Tardbp in vivo, we show that oligodendrocyte lineage cells are differentially sensitive to loss of TDP-43. While OPCs depend on TDP-43 for survival, with conditional deletion resulting in cascading cell loss followed by rapid regeneration to restore their density, oligodendrocytes become less sensitive to TDP-43 depletion as they mature. Deletion of TDP-43 early in the maturation process led to eventual oligodendrocyte degeneration, seizures, and premature lethality, while oligodendrocytes that experienced late deletion survived and mice exhibited a normal lifespan. At both stages, TDP-43-deficient oligodendrocytes formed fewer and thinner myelin sheaths and extended new processes that inappropriately wrapped neuronal somata and blood vessels. Transcriptional analysis revealed that in the absence of TDP-43, key proteins involved in oligodendrocyte maturation and myelination were misspliced, leading to aberrant incorporation of cryptic exons. Inducible deletion of TDP-43 from oligodendrocytes in the adult central nervous system (CNS) induced the same progressive morphological changes and mice acquired profound hindlimb weakness, suggesting that loss of TDP-43 function in oligodendrocytes may contribute to neuronal dysfunction in neurodegenerative disease.Generation of oligodendrocytes in the adult brain enables both adaptive changes in neural circuits and regeneration of myelin sheaths destroyed by injury, disease, and normal aging. This transformation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes requires processing of distinct mRNAs at different stages of cell maturation. Although mislocalization and aggregation of the RNA-binding protein, TDP-43, occur in both neurons and glia in neurodegenerative diseases, the consequences of TDP-43 loss within different stages of the oligodendrocyte lineage are not well understood. By performing stage-specific genetic inactivation of  The distribution of myelin in the adult brain is not static, as new oligodendrocytes continue to be generated in response to changes in experience, such as enhanced sensory input and intense motor learning . As a reTARDBP are responsible for both familial and sporadic forms of ALS (TDP-43 (TAR DNA-binding protein 43 kDa) is a DNA/RNA-binding protein that serves as a master regulator of RNA metabolism, controlling transcriptional integrity, post-transcriptional stability, and localization of mRNAs . Within s of ALS , suggests of ALS , suggestTardbp in microglia enhanced expression of NF-κB and NLRP3, leading to a more activated phenotype characterized by enhanced phagocytosis  mice, by administering tamoxifen at P90 and then collecting brain tissue 2 weeks later (P90 + 14) (Rosa-CAG-LSL-EGFP) allows the visualization of the morphology of Cre-recombined cells (n = 4) (panel i) . However, when the interval between tamoxifen administration and sacrifice was increased to 30 days (P90 + 30), OPCs in gray matter were also depleted (panel ii) . Remarkably, OPCs in white matter had repopulated by this time, and highly polarized OPCs were visible migrating out of the corpus callosum into the gray matter   . Inclusied cells . Immunol (n = 4) , resulti (n = 4) , panel i80 + 14) , indicatTardbp from these cells using two constitutive Cre lines which differ in their timing of expression during oligodendrocyte maturation: iCreMog, which has been used extensively to manipulate mature oligodendrocytes   that has been shown to promote oligodendrocyte differentiation  . Single-ng stage . Gene exntiation , suggestfl/fl;RCEMobp-iCre;Tardbp) (Mobp-TDP43 KO) began to exhibit spontaneous seizures at ~P110 , similar to the phenotype exhibited by Cre;Tardbpfl/flCnp mice  (Mog-TDP43 KO) did not exhibit spontaneous seizures or other gross behavioral abnormalities and survived as well as controls (blue lines).Abolishing TDP-43 expression at these different stages resulted in very different outcomes. Mice in which TDP-43 was constitutively removed from premyelinating oligodendrocytes , whereas deletion of TDP-43 from mature oligodendrocytes (Mog-TDP43 KO) was not associated with a significant change in OPC proliferation . Moreover, there was an increase in the number of newly formed oligodendrocytes in Mobp-TDP43 KO, as assessed by the emergence of cells that expressed lncOL1 . Myelinated axons in Mobp-TDP43 KO and Mog-TDP43 KO mice had significantly higher G ratios than controls at both P90 and P180, with thinner sheaths formed , in mice with TDP-43 KO oligodendrocytes, nodes often consisted of Caspr triads and βIV-spectrin doublets (red arrowheads). EM analysis revealed that some axons exhibited double myelination  . HistoloAs the cuff-like structures are reminiscent of astrocyte endfeet , we labeMobp-iCreER mice, which allow selective Cre-dependent recombination in oligodendrocytes in the adult CNS , ASPA+ oligodendrocytes were present in the motor cortex with morphologies comparable to controls  (yellow arrowhead), indicating that TDP-43 is also required to maintain oligodendrocyte structure in the adult CNS. However, the behavioral consequences of TDP-43 deletion in the mature CNS were distinct, as these mice exhibited progressive motor problems, including limp tails, hindlimb clasping  . Howeverclasping , Video 4clasping , but notclasping . Althougclasping , the spiclasping , indicatclasping . Thus, aclasping , triggerclasping .TDP-43 is a highly conserved, multifunctional protein that regulates key aspects of RNA processing. Previous studies have shown that loss of nuclear TDP-43 function, through genetic deletion or disease-induced mislocalization, is sufficient to cause aberrant incorporation of intronic sequences (cryptic exons) into mature mRNA that can alter protein expression and function . Due to Ermn (Ermin), an oligodendrocyte-specific cytoskeletal protein that regulates process branching and sheath extension  Mobp-iCre, (3) iCreMog, and (4) Mobp-iCreER, allowing Tardbp inactivation at discrete stages within the oligodendrocyte lineage in both the developing and mature CNS. Bulk RNA-Seq of purified oligodendrocytes from Mobp-iCre and iCreMog mouse lines showed that this approach was able to target different stages of oligodendrocyte development. In particular, oligodendrocytes labeled by Mobp-iCre showed higher expression of genes enriched in newly formed oligodendrocytes, such as Gpr37 and Mag, whereas those labeled by iCreMog had higher expression of genes enriched in myelin-forming and mature oligodendrocytes, such as Gamt and Opalin  d Opalin . Therefoate OPCs , allowedate OPCs .Tardbp within OPCs resulted in their rapid degeneration, with loss first detected in the white matter of the corpus callosum 2 weeks after tamoxifen exposure, followed closely by OPC depletion in cortical gray matter, demonstrating that these progenitors require TDP-43 for survival. OPCs represent the largest population of actively dividing cells in the brain outside the subventricular and subgranular germinal zones , surviving cells that retain TDP-43 are expected to proliferate to reconstitute the population. Consistent with this hypothesis, all OPCs after 30 days of recovery were EGFP negative  expressed at different stages (early and late) of the maturation of postmitotic oligodendrocytes to determine whether the effects of TDP-43 loss depend on their state of differentiation. In contrast to TDP-43-deficient OPCs, oligodendrocytes were generated, and myelin sheaths were formed in both lines despite inactivation of Tardbp. As the half-life of TDP-43 in vitro was measured to be 12–34 hr (CreCnp-mediated deletion (Mobp-iCre) resulting in oligodendrocyte degeneration, seizures, and premature death by 6 months of age. This phenotype is similar to, but not as severe as that observed in Cre;Tardbpfl/flCnp mice, in which mature oligodendrocytes, OPCs, premyelinating oligodendrocytes, and Schwann cells were genetically manipulated  and enhanced proliferation of OPCs, indicative of oligodendrocyte degeneration, similar to the phenotypic changes observed after inducible deletion of oligodendrocytes in the adult CNS  led to a strikingly different outcome: oligodendrocytes survived, OPC proliferation was not enhanced and there was no gliosis. Correspondingly, these animals also did not exhibit seizures or premature death. Bulk RNA-Seq transcriptional analysis of oligodendrocytes with early and late deletion, even prior to the phenotypic changes in Mobp-iCre mice (P30), revealed that pathways involved in gene expression, cellular localization, cell adhesion, and metabolism were affected. Further, differential gene expression analysis of early, premyelinating and myelinating oligodendrocytes from obtained through single-cell RNA sequencing , change in gene expression as oligodendrocytes mature  led to distinct pathological changes, such as hindlimb weakness, hindlimb clasping, and limp tail, phenotypes not observed with developmental Tardbp inactivation, and these mice did not exhibit seizures or premature death. Although these distinct phenotypes may arise from differences in the proportion of oligodendrocytes affected, they mirror functional differences observed in neurodevelopmental and neurodegenerative diseases. In neurodevelopmental disorders, such as autism spectrum disorders and schizophrenia, abnormal myelination and altered density of oligodendrocytes have been correlated with neural hyperexcitability and circuit dysfunction , including inert nanofibers and fixed axons . HoweverErmn KO mice . Of the other founder lines, the line with highest tamoxifen-dependent recombination efficiency was selected and maintained as a conditional Mobp-iCreERT2 mouse line   .Both female and male mice were used for experiments and randomly assigned to experimental groups. Mice were maintained on a 12 hr light/dark cycle, housed in groups no larger than 5, and food and water were provided ad libitum. All animal experiments were performed in strict accordance with protocols approved by the Animal Care and Use Committee at Johns Hopkins University. All experiments were initially performed with a sample size of 3  for quantification and statistical analyses, and power analysis was then performed to determine the minimum sample size to achieve sufficient statistical power to detect the effect of TDP-43 loss in different transgenic mouse lines at different timepoints. The following transgenic mouse lines were used in this study:Pdgfra-CreER (ra-CreER Tardbpfloxed (bpfloxed Rosa-CAG-LSL-EGFP) (JAX MMRRC stock #32038) (RCE ( #32038) Mobp-iCreMobp-iCreERiCreMog  dissolved in sunflower seed oil (Sigma) and administered by intraperitoneal injection daily for five consecutive days at a dose of 100 mg/kg body weight. Mice were perfused 14, 30, 60, and 90 days from the last day of tamoxifen injection.To induce 2), and their body temperature was maintained at 37°C with a thermostat-controlled heating plate. The skin over the right hemisphere was removed and the skull cleaned. A 3 × 3 mm region of skull over motor cortex was removed using a high-speed dental drill. A piece of cover glass  was placed in the craniotomy and sealed with VetBond (3 M), then dental cement (C&B Metabond) and a custom metal plate with a central hole was attached to the skull for head stabilization. In vivo imaging sessions began 3 weeks after cranial window procedure in order for the surgery-induced inflammation to subside. During imaging sessions, mice were anesthetized with isoflurane and immobilized by attaching the head plate to a custom stage. Images were collected using a Zeiss LSM 710 microscope equipped with a GaAsP detector using a mode-locked Ti:sapphire laser (Coherent Ultra) tuned to 920 nm.Cranial windows were prepared as previously described . Briefly3 and kept in 4°C until use. Free-floating sections were preincubated in blocking solution  for 1 or 2 hr at room temperature, then incubated for 24–48 hr at 4°C or room temperature in primary antibody. Secondary antibody incubation was performed at room temperature for 2–4 hr. Sections were mounted on slides with Aqua Polymount (Polysciences). Images were acquired using a confocal laser-scanning microscope . The following primary antibodies were used in this study: rabbit anti-ASPA , chicken anti-GFP , goat anti-GFP , mouse anti-MBP , chicken anti-MBP , rabbit anti-GFAP , Alexa Fluor 647 isolectin GS-IB4 (Thermo Fisher), rabbit anti-Aquaporin 4 , mouse anti-NeuN , mouse anti-CC1 , mouse anti-Ermin (Gift from E. Peles), guinea pig anti-Caspr (Gift from M. Bhat), rabbit anti-BIV-spectrin (Gift from M. Rasband), goat anti-ChAT , guinea pig anti-NG2 (Generated in D.E. Bergles lab), and rabbit anti-Ki-67 . Appropriate donkey secondary antibodies conjugated to Alexa Fluor 488, Cy3 or Alexa Fluor 546, and Cy5 or Alexa Fluor 647 were used at 1:1000–2000.Mice were deeply anesthetized with sodium pentobarbital (100 mg/kg) and perfused transcardially first with 15–20 ml room temperature PBS (pH 7.4) followed by 10 ml ice-cold 4% paraformaldehyde . CNS tissues were dissected  and postfixed in 4% PFA at 4°C for 4–6 or 12–20 hr depending on the antibodies to be used for immunohistochemistry. The tissues samples were transferred to 30% sucrose solution  for cryoprotection for at least two overnights before sectioning. Tissue samples were frozen in TissueTek and sectioned at 35 μm thickness on a cryostat (Thermo Scientific Microm HM 550) at −20°C. Tissue sections were collected into a 24-well plate with PBS with 0.1% NaNG ratio analysis 10 random micrographs covering an area of ~180 µm2 each were taken at a magnification of ×6000 using a LEO912 transmission electron microscope  with an on-axis 2k CCD camera . For random selection of axons the images were overlaid with a grid and axons on grid crossings were analyzed with a minimum of 100 axons per animal. For G ratio determination the area of the axon and the corresponding fiber including the myelin sheath were measured using Fiji/ImageJ  and postfixed at 4°C for two overnights. Subsequent TEM processing was performed according to a previously described protocol . For G ri/ImageJ  and the G ratio quantifications were performed by a blinded observer. Longitudinal image stacks were registered using FIJI plugin ‘Correct 3D Drift’ . Tissue was stored at 4°C for 48+ hr before sectioning. Flatmounted hemicortex were extracted, frozen in TissueTek, and sectioned at 40 µm thickness on a cryostat (Thermo Scientific Microm HM 550) at −20°C. Free-floating immunohistochemistry against MBP and EGFP was performed on the first section of the flatmount, which consists of the layer I of the cerebral cortex, as described previously . n represents the number of animals  used in each experiment. Data are reported as mean ± standard error of the mean and p value <0.05 was considered statistically significant.Statistical analyses were performed with GraphPad Prism 7 or Excel (Microsoft). All technical replicates  for each sample were averaged as a single data point. Significance was determined using one-way ANOVA test with Tukey’s correction for multiple comparisons or unpaired, two-tailed Student’s 2+ and Mg2+. Only the cortex was dissected out for downstream processing and mechanically dissociated with a razor blade on ice. The Miltenyi Neural Tissue Dissociation kit was used to perform enzymatic dissociation of the tissue into a single-cell suspension, and Myelin Debris Removal solution (Miltenyi) to remove myelin debris. The cells were resuspended in FACS sorting buffer (5 ml of 1 M HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) + 2 ml of Fetal Bovine Serum (FBS) + 800 μl of 0.5 M EDTA (Ethylenediaminetetraacetic acid) in 200 ml HBSS w/o Ca2+, Mg2+) and filtered through 35 μm strainer. The cells were sorted for endogenous GFP fluorescence on BD FACS Aria IIu Cell Sorter using a 100 μm nozzle at the Ross Flow Cytometry Core Facility at Johns Hopkins University School of Medicine.Male and female Mobp-TDP43 and Mog-TDP43 mice aged at P30 were cardiac perfused with ice-cold 1× Hank's Balanced Salt Solution (HBSS) w/o CaRNA was isolated from FACS purified oligodendrocytes (~5000 cells/sample) using QIAGEN RNeasy Micro Kit following the manufacturer’s instructions. cDNA was synthesized by using QIAGEN QuantiTect Reverse Transcription Kit. Due to low amount of cDNA, Nugen Single-cell RNAseq kit was used to generate cDNA libraries for 100 bp paired-end sequencing on Illumina HiSeq (70–100 million paired reads/sample).Reads were aligned to the latest mouse mm10 reference genome using the STAR spliced read aligner with default parameters. Total counts of read fragments aligned to known gene regions within the mouse mm10 RefSeq reference annotation were used as the basis for quantification of gene expression. Fragment counts were derived using HTS-seq program using mm10 Ensembl transcripts as the model. Various QC analyses were conducted to assess the quality of the data and to identify potential outliers . Principal component analysis correction was performed to correct for batch effects and differences in sample RNA integrity number. Differentially expressed genes were identified using EdgeR Bioconductor package and ranked based on adjusted p values  of <0.05.Ermn and cryptic exon-specific Ermn transcripts were designed using Benchling and Primer3.RNA was isolated from whole mouse brain tissue using ReliaPrep RNA Tissue Miniprep System (Promega) following the manufacturer’s instructions. cDNA was synthesized by using QIAGEN QuantiTect Reverse Transcription Kit. RT-PCR primers against normal Whole mouse brain tissue was homogenized in 1.5 ml Eppendorf tube with a handheld homogenizer in Pierce RIPA buffer (Thermo Fisher) with Sodium Orthovanadate (Vanadate). After centrifugation at 4°C, supernatant was collected, and the protein concentration was determined using Pierce BCA Protein Assay Kit (Thermo Fisher). 50 μg of protein was loaded into each lane of Mini-Protean TGX gels (BioRad) and ran at 120 V for 1.5 hr. Protein was transferred onto Immobilon-FL PVDF Membrane (Millipore) at 100 V for 1.5 hr. Intercept (TBS) Blocking Buffer (Li-Cor) was used for blocking, and the membrane was incubated in primary antibody solution at 4°C overnight with gentle shaking. Mouse-Ermin  and Rabbit-GAPDH  primary antibodies were used. IRDye 680RD Goat anti-Mouse IgG (H + L) (Li-Cor) and IRDye 800CW Goat anti-Rabbit IgG (H + L) (Li-Cor) were used as secondary antibodies for imaging on Odyssey Imaging Workstation (Li-Cor). Heo et al., 2021 investigated the role of the DNA/RNA-binding protein TDP-43 on oligodendrocyte maturation and showed that deletion of this protein induces deleterious effects on oligodendrocyte function in transgenic mouse models. These findings are exciting and support TDP-43 as a key regulator of oligodendrocyte function which may go awry in neurodegenerative disorders. public reviews designed to be posted alongside the preprint for the benefit of readers; ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Gary Westbrook as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Maria D Purice (Reviewer #3).Thank you for submitting your article "Stage-specific control of oligodendrocyte survival and morphogenesis by TDP-43" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission with specific attention to the essential revisions.Essential revisions:The majority of the comments are focused on the clarity of the written document, as well as suggestions for making the figures more informative as well as specific recommendations for increasing the clarity of the figures and providing quantification of a few of the figures.– There were a few additional experimental data requested centered on reporter data on recombination efficiency (see reviewer 2 point #3)- and neurofascin splicing (see rev 2 point 5).– Please provide more details and quantification of the immunohistochemistry images shown as noted by reviewer 1 in legends and methods .– Please address reviewer 3 comment regarding potential loss of OPC identity. All reviewers agreed this is important especially since one marker (NG2) was used to define OPCs. Because the absence of TDP-43 alters splicing, it is possible that NG2 is not being expressed. This could also help explain the lack of astrogliosis.Reviewer #1 (Recommendations for the authors):Major Strengths:• Mog-TDP43 and Mobp-TDP43 KO mice represent useful tools for the field to study early and late stages of oligodendrocyte maturation• The stage-dependent role of TDP-43 in oligodendrocyte maturation, morphology, and function is particularly novel.• Transcriptomic analysis  is particularly strong, showing that TDP-43 KO oligodendrocytes exhibit missplicing of cytoskeletal-associated genesMajor Weaknesses:• Very minimal quantification of immunohistochemistry images . Quantification of all images must be included with details in methods/legends• Several figures could be split up into separate figures to enhance clarity. Schematics should also be included in separate figures to provide additional visuals of experiments• No stain of TDP-43 in immunohistochemistry images. Please address.• No quantification of behavior/hindlimb weakness • Narrative is lost through the vast amount of data shown, especially within each figure• Figure 3 axon thinning and density could be shown via A,B,D,E,F and without C and G• Figure 4 does not need examples of abnormal structures (could be put in supplement), and lacks quantification• Both Figures 3 and 4 could be condensed or moved to supplementalReviewer #2 (Recommendations for the authors):The following points are suggestions to improve the overall presentation and impact of the manuscript.1. In the first section of the results the authors mention assessing the efficiency of Cre-mediated recombination in the PDGFRa-TDP43 mice by measuring EGFP expression in NG2-positive OPCs. It is likely that a sentence got deleted that described PDGFRa-CreER mice mated to EGFP Cre reporter (RCE) mice to allow for this assessment. It is mentioned in the figure 1 legend.2. The data presented clearly shows that TDP-43 is required for the survival of OPCs in young and older adult animals. Strikingly, the authors also show that the population of OPCs is quickly restored following their loss. The authors make the reasonable suggestion that the repopulated cells are derived from the minor population of OPCs that did not originally delete TDP-43. Nevertheless, it also seems possible that these cells are derived from a "pre-OPC" progenitor pool that through cell division and maturation are able to replenish the depleted OPC pool. The authors' interpretation is based on their extensive, groundbreaking work on how OPCs replenish lost oligodendrocytes, but it might be possible that distinct mechanisms respond to OPC loss. It might be worth mentioning this alternative possibility in the discussion.3. The authors have done an excellent job demonstrating recombination efficiency and specificity using the PDGFRa and Mobp Cre drivers. Nevertheless, they have not shown similar data for the Mog Cre driver. This data would be helpful when considering the apparent lack of impact on oligodendrocyte viability, in contrast with the other drivers, in the Mog-Cre mice.4. The sentence starting the new section on page 8 begins "Despite the eventual vulnerability of oligodendrocytes to loss of TDP-43, in both Mobp-TDP43 KO and Mog-197 TDP43 KO mice", which is confusing. The authors have clearly shown oligodendrocyte "vulnerability" in the Mobp animals, but up to this point in the manuscript all of the assays presented appear to show unperturbed oligodendrocytes in the Mog animals. Subsequently they demonstrate thinner myelin in these mice, but the introductory statement should be clarified or re-written.5. Chang et al. (2021) recently demonstrated that the loss of TDP-43 in myelinating Schwann cells resulted in aberrant splicing of the paranodal component Neurofascin, which is also expressed by oligodendrocytes. The authors of the current study also demonstrate aberrant splicing in the TDP-43 deficient oligodendrocytes. It would be interesting to know whether Neurofascin RNA is also abnormally processed in the oligodendrocyte mutants. Paranodal abnormalities might contribute to the clinical phenotype of the mutant animals.Reviewer #3 (Recommendations for the authors):Using their mouse models, the authors are able to show the role of TDP-43 in OPC survival, and the role of TDP-43 in early and late stages of postmitotic oligodendrocytes. It would be useful to have a summary figure that shows the role of TDP-43 in OPC/oligodendrocyte development.In lines 164-66, the authors mention that their results are "similar to the phenotype exhibited by Cnp-Cre;Tardbp 165 fl/fl mice , in which recombination occurs in OPCs, early postmitotic oligodendrocytes, and Schwann cells in the PNS ." This statement and citations are confusing. In J. Wang et al., 2018 they confirmed that Cre-mediated recombination occurs for over 95% of mature oligodendrocytes, which are labeled with APC-CC1 and they saw no colocalization of GFP with NG2-positive OPCs, thus suggesting that Cnp-Cre-mediated recombination in the spinal cord is restricted to mature oligodendrocytes. In addition, Lappe-Siefke et al. 2003 does not look at Schwann cells in the PNS. Please clarify these citations.Lines 343-4. The sentence "Thus, death of only a few proliferating OPCs upon removal of TDP-43 is likely to induce a cascading depletion, capturing neighboring cells  as they are induced to proliferate" is confusing. Can the authors clarify what they mean by a cascading-depletion of TDP-43 in other neighboring cells?The methods are easy to follow, however for the RNA isolation section (line 569), it would be helpful to include the number of oligodendrocytes that were sorted and used for RNA extraction. Essential revisions:The majority of the comments are focused on the clarity of the written document, as well as suggestions for making the figures more informative as well as specific recommendations for increasing the clarity of the figures and providing quantification of a few of the figures.– There were a few additional experimental data requested centered on reporter data on recombination efficiency (see reviewer 2 point #3)- and neurofascin splicing ( see rev 2 point 5).Mog-iCre mice was quantified by crossing these mice to a Cre-dependent EGFP reporter mouse line (“RCE”). Analysis of these mice [add experimental details – timing of tam and sac], revealed that 97.3±0.7% of ASPA+ oligodendrocytes were EGFP+ in the cerebral cortex ( = recombination efficiency), whereas 95.9±0.5% of EGFP+ cells were ASPA+ ( = recombination specificity). A small percentage of neurons exhibit Cre activity in this animals . These data have been added as new Figure 2—figure supplement 1.The efficiency of Cre recombination in Nfasc splicing in cortical oligodendrocytes with Tardbp deletion. These data are consistent with observations recently reported by Chang et al. (2021), in which Nfasc missplicing was observed in sciatic nerve, but not in the spinal cord after Cnp-Cre-mediated deletion of Tardbp . These observations have been added to the Discussion section of the manuscript (p. 18).We did not observe any changes in – Please provide more details and quantification of the immunohistochemistry images shown as noted by reviewer 1 in legends and methods .3) of abnormal association of TDP-43 KO oligodendrocytes with non-axonal structures in Figure 4—figure supplement 4. The method of quantification is now described in the Figure 4—figure supplement 4 legend and the Materials and Methods section.We have added quantification of the prevalence  was used to define OPCs. Because the absence of TDP-43 alters splicing, it is possible that NG2 is not being expressed. This could also help explain the lack of astrogliosis.+ TDP-43 KO OPCs disappear, rather than downregulate NG2. We also show that PDGFRα+ cells also disappear upon Cre recombination. These data have been added to Figure 1—figure supplement 1D. Of note, a previous study that used diphtheria toxin to ablate OPCs reported that widespread apoptosis of OPCs does not induce microglial activation or pro-inflammatory changes .We agree this is an important issue. In these experiments, a Cre-dependent EGFP reporter transgene (RCE) was included to follow the fate of Cre-recombined OPCs over time. Immunolabeling for EGFP in these mice revealed that all EGFPReviewer #1 (Recommendations for the authors):Major Strengths:• Mog-TDP43 and Mobp-TDP43 KO mice represent useful tools for the field to study early and late stages of oligodendrocyte maturation• The stage-dependent role of TDP-43 in oligodendrocyte maturation, morphology, and function is particularly novel.• Transcriptomic analysis  is particularly strong, showing that TDP-43 KO oligodendrocytes exhibit missplicing of cytoskeletal-associated genesMajor Weaknesses:• Very minimal quantification of immunohistochemistry images . Quantification of all images must be included with details in methods/legendsQuantification of abnormal association with non-axonal structures has been quantified . The methods of quantification have been added to the Material and Methods section.• Several figures could be split up into separate figures to enhance clarity. Schematics should also be included in separate figures to provide additional visuals of experimentsFigure 3 and 4 have been split and a summary figure has been generated  outlining the distinct role of TDP-43 at different stages of oligodendrocyte development.• No stain of TDP-43 in immunohistochemistry images. Please address.In the mouse brain, TDP-43 protein is most highly expressed by neurons. With available antibodies, immunostaining for TDP-43 only resulted in labeling of neuronal somata. Although TDP-43 is below the limit of detection within oligodendrocyte lineage cells in tissue sections, mRNA for TDP-43 is consistently detected using RNA sequencing approaches and the profound phenotypes we observe indicate its importance in promoting the survival of OPCs, oligodendrocyte maturation, and oligodendrocyte stability.• No quantification of behavior/hindlimb weakness Given the similarity of the motor abnormalities  exhibited by TDP43 cKO mice to that observed in mice subjected to experimental autoimmune encephalomyelitis (EAE), we used the well-established EAE 0-5 severity scoring scale to quantify the behavior of these mice. These data are now included in Figure 5E.• Narrative is lost through the vast amount of data shown, especially within each figure• Figure 3 axon thinning and density could be shown via A,B,D,E,F and without C and G• Figure 4 does not need examples of abnormal structures (could be put in supplement), and lacks quantification• Both Figures 3 and 4 could be condensed or moved to supplementalWe have now separated the main figures  and moved the data indicated into Supplementary Data.Reviewer #2 (Recommendations for the authors):The following points are suggestions to improve the overall presentation and impact of the manuscript.1. In the first section of the results the authors mention assessing the efficiency of Cre-mediated recombination in the PDGFRa-TDP43 mice by measuring EGFP expression in NG2-positive OPCs. It is likely that a sentence got deleted that described PDGFRa-CreER mice mated to EGFP Cre reporter (RCE) mice to allow for this assessment. It is mentioned in the figure 1 legend.Indeed, all of the mice used in this study expressed a Cre-dependent EGFP reporter (RCE) to assess Cre recombination efficiency and to allow visualization of the morphology of TDP-43 KO cells. This information is now included in the Results (p.5).2. The data presented clearly shows that TDP-43 is required for the survival of OPCs in young and older adult animals. Strikingly, the authors also show that the population of OPCs is quickly restored following their loss. The authors make the reasonable suggestion that the repopulated cells are derived from the minor population of OPCs that did not originally delete TDP-43. Nevertheless, it also seems possible that these cells are derived from a "pre-OPC" progenitor pool that through cell division and maturation are able to replenish the depleted OPC pool. The authors' interpretation is based on their extensive, groundbreaking work on how OPCs replenish lost oligodendrocytes, but it might be possible that distinct mechanisms respond to OPC loss. It might be worth mentioning this alternative possibility in the discussion.Thank you for this suggestion. Although we favor the conclusion that repopulation occurs primarily from surviving cells, based on the recombination efficiency and qualitative assessments of proliferation of glial progenitors around the lateral ventricles, we now note this possibility in the Discussion (p. 14).3. The authors have done an excellent job demonstrating recombination efficiency and specificity using the PDGFRa and Mobp Cre drivers. Nevertheless, they have not shown similar data for the Mog Cre driver. This data would be helpful when considering the apparent lack of impact on oligodendrocyte viability, in contrast with the other drivers, in the Mog-Cre mice.We now provide these data in Figure 2—figure supplement 1.4. The sentence starting the new section on page 8 begins "Despite the eventual vulnerability of oligodendrocytes to loss of TDP-43, in both Mobp-TDP43 KO and Mog-197 TDP43 KO mice", which is confusing. The authors have clearly shown oligodendrocyte "vulnerability" in the Mobp animals, but up to this point in the manuscript all of the assays presented appear to show unperturbed oligodendrocytes in the Mog animals. Subsequently they demonstrate thinner myelin in these mice, but the introductory statement should be clarified or re-written.We have removed the text referring to the vulnerability and modified the introductory statement to the following: In both Mobp-TDP43 KO and Mog-TDP43 KO mice, oligodendrocytes were able to mature and form myelin sheaths, global myelin tracts within the brain were established , and there was no evidence of astrogliosis early in development (P30) .5. Chang et al. (2021) recently demonstrated that the loss of TDP-43 in myelinating Schwann cells resulted in aberrant splicing of the paranodal component Neurofascin, which is also expressed by oligodendrocytes. The authors of the current study also demonstrate aberrant splicing in the TDP-43 deficient oligodendrocytes. It would be interesting to know whether Neurofascin RNA is also abnormally processed in the oligodendrocyte mutants. Paranodal abnormalities might contribute to the clinical phenotype of the mutant animals.Nfasc was aberrantly spiced after deletion of Tardbp. Of note, Chang et al. (2021) observed that aberrant splicing of Nfasc occurred in the sciatic nerve but not in the spinal cord in Cnp-Cre cKO mice, which recombine in both the CNS and PNS. This result is now reported in the Discussion (p. 18).We examined our RNAseq datasets and did not observe that Reviewer #3 (Recommendations for the authors):Using their mouse models, the authors are able to show the role of TDP-43 in OPC survival, and the role of TDP-43 in early and late stages of postmitotic oligodendrocytes. It would be useful to have a summary figure that shows the role of TDP-43 in OPC/oligodendrocyte development.We have added a new summary figure to accompany the manuscript .In lines 164-66, the authors mention that their results are "similar to the phenotype exhibited by Cnp-Cre;Tardbp 165 fl/fl mice , in which recombination occurs in OPCs, early postmitotic oligodendrocytes, and Schwann cells in the PNS ." This statement and citations are confusing. In J. Wang et al., 2018 they confirmed that Cre-mediated recombination occurs for over 95% of mature oligodendrocytes, which are labeled with APC-CC1 and they saw no colocalization of GFP with NG2-positive OPCs, thus suggesting that Cnp-Cre-mediated recombination in the spinal cord is restricted to mature oligodendrocytes. In addition, Lappe-Siefke et al. 2003 does not look at Schwann cells in the PNS. Please clarify these citations.We have revised the reference to the following prior studies: Chang et al. 2021 and Tognatta et al. 2017 (p. 7).Lines 343-4. The sentence "Thus, death of only a few proliferating OPCs upon removal of TDP-43 is likely to induce a cascading depletion, capturing neighboring cells  as they are induced to proliferate" is confusing. Can the authors clarify what they mean by a cascading-depletion of TDP-43 in other neighboring cells?We have clarified this statement .The methods are easy to follow, however for the RNA isolation section (line 569), it would be helpful to include the number of oligodendrocytes that were sorted and used for RNA extraction.This method has been clarified.ReferencesEmbo J 30:277–288.Ayala YM, De Conti L, Avendano-Vazquez SE, Dhir A, Romano M, D'Ambrogio A, Tollervey J, Ule J, Baralle M, Buratti E, Baralle FE (2011) TDP-43 regulates its mRNA levels through a negative feedback loop. Neuron, 88(5), 941–956. https://doi.org/10.1016/j.neuron.2015.10.046.Birey, F., Kloc, M., Chavali, M., Hussein, I., Wilson, M., Christoffel, D. J., Chen, T., Frohman, M. A., Robinson, J. K., Russo, S. J., Maffei, A., and Aguirre, A. (2015). Genetic and Stress-Induced Loss of NG2 Glia Triggers Emergence of Depressive-like Behaviors through Reduced Secretion of FGF2. Life 10 (March). https://doi.org/10.7554/elife.64456.Chang, Kae Jiun, Ira Agrawal, Anna Vainshtein, Wan Yun Ho, Wendy Xin, Greg Tucker-Kellogg, Keiichiro Susuki, Elior Peles, Shuo Chien Ling, and Jonah R. Chan. 2021. “TDP-43 Maximizes Nerve Conduction Velocity by Repressing a Cryptic Exon for Paranodal Junction Assembly in Schwann Cells.” eNature Neuroscience 16 (6): 668–76. https://doi.org/10.1038/nn.3390.Hughes, Ethan G., Shin H. Kang, Masahiro Fukaya, and Dwight E. Bergles. 2013. “Oligodendrocyte Progenitors Balance Growth with Self-Repulsion to Achieve Homeostasis in the Adult Brain.” J Clin Invest 121:726–738.Igaz LM, Kwong LK, Lee EB, Chen-Plotkin A, Swanson E, Unger T, Malunda J, Xu Y, Winton MJ, Trojanowski JQ, Lee VM (2011) Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice. Life 9: 1–61. https://doi.org/10.7554/eLife.56621.Orthmann-Murphy, Jennifer, Cody L Call, Gian Carlo Molina-Castro, Yu Chen Hsieh, Matthew N Rasband, Peter A Calabresi, and Dwight E Bergles. 2020. “Remyelination Alters the Pattern of Myelin in the Cerebral Cortex.” eNat Neurosci 14:459–468.Polymenidou M, Lagier-Tourenne C, Hutt KR, Huelga SC, Moran J, Liang TY, Ling SC, Sun E, Wancewicz E, Mazur C, Kordasiewicz H, Sedaghat Y, Donohue JP, Shiue L, Bennett CF, Yeo GW, Cleveland DW (2011) Long pre-mRNA depletion and RNA missplicing contribute to neuronal vulnerability from loss of TDP-43. Glia 65(2), 342–359. https://doi.org/10.1002/glia.23095.Tognatta, R., Sun, W., Goebbels, S., Nave, K. A., Nishiyama, A., Schoch, S., Dimou, L., and Dietrich, D. 2017. "Transient Cnp expression by early progenitors causes Cre-Lox-based reporter lines to map profoundly different fates". Nature Neuroscience, 19(1), 65–74. https://doi.org/10.1038/nn.4193.Traka, M., Podojil, J. R., McCarthy, D. P., Miller, S. D., and Popko, B. (2016). Oligodendrocyte death results in immune-mediated CNS demyelination.


A high performance humidity sensor using tilted fiber Bragg grating (TFBG) and functional graphene oxide (GO)/multi-walled carbon nanotubes (MWCNTs) hybrid nano-materials was proposed. The humidity-sensitive material with three-dimensional (3D) structure was synthesized by the MWCNTs and GOs. Comparing with traditional two dimensional (2D) GOs film, water molecules could be absorbed effectively due to the larger ripples and more holes in GO/MWCNTs layers. The water molecule will fill the entire space in the 3D structure instead of air, which further enhances the absorption efficiency of the hybrid nanomaterial. TFBG as a compact and robust surrounding complex dielectric constant sensing platform was utilized. The mode coupling coefficient or the amplitude of TFBG cladding mode will vary sharply with the imaginary part of permittivity of the hybrid nanomaterial, realizing the high performance RH sensing. In the experiments, we successfully demonstrated that this 3D structural nanomaterial composed by the MWCNTs and GOs has significant advantages for expanding the range of humidity detection (range from 30% to 90%) and enhancing the detection sensitivity (0.377 dB/% RH is twice more than humidity sensor with 2D GO film). The TFBG-based RH sensor also exhibits good repeatability and stability. Our proposed humidity sensor has potential application in environmental and healthy monitoring fields. Humidity monitoring has taken an important role in numerous areas such as corrosion protection, precision electronics manufacturing, food processing, and other industries ,2,3. Tra2 nanoparticles coated S-Taper fiber as an RH sensor achieving the sensitivity of 0.441 dB/% RH and a range of 83.8% RH to 95.2% RH [2 coated side polished fiber (SPF) with a good linear correlation coefficient of 99.39% and a sensitivity of 0.1213 dB/% RH [2 = 0.980) [On the other hand, it is crucial to select a suitable humidity-sensitive material for RH measurement. The humidity-sensitive materials with a lot of hydrophilic functional groups, good swelling degree, and large specific surface area are attractive for measuring the RH. The widely used humidity sensing materials are porous ceramics, semiconducting materials, polymers , and novel carbon materials  ,23,24,2595.2% RH . Y. Luo  dB/% RH . However dB/% RH ,30. For  dB/% RH . K. Prab= 0.980) . The S t= 0.980) . In thosHerein, we proposed a TFBG-based humidity sensor with GO and evenly dispersed multi-walled carbon nanotubes (MWCNTs) hybrid humidity-sensitive nanomaterial. TFBG could couple most of the lights in the core to the cladding and make the lights directly interact with the humidity-sensitive material. The humidity sensing film was synthesized by GO/MWCNTs hybrid nanomaterial with three-dimensional (3D) structure. The MWCNTs play an essential role in supporting and expanding the space between the GO sheets. Via the supporting MWCNTs and GO films, the configuration makes water molecule fill the entire space instead of air which might strengthen the absorption effect of the molecule onto the hybrid nanomaterial. We will creatively combine TFBG with GO/MWCNTs hybrid nanomaterial to measure the relative humidity, and experimentally study the perturbation mechanism of the material’s dielectric constant. The proposed sensor with good performance will show good potential applications for health and environmental monitoring in many fields.rλ meeting the phase matching condition could be expressed as:θ is the tilt angle of the TFBG, Λ is the period of TFBG. rλ, respectively [For a TFBG, the resonance wavelength ectively .R of the TFBG cladding modes are related to the length L of the TFBG and the coupling coefficient κ between the exciting cladding mode and core mode. It can be expressed as follows:The intensity C is a constant depended on the electric field distribution of core mode coreE and cladding mode rE. The coupling coefficient is calculated as follows:e is the unit charge, cμ is the change of the chemical potential, h is Planck’s constant, and Bk is Boltzmann’s constant. T and τ are the environment temperature and vibration frequency [The conductivity σ of GO could be calculated through Equation (4):requency .GO or GO/MWCNTs film will absorb more and more water molecules with the increasing of RH, and the GO layer is filled by the absorbed water molecules. The density of the GO increases with the increasing humidity, which leads to the decrease of the conductivity (Im(nGO)) . For tigIm(nGO)) ,31. WhilIm(nGO)) . For theThe experimental system is shown in The physical precipitation method is selected to coat the GO or GO/MWCNTs on the fiber surface. First, the TFBG sensor is immersed into hydrochloric acid solution (20 min) and then flushed with plenty of deionized water and ethanol. Second, the sensor is immersed in GO aqueous solution or GO/MWCNTs solution to obtain GO or GO/MWCNTs film. At the same time, the sensor with GO aqueous solution or GO/MWCNTs solution is dried in a drying oven for one hour. Finally, to remove unsecured GO or GO/MWCNTs film from the TFBG’s surface, the sensor is washed with ethanol or deionized water and blown dry with nitrogen. The GO/MWCNTs aqueous solution was prepared by simple sonication. CNTs could adhere to the flat layers of GO through strong π-π stacking interaction ,35. TherIn this paper, the humidity response of GO coating TFBG was first performed. 2 of 99.19%. Comparing with the humidity sensor based on TFBG and GO film, the sensitivity under GO/MWCNTs film coating is twice more than that under GO film condition and the sensor detection range of RH is effectively expanded by coating with GO/MWCNTs film as shown in To evaluate the repeatability and reusability of the proposed sensor, three times repeat tests with different relative humidity were performed. It can be seen in Finally, the stability and the response time of the proposed fiber optic humidity sensor were also investigated. First, the TFBG coated with GO/MWCNTs film humidity sensor is placed in a constant temperature and humidity chamber . In this paper, a GO/MWCNTs-based TFBG humidity sensor with high sensitivity and large dynamic range has been demonstrated. In this humidity measurement, the light coupling element is the TFBG which easily excites cladding modes by coupling the core light to the cladding and interacts with the humidity-sensitive material on the fiber surface. The addition of MWCNTs enlarges the distance between the GO sheets and promotes the absorption of water molecules by the enriched surface/ripples/holes of both GO and MWCNTs. Thus, the composite of GO and MWCNTs has obvious advantages for expanding the humidity detection range and improving the detection sensitivity. The experimental results show that the sensitivity of RH detection sensor is 0.377 dB/% RH, which is twice more than that with GO film and the range of humidity detection is 30–90%. Moreover, the proposed humidity sensor possesses excellent repeatability, stability, and fast response characteristics. Therefore, this humidity sensor has potential applications for health and environmental humidity monitoring.


We present the design, fabrication, and testing of a drone-mountable gas sensing platform for environmental monitoring applications. An array of graphene-based field-effect transistors in combination with commercial humidity and temperature sensors are used to relay information by wireless communication about the presence of airborne chemicals. We show that the design, based on an ESP32 microcontroller combined with a 32-bit analog-to-digital converter, can be used to achieve an electronic response similar, within a factor of two, to state-of-the-art laboratory monitoring equipment. The sensing platform is then mounted on a drone to conduct field tests, on the ground and in flight. During these tests, we demonstrate a one order of magnitude reduction in environmental noise by reducing contributions from humidity and temperature fluctuations, which are monitored in real-time with a commercial sensor integrated to the sensing platform. The sensing device is controlled by a mobile application and uses LoRaWAN, a low-power, wide-area networking protocol, for real-time data transmission to the cloud, compatible with Internet of Things (IoT) applications. Detection and identification of volatile compounds in remote locations for air quality monitoring are crucial to establish and enforce environmental policies on greenhouse emission or provide early warning for dangerous air-borne agents. To enable these applications, gas sensors must not only feature high sensitivity and selectivity to specific molecules, but also have practical in-field characteristics, such as being compact, light-weight, deployable, and low cost ,2,3. OnePrevious work demonstrated that, when exploring new compact sensing technologies, graphene-based chemiresistors satisfy many requirements for gas sensing applications ,10. For In parallel to the development of smaller gas sensing devices, programmable 32-bit microcontrollers (μC), such as ESP32, are increasingly used to achieve compact and portable processing platforms ,22,23,24Here, we report a drone-mountable sensing platform using a graphene chemiresistor array fabricated integrated to a battery-powered μC development board and accessible with short- and long-range wireless communication systems. More importantly, we demonstrate the possibility to integrate graphene-based sensing technology into a drone-mountable platform for real-life applications. We propose a compact design and explore some of the main limitations, including environmental noise caused by temperature and humidity fluctuations. In a lab setting, the device reading accuracy is similar to the one provided by a state-of-the-art multi-meter . Tests performed during flights and under rapidly changing environmental conditions show a significant increase in noise level. We were able to reduce signal fluctuations by about an order of magnitude when normalizing the signal against independent temperature and humidity readings from a commercial sensor also integrated into the sensing platform.2 substrate with parallel gold electrodes deposited on the surface. The chemiresistors are fabricated following the technique described in [We design a compact and light-weight (185 g) sensing platform using commercially available μC devices and compatible components see . The funribed in . AlthougA high-accuracy 32-bit analog-to-digital converter (ADC) monitors real-time resistance of the four devices by measuring the voltage across four reference resistances directly mounted on the sensor board. Note that the ADS1262 ADC model used in this configuration could allow up to eight channels to be read simultaneously. The μC performs a circuit calculation using these voltages to retrieve the resistance of each three chemiresistors and the static resistor  sensor (model BME280) monitors environmental fluctuations. Its readings are used to normalize the graphene sensors’ response as we demonstrate later. A comprehensive functional diagram including all device components can be found in The sensing platform architecture is adapted to the drone size and shape see . The 3D We demonstrate the practicality of the device by testing it under different experimental conditions: (1) in a laboratory setting, inside an enclosure under a controlled environment, (2) mounted on a drone during an indoor flight, and (3) outdoor while being exposed to wind and sun. In the laboratory, experiments are conducted with a standard configuration see  for evalR/R0 = 0.012%. The response ΔR/R0 provided by a sensor is defined as the change of resistance ΔR of the graphene sheet divided by its resistance R0 at the beginning of the experiment. We performed a similar series of measurements using a state-of-the-art sensor monitoring system. This system extracts the electrical resistance from the linear I-V characteristic curve obtained by a multimeter (Keithley 2400) by keeping the voltage constant and reading the current. Results shown in R/R0 = 0.006%). Considering that the standard laboratory data acquisition equipment is significantly more bulky and expensive than our compact sensing platform, we believe this comparison not only demonstrates the ability to use μC-based infrastructure for sensitive remote gas sensing, but also its potential to replace standard data acquisition equipment to save space and costs. All data acquisition in In a first experiment, the sensing platform is placed inside a plexiglass enclosure under steady conditions and we investigate the optimal sensitivity of the device by analyzing the noise characteristics with a series of continuous measurements. G1, G2, and G3, and the static resistance, StaticR, (top panel), as well as the recorded relative humidity and temperature provided by the TH sensor (bottom panel). In this experiment, water vapour is first introduced in the enclosure. We see a correlation between the humidity level (blue line) and the signal (ΔR/R0) provided by the three graphene sensors, where R0 is the resistance at time 0 s. The fact that StaticR remains constant during the experiment demonstrates the stability of the electronic configuration and reliability of the resistance readings.In another experiment, also performed in the laboratory setting described in G = ΔR/R0 of a graphene sensor depends on ambient humidity and temperature, which are varying parameters difficult to control in real-life applications. In practice, this can result in signal fluctuations reducing the sensor’s sensitivity to a targeted gas. Here, we independently measure humidity and temperature and use signal processing to account for the effects of these two environmental parameters on the sensor. We build a model, based on previous work [modG, the projected signal derived from humidity and temperature readings only:T is the temperature in K, A kBN (referred as the gas constant 8.314 J/mol K) is the Avogadro’s number multiplied by the Bolzmann constant, and dE is the energy for water desorption  depends on intrinsic graphene electrical properties, such as the doping level [The signal ous work ,31, to c04 J/mol ). The vang level . These tG. After adjustment, most of these modulations are cancelled out and we are left with a signal perfectly correlated with the exposure times to ethanol. More strikingly, the slope of ΔR/R0 (green dashed lines) is changing accordingly to the ethanol concentration, which is equal to 7800 ppm in the light red region and 13,700 ppm in the darker red regions. Chemiresistors used in this work have a response time of ~20 min [R/R0 reaching 90% the maximum signal associated with a constant analyte concentration. For real-life applications, other features, such as the maximum slope of the time-dependent signal, or a comparison between the responses provided by different types of sensors, could be monitored to reduce the time necessary to detect and identify chemicals [ ~20 min , which ihemicals . Furtherhemicals , implemehemicals , or usinhemicals . The tesAs a first step towards assessing the performance of the drone platform in real-life environments, we mounted the sensing platform on a drone while collecting data with the sensors on-board. adjG and significantly reduce the effects of fluctuating humidity.We also investigated the response of the graphene sensors to a sudden increase in humidity during in-flight tests by approaching the flying drone to a humidifier releasing a jet of water vapour b. IntereadjG. The standard deviation of the three signals from the graphene sensors  decreases, on average, from 0.53% to 0.08%. In other words, the noise decreases by a factor of ~7. For the experiment shown in In a final experiment, we investigate the response of the sensing platform when it is left outdoor , while being exposed to fluctuating environmental conditions. The results are presented in G (ΔR/R0) with relatively low noise (0.06%), only two times higher than the one measured with standard, state-of-the-art laboratory equipment. When exposed to water and ethanol vapours, graphene sensors show a clear response. We build a model of the graphene response as a function of monitored changes in humidity and temperature, and use it to obtain an adjusted signal adjG independent of these two environmental parameters and distinctively revealing the presence of specific gases. We demonstrate this concept by exposing our sensors to fluctuating water vapour concentrations while detecting ethanol. Indoor flight tests show that the flying drone motion does not significantly affect the sensing response, especially when the platform temperature has reached an equilibrium. Finally, we demonstrate that fast-varying outdoor environmental conditions increase graphene-based chemiresistor noise. This poses a major obstacle to the deployment of sensing technologies in the field. However, cross-data analysis on data provided by a commercial temperature/humidity (TH) sensor and graphene sensors can be used in real-time to decrease unwanted signals created by environmental fluctuations. We demonstrate a noise reduction approaching an order of magnitude with room for further improvements, which can be achieved by perfectly matching the time response of the TH and graphene sensors. In our experiment, three graphene chemisensors fabricated with the same technique are tested simultaneously to demonstrate reproducibility. This compensation method for temperature and humidity is software-driven and can be easily applied to graphene-based sensor systems. In a lab-setting, we demonstrate that our platform can achieve a signal-to-noise comparable to the one obtained with high-quality commercial multimeters. Previous work has established correlations between air flow and the relative positioning of the sensors. The fan included in our device architecture helps sample the surrounding area but a more in-depth study of the placement and orientation of the air intake could still improve the overall performances of our gas sensing platform [We demonstrate the use of a microcontroller (μC) and related components to produce a lightweight, compact, and low-cost platform, which can be mounted on a drone for remote gas monitoring over distances of 1 km. Under controlled conditions, our platform can relay a signal platform ,39,40. Iplatform  to enhanplatform .


In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN’s sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes ‘compression’ near the cleavage site. This provides multiple advantages for L1-EN’s role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics.Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising ∼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1’s ORFp2 protein (L1-EN) initiates   The autonomous non-LTR retrotransposon long interspersed nuclear element-1 (LINE-1 or L1) is one the most abundant mobile elements in the human genome, comprising 17–20% of its content . L1 elemExpression and/or new insertions of L1 can have various DNA damaging effects ranging from introduction of DNA breaks to oncogene activation. The process also impacts the immune system and is associated with multiple significant human diseases including metabolic, neurological and autoimmune disorders  and cancin vitro in cultured human cells  and DNase I ,47, whican cells  and in vf cancer . The mecf cancer ,21,52, if cancer ,7,21. Inf cancer  and doesf cancer . These df cancer ,53. Howef cancer  suggestef cancer . Overall2+ ion. Two mutations were introduced into L1-EN to facilitate crystallization of the enzyme complexed with DNA: D145A to prevent DNA cleavage . Cell cultures were grown in Terrific Broth with shaking at 37°C to OD600 = 1.5. Protein expression was then induced by adding IPTG to 1 mM final concentration and incubating cells with shaking at 16°C overnight. The cell pellet was suspended in purification Buffer A phosphine hydrochloride (TCEP.HCl) containing 50 μg/ml leupeptin, 50 μg/ml aprotinin, 1 mM PMSF, 2 mM CHAPS and 0.1% Brij35. The cell suspension was frozen in liquid nitrogen and then cells were lysed by thawing followed by four cycles of sonication at 50% power, 50% pulsar setting for 3 min each. The lysate was clarified by centrifugation at 16,000 rpm for 50 min using a Sorvall RC5C centrifuge. The supernatant was mixed with 5 ml His60 NiNTA Superflow resin (TaKaRa) equilibrated with Buffer A. The solution was incubated on a rotator for 30 min at 4°C and then loaded onto a gravity column. After washing the resin with 300 ml of Buffer A containing 20mM imidazole, the protein was eluted with Buffer A containing 200 mM imidazole in two fractions of 10 ml each. The 6xHis-SUMO tag was cleaved from the protein with Ulp1 peptidase overnight at 4°C. The cleaved protein was diluted 5-fold with Buffer A without NaCl to a final concentration of 200 mM NaCl and applied to a 5 ml Hi-Trap heparin affinity column . The protein was eluted with a gradient of NaCl (100 mM to 1 M) at ∼700 mM NaCl concentration.An N-terminal fragment of human LINE-1 encoding amino acids 1–238 of ORF2  was cloned into the pET28b+ based pSMT3 vector  using the Gibson Assembly protocol. The resulting plasmid, pSMT3-LINE-EN, directs expression of LINE-1 endonuclease (L1-EN) with an N-terminal 6xHis-SUMO tag for use in protein purification. pSMT3-LINE1-EN was transformed into BL21* http://www.rasmb.org/sednterp) were 1.04913 ρ and 0.01436 η, respectively. These values were used to fit the data to the Lamm equation in SEDFIT software  was used to measure potential protein oligomerization in solution. Purified protein was concentrated and extensively dialyzed against AUC buffer . Sedimentation velocity studies were performed in a Beckman XL-A analytical ultracentrifuge at 20°C and 35 000 rpm. Absorbance at 280 nm was measured every 4 min for a total of 200 scans. The buffer viscosity and density as calculated by Sednterp  protocol adjusted to have partially-overlapping primers with 3′-overhangs (ThermoFisher Scientific). Mutations were confirmed by sequencing before transformation of constructs into BL21* cells. Mutant L1-EN proteins were expressed and purified as described above for WT L1-EN.in vitro using a fluorescent DNA probe created by annealing two labeled oligonucleotides: 5′-/56-FAM/CCTTTTTTTTTAACCGC-3′ and 5′-GCGGTTAAAAAAAAAGG/3IABkFQ/-3′ (Integrated DNA Technologies) resulting in quenching of the FAM fluorescence signal. For each reaction, the indicated amount of recombinant L1-EN protein (50–400 ng) was incubated with 100 nM DNA probe in 20 mM HEPES pH 7.0, 50 mM NaCl, 1 mM MgCl2, 1 mM DTT, 0.1 mg/ml BSA, 10% glycerol and fluorescence was measured for 60 min at 37°C. In this assay, L1-EN-mediated nicking of the DNA probe results in separation of a fluorescein labeled product from the probe containing the quencher. The product was quantified by measuring fluorescence ; emission wavelength: 520 nm (10 nm bandwidth)). A linear range of the fluorescence change curve  was used to calculate protein activity; results are expressed as relative fluorescent units (RFU) per min per ng protein and are shown as the mean ± standard deviation (SD) for 4–5 replicates, as indicated. Data were collected and averaged for several protein concentrations  with duplicates for each concentration.Endonuclease activity of WT and mutant L1-EN proteins was measured dna14 and dna17 DNA substrates  and (iii) for co-crystallization of D145A/Y226K mutant L1-EN with dna14: 0.1M sodium acetate pH 4.5, 0. 2M ammonium acetate, 15% PEG 4000 from ProPlex screen (Molecular Dimensions). Crystals were cryoprotected by adding 10% glycerol and 10% ethylene glycol and flash frozen either in liquid nitrogen or on a cold nitrogen gas stream. To assess Mg2+ ion coordination, crystals of WT L1-EN protein without DNA were soaked with 20 mM MgCl2 during cryoprotection for 30–60 s before freezing.WT and mutant L1-EN proteins were concentrated to 16–24 mg/ml in 300 mM NaCl, 15 mM HEPES pH 7.5, 2% glycerol, 1 mM TCEP. For co-crystallization with dna14 complex and for WT L1-EN coordinated with Mg2+ were collected using a home source X-ray generator Rigaku-MicorMax-007 HF with Raxis IV++ detectors and X-Stream nitrogen cooling system  School of Medicine, St. Louis, MO). Data for the L1-EN D145A/Y226K-dna17 complex were collected on the beam line 23-ID-B at the Advanced Photon Source, Argonne National Lab . Data were integrated and scaled using the HKL2000 program and structures were solved using the PHENIX software suite  was applied to protein molecules during model building and initial refinement cycles with torsion NCS restraints automatically defined in PHENIX. NCS restraints were omitted from the final refinement cycles. Crystals of L1-EN with dna14 belong to P6122 space group with one complex per asymmetric unit. Data collection and refinement statistics are presented in Table Data for the L1-EN D145A/Y226K-re suite ,58 with re suite  as a seare suite . Crystal2+ ion-bound L1-EN structure and dna14-bound structures into a chimeric model containing the Mg2+ ion and DNA substrate including conformational changes associated with DNA binding. First, the chimeric model was built by ‘morphing’ the Mg2+ bound structure outside the immediate vicinity of the Mg2+ ion (12 Å) into the DNA-bound conformation. To accomplish this, harmonic tethers to the DNA-bound structure were applied and constrained force-field relaxation  the B-form conformation observed in all complexes, (ii) the alternative rotated conformation of DNA observed in the complex with dna14 or (iii) an alternative model of the polynucleotide chain containing the -O-[PO3H]–2-O- (holophosphoric acid diester) link in the transition state based on the alternative rotated conformation. Trigonal bipyramidal geometry was derived from the small molecule crystallographic data found in the Crystallography Open Database  in inteDatabase ,64 and kWe modeled a putative path for the conformational transition of the scissile bond phosphate from the B-form conformation to an alternative conformation and a holophosphate intermediate/transition state. Torsion driving was performed, changing alpha and gamma angles of the phosphate in steps of 1/100 of the total change from the initial to final conformation. Other torsions were relaxed with constraints . All procedures were performed in ICM-Pro  and implemented as scripts (available upon request).dna14 and dna17, and structures were solved to a resolution of 2.8 Å  with accession numbers of PBD 7N8S for the L1-EN-D145A/Y226K/dna14 complex and PDB 7N94 for the L1-EN-D145A/Y226K/dna17 complex. L1-EN-D145A/Y226K will be referred to hereafter as simply L1-EN in the context of these protein–DNA complexes.We attempted to co-crystallize L1-EN with DNA using a mutant form of L1-EN in which a residue essential for catalysis, Asp 145, is mutated to alanine (D145A) to prevent DNA cleavage. A set of short double-stranded DNA substrates containing the target sequence TTTTTAA and single nucleotide T/A overhangs to facilitate crystal lattice formation was used for crystallization trials Figure . Severaldna17 and one with dna14), the DNA substrate was bound to the protein in the same orientation and with similar distortions of the double helix conformation at the recognition site, as described below in more detail . The limited number of L1-EN interactions with bases in these structures rules out a conventional sequence recognition mechanism involving a network of nucleotide-specific hydrogen bonds. Base interactions by Pro197 and His198 of the βB5–βB6 β-hairpin loop and by Asn118 may contribute to recognition of two base pairs but cannot explain L1-EN’s preference for the entire target site. Consistent with this, previously published mutagenesis of this loop, including its replacement with loops from other retrotransposons did not result in altered sequence specific nicking  near the scissile bond (nucleotide positions +3 to +1)  Figure . The sug) Figure . This pr0 Figure . Both ma0 Figure ,53 basedOur crystal structures show that the conformation of DNA bound to L1-EN deviates significantly from that of a canonical B-form helix. Remarkably, the observed deviations in geometrical parameters were almost identical in all complexes of L1-EN with DNA for the middle part of the DNA substrates, particularly base pairs in positions –4 to + 4 relative to the scissile bond Figures . In cont+2 and G+3, pushing the latter away from the position that it would occupy in an ideal B-form helix and forcing widening of the minor groove to such an extent that its width becomes equal to that of a canonical major groove. The extensive contacts between the strand 1 sugar-phosphate backbone at positions +1 to +3 and the cleft formed by the βB5–βB6 and αB1–βB3 loops of L1-EN  was well defined and the significant extension around the scissile bond phosphate cannot be attributed to low-resolution noise. Moreover, water or other solvent molecules cannot be fitted and refined in the extra density. Instead, we modeled two alternative conformations for this phosphate group with one corresponding to the typical conformation expected for a B-form helix   ,72 (SuppDB:431D) . The phoDB:431D) .2+ ion  (2+ ion in a DNase I structure (2 during cryoprotection and refined the structure to 2.0 Å resolution. The structure of Mg-bound L1-EN that we obtained (PDB 7N8K) was almost identical to the published L1-EN structure (PDB: 1VYB)  and Asn147 (2.7 Å). This water molecule coordinated by Asp145 and Asn147 is also present in previously published structures of L1-EN (1VYB)  . Similartructure , where itructure ,67,75, atructure . To confB: 1VYB) , with a B: 1VYB) . The reftructure  by just  in APE1 ,67,75 an in APE1 . One of N (1VYB) . NotablyN (1VYB) ,66. Upone Figure , C. Modedna14 bound to L1-EN, the rotated-out phosphate group is even closer to Glu43  complexes showed that the phosphate group of the scissile bond in the rotated conformation is located exactly between the 5′ and 3′ ends of the cleaved product DNA  with active site amino acids around Mg2+ coming from our structure of Mg2+-bound WT L1-EN without DNA and the rest of the amino acids coming from our structure of L1-EN-D145A/Y226K complexed with dna14. These two crystal structures are quite close in the intermediate layer of 10–14 Å from the Mg2+ ion, with RMSD = 0.67 Å for all heavy atoms. This allowed a smooth transition without any significant distortions such as clashes, deviations of phi/psi pairs from favorable regions of the Ramachandran plot or unfavorable omega angles. Covalent geometry, i.e., bond lengths and angles, was automatically preserved in torsion-only relaxation. When DNA was introduced into the model of the complex with the scissile bond phosphate in the conformation of B-form DNA, one of the water molecules solvating Mg2+  and displaces two water molecules from the Mg2+ coordination shell  as a favorable transition state of the hydrolysis reaction in place of the regular ortho-phosphate (-O-[PO2]–-O-). Indeed, when modeled into the rotated phosphate DNA conformation, the transient holo-phosphate moiety became strongly coordinated by the Mg2+ ion via two of its oxygen atoms (at 2.0 Å and 2.1 Å from Mg2+), replacing the two water molecules and maintaining near-octahedral Mg2+ coordination  with respect to the typically much more stable PO4 which would only contribute one oxygen atom to the Mg2+ coordination shell  state, but we can speculate that from this point, phosphate O3′ may accept a proton, potentially, from an adjacent Mg product  indicate product .dna14 complex revealed rotation of the scissile bond phosphate group leading to two distinct states for the nucleophilic attack and a subsequent transition state. Similar local rotation is possible in a flipped-out conformation of DNA bound to APE1. Indeed, differences comparable to the phosphate rotation in L1-EN-bound DNA exist between conformations of APE1-bound DNA substrate  and DNADB 1DE8, ).2+ ion to attract negatively charged phosphate and stimulate its rotation. Therefore, we propose that this rotation is a result of the DNA helix distortion imposed by protein interactions leading to conformational tension around the scissile bond which forces the phosphate group out of the stable B-form conformation  with accession numbers of PBD 7N8S for the L1-EN-D145A/Y226K/gkab826_Supplemental_FilesClick here for additional data file.


The prepared glass-ceramics contain from 2 to 25 wt.% of crystalline NaMoO2PO4. The sodium-ion conductivity in these materials decreases up to one order of magnitude with an increase in the degree of crystallization due to the immobilization of sodium ions in crystalline NaMoO2PO4. The transport of sodium ions in these materials occurs primarily through the dominant continuous glassy phase, and it is weakly affected by the sporadically distributed crystalline grains. However, the prepared glass-ceramics exhibit high proton conductivity in a humid atmosphere and remarkable humidity-sensing properties; this could be related to crystalline NaMoO2PO4, which provides sites for water adsorption. The glass-ceramic prepared at 450 °C for 24 h shows the best humidity-sensing performance among all samples, showing an increase in proton conductivity for more than seven orders of magnitude with the increase in relative humidity from 0% to 95%. Under a highly humid atmosphere (95% relative humidity and 25 °C), the proton conductivity of this glass-ceramic reaches 5.2 × 10−3 (Ω cm)−1. Moreover, the electrical response of these materials on the change in the relative humidity is linear and reversible in the entire range of the relative humidity, which indicates that they are novel promising candidates for application as humidity sensors. A series of glass-ceramics were prepared by heat-treatments of 40Na The controlled crystallization of glasses is an advantageous procedure for the preparation of glass-ceramics with specific physicochemical properties ,2. AccorIn the last several decades, a significant research effort has been directed toward the preparation and development of glass-ceramics with high electrical conductivity. Indeed, many studies have shown that ionic ,9,10,11,2O-xMoO3-(60-x)P2O5, 0 ≤ x ≤ 50 mol% and showed that the replacement of the conventional glass former (P2O5) with the conditional glass-forming oxide (MoO3) causes a significant increase in sodium-ion conductivity with the maximum in conductivity at x = 30 mol% , where ω = 2πf, f is the measuring frequency, i = (−1)1/2 is the imaginary unit, A is the constant, and α is a power law exponent with a value in the range of 0 < α < 1 [α = 1, the CPE acts as an ideal capacitor, whereas for α = 0, it is a resistor. The fitting parameters (resistance R and parameters A and α) at various temperatures for the 490 C-12 h glass-ceramic are given in A1, representing a pseudo-capacitance, ranges from 10−12 to 10−10 sα Ω−1, whereas parameter A2 exhibits significantly larger values, 10−7–10−5 sα Ω−1, which is in agreement with the capacitance of the bulk and the sample–electrode interface, respectively [The corresponding equivalent electrical circuit for the impedance plots shown in  < α < 1 . For α =ectively .R) and electrode geometry, DC conductivity was calculated as explained in the Materials and Methods Section. For all samples, the DC conductivity exhibits Arrhenius temperature dependence  vs. 1000/T using the equation σDCT = σ0exp(−EDC/kBT), where σDC is the conductivity, σ0 is the pre-exponential factor, kB is the Boltzmann constant, and T is the temperature (K). The DC conductivity at 30 °C and the determined activation energy, EDC, for the parent glass and prepared glass-ceramics are shown in EDC, for the glass-ceramics are nearly constant and very close to the activation energy for the parent glass, which supports our conclusion that the conduction process in all glass-ceramics takes place primarily through the amorphous glass matrix. From the values of resistance −1 (see σDC = 5.2 × 10−3 (Ω cm)−1 at RH = 95%. This huge increase in conductivity is related to the transport of protons due to adsorbed water on the surface of the glass-ceramic.In the course of the electrical characterization of the glass-ceramics prepared at 450 and 490 °C, it was found that their electrical conductivity differed significantly depending on whether the measurements were performed in ambient air or dry nitrogen. While conductivity measured in dry nitrogen was of the order of ~102O ⟷ H3O+ + OH−, and charge transport occurs when H3O+ releases a proton to a neighboring water molecule, which accepts it while releasing another proton, and so forth (Grotthuss chain reaction). However, at high humidity, liquid water condenses in the pores and electrolytic conduction takes place along with the proton transport in the adsorbed layers [It is known that, in porous ceramics, the mechanism of water-related conduction includes the physical adsorption of water molecules at the grain surface and water condensation in the pores depending on the relative humidity ,32. At ld layers .2PO4, which provides sites for the adsorption of water (note that the fully amorphous samples prepared by heat-treatment at 380 °C do not exhibit humidity-dependent conductivity) and porous microstructure. In line with this, we observed that the 450 C-24 h sample showed the best humidity-sensing performance in terms of the change in magnitude of conductivity with the change in relative humidity  glass at 380 (Tg), 450 and 490 °C (Tc) and for various times . The XRPD analysis shows that the heat-treatment at 380 °C, irrespective of the heat-treatment time, does not induce crystallization processes, whereas at higher temperatures, NaMoO2PO4 crystallizes. With the increase in the heat-treatment temperature from 450 to 490 °C, the amount of crystalline phase increases and reaches a maximal value of 25 wt.%; however, at both temperatures, the crystallization process completes within 12 h. The sodium-ion conductivity decreases for up to one order of magnitude with the increase in the fraction of crystalline NaMoO2PO4, which is related to the immobilization of the fraction of sodium ions in the crystals. The prepared glass-ceramics show excellent humidity-sensing properties due to NaMoO2PO4 crystals, which provide sites for the adsorption of water. The proton conductivity of the best-performing glass-ceramic prepared at 450 °C and for 24 h increases by more than seven orders of magnitude with the increase in relative humidity from 0% to 95%. Based on the high proton conductivity, as well as the linear and reversible electrical response over the entire relative humidity range, these glass-ceramics appear to be excellent novel candidates for application as humidity sensors.A series of glass-ceramics were prepared by heat-treatments of 40Na


Triticum aestivum) obtained by fermentation with selected lactobacillus and natural yeast strains, we used an in vivo model of hyperglycemia-induced retinal damage, the fruit fly Drosophila melanogaster fed with high-sucrose diet. Lisosan G positively affected the visual system of hyperglycemic flies at structural/functional level, decreased apoptosis, and reactivated protective autophagy at the retina internal network. Also, in high sucrose-fed Drosophila, Lisosan G reduced the levels of brain ROS and retina peroxynitrite. The analysis of oxidative stress-related metabolites suggested 7,8-dihydrofolate, uric acid, dihydroorotate, γ-L-glutamyl-L-cysteine, allantoin, cysteinyl-glycine, and quinolate as key mediators of Lisosan G-induced inhibition of neuronal ROS, along with the upregulation of glutathione system. Of note, Lisosan G may impact oxidative stress and the ensuing retinal cell death, also independently from autophagy, although the autophagy-ROS cross-talk is critical. This study demonstrated that the continuous supplementation with the alimentary integrator Lisosan G exerts a robust and multifaceted antioxidant effect on retinal neurons, thus providing efficacious neuroprotection of hyperglycemic eye.Aberrant production of reactive oxygen species (ROS) is a common feature of damaged retinal neurons in diabetic retinopathy, and antioxidants may exert both preventive and therapeutic action. To evaluate the beneficial and antioxidant properties of food supplementation with Lisosan G, a powder of bran and germ of grain ( Retinal cells are extremely metabolically active and enriched of mitochondria, the predominant intrinsic sources of reactive oxygen species (ROS) ,2. In adDrosophila melanogaster fed with high-sucrose diets as an in vivo model of hyperglycemia-induced neuronal damage . N. N22]. NOxidative stress was then determined using the DCFH-DA probe. In fly brains of Drosophila, the levels of DCF fluorescence induced by high-sucrose regimens were restored by Lisosan G, indicating a significant reduction of ROS generation C.p < 0.05; These data prompted us to further dissect the oxidative stress-related machinery modulated by Lisosan G in our system. We performed an untargeted metabolomics analysis to identify changes in metabolites linked to cellular redox state. A total of 146 metabolites was annotated. Multivariate PLS-DA statistical analysis was carried out A. ResultFinally, we calculated the intracellular concentrations of the reduced form of glutathione (GSH) and oxidized form of GSH (GSSG)  since thmelanogaster were attracted by light, avoiding shaded areas. The investigation of visual system response by a phototaxis assay may thus provide a functional correlation to a structural/molecular alteration of an eye [When given a choice, adult D. f an eye ,22,23. ID. melanogaster raised in high-sucrose diets displayed typical neurodegenerative features, namely apoptosis and dysfunctional autophagy flux, primarily affecting the internal network of the retina [The hyperglycemic eye of adult e retina . As showe retina ,22 remarSimilarly, the analysis of LC3 and p62 autophagy proteins in 35% sucrose-treated flies revealed a large amount of LC3/p62 clusters in the lamina when compared with 5% normoglycemic control , while tIn this respect, the accumulated autophagic vesicles with double or multiple-membrane containing electron-dense material , vacuoleIn another set of experiments, 35% sucrose-treated flies were reared for 10 days on a diet supplemented with rapamycin, a widely used autophagy activator. Rapamycin was used at the optimum concentration of 10 nM, already shown to be effective in Drosophila eyes . As expeMoreover, autophagy activation improved the responsiveness to the light of hyperglycemic Drosophila A. In parAccordingly, in the 35% sucrose + rapamycin-treated flies, the array of ommatidia and rhabdomeres in longitudinal and cross eye sections appeared similarly restored both in the absence and in the presence of Lisosan G B,C.Previous observations in adult flies fed for 10 days with 35% sucrose diets showed a clear decrease in light responsiveness as a consequence of vision defects . HyperglD. melanogaster provides further evidence that Lisosan G has potential nutraceutical profiles against hyperglycemia-induced degeneration of retinal neurons. There is a general agreement that nutrients could offer a new line of defense against DR, with the efficacy of different formulations and administration as a key aspect to be considered [In streptozotocin-induced diabetic rats, the natural substance Lisosan G, commercialized as a nutritional supplement, showed anti-inflammatory effects when administered for oral gavage, with protective actions against neural/functional and vascular defects characteristic of DR . Our in nsidered . In thisnsidered ,50, Lisonsidered .At the functional level, Lisosan G-supplemented food ameliorated the visual response of 35% sucrose-treated Drosophila and decreased the presence of cell death/apoptotic features in the retina internal network. These data point to the correlation between the anti-apoptotic effects of Lisosan G and functional recovery. Besides, we provide further mechanistic aspects since Lisosan G reactivated autophagy in the retina of hyperglycemic flies. Accordingly, typical hallmarks of neuroretina degeneration, for instance, vacuoles and damaged mitochondria, were not detectable at the ultrastructural level in eye neurons. Cell death-suppressing and differentiating effects of autophagy are required for Drosophila eye formation . Also, dAmong human tissues, the retina is one of the highest consumers of oxygen and is prone to oxidant burden. Aberrant ROS are responsible for retinal injury and a common feature of retinal neurons in DR ,7,8,9,112O2 generation enough to have detrimental cellular effects as previously reported [D. melanogaster, uric acid is metabolized to allantoin by the enzyme urate oxidase [In our system, we also attempted to profile the mechanisms of oxidative stress-related perturbations in the Drosophila brain tissue at the level of metabolite composition. Among the considerably accumulated metabolites in hyperglycemic flies, we detected quinolate, which represents one of the most neurotoxic metabolites causing the formation of lipid peroxidation products in nervous tissue . Toxic creported . We obsereported . GGSH isreported . Hyperglreported . Moreovereported . Not onlreported . Our datreported , which rreported . Unlike  oxidase . In urat oxidase . InteresEmerging evidence reveals that oxidative stress-induced disruption of autophagy in retinal cells is closely related to the pathogenesis of DR . In thisExogenous antioxidants of natural origin may be used to preserve redox homeostasis in the diabetic retina ,26,27. TD. melanogaster is considered a very potent in vivo tool to study human neurodegenerative diseases, including eye conditions, to complement more traditional vertebrate systems [mdx mice, the most commonly employed models in Duchenne muscular dystrophy research [ systems ,69,70,71research . Drosoph