The misfolding of transactive response DNA-binding protein (TDP-43) is a major contributor to the pathogenesis of TDP-43 proteinopathies, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 inclusions, but also plays a role in other neurodegenerative diseases including Alzheimer disease. It is thought that different truncations at the N- and C-termini of TDP-43 contribute to its misfolding and aggregation in the brain, and that these aberrant TDP-43 fragments contribute to disease. Despite this, little is known about whether different truncation events influence the protein’s transmissibility between cells and how this cell-to-cell transfer occurs. In this study, we use a well-established cellular model to study the efficiency by which full-length and truncated TDP-43 fragments are transferred between neuron-like cells. We demonstrate that preservation of the N-terminus of TDP-43 enhances its transmissibility between cells and that this protein transmission occurs in a manner exclusive of extracellular vesicles, instead requiring cellular proximity for efficient propagation. These data indicate that the N-terminus of TDP-43 might be a useful target in the generation of therapeutics to limit the spread of TDP-43 pathology. Transactive response DNA-binding protein 43 (TDP-43) is known to play a key role in the pathogenesis of canonical TDP-43 proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontal temporal lobar dementia (FTLD) but is also frequently observed in more common neurodegenerative diseases (NDs) including tauopathies, synucleinopathies, and up to 57% of Alzheimer’s disease (AD) patients . TDP-43 A number of disease-related posttranslational modifications have been described, including hyperphosphorylation at the C-terminus and truncation events resulting in the aggregation prone, cytotoxic fragments found in the inclusions of ALS and FTLD brains . C-termiTDP-43 proteinopathies share a number of similarities with other NDs, including progressive involvement of interconnected brain regions as disease progression occurs, as well as the ability of TDP-43 aggregates to seed further aggregation . The dirTo elucidate whether TDP-43 and TDP-43 truncated fragments are transferred directly between cells and, if so, which regions of the protein are important to the transfer process, we generated a number of cell lines expressing different fragments of TDP-43. Using our well-established, highly differentiated three-dimensional coculture cellular model, we studied the ability of each expression construct in their propensity to transfer between cells and their downstream effects upon the recipient cell.170– 414, construct 4216– 414, and construct AA51–414) were highly cytotoxic and resulted in cell death within 72 h, so we were unable to generate stable expressing cell lines with these constructs. The naming scheme of the constructs was adapted from Plasmids expressing full-length TDP-43 and fragments of TDP-43 were obtained from Addgene. Plasmids deposited to Addgene by L-glutamine . Differentiation and coculture were performed as previously described , supplemented with 10% fetal bovine serum , 50 μg/mL streptomycin , 50 U/mL penicillin , and 2 mM escribed . In ordeTo definitively demonstrate that passage of TDP-43 and its fragments were transferred in both directions during coculture , additional experiments were performed involving the use of Qdot-800  to further label the donor cell population. Qtracker was chosen because it has minimal impact on cellular processes and does not leak out of cells and because of its favorable emission spectrum. The coculture process was performed similarly to that described above with minor modifications. In this circumstance, donor cells are differentiated in the same way but, on day 6 of differentiation, were supplemented with Qtracker-800 according to manufacturer’s instructions. The following day, cells were sorted to collect those that had taken up Qdot-800 nanocrystals and resuspended onto the fully differentiated (including growth factor cocktail), ECM-suspended acceptor cells, where they were left for 24 h before performing further analysis. Data corresponding to these experiments are depicted in their respective figures as the proportion of protein transfer events that have occurred through “anterograde transfer” (donor cell to acceptor cell) and “retrograde transfer” (acceptor cell to donor cell). A flowchart of the differentiation and experimental processes for the Qdot-labeled coculture studies can be found in For Transwell coculture experiments, donor and acceptor cells were cultured in the same manner as described previously. Rather than seeding donor cells directly onto the acceptor cells in ECM gel, they were plated into Transwell plate inserts with a 0.4-μm pore , using the same 7:10 (donor:acceptor) cell ratio used in all experiments. This configuration permits donor and acceptor cells to secrete cell products into a shared extracellular milieu, while maintaining a physical barrier between both populations. Data corresponding to these experiments are depicted in their respective figures as the proportion of acceptor cells that were double labeled, indicative of protein transmission. A flowchart of the experimental process for the Transwell experiments can be found in 2 and incubated with 1,000 ng EV isolated protein (see separate EV isolation section) for 24 h. Following this incubation, cells were analyzed by flow cytometry. Data corresponding to these experiments are depicted in their respective figures as the proportion of wild-type (WT) unlabeled acceptor cells that were fluorescently labeled, indicative of protein transmission. A flowchart of the experimental process for the EV experiments can be found in Treatment of acceptor cells with extracellular vesicles (EVs) was done as previously described . In ordeWhen measuring cytotoxicity in cocultures, lactate dehydrogenase (LDH) assay kit  was used according to manufacturer’s instructions, measured at 490 and 650 nm.g for 10 min to remove cellular debris. The resulting supernatant was sequentially centrifuged: 10,000 × g (30 min), 100,000 × g (120 min); the pellet resuspended/washed in PBS and spun again at 100,000 × g . The final pellet was resuspended in PBS. To ensure that equal amounts of EV isolated protein were added to acceptor cells in downstream experiments, the protein content of the EV resuspension was quantified using Quant-iT Protein Assay Kit (Invitrogen) according to manufacturer’s instructions. Cells were plated onto 24-well plates at 25,000/cm2 and incubated with 1,000 ng of isolated EV protein for 24 h.TDP-43 expressing cell lines were differentiated for 7 days with RA. After the differentiation process, cells were washed thoroughly with phosphate-buffered saline (PBS) to ensure the removal of potentially contaminating EVs of fetal bovine serum origin. Following this important washing step, the media was replaced with fresh SFM and incubated for 48 h to allow release of EVs into the media. This conditioned media was collected for isolation by differential ultracentrifugation as described previously . CultureTo confirm the validity of our EV preparations from cell culture, we isolated and analyzed EVs from human tissues. Fresh-frozen postmortem brain samples of temporal neocortex from healthy (non-demented) donors were provided by the brain bank at Uppsala University, Sweden. The collection and use of the tissue were approved by the Regional Ethical Committee in Uppsala, Sweden . Isolation of brain exosomes from freshly frozen human brain tissues (250 mg) was performed by tissue dissociation with papain  followed by filtration through 40-μm mesh filter  and 0.2-μm syringe filter . The crude exosomes were then isolated by differential centrifugation as described above.2O) and ECL reagent . A list of antibodies used in this study indicating manufacturer and dilutions is provided in Confirmation of EV preparations and TDP-43 expression in each construct-expressing cell line was performed by Western blotting. For EV preparations, EVs were pelleted and lysed in RIPA buffer , followed by vigorous vortexing. Extracellular vesicle lysates were mixed with Laemmli loading buffer and dithiothreitol (DTT), boiled at 95°C for 5 min, and loaded into a 4–20% SDS ClearPage gel , and transferred onto nitrocellulose membranes . Extracellular vesicle blots were visualized using SuperSignal West Femto ECL substrate (Thermo). For confirmation of TDP-43 expression in construct-expressing cell lines, the preparation was the same as above, except for the use of a different lysis buffer  according to manufacturer’s instructions. Analysis and cell sorting were carried out simultaneously using a FACSAria III (BD Biosciences), equipped with the following laser and filter configurations for the detection of each fluorophore: GFP: 488 nm laser, 530/30 emission filter; td-tomato: 561 nm laser, 610/20 emission filter; Qdot-800: 405 nm laser, 780/60 emission filter. Gating strategies are provided in While previous studies have demonstrated the propagation of TDP-43 pathology in the brain, it remains unclear by which manner this propagation occurs, as reviewed in 1– 414). After 24 h, the coculture was analyzed by flow cytometry (1– 314 and WT-TDP-GFP1– 414 (full-length TDP-43 fused to GFP) were found to be transferred most readily compared to the other TDP-43 constructs. We utilized CD63–GFP (a marker of EVs) and actin–GFP as positive and negative control cell lines, respectively. Both of these control cell lines also demonstrated cell to cell transfer, with CD63–GFP showing the greatest degree of transfer among all permutations. The full analysis of variance (ANOVA) table corresponding to the transfer experiment in This coculture model has been used previously to demonstrate the cell-to-cell transfer of Aβ and α-synuclein but had not been used to examine the transfer of TDP-43 between cells. In order to assess the suitability of this coculture model for the study of cell-to-cell transmission of TDP-43, we initially confirmed that full-length TDP-43 was efficiently transferred from donor cells to acceptor cells . After uytometry . We obse1– 414 cells were used as donor cells. In this way, we validate that the transfer is a bona fide event while also illuminating whether this transfer is skewed in one direction or another . In this reversed scenario, we noted a similar trend for TDP-43 transfer . We speculate that the minute differences in transfer between In addition to these initial transfer experiments, we also examined this phenomenon with the donor and acceptor cell pairs reversed. That is, the TDP-43 fragment (GFP)–expressing cells were differentiated into the acceptor cells, whereas the td-tomatoTDP43transfer . While t86– 414 was found primarily in the nucleus, but also to a lesser extent uniformly distributed throughout the cell soma  and nuclear export sequence (NES) intact, construct 5257– 414 has both regions omitted, perhaps having a deleterious effect upon its localization, whereas construct 61– 314 displays two unique phenotypes: cytoplasmic and nuclear puncta (1– 414 puncta of the acceptor cells (Pearson correlation coefficient = 0.48). Construct 101– 105 was localized entirely to the nucleus, which was anticipated given that it contains only a short N-terminus fragment containing the NLS and no NES (1– 414 (1– 414 (Pearson correlation coefficient = 0.82). This enhanced colocalization of CD63 with td-tomatoTDP431– 414 suggests that, at the very least, TDP-43 is sequestered to subcellular compartments containing burgeoning EVs. The observed colocalization between TDP-43 and CD63 suggests that transfer of TDP-43 between cells might occur via an EV-mediated mechanism, which is investigated later in this study. The images provided in 1– 414 as the donor cells, GFP-tagged fragments as the acceptor cells) are provided in In addition to confirming that protein transfer occurs between donor and acceptor cells, we also determined the compartmentalization of the transferred proteins, as aberrant localization of TDP-43 is a marker of TDP-43 pathology. As an RNA-binding molecule, TDP-43 is natively found in the nucleus, whereas pathogenic TDP-43 can be found in the cytoplasm . Each TDell soma . Similarell body . While cr puncta . While md no NES . Of part , we wanted to further investigate the direction of transfer in order to unequivocally confirm that transmission of TDP-43 between cells is not restricted only to full-length protein or fragmented protein. To investigate if the transfer events occurred primarily “anterograde” (donor cell to acceptor cell), primarily “retrograde” (acceptor cell to donor cell), or a combination of both, we modified the coculture model by further labeling the donor cells with Qtracker-800 prior to the formation of the coculture. These Qdot-800 nanocrystals do not interfere with cellular processes and are not passed between adjacent cells , making 86– 414 and 101– 105 showed the least amount of anterograde transfer, compared to constructs 61– 314 and WT-TDP-GFP1– 414, which showed the greatest degree of anterograde transfer among the TDP-43 constructs. Like our previous experiments, we also reversed the donor/acceptor pairs (1– 105 expressing acceptor cells displayed the least amount of anterograde transfer (full-length td-tomatoTDP431– 414 proteins transferred toward construct 101– 105 acceptor cells) in this experimental paradigm, therefore skewing the proportion of transfer events toward retrograde (acceptor cell to donor cell) in this circumstance. Taking Using this Qdot-800 coculture system, we determined that, as a proportion of all TDP-43 transfer events, the majority of TDP-43 is transferred from donor to acceptor cell (“anterograde”), regardless of which TDP-43 fragment was examined . As in For pairs . Much lior pairs , construWe have previously used this cellular coculture model to show that Aβ and α-synuclein can be transferred between cells via EVs . Given tExtracellular vesicles were isolated from the conditioned media of each TDP-43 construct–expressing cell line using the differential ultracentrifugation method. The presence of EVs in the isolations was first confirmed by the detection of flotillin-1 by Western blot, and the preparations were then analyzed for TDP-43 . Because1– 414, which expresses a C-terminal td-tomato tag) . As expected, td-tomatoTDP431– 414 was not detected here as it does not express GFP. Construct 101– 105 shows very little if any staining at the appropriate size (≈39 kDa). The absence of construct 101– 105 in the EV-enriched fraction could be related to its hyperlocalization to the nucleus (257– 414 was not detected in EVs at the expected molecular weight (≈42 kDa). Construct 5257– 414 lacks the TDP-43 NLS and the NES and displayed similar levels of protein transfer compared to construct 101– 105 . Conversely, construct 61– 314 was detected abundantly in EVs, with a prominent smear appearing at the predicted weight, ≈60 kDa, as well as a fainter band at ≈120 kDa and a fainter band still greater than 150 kDa. We postulate that these bands correlate to construct 61– 314 singlets, doublets, and triplets, respectively. The full-length TDP-43–expressing cell line WT-TDP-GFP1– 414 also demonstrated a significant propensity for incorporation into EVs, showing a prominent band at the expected molecular weight of ≈70 kDa. Interestingly, the two most readily TDP-43 proteins incorporated into EVs (construct 61– 314 and WT-TDP-GFP1– 414) were also the two constructs that demonstrated the highest amount of protein transfer  . GFP was nucleus , likely  nucleus . Likewistransfer . CD63–GFtransfer , but alstransfer .The isolated EVs were then added to WT RA differentiated cells 24 h before analysis by flow cytometry. In this circumstance, acceptor cells were differentiated with RA without ECM gel, to eliminate the possibility that EV motility would be hindered by the extracellular gel matrix. Remarkably, despite the detection of TDP-43 within EVs generated by these cells, analysis of the acceptor cells indicated that no appreciable protein transfer had occurred for any of the constructs . A visua1– 414 was used as a donor cell and the TDP-43 fragments assumed the role as acceptor  are packaged into EVs more favorably. This hypothesis, that the N-terminus of TDP-43 is important for its packaging into EVs, coincides with the observations that the N-terminus was related to increased protein transfer, alluding to the importance of the TDP-43 N-terminus in this system. Further, these data indicate that transfer of TDP-43 and TDP-43 truncated fragments occurs by a mechanism dependent upon cellular proximity.in vitro , display significantly reduced propensity for protein transfer. Previous studies allege that the RRM2 region of TDP-43, particularly AA216–257, was required for TDP-43 aggregation , but these constructs proved to be highly lethal, where transfection with each of these plasmids led to cell death within 72 h. While we were unable to create stable cell lines with these constructs, we were able to perform transfer studies by transient transfection and confirmed that they also successfully transferred their proteins to acceptor cells, but given the rarity of the construct AA51–414, construct 3170– 414 and construct 4216– 414 fragment–expressing cells surviving the transfection, coculture, and FACS process, we did not include these data.We show that construct 6regation , whereasregation . Constru1– 105 would transfer most efficiently; however, we observed that it is comparable to the rate by which constructs 286– 414 and 5257– 414 were transferred. It is likely that, because this protein fragment is so tightly localized to the nucleus by the presence of the NLS while also lacking the NES, this impedes its ability to transfer with greater efficiency. This is further exemplified by the equitable levels of transfer between the aforementioned construct 101– 105 and construct 5257– 414, which lacks both the NLS and NES, and is found primarily in the nucleus. In a previous report, the extreme N-terminus (AA1–10) was found to be most crucial for the native function of TDP-43, which requires homodimerization, as well as a necessity in the formation of TDP-43 aggregates (86– 414 and 5257– 414) had diminished protein transfer propensity, but also that these lines did not form punctate structures. In contrast, construct 61– 314 but also the full-length TDP-43 cell lines displayed the highest degree of protein transfer between cells, as well as the spontaneous formation of puncta in donor and acceptor cells in all scenarios. Taken together, we suggest that these data signify the importance of the N-terminus, presumably AA1–50, as the region most involved in promoting TDP-43 transfer, whereas preservation of the C-terminus (AA315–414) inhibits TDP-43 transfer.Because the C-terminal fragments all resulted in significantly diminished TDP-43 protein transfer, it would be expected that construct 10gregates . We obse1– 314 and the full-length WT-TDP-GFP1– 414) also displayed a higher degree of incorporation into EVs. However, ultimately EVs were not shown to be an efficient mechanism of transfer of fragmented or full-length TDP-43. In concordance with these data, when donor cells were physically separated from acceptor cells but shared a common extracellular environment, TDP-43 transfer was similarly abolished, in accordance with previous reports  donors were provided by the brain bank at Uppsala University, Sweden. The collection and use of the tissue was approved by the Regional Ethical Committee in Uppsala, Sweden . The patients/participants provided their written informed consent to participate in this study.CS and MH: study concept and design, interpretation of data, and manuscript preparation. CS and VS: experimental work. CS: analysis of data. All authors critically read and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Zea mays L.) production. Therefore, using amplicon sequencing, we aimed to clarify how AMF communities and their diversity in maize roots vary under different cover cropping systems and two types of tillage (rotary and no tillage). Two kinds of cover crops (hairy vetch and brown mustard) and fallow treatments were established with rotary or no tillage in rotation with maize crops. Tillage and no tillage yielded a set of relatively common AMF operational taxonomic units (OTUs) in the maize crops, representing 78.3% of the total OTUs. The percentage of maize crop OTUs that were specific to only tillage and no tillage were 9.6% and 12.0%, respectively. We found that tillage system significantly altered the AMF communities in maize roots. However, the AMF communities of maize crops among cover cropping treatments did not vary considerably. Our findings indicate that compared with cover cropping, tillage may shape AMF communities in maize more strongly.Our understanding regarding the influence of intensive agricultural practices, including cover cropping and tillage, on communities of arbuscular mycorrhizal fungi (AMF) is lacking. This would prove to be an obstacle in the improvement of current maize ( In general, conservation tillage can improve soil aggregation, increase the amount of soil organic carbon in the surface layer, and reduce erosion3. Moreover, conservation tillage enhances soil microbial diversity and the abundance of beneficial functional soil microorganisms4. The soil microbial communities stimulated by conservation tillage can play important roles in soil aggregation, soil carbon sequestration, and soil nutrition; improve water use efficiencies; and influence crop yields6.Growing cover crops and practicing conservation tillage are agricultural practices worldwide that have been recommended to promote soil fertility. In particular, Williams 7, enhances soil organic matter8, inhibits weed establishment9, and increases the abundance and activity of arbuscular mycorrhizal fungi (AMF)10. Indeed, crops with AMF have a greater capacity to take up phosphorus (P), zinc (Zn), and water10 than crops without AMF. This greater capacity generally results in robust crop growth under conditions of limited nutrient availability or drought. AMF also contribute to increasing plant resistance against pathogens and help make crops generally healthier13. AMF also contribute to crop health indirectly because of their effects on soil processes in agricultural settings15. In particular, they can contribute to soil health, soil aggregate formation, and soil stability by increasing the soil’s nutrient cycling and organic matter content17. However, certain agricultural practices have negative influences on AMF abundance and functions19. For example, conventional tillage systems, including chisel plowing, rotary tillage, and disc harrowing, can disrupt the AMF hyphal network20, inhibit AMF development and decrease AMF abundance in soil21. Fallowing during the winter also decreases the AMF abundance in soil and reduces the benefit of AMF on crops23. In contrast, no-tillage or reduced tillage systems can increase the abundance and hyphal network of AMF in soil24. Growing cover crops as pre-crops can increase indigenous AMF abundance in soil and root colonization by AMF, thus improving the growth and yield of subsequent crops27. Furthermore, the introduction of mycorrhizal pre-crops combined with a no-tillage system increases early stage maize (Zea mays L.) growth and AMF colonization to a greater extent than that of mycorrhizal pre-crops combined with a rotary-tillage system28. Thus, combining cover crop systems with conservation tillage may provide many benefits that improve subsequent crop performance.The practice of cover cropping potentially reduces soil and wind erosion29. Increased AMF richness in agricultural ecosystems has been suggested to improve crop performance30, while co-inoculation of AMF taxa belonging to different families improves plant biomass and mineral nutrition more efficiently than mono-inoculation33. In terms of agricultural management practices, tillage35 and cover cropping36 can alter the AMF community structure and diversity in soil and roots. For instance, Oehl and Koch37 reported AMF spore communities in conventional tillage are distinct from those in no-tillage systems in a vineyard farm, based on AMF spore morphology. Morimoto et al.38 showed that AMF communities in the roots of subsequent soybean crops differed, depending on their rotation with winter wheat or fallow. Other studies have shown that AMF communities can be shaped in subsequent crops, and the effect of the identity of the host crop is stronger than that of the cover crops40. Aside from these conflicting results, most other studies have investigated only the impact of cover cropping on AMF communities in subsequent crops40 and there is a lack of information about the impacts on AMF communities in subsequent crops under different agricultural management systems, such as the combination of cover cropping with tillage systems. Given our lack of knowledge regarding AMF communities in combined cover crop rotations with different tillage systems, it is imperative to understand how combining cover cropping with different tillage systems change AMF communities in subsequent crops using next-generation sequencing techniques .Moreover, there is clear evidence that AMF community structure and diversity improve plant growth performanceThe AMF communities of subsequent crops in cover cropping combined with rotary tillage are distinct from those in cover cropping combined with no tillage.The AMF communities of subsequent crops in a rotary-tillage system will have relatively more taxa that can tolerate stress in the presence of disturbance compared to those in a cover cropping system combined with no tillage.It is currently unclear which factor, cover cropping or tillage, has a greater influence in the shifts in the AMF communities in the roots of subsequent crops under cover crop rotational systems. Little is also known about how the combination of cover cropping with different types of tillage (rotary tillage and no tillage) drives shifts in the AMF communities in the roots of subsequent crops. Knowing whether such management affects the soil status and its connections to AMF taxa may help to determine the proper practices under specific agricultural settings. We hypothesized that:45. Thus, we used this technique to investigate how and whether AMF communities in the roots of maize crops change in different types of cover cropping systems combined with rotary or no tillage.Illumina amplicon sequencing is a commonly used and helpful technique for AMF community analysisVicia villosa Roth., var. Fujiemon) and brown mustard (Brassica juncea (L.) Czern & Coss., var. Karajin)] and one bare fallow treatment with rotary or no tillage as our experimental design . We combined two different cover crops [hairy vetch  randomly collected using a core sampler . On May 17, 2017, we pooled the soil samples into one composite soil sample. The roots of maize crops were collected at the stage of eight fully emerged leaves (V8 stage) on July 3, 2017. We collected maize root samples from nine plants  per plot. After collecting the root samples, we stored them at −80 °C for subsequent staining and DNA extraction. The roots were stained with a 3,3′-diaminobenzidine (DAB) solution3-N) content was measured using a LAQUA Twin nitrate meter . Extractable P (available P) from all soils, regardless of tillage management, was obtained as previously described48 and measured using molybdenum blue method at 710 nm using a UV-1700 Spectrophotometer . Soil acid phosphatase (ACP), alkaline phosphatase (ALP), and β-glucosidase activities in the soils were measured according to Ishii and Hayano49 and Hayano50.Soil biochemical properties were measured after cover cropping. We analyzed soil pH (soil:water ratio of 1:2.5 w/v) with a digital pH and conductivity meter , and soil nitrate-nitrogen  region using a previously described PCR method27. We selected the primer pair AM151/NS3152 for the first PCR to amplify a partial region of the 18S rDNA of AMF taxa. The first PCR was performed in 10-μl reactions, each consisting of 2 × Platinum™ Green Master Mix (Thermo Fisher Scientific Inc), 1 µl of template DNA, and 0.4 μM of AM1/NS31 primers. For the second PCR procedure, we diluted the amplicons from the first PCR 10-fold and used these amplicons as a template for the AMV4.5NF/AMDGR53 primers that are attached via the Illumina MiSeq adapter sequences. The second PCR was performed in 10 μl reactions comprising 1 µl of template DNA, 2 × Platinum™ Green Master Mix, and 0.3 μM of AMV4.5NF/AMDGR primers. Then we added both the Illumina MiSeq adapter sequences and an 8-bp barcode sequence as an index to distinguish each PCR amplicon.We froze 100 mg of fresh maize roots from all plots and retrieved total genomic DNA from the roots using the DNA suisui-P kit et al.54. Briefly, we washed and cleaned the amplicons from the second PCR before amplicon sequencing. These products were paired-end (PE) sequenced (2 × 300 bp) using an instrument of Illumina MiSeq amplicon sequencing at the Bioengineering Lab Co., Ltd. We performed the processing of sequence reads using QIIME version 1.9.155. The PE reads were truncated at any site that received an average quality score of <20 over a 40-bp sliding window, and truncated reads shorter than 40 bp were discarded using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html). We assembled the PE reads through their overlapping sequences that had a minimum overlap length of 10 bp and we deleted the reads that could not be assembled. We used Fast length adjustment of short reads (FLASH) ver. 1.2.11 to assess the clean sequences. Chimeric sequences were removed using UCHIME in USEARCH ver. 10.0.240. We then utilized the BLAST function in the MaarjAM database (https://maarjam.botany.ut.ee/) to determine and group the operational taxonomic units (OTUs) at 97% similarity. Additional taxonomic assignment was based on phylogenetic relationships. Representative sequences were aligned with known AMF taxa from NCBI Genbank and a neighbor-joining phylogenetic tree with 1000 bootstrap replicates was created using MEGA 756 . Before molecular bioinformatic analysis, we resampled according to the lowest number of reads to equalize the assessment among all treatments regardless of Illumina amplicon read depth. After rarefaction analysis, we calculated the Hill number 0D as OTU richness (q = 0), the Shannon index , and the Simpson index  from data on the diversity of AMF communities using the “renyi” function58 of the vegan package59. Additionally, to test whether AMF OTUs were significantly related to rotary tillage and no tillage with cover cropping, we performed a species indicator analysis. We also estimated values of indicator species using “multipatt” function60 in the indicspecies package to test for statistical significance of the highest OTU association in each plot.Root staining data reflecting AMF colonization was transformed into the arcsine-square root values for normalization. The differences in the mean values of each parameter among tillage and cover cropping systems were evaluated by Tukey’s test and then two-way analysis of variance (ANOVA) was conducted using the vegan package in R.3.6.159. Goodness-of-fit (R2) for measured factors fitted to the db-RDA ordination of the AMF communities were calculated using the “envfit” function in the vegan package with P-values based on 999 permutations59. A permutational multivariate analysis of variance (PERMANOVA) was performed with 9999 permutations using the “adonis”59 function in the vegan package to investigate if AMF communities differed significantly in tillage management and cover cropping. In addition, a multinomial species classification method61 using the package vegan were introduced to recognize “specialist OTUs” and “generalist OTUs” for tillage or no tillage.To demonstrate the variations in the AMF communities in maize crops among tillage management and cover cropping, we performed distance-based redundancy analysis (db-RDA) in the 3-N content in soil was significantly influenced by cover cropping, but soil pH was not affected by either tillage or cover crop management  .We detected a total of 148 AMF OTUs in the roots of maize Fig. . Regardlly) Fig. . The relGlomus and uncultured Glomeromycotina taxa in the rotary-tillage system  and brown mustard  significantly affected the AMF communities  did not affect the AMF communities of maize crops. Among the no-tillage treatments, only brown mustard  significantly affected the AMF communities . However, cover crops did not significantly affect the AMF communities in no-tillage treatments .Among rotary-tillage treatments, bare fallow   and no tillage  had significant effects on the AMF communities in maize crops. Moreover, PERMANOVA shows that tillage management rather than cover cropping or the interaction between tillage management and cover crop type significantly affected the AMF communities in maize crops .The db-RDA also found that tillage management alters the AMF communities of maize crops, while only brown mustard pre-crop significantly affected the AMF communities (Monte-Carlo permutation test: Glomus and Rhizophagus) and Claroideoglomeraceae (Claroideoglomus) are the main genera in maize roots  rDNA of AMF are strongly biased toward Glomeraceae. Because our results show that Glomeraceae are dominant in maize roots regardless of tillage and cover cropping, further studies should carefully select primer pairs that avoid potential PCR and primer bias as well as technical sequencing errors.Our findings indicate that representatives of Glomeraceae  may be the key in shaping AMF communities in roots.The beneficial impacts of cover cropping may differ among the AMF communities, and the question of whether cover crop type changes the AMF communities in the roots of maize crops remains to be investigated. Higo ngs Fig. . However74. Moreover, tillage management induced more changes to the AMF communities in maize roots than the type of cover crops. However, the questions of how and whether the level of tillage intensity alters the AMF communities in agricultural settings remain unanswered. Future studies on the interactions between the AMF communities and agricultural settings should focus on how tillage intensity affects the AMF communities in maize crops rotated with cover crops.In addition, AMF communities in maize crops varied distinctly, indicating that tillage practice altered the AMF communities in maize crops Fig. , in part76. In general, cover crop residues that have been incorporated into the soil surface by conventional tillage systems release allelopathic substances that can inhibit weed germination, establishment, and growth77. In contrast, in conservation tillage systems (including no tillage and reduced tillage), the nutrients from surface-broadcasted fertilizers and surface-applied crop residues accumulate and concentrate in the topsoil, where their movements are reduced78. In no-tillage systems, however, crop residue decomposition rates and nutrient release into the soil are slow compared to conventional tillage systems, including rotary tillage and chisel plow79, which may influence the soil microbial community structures80. These combined findings indicate that the differences between the effects of rotary and no-tillage management systems on the AMF communities in the roots of maize crops can be explained by cover crop residues releasing allelopathic substances and nutrients due to enhanced decomposition rates , ambient soils (20 °C), and warmed soils (26 °C)83. Heinemeyer et al.84 reported that shading and soil temperature changed AMF communities in roots, with some AMF taxa being replaced  by others and some disappearing throughout winter, summer and autumn. Higo et al.85 also reported on seasonal variation, finding a distinct difference between winter and spring AMF communities in the roots of winter cover crops. However, we did not investigate the effect of tillage systems and cover crop residues on soil temperature fluctuations. Future research should investigate how and whether AMF communities respond to environmental changes due to cover cropping and tillage management systems.Moreover, tillage can affect soil temperature by changing soil surface conditionsTillage management may be more important than cover cropping in shaping the AMF communities in maize crops. In addition, various kinds of cover cropping systems may produce various types of effects on the AMF communities in maize crop roots. The synergistic effects of tillage and cover cropping are partially responsible for shaping the AMF communities. Knowledge in this connection should be valuable in future experiments. This is because variations in the AMF communities in roots may influence crop growth performance in cropping systems with tillage management. However, we still need to clarify how the combination of tillage intensity and cover crop type is associated with individual AMF taxa in agricultural settings. Such an investigation will provide beneficial information on the appropriate cover crop selection and tillage practice for improving the functions of AMF taxa in cropping systems.Supplementary information 1.Supplementary information 2.


Computational studies of the Co-catalyzed remote nitration of 8-aminoquinolinamides have found the mechanism to operate through an unexpected high-spin induced remote radical-coupling. via a subtly different mechanism despite having a similar chelate environment.Previously, an unexpected Co-catalysed remote C–H nitration of 8-aminoquinolinamide compounds was developed. This report provided a novel reactivity for Co which was assumed to proceed through the mechanistic pathway already known for analogous Cu-catalysed remote couplings of the same substrates. In order to shed light into this intriguing, and previously unobserved reactivity for Co, a thorough computational study has now been performed, which has allowed for a full understanding of the operative mechanism. This study demonstrates that the Co-catalysed remote coupling does not occur through the previously proposed Single Electron Transfer (SET) mechanism, but actually operates through a high-spin induced remote radical coupling mechanism, through a key intermediate with significant proportion of spin density at the 5- and 7-positions of the aminoquinoline ring. Additionally, new experimental data provides expansion of the synthetic utility of the original nitration procedure towards 1-naphthylpicolinamide which unexpectedly appears to operate  However, the already known rich mechanistic diversity of Co in the field of C–H functionalisation makes this metal stand out as an exciting candidate for the basis of novel protocols.Functionalisation of quinolines beyond traditional C2 modification has recently become a topic of increasing interest10One novel approach to the functionalisation of 8-aminoquinolinamides has been the remote (non-proximate) C–H bond chlorination catalysed by Cu at the C7 position, which was originally reported by Stahl/Ertem and co-workers in 2013 .11 Non-p14tert-butyl nitrite (TBN) as nitro source  source” in the mechanism. Given the use of AcOH as solvent, the experimental conditions would allow for extensive counter-ion ligand exchange, leading to a number of potential Co(ii) species. Relative free energies for ligand exchange 2 over Co(OAc)2 in the original protocolin situ formed HNO3 in an acidic environment.The initial approach in this study was to identify the nature of the starting “Co, where this report revealed the highly complex mechanisms operative in Co-catalyzed C–H activation processes.After this initial study on catalyst species we embarked on full elucidation of the reaction mechanism. As a result of the moderate to large size of the catalytic system being studied, DFT calculations would traditionally be the method of choice. However, due to the potential for different spin states to be involved in the mechanism, which pose a significant challenge for DFT based methods, the recently implemented open shell DLPNO-CCSD(T) method was utilised.in situ formed tBuO˙ radical leading to the radical of the 8-aminoquinolinamide ligand, 1L. Coupling 1L to the [Co(AcO)2] pre-catalyst forms the initial catalytic species 1Int1·L. This species can exist in three possible spin states; low spin singlet , medium spin triplet  and high spin quintet . With the DLPNO-CCSD(T) method the high spin quintet state (5Int 1·L1) proved to be the ground-state species, which is in full agreement with previous work from Chen and co-worker.1Int 1·L1 and 3Int 1·L1 are Co(iii) complexes whereas the 5Int 1·L1 is actually a high-spin quartet Co(ii) species with the radical still residing on the quinoline moiety of the ligand. Cumulative spin densities on the aminoquinoline ligand, calculated using DLPNO-CCSD/def2-tzvp,1Int 1·L1, 3Int 1·L1 and 5Int 1·L1 are 0, 0.18 and 0.72, respectively. 5Int 1·L1. A similar spin population distribution was previously observed by Chen and co-worker leading to the description as a quartet Co(ii) ferromagnetically coupled with a ligand radical.20The first step of the reaction is hydrogen abstraction by the 1Int 1·L1 and 3Int 1·L1 being Co(iii) whereas 5Int 1·L1 being high spin Co(ii) with an associated radical ligand, based on the spin density analysis.Topological analysis using Quantum Theory of Atom in Molecules (QTAIM)2 radical coupling at the 5- and 7-positions starting from 5Int 1·L1 lie at 4.7 and 5.2 kcal mol–1 respectively  methods,–1 at most and therefore as a result we are confident this reaction proceeds via the proposed high spin pathway.The transition states for the remote NOectively . This lo1Int 1·L1. The first excited state transition, corresponding to the excitation of an electron from the 8-aminoquinolinamide ligand to the Co(iii) centre, is found to be in the range of 37–43 kcal mol–1, depending on the choice of DFT functional and amount of HF exchange included (see ESI for full detailsii) complex.1Int 1ES·L1, with the hybrid functional PBE0 provides a value of 24.4 kcal mol–1 above 1Int 1·L1. Although not directly comparable due to the differing methodologies used, both the transition energy and the optimised excited state complex is significantly higher in energy than the transition state on the proposed high-spin mechanism starting from the quintet ground state, 5Int 1·L1. As a result of the requirement for a coupling transition state after 1Int 1ES·L1, this precludes this mechanism on the grounds of it being energetically unfeasible.To ensure the previously proposed intramolecular Singlet Electron Transfers (SET) mechanism was not involved in the reaction, LR-TD-DFT  calculations were performed on 5Int 1·L1 results in the formation of the intermediate complex, 4Int 2·L1. This intermediate is 8.0 kcal mol–1 lower in energy than the preceding starting complex. The lower energy pathway leading to nitration at the 5-position forms 4Int 2·LA1 which is 3.8 kcal mol–1 more stable than the corresponding 7-substituted product (4Int 2·LB1), and 16.6 kcal mol–1 more stable than the equivalent low spin doublet mechanism (2Int 2·LA1). A number of different pathways for the deprotonation of 4Int 2·L1 at the 5- and 7-positions were explored. Addition of [Co(AcO)3]– to 4Int 2·L1 forms 5Int 3·L1, this step is exergonic by 9.7 kcal mol–1. Cobalt assisted deprotonation by acetate anion has a barrier of 9.8 kcal mol–1, leading to the final intermediate 4Int 4·L1]––– furnishes the chelate species of the final product, which can then undergo proto-demetallation by acetic acid to reform the initial Co(OAc)2 catalyst, releasing the final nitrated 8-aminoquinolinamide product.As a result of this new in-depth study, in adjustment to our previously proposed SET mechanism for the Co-catalysed remote nitration of 8-aminoquinolamide substrates, we now propose the mechanism shown in 2L,  with the quintet (S = 2) and triplet (S = 1) states being 2.2 and 3.7 kcal mol–1 higher in energy respectively  calculations suggest the ground state of the 1-naphthylpicolinamide complex is the singlet (ectively . TDDFT c5Int 1·L2 showed a significant proportion of density at the observed reactive 2- and 4-positions of the 1-aminonaphthyl moiety (via either a low spin (S = 1/2) or high spin (S = 3/2) pathway. As with the 8-aminoquiolinamide mechanism the high spin pathway, through 4TS 1-2·L2, is significantly more favourable. This suggests a Multi-State Reactivity (MSR) mechanism with crossing between potential energy surfaces,4Int 2·LA/B2, are approximately 25 kcal mol–1 more energetically stable compared to the equivalent low spin intermediate, 2Int 2·LA/B2. Interestingly the observed substitution at the 4-position is kinetically favoured, while the 2-position is the thermodynamic intermediate. This can be accounted for by the additional long range hydrogen bonding (2.45 Å) between the hydrogen at the 2-position of the nitrated naphthyl moiety and the Co bound acetate. The calculated higher energy barriers for 1-naphthylpicolinamide compared to 8-aminoquinolinamide agrees with the experimental requirement for more forcing reaction conditions . Increased spin density on the Co and decreased spin density on the 1-aminonaphthyl moiety further support this decrease in reactivity . Overall, the distinct mechanistic pathways shown in this work provide a complimentary tool for the remote C–H functionalisation of both 8-aminoquinolinamides and 1-naphthylpicolinamides. This new mechanistic understanding should now inspire others to develop new synthetic protocols for remote functionalisation using Co-catalysis.In summary, we have studied the mechanism of the remote nitration of 8-aminoquinolinamides catalysed by Co using the robust DLPNO-CCSD(T) methodology and found there to be a significant difference in mechanism to the well-studied analogous Cu-catalysed remote functionalisation protocols of 8-aminoquinolinamides. The results reported within this work identify an unusual mechanism which does not pass through the initially proposed SET process, but instead, the reaction takes place There are no conflicts to declare.Supplementary informationClick here for additional data file.


Amyotrophic lateral sclerosis (ALS) is a terminal late-onset condition characterized by the loss of upper and lower motor neurons. Mutations in more than 30 genes are associated to the disease, but these explain only ~20% of cases. The molecular functions of these genes implicate a wide range of cellular processes in ALS pathology, a cohesive understanding of which may provide clues to common molecular mechanisms across both familial (inherited) and sporadic cases and could be key to the development of effective therapeutic approaches. Here, the different pathways that have been investigated in ALS are summarized, discussing in detail: mitochondrial dysfunction, oxidative stress, axonal transport dysregulation, glutamate excitotoxicity, endosomal and vesicular transport impairment, impaired protein homeostasis, and aberrant RNA metabolism. This review considers the mechanistic roles of ALS-associated genes in pathology, viewed through the prism of shared molecular pathways. Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease (MND), with an estimated ~223,000 patients being affected globally in 2015 . ApproxiFused in Sarcoma (FUS), Superoxide dismutase (SOD1), TAR DNA-binding protein 43 (TDP-43), and Chromosome 9 open reading frame 72 (C9orf72) . utgrowth , while md miR192 , which ad miR192  or senesd miR192 .Altogether, these findings are consistent with defective miRNA processing in ALS patients, which may affect downstream pathways with an impact on motor neuron survival.FUS and TDP-43 can increase the persistence of stress granules in the cytoplasm, resulting in a possible toxic gain-of-function [A232E and VCPR155H mutations showed a constitutive appearance and accumulation of stress granules containing TDP-43 [In response to stressful conditions, RNA granules, also known as stress granules, are generated and can recruit FUS and TDP-43 ,308. Mutfunction  by inhibfunction ,309. Mutfunction ,311. Strg TDP-43 . Concordg TDP-43 .C9orf72 patients, stress granules are also involved in the sequestration of proteins required for effective nucleoplasmic transport, such as RAN GAP [C9orf72 ALS cells is controverisal. Indeed, while some studies observed that newly formed DPRs such as poly-PR can bind to nuclear pore transporters, thereby impairing the subsequent translocation of molecules [In  RAN GAP , or impo RAN GAP ,41. The olecules , other solecules . Howeverolecules .Over 150 years have passed since ALS was first reported by Charcot, and still, the etiology of the disease remains elusive. Although research is progressing and genetic studies continue to identify novel gene mutations in familial cases of ALS , many quBy detailing, as has been done in this review, the molecular events of the various pathways that are implicated in ALS, it becomes clear that these pathways can be linked to each other—in some cases, with one leading to another. For example, disrupted axonal transport can lead to an accumulation of nonfuctional mitochondria, while ATP deficiency and increased oxidative stress may damage proteins and DNA, which, in turn, could exacerbate the disruption of cellular homeostasis, leading to motor neuron death . These p


Neuronal cytoplasmic inclusions containing TAR DNA-binding protein 43 (TDP-43) are a neuropathological feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer’s Disease (AD). Emerging evidence also indicates that systemic inflammation may be a contributor to the pathology progression of these neurodegenerative diseases.E. coli lipopolysaccharide (LPS) administration challenge for 2 weeks to mimic a chronically altered low-grade systemic inflammatory state. Mice were then subjected to neurobehavioral studies, followed by biochemical and immunohistochemical analyses of the brain tissue.To investigate the role of systemic inflammation in the progression of neuronal TDP-43 pathology, AAV9 particles driven by the UCHL1 promoter were delivered to the frontal cortex of wild-type aged mice via intracranial injections to overexpress TDP-43 or green fluorescent protein (GFP) in corticospinal motor neurons. Animals were then subjected to a low-dose (500 μg/kg) intraperitoneal In the present study, we report that elevated neuronal TDP-43 levels induced microglial and astrocytic activation in the cortex of injected mice followed by increased RANTES signaling. Moreover, overexpression of TDP-43 exerted abundant mouse immunoglobulin G (IgG), CD3, and CD4+ T cell infiltration as well as endothelial and pericyte activation suggesting increased blood-brain barrier permeability. The BBB permeability in TDP-43 overexpressing brains yielded the frontal cortex vulnerable to the systemic inflammatory response following LPS treatment, leading to marked neutrophil infiltration, neuronal loss, reduced synaptosome-associated protein 25 (SNAP-25) levels, and behavioral impairments in the radial arm water maze (RAWM) task.These results reveal a novel role for TDP-43 in BBB permeability and leukocyte recruitment, indicating complex intermolecular interactions between an altered systemic inflammatory state and pathologically prone TDP-43 protein to promote disease progression. TARDBP gene, is a 43-kDa nuclear protein that belongs to the heterogeneous nuclear ribonucleoproteins (hnRNPs) family that binds RNA [TAR (transactive response) DNA-binding protein 43 kDa (TDP-43), encoded by the inds RNA . Severalinds RNA . For exainds RNA . In 2006inds RNA , 8. In sinds RNA . Furtherinds RNA . And mosinds RNA .C9ORF72 mutation carriers [E. coli lipopolysaccharides (LPS)-induced inflammation can exacerbate pathological accumulation of disease-associated proteins, including tau and β-amyloid [Notably, many patients suffering from neurological disorders present a persistently heightened systemic inflammatory state referred to as low-grade systemic inflammation which is a risk factor for morbidity and mortality in the elderly . Older pcarriers . High lecarriers . ALS blocarriers –18. Furtcarriers . Combine-amyloid , 21. Mor-amyloid . TDP-43 -amyloid , 24.E. coli LPS, in the progression of TDP-43 proteinopathies in neurons of the frontal cortex, a brain region affected by many of these diseases. Through neurobehavioral, biochemical, and histochemical assessments, our findings suggest that elevated TDP-43 levels render the brain vulnerable to the systemic immune response during inflammation by promoting BBB permeability and impairing components of the neurovascular unit.Although TDP-43 is linked to several diseases that present an altered systemic inflammatory response , the relationship between the two has not been fully explored. Here, we investigate the effect of chronic low-grade systemic inflammation, using TDP-43 (FLAG-tagged) or GFP were cloned into the Hind III and Sac I sites of the rAAV vector pTR-MCS under the control of the ubiquitin carboxy-terminal hydrolase L1 (UCHL1) promoter. rAAV serotype 9 viruses were generated using pAAV9 and pXX6 in Hek293 cells as described previously .12 viral particles/mL) at a constant rate of 2.5 μL/min through a CED needle in each placement. The needle was kept in place for 1 min following the injection and then was raised slowly. Either rAAV9-GFP or rAAV9-TDP-43 under the UCHL1 promoter was injected bilaterally in the cortex of wild-type mice at 10.5 months (n = 6 mice/group). Virus transduction through anterior cortical layer V and IV neurons was determined based on cell type and boundaries of cortical layers I–VI following Allen Mouse Brain Atlas (Institute 2011)  were bred in the vivarium of the Byrd Alzheimer's Institute, USF Health. The injection procedure was performed using the convection-enhanced delivery method as described previously . BrieflyAnimal procedures were performed in accordance with the recommendations of the National Research Council’s “Guide for the Care and Use of Laboratory Animals” and were previously approved by the University of South Florida Institute of Animal Care and Use Committee (IACUC).E. coli lipopolysaccharide (LPS) treatment regime as previously published [E. coli lipopolysaccharides  every 3 days. This way, four treatment groups were created: GFP AAV9 , GFP AAV9 (i.p. LPS), TDP-43 AAV9 , and TDP-43 AAV9 (i.p. LPS) (n = 6 mice/group).To model low-grade systemic inflammation, we challenged the mice with a modified ublished . BrieflyMouse behavior was performed during the last week of LPS treatment prior to sacrifice. All behavioral tests are blinded to genotype and treatment to the investigator.Motor coordination and learning performance were assessed by placing mice onto an accelerating circular rod . The time until falling was recorded for each mouse. Mice were given four trials each day for 2 consecutive days. Performance in the first trial of day 1 serves as a measure of baseline locomotor coordination, whereas improvement in the latency to fall within each day (short-term learning) and between days (long-term learning) serve as measures of motor learning.Grip strength was measured using a grip strength meter . Mice were held by the base of the tail and allowed to grasp the bar of the meter with their front or back paws. The mouse was then pulled gently backward away from the bar slowly to allow the mouse to develop a resistance against the pulling force. The peak force given by the meter was recorded from their front and back paw measurements.The radial arm water maze consists of a 1-m pool with six swim paths radiating out of an open central area, with a hidden escape platform located at the end of one of the arms. On each trial, the mouse was allowed to swim in the pool for up to 60 s to find the escape platform. Incorrect arm entries or failure to select an arm for 15 s were counted as errors. For a given mouse, the platform was located in the same arm on each trial, but the start arms were varied for each trial so that mice rely upon spatial cues to solve the task. On day 1, mice were given 15 trials, each block consisting of three trials  alternating between a visible platform and a hidden platform. The following day, mice were given 15 additional trials (five blocks), all using a hidden platform. The goal arm location for sequential mice was different to avoid odor cues from revealing the goal arm. On the last day of testing, all animals were tested in the open pool task with a visible platform to ascertain their vision and ability to climb on the platform. To do so, the platform was elevated above the water surface and had an attached flag. Additionally, all visual cues were removed so that mice relied only on their sight to find the platform. Latency to find and ascend the platform was recorded (60 s maximum).Two months post intracerebral injection, mice were weighed, overdosed with a euthanizing solution containing pentobarbital, and perfused with 25 mL of 0.9% saline solution. Brains were collected following saline perfusion and were hemisected down the sagittal midline. One hemisphere was dissected and frozen on dry ice for biochemical studies. The second hemisphere was immersion fixed in 4% paraformaldehyde for 24 h and cryoprotected in successive incubations of 10%, 20%, and 30% solutions of sucrose for 24 h in each solution. Subsequently, the fixed hemispheres were frozen on a cold stage and sectioned in the horizontal plane (25 μm thickness) using a sliding microtome. Brain sections were stored in Dulbecco’s phosphate-buffered saline (DPBS) with 10 mM sodium azide solution at 4 °C for immunohistochemistry.2O2 in PBS, 15 min). Tissue samples were permeabilized  and incubated overnight in primary antibody for HRP-conjugated mouse IgG , CD3 , CD4 , Ly6B.2 , or biotinylated NeuN . Sections were rinsed in PBS, then incubated in corresponding biotinylated secondary antibodies for 2 h. Sections incubated with biotinylated NeuN proceeded directly to the enzyme conjugation step. Following incubation, tissue sections were rinsed in PBS and incubated with Vectastain Elite ® ABC kit  for enzyme conjugation. Finally, sections were developed using 0.05% diaminobenzidine, 0.5% Ni++, and 0.03% H2O2. Tissue sections were then mounted onto slides, dehydrated, and cover-slipped. Each immunohistochemical stain omitted some sections from primary antibody incubation to evaluate the nonspecific reaction of the secondary antibody. NeuN slides were counterstained with 0.05% cresyl violet.Six sections, representative of the brain, were chosen for histochemical analyses. For bright-field microscopy, floating sections from all animals were placed in multi-sample staining trays, and endogenous peroxidase activity was blocked , TDP-43 , Iba1 , CD11b , MHCII , GFAP , and CD45 . The next day, tissue sections were washed and placed in respective Alexa Fluor secondary antibodies  for 2 h. Then, tissue sections were washed, mounted, and cover-slipped using ProLong Gold anti-fade reagent (Thermo Scientific) and counterstained with Hoechst 33342 where indicated.Tissue sections were mounted on slides and dried overnight. The next day, sections were rehydrated in distilled water for 30 s then incubated in a solution containing 2% concentrated HCl and 2% potassium ferrocyanide for 15 min. Thereafter, sections were rinsed two times in distilled water for 30 s followed by rinsing in tap water for 5 min. Finally, slides were dehydrated by dipping 8 times in 95% ethanol and 8 times through two changes in 100% ethanol and cleared in xylenes 3 times for 5 min followed by cover-slipping using DPX.Tissue sections stained for CD4 and NeuN were imaged using the Carl Zeiss AxioImager.Z1 microscope  using a × 20 objective. Tissue sections stained for TDP-43, GFAP, Iba1, CD11b, and CD45 were imaged using the Zeiss LSM 880  at × 3 objective. GFAP, Iba1, CD11b, MHCII, and CD45 levels (integrated density) in tissue sections were quantified using ImageJ analysis software with six representative regions of interest per mouse . To determine the levels of extranuclear TDP-43, × 63 images from brain sections stained with TDP-43 and DAPI were converted to 8-bit and processed by Gaussian blur to remove background (radius = 50). Then a mask was created using the DAPI channel and subtracted from the TDP-43 channel to remove the nuclear signal. Finally, integrated density values were calculated from these images using a threshold of positive staining that was kept constant throughout the analysis. The number of neurons stained with NeuN and cresyl violet in the frontal cortex of mice was counted manually by a blind investigator in at least six 300-μm regions of interest per mouse around the site of injection. Ly6B.2, CD3, and CD4+ cells were counted manually in whole-brain sections from at least six sections per mouse used as representative.g for 30 min. The supernatant was used for biochemical analyses. The protein concentration of each sample was measured using BCA assay. To obtain insoluble TDP-43 fraction, pellets were washed in RIPA buffer to eliminate remaining soluble proteins and centrifuged at 100,000g for 30 min at 4 °C. Thereafter, the supernatant was discarded and urea buffer  was added to the pellet, sonicated, and centrifuged at 100,000g for 30 min at room temperature. Protein concentration was determined by Bradford assay. Lysates were separated by SDS-PAGE and transferred to a PVDF membrane. Human TDP-43 (Sigma), total TDP-43 (ProteinTech), GFAP , PSD95 , SNAP-25 , SNAP-23 , Syntaxin-1A , laminin (ab11575), ICAM1 , occludin , ZO-1 , claudin 5 (1:1000), VCAM (1:1000), caveolin , CD13 , PDGFRβ (Abcam), GAPDH , and actin  levels were assessed by addition of primary antibodies, species-appropriate secondary antibodies , and exposed to ECL for chemiluminescent detection or imaged using a LICOR machine.The frontal cortex and hippocampus of mice were homogenized in ice-cold RIPA buffer and sonicated for 30 s. Thereafter, the tissue homogenate was centrifuged at 38,000×The concentrations of eotaxin, IL-1α, Il-1β, IL-10, VEGF, MIP-1α, RANTES, and KC-GRO were measured using the mouse cytokine/chemokine panel in mouse frontal cortex tissue lysate . Briefly, the Bio-Plex Suspension Array System (Bio-Rad Laboratories) was calibrated using CAL2 with the high PMT setting of the Bio-Plex calibration kit, and standard sample preparation was performed according to the manufacturer’s directions. The filter plate was prewetted with wash buffer and vacuum-filtered before adding standard, control, or study samples to the appropriate wells. Mixed capture beads were then added to each well, and the plates were incubated overnight at 4 °C with shaking. After two washes, 25 μL of detection antibody was added to each well, incubated for 1 h at room temperature, and then treated with 25 μL of streptavidin-phycoerythrin for 30 min at room temperature. The plate was washed twice, and 150 μL of the Bio-Plex sheath fluid assay buffer was added to each well and read using the Bio-Plex Suspension Array System software (Bio-Rad Laboratories) per the kit instructions. The concentration of each analyte was calculated according to the standard curve.t test, one-way ANOVA or two-way repeated-measures ANOVA with Fisher’s Least Significant Difference (LSD), and Tukey or Bonferroni’s post hoc tests were used as detailed in the figure legends. Values were considered significant if p < 0.05. Graphs were generated using GraphPad Prism 8.0 analysis software. N-value in this study is depicted as the number of animals and indicated in the figure legends.Two-tailed Student’s E. coli lipopolysaccharide (LPS) treatment regimen as described in the “Methods” section and Fig. atlas.brain-map.org) allowed us to determine viral transduction throughout layers of the cortex  viruses to drive the overexpression of FLAG-tagged wild-type TDP-43 (TDP-43 AAV9) or GFP under the UCHL1 promoter in the frontal cortex 10.5-month-old wild-type mice for 2 months. UCHL1 is abundantly expressed in corticospinal motor neurons (CSMN) that become affected in TDP-43 proteinopathies which made its promoter ideal for our study –29. NextTo characterize the TDP-43 pathology achieved by AAV overexpression, we analyzed RIPA-soluble and urea-soluble fractions from homogenized frontal cortices. Western blotting revealed soluble and insoluble FLAG-TDP-43 levels were significantly increased, confirming the expression and formation of pathology upon TDP-43 overexpression Fig. a, b. MorVarious TDP-43 mouse models have reported muscle weakness, impaired motor learning, and deficits in learning and memory , 34. We Cognitive dysfunction is associated with neuronal loss and synaptic dysfunction , 36. At Thus far, our data suggested that low-grade systemic inflammation can promote TDP-43-induced neurodegeneration, resulting in cognitive impairment. Given that the deregulation of TDP-43 can induce neuroinflammation and increase neuronal death through NF-κB signaling , 44, we Given the presence of mononuclear CD45-positive cells, we surmised that TDP-43 overexpression could induce peripheral cell infiltration. Therefore, brain tissue sections were labeled with CD3 and CD4 receptor antigens, both found in T lymphocytes. As suspected, TDP-43 overexpression, regardless of systemic inflammation, led to marked increases in CD3+ Fig. a, b and Next, we measured the levels of a range of inflammatory cytokines in frontal cortex homogenates, similar to our recent report . Low-graThe etiology of several neurodegenerative diseases, including ALS, FLTD, and AD, remains elusive. Nevertheless, research has identified several mutations and proteins in the brain that accelerate disease progression and have become key pathological features, such as TDP-43. Additionally, further studies have consistently observed systemic inflammatory changes in these diseases, giving rise to the idea of, not only systemic inflammation as a trigger for neurodegeneration, but also an intrinsically altered immune system . With thThe radial arm water maze (RAWM) is generally known to test spatial learning, which is strongly tied to hippocampal function; however, we observed significant learning impairment in the cortical TDP-43 overexpressing mice subjected to LPS. In support of our findings, studies indicate that spatial memory is not strictly tied to the hippocampus as it is not affected following hippocampal insults in certain tasks –58, contTDP-43 overexpression increased IgG, as well as CD3+ and CD4+ T cell infiltration, which were associated with increased endothelial signaling since both ICAM1 and caveolin 1 facilitate leukocyte infiltration in the brain , 71. FurThe extravasation of T cells could exacerbate TDP-43 induced neurodegeneration. This is in agreement with studies showing that CD3+ T cell infiltration in the hippocampus promotes neuroinflammation and cognitive dysfunction in tau overexpressing models . InteresAlthough TDP-43 overexpression alone led to peripheral cell infiltration, low-grade systemic inflammation in TDP-43 AAV9 mice increased the infiltration of neutrophils into the brain through KC-GRO and MIP-1α signaling , 81. NeuWild-type TDP-43 neurotoxic effects have been extensively studied in various models of overexpression , 34, 53,Overall, our results uncover a novel role of TDP-43 in blood-brain barrier permeability and leukocyte recruitment which can compromise the brains of patients with TDP-43 proteinopathies and alter disease progression, especially in association with other inflammatory conditions. The data also shed light on TDP-43 as a factor explaining the BBB abnormalities observed in patients diagnosed with neurodegenerative diseases while also supporting a new body of research regarding the role of TDP-43 in alterations of the neurovascular unit.Additional file 1: Figure S1. TDP-43 oligomers and c-terminal fragments are not altered by TDP-43 overexpression. (A) Western blots of RIPA and urea-soluble brain homogenates probed for total TDP-43 at a high exposure to visualize oligomers and C-terminal fragments. (B) Quantification of oligomers and C-terminal fragments in the RIPA and urea-soluble fractions (n = 4 mice/group). Statistical analysis was carried out using a one-way ANOVA with Bonferroni post-hoc test.Additional file 2: Figure S2. TDP-43 overexpression increases astrocytic activation. (A) Representative images depicting GFAP levels (red channel) in the frontal cortex of mouse brain tissue. Scale bar = 20 μm. (B) Quantification of GFAP levels. Statistical analysis was carried out using a one-way ANOVA with Bonferroni post-hoc test . (C) Representative blots probed for GFAP, aquaporin 4, and actin. (D) Quantification of GFAP and (E) aquaporin 4 levels normalized to actin. Statistical analysis was carried out using a one-way ANOVA with Bonferroni post-hoc test .Additional file 3: Figure S3. Regional differences in LPS-driven neuroinflammation. (A) Representative blots probed for p-CamKIIβ, RGS14, and iNOS in hippocampal and frontal cortex tissue lysates. (B) Quantification of p-CamKIIβ, RGS14, and iNOS normalized to actin. Statistical analysis was carried out using one-way ANOVAs with Bonferroni post-hoc test (n = 4 mice/group).Additional file 4: Figure S4. TDP-43 overexpression induces pericyte activation and does not alter tight junction protein levels. (A) Representative blots probed for CD13, PDGFRβ, and GAPDH. (B) Quantification of CD13 and PDGFRβ levels normalized to GAPDH. Statistical analysis was carried out using a one-way ANOVA with Bonferroni post-hoc test . (C) Representative blots probed for laminin B, ZO-1, occludin, claudin 5, claudin 3, and actin. (D) Quantification of laminin B, ZO-1, occludin, claudin 5, and claudin 3 levels normalized to actin. Statistical analysis was carried out using a one-way ANOVA with Bonferroni post-hoc test (n = 4 mice/group)


Fluorinated graphene has a tunable band gap that is useful in making flexible graphene electronics. But the carbon–fluorine (C–F) bonds in fluorinated graphene can be easily broken by increased temperature or electron beam irradiation. Here, we demonstrate that the stability of fluorinated graphene is mainly determined by its C–F configuration. The double-sided fluorinated graphene has a much stronger stability than the single-sided fluorinated graphene under the same irradiation dose. Density functional theory calculations show that the configuration of double-sided fluorinated graphene has a negative and low formation energy, indicating to be an energetically stable structure. On the contrary, the formation energy of single-sided fluorinated graphene is positive, leading to an unstable C–F bonding that is easily broken by the irradiation. Our findings make a new step towards a more stable and efficient design of graphene electronic devices. Utilizing the superior properties of this two-dimensional (2D) material, such as the exceptionally high charge mobility2, high optical transparency3, excellent flexibility4, ultrahigh mechanical strength and thermal conductivity9, pristine graphene can be used in a wide range of applications. However, the special feature of zero bandgap in graphene has greatly limited its application as a semiconductor12. Consequently, it is important to open the bandgap of pristine graphene through the bandgap engineering and explore its utilization as a novel 2D semiconductor. Fluorination has been proved to be an efficient method to modify the electronic structures of graphene. The band gap in the fully fluorinated graphene (F/C ratio is 50%) can be opened up to 3.07 eV based on the theoretical calculation13. The rise of fluorinated graphene makes the flexible and transparent all-graphene-electronics possible14. By simply changing the fluorine/carbon (F/C) ratio of this atomic layer, the electrical characteristic of fluorinated graphene can be tuned from conductive, semiconductive to insulating. The electron beam lithography (EBL) and direct electron beam irradiation have been practically used to open the nanometer-sized conductive channels in the fluorinated graphene and fabricate the all-graphene-electronic devices16.Graphene has attracted intense attention since its discovery20. The conventional synthesis methods include chemical fluorination and physical exfoliation: the former includes direct gas fluorination21, plasma fluorination24, hydrothermal fluorination25, etc.; the latter includes sonochemical exfoliation27, modified Hummer’s exfoliation28, thermal exfoliation29, etc. The F/C ratio in the fluorinated graphene depends on the fluorine agents or exfoliation solvents, the reaction temperature and time. By using the same synthetic method, higher reaction temperature and longer reaction time will result in a higher F/C ratio.Synthesis of fluorinated graphene and its stability are important for designing the flexible and printed electronic devices31 and electron beam irradiation14. A short time of annealing at temperatures below 400 °C or low-dose electron beam irradiation will partially recover fluorinated graphene to pristine graphene. A direct proof for the recovery of fluorinated graphene is the decrease in the D-band Raman spectra and significant increase in the electrical conductivity. Prolonged annealing time at high temperature and high-dose electron beam irradiation may remove both C and F atoms in the fluorinated graphene31. On the other hand, single-sided fluorinated graphene with low F/C ratio prepared by using XeF2 gas will lose 50–80% initial fluorine coverage over 10 days before reaching a saturated state32. All these facts show that the fluorinated graphene possesses a poor stability over storage time, temperature and irradiation, which severely limits the practical utilization of fluorinated graphene as a candidate of 2D semiconductor. It is interesting to know that the stability of fluorinated graphene is related to the F/C ratio of sample. A highly fluorinated graphene on substrate remains insulating after 10 days while a mildly fluorinated sample is partially recovered to its pristine state. It reflects the fact that stability of fluorinated graphene depends on the site and structure of C–F bonds, but the physical mechanism remains unclear. Understanding the stability of C–F bonding at molecular level and its electrical property is the key for the practical design of fluorinated graphene-based 2D semiconductors.So far, there are a lot of works focused on the synthesis of the fluorinated graphene, while less attention has been paid to the stability of fluorinated graphene. Recently, researchers found that the stability of fluorinated graphene is sensitive to both temperature2 gas. Subsequently, the electron beam irradiation was used to selectively remove the fluorine atoms on graphene, and the resultant variation of the sample’s electrical conductivity was monitored continuously. The measurement results show that the electrical conductivity of double-sided fluorinated graphene remained unchanged after electron beam irradiation, while the electrical conductivity of single-sided sample was significantly increased due to the loss of fluorine atoms. Such comparison demonstrates that the structure of C–F bonding at both sides of graphene has much stronger stability than that at the single side. First-principle calculations further confirmed the symmetric charge distribution and stable formation energy in the double-sided fluorinated graphene, which explains the strong stability of double-sided C–F bonding at molecular level. This work highlights the importance of double-sided fluorination to achieve stable chemically functionalized graphene samples, which can be used in the practical design of all-graphene transparent and flexible electronics.In this work, we have synthesized both single-sided and double-sided fluorinated monolayer graphene by using XeF31. At first, a monolayer graphene was grown on copper foil by using a conventional chemical vapor deposition method. Then, a thin layer of polymethyl methacrylate (PMMA) was spin-coated on the surface and used as supporting layer during the transfer process. The copper foil was etched away and the graphene/PMMA layer was transferred onto a SiO2/Si substrate. After that, the graphene was cut into micro-ribbons following the EBL patterning and O2 plasma etching. In order to measure the electrical properties of functionalized graphene sample, a thin metallic film (10 nm thick Cr and 100 nm thick Au) was deposited on the graphene ribbon by using an electron beam physical vapor deposition method. The patterned metallic electrodes were made by using EBL and a standard lift-off method. Then, the graphene was chemically modified following two schedules: (1) For the single-sided fluorinated graphene, the graphene supported on SiO2/Si substrate was directly put into a XeF2 gas etcher (BP-3F Samco Inc.). The thin SiO2 passivation layer blocked the contact between XeF2 gas and backside of graphene, thus only the top surface of graphene was fluorinated in the XeF2 gas. (2) For the double-sided fluorinated graphene, the thin SiO2 layer was removed in a buffered hydrofluoric solution. Then, the graphene ribbon partially supported on silicon was put into the XeF2 gas etcher and the silicon substrate was etched by a depth around 8 μm. Finally, the graphene ribbon was suspended between two metallic electrodes above the substrate. In this way, both top and bottom surfaces of graphene were fluorinated in the XeF2 gas. The fluorination of graphene was done at room temperature. The F/C ratio of fluorinated graphene can be tuned by controlling the exposure time in the XeF2 gas. Scanning electron microscope (SEM) images of tested samples can be found in Fig. The fabrication process of fluorinated graphene is given as following37. In the fabrication process, the SiO2 layer was removed by using a buffered hydrofluoric acid (BHF) through a micrometer sized window opened in the polymer resist layer. Due to the isotropic etching behavior of BHF, a part of SiO2 below graphene and metal was removed. The length of over-etching was measured to be 2.5 μm in the SEM image of device taken after released from the silicon substrate by using XeF2 etching. So it is seen in Fig. 2 under the metallic source, SiO2 between source and drain, and a 2.5 μm wide SiO2 section under the metallic drain were removed. But SiO2 in the middle of the drain pad remains and forms an Au/Graphene/SiO2 sandwich structure, shown as the light green part in the figure. XeF2 gas cannot permeate the SiO2 layer, so the graphene between Au and SiO2 still remains its pristine crystalline structure. In Fig. 38. This indicates that the fluorinated graphene is almost transparent in the range of visible light with energy from ~ 1–3 eV38. The absorption spectrum of partially fluorinated graphene is higher than that of the fully fluorinated graphene, and slowly increases as the photon energy increases. Hence, the fluorinated graphene with low F/C ratio appears semi-transparent under the light. As seen in Fig. Figure Raman spectra, X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) data were collected on the fabricated samples.2 gas on the top surface of supported graphene, the 2D-band peak is dramatically decreased, while the D-band peak increased and exceeds the G-band peak. All the Raman peaks of fluorinated graphene are widened and the baseline has been raised, comparing with the Raman spectrum of pristine graphene. These features indicate that the fluorination will break the symmetrical two-dimensional lattice structure of graphene and increase its defect level. In the case of DSFG, the baseline of Raman spectrum keeps increasing and the ratio between 2D-band and G-band peaks is further decreased. This indicates a higher level of fluorination to the graphene lattice.Figure 2 layer under graphene. The Si and O signals were missing for the suspended DSFG sample.Figures Figure 14. It is an effective and precise tool to investigate the stability of C–F bonding. In order to conduct in-situ electrical characterization of fluorinated graphene in the SEM chamber, four-lead wires were fed through to connect the graphene sample and two high-precision Keithley digital multimeters (2002 series). The I-V curve of fluorinated graphene was measured by using a four-probe method. Electrical measurement inside the SEM chamber (< 10–4 Pa) prevents contamination or dopant in the atmospheric environment, which may further change the resistance of graphene sample. A low energy electron beam  was used for observation in the experiment, which does not significantly affect the electrical property of graphene14.It has been reported that the electron beam irradiation can be used to remove the fluorine atoms on graphene and regain its electrical conductivityI-V curve of SSFG sample #4. The whole SSFG ribbon between source and drain was uniformly irradiated by an electron beam, with a dose gradually increased from 0 to 0.40 C/cm2. A similar experimental result14 of single-sided fluorinated graphene with a dose up to 1.00 C/cm2 is also included in Fig. I-V curve of SSFG sample shows a typical symmetric behavior with respect to the bias voltage ranging from − 9 V to 9 V. Higher electron beam irradiation dose removes more fluorine atoms from graphene surface, and the source-drain current Isd increases monotonically. Hence, the resistance per square of SSFG decreases quite rapidly with the increasing irradiation dose, as shown in Fig. 14. The fluorine atoms on graphene skeleton reduce the charge in the conducting orbitals and introduce more scattering centers. Thus, the resistivity of fluorinated graphene is much larger than its pristine state, and is highly sensitive to the F/C ratio. Although four SSFG samples have almost the same geometric size and crystalline quality as a pristine graphene before fluorination, the fluctuation in the XeF2 gas pressure and reaction time may cause significant difference in the resistances of SSFG samples. As for SSFG #3, the resistance was decreased by 200 times after electron beam irradiation and approached a saturation value of the pristine graphene. This result demonstrates that the single-sided fluorinated graphene can be easily tuned from an insulator to a conductor by simply applying electron beam irradiation. However, the electrical property of SSFG is not so stable in two aspects: (i) the resistivity of sample is highly sensitive to the synthesis process of fluorination, and (ii) the resistivity of partially fluorinated graphene may change due to various reasons, such as irradiation, temperature, dopant in the atmospheric environment or storage time32. This can be understood by the fact that even a low energy electron beam irradiation can alter the resistance of SSFG, and the other factors  at the same energy level will have the similar impact.Figure I−V curve and resistance per square of DSFG samples. The I−V curve in DSFG sample represents a typical asymmetric diode characteristic, which is totally different from the metallic symmetric electrical behavior of SSFG shown in Fig. 43, a charge rectification behavior is noticed in the metal/fluorinated graphene (MFG). The graphene below metallic film was fluorinated at the bottom surface with an opened band gap up to 2.8 eV13. The difference in the work functions between metal and fluorinated graphene causes a potential barrier at their interface, leading to the charge rectification phenomenon. Here, the thickness of MFG heterojunction is mainly decided by the metallic film, because the fluorinated graphene only has a thickness less than 1 nm. It has a potential to create a vertically stacked diode within several nanometers by reducing the thickness of metallic film. For the SSFG samples supported on SiO2/Si, no charge rectification behavior was shown in Fig. 2 has a zero band gap. This result proves the validity of building nanoscale diodes by using fluorinated graphene with a tunable band gap.Figure I-V curve of DSFG was measured from − 8 V to 8 V at each irradiation dose. Extreme caution should be paid to load the bias voltage on DSFG, and it took a relatively long time to obtain a single experimental data point in Fig. 2 dose irradiation. In contrast, the same electron beam irradiation didn’t cause any reduction in the resistance of DSFG, but instead slightly increased the resistance. These results all proves the fact that the C-F bonding in DSFG has a much stronger stability than that in SSFG.In addition to the charge rectification at the MFG interface, the main finding in Fig. 45. The electron–ion interactions are described by the projector-augmented-wave (PAW) method46. The Perdew-Burke-Ernzerhof (PBE) functional within the generalized-gradient approximation (GGA) is used to treat the exchange–correlation interaction of electrons47. A kinetic energy cutoff of 400 eV for the planewave basis and a convergence criterion of 10–7 eV for the total energies are adopted for all ab initio calculations. The van der Waals (vdW) interaction is included according to the Tkatchenko and Scheffler method48.In order to understand the mechanism responsible for the ultra-strong stability observed in DSFG, first-principle calculations were carried out by using the Vienna Ab initio Simulation Package (VASP) based on density functional theory (DFT) and planewave basiseF) by performing ab initio calculations. Take the suspended graphene for instance, the eF is calculated as49:EFG, EF and EG are the total energy of fluorinated graphene, one fluorine atom, and pure graphene sheet, respectively. NF is the number of fluorine atoms in the calculation cell.To gain insight into the stability of fluorinated graphene, we calculated the averaged formation energy per fluorine atoms  and silicon dioxide (SiO2) are used as the substrate in our calculations, where are two typical dielectric substrates in literatures51. The stability of SSFG supported on the substrate of h-BN and amorphous SiO2 was studied respectively. As shown in Fig. h-BN substrate is relatively flat, while it is notably curved on amorphous SiO2 substrate. The eF of the supported graphene was calculated similar as Eq. (3) after replacing EFG with the total energy of fluorinated graphene with substrate, and EG with the total energy of pure graphene with substrate. The eF of the supported fluorinated graphene on h-BN and SiO2 substrates are 1.4854 eV and 1.5144 eV, respectively. Obviously, the positive values of eF indicate an unstable C–F bonding. Therefore, the defluorination in the single-sided fluorinated graphene can be easily induced by the electron beam irradiation as observed in experiment. In addition, the rough surface of amorphous SiO2 substrate causes fluctuation in the supported SSFG structure, which makes the defluorination process even easier.For the case of DSFG, we consider the structure of the fully fluorinated graphene (F/C ratio is 100%). The optimized crystal structure is shown in Fig. 52. As shown in Fig. h-BN and graphene leads to the charges in graphene dangling on the substrate side, as shown in the DCD of Fig. eF between neighbor C atoms in DSFG is − 3.380 eV, which is higher than the eF between fluorinate atoms and graphene layer (− 1.0534 eV). This calculation indicates the defluorination can be achieved without damaging the graphene layer as observed in the previous study13, which further makes the resistance of fluorinated graphene approaching to the value of pristine graphene during the defluorination process.To further understand the bonding nature and stability in the fluorinated graphene, we calculated the deformation charge density (DCD)Fluorination has been proved to be an efficient way to tune the band gap of graphene and control its electrical properties. Furthermore, electron beam irradiation was proved to be an efficient method in patterning the conductive channels in the fluorinated graphene, wherein the stability of fluorinated graphene is an important factor. In this report, we have fabricated electronic device with fluorinated graphene in two different configurations, double-sided fluorinated graphene and single-sided fluorinated graphene. The results show that the double-sided fluorinated graphene has an ultra-strong stability against the electron beam irradiation, while the single-sided fluorinated graphene loses the fluorine atoms easily even at a low irradiation dose. The first-principle calculations reveal that there exists a strong covalent bonding between the fluorine and carbon atoms in the double-sided fluorinated graphene, which well explains the ultra-strong stability observed in the experiment. Furthermore, a significant charge rectification behavior was observed at the interface between metal and fluorinated graphene. Our study provides important insights to the understanding of the unique chemical bonding characteristics in suspended graphene sheet, which is crucial for the fabrication of more stable and efficient graphene electronic devices.