Recently, it was reported that CK-1δ (protein casein kinase-1δ) is able to phosphorylate TDP-43. Here, the preclinical efficacy of a benzothiazole-based CK-1δ inhibitor IGS-2.7, both in a TDP-43 (A315T) transgenic mouse and in a human cell-based model of ALS, is shown. Treatment with IGS-2.7 produces a significant preservation of motor neurons in the anterior horn at lumbar level, a decrease in both astroglial and microglial reactivity in this area, and in TDP-43 phosphorylation in spinal cord samples. Furthermore, the recovery of TDP-43 homeostasis  in a human-based cell model from ALS patients after treatment with IGS-2.7 is also reported. Moreover, we have shown a trend to increase in CK-1δ mRNA in spinal cord and significantly in frontal cortex of sALS cases. All these data show for the first time the  Despite the severity of the disease and the high health care and social costs, no cure or viable long-term effective treatment has been identified, with only two therapeutic agents (riluzole and edaravone) already approved by FDA (Food and Drug Administration) but having limited efficacy or serving only for specific groups of patients2.Amyotrophic Lateral Sclerosis (ALS) is a fatal progressive neurodegenerative disease, which results in the destruction of upper and/or lower motor neurons in the brain and spinal cord. It usually affects people between 40 and 60-year-old and the average survival from onset to death is 3–4 years3. Mutations in this protein have been associated with cases of ALS (accounting for approximately 5% of genetic cases), but also have been associated with tau protein-independent cases of frontotemporal dementia (FTD), generating the idea of a pathological spectrum between ALS and FTD based on alterations in TDP-43 and other related proteins4. Today, it is recognized that TDP-43 proteinopathy, characterized by hyperphosphorylation, truncation, ubiquitination, and/or nuclear depletion in neurons, is the prominent and common pathological feature of sporadic and familiar ALS1. Furthermore, it is present in rare diseases such as Perry syndrome or Alexander disease, but also in the prevalent Alzheimer’s disease (AD) and recently in the limbic-predominant age-related TDP-43 encephalopathy (LATE)3. In addition, TDP-43 pathology is a secondary feature of several other neurodegenerative disorders, including Parkinson’s disease (PD), and Huntington’s disease (HD), where its presence may aggravate the primary existing proteinopathy4.In 2006, the trans-activating response region DNA binding protein of 43 KDa, known as TDP-43, was identified as one of the main hallmarks of sporadic and familial ALS, showing accumulations in the cytoplasm of cortical and spinal motor neurons6. In fact, phospho-TDP-43 is distributed in brain areas of FTD and ALS patients5. Different kinases have been recently involved in TDP-43 phosphorylation. Among these, protein casein kinase-1 (CK-1)7, tau tubulin kinase 1 (TTBK1)6 and cell division cycle kinase 7 (CDC7)7 are the best characterized.The study of the post-translational regulation of TDP-43 has placed the phosphorylation of this protein at specific residues, which is dependent on certain protein kinases, as a key event for regulating its cellular activities and also for its dysregulation associated with pathological conditionsin vivo in more than 29 different sites, being 18 of them located in the C-terminal glycine-rich region7. Moreover, different stress signaling cause CK-1-dependent phosphorylation of TDP-43 triggering its cytosolic mislocalization and accumulation9. Furthermore, TDP-43 binds directly to and regulates the expression of CK-1ε mRNA10.CK-1 was the first kinase identified to phosphorylate TDP-43 1–3,δ and ε)8. It is tightly regulated in cells because its role in crucial cellular processes. However, its dysregulation leads to different pathologies including cancer and neurodegenerative diseases11. Recently, inhibition of CK-1, mainly the δ and ε isoforms, has been proposed as a potential treatment for different neurodegenerative diseases including ALS, FTD9 and Alzheimer’s disease12.CK-1 is a highly conserved Ser/Thr kinase, constitutively active and ubiquitously expressed in eukaryotic organisms, with different human isoforms characterized  mutation12. Therefore, our working hypothesis is that inhibitors of CK-1δ able to modulate TDP-43 proteinopathy in vivo, may be a good therapeutic strategy for the severe ALS. We have treated the TDP-43 (A315T) transgenic mice, one of the first experimental models of ALS based on mutations in TDP-43 protein13, with the brain permeable CK-1 inhibitor IGS-2.7. Our results show, for the first time, an in vivo decrease of TDP-43 phosphorylation, together with motor neuron survival and decrease of both astroglial and microglial reactivity. Furthermore, using lymphoblasts from sALS patients we have showed the recovery of TDP-43 homeostasis  after the treatment with the CK-1δ inhibitor. Moreover, an increase of CSKN1D mRNA in spinal cord and frontal cortex from patients of sporadic ALS (sALS) are also shown. All these data may serve as a solid proof of concept for the potential therapy of ALS with CK-1δ or dual CK-1δ/ε inhibitors showing a promising neuroprotective effect.We have discovered and synthetized new families of potent CK-1δ and dual CK-1δ/ε inhibitors with high selectivity score over other kinases based on the modification of the benzothiazole scaffoldThe first objective of our study was to explore the effects of a chronic treatment with the compound IGS-2.7 in the TDP-43 A315T) transgenic mouse model. To this end, TDP-43 (A315T) transgenic mice and wild-type animals were daily treated with the inhibitor IGS-2.7 or vehicle from the age of 65 days up to 90 days. First, we observed the expected progressive decrease in animal weight in TDP-43 (A315T) transgenic mice, which was partially delayed after the treatment with IGS-2.7. This effect of IGS-2.7 in transgenic TDP-43 (A315T) mice weight was statistically significant from the day 19 of treatment  transgenic mice. A reduction in the number of motor neurons was evident in the anterior horn at lumbar level by using both Nissl staining and choline acetyl transferase (ChAT) immunohistochemistry  transgenic mice and wild-type animals by immunoblotting with specific antibodies. We observed a robust increase in the ratio of pTDP-43/TDP-43 in TDP-43 (A315T) transgenic mice compared with wild-type animals  and anti-TDP-43 antibodies at two different compound concentrations. Figure Previous work from our laboratory demonstrated that lymphoblasts derived from FTD and ALS patients, easily available, could represent a suitable platform to search novel disease-modifying drugsNext, we addressed whether the treatment of IGS-2.7 on ALS lymphoblasts could normalize the mislocalization of TDP-43, thus recovering the TDP-43 homeostasis. Subcellular distribution of TDP-43 was assessed by immunostaining using a phospho-independent anti-TDP-43 antibody. Figure CSNK1D and CSNK1E genes and the kinase abundance of CK1 in spinal cord of control and sporadic ALS patients. Figure CSNK1D and CSNK1E mRNA in the anterior horn of the spinal cord and frontal cortex area 8 in sALS cases. A tendency to increase of CSNK1D mRNA levels was observed in the anterior horn of spinal cord (p = 0.07) in sALS cases when compared with controls   Fig. . In contImmunohistochemistry for CK-1δ was performed thereafter in human spinal cord sections of 6 control and sporadic ALS cases. Figure 8. Since TDP-43 protein plays an important role in RNA metabolism, apoptosis, cell division and plasticity21, it is likely that TDP-43 aggregation can promote neurodegeneration22. Therefore, strategies that prevent TDP-43 misfolding, aggregation and/or enhance clearance of pathological TDP-43 may serve as an important neuroprotective mechanism to counteract disease progression in ALS. With this purpose, we have explored here the therapeutic potential of inhibiting one of the key protein kinases involved in TDP-43 phosphorylation, protein kinase CK-1δ. We have tested the effectiveness of the brain permeable small molecule named as IGS-2.7, a potent inhibitor of CK-1δ with some residual inhibitory activity on CK-1ε, in an in vivo preclinical study using a TDP-43 transgenic mice model of ALS. It is important to remark that it is the first attempt to investigate a pharmacological treatment based on kinase inhibition in this mouse model of ALS. Moreover, we have extended our study to lymphoblasts from sporadic ALS patients, as we have shown the versatility of this human-cell based assay for drug discovery programs including FTD23.ALS is a fatal motor neurodegenerative disorder devoid of an effective therapy. The development of innovative drugs displaying neuroprotective and anti-inflammatory activities that might delay disease onset, slow its progression and/or prolong survival is a major therapeutic goal in ALS disorder. Deposition of intracellular aggregates of phosphorylated TDP-43 is a hallmark lesion in almost all cases of ALS, as well as in FTDin vivo in the spinal cord of TDP-43 transgenic mice, being this effect associated with an attenuation of most of the events that reflect the worsening of the pathological phenotype. This chronic treatment has no effect in the healthy animal (wild-type), despite the fact that CK1-δ is constitutively expressed. It seems that IGS-2.7 selectively prevents the aberrant hyperphosphorylation found in the transgenic TDP-43 mice. Whether the overactivation of CK-1δ in pathological conditions, is due to changes in mRNA, protein expression levels, posttranslational modifications, or in the sequestration of the enzyme in subcellular compartments24 remains to be elucidated. Among the pathological events ameliorated by CK-1δ inhibitor treatment, IGS-2.7 attenuated the typical animal weight losses, prevented the death of spinal motor neurons and reversed the glial reactivity affecting both microglial cells and especially astrocytes, which are particularly abundant in this experimental model. All these data strongly suggest that the inhibition of CK-1δ with the benzothiazole derivative IGS-2.7 may modulate TDP-43 toxicity in vivo by limiting TDP-43 phosphorylation, which situates this event in a key position to explain the benefits observed with this drug candidate in the preservation of spinal motor neurons. It is true that the recovery of these parameters is not complete but partial, which stresses the need to better optimize this treatment, either by elevating the active dose used or by combining IGS-2.7 with another neuroprotective agent (e.g riluzole). Furthermore, these rescued motor neurons should be involved in the muscular junction as choline acetyl transferase (ChAT) immunohistochemistry, an enzyme responsible for the synthesis of the neurotransmitter acetylcholine, is used in their quantification25.Our results, shown that a chronic treatment with the compound IGS-2.7 effectively prevents the phosphorylation of TDP-43 23. Recently, we have shown that immortalized lymphocytes from sporadic ALS patients recapitulate pathological TDP-43 features17. Here, we report that treatment with the CK-1δ IGS-2.7 inhibitor improved TDP-43 homeostasis in this human cell-based sALS model. Benzothiazole IGS-2.7 not only decreases TDP-43 phosphorylation in cells derived from ALS patients but also corrects the subcellular localization of TDP-43, preventing the abnormal cytosolic TDP-43 accumulation in ALS lymphoblasts. This finding is in consonance with a previous work in which the effect of IGS-2.7 on the subcellular localization of TDP-43 was additionally evaluated in fractionated nuclear and cytoplasmic extracts from FTD-derived lymphoblasts16.Moreover, we used lymphoblastoid cell lines derived from sALS patients to mimic the central nervous system (CNS) dysfunction and investigated the effect of IGS-2.7 on TDP-43 pathology. Lymphoblasts from patients may reflect changes occurring in the CNS and represent a novel, easily accessible human-based cell model for drug screening and investigationin vivo model, we analysed the expression of CK-1δ and CK-1ε in the anterior horn of the spinal cord and frontal cortex area 8 in healthy subjects and sALS patients by quantitative RT-PCR. We have found a tendency to increase in the mRNA levels of CSNK1D in the anterior horn of the spinal cord and a significant increase in the frontal cortex area 8. In contrast, no differences in CSNK1E expression were found in the spinal cord and frontal cortex in sALS when compared with controls. Moreover, we analysed CK-1δ immunoreactivity in the anterior horn of spinal cord in control and ALS cases. CK-1δ expression was present in the cytoplasm of motor neurons of anterior horn without differences between control and ALS cases, with the exception of apparent increased immunoreactivity in shrunken neurons. However, we observed increased CK-1δ immunoreactivity in reactive glial cells in the anterior horn of ALS cases. This increase was not observed in control samples. These observations suggest the involvement of CK-1δ in human ALS pathology. The selective immunoreactivity of CK-1δ found in ALS reactive glial cells may also well correlate with the in vivo effect observed after the treatment with IGS-2.7 of reversion of the glial reactivity both in microglial cells and especially astrocytes.Finally, and in order to enhance the translational value of the results found in the in vivo, in TDP-43 mouse model, in which the inhibitor preserved motor neurons in ventral horn of spinal cord, and also in vitro using a human cell-based ALS model (lymphoblasts derived from sALS patients). In that model, IGS-2.7 treatment recovers TDP-43 homeostasis by decreasing its phosphorylation and enhancing the nuclear localization. Moreover, up-regulation of CK-1δ is shown in brain  and spinal cord of sALS patients. Altogether, our results indicate that the inhibition of CK-1δ with selective inhibitors, specifically with the benzothiazole derivative IGS-2.7 used here, might represent a good therapeutic approach for disorders involved in TDP-43 protein alterations including ALS and FTD, among others. Only future clinical studies can confirm these results in patients. Moreover, it is worth mentioning that this CK-1δ inhibitor does not produce mutations, which makes it a good candidate for future pharmaceutical development26. Only future clinical trials will be able to confirm these results in patients.In conclusion, our results show strong evidence to support that IGS-2.7 has neuroprotective properties, derived from its capability to reduce the phosphorylation of TDP-43. This has been seen N-benzothiazolyl-2-phenyl-acetamides derivative, CK-1δ inhibitor, IGS-2.7 was synthesized as previously described27 in our laboratory. Antibodies against human TDP-43 (10782-2-AP) and phospho (Ser409/410)-TDP-43 (22309-1AP) were purchased from Proteintech . Antibodies against α-tubulin (sc-23948), and GAPDH (sc-25778) were obtained from Santa Cruz Biotechnologies , anti-GFAP antibody was obtained from DAKO , anti-Iba-1 (019-19741) was purchased in Wako Chemicals , and anti-ChAT antibody (PA5-29653) was obtained from Life Technologies . Dilutions are shown in Table All reagents for cell culture assays were obtained from Invitrogen . For Western blots (WB) experiments, PVDF (polyvinylidene difluoride) membranes were obtained from Bio-Rad . Chemiluminescent reagent (ECL) for WB detection was purchased from Amersham . All other reagents were of molecular grade. The Prp-hTDP-43(A315T) transgenic mice and non-transgenic littermate sibling were used as model in all the experiments. Mice were breeding in our animal facilities from initial breeders were purchased to Jackson Laboratories .13. Mice were housed in an environment with controlling temperature (22 ± 1 °C) and light (12-h light/dark cycle) with food and water available ad libitum. Experimental designs and procedures were approved by the ethical committees of the Complutense University and the regulatory institution (ref. PROEX 059/16) in accordance with the European Commission regulations (2010/63/EU) for the use of laboratory animals. Then, once genotyped, wild-type and transgenic mice were identified by numbered earmarks and, prior to the start of the different experiments, they were randomly allocated to the different treatment groups. For data collection, in all behavioral and histological analyses, researchers were blinded to the animal treatment, whereas for the biochemical analyses, due to the form in which the data are collected, blinding was not considered necessary, as previously described28.Offspring were genotyped for the TARDBP transgene which contain the A315T mutation following a procedure described previously29. All patients were diagnosed by applying the revised El Escorial criteria30 in the Hospital Doce de Octubre . All procedures were approved by the Hospital Doce de Octubre and the Spanish Council of Higher Research Institutional Review Board and are in accordance with National and European Union Guidelines. Informed written consent was obtained from all the participants. Demographic and clinic characterization of individuals enrolled in this study can be found in Table Peripheral blood samples from 4 sALS patients and 4 age-matched control subjects were used for peripheral blood mononuclear cells (PBMCs) isolation on Lymphoprep™ density-gradient centrifugation according to the instructions of the manufacturer . Establishment of lymphoblastoid cell lines was performed in our laboratory by infecting peripheral blood lymphocytes with the Epstein–Barr virus as described previously2 incubator at 37 °C. Fluid was routinely changed every three days by removing the medium above the settled cells and replacing it with an equal volume of fresh medium as previously described17.Lymphoblastoid cells lines were grown in suspension in T flasks in an upright position, in approximately 8 ml of RPMI-1640 medium that contained 2 mM L-glutamine, 100 μg/ml streptomycin/penicillin and 10% (v/v) fetal bovine serum and maintained in a humidified 5% CO50 value for CK-1δ of 23 nM and a residual IC50 of 840 nM for CK-1ε27. Mice (10 per group) were treated starting at the age of 65 days old, intraperitoneally, with IGS-2.7 (1 mg/kg of body weight) in 6.2% Tween 20 and 4% DMSO in saline buffer daily until sacrifice at the age of 90 days old. The rationale for this range of age has been explained in several studies31 based on the fast progression of this model compared to other TDP-43-based model32. Vehicle injections were administered to control animals. This dose was selected based on previous pharmacokinetic results of compound IGS-2.7, that shows good levels of the drug in plasma and brain after intraperitoneally and oral administration16. During all the treatment, we also recorded the physical appearance and the animal weight gain.TDP-43 (A315T) transgenic male mice and wild-type mice were used to register disease progression and spinal cord decline in a pharmacological study using the CK-1δ inhibitor, IGS-2.7. This compound was synthetized in our laboratory following described procedures showing an IC28, after 25 days of chronic treatment, TDP-43(A315T) transgenic male mice and their corresponding wild-type animals were euthanized 24 h after the last injection of IGS-2.7 by decapitation and their spinal cords were dissected and rapidly removed. The spinal samples to be used for western blot analyses were frozen in 2-methylbutane cooled in dry ice and stored at −80 °C. The spinal samples to be used for histological analyses were fixed for one day at 4 °C in fresh 4% paraformaldehyde prepared in 0.1 M phosphate buffered-saline, pH 7.4. Samples were then cryoprotected by immersion in a 30% sucrose solution for a further day, and finally stored at −80 °C. Fixed spinal cords were sliced with a cryostat at the lumbar level (L4-L6) to obtain coronal sections (20 μm thick) that were collected on gelatine-coated slides. Sections were used for procedures of Nissl-staining and immunofluorescence.As we described previously28. We used cresyl violet 0.25% with 0.1% of acetic acid. Slices were dehydrated by an increased ethanol concentration series, sealed and cover-slipped with the non-aqueous mounting medium DPX . A Leica DMRB microscope  and a Leica DFC300FX camera were used for slice observation and for taking photographs of the tissues. The number of total motor neurons in the ventral horn was counted in at least six slices to a minimum of 6 animals per experimental group. To count the number of stained motor neurons (>400 μm2) in the ventral horn, high resolutions photomicrographs were taken with a 10X objective under the same conditions of light, brightness and contrast. The final value for each group is the mean of all animals included in the study.Sections of the spinal cord at the lumbar level (L4-L6) were used for Nissl staining following the protocol previously described28. Tissue sections were pre-incubated for 1 h with Tris-buffered saline with 1% Triton X-100 (pH 7.5) for permeabilization. Then, sections were incubated overnight at 4 °C with a polyclonal anti-ChAT , anti-Iba-1  or anti GFAP  antibody. After several washes with Tris-buffered saline, tissues were incubated with an Alexa 488 secondary antibody conjugate  for 2 h at 37 °C. For slice observation and photography, a Leica DMRB microscope and a Leica DFC300FX camera were used. Quantification of immunofluorescence was carried out on high-resolution photomicrographs that were taken with the 10X objective, under the same conditions of light, brightness and contrast. The software of analysis Image J  was used to quantify the mean density of labelling in a selective area. Six sections coming from at least six animals were analysed to establish the mean value for each group included in the study. The final data were normalized and were expressed relative to wild-type animals. Subcellular localization of TDP-43 was assessed in human lymphoblasts by immunofluorescence as previously described17. Cells (1 × 106 ml−1) were incubated with and without IGS-2.7 (5 µM) for 24 h and then fixed for 30 min in 4% paraformaldehyde in PBS and blocked and permeabilized with 0.5% TritonX-100 in PBS-0.5% BSA for 60 min at room temperature. Cells were attached to poly-L-lysine coated coverslips using the Cytospin centrifuge at 700 rpm for 7 minutes before they were incubated overnight with the anti-TDP-43 polyclonal antibody. After removing the primary antibody, cells were washed with PBS and were incubated with Alexa Fluor 488-conjugated anti-rabbit antibody. Preparations were mounted on ProLong® Gold Antifade Reagent with DAPI (Thermo Fisher) allowing nuclear visualization. High-resolution images were acquired for ∼30 cells per group in n = 4 independent experiments using a confocal microscope Zeiss 510 equipped with a META detection system and a 63x oil immersion objective. Cytosolic TDP-43 levels per cell were quantified using the Volocity software .Slices from TDP-43 (A315T) mice model and controls were used for immunofluorescence study. The protocol used was as described previously2O2 for 15 min, followed by 3% normal horse serum incubation and washed in distilled water. The sections were incubated at 4 °C overnight with the mouse monoclonal anti-CK-1δ antibody  used at a dilution of 1:100. Following incubation with the primary antibody, the sections were incubated with EnVision+ system peroxidase  for 30 min at room temperature. The peroxidase reaction was visualized with diaminobenzidine and H2O2. Control of the immunostaining included omission of the primary antibody and no signal was obtained following incubation with only the secondary antibody. Finally, the sections were slightly counterstained with haematoxylin.Tissue sections from 6 ALS cases and 6 age-controls were used for immunostaining study. Formalin-fixed, paraffin-embedded, de-waxed 4 µm-thick sections of human anterior horn of the spinal cord at the level of upper lumbar were boiled in citrate buffer for 20 min to retrieve protein antigenicity. Endogenous peroxidases were blocked by incubation in 10% methanol and 1% H33.Samples of spinal cords from TDP-43 (A315T) mice and controls were used to assess TDP-43 phosphorylation state as previously describedMouse spinal cords were lysed in RIPA buffer supplemented with a protease and phosphatase inhibitor cocktail . To prepare whole-cell extract, cells were harvested, washed in PBS and then lysed in ice-cold lysis buffer as described. The protein content of the extracts was determined by the Pierce BCA Protein Assay kit (Thermo Scientific). 20–50 μg of protein were fractionated on SDS poly-acrylamide gel and transferred to poly-vinylidene fluoride (PVDF) membranes . The membranes were then blocked with 5% bovine serum albumin (BSA) (Sigma) for 1 h and then incubated overnight at 4 °C, with primary antibodies at the dilutions indicated in Table Post-mortem fresh-frozen lumbar spinal cord (SC) and frontal cortex (FC) (Brodmann area 8) samples were obtained from the Institute of Neuropathology HUB-ICO-IDIBELL Biobank following the guidelines of Spanish legislation on this matter  and approval of the local ethics committee of the Bellvitge University Hospital-IDIBELL. The post mortem delay varied from 2 hours and 15 minutes to 17 hours. One hemisphere was immediately cut in coronal sections, and selected areas of the encephalon were rapidly dissected, frozen on metal plates over dry ice, placed in individual labeled air-tight plastic bags, and stored at −80 °C until use for biochemical studies. The other hemisphere was fixed by immersion in 4% buffered formalin for no less than 3 weeks for morphological studies. The lumbar anterior spinal cord was dissected on a dry-ice frozen plate under a binocular microscope at a magnification x4. Cases with concomitant pathologies were not considered in the present study. The series included 18 sALS cases and 30 controls. The anterior horn of the spinal cord was available in 14 sALS  and the frontal cortex area 8 in 15 sALS . The spinal cord and frontal cortex were available in 11 cases34. Mutations related to ALS and/or FTD, including SOD1, C9ORF72, TARDBP and FUS, were excluded in every case. Age-matched control cases had not suffered from neurologic and psychiatric disorders and did not show alterations34.CSNK1D) and epsilon (CSNK1E) genes was assessed using the following TaqMan probes: Hs01095996_m1 and Hs01017895_m1, respectively. The values of glucuronidase Beta (GUS-β) gene (Hs00939627_m1) were used as internal controls for normalization of frontal cortex samples34 and Hypoxanthine Phosphoribosyl transferase 1 (HPRT-1), (Hs02800695_m1) values were used as internal controls34 for normalization of spinal cord samples. The parameters of the reactions were 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 sec and 60 °C for 1 min. Finally, capture of all TaqMan PCR data used the Sequence Detection Software . The double-delta cycle threshold (ΔΔCT) method was used to analyse the data; and statistical study was performed using T-student test. The significance level was set at *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control group34.RNA from frozen spinal cord and frontal cortex area 8 was extracted following the instructions of the supplier . RNA integrity and 28S/18S ratios were determined with the Agilent Bioanalyzer  to assess RNA quality, and the RNA concentration was evaluated using a NanoDrop™ Spectrophotometer (Thermo Fisher Scientific). Complementary DNA (cDNA) preparation used High-Capacity cDNA Reverse Transcription kit  following the protocol provided by the supplier. Parallel reactions for each RNA sample run in the absence of MultiScribe Reverse Transcriptase to assess the lack of contamination of genomic DNA. TaqMan RT-qPCR assays were performed in duplicate for each gene on cDNA samples in 384-well optical plates using an ABI Prism 7900 Sequence Detection system . For each 10 μL TaqMan reaction, 2.25 μL cDNA was mixed with 0.25 μL 20x TaqMan Gene Expression Assays and 2.50 μL of 2x TaqMan Universal PCR Master Mix (Applied Biosystems). Relative expression of casein kinase 1 isoform delta . Significant differences between the groups were evaluated using two-way ANOVA test followed by a Bonferroni Supplemmentary figures.


Results show that spherical particles, semi-graphitic particles, and graphene flakes coexist in products under an Ar atmosphere. Under an He atmosphere, all products are graphene flakes. Graphene flakes with fewer layers, higher crystallinity, and a larger BET surface area are prepared in Ar-H2 and Ar-N2. Preliminary analysis reveals that a high-energy environment and abundant H atoms can suppress the formation of curved or closed structures, which leads to the production of graphene flakes with high crystallinity. Furthermore, nitrogen-doped graphene flakes with 1–4 layers are successfully synthesized with the addition of N2, which indicates the thermal plasma process also has great potential for the synthesis of nitrogen-doped graphene flakes due to its continuous manner, cheap raw materials, and adjustable nitrogen-doped content.A thermal plasma process at atmospheric pressure is an attractive method for continuous synthesis of graphene flakes. In this paper, a magnetically rotating arc plasma system is employed to investigate the effects of buffer gases on graphene flakes synthesis in a thermal plasma process. Carbon nanomaterials are prepared in Ar, He, Ar-H Graphene is a novel nanomaterial with a single layer of carbon atoms packed in a hexagonal lattice. Since the first synthesis in 2004, graphene has emerged as highly active research field due to its fascinating physical, chemical, and mechanical properties ,2,3,4. I2 may promote the transformation of products from spherical particles to graphene flakes. Kim et al. [In recent years, a thermal plasma process at atmospheric pressure was developed for continuous synthesis of graphene flakes . In thism et al.  and Suh m et al. ,30 propom et al. ,32,33. Tm et al. ,38,39,40m et al. ,42,43.2, and Ar-N2) via propane decomposition. Transmission electron microscopy (TEM), Raman spectroscopy, X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), and the Brunauer–Emmett–Teller (BET) method are employed to analyze the microstructure of the products. Based on product characterization, the influence mechanism of buffer gases on the graphene flakes formation is discussed.In this paper, a magnetically rotating arc plasma system is applied to study the effects of buffer gases on graphene flakes synthesis in the thermal plasma process. Carbon nanomaterials are synthesized under different buffer gases .During the course of the experiment, the argon is first injected into the plasma generator, and the arc is ignited under a pure Ar atmosphere. Then the argon is replaced by the buffer gas. Lastly, the propane is mixed with the buffer gas in the mixing cavity and then introduced into the plasma generator for decomposition. The typical duration time of each test is 20 min. A mass of solid products are deposited on the inner wall of the deposition chamber. The solid products are powdery and pile together loosely. The packing density of resultant solid products is 0.1–0.12 g/cm−1. X-ray diffraction (XRD) measurements are performed using a Cu-Kα radiation in the 2θ range of 10 to 70°. The elemental analysis of the products is conducted using X-ray photoelectron spectroscopy . XPS spectra are obtained via a monochromatic Al irradiation source in the range of 200–700 eV. Nitrogen adsorption–desorption isotherm measurements of the products are performed using a surface area analyzer . The Brunauer–Emmett–Teller (BET) method is used to calculate the specific surface area of the products. The product morphology is characterized by transmission electron microscopy  and high-resolution transmission electron microscopy . The crystalline structure of the products is analyzed using an X-ray diffractometer  and a Raman spectrometer . Raman spectrum measurements are carried out by the 532-nm line of an He-Ne laser as the excitation source in the spectral range of 500–3000 cmTypical TEM images of the products obtained under an Ar atmosphere are shown in 2, more transparent graphene flakes with a size range of 50–200 nm are obtained, as shown in 2. The HRTEM image in 2, all products are graphene flakes that exhibit a smaller size , as shown in 2 are very straight, which suggests a well-ordered graphitic structure in the graphene flakes.When the buffer gas changes, the product morphology demonstrates clear variation, as shown in D band at about 1350 cm−1, the G band at about 1580 cm−1, and the 2D band at about 2700 cm−1, respectively. The presence of the D band corresponds to a disordered or defective structure in carbon materials. The G band is related to phonon vibrations in sp2 carbon materials, which reflects the ordered graphitic sheet [2D band is from the overtone of the D band, and it is closely related to the band structure of graphene layers [D band to the G band (DI/GI), the peak position, and the full-width at half-maximum (FWHM) of the G band are widely used for characterizing the defect quantity in the samples [2D band to the G band (2DI/GI) is used to determine the thickness of graphene flakes, whereas a high value of 2DI/GI indicates fewer graphene layers [G band, the ID/IG value, and the 2DI/GI value. G band as well as the values of ID/IG and I2D/IG are sensitive to the buffer gas. The ID/IG value under an Ar atmosphere is 0.54 and the FWHM of the G band is 42.37 cm−1, which is higher than the others. This can be attributed to the existence of spherical particles in the products. These spherical particles have an amorphous structure, which carries a high degree of defects and disorder. This results in low product crystallinity. The ID/IG value is 0.46 for He, 0.37 for Ar-H2, and 0.25 for Ar-N2, which implies increasingly high product crystallinity as the buffer gas changes. Meanwhile, the FWHM of the G band decreases slightly with the changes in buffer gases, which also confirms the increasingly high crystallinity. This phenomenon aligns with the variation tendency of TEM images in 2D/IG value increases as the buffer gas changes. As such, the number of layers of graphene flakes diminishes gradually, in accordance with the TEM results in 2, and Ar-N2, all products are graphene flakes, but the FWHM of the G band is about 30 cm−1, which is higher than the single-layer graphene (~14 cm−1) or graphite (~12 cm−1) [G band, the crystal size (La) of graphene flakes can be estimated, which is about 20 nm. The G band for Ar-N2 presents a small blue shift with respect to those for other buffer gases. This phenomenon may be due to the incorporation of nitrogen into the graphene lattices, which results in the compressive/tensile strain in the C-C bonds [Raman spectrum analysis of products under different buffer gases is displayed in ic sheet . The 2D e layers ,58. The  samples ,60. The 12 cm−1) . The rel-C bonds ,63.θ ≈ 26° and 2θ ≈ 43°, which correspond to the 002 and 100 diffraction peaks of carbon, respectively. Generally, the peak intensities of 002 and 100 are characteristic of a graphitic structure [2 and N2 are added to the buffer gas, the products have 002 peaks with d-spacing of 3.406 Å and 3.397 Å at 26.14°and 26.21°, respectively. The shift of the 002 peak to a higher diffraction angle and declining d-spacing value further confirm a higher level of crystallinity in the products as the buffer gas changes. According to the TEM images  to distinguish different regions in the 002 peak. XRD analysis in θ ≈ 26°, which reflects a graphitic structure, and the other is on the left, which corresponds to the disordered structure. The area from the fitting peak can describe the relative content of graphitic and disordered structures [2, the graphitic structure content is 69.3%, and increases to about 75% with the addition of N2. Combined with the morphology in TEM images, an increase in the graphitic region is mainly due to the transformation of products from spherical particles to graphene flakes, or due to the straighter graphitic layers as the buffer gas changes.XRD is a useful method for the characterization of the crystal structure of nanoparticles, which is a supplement to the Raman result. XRD spectra of products obtained under different buffer gases are shown in tructure . XRD speM images , the graructures . Figure 2 are basically the same, and are not displayed in this case. When N2 is added, a small N1s (nitrogen) peak at 399.9 eV is also presented besides the C1s peak and O1s peak, as shown in 2, the carbon content is more than 98.7%, and the oxygen content is less than 1.3%. In our opinion, the oxygen is from the ambient air because the products are prepared in the absence of oxygen. When the products are exposed to the ambient air, the products can be oxidized by H2O/O2 species due to the existence of active sites, so as to form C-O, C-O-C, or C=O groups. For Ar-N2, the total amount of nitrogen incorporated into the graphene flakes is about 1.9%. The oxygen content is up to 2.3%, which is higher than those in other buffer gases. Usually, nitrogen-doped graphene flakes have more active sites [2 own a high level of oxygen content. However, the oxygen content measured by the XPS method is likely a little higher than true values. This is because the oxidation mainly occurs at the surface and edge of the products instead of the internal region. In order to distinguish the nitrogen-doped types, high resolution analysis of the N1s peak is depicted in N (peak at 398.4 eV), pyrrolic N (peak at 399.8 eV), graphitic N (peak at 400.9 e V), and oxidized N (peak at 402.2 eV). Their content is 0.43%, 0.85%, 0.36%, and 0.26%, respectively.The XPS spectrum can effectively reflect the elemental components of the sample surface. In our experiment, the products are prepared by an in situ synthetic method. Thus, the elemental components of the internal region and surfaces are basically the same, in principle. In this section, XPS spectra are employed to analyze the element composition of products under different buffer gases, as shown in ve sites . This is2 flow rates are summarized in 2, and the total buffer gas flow is controlled to be 35 slm. 2 flow rate. The nitrogen-doped content is only 0.7% for 1 slm of N2, and rises to 1.9% for 3 slm of N2 and to 2.8% for 5 slm of N2. Noticeably, the pyrrolic N dominates the N-bonding types, which is more than 40% of total nitrogen-doped contents. The pyrrolic N is considered to be easily formed in a rich H environment [N is due to the abundant H atoms, which come from propane decomposition. Moreover, the pyrrolic N and pyridinic N are mainly located at the edges of graphene [Nitrogen-doped contents for different Nironment , so the graphene ,71. The 2 adsorption–desorption isotherms of the products prepared under different buffer gases are revealed in 0), and presents a typical hysteresis loop in the desorption branch. The characteristic of N2 adsorption–desorption isotherms indicates the formation of mesopores in the products [P/P0 > 0.9 may occur on its external surface and the internal surface of pores is formed by the re-stacking of graphene flakes. Textural data of products obtained in different buffer gases is shown in 2, and Ar-N2 is 138.26 m2/g, 172.63 m2/g, 281.94 m2/g, and 353.77 m2/g, respectively. All products have an average pore size in the range of 13–20 nm. The products in Ar have the smallest BET surface area, which is mainly due to the presence of spherical particles and semi-graphitic particles. Usually, the specific surface area of graphene increases as the layer number decreases. As suggested by TEM images and Raman results, the layer number of graphene flakes in Ar-N2 is fewer than those in He and Ar-H2. Thus, the BET surface area of graphene flakes in Ar-N2 is the largest. In addition, the graphene flakes in Ar-N2 have the smallest average pore size. The possible reason is that the smaller graphene flakes in Ar-N2 are easier to form smaller mesopores compared with those produced in He and Ar-H2.The Nproducts ,72. Cons2 is 353.77 m2/g, which is lower than the theoretical surface area for the four-layer graphene flakes (about 660 m2/g). The significant loss of accessible surface area is likely a consequence of the inhomogeneity as well as overlap or severe aggregation of graphene flakes [2/g [The BET surface area of graphene flakes prepared in this paper is lower than the theoretical value. For example, the BET surface area of graphene flakes in Ar-Ne flakes . Howeverkes [2/g . This ph2 is added, the synthesis rate is less than 200 mg per minute, and only about 100 mg per minute with the addition of N2. Accordingly, the yield of carbon nanomaterials decreases from 18% to 7%. This result indicates H2 and N2 may be involved in forming more gaseous products.The synthesis rate/yield of the target product is an important indicator to evaluate the thermal plasma process. 2 radicals are the main precursor species of graphene flakes [2 atmosphere, the formed C2 radicals can be consumed due to the formation of hydrocarbons by collision with the hydrogen molecules through the reaction [2 + H2 → C2H + H, so the synthesis rate of graphene flakes decreases. Similarly, the presence of N2 in plasma favors the formation of cyanides due to the interaction of nitrogen molecules with C2 radicals through the reaction [2 + N2 → CN + CN. The formation of CN species suppresses the production of C2 radicals. Thus, the synthesis rate of graphene flakes is also reduced. Even so, the synthesis rate/yield of graphene flakes in this paper can still compete with that in the microwave plasma process [Recent research suggests that the Ce flakes ,25,28. Ireaction ,74: C2 +reaction ,76: C2 + process ,21 in whFormation of carbon nanomaterials in the thermal plasma process is a complicated activity because the plasma environment is a complex, multi-component system including electrons, ions, atoms, molecules, and more. The formation mechanism remains unclear although some meaningful work has been made in recent years ,32,52,542, and Ar-N2 is clearly higher than that for Ar because of the higher enthalpy for He, H2, and N2, even though the average gas temperature is always controlled to be 3500 K. Hence, the addition of high-enthalpy gas indicates an environment of high energy. In a recent study, Whitesides et al. [As listed in s et al.  investigs et al. ,23,27,312 and N2 indicate that H2 and N2 also play key roles in graphene flakes synthesis. Several experimental studies have confirmed that H atoms are beneficial to graphene formation [2 can be easily cracked to H atoms, and the existence of H atoms has been reported in relevant research [2 facilitates the formation of straighter graphene flakes. Moreover, the thickness of the graphene flakes depends on the cooling rate in the formation process, and, normally, the faster cooling rate leads to thinner graphene flakes [2 has a more efficient quenching capability because its thermal conductivity is larger than that of Ar and He [2.The thinner and straighter graphene flakes with the addition of Hormation ,39,78. Iormation . Thus, tresearch ,29. As ae flakes . H2 has r and He . This ma2 usually results in the formation of products with low crystallinity, such as spherical particles [2. The variation of the synthesis rate/yield in 2 may increase the relative content of H atoms since many C2 radicals are removed via CN species formation. The existence of large quantities of pyrrolic N in graphene flakes also indicates a rich H environment. Given this, formation of thin and straight graphene flakes in the Ar-N2 atmosphere is likely attributed to three factors. First, high enthalpy N2 can maintain a high-energy environment. Second, abundant H atoms are produced in the plasma region through propane decomposition. Third, the addition of N2 may improve the relative content of H atoms due to the CN species formation. Thus, the product morphology with N2 addition is very similar to nitrogen-doped graphene flakes produced by the arc-discharge method under an atmosphere that contains NH3 [The formation of nitrogen-doped graphene flakes shows an interesting result. In the arc-discharge method, the addition of Narticles  and carbarticles ,81, becaarticles ,83. On tains NH3 . Nitrogeains NH3  such as 2, and Ar-N2) are characterized by TEM, HRTEM, Raman spectra, XRD, XPS, and the BET method. Experimental results indicate that the product microstructure depends on buffer gases. Under an Ar atmosphere, three kinds of products  coexist. Under a He atmosphere, all products transform into graphene flakes. For Ar-H2 and Ar-N2, graphene flakes with fewer layers, higher crystallinity, and larger BET surface area are obtained. As such, the buffer gas with high enthalpy, and the addition of some reactive molecules , can promote the graphene flakes formation. Initial analysis indicates that a high-energy environment and abundant H atoms can prevent the formation of curved or closed structures, which produces graphene flakes with high crystallinity. In particular, nitrogen-doped graphene flakes with 1-4 layers and adjustable nitrogen-doped contents are successfully synthesized with the addition of N2. In summary, this study reveals that the thermal plasma process has great potential for graphene flakes synthesis because the morphology and composition of products can be effectively regulated via changes in buffer gases.In this paper, a magnetically rotating arc plasma system is used to prepare carbon nanomaterials by propane decomposition. The products obtained in different buffer gases (i.e., Ar, He, Ar-H


Neurosurgery on individuals with lesions around language areas becomes even more complicated when the patient is bilingual. It is thus important to understand the principles that predict the likelihood of convergent versus separate neuroanatomical organization of the first (L1) and the second language (L2) in these individuals. We reviewed all English-language publications on neurosurgical language mapping in bilinguals before January 2020 in three databases . Our search yielded 28 studies with 207 participants. The reviewed data suggest several principles of language organization in bilingual neurosurgical patients: (1) separate cortical areas uniquely dedicated to each language in both anterior and posterior language sites are the rule rather than occasional findings, (2) In cases where there was a convergent neuroanatomical representation for L1 and L2, two factors explained the overlap: an early age of L2 acquisition and a small linguistic distance between L1 and L2 and (3) When L1 and L2 diverged neuroanatomically, more L1-specific sites were identified for early age of L2 acquisition, high L2 proficiency and a larger linguistic distance. This work provides initial evidence-based principles predicting the likelihood of converging versus separate neural representations of L1 and L2 in neurosurgical patients. Because over half of the world’s population communicates in two or more languages , understBased on neuroimaging studies involving group analyses of healthy individuals, we know that the amount of neuroanatomical overlap between L1 and L2 can be modulated by a variety of factors, including the age of L2 acquisition ,12, profWe present a comprehensive systematic review of the factors that have been separately argued to modify the neural architecture in healthy bilinguals. In this work, we solely focused on clinical language mapping studies that included data from bilingual neurosurgical patients. Most of the reviewed reports performed an invasive verification of the representation of L1 and L2 through techniques such as direct cortical stimulation. Invasive techniques offer more precise information on language organization in the brain than neuroimaging methods. While our main focus is studies that performed direct cortical stimulation, we also include reports that used other techniques, such as Wada testing and single-subject functional magnetic resonance imaging (fMRI), for validation. Similarly, our target group is bilingual patients with brain tumors who underwent direct cortical stimulation but we verify the results by extending our analyses to a broader population of bilingual patients who received clinical language mapping . Our work provides multiple evidence-based principles that predict the conditions under which languages are more likely to have converging neural representations of L1 and L2 in neurosurgical patients.n = 43). Next, we applied the following exclusion criteria included: not bilingual or multilingual (n = 12), not pre- or intraoperative language mapping (n = 8), a review article (n = 1), not in English (n = 2), insufficient biomedical, linguistic and methodological information (n = 2), not examining languages separately to map areas of overlap  (n = 1), not an article (n = 1) and a positron emission tomography (PET) study (n = 1)  patient characteristics: lesion etiology, lesion location, age, handedness, gender, presence or absence of aphasia and the age of disease onset, (2) language information: the number of languages beyond L1 (L2+), proficiency level of L2, the age of L2 acquisition, the manner of L2 acquisition, the amount of exposure to L1 and L2, the linguistic distance between L1 and L2 and the modality of acquisition and (3) acquisition techniques of language mapping: the method of acquisition and types of employed languages tasks. We provide a narrative synthesis to summarize and explain both the characteristics and results of the included studies.n = 16) (a) on patients with brain tumors, (b) that performed ESM and (c) used an object naming task (with or without other tasks). We then expanded the analyses to other diagnoses , aneurysms), methods (fMRI and the Wada test) and tasks to both verify the initial findings and to determine whether there was additional information that was not present in the homogenous study set. Because there was insufficient information and very substantial variability in the sample for our variables of interest, a meta-analysis could not be conducted on the homogenous set of 16 studies. The variables included lesion location, tumor type and grade, patients’ age (pediatric cases were included) and task differences . In addition, there was insufficient information on the possible impact of aphasia on the neuroanatomical representation of L1 and L2 in the reviewed sample. Further, many studies did not include the effect of the linguistic variables of interest  on the neural organization of L1 and L2 in their results.As illustrated in Since the majority of patients in the reviewed studies were bilingual, the term “bilingual” will be used throughout the paper except when discussing cases that used more than two languages. Individuals speaking more than two languages (15.9% of the reviewed cases) will be referred to as “multilingual.” Finally, “L2+” will be used as an umbrella term for any language that was acquired after one’s first language, particularly in the context of multilinguals.The homogenous set of 16 ESM studies focusing on patients with brain tumors all reported at least some areas in the brain with separate representation for L1 and L2. In addition, 14 of the 16 studies found areas where L1 and L2 converged neuroanatomically. The remaining two studies did not identify regions of overlap.As the next step, we expanded the analyses by adding the remaining 12 studies that included more diverse diagnoses, brain mapping methods and tasks. As presented in Among the 16 homogenous studies, thirteen examined language-specific regions in the frontal versus posterior language sites. Twelve of the thirteen studies identified divergent representation for each examined language in both anterior and posterior language areas. Four additional studies were restricted to one of the areas due to limited ESM coverage—two reports assessed language function only in the anterior language areas and two in the posterior areas. These studies also noted separate language organization in the respective regions.The results remained similar for the complete set of 28 studies. Fourteen of the 28 studies focused on language-specific areas in the frontal versus posterior language areas. Eleven of the 14 studies (61%) reported a distinct organization for each examined language in both anterior and posterior language areas. Three studies (17%) found separate representation for L1 and L2 in either the anterior versus posterior language sites. Once again, four additional studies had restricted ESM coverage within language sites and they showed separate neural representation for L1 and L2 in their respective areas . The bottom panel of We found sufficient data to draw conclusions on three factors: age of L2 acquisition, proficiency level of L2 and the linguistic distance between L1 and L2 (see the sections below). There was insufficient data to determine the effects of the following three factors on the amount of neuroanatomical co-localization between L1 and L2: the amount of exposure to L1 and L2, whether languages are acquired formally or informally or whether the languages are oral or signed.When it was possible, we defined early acquisition as below age five and late acquisition as above age five based on neuroimaging developmental data . HoweverThe majority of the reviewed studies did not explicitly evaluate the impact of the age of L2 acquisition on the degree of neuroanatomical overlap between L1 and L2 in the brain. However, three ESM studies and one fMRI study have provided compelling evidence that languages are more likely to co-localize in the brain in patients who acquired their L2 earlier in life. Those studies included both bi- and multilingual patients. Specifically, Walker et al.  reportedWhile the three studies focused on within-hemisphere localization of L1 and L2, one fMRI report  examinedThe remaining paragraphs of this section will report results from bi- and multilingual patients. Since in several studies bilinguals and multilinguals were clustered as a single group, we will use the term “L2” to describe L2 in bilinguals and languages beyond L2 in multilinguals. Among the homogenous set of 16 studies, seven reported whether there were more L1-specific or L2-specific sites in cases where L1 and L2 diverged neuroanatomically in patients with early and/or late age of L2 acquisition (42 cases). Two larger studies investigated bilingual patients  who acquired their L2 early. The studies found more L1- specific regions than L2-specific areas. There were no cases of more L2-specific than L1-specific sites in the early bilinguals.The results were similar for the complete set of 28 studies. The more divergent sample included ten studies  that reported whether there were more L1-specific or L2-specific regions in cases where L1 and L2 diverged neuroanatomically in patients with early and/or late age of L2 acquisition. The ten studies used ESM, one—fMRI and one—both ESM and fMRI. Four of the ten studies  observed that bilingual patients who acquired their L2 early, had more L1- than L2-specific regions. As was the case with the pool of the homogenous studies, we did not find data in any of the heterogeneous studies that would contradict the result of there being more L1-specific sites, compared to L2-specific regions.Further, within the set of the 16 homogenous studies, five investigated the prevalence of L1- versus L2-specific sites in patients who acquired their L2 late (12 cases). Among the 12 cases of late bilingualism, 11 displayed more L1-specific areas and two patients had more L2-specific regions.We verified the results for the complete set of 28 studies. The findings were largely similar and are shown in In sum, the reviewed studies suggest that the age of acquisition of L2 seems to be a robust variable affecting the amount of overlap between L1 and L2. The second language acquired early is more likely to neuroanatomically overlap with L1, while L2 learned late is more likely to be organized separately from L1. Moreover, early L2 acquisition is associated with a greater volume of activation in the right hemisphere for L1 and L2, compared to monolinguals. Finally, we examined the prevalence of L1- versus L2-specific sites in early versus late bilingual and multilingual patients. We analyzed a set of 16 homogenous studies separately and then combined it with a more heterogeneous set of additional 12 studies. Each of the analyses showed that in cases where languages diverged neuroanatomically, more L1-specific areas were found in early bilinguals and multilinguals. Although late bilinguals and multilinguals also displayed considerably more L1 than L2-specific sites, about a third of the reviewed cases had more L2-specific regions. The frequency of occurrence of L1- vs. L2-specific areas may be modulated by proficiency in late bilinguals and multilinguals. These results suggest the importance of mapping both languages in neurosurgical patients who learned their L2 later in life.The most basic classification of language proficiency has three major levels of proficiency that apply to language production, language comprehension, writing and reading: (a) “low/basic user”—the speaker has basic communicative abilities in L2, (b) “intermediate/independent user”—the speaker can participate in a conversation about familiar matters using connected text and (c) “high/proficient user”—the speaker is very fluent in their L2 and is able to interact with native speakers naturally (based on the Common European Framework of Reference for Languages) .n = 191), 25 had an intermediate and 11 had low proficiency  that compared the frequency of occurrence of L1- versus L2-specific sites in individuals with a high L2 proficiency level when L1 and L2 diverged neuroanatomically. Four of the seven studies (38 patients) found more L1-specific regions than L2-specific regions. The two remaining studies (10 patients) demonstrated more L2-specific than L1-specific sites.The results were confirmed for the complete set of 28 studies. The number of studies that looked at L1- versus L2-specific sites in individuals with a high L2 proficiency level now increased to nine (71 patients). Among these nine studies, six  reported more L1-specific areas and three  found more L2-specific sites. The reviewed 28 studies provided only scarce data on the frequency of occurrence of L1- versus L2-specific sites in individuals with a moderate and low L2 proficiency level when L1 and L2 diverged neuroanatomically. Since there were only three case studies with one case per report, we combined moderate and low proficiency of those patients into a single “lower proficiency” category. Because the data on lower proficiency was so limited, we did not present them in In sum, the degree of proficiency does not seem to predict the amount of neuroanatomical overlap between L1 and L2 in the reviewed studies. Both patients with low and high L2 proficiency had numerous divergent sites subserving L1 and L2. Individuals with high L2 proficiency were more likely to have more L1-specific than L2-specific regions, which was confirmed by both the homogenous and the heterogeneous study set.A linguistic distance is a measure of typological similarity between languages. For this review, a linguistic distance was determined using a simple measure: the number of branches between two languages on the language family tree see . In simpAmong the reviewed studies, 40 languages from nine major families were examined see . AdditioWe determined the linguistic distance based on the actual languages reported in the reviewed studies. The linguistic distance was assessed between each L1 and each L2+, which totaled 66 language pairs across the reviewed studies. Seven languages were linguistically very close to the patients’ L1, nine were close, 19 were moderately distant and 29 were distant.There were insufficient data in the homogeneous study set on the effect of the linguistic distance on the amount of neuroanatomical overlap between L1 and L2. We will thus report findings only from the complete set of 28 studies. Rapport et al.  performeCheung et al.  used fMRTaken together, the linguistic distance impacts the cerebral organization of languages. The more distant L1 is from L2+, the more separate neural organization may be expected. More L1-specific regions are predicted when L1 is a language from the Indo-European family and L2 is a non-Indo-European language.The reviewed data suggest several principles of language organization in bilingual neurosurgical patients. The first major principle is that separate cortical representation uniquely dedicated to each language is the rule rather than an occasional finding. Second, divergent neuroanatomical organization of L1 and L2 is found both in the anterior and posterior language areas. This finding is clinically significant because it highlights the need to map L1 and L2 both anteriorly and posteriorly for surgical purposes. Third, in cases where there was a convergent neuroanatomical representation for L1 and L2, two factors explained the overlap: an early age of L2 acquisition and a small linguistic distance between L1 and L2. On the other hand, when L2 was acquired late and when there is a large linguistic distance between L1 and L2, languages are more likely to be organized separately. This finding suggests the importance of mapping both languages in neurosurgical patients who learned their L2 later and when the linguistic distance between L1 and L2 is larger. Finally, when languages diverge neuroanatomically, there are typically more L1-specific regions in: (1) early, high proficient bilinguals, (2) individuals with high L2 proficiency and (3) in cases where L1 was an Indo-European language and L2 was non-Indo-European language. The number of L1- and L2-specific regions appears more evenly distributed among late bilinguals and this neural organization might be further modulated by proficiency. In sum, this work provides initial evidence-based principles predicting the likelihood of converging versus separate neural representations of L1 and L2 in neurosurgical patients.As can be seen in n = 7) despite performing a range of language tasks during ESM was a report by Bello et al. [Nearly 16% of the patients examined in this review were multilingual. Differences in the neural organization of languages in bilinguals versus multilinguals are complex and not well understood. Multilinguals have mainly been investigated in case studies . One of o et al. . The pato et al. . It is pBecause the reviewed studies were heterogeneous in terms of diagnoses, as well as language mapping techniques and tasks used, wherever possible, we first analyzed the data using a homogenous set of 16 studies separately. Next, we verified the findings by adding 12 heterogeneous studies to the set of 16 studies, totaling 28 studies. In all the instances, the pool of the 28 studies confirmed the results from the homogenous study set. However, we note that the different medical conditions included in the heterogeneous study set  may impact the results of language mapping to a certain degree. For instance, in patients with pediatric epilepsy and seizure focus in the language-dominant hemisphere , we frequently observe functional reorganization of language to a contra-lesional hemisphere  . AVMs 5 and braiSimilar to the various diagnoses, different acquisition techniques used in the heterogeneous study set may have influenced the results of clinical language mapping in the reviewed sample. Each of the language mapping methods has its idiosyncrasies ,56,57. Fn = 24) performed an object naming task and 17 of the studies used the task in combination with other language tasks. Those studies rarely reported results for individual tasks. Four studies [This review has a few limitations that should be considered when interpreting the presented findings on the organization of L1 and L2 in the brain. First, knowing that different language tasks can give different mapping results ,58, it s studies ,25,28,35 studies ,61,62,63 studies , which i studies . Another studies ,65.The goal of this paper was to review the available single-subject data on bilingual neurosurgical patients to provide an initial set of evidence-based principles predicting the likelihood of converging neural representations of L1 and L2 in neurosurgical patients. We suggest considering these principles when planning neurosurgery in bilingual patients.


Graphene is an attractive soft material for various applications due to its unique and exclusive properties. The processing and preservation of 2D graphene at large scales is challenging due to its inherent propensity for layer restacking. Three-dimensional graphene-based nanomaterials (3D-GNMs) preserve their structures while improving processability along with providing enhanced characteristics, which exhibit some notable advantages over 2D graphene. This feature article presents recent trends in the fabrication and characterization of 3D-GNMs toward the study of their morphologies, structures, functional groups, and chemical compositions using scanning electron microscopy, X-ray diffraction, Raman spectroscopy, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. Owing to the attractive properties of 3D-GNMs, which include high surface areas, porous structures, improved electrical conductivity, high mechanical strength, and robust structures, they have generated tremendous interest for various applications such as energy storage, sensors, and energy conversion. This article summarizes the most recent advances in electrochemical applications of 3D-GNMs, pertaining to energy storage, where they can serve as supercapacitor electrode materials and energy conversion as oxygen reduction reaction catalysts, along with an outlook. It has been demonstrated that graphene is one of the thinnest and strongest materials known, having excellent flexibility and transparency, which also exhibits exceptional electrical and thermal conductivity, charge carrier mobility, as well as an extensive surface area [Graphene is a 2D material that possesses space area ,2. Its pace area . Due to ace area ,6,7,8,9.ace area ,11,12,13ace area ,16,17,18Various nanostructures can also be derived from 2D graphene. In its 2D form, graphene has a tendency to reaggregate due to weak van der Waals forces that can link its layers, which can alter its performance ,20. To oElectrochemical capacitors are electrochemical energy storage devices that bridge the gap between conventional electrolytic capacitors and batteries. Supercapacitors could store more energy than conventional capacitors. Unlike batteries, supercapacitors have their charge storage not limited by the diffusion of ions within bulk electrodes; thus, they can deliver high power and rapid charge/discharge cycles through ion adsorption and desorption . TypicalThe oxygen reduction reaction (ORR) plays a vital role in many electrochemical energy conversion and storage devices, such as fuel cells and metal–air batteries. The sluggish kinetics of ORR is a limiting factor of device performance; hence, it is essential to develop a stable, high-performance electrocatalyst for improving the efficiency and reducing the cost of devices ,43. AlthIn this feature article, the main strategies for the fabrication of 3D graphene-based nanomaterials are presented. The characterization of the morphologies, structures, and compositions of the fabricated nanomaterials using scanning electron microscopy (SEM) images, X-ray diffraction (XRD), Raman spectroscopy, Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS) is addressed. The synthesis and characterization of the novel 3D graphene-based nanomaterials including interconnected reduced graphene oxide (IC-rGO), N-doped IC-rGO, and Pd-decorated N-IC-rGO as well as their promising applications in energy conversion and storage are highlighted. An outlook of the development of advanced 3D graphene-based soft nanomaterials for energy and environmental applications is discussed.The synthesis and assembly of 3D graphene-based nanomaterials provide compelling research, particularly related to the control of their morphology and properties for practical applications. In this section, we will focus on the fabrication of 3D graphene architectures. As illustrated in Chemical vapor deposition (CVD) is a well-established process for the synthesis of various carbon-based nanostructures, including carbon nanotubes, graphene, and other fullerenes, etc. . Cheng e3, and MnO2 have been employed as scaffolds [Freestanding 3D graphene superstructures can also be prepared using a template-assisted chemical method. This process is similar to the above-discussed methodology; however, the starting materials are different. In general, the functionalized graphene is used as a precursor for this process, particularly graphene oxides and reduced graphene oxides . The funcaffolds . In somecaffolds ,72,73. TThe Template-Free Chemical Method is also referred to as the chemical self-assembly method . Three-dOur research group developed an innovative one-pot approach for the synthesis of interconnected reduced graphene oxide (IC-rGO) without the addition of cross-linked molecules, which we referred as a Streamlined Hummers Method (SHM) . As show−1) is dispersed in water and sonicated for 30 min. Then, ammonium fluoride (2 M) was poured into the dispersion while stirring; after that, the mixture was transferred to a 40 mL Teflon vessel and kept in an autoclave to be treated at 180 °C for 6 h. After cooling to room temperature, the obtained N- IC-rGO was carefully washed with pure water and ethanol, and then, it was dried in a hot air oven overnight. To further modify the N-IC-rGO, Pd nanoparticles were deposited using the soft alcohol reduction (SAR) method [The structural stability of the formed IC-rGO was impressive due to existing covalent bonds between the graphene sheets. We also attempted to modify the IC-rGO with various dopants such as nitrogen and palladium . The N-d) method , where tAs 3D-GNMs were synthesized from graphite, the morphological transformation was examined using Scanning Electron Microscopy (SEM) (FEI Quanta FEG 250). 1D linear detector. Typically, X-ray diffractograms of graphite exhibit two characteristic peaks at 2θ = 26° and 43°, corresponding to (002) and (100) plane reflections, respectively (JCPDS No. 01-0646) [X-ray diffraction (XRD) is one of the most valuable techniques that is extensively used for the characterization of carbon-based materials including carbon nanotubes, diamond, activated carbon, as well as 2D and 3D graphene ,85. As a01-0646) , which i01-0646) . Upon th2 vibrations, and stacking orders [−1, respectively. Graphite without disorder displays only the G band at approximately 1580 cm−1. When disorder is introduced within graphite, a defect-induced (D) band appears at approximately 1350 cm−1 [G/ID) relation, as the in-plane crystallite size is inversely proportional to the defect density; thus, smaller defect density corresponds to a larger crystallite size and vice versa.Raman spectroscopy is employed extensively for the structural and electronic characterization of graphitic materials. Raman spectra provide some useful information on defects, carbon spg orders . All Ram350 cm−1 . For exa−1 , 1730 cm−1 , 1610 cm−1 (C=C), 1226 cm−1 (epoxy), and 1049 cm−1  were evidenced for GO. However, upon reduction by a thermal method, the formation of T-rGO and IC-rGO resulted in a weakened OH peak, which confirmed the efficient removal of oxygen functional groups. The extensive removal of oxygen functional groups during heat treatment under an inert environment results in 2D crumpled sheet-like morphologies. Compared with 2D T-rGO, the FTIR spectrum for 3D IC-rGO displayed a strong absorption peak at 1209 cm−1, which could be assigned to the formation of ethers and esters (C–O–C). The formation of ether and ester groups may play an essential role toward interconnecting functionalized GO sheets to form 3D IC-rGO. In N-IC-rGO, the C–N stretching bonds appeared at approximately 1100 and 1570 cm−1, which confirmed successful N-doping [Fourier transform infrared (FTIR) spectroscopy is a useful technique for qualitatively identifying surface functional groups that are present in graphene-based nanomaterials. FTIR spectra were recorded using a Thermo Scientific spectrometer. An example is demonstrated in N-doping ,89. In aThe accurate chemical composition of 3D-GNMs was determined using XPS measurements, which were performed using a Thermo Scientific K-α XPS spectrometer. All prepared samples were run at a takeoff angle of 90°. A monochromatic Al Kα X-ray source was employed with a spot size of 400 μm. The charge compensation was measured, and the location of the energy scale was adjusted to place the main C1s feature (C–C) at 284.6 eV. All data analyses were carried out using XPS peakfit software. A high-resolution C1s spectrum can be used to understand the type of functional groups of carbon-based nanomaterials. 2) and dried at room temperature overnight before the electrochemical measurements. A polycrystalline Pt wire and Ag/AgCl (saturated KCl) were used as the counter and reference electrodes, respectively.The 3D graphene-based nanomaterials possess some unique properties and enable various environmental, energy, and sensing applications. For instance, supercapacitors are attractive devices for energy storage with a wide range of applications from portable electronics to electric vehicles. The fabricated 3D-GNMs were tested for supercapacitor applications, where a CHI 660E potentiostat was employed for all electrochemical experiments. A three-electrode electrochemical cell was utilized. The pre-determined active materials  were added to 950 μL of pure water and 50 μL of a 10% Nafion binder. After being ultrasonicated for 30 min, an aliquot (3.0 μL) of the homogenous dispersion was cast on a well-polished glassy carbon electrode (GCE) surface (area 0.07 cm2SO4. Cyclic voltammogram (CV) was used to understand the capacitance and the mechanism of energy storage. Specific capacitance could be estimated from the area of cyclic voltammogram. −1, which was significantly higher compared to 2D T-rGO (54.2 F g−1) and GO (2.3 F g−1). As seen in the SEM images, the 3D interconnected structure of IC-rGO increased the active surface area and minimized restacking while processing as an electrode compared to the 2D T-rGO. Constant current charge/discharge studies are another useful technique to assess the specific capacitance. Here, the capacitance is proportional to the discharge duration. −1 at 1 A g−1 and 176 F g−1 at 10 A g−1, which is double that of the capacitance calculated for 2D T-rGO, which is 93 F g−1 at 1 A g−1 and 70 F g−1 at 10 A g−1. Furthermore, the longevity of the energy storage material is assessed through cycle life testing. Cycle life testing is continuously performing charge/discharge at a specific current for a prolonged duration. −1 for 5000 cycles, and the insert shows the stable charge/discharge pattern at different stages of the testing procedure. It is evident that the capacity of IC-rGO was retained without any noticeable loss over 5000 cycles.All the electrochemical experiments were performed in 0.5 M H−1 scan rate. The calculated capacitance of N-IC-rGO was 327 F g−1, which was significantly higher compared to that of IC-rGO. −1. The significant increase in capacitance was attributed to the introduction of N, which altered the electronic structure of the 3D IC-rGO.So far, the 3D IC-rGO demonstrated higher stability and higher capacitance compared to its 2D T-rGO counterpart. Next, we investigated the effect of N doping on the capacitance. 2. −1. The shape of the CV pattern was consistent at different scan rates, which was indicative of rate capability—that is, retaining capacitance over fast charge/discharge conditions. N-doping not only increased the capacitance but also enhanced the rate capability. The improved rate capability was attributed to the smaller resistance, which was attributed to the introduction of graphitic N. Furthermore, the long-term performance stability of N-IC-rGO was examined through cycle life tests at 10 A g−1 charging and discharging. The presence of pyrrolic N and pyridinic N is known to improve pseudo-capacitance, whereas graphitic N increases electronic conductivity. This excellent performance motivated us to investigate the performance in a symmetric two-electrode capacitor. Graphite rods were used as current collectors, polyvinyl alcohol–phosphoric acid gel was used as the electrolyte, and filter paper was used as a separator. Active material loading on the electrodes was approximately 1 mg cm 2), metal chalcogenides (MoS2), and conducting polymers (polyaniline) have also been reported as supercapacitor materials. 3D graphene facilitates the uniform distribution of the metal oxides/chalcogenides, minimizes the interfacial resistance, and improves the stability of the hybrid nanocomposites. The hierarchical structures of metal oxides/chalcogenides via their pseudo-capacitance sites increase the ion accessible area, which contributes to the energy density and stability of the energy storage device. So far, the energy storage performance of the 3D-GNMs was demonstrated based on the IC-rGO and N-doped IC-rGO. Several other 3D graphene structures such as aerogels and hydrogels have also been explored for supercapacitor materials. Besides N-doping, the co-doping of heteroatoms (N and S) for energy storage has also been reported. Doping single heteroatom improves capacitance as a result of tuning the electronic structure of graphene, whereas dual heteroatoms provide unique features of both heteroatoms, which further enhances the energy storage performance. Furthermore, 3D-GNMs combined with metal oxides , water splitting, carbon dioxide reduction, etc. ORR is a cathodic reaction in fuel cells and air batteries. In this section, the catalytic activity of 3D-GNM is demonstrated using two examples—namely, N-IC-rGO and Pd-N-IC-rGO for oxygen reduction reaction.2 with active material loading 0.21 mg cm−2 in 0.1 M KOH electrolyte. The CV technique is useful to confirm the catalytic activity. −1 at 1600 RPM. −2 and −0.18 V, respectively. Further, the Tafel slopes of N-IC-rGO, IC-rGO, and N-rGO were determined to be 80 mV, 128 mV, and 97 mV, respectively. The N-IC-rGO exhibited a significantly more positive onset potential and half-wave potential, increased the current density, and had the smallest Tafel slope in contrast to IC-rGO and N-rGO. This catalytic activity was entirely attributed to the N functionalities on the 3D structured IC-rGO. The presence of graphitic N improved the conductivity and electron density on the IC-rGO, which provided active sites for the ORR.All measurements were performed using a rotating disc electrode (RDE) of 0.19 cmk = kinetic current density, Jd = diffusion current density, 1/B is the slope, and ω = angular rotation speed.To understand catalyst selectivity, it is important to study the ORR reaction pathway. A set of LSVs recorded with different RPMs are shown in −1 Vs ω−0.5) constructed from the LSV data at different revolutions per minute (RPM). Using the slope (1/B), the number of transferred electrons was determined using Equation (2), where n = number of electrons, F = Faradaic constant, A = area of the electrode, D(O2) = diffusion coefficient of oxygen gas, ϑ = kinetic viscosity of solution (0.01 cm2/s), and C(O2) = concentration of dissolved oxygen (1.26 × 10−3 M).From the above calculations the number of electrons transferred during the ORR on the N-IC-rGO was found to be 4.0, which is shown in the insert of Furthermore, we investigated the effects of metal nanoparticle deposition on the 3D N-IC-rGO. Pt/C is a state-of-the-art catalyst for ORR; however, it is expensive and scarcely available. Therefore, we selected Pd, which is cheaper than Pt. We aimed to improve the mass activity of Pd by anchoring it on an active 3D N-IC-rGO catalyst. −1 scan rate. The Pd-N-IC-rGO apparently showed improved ORR activity onset potential and limiting current density. The onset potential, half-wave potential, and limiting current density of Pd-N-IC-rGO from the LSV were 0.02 V, −0.16 V Vs Ag/AgCl, and 3.34 mA cm−2. Furthermore, the reaction pathway was studied using a K-L plot. 3O4), mixed metal oxides , alloy nanoparticles (Ni-Fe), etc. N-doping on the 3D IC-rGO increased the ORR activity compared to 2D N-rGO. Pd nanoparticle decoration onto N-IC-rGO further improved the ORR activity.Firstly, Pd decoration on N-IC-rGO was accomplished with an environment-friendly soft alcohol reduction method (SARM) . The decIn this feature article, we initially discussed recent advances in the fabrication of 3D graphene-based nanomaterials. Subsequently, the characterization techniques used to study the 3D materials were discussed, using the IC-rGO and N-IC-rGO as models. Morphological transformations were characterized using SEM, whereas the structural changes and chemical compositions were elucidated using XRD, Raman, FTIR, and XPS techniques. The aggregation of 2D graphene sheets could potentially inhibit ion transport into its structure, which was overcome with 3D network macrostructures such as IC-rGO and N-IC-rGO. The advantages of the 3D morphology were elucidated through the electrochemical energy storage performance of IC-rGO, while the benefits of heteroatom doping onto 3D graphene were investigated through N-IC-rGO. N-doping on a 3D-GNM enhanced the specific capacitance and the rate capability. Moreover, N-doping on the 3D-GNM was shown to have catalytic activity for the ORR. In addition to the N-doping effect, we also demonstrated that the decoration onto the 3D N-IC-rGO with Pd nanoparticles further improved the ORR performance. Overall, this article summarized recent progress in the fabrication of 3D graphene hierarchical structures and their characterization, as well as their electrochemical energy storage and oxygen reduction catalysis applications.The rising global demand for energy storage and production makes graphene-based nanomaterials important candidates for environmentally compatible energy storage and conversion devices. In its pure form, graphene has remarkable properties; however, it suffers limitations for large-scale production due to the re-stacking issues. This key challenge can be addressed through the development of 3D hierarchical structures. Various 3D-GNMs have been reported, including IC-rGO, hydrogel, aerogel, sponge, foam, etc. In addition, pore architectures  were evolved to improve the performance of graphene-based materials. Current trends involve the development of hybrid materials to exploit the synergies resulting from the combination of 3D-GNM with functional candidates, such as carbon materials (CNT), metal/alloy nanoparticles, metal oxides, metal hydroxides, metal sulfides, etc., for energy storage and catalytic applications. Based on this, possible future research directions include (1) exploration of novel materials considering the practical needs of device energy/power outputs such as supercapacitors, air batteries, and fuel cells; and (2) the development of promising materials for flexible and compact energy storage devices, which would advance a myriad of applications in wearable electronics and biomedicine. The outlook for 3D-GNMs is to identify beneficial synergies via a fundamental understanding of their formation mechanisms, which will lead to breakthroughs toward the development of custom materials for specific needs that can meet energy demand challenges. In view of the early improvements demonstrated here, we believe that the future potential of 3D graphene-based materials is immense.


Also, siRNA-mediated knockdown of TDP-43 had no effect on BACE1 immunoreactivity. Taken together, these data indicate that TDP-43 function and/or dysfunction in AD is likely independent from dysregulation of APP expression and proteolytic processing and Aβ generation.Alzheimer’s disease (AD) is a neurodegenerative condition, of which one of the cardinal pathological hallmarks is the extracellular accumulation of amyloid β (Aβ) peptides. These peptides are generated via proteolysis of the amyloid precursor protein (APP), in a manner dependent on the β-secretase, BACE1 and the multicomponent γ-secretase complex. Recent data also suggest a contributory role in AD of transactive response DNA binding protein 43 (TDP-43). There is little insight into a possible mechanism linking TDP-43 and APP processing. To this end, we used cultured human neuronal cells to investigate the ability of TDP-43 to interact with APP and modulate its proteolytic processing. Immunocytochemistry showed TDP-43 to be spatially segregated from both the extranuclear APP holoprotein and its nuclear C-terminal fragment. The latter (APP intracellular domain) was shown to predominantly localise to nucleoli, from which TDP-43 was excluded. Furthermore, neither overexpression of each of the APP isoforms nor siRNA-mediated knockdown of APP had any effect on TDP-43 expression. Doxycycline-stimulated overexpression of TDP-43 was explored in an inducible cell line. Overexpression of TDP-43 had no effect on expression of the APP holoprotein, nor any of the key proteins involved in its proteolysis. Furthermore, increased TDP-43 expression had no effect on BACE1 enzymatic activity or immunoreactivity of Aβ It is t APP770) ,4.1-40 and Aβ1-42 are the most significant), which may play a role in the pathogenesis of AD [There are two predominant proteolytic pathways of APP processing, though other pathways have been identified . In neuris of AD . The secis of AD . This clis of AD ,12, precis of AD . The mosis of AD .There is an emerging view that transactive response DNA binding protein 43 (TDP-43) plays a key role in AD pathogenesis. TDP-43 was first linked to neurodegenerative disease through its discovery as a component of classical ubiquitination inclusions in motor neuron disease and frontotemporal dementia . Since tTDP-43 inclusions have been identified in AD brains in the form of high molecular weight  and phosHowever, despite evidence showing the presence of TDP-43 inclusions in AD cases and an inverse correlation with cognitive performance, mechanistic insight is sparse. It is neither clear why TDP-43 forms inclusions in neurodegenerative disease generally, nor what specific effects this might have in AD. Overexpression of TDP-43 in AD transgenic mice has been shown to reduce Aβ plaque number, through mechanisms unclear , althougDespite the centrality of APP, its proteolytic processing and metabolites in AD, there has only been one investigation, in mice, of the ability of TDP-43 to modulate APP processing . To thisAll chemicals were purchased from Fisher Scientific  unless otherwise stated. Actinomycin D was from Sigma–Aldrich .Primary antibodies used: for APP , TDP-43 , AICD , Tip60 , Fe65 , FLAG , ADAM10 , BACE1 , nicastrin , Pen2 , PS1 , PS2 , fibrillarin  and GAPDH .2 in a humidified atmosphere. For differentiation, cells were seeded and cultured in full growth medium (above) for 24 h, then differentiated for 7 days in DMEM F12 supplemented with 1% FBS , 100 U/ml penicillin, 100 µg/ml streptomycin, 1% non-essential amino acids and 1 µM retinoic acid. SH-SY5Y cells overexpressing APP isoforms were generated as previously described [SH-SY5Y cells (RRID:CVCL_0019) were cultured in 1:1 Dulbecco’s modified Eagle’s medium: Ham’s F12 with UltraGlutamine , supplemented with 10% foetal bovine serum (FBS) , 100 U/ml penicillin, 100 µg/ml streptomycin (both Life Technologies) and 1% non-essential amino acids (Sigma–Aldrich). Cells were incubated at 37°C and 5% COescribed .2, 95% air atmosphere. iPSC pluripotency and successful cortical neuron differentiation were confirmed using immunofluorescence microscopy with appropriate markers [l-glutamine, 100 µM 2-mercaptoethanol, 25 µM insulin, 100 U/ml penicillin, 100 µg/ml streptomycin  and 0.5% non-essential amino acids.The iPSC line, OX1-19  was maintained on Matrigel  in mTeSR1 medium  containing 50 U/ml penicillin and 50 µg/ml streptomycin (Life Technologies) in a humidified incubator at 37°C in a 5% CO markers . The iPS markers ,45, usinFor the generation of the inducible cell line, the TRE-x system was used (Thermo Fisher Scientfic). Two plasmid vectors were used: the regulatory pcDNA6/TR and pT-Rex-DEST30 (expressing FLAG-TDP-43) . Both vectors were transfected into SH-SY5Y cells  using the Amaxa 4D-Nucleofector on programme CA-137 (Lonza). After 48 h, cells stably expressing both plasmids were selected using blasticidin (5 µg/ml) and G418 (500 µg/ml), selecting for the regulatory and expression vectors, respectively. TDP-43 expression was induced using 500 ng/ml doxyxlycline hyclate (Sigma–Aldrich) for 72 h. Expression of FLAG-TDP-43 was verified using immunoblotting.SH-SY5Y cells were differentiated for 7 days as described and then nucleofected with siRNA duplexes targeting either APP , TDP-43  or a non-targeting control  according to the manufacturer’s instructions . Cells were further cultured for 72 h and target knockdown verified using immunoblotting.Cells were cultured on gelatin-coated glass coverslips . After treatment, cells were fixed in 4% paraformaldehyde for 10 min then washed in phosphate-buffered saline (PBS). Cells were subsequently permeabilised in 0.5% Triton X-100, washed in PBS and incubated with blocking buffer  in PBS) for 1 h. Coverslips were incubated with primary antibody for 1 h , washed in PBS and incubated for 1 h in secondary antibody , Alexa Fluor 555  or CF 647 . After washing, coverslips were incubated with 1 µg/ml DAPI  for 5 min and mounted on microscope slides with Prolong Diamond mounting medium (Thermo Fisher Scientific). Images were acquired on an Olympus IX83 inverted microscope using Lumencor LED excitation, a 40× or 60× objective and the Sedat QUAD filter set . The images were collected using a Retiga R6 Q-Imaging CCD camera and Metamorph v7.8.4.0 . Images were then processed and analysed using ImageJ .g for 5 min (4°C) and re-suspended in 6× volume of lysis buffer (RIPA buffer: 50 mM Tris/HCl (pH 8.0), 150 mM sodium chloride, 1% Igepal CA-630 (Sigma–Aldrich), 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium fluoride, 1 mM sodium orthovanadate, and Complete Protease Inhibitor cocktail ). Lysis was performed for 30 min on ice, followed by centrifugation at 3000×g for 5 min (4°C) to yield the RIPA-soluble fraction as the supernatant, which was used for immunoblotting.Cells were washed twice in ice-cold PBS and harvested in PBS. Cells were pelleted at 3000×Protein concentration in the RIPA-soluble fraction was determined using the bicinchoninic acid (BCA) method , using aProtein samples were separated by electrophoresis at 120 V for 90 min on a polyacrylamide gel. After SDS/PAGE, proteins were transferred to polyvinylidene fluoride (PVDF) membranes . Blots were incubated for 2 h in blocking solution (5% (w/v) milk power, 2% (w/v) BSA in TBS + 1% (v/v) Tween-20 (TBST)). The blots were then incubated overnight in primary antibody (5% (w/v) milk powder in TBS). Blots were washed 4 × 10 min with TBST before the addition of secondary antibody  milk powder in TBST, 1:5000 (Thermo Fisher Scientific)) for 1 h, followed by 4 × 10 min washes with TBST. Protein bands were visualised by chemiluminescence  using a G:BOX and GeneTools software .CT method with succinate dehydrogenase complex, subunit A (SDHA) as a reference gene. Primer sequences were as follows: BACE1  and SDHA .RNA was isolated from differentiated SH-SY5Y cells using the RNeasy Mini Kit according to the manufacturers’ instructions (Qiagen). cDNA was subsequently prepared using the Applied Biosystems High Capacity cDNA Synthesis Kit after which quantitative PCRs (qPCRs) were prepared as follows : 1 µl cDNA, 500 nM each of forward and reverse primers with PowerUp SYBR Green Master Mix (Thermo Fisher Scientific). Thermal cycler  parameters were set as follows: 2 min @ 50°C, 2 min @ 95°C and 40 cycles of 15s @ 95°C, 15s @ 53°C and 60s @ 72°C and data analysed using the ΔΔg, 5 min) and protein quantitation of the supernatant. Subsequently, 2 µl of BACE1 substrate (Abcam) was added to 50 µl supernatant  followed by kinetic output measurement on a fluorescent microplate reader at Ex/Em = 335/495 nm . Rate (ΔRFU) was calculated in the linear reaction phase.Differentiated SH-SY5Y cells were lysed in assay buffer  for 30 min on ice, before clarification by centrifugation . The resultant supernatant was concentrated ten-fold using a Vivaspin 2 (2000 MWCO) and the protein concentration assessed. Conditioned media samples were then added to enzyme-linked immunosorbent assay (ELISA) plates to quantify Aβ1-40  and Aβ1-42  (both Thermo Fisher) ELISAs were performed according to the manufacturer’s instructions, including incubation of condition medium with biotinylated detection antibody, followed by HRP–conjugated secondary antibodies, chromagen and finally the stop solution, resulting in a colorimetric response proportional to target abundance, the absorbance of which was measured on a microplate reader at 450 nm .Conditioned media samples (48 h) were isolated from differentiated SH-SY5Y cells and centrifuged at low speed to remove cell debris  or Kruskal–Wallis with Dunn’s post hoc test as indicated; P<0.05 (*), P<0.01 (**), P<0.001 (***) or P<0.0001 (****). Error bars indicate standard deviation. All statistical analyses were performed using GraphPad Prism 8 .All experiments are In order to investigate a possible direct relationship between APP and TDP-43, putative co-localisation was assessed using immunofluorescence microscopy. Using differentiated SH-SY5Y cells and, separately, OX1-19 iPSC-derived neurons (iPSNs), the localisation of the APP holoprotein and TDP-43 was investigated. As expected, TDP-43 was localised exclusively in the nucleus, whereas the APP holoprotein was excluded from the nucleus A. AICD tIn order to investigate the identity of these AICD-positive, TDP-43-negative subnuclear structures, we assessed whether they represented the AFT (AICD-Fe65-Tip60) transcriptional regulatory complex. However, there was no evidence of Fe65 or Tip60 co-staining with these structures. Moreover, Tip60 immunostaining is absent from these AICD-positive puncta in a similar manner to TDP-43 A. It is Given the large size of the AICD-positive structures, it was hypothesised that they may represent nucleoli. Indeed, the nucleolar marker fibrillarin showed strong co-localisation with AICD, indicating that AICD may be localised to the nucleolus B. LocaliAPP has been shown to regulate the expression of numerous genes, both directly through AICD and indirectly via its binding partners ,55,56. IAn inducible cell line was generated, whereby TDP-43 was only expressed when activated by doxycycline. Incubation of cells with doxycycline resulted in increased expression of TDP-43, combined with FLAG immunoreactivity A, indica1-40 or Aβ1-42, nor did it affect the ratio between the two peptides (1-40) or 0.076 pg/µg protein (Aβ1-42) and in the presence of doxycycline was 2.73 pg/µg protein (Aβ1-40) or 0.073 pg/µg protein (Aβ1-42). In addition, there was no effect on non-amyloidogenic processing of APP, as immunoblotting conditioned medium for sAPPα showed no change upon TDP-43 induction (APP processing is an enzymatic process and, as such, may be modulated by changes in secretase activity which are independent of protein expression. In order to assess this, β-secretase activity was assessed using a fluorescent substrate. Doxycycline-induced expression of TDP-43 had no effect on β-secretase activity B. Increapeptides C–E. The nduction F,G. Toge1-40 or Aβ1-42. In addition, TDP-43 did not directly affect BACE1 activity. Our data strongly suggest that TDP-43 is not directly involved in any AD-linked alterations in APP expression or proteolytic cleavage. Given these findings and the notable regional differences between TDP-43 and Aβ pathology in AD [In the present study, we have focused on the interaction of TDP-43 with APP proteolytic processing. TDP-43 did not influence APP expression, nor did APP modulate TDP-43 expression. TDP-43 had no effect on the non-amyloidogenic cleavage of APP and did not alter the production of either Aβgy in AD , it is h