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.


Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease for which age is the most important risk factor. Different mechanisms associated with aging, including stem cell dysfunction, have been described to participate in the pathophysiology of IPF. We observed an extrapulmonary effect associated with IPF: increase in cell senescence of bone marrow-derived mesenchymal stem cells (B-MSCs).INK4A, p21, and β-galactosidase activity. Mitochondrial function and DNA damage were measured. Paracrine induction of senescence and profibrotic responses were analyzed in vitro using human lung fibroblasts. The reparative capacity of B-MSCs was examined in vivo using the bleomycin-induced lung fibrosis model.B-MSCs were obtained from vertebral bodies procured from IPF patients and age-matched normal controls. Cell senescence was determined by cell proliferation and expression of markers of cell senescence p16In our study, we demonstrate for the first time that B-MSCs from IPF patients are senescent with significant differences in mitochondrial function, with accumulation of DNA damage resulting in defects in critical cell functions when compared with age-matched controls. Senescent IPF B-MSCs have the capability of paracrine senescence by inducing senescence in normal-aged fibroblasts, suggesting a possible link between senescent B-MSCs and the late onset of the disease. IPF B-MSCs also showed a diminished capacity to migrate and were less effective in preventing fibrotic changes observed in mice after bleomycin-induced injury, increasing illness severity and proinflammatory responses.We describe extrapulmonary alterations in B-MSCs from IPF patients. The consequences of having senescent B-MSCs are not completely understood, but the decrease in their ability to respond to normal activation and the risk of having a negative impact on the local niche by inducing inflammation and senescence in the neighboring cells suggests a new link between B-MSC and the onset of the disease.The online version of this article (10.1186/s13287-018-0970-6) contains supplementary material, which is available to authorized users. Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease characterized by a progressive and irreversible loss of lung function though accumulation of scar tissue –3. Its aAging is considered the main risk factor for IPF –11. Alonn = 7), old donors , and IPF patients   of the University of Pittsburgh. As previously described, B-MSCs were isolated from bone marrow fragments from cadaveric vertebral bones . B-MSCs Female 11-week-old C57BL/6 mice  were treated with 2 U/kg bleomycin hydrochloride solution  dissolved to 1 U/ml in sterile saline and delivered by direct injection into the trachea using a 0.9-mm needle. While under anesthesia, a cell suspension of 500,000 human B-MSCs in 100 μL culture medium was injected intravenously. Control group mice received the same volume of sterile medium solution  and STATA version 13 (Stata Corporation). Comparisons between control and IPF B-MSCs were made using a Mann-Whitney test. Kruskal-Wallis and Dunn tests were used for between-group comparisons. For time-dependent observations, we used mixed-effect models with a robust variance estimator to calculate the difference between groups in experimental outcomes.B-MSCs isolated from IPF patients showed morphological changes characterized by increased cell size accompanied by replicative senescence in comparison with B-MSCs from age-matched controls. Quantification of B-MSC proliferation by measurement of DNA staining showed decreased cell proliferation Fig. , which wINK4A and p53 were moderately increased when compared with age-matched controls [To confirm the senescence phenotype of B-MSCs from IPF patients, we investigated the presence of other markers of senescence. Measurements of senescence-associated βgalactosidase  activity showed significantly higher positivity in B-MSCs derived from IPF patients compared with the control group Fig. . Furthercontrols , 20 , and maximal respiratory capacity on the injection of the mitochondrial inner membrane uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Finally, the complex I inhibitor rotenone was administered to determine the nonmitochondrial oxygen consumption rate (OCR). IPF B-MSCs showed lower OCR at basal and maximal respiration conditions and after injection of mitochondrial complex inhibitors in comparison with control cells Fig. .Fig. 6Mi. As a consequence of lower oxygen consumption and glycolytic rates, total adenosine triphosphate (ATP) content in IPF patients decreased compared with the control group  readings at baseline and after FCCP treatment were lower in IPF B-MSCs compared with controls, suggesting there was no glycolytic reprograming in these cells Fig. . In factoup Fig. .d-glucose (2-DG)  Fig. , whereasDG) Fig. . These rDG) Fig.  and IPF DG) Fig. .To assess changes in the capabilities of human B-MSCs to alter the severity of the lung injury, we evaluated the ability of age-matched control and IPF B-MSCs to prevent the development of bleomycin-induced lung fibrosis and then compared this with the response mediated by B-MSCs isolated from young donors. Two regimens of cell infusion were examined: a preventive regimen with infusion of cells 2 h after bleomycin injection, and a therapeutic regimen with cell infusion at day 7 post-bleomycin.Weight loss was used as a measurement of illness severity. As previously reported, mice in the preventive and therapeutic regimens that received young B-MSCs were protected against weight loss compared with bleomycin-treated mice without B-MSC infusion Fig. . Mice reINK4A and p53  from IPF B-MSCs or age-matched controls. After 48 h of treatment, human lung fibroblasts showed increased expression of markers of senescence, including β-galactosidase activity Fig. , and uprp53 Fig. . In parap53 Fig. .Fig. 8StIPF is an age-related systemic disease with a predominant lung phenotype. There is compelling evidence that, for unknown reasons, the lung can be the main target of systemic alterations such as telomere mutations, alterations in proteostasis, and mitochondrial dysfunction. It has been proposed that the reparative capacity of B-MSCs may be decreased with age . In our Aging is a process that affects all cells, including mesenchymal stem cells. It has been suggested that, in aged mesenchymal stem cells and aged lungs, several pathways are altered that could increase the risk of IPF . We haveA decrease in cell proliferation, mitochondrial dysfunction, telomere attrition, and cellular senescence are identified as hallmarks of aging . Our resIn the present study, we have demonstrated that B-MSCs from IPF patients have important differences in mitochondrial function, increases in DNA damage that result in cell senescence, and defects in critical cell functions when compared with age-matched controls. IPF B-MSCs show signs of accelerated senescence that suggests a link between aging and the late onset of the disease. Given that MSCs exhibit decreased function with age and disease, this confirms the possible risk of the use of autologous stem cells in patients with IPF.MSCs, like other cells in IPF patients, have multiple defects that can result in an increase in the severity of the disease. We have identified extrapulmonary changes in the bone marrow-derived mesenchymal stem cells (B-MSCs). Although there is evidence in animal models, no human studies have assessed the function of IPF B-MSCs compared with age-matched old control donors. In our study, we demonstrate for the first time that B-MSCs from IPF patients are senescent with significant differences in mitochondrial function and accumulation of DNA damage resulting in defects in critical cell functions when compared with age-matched controls. Senescent IPF B-MSCs have the capability to stimulate paracrine senescence by inducing senescence in normal aged fibroblasts, suggesting a possible link between senescent B-MSCs and the late onset of the disease. Despite IPF being a disease with a respiratory phenotype and a major representation in the lung, our results show systemic consequences of the disease.Additional file 1:Supplementary figures. (ZIP 623 kb)


We identify graphene layer on a disordered substrate as a system where localization of phonons can be observed. Generally, observation of localization for scattering waves is not simple, because the Rayleigh scattering is inversely proportional to a high power of wavelength. The situation is radically different for the out of plane vibrations, so-called flexural phonons, scattered by pinning centers induced by a substrate. In this case, the scattering time for vanishing wave vector tends to a finite limit. One may, therefore, expect that physics of the flexural phonons exhibits features characteristic for electron localization in two dimensions, albeit without complications caused by the electron-electron interactions. We confirm this idea by calculating statistical properties of the Anderson localization of flexural phonons for a model of elastic sheet in the presence of the pinning centers. Finally, we discuss possible manifestations of the flexural phonons, including the localized ones, in the electronic thermal conductance. Usually, FPs are considered in the context of the suspended graphene. Here we argue that graphene layer placed on the top of the supporting SiO2 substrate gives an opportunity to observe Anderson localization2 for the FPs.Most of the research in graphene emphasizes the relativistic character of its electron spectrum. However, graphene is also interesting due to its out-of-plane  vibrational phonon modes. Flexural phonons (FPs) are a unique addition that Van der Waals heterostructures have brought into microscopic physics3. If the velocity component normal to the surface of the cylinder vanishes, i.e., σR is proportional to a(ka)3, where a is the radius of the cylinder and k is the wave vector. This is the conventional Rayleigh scattering result4 for two dimensional (2d) geometry. However, when pressure is constant, the boundary condition reads Φ(a) = 0, and this influences forcefully the scattering. Unlike the Rayleigh scattering, the zero angular harmonic is involved, and as a result the cross section diverges at small k as ∝To get an idea, let us recall the known facts about a long-wave acoustic wave scattering on a cylinder. The output depends drastically on the boundary conditions for the velocity potential Φ on the surface of the cylinder2 substrate, it has been concluded that in this system the layer is suspended between hills of the substrate8. We have checked that scattering of the FPs from areas attached to the substrate is similar to the scattering from a rigid obstacle9. The zero harmonic is also involved, and the scattering cross-section diverges as σfl = 4/k.In graphene, the substrate cannot scatter effectively the usual acoustic waves, longitudinal and transverse, because graphene itself is one of the most rigid substances. The other thing are the out of plane phonons. From the analysis of intrinsic and extrinsic corrugation of monolayer graphene deposited on SiOni. Taking into consideration that the spectrum of the FPs ω(k) = αk2 is quadratic, i.e., velocity is linear in k, one obtains a scattering rate τ−1 = vσflni, that is finite in the low-energy limit. Thus, for the FPs one may expect localization of the low-energy modes with ω(k) < τ−1. This is in striking contrast with localization of acoustic modes, which is known to happen only at high enough frequency14.In this work we study statistical properties of the out of plane excitations for a pinned-suspended flexible sheet. Whether pinning centers are located in the vicinity of the maximal heights of the substrate where the interaction with the layer is the strongest, or there are charges on the substrate which interact strongly with their images, will be not important for our purposes. First of all, we are interested in the scattering rate of the FPs in the presence of the randomly located pinning centers with concentration 15 indicate, however, towards a possibility of the detaching a graphene sheet from a substrate to relieve its strain by slipping. (This is manifest by straightening of the cantilever). In the case of the SiO2 substrate, both experiment and theory agree that for typical magnitude for corrugations, the graphene layer is partially detached from the substrate. Moreover, the theoretical considerations17 justify the use of a contact force that is finite when graphene is conforming to the substrate and zero otherwise. The basic experimental facts7 which lead to the conclusion that graphene layer deposited on SiO2 is partly freely suspended are as follows: The long-range corrugation of the substrate with the correlation length of about 25 nm is also visible on the graphene sheet, but with a smaller amplitude than on the substrate. Mesoscopic corrugations with smaller length of about 15 nm not induced by the substrate were also identified. These short range corrugations are similar in height and wavelength to the ones observed on suspended graphene19. In addition, the picture of partially suspended graphene, and the presence of the FPs in the graphene on the SiO2 substrate, has been confirmed by the transport20 and thermal measurements22.Let us comment upon the graphene layer deposited on the top of the corrugated substrate. Naively, the membrane-like layer either follows the substrate or hovers over the surface at some distance. Measurements with the use of cantileversHere, we demonstrate that the FPs in a pinned-suspended flexible sheet are more similar to disordered electrons rather than to acoustical phonons. The main point here is that the pinning centers are effectively rigid obstacles for the FPs. As we have already explained, this leads to the non-vanishing scattering rate at small energies. Let us touch upon this point in more detail.h is displacement in the out of plane direction; the term describing the barrier is on the right-hand side of the above equation. As a result, a flexural phonon with an energy smaller than ω0 cannot enter the area of pinning. We have checked that such an inclusion is equivalent to a rigid obstacle. For not small ka, the cross-section σfl(k) = 4f(ka)/k. Interestingly enough, for f(ka) ≈ ka, for a discussion see Section I in METHODS. Therefore, the limiting cross-section is ≈4a, i.e., twice larger than the width of the obstacle3. (This form of σfl(k) is, of course, valid only when the energy of the phonon is much less than the pinning potential, i.e., 23 we assume that the pinning potential ω0 is about few meV).Usually by a rigid obstacle one understands an inclusion with the Young’s modulus much higher than that in surrounding area. For graphene, which itself is very rigid, this is not an issue. However, the pinning potential introduces an energy barrier of a finite height for the flexural modes:T2. The rate of energy exchange between electrons and FPs, as discussed below in section “Thermal transport”, is proportional to T3. Therefore, the openness of the FPs, cannot effectively destroy their localization at low temperatures.Let us now touch upon a subtle question of the openness of the ensemble of the FPs which can be relevant for their localization. In the discussed model, the low-energy FPs cannot penetrate into the areas of strong contact with the substrate. Therefore, the most relevant channel connecting the FPs with the other degrees of freedom is the interaction with conducting electrons. The effect of this interaction can be estimated by comparing the amount of heat stored in the FPs with the rate of cooling of electrons. At low temperatures, most of the heat in the graphene layer is stored by the FPs, as they are the softest modes. The amount of this heat is ∝W. Instead, there is concentration of the pinned sites. Furthermore, the FPs are described by the square of the Laplacian, rather than by the Laplacian in the case of electrons. To understand the general properties of the FPs in the presence of random pinning scatterers, we solve the equation of motion for the out-of-plane displacements using finite difference method on a 2d square lattice. We study a model in which the graphene sheet is completely attached at the pinning centers. For that, we use discretized LHS of Eq. h = 0 at randomly chosen pinned sites, so that one pinned site represents an attached area of the size ≈2a 2 which approximately equals 0.08 K for the parameters mentioned above. We found out here that typical energy scale for strong localization, 2 substrate We estimate the size of an attached area to be mentclass2pt{minimd is that the critical point is at e2/(2πħ). At a finite lloc, which is analogue of a correlation length at a quantum phase transition. A sample of size 24. A consequence of strong fluctuations of wave function amplitude in the critical region is the multifractality .As is well known, localization is a quantum critical phenomenon. The peculiarity of 2i.e., the “metal-insulator” transition with the mobility edge), or there is a crossover from strong to weak localization (WL)?Turning back to disordered FPs, the first question that needs to be answered is: Is there a transition to delocalized states at a certain energy . From our simulations, it is clear that low-energy modes are localized: the IPR scales with the sample size to a finite value. For higher energies, the behavior of the wave functions changes, see Fig. lloc starts to exceed the sample size. We are particularly interested in studying the FPs in this region when d Anderson model does not constitute a truly critical system, thanks to exponentially large (but still finite) localization length at d electrons at large 26. As we shall see, similar physics holds also for our system of the FPs.To figure this out, we first studied the dependence of the Inverse Participation Ratio (IPR) on the sample size We proceed as follows: From the size dependence of the IPR at a given energy as shown on the Fig. 28 equal to L is due to the WL corrections. Thus, for each concentration of the pinned sites we can prescribe for different energies ω the corresponding value of the phonon “conductance” gph, using the expression for the fractal dimension D2 for electrons. We defer the discussion of the dependence of D2 on the sample size, D2(L), to the end of section “Weak multifractality of eigenfunctions”.d, the well developed theory connects the behavior of various physical quantities with the value of the conductance25. We have calculated numerically the same quantities for the FPs, using the values of gph extracted from IPR, and found a very good agreement with the theoretical predictions existing for the disordered electrons in the case of the GOE. Below we present some results of such calculations.For disordered electrons in 2P(s) for localized states has almost Poissonian statistics, while for metallic states it is of the Wigner-Dyson form. Next, in 2d it becomes especially interesting to study the variance N in a strip of width Ω around the energy ω: d = 1 and 3, in two dimensions this quantity is directly related to the WL corrections29. Figure d = 2; for details the reader is referred to section “Energy level statistics”.First, we have checked  are of comparable strength for the discussed scales.Let us estimate energy of the FPs at which a crossover from strong to WL occurs. Strong localization, mentclass2pt{minimf ≈ ka > 1, the energy of the strongly localized FPs can be a few times larger. As we have already mentioned, a reasonable estimate for 2 is ≈0.3 K. Furthermore, effects of the weak localization noticeably expand localization of the FPs. One may easily show that, as compared to the strong localization, the weak localization increases momenta of the FPs which undergo localization by a factor ln(L/l). . Correspondingly, weak localization boosts the energy of the localized FPs by a factor ln2(L/l). For a standard micron size sample, So far, we have discussed point-like pinning centers only. In reality, the size of the attached areas can be comparable with the distances between them. Then, owing to the factor 36. For graphene on a random substrate, FPs not only scatter from the points of contact with the substrate, but also excite acoustic phonons. One may check, following the calculations of ref.37 for disorder-assisted scattering, that at low temperatures the effect is vanishingly small.In writing Eq.  we have Y0), while a bending rigidity κ0 is relatively modest. The anharmonicity transfers the strong in-plane rigidity into the bending one. The effect is controlled by the temperature, and is of the infrared origin. For small wave vectors κR(q) ~ κ0(q/qth)η− with the scaling exponent η ≈ 0.8–0.8539. The transition scale is given by qth ≈ 1.6 nm−1. As a result, effective bending rigidity of an atomically thin graphene ribbon that are 10–100 micrometers in size at room temperature can be thousands times larger than at T = 043. .Next, as it is well known anharmonicity yields a strong effect on the bending rigidity in graphene. The point is that the in-plane rigidity of graphene is extremely high  ≈ κ0. In graphene, the energy of a FP with the wave vector qth is equal to 2 substrate. Furthermore, in our analysis, we were mostly interested in FPs with the frequency exceeding Strong effects caused by the renormalizations, which have been mentioned above, correspond to the vanishing wave vector 2 substrate. We argue that traces of localization of the FPs may have been observed in the experiments at low temperatures.We shall discuss now the implications of the FPs on the thermal transport of a graphene layer placed on a corrugated SiO2 at low temperatures, refs49, has not been fully understood, yet. Theoretically, the heat flux from electrons to the FPs is known37 to be ∝δ = 3 observed in refs46 at low temperatures and far away from the neutral point. At concentrations of the electric carriers, electrons or holes, n ~ 1012 cm−2 the alternative explanation of δ = 3 with the use of the result obtained for the case of the unscreened deformation potential49 is not realistic; a discussion of this point is given below in section “Electron-phonon interaction at low temperatures”.The temperature behavior of overheating in graphene on the top of SiO46 for cooling rate of graphene at the lowest temperatures 52 that neither the Fermi liquid nor renormalization-group corrections in disordered 2d electron systems can modify the Lorenz number imitating the electron heat diffusion contribution, Moreover, we believe that the existence of localized FPs may explain the experimental result of ref.four powers of the FP-momenta. However, the localized FPs are not goldstone modes anymore. For localized FPs, the momenta that enter into the matrix elements of the electron-FP interaction should be substituted by the inverse of the localization length. As a result, two powers of frequencies in the expression for the heat flux P saturate at ω−2. The diverging integration should be cut-off at energies typical for the localized FPs. As a result one gets a contribution to the cooling rate of the order Let us comment upon 59. However, a layer placed on the top of a corrugated substrate, which has been discussed in the present work, is very different from the disordered membranes considered so far. First, disorder here is external rather than internally quenched. Next, disorder pins rigidly the height of randomly chosen points of the layer, rather than acting on the metric and curvature tensors describing the deformation of the membrane. Questions of localization of the FPs to the best of our knowledge have not been addressed previosly.Studying effects of disorder on the properties of elastic membranes has a long history2 substrates are expected to play an important role in applications related to thermal transport and for ultrasensitive bolometry. While the graphene sheet is pinned at random points, the FPs may exist in between due to the corrugation typical for this substrate surface. We have argued that in this system Anderson localization of low-energy FPs develop. We showed that the ensemble of flexural phonons in a pinned-suspended flexible sheet is statistically identical to an ensemble of disordered electrons, despite the very different underlying mathematical descriptions. Localization of flexural phonons should be important for thermal transport in such hybrid systems even at not very low temperatures. Traces of localization of the FPs may have been already observed in the experiments. Let us note that we have already shown that FPs give a significant contribution to dephasing rate of electrons in graphene60. Here, we have argued that localization of the FPs opens interesting perspectives for thermal transport.Graphene layers on top of SiO9, the scattering cross-section of the FP by a rigid obstacle of the radius a equals f(z) is given by the following expression:f(z) with asymptotes f(0) = 1 and f is important for the effectiveness of the weak localization in samples of large size.According toThe spatial distribution of wave functions is conveniently characterized by inverse participation ratios:Pq〉 shows the scaling behavior with the system size L:After sample average, 〈Dq = 0, while in a metal Dq = d. At a critical point, Dq is a fractional which leads to anomalous scaling behavior in 〈Pq〉. This is a manifestation of the wave function multifractality, which is a consequence of the spatial correlations of the wave function.Obviously, in the insulating state d, the inverse participation ratios scale asIn 2Dq from dimension 2 is determined by a small parameter 1/πg. The above result 6 was first obtained by Wegner27 via the renormalization group calculations for a system of disordered (non-interacting) electrons; see also28. The dimensionless parameter g is a conductance of a sample measured in quantum units. In analogous to weak localization, the phenomena is coined “weak multifractality”.Here, the deviation of q = 2. The corresponding inverse participation ratio, Pq = 2, was denoted as IPR. This quantity allowed us to extract the phononic “conductance”, which we used for the statistical analysis of our system. Note that, because of the absence of the genuine critical point in 2d, g becomes size-dependent, i.e., g(L) = g0 − (1/π) ln L/l where l is the mean-free path of FPs at a given energy, and g0 = g(l). This implies that for each scale L one can use the standard formula given above, but with slowly varying g(L) in the exponent. This is possible because corrections to g are not large in a finite size sample, and owing to the slow dependence of g on spatial scale L. With this procedure, we have obtained an excellent agreement between the theory of the logarithmic corrections to the conductivity and our numerical results as it is shown in Fig. We study a model of random pinning centers, in which the graphene sheet is completely attached at the pinning centers; see Fig. ωn, such as the distribution function of level spacing P(s), and the level number variance Several quantities are introduced to measure the fluctuations of energy levels P(s) = exp(−s). In our system of the FPs the crossover from the localized to delocalized behavior is illustrated on Fig. Random Matrix Theory (RMT) could be used to describe these quantities in ergodic systems . Here, we will focus on the case of Gaussian Orthogonal Ensemble (GOE). Then the distribution function of level spacing is well described by the Wigner surmise: N〉 = Ω/Δ, and cβ is a known constant. This, however, is far from the true behavior  and, hence, D → D + δD, where Kravtsov and Lerner recognized in29), we use Eq. Q and s, this yields11 for fitting the numerical data presented in Fig. Now, instead of splitting this expression into two parts as it was done iny:y〉 = 1.Porter and Thomas were first who studied the distribution of eigenfunction amplitudes within the RMT framework. Their result shows simply Gaussian distribution, which leads to the following distribution of the intensities d-dimensional disordered system. The eigenfunction intensity r0 is distributed as:The supersymmetric field theory was applied to the study of the eigenfunction statistics in a d = 2, and not too large y, one can calculate perturbatively the deviations from the RMT distribution P(y). The corrected distribution function was found by Fyodorov and Mirlin in (31):y can be seen in Fig. For 46 the T3 law has been observed down to 0.5 K for sample D3. Such a behaivior corresponds to the dirty regime with negligible screening of the e-ph deformation potential according to the Table 1 presented in49. We believe, however, that applicability of the screenless approximation for the e-ph interaction at such low temperatures and concentration of carriers n ~ 1012 cm−2 is highly questionable.Some recent experiments interpreted the cooling rate in terms of the weakly screened electron-phonon (e-ph) interaction down to very low temperatures. In particular, in49, temperature above which screening becomes irrelevant in the dirty regime 49, one finds that crossover from the T5 to the observed T3 behavior should happen at κ for an effective dielectric constant . Here we expressed the result in terms of TBG using the fact that in graphene the inverse of the screening radius is of the order of the Fermi momentum. The unscreened T3 behavior may occur at temperatures higher than Tdis. (For samples D1 and D3 in (46) one has Tdis ≈ 40 K).To illustrate our point, let us estimate, followingTBG ≈ 80 K. Thus, one may expect for the crossover temperature κ. We, therefore, believe that this mechanism may be discarded for explanation of the T3 scaling of the electron-phonon heat flux observed at low temperatures.For the aforementioned sample D3,