, meaning CaM is crucial for selective detection. Also, other heavy metal detection experiments were progressed by functionalizing RGO with metallothionein type II protein (MT-II), which binds with Zn, Cu, Se, or Cd, and Hg selectively. As shown in 2+ at a detection limit of 1 nM. Comparing the detection of Ca2+ and Hg2+, mercury has much higher sensitivity than calcium, which indicates the fact that mercury has better binding affinity to MT than calcium to CaM. MT-II is negatively charged in pH 7.0, and metal binding brings MT-II closer to the RGO surface and increases the field effect from highly negative charged MT-II to RGO. As shown in + ions increase on the graphene surface by the addition of NaCl solution, the positive charge accumulates on the graphene channel. On the other hand, when Na+ ion concentration is low, these ions adsorb to the hydroxyl group. Consequently, the charge neutrality point shifts toward the negative direction as the concentration increases. Transfer curves in diverse Na+ concentrations were plotted and curve shifting happened. Also, the current–time graph was plotted and the drain current decreased as Na ion concentration increased. Wen et al. detected lead ions using Au nanoparticles and DNAzyme functionalization onto graphene  due to p-type doping effect. DNA molecule induces n-doping effects to graphene and curves shift to the negative direction. Consequently, the DNAzyme/Au nanoparticles/graphene curve is in the intermediate of graphene and Au nanoparticles/graphene curves. When lead ions become adjacent to the device, the transfer curves shift to the right side again and the shifting is proportional to the concentration of lead.Graphene may be used as the sensing material in the channel positioning in between the drain and source electrodes. The gate potential can be applied from the top or bottom, in which the former is applied to the electrical double layer in the electrode and the latter is applied through a typical thin SiO2-based FET to sense mercury ions. MoS2 has a high affinity to Hg2+ due to their sulfur sites that are bonded extremely strong  microfluidic channel was introduced to the device for Hg2+ solution delivery. The Hg2+ solution was made by dissolving Hg(ClO4)2 in DI water. In 2+ solutions possessing various concentrations flowed into the device. As the concentration increased, the conductance declined due to charge impurity-induced scattering effects. As Hg2+ increases, the p-type doping effect also increases (transfer curves shift to the right as a higher concentration of Hg2+ is introduced), leading to electrons to decrease, resulting in the reduction of carrier mobility. Also, in 2+ with MoS2 by decorating specific DNA functional groups. 2/DNA-Au nanoparticle hybrid structure. MoS2 nanosheets were formed by Li ion exfoliation at RT. MoS2 powder was intercalated by lithium ions and then they were sonicated in water. MoS2 crystals were formed and then immersed into a butyllithium solution for a week filled with Ar gas. LixMoS2 was retrieved by centrifugation and was washed to eliminate the residues. Ultrasonication was processed for exfoliation to form nanosheets. Then, the film was transferred onto a SiO2 substrate as shown in 2+ is introduced to the device, T-(Hg2+)-T chelates, formed by the reactions between Hg2+ and the thymidine of the DNA molecules resulting in electrical properties to change MoS2. Real-time detections of Hg2+ in water are illustrated in 2/DNA-Au nanoparticles, the purple dash curve indicates MoS2-Au nanoparticles, and the blue dash curves indicate MoS2 only. From the curves in 2 has higher conductivity as Hg2+ concentration increases. The results are opposite from Jiang et al., which explains that MoS2 from Zhou et al. is a p-type semiconductor. From the fabricating process, MoS2 was exposed to an oxygen-containing atmosphere and the oxygen molecules were adsorbed onto the defects or sulfur sites on the layer of MoS2 film and eventually worked as p-type dopants by trapping electrons  and hole density reduces, resulting in BP conductivity decline  Hg2 substrate was modified with 3-aminopropyltriethoxysilane (APTES) to supply a positively charged surface. This process provides the uniformity of GO patterns and prevents aggregation on the substrate. Vg = 0.6 V, −0.6 V. Each step indicates the addition of dopamine increasing the concentration ranging from 1 to 8 mM. Dopamine can easily bind with RGO through strong π-π interactions. Also, the transfer curves shifted to the right, showing that they induce p-doping or incline the hole concentration in the RGO film , graphene, and graphene/Pt nanocomposite electrodes. Anodic peak currents were 102 μA (GC), 183 μA (graphene), and 371 μA (graphene/Pt). From these results, both graphene and graphene/Pt-modified electrodes increase the electrochemical sensitivity. The amperometric responses of AA, DA, and UA were measured. All experiments were progressed in a 0.1 M phosphate buffer solution at 0.5 V and the detection limits were 0.15 μM (AA), 0.03 μM (DA), and 0.05 μM (UA). Sheng et al. doped nitrogen on graphene for the simultaneous determination of AA, DA, and UA. After mixing GO and melamine, the mixture was annealed to 800°C for 1 h in Ar atmosphere. Then, the final product was taken out from the tube. The electrochemistry of each solution was measured. All three cases had excellent electrocatalytic activity toward the oxidation of AA, DA, and UA. The interactions between the target biomolecules (hydrogen bonds), nitrogen-doped graphene (π-π stack), structure of the molecules, and the unique structural and properties of nitrogen-doped graphene influenced the electrocatalytic activity.Dopamine (DA), ascorbic acid (AA), and uric acid (UA) are very essential to human metabolism because they influence the central nervous system and serum by coexisting. The lack or excess of DA, AA, and UA may lead to various disorders that can be fatal to life. The importance of direct, simultaneous, quick sensing of DA, AA, and UA cannot be emphasized. Overall, 2D material-based DA, AA, and UA sensors accompany doping, metal nanoparticle decoration, and reduction process to enhance selectivity and sensitivity. He et al. manufactured a dopamine device based on RGO FET by reducing patterned GO films. 2 nanosheets for the detection of AA, DA, and UA. In 2 nanosheets were prepared by the intercalation exfoliation method. The Au nanoparticles were decorated by the electrochemical deposition method. The synergetic effects of MoS2 and Au nanoparticles enhanced the oxidation reactions of AA, DA, and UA. 2 and Au nanoparticle-decorated MoS2 nanostructures on ITO. The detection limits for AA, DA, and UA were 100, 0.05, and 10 μM, respectively. Wu et al. electrochemically reduced MoS2 single-layered nanosheets to detect various biomolecules such as DA in the presence of AA and UA. The oxidation peak of AA was much lower than that of DA or UA. The DA oxidation signal being stronger than AA or UA is due to the negative charges existing on reduced MoS2, which repel the anionic AA and UA but attract the cationic DA. The peak current of DA increased linearly with the DA concentration from 1 to 50 μM  was emerging for label-free biological detection Homola, . Recentl2, providing ultrasensitivity to DNA. The role of graphene is to protect the reaction between MoS2 and the surrounding environment and to be a biocompatible superficial layer to immobilize DNA molecules on its surface. The photoluminescence intensity of the MoS2 from the stack of graphene/MoS2 increased as the concentration of DNA increased by adding them. Real-time measurements were available in a few minutes and the detection limit was reached to the level of aM. In 2, which enhances its PL intensity. Also, graphene is not affected by the DNA sensing by confirming the Raman spectra, suggesting that the Fermi energy of graphene is not changed by DNA adhering. This is due to the electron transfer of MoS2 to graphene, which means any induced hole of graphene is filled by the electron from MoS2, resulting in MoS2 becoming reduced due to the loss of electrons. Loan et al. designed a heterostructural structure by stacking graphene on MoS2-modified poly (PDDA)–mesoporous carbon (MC) composites to detect L-Cys. The fabrication process is illustrated in 2 nanocubes were decorated onto PDDA-MC by the reduction process of (NH4)2MoS4 using the hydrothermal method. L-Cys was demonstrated with amperometric responses plotted in Protein is the basic unit that makes up any living organism. Protein and amino acid detection are crucial in medical diagnosis (Yoo et al., The promising physical and chemical properties of 2D materials have encouraged lots of studies on developing highly selective ion/molecule sensors utilizing surface defects, large specific surface area, and easy surface functionalization. Many researchers are seeking to use 2D materials as ion/molecule sensors for various reasons such as environmental monitoring, real-life healthcare, and food intake applications. Although studies on 2D material-based ion/molecule sensors are in the early stage compared to oxide-based sensors , variousThe manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Streptococcus suis is a prominent pathogen causing septicemia and meningitis in swine and humans. Bacitracin is used widely as a growth promoter in animal feed and to control the spread of necrotic enteritis in most developing countries. This study aimed to characterize a novel membrane transporter module Sst comprising SstE, SstF, and SstG for bacitracin resistance.bceAB and bceRS. A four-fold decrease in bacitracin susceptibility was observed in sstFEG deletion mutant comparing with S. suis wildtype strain CZ130302. Further studies indicated that the bacitracin tolerance mediated by SstFEG is not only independent of the BceAB transporter, but also regulated by the two-component system BceSR. Given that SstFEG are harbored by almost all virulent strains, but not in the avirulent strains, we managed to explore its potential role in bacterial pathogencity. Indeed, our results showed that SstFEG is involved in S. suis colonization and virulence in animal infection model by its potential competitive survival advantage against host bactericidal effect.Comparative genomics and protein homology analysis found a potential efflux pump SstFEG encoded upstream of well-known bacitracin-resistance genes S. suis to provide evidence regarding the important roles of the novel ABC transporter system SstFEG with respect to drug resistance and virulence.To our knowledge, this is the first study to functionally characterize the bacitracin efflux pump in  Streptococcus suis is an emerging zoonotic pathogen imposing a serious burden on the porcine industry as well as a severe public health concern .Additional file 1. Identification of mutant strains via PCR.Additional file 2. Comparison of growth curves between wild-type CZ130302 and mutant strain CZ13-△sstEFG.Additional file 3. Genes involved in bacitracin transport.Additional file 4. Primers used in this study.