Pathological forms of TAR DNA-binding protein 43 (TDP-43) are present in motor neurons of almost all amyotrophic lateral sclerosis (ALS) patients, and mutations in TDP-43 are also present in ALS. Loss and gain of TDP-43 functions are implicated in pathogenesis, but the mechanisms are unclear. While the RNA functions of TDP-43 have been widely investigated, its DNA binding roles remain unclear. However, recent studies have implicated a role for TDP-43 in the DNA damage response.2O2 treatment. We investigated the consequences of depletion of TDP-43 on DNA repair using small interfering RNAs. Specific non homologous end joining (NHEJ) reporters (EJ5GFP and EJ2GFP) and cells lacking DNA-dependent serine/threonine protein kinase (DNA-PK) were used to investigate the role of TDP-43 in DNA repair. To investigate the recruitment of TDP-43 to sites of DNA damage we used single molecule super-resolution microscopy and a co-immunoprecipitation assay. We also investigated DNA damage in an ALS transgenic mouse model, in which TDP-43 accumulates pathologically in the cytoplasm. We also examined fibroblasts derived from ALS patients bearing the TDP-43 M337V mutation for evidence of DNA damage.We used NSC-34 motor neuron-like cells and primary cortical neurons expressing wildtype TDP-43 or TDP-43 ALS associated mutants , in which DNA damage was induced by etoposide or HWe demonstrate that wildtype TDP-43 is recruited to sites of DNA damage where it participates in classical NHEJ DNA repair. However, ALS-associated TDP-43 mutants lose this activity, which induces DNA damage. Furthermore, DNA damage is present in mice displaying TDP-43 pathology, implying an active role in neurodegeneration. Additionally, DNA damage triggers features typical of TDP-43 pathology; cytoplasmic mis-localisation and stress granule formation. Similarly, inhibition of NHEJ induces TDP-43 mis-localisation to the cytoplasm.This study reveals that TDP-43 functions in DNA repair, but loss of this function triggers DNA damage and is associated with key pathological features of ALS. Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder associated with atrophy of both upper and/or lower motor neurons, leading to muscle wasting due to denervation . ALS oveTDP-43 is a RNA- and DNA-binding protein normally located in the nucleus, where it is involved in transcription, splicing, RNA metabolism and miRNA biogenesis . Whilst The genome is under constant attack from both environmental agents and normal metabolic processes. Thus, maintaining genomic integrity is essential because damage to DNA can induce mutations and seriously compromise cellular viability. DNA damage induces the ‘DNA damage response (DDR)’, signalling pathways that aim to detect and repair the damage. However, persistent DNA damage and defects in DNA repair lead to apoptosis. The most cytotoxic lesions are double-stranded DNA breaks (DSBs) which are primarily repaired by error-prone non-homologous end joining (NHEJ) in neurons , unlike SETX, VCP, NEK1, C21orf2, and CCNF [C9ORF72, which cause a major proportion of familial ALS cases, induce DNA damage in neuronal cells and motor neurons of ALS patients [Fused in Sarcoma (FUS) has striking functional and pathological similarities to TDP-43 in ALS, and it normally performs important functions in DNA repair . Interesand CCNF –27. Recepatients . This wapatients . TDP-43 patients  and two patients , 32. It patients , and expIn this study we demonstrate that TDP-43 is a DNA repair protein that functions in classical, but not alternative, NHEJ in neuronal cells. However, ALS TDP-43 mutants A315T and Q331K lack this normally protective function, leading to the accumulation of DNA damage. Furthermore, in mouse models displaying pathological TDP-43 that develop features of ALS , DNA damage is present before the onset of disease. These mice express cytoplasmic, wildtype TDP-43 and thus bear semblance to sporadic ALS. We also show that DNA damage induces features typical of TDP-43 pathology: mis-localisation to the cytoplasm and SG formation. Moreover, inhibition of classical NHEJ repair also leads to TDP-43 mis-localisation to the cytoplasm. This study therefore places aberrant DNA damage responses onto the pathophysiolology of TDP-43, which is associated with almost all ALS cases. It therefore highlights the DDR as a novel therapeutic target for ameliorating TDP-43 pathology in ALS.Details of the patients used in this study are summarised in Table Monogenic B6;C3-Tg (NEFH-tTA)8Vle/J  mice and monogenic B6;C3-Tg (tetO-TARDBP*)4Vle/J  mice were obtained from the Jackson Laboratory (USA) and maintained on a mixed B6/C3H background  and were crossed to obtain bigenic rNLS and littermate control mice. All the mice were genotyped according to the Jackson Laboratory protocols. Expression of the hTDP-43–rNLS transgene was confirmed by cytoplasmic TDP-43 staining and increased levels of human TDP-43 by immunoblotting. Mice were provided with feed containing 200 mg/kg doxycycline  until 6–8 weeks of age.Fibroblasts from both ALS patients and pre-symptomatic carriers bearing the M337V mutation were obtained and cultured as previously described [2. Neurons were transfected with Lipofectamine 2000 at 3 or 5 DIV and fixed with 4% paraformaldehyde after 24 h.Primary cortical neuronal cultures were prepared from mouse C57BL/6 E16 brains. After isolation, cells were plated on glass coverslips covered with poly-L-lysine (Sigma-Aldrich), in B27 neurobasal medium supplemented with 1% glutamate and 1% penicillin/streptomycin and incubated at 37 °C and 5% CO2.NSC-34/CHO-9/XR-C1 or Neuro2A cells were cultured in DMEM medium with 10% FBS at 37 °C and 5% CO2O2 for 1 h. For inhibition of DNA-PK activity, a specific inhibitor was used, NU 7441 .DNA damage was induced by treatment with 13.5 μM etoposide or 100 μM HAfter washing with 1 x PBS, cells were permeabilised in 0.1% Triton X-100 in PBS for 5 min and blocked in 1% BSA in PBS for 30 min. Staining was performed overnight with the following primary antibodies at a concentration of 1:500: rabbit anti-γH2AX ; rabbit anti-53BP1 , mouse anti-DNA-PKcs (Abcam), rabbit anti-TDP-43 C-terminus (405–414) , and rabbit anti-phospho-TDP-43 . The following secondary antibodies were used: anti-rabbit Alexa Fluor 488, (Life technologies), anti-mouse Alexa Fluor 594 (Life technologies) or anti-rabbit Alexa Fluor 647 antibodies  for 2 h. The nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich) or DAPI (Sigma Aldrich).The Comet assay was adapted from a previous method . BrieflyTwo small interfering RNAs (siRNAs) described previously , targetiAfter deparaffinisation, antigen retrieval in 10 mM citrate buffer with 0.1% Tween for 30 min at 80 °C was performed. Sections were blocked with normal donkey serum for 30 min and then stained with anti-γH2AX  or anti-human TDP-43 antibodies  for 24 h at 4 °C, and after three washes with PBS, the tissues were incubated with AlexaFluor 488/564  conjugated secondary antibodies for 2 h at 4 °C. The nuclei were counterstained with Hoechst 33342 (Sigma-Aldrich).Fluorescently labelled cells were acquired using a Zeiss AxioImager epi-fluorescence microscope (Zeiss) or a Zeiss LSM 880 confocal microscope.The following mammalian expression plasmids were used as described previously: pimEJ5GFP (Addgene # 44026) and EJ2GFP-puro (Addgene # 44025) were a gift from Jeremy Stark, TDP-43 wildtype-EGFP, TDP-43 A315T-EGFP, TDP-43 Q331K-EGFP, pEGFP-N1 , TDP-43 Mouse brain tissues were thawed on ice and sonicated in 5x volumes of BICARB buffer . Samples were centrifuged at 100,000 x g at 4 °C for 30 min. Supernatants were collected as BICARB soluble fractions. The human fibroblasts or NSC-34 cells were lysed in 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% (wt/v) SDS, 1% protease inhibitor cocktail (Sigma) and 1% phosphatase inhibitor cocktail (Sigma). Protein concentrations were determined using a BCA protein assay (Pierce). Twenty micrograms of soluble fractions were analysed by 4–15% gradient SDS-PAGE and blotted onto nitrocellulose membranes (Bio-Rad). Blots were blocked with 5% skim milk and incubated with primary antibodies diluted in blocking buffer overnight at 4 °C. The next day, unbound primary antibodies were removed by washing and blots were incubated with HRP-conjugated secondary antibodies for 1–2 h at room temperature. Images were developed with ECL blotting substrates (Bio-Rad) and a ChemiDoc Gel imaging system (Bio-Rad). Antibodies used for immunoblotting were: rabbit anti-γH2AX , mouse anti-GAPDH , rabbit anti-GAPDH , rabbit anti-TDP-43 , goat anti-mouse IgG and IgM HRP conjugate , and goat anti-rabbit IgG antibodies, peroxidase conjugated .Immunoprecipitation was performed using GFP-Trap MA (Chromotek), which utilizes small recombinant alpaca antibody fragments covalently coupled to the surface of agarose magnetic beads. Briefly, NSC-34 transfected cells were lysed in lysis buffer  with a cocktail of protease inhibitors (Roche) and 1 m PMSF for 30 min. on ice. Then, cell lysates were centrifuged at 20,000 x g for 10 min at 4 °C. After centrifugation, the lysates were transferred to a pre-cooled new tube. The protein concentration was quantified using a BCA kit (Thermo Fisher Scientific). Five hundred microgram of the lysate was taken for further processing. Fifty microliter of diluted lysates were retained for immunoblotting analysis for the input samples. Twenty five microliter of equilibrated GFP-Trap MA beads were added to diluted lysates and rotated end-over-end overnight at 4 °C. The following day, the beads were separated magnetically from the supernatant and washed three times. Magnetically separated beads were resuspended in 100 ul 2x SDS-sample buffer and boiled for 10 min at 95 °C to dissociate immunocomplexes from GFP-Trap MA beads. The beads were separated from the supernatant magnetically and the supernatant was subjected to immunoblotting.NSC-34 cells were transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturers’ protocol.NHEJ reporters, pimEJ5GFP or EJ2GFP-puro, were pre-digested with I-SceI restriction enzyme and the digestion products following purification were run on a 1% agarose gel to confirm that each reporter had been digested. Cells were co-transfected with mCherry-tagged TDP-43 wildtype, A315T, or Q331K, and pre-digested NHEJ reporter by nucleofection using Cell Line Nucleofector Kit V or Human Dermal Fibroblast Nucleofector™ Kit, according to the manufacturers protocol (Lonza). The efficiency of GFP restoration through NHEJ was estimated as the percentage of cells with both mCherry and GFP signals, in proportion to the total number of cells with mCherry expression , 38.NSC-34 cells were cultured in DMEM media (Gibco) on 13 mm coverslips (Menzel-Glasser) in 24 well plates and transiently transfected with EGFP-tagged TDP-43 using Lipofectamine 2000 (Invitrogen). To induce DNA damage, cells were treated with 13.5 μM etoposide  or DMSO as a control. After 48 h, cells were fixed with 4% PFA and mounted with Dako fluorescence mounting media. Images were taken with an epi-fluorescent microscope (Zeiss). For identification of SGs containing TDP-43, immunocytochemistry using a mouse anti-HuR (Invitrogen) antibody was performed. Primary cortical neurons were cultured in B27 neurobasal medium supplemented with 1% glutamate and 1% penicillin/streptomycin. Primary neurons were transfected with EGFP-tagged TDP-43 with Lipofectamine 2000 at 3 or 5 DIV. After 24 h neurons were treated with 13.5 μM  or DMSO as a control for 1 h and fixed with 4% paraformaldehyde. Slides were mounted with Dako fluorescence mounting media. Images were taken with an epi-fluorescent microscope (Zeiss).NSC-34 cells and primary neurons were selected for imaging only using the fluorescence/confocal microscopy channel displaying expression of GFP or mCherry-tagged TDP-43. Human fibroblasts were selected for imaging using the confocal microscopy channel displaying Hoechst staining of the nuclei. Hence, the channel displaying the presence of DNA damage foci was invisible to the researcher when the cells were selected for analysis. This strategy allowed us to identify transfected cells for analysis, but also it ensured that the researcher was blinded to the presence of DNA damage foci. Similarly, after imaging, cells were selected for analysis based only on the fluorescence microscopy channel displaying TDP-43 expression. Analysis of DNA damage foci was performed by three people. DNA damage images on immunofluorescently labelled NSC-34 cells or human fibroblasts were collected using a Zeiss LSM 880 confocal microscope with objective Plan-Apochromat 63x/1.4 Oil DIC M27 and z-stacks with intervals of 0.25 μm. Imaris software was used for three-dimensional reconstructions and quantification of the total area and volume of foci in NSC-34 cells. DNA damage analysis in primary cortical neurons or human fibroblasts was evaluated as the average number of γH2AX/53BP1 foci using ImageJ software. To quantify DNA damage in the TDP-43 rNLS mouse model, neurons specifically expressing the transgene were identified by double immunohistochemistry using an γH2AX antibody and an antibody specific for human, rather than mouse, TDP-43, because this model expresses human rNLS TDP-43 via a neuronal-specific promoter. Neurons were chosen at random within the cortex at different depths of coronal cross sections, and at similar levels in TDP-43 rNLS and control mice. Similar to the primary neurons, DNA damage was evaluated as the average number of γH2AX foci using ImageJ software.Fixed NSC-34 cells prepared as described above on coverglass were imaged using a custom home-built SR microscope as previously described , 40. ThiOutput TIFF stacks were processed using the ImageJ plugin ThunderSTORM with default settings and then refined further as detailed by the plugin authors . Specifip value < 0.05 was considered statistically significant.Data represent mean values and the standard error of the mean. Results were analysed using one-way or two-way analysis of variance (ANOVA) followed by Sidak, Tukey or Dunnett C post hoc tests, t-Student test or Mann Whitney test. The statistical analyses were performed using OriginPro 8.0, IBM SPSS or GraphPad Prism 7 software, and 2O2, which induces DSBs by oxidative stress [2O2 for 1 h, 24 h after transfection. The concentration of H2O2 was used as previously [2O2 treatment, foci of significantly smaller area and volume were detected in cells expressing wildtype TDP-43 compared to untransfected cells  or those expressing EGFP only , Fig. 202), or mutants (detailed below)). This result demonstrates that over-expression of wildtype TDP-43 is protective against persistent DNA damage induced by two different mechanisms. The DNA damage foci observed were all present in the nucleus, indicating that TDP-43 was protective against DNA damage in the nucleus, rather than in mitochondria.When DNA damage is induced and DSBs form, phosphorylation of the histone variant H2AX follows . The rese stress , a proceeviously . Followip < 0.05, Q331K, **p < 0.01; volume: A315T, *p < 0.05, Q331K, **p < 0.01) or H2O2 treatment  or those expressing mCherry only  and Q331K cells ) and immunoblotting ). Similar findings have been reported for FUS, which was shown to be involved in the recognition of DSBs by inhibiting H2AX phosphorylation [Next, we examined if TDP-43 knock down has the opposite effect to TDP-43 overexpression. We used two previously described specific siRNAs , targetirylation . Our datp < 0.001, Fig. p < 0.05) or Q331K (***p < 0.001) mutants compared to those expressing wildtype TDP-43 after etoposide treatment  and Q331K (****p < 0.0001). This indicates that fewer DSBs were present in wildtype TDP-43 cells, but not in mutant expressing cells, compared to control cells  of each comet is defined as the product of the comet tail length and the fraction of total DNA within the tail. Increased values indicate that more prominent DNA damage is present. This analysis revealed significantly lower OTM values for wildtype TDP-43 expressing cells after etoposide treatment compared to TDP-43 mutants A315T (*dSTORM SR imaging of immunolabelled 53BP1 [a-e). In contrast, mCherry consistently displayed a low Interaction Factor with 53BP1, indicative of low or random colocalization . This confirmed that minimal colocalization artefacts were present due to crosstalk, bleed-through, or photoconversion, which we minimized using previously described methods [p < 0.001, Fig. 5a-e).These results taken together suggest that TDP-43 functions in DNA repair. However, it is unclear whether TDP-43 is recruited to DNA damage foci, where it could perform a specific DNA repair role at the DSB. Alternatively, it could perform a more indirect function in DNA repair, and therefore may not be recruited to sites of DNA damage. Hence, next we investigated the recruitment of TDP-43 to DNA damage 53BP1) foci in more detail by super-resolution microscopy (SR), using an assay previously developed for probing individual DSB sites . In celled 53BP1 . We analed 53BP1 . Wildtyp3BP1 foci methods . InteresTo confirm that TDP-43 is recruited to sites of DNA damage, we performed immunoprecipitation of lysates from NSC-34 cells expressing TDP-43 or controls expressing GFP only. We immunoprecipitated GFP-tagged TDP-43 or GFP lysates using ChemoTek GFP-Trap magnetic beads and performed immunoblotting for 53BP1. 53BP1 precipitated together with wildtype, A315T and Q331K TDP-43, but not with GFP only  or Q331K mutants (*p < 0.05) were undergoing classical NHEJ compared to those expressing wildtype TDP-43, demonstrated by fewer GFP-positive cells   Fig. c. These p < 0.001, Fig. p < 0.001), or Q331K (***p < 0.001), compared to wildtype TDP-43 , consisting of Ku and DNA-PK catalytic subunits (DNA-PKcs), is a critical component that is recruited early in classical NHEJ . To furt-43 Fig. a. Hence,p < 0.001, Fig. To confirm these findings, we next examined the recruitment of endogenous TDP-43 to DNA-PKcs foci in cells after etoposide treatment using SR microscopy as above. The Interaction Factor was calculated between TDP-43 and DNA-PKcs. Little colocalization was detected in DMSO treated cells, as expected (Interaction Factor < 0.2). However, this increased significantly upon induction of DNA damage by etoposide, indicating that TDP-43 is recruited to DNA-PKcs specifically when DNA damage is induced, and hence to regions of DSBs (Interaction Factor > 0.4). These data confirm that TDP-43 is recruited to sites of NHEJ repair  compared to controls, and elevated expression of γH2AX was also present in tissue lysates from these mice , revealing the accumulation of DNA damage. These data therefore indicate that DNA damage is already present, together with the onset of TDP-43 accumulation in the cytoplasm, in this mouse model of ALS. In addition, they reveal that DNA damage is triggered at early disease stages, implying it is relevant to pathophysiology and not simply a consequence of neurodegeneration. Furthermore, these findings suggest that DNA damage is associated with pathological localisation of TDP-43 in the cytoplasm, as well as familial ALS-associated mutant TDP-43, thus placing DNA damage onto the broad pathophysiology of ALS, not just cases caused by TDP-43 mutations.Next, we examined the relationship between DNA damage and TDP-43 in a transgenic TDP-43 mouse model , which ep < 0.01, Fig. . However, mutants TDP-43 Q331K and A315T were recruited to a lesser extent than wildtype TDP-43 . These results indicate that TDP-43 is recruited to SGs, which are implicated as precursors of inclusions in ALS [p < 0.05)  formation. We examined this possibility in NSC-34 cells and primary mouse cortical neurons expressing wildtype or mutants Q331K or A315T TDP-43, treated with 13.5 μM etoposide for 48 h. In NSC-34 cells, induction of DNA damage by etoposide triggered the formation of SGs, confirmed by immunocytochemistry for HuR  Fig. b. Similap < 0.05, Fig. p < 0.05, Fig. As our results implicate TDP-43 in NHEJ DNA repair and DNA damage in the formation of TDP-43 pathology, we next examined whether inhibition of classical NHEJ DNA repair can directly induce TDP-43 pathology. Therefore, we inhibited DNA-PK activity pharmacologically using selective DNA-PK inhibitor NU 7441  in cellsTo confirm that our findings are related to human ALS, and are not an artefact of protein overexpression systems, we next investigated DNA damage, NHEJ repair and TDP-43 pathology in previously described fibroblasts obtained from subjects bearing the TDP-43 M337V mutation [p < 0.05, Fig. (i)). Moreover, the number of 53BP1 foci/cell was significantly increased further in the affected ALS patient fibroblasts compared to those from control individuals ) and the pre-symptomatic patient (*p < 0.05). These data therefore imply that DNA damage is present in mutant TDP-43-associated ALS and may increase during disease course. The fibroblasts obtained from the symptomatic M337V patients were difficult to culture because they were prone to undergo cell death. Hence the next experiments were performed on fibroblasts from pre-symptomatic cases only, which could be readily cultured. DNA damage was examined further by immunoblotting for DNA damage markers 53BP1 and γH2AX in the pre-symptomatic TDP-43 M337V fibroblasts, compared to control individuals  and (iii), respectively). More expression of 53BP1 and γH2AX was present in TDP-43 M337V fibroblasts, compared to controls. These results therefore reveal the presence of DNA damage in human ALS fibroblasts with the TDP-43 M337V mutation.DNA damage was first examined by immunocytochemistry for 53BP1. The average number of 53BP1 foci per cell was significantly elevated in fibroblasts derived from the pre-symptomatic cases compared to the control lines . These results therefore imply that there is an association between TDP-43 pathology and DNA damage in cells without artificial protein overexpression. These data together confirm the relationship between impaired DNA repair and human ALS associated with TDP-43 mutations.Next, we investigated NHEJ repair in fibroblasts from pre-symptomatic TDP-43 M337V patients using the EJ5GFP reporter, to quantitate total NHEJ as above. TDP-43 M337V or control fibroblasts were co-transfected with pre-digested EJ5GFP reporter and empty mCherry vector as a control for transfection efficiency. NHEJ repair was found to be impaired in TDP-43 M337V fibroblasts compared to controls, demonstrated by fewer fibroblasts with GFP expression, indicating restoration of the EJ5GFP reporter  are most relevant to neurodegeneration in ALS. Our findings point to a loss of nuclear function of TDP-43 as a pathogenic process in ALS. Whilst the RNA-related functions of TDP-43 are well described, this study also demonstrates that an important DNA-specific function is perturbed in ALS. In addition, induction of DNA damage leads to mis-localisation of TDP-43 to the cytoplasm, and the formation and recruitment of TDP-43 to SGs, revealing that DNA damage can induce features characteristic of TDP-43 pathology. SGs positive for mutant TDP-43, Q331K and A315T, were also formed upon induction of DNA damage. Interestingly, however fewer SGs were formed in these cells compared to those expressing wildtype TDP-43. The results are consistent with the notion that functional TDP-43 is required for efficient SG dynamics .Similarly, inhibition of NHEJ leads to TDP-43 mis-localisation to the cytoplasm. Therefore, this raises the possibility that persistent DNA damage and defective DNA repair could further perturb the nuclear functions of TDP-43 in DNA repair, thus exacerbating nuclear DNA damage. Surprisingly, TDP-43 knockdown prevented H2AX phosphorylation, an important event in the detection of DSBs, suggesting that TDP-43 functions in the recognition of DSBs. Similar findings were reported for FUS, implying that it functions in the detection of DSBs .The formation of 53BP1 foci in cells expressing TDP-43 implicates NHEJ, the only DSB repair mechanism available to neurons, as the mechanism of DNA repair mediated by TDP-43, and that which is lost from mutant TDP-43 in ALS. NHEJ is already more error-prone than HR , and whe, Fig. To obtain more detailed insights into TDP-43 function in NHEJ, we examined the formation of DNA-PKcs foci in NSC-34 cells following DNA damage. Classical NHEJ is initiated when a Ku70/80 heterodimer binds to DSBs, which recruits DNA-PKcs to create the DNA-PK complex, forming DNA damage foci. This event is therefore early in classical NHEJ and it facilitates binding of the other DNA repair proteins involved in this process , 64. We 52O2, a source of oxidative DNA damage. Moreover, we also demonstrate that impaired NHEJ was present in fibroblasts bearing the M337V TDP-43 mutation.In this study we also demonstrate that TDP-43 is involved in the phosphorylation of γH2AX, further implying that TDP-43 functions upstream of 53BP1. In addition, DNA damage was detected in mutant TDP-43 Q331K patient tissues, and in vitro, TDP-43 Q331K mis-localisation to the cytoplasm prevented nuclear translocation of the XRCC4-DNA Ligase 4 complex . In thisFurther SR imaging demonstrated that the TDP-43 mutants are recruited less efficiently to DNA damage foci than wildtype TDP-43, implying that this is one mechanism triggering impairment of NHEJ in ALS. However, it is important to note that the TDP-43 mutants were recruited more than mCherry only to DNA damage foci, revealing that at least a proportion of mutant TDP-43 does reach the sites of DNA damage. It is therefore possible that additional deficits also exist for mutant TDP-43, such as functional deficiencies in the repair of DSBs, which combined with impaired recruitment of TDP-43 to these sites, enhances the lack of DNA repair and thus the accumulation of DNA damage. Together, these results therefore reveal that TDP-43 is involved in DNA-PKcs-dependent DDR, confirming its role in NHEJ. Importantly, the TDP-43 mutants (Q331K and A315T) were defective in this process, revealing that they lose this important and specific cellular function, resulting in accumulation of DNA damage in ALS.Our study also describes a compelling link between DNA damage, defects in DNA repair and TDP-43 pathology in ALS. The presence of pathological forms of TDP-43 in affected neurons is a characteristic feature of 97% of ALS , 66. A pConsistent with this notion, DNA damage was present at early stages of disease in TDP-43 rNLS mice, coincident with TDP-43 accumulation in the cytoplasm and prior to the presence of neurodegeneration in this mouse model. Importantly, TDP-43 rNLS mice express mis-localised wildtype TDP-43 rather than mutant TDP-43, and therefore arguably this model is closer to sporadic ALS than TDP-43-associated familial ALS. Since these mice show an early decrease in levels of normal endogenous TDP-43 in the nucleus of neurons due to auto-regulation of expression , the incIncreasingly DNA damage is becoming linked to neurodegenerative diseases . ApoptosFinally, we investigated the relevance of our findings to human ALS using fibroblasts from an ALS patient bearing the TDP-43 M337V mutation and two pre-symptomatic TDP-43 M337V carriers. Enhanced DNA damage was present in affected and pre-symptomatic fibroblasts. Importantly, significantly more DNA damage was present in pre-symptomatic affected fibroblasts compared to presymptomatic fibroblasts, implying an active role for DNA damage in the development of ALS. However, it is possible that the presence of the TDP-43 M337V mutation renders pre-symptomatic fibroblasts more vulnerable to DNA damage in vitro. Similarly, total NHEJ was impaired in TDP-43 M337V pre-symptomatic fibroblasts compared to control fibroblasts. The upregulation of 53BP1, which promotes classical NHEJ , is alsoThe findings of our study raise the possibility that failure of DNA repair processes by mutant TDP-43 may trigger a vicious cycle, in which the resulting accumulation of DNA damage triggers TDP-43 mis-localisation in the cytoplasm, inducing more loss of nuclear TDP-43. This would enhance existing DNA damage and perturb DNA repair even further, leading to significant accumulation of DNA damage during the normal human lifespan, triggering neurodegeneration. Figure In this study we demonstrate that TDP-43 mediates DNA repair via the NHEJ pathway. In contrast, TDP-43 ALS mutants Q331K and A315T lose this function, contributing to the accumulation of DNA damage. Our findings also reveal a link between DNA damage and the formation of pathological forms of TDP-43 in ALS. Whilst the RNA-related functions of TDP-43 are well described, this study identifies an important DNA-specific function that is perturbed in ALS.Additional file 1: Supplementary Figure 1. A315T and Q331K mutants do not prevent the formation of 53BP1 foci (EGFP-tagged constructs). Top panels; Confocal microscopy of NSC34-cells expressing wildtype TDP-43 display less DNA damage compared to controls; untransfected (Un) or EGFP only cells (EGFP), determined by (i) total area, (ii) and total volume of 53BP1 foci after treatment with 13.5 μM topoisomerase II inhibitor etoposide. In contrast, cells expressing ALS-associated mutants A315T and Q331K are not protected from damage compared to wildtype TDP-43. Scale bar 10 μm. Middle panels: Quantification was performed on 3D reconstructions of z-stack images using Imaris software. 2-way ANOVA with Sidak correction for multiple comparison. Mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001. At least 20 cells/group were analyzed. Bottom panels; Representative 3D reconstruction of confocal images of cells illustrating 53BP1 foci, Scale bar 5 μm.Additional file 2: Supplementary Table 1. Q331K and A315T TDP-43 mutants cause cell death in cells with DNA-PK deficiency. Expression of Q331K and A315T TDP-43 mutants in XR-C1 cells lacking DNA PK was lethal in all three replicates (crosses). Wildtype CHO-1 cells with Q331K and A315T expression survived in 2 out of 3 replicates (check marks).


Fast-response humidity sensors using nanomaterials are attractive and have been intensively studied. Among the various nanomaterials, nonporous inorganic nanoparticles are suitable for use in humidity sensitive films for sensors. Here, we focus on a nonporous inorganic nanoparticle film and investigate a humidity sensor using the film. Hysteresis error and a dynamic response to a change of humidity are fundamental specifications of humidity sensors. A humidity sensor using a 50 nm silica nanoparticle film shows a hysteresis error of 2% at 85% RH and a response/recovery time of 2.8/2.3 s in 30% RH to 70% RH. We also summarize response/recovery times and hysteresis errors of state-of-the-art humidity sensors. As compared to those of commercial sensors and porous nanoparticle-based sensors evaluated using saturated salt solutions, the fabricated sensor shows a comparative hysteresis error and shorter response time. Humidity sensing has been of great importance in environmental monitoring, planting in factories, and human care ,2. HumidRecently, there have been intensive studies to improve the dynamic response of humidity sensors. Borini et al. reported a pioneering work demonstrating the ultrafast response (≈30 ms) of humidity sensors using a graphene oxide film with a 15-nm thickness . RecentlAmong the several kinds of nanomaterials, we focus on inorganic nanoparticles as a component of humidity sensitive films. Size-regulated inorganic nanoparticles are chemically stable in air and a thin film can be formed by solution processes using colloidal nanoparticles. In previous studies, porous inorganic nanoparticles were used for humidity sensors to increase sensitivity because the nanopores inside nanoparticles easily condense water molecules ,10,11,12In this study, we investigate the humidity hysteresis and response time of a nonporous inorganic nanoparticle-based humidity sensor. Nonporous silica nanoparticles with 50 nm and 200 nm diameters were used to form a humidity-sensitive film. For humidity sensing, we utilized surface conduction via water molecular layers on the nanoparticles. We evaluated the humidity hysteresis in ascending/descending humidity levels and response times relative to dynamic changes in humidity. A remote detection of respiratory air by the sensors was shown. We summarized the response/recovery time and hysteresis error of state-of-the-art humidity sensors.We prepared gold interdigitated electrodes (line and spacing: 20 μm) on substrates as follows: gold with a titanium adhesive layer (100 and 10 nm) was sputtered  on a silicon wafer . The gold/titanium layer was patterned into interdigitated structures with photolithography and dry-etching processes. The finished electrodes were electrically isolated under high humidity.Colloidal silica nanoparticles were purchased from Sigma-Aldrich (product no.: “803073-1ML” and “803847-1ML”). The densities of the 50 and 200 nm nanoparticle colloids were diluted by adding ethanol 10 and 100 times, respectively. The colloids were spin-coated on bare interdigitated electrodes in ambient air. After the coating process, the substrate was heated to evaporate the solvent from the film.Static humidity dependence of impedance was obtained in an environment-controlled chamber . The chamber was controlled at 34% to 90% relative humidity (RH) with 3% accuracy at 20 °C. The test pressure was 1 atm and the temperature variation was ±0.3 °C. The impedance was measured by an LCR meter , which was controlled using LabVIEW . A voltage amplitude for impedance measurements was 1.0 V and a voltage frequency was 0.5 Hz to 1 kHz.2CO3, Mg(NO3)2, NaBr, NaCl, KCl, and KNO3 salts were used in this study. Abrupt changes in humidity were given by the sensor which was placed in and removed from the vessel. The zero condition of the change is in ambient air without airflow. The temperature, relative humidity, and gas pressure are 20 °C, 30% RH, and 1 atm, respectively. The actual humidity in the vessels was simultaneously monitored by a commercial humidity sensor . The measured humidity levels above the salt solutions, using K2CO3, Mg(NO3)2, NaBr, NaCl, KCl, and KNO3, were 36, 46, 52, 61, 66, 70% RH, respectively. It should be noted that we only investigated the static response time without gas flow. The sampling period of the sensor impedance was 1 s and the applied voltage was 1 V at 1 Hz.A dynamic response of impedance to a change of humidity was observed by using vessels with salt solutions. The volume of the vessels was 50 mL and the detailed dimensions are shown in the We also tested the availability of the nanoparticle-based sensor for the detection of respiration. Exhaled air has high moisture and temperature, is close to body temperature, and therefore, cyclically changed both the moisture and temperature of the air in front of the face. A mockup with a portable data logger  was packaged in a plastic test case  and a tablet with a custom-made Android API (an intrinsic source code is available from MbientLab) was used. Interdigitated electrodes on a glass (line and spacing: 100 μm) were used for the substrate of the sensor. A 50 nm nanoparticle coating was carried out by using the same method as above. A change of resistive impedance was converted to a voltage by using a voltage divider comprised with 10 MΩ. Collected data were transmitted to the tablet via Bluetooth. The measurement was approved by the internal review board.2 surface.A morphology and thickness of nanoparticle films were observed by a field-emission scanning electron microscope . Top-view and cross-section SEM images are shown in On the contrary, water molecules physically form multilayers on surface hydroxyls via hydrogen bonds. This physisorption is reversible and the thickness of the multilayers is dependent on humidity in air. Using attenuated total reflection infrared (ATR-IR) spectroscopy, Chen et al. investigated the thickness of physically adsorbed water layers over hydroxyls on a silicon wafer . They fof) dependence of impedance in a 50 nm nanoparticle film as a function of humidity (a): absolute value and (b) phase angle of impedance). Solid and dashed lines represent the measurements in the ascending and descending humidity levels, respectively. As a humidity increases from 40% RH to 90% RH, the impedance at a lower frequency decreases and shows a plateau region. This plateau region is attributed to a resistive impedance of proton conduction . A linear decrease in impedance (Z), which is independent on humidity, is due to a parasitic capacitance (pC) of the measurement system: i.e., |Z|∝(pωC)−1 = (2πfCp)−1 where |Z| is an absolute value of impedance and ω is an angular frequency (ω = 2πf) of an AC voltage.H is a relative humidity in percentage. In other words, we express this relation as This exponential relation can be attributed to the formation of water layers. According to Asay et al., a structural evolution of water molecular formation has three steps on a flat silicon-oxide surface . First, The hysteresis error is calculated by the difference between the experimental results and the empirical fitting line. We evaluated maximum humidity hysteresis at 6% at 40% RH and 2% at 85% RH in the ascending/descending humidity levels. At a lower humidity, the impedance becomes larger and its accuracy is reduced. This nanoparticle humidity sensor based on proton conduction is suitable for detecting higher humidities. It should be noted that a humidity sensor using 200 nm silica nanoparticles shows 10 times larger impedance although an exponential dependence appears a,b. BecaH1 and H2 represent as relative humidity in and out of the vessel, respectively. τ1) and recovery time (τ2) in 30% RH to 70% RH are evaluated to be 2.8 and 2.3 s, which are defined as t90 and t10  are shown in t90 and t10 were evaluated using a graphical readout. We assumed that the uncertainty of the measured response times was up to 1 s because the sampling period was approximately 1 s.A schematic setup for measuring a dynamic response to a humidity change is shown in Vout). Expiratory air is successively detected in 2 s with sharp voltage signals, as shown in Vout to actual relative humidity because temperature sensing is required for the conversion. In this proof-of-concept, the sensor does not measure relative humidity of exhaled air but only detects the existence of water vapor qualitatively. The measurement of relative humidity in breath air is a cutting-edge topic and further developments are required in our system [Using this fast response/recovery time, we show a proof-of-concept test for practical applications of a nanoparticle humidity sensor. Because a common respiratory rate ranges from 12 bpm to 20 bpm (0.05 Hz to 0.083 Hz), a humidity sensor with a 2.8/2.3 s response/recovery time can be used for sensing respiratory rate. A setup of remote sensing of respiration is shown in r system .τ) is equivalent to the total of the mixing time to obtain certain humidity in the chamber (mτ), the adsorption time of water molecules onto the sensor (aτ), and the time required for the change of the sensor property (cτ): τ = mτ + aτ + cτ. The value of mτ is related to extrinsic response and aτ and cτ are attributed to intrinsic response.It should be noted that there are two types of response times regarding diffusion of water molecules: dominated by intrinsic (sensor related) and extrinsic (environment related) processes . Sensor mτ is reduced and the evaluated recovery time is dominated by the intrinsic aτ and cτ. On the other hand, the evaluated response time may include the effect of mτ because the response time (2.8 s) is longer than the recovery time (2.3 s), as shown in In the case of the recovery process, because the volume out of the vessel (ambient air) is much larger than that of the moist air in the vessel (50 mL), mτ to obtain certain humidity is included in the evaluation of response/recovery time. In case of using a saturated salt solution, the effect of extrinsic mτ can be reduced because the humidity is stable at certain values in the vessel [We finally discuss sensor specifications, a response/recovery time, and a humidity hysteresis with state-of-the-art humidity sensors using nanomaterials. e vessel . TherefoIt should be noted that the specifications in In order to guarantee practical usability, it is essential to achieve repeatability of sensors. Therefore, we show the humidity dependence of impedance on other two sensor chips in the In summary, we investigated a humidity hysteresis and a response time of a nonporous inorganic nanoparticle-based humidity sensor. A nonporous nanoparticle film was potential to enhance fast humidity sensing with small hysteresis. A humidity hysteresis and a response/recovery time of a 50 nm nanoparticle film were evaluated to be as 2% to 6% (at 85% RH to 40% RH) and 2.8/2.3 s (a change of humidity between 30% RH to 70% RH), respectively. We demonstrated a remote sensing of respiration. According to the specifications with state-of-the-art sensors, the nonporous nanoparticle sensor showed a faster response than commercial humidity sensors and porous nanoparticle-based sensors evaluated by using saturated salt solutions. Repeatability of the sensors should be improved in future works towards practical usage.


Further, ZnSk displayed superb antibacterial activity against the tested model gram-negative Escherichia coli bacterium. The produced ZnSk has huge scope to be used for real-world water contaminants remediation applications. We have fabricated ZnO nanoflake structures using degummed silk fibers as templates, via soaking and calcining the silk fibers bearing ZnO nanoparticles at 150 °C for 6 h. The obtained ZnO nanostructures were characterized using scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), and UV-vis and fluorescence spectroscopic analysis. The size (~500–700 nm) in length and thicknesses (~60 nm) of ZnO nanoflakes were produced. The catalysis performances of ZnO nanoflakes on silk fibers (ZnSk) via photo-degradation of naphthalene (93% in 256 min), as well as Rose Bengal dye removal (~1.7 mM g ZnO nanoparticles have significant technological applications, as they are well known for their catalytic, electrical, optoelectronic, antimicrobial properties, etc. . They po2, Zirconia, ZnO, hematite, germanium oxide composites, etc. [Hippospongia communis was used to produce TiO2 3D composite structures using hydrothermal conditions (120 °C for 3 h) [Aplysina cauliformis were used to produce novel chitin–GeO2 photoluminescent nanocomposites, using a hydrothermal reaction with a temperature of 185 °C [Sepia officinalis to synthesize a template for the in vitro formation of ZnO nanostructure composites at 70 °C, which are extreme for the used biological material [Taking a practical science point of view into consideration, recent research has been accelerated towards the development of novel composite materials using thermostable biopolymers  and selected inorganic phases ,7. Usinges, etc. ,9,10. Fofor 3 h) . Likewismaterial ,9,10. AmTemplate directed synthesis of ZnO materials is gaining significant interest in research communities, and various porous and non-porous materials of ZnO have been synthesized ,5. PolyeNatural fibers from silkworms are well known for their biomedical applications, and recent studies display their potential as templates for nanoparticle synthesis ,14 DegumE. coli bacteria may be useful for large-scale wastewater treatment applications in real-time settings.Our work focused on introducing a facile template assisted synthesis strategy to attain flake type ZnO nanoparticles. In this study, a green template source (silk fibers) was utilized with zinc acetate precursor, with a reasonably low temperature annealing process, to attain ZnO nanoflakes on silk fibers (ZnSk). The synthesized ZnSk were used for pollutant remediation studies, such as naphthalene degradation and Ross Bengal (RB) dye removal from the aqueous system successfully. The stated chemicals were selected as model pollutants, as they have profound effects on the environment. For instance, the photo-degradation of naphthalene is slow, with photochemical half-life usually beyond 100 h , and RB,Bombyxmori silkworm were gifted by Silkworm Culture and Reeling Unit, Chebrolu, Andhra Pradesh, India. Further degumming steps were adopted from a reported study [2CO3 solution at 100 °C for 30 min. The process was repeated until the silk fibers tuned to white from pale yellow and the degumming solution stayed clear. Next, the samples obtained were washed with deionized water and naturally dried to get degummed silks.Dried cocoons of ed study . In brie3COO)2·2H2O solution was prepared using a mixture of H2O: methanol: ethanol in the volumetric ratio of 5:4:6. A small amount of acetic acid was added to adjust the pH value to ~4.8 to prevent the formation of hydroxides. Then, the solution was homogeneously mixed using a sonicator and 1 mL of it added to degummed silk fibroin of 56.8 mg. The wetted silk fiber was transferred to a glass crucible and kept in an oven at 150 °C for 6 h. The solution without the silk fiber was kept as a control with the above ascribed conditions. The obtained ZnSk structures were further analyzed.ZnO nanostructures were obtained on silk fibers by modifying the approach presented in a reported study using zinc acetate as a starting material . In a tyThe photocatalytic activity of the obtained ZnSk was investigated by means of naphthalene degradation experiment. Primarily, 80 mg of the ZnSk sample  was added to 30 mL naphthalene solution of 43 mg/L in a 100 mL wide mouth glass flask. The investigation was performed at room temperature (34 °C), with continuous stirring (800 rpm) in a rotary shaker incubator. During incubation, the obtained suspensions were irradiated from top under UV lamp (11W), Philips PL-S, Hyderabad, India, a compact UVC source with intensity of 22 lux. Samples for analysis were taken periodically and the decomposition of naphthalene versus irradiation time was monitored by UV–visible spectrophotometer.For reference measurements, silk fibers devoid of ZnO were also exposed to naphthalene, in the same way as the above stated experimental conditions, and monitored spectrally.Adsorption experiments were carried out by maintaining a ratio of RB/(ZnSk or silk fibers) as 2.45 mg/g. An orbital incubator shaker  was used for the study, and samples were monitored till 400 min. A pH of ~6.5 and a temperature of ~34 °C were used for investigations devoid of light. The concentration of residual RB was determined using a UV-visible spectrophotometer , where the measurements were made at 545 nm, which corresponds to its maximum absorbance. Silk fibers devoid of ZnO are used as control. The change in RB dye amounts were calculated from the concentrations in solutions before and after dye adsorption. The mechanism of adsorption by silk and ZnO deposited fibers were estimated by using the pseudo-first and second-order kinetic models . AdditioE. coli, using the zone of inhibition (ZOI) method. Bacterial cell suspension of 0.1 mL (from 2% w/v) was plated uniformly, with the help of spreader on nutrient agar plates. Using a sterile cork borer, 10 mm wells were made on agar plates, and ZnSk composite (of 20 mg) was placed in the well. Here, degummed silk fibers were used as a control and agar plates with samples were then incubated at 37 °C for 24 h. The diameters of the resulting ZOI in mm due to microbial growth were measured using imageJ software on digital images, and the antibacterial activity was determined.The produced ZnSk composite was tested for bactericidal activity against Gram negative UV-visible spectra of the samples were observed using Cyberlab UV100, UV-vis spectrophotometer. The scanning electron microscopy (SEM) observations were performed on JEOL-2100F machine . X-ray diffraction (XRD) measurements were done by X-ray diffractometer , with an accelerating voltage of 35 kV, with a current of 30 mA. The photoluminescence (PL) spectra were measured using spectrofluorometer  of 274 nm is observed, with a decreasing trend in λmax values and up to 93% degradation is observed in 256 min. Silk fibers devoid of ZnO did not displayed any change in λmax value at 274 nm upon UV irradiation, indicating no effect on naphthalene degradation. The prepared ZnSk composites, after repeated use (three times), displayed a decreasing trend of naphthalene absorbance, and the average λmax values at 274 nm with increasing time is shown in Next, we studied degradation study, where 0/C) vs irradiation time, fit better with the first-order kinetic model of the rate law  dose, the degradation behavior of naphthalene is comparable or superior to other reported studies with different nanoparticles (like TiO2) and other approaches [The observed data, in accordance with the ln ZIn addition to the above, complementing our study and observations, the enhanced photocatalytic properties of anisotropic ZnO materials are attributed to the high surface area and the nanofeatures ,34. ThesThe synthesized ZnSk structures have huge scope to be used in real time studies, as they are template based and firmly associated with silk fibers after repeated aqueous washings (observed Zn content is 118 mg/gm ZnSk with net loss in terms of Zn being ~4.8% after 12 washings). The application of ZnSk will prevent the leaching out of ZnO, and these structures can be recovered and reused easily, without any trouble. The consistent degradation ability (>97%) of ZnSk composites after 12 cycles c furtherRB, an anionic dye, was used as a model pollutant to evaluate the adsorption properties of the produced ZnSk composite. As soon as the fibers were exposed to the RB solution, the rose color slowly disappeared, and the typical visible spectrum peak of RB at ~550 nm progressively decreased a. The raTo investigate adsorption controlling mechanism, a pseudo-first order kinetic model was utilized to evaluate the experimental data . The rate (mg/g) is adsorption capacity, qt is adsorption at time t and k1 (min−1), k2 (g mg−1 min−1) are pseudo-first and pseudo-second order rate constants, and h (mg g−1 min−1) is the initial adsorption rate, corresponding to the second-order kinetic model.Furthermore, the pseudo-second order model is presented as follows:2) values for the pseudo-first order kinetics are 0.86 and 0.75 for ZnSk and silk fibers respectively, while R2 values for the second-order model are 0.99 and 0.99 for the same. A better correlation between the modelled and experimental data was observed using the pseudo-second order kinetics model. This favors chemisorption, and this behavior is the rate controlling factor for the adsorption of RB onto silk fibers and ZnSk composite used. The observed performance might be true, as the degummed silk fibers have the ability to fix anionic dyes through ionic bonding. Here, positive –NH2 group of the fiber will bind to acid group of dye [The adsorption kinetics was estimated using Equations (1) and (2) to get information on the adsorption behavior with respect to time. Batch adsorption data using the pseudo-first and second-order kinetic models are presented in p of dye .e is equilibrium dye concentration on adsorbent (mg g−1), Qm is monolayer capacity of the adsorbent (mg g−1), K is adsorption constant (L mg−1), and Ce is equilibrium dye concentration in solution (mg L−1).Furthermore, for examination of the adsorption behavior with different RB dye concentration, a quantitative Langmuir adsorption isotherm model was utilized to evaluate the experimental data. Langmuir equation in its linear form is usually expressed as:e/Qe versus Ce should be a straight line with a slope 1/Qm and intercept 1/QmK.According to the above equation, a plot of Ce/Qe vs. Ce is given in −1), and Qm values are found to be ~1.706 and 0.679 mg g−1 for ZnSk and silk fibers, respectively. The observed Qm values are promising and better than reported values using bottom ash type adsorbents [−1 in the former case, and it is 0.868 mg/g in the later reported study. The reusability of ZnSk composites are tested by exposing them to 0.006 mM RB with a ZnSk dose of 0.287 g in 50 mL solution  for 12 cycles, and results are shown in The equilibrium adsorption isotherm is of primary importance in the design of adsorption process. The Langmuir adsorption model is applied to the experimental data obtained from various RB concentrations and its adsorption a. A plotsorbents  and Fe production, hydrogen peroxide generation, attachment to bacterial cell surfaces, etc. [The antimicrobial activity of as synthesized ZnSk composite was tested on es, etc. ,38,39,40−1 of ZnSk composite material, and the experimental data fitted better to the Langmuir model. In addition to the above, with a potent antimicrobial activity against the tested E. coli at ambient conditions, results suggest that the multifunctional use of the produced ZnSk composites could have huge scope to be used in environmental, industrial, and wastewater treatment applications.In this study, biopolymer, silk fiber was used as a template to synthesize ZnO nanoflakes with zinc acetate as a starting material. This biomimetic approach uses a simple hydrothermal process (with reaction temperature of 150 °C for 6 h) for the growth and formation of ZnO- silk fiber (ZnSk) composites. The ZnSk composite structure was visualized through the electron microscopy, and the results reveal the size of and morphology of the nanoflakes . The deposition of ZnO was confirmed by XRD, SEM, UV, and fluorescence studies. The efficiency of ZnSk composites in naphthalene degradation is demonstrated. The preliminary measurements of the photocatalytic property of ZnSk composites demonstrate more than 97% degradation ability, along with the reusability of the photocatalyst. Furthermore, taking the advantage of ZnSk composite’s anionic dye adsorption capacity, we used RB for adsorption studies. The maximum RB adsorption capacity was found to be ~1.706 mg g


Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease with limited therapeutic options. The current model suggests that chronic or repetitive “micro-injuries” of the alveolar epithelium lead to activation and proliferation of fibroblasts and excessive extracellular matrix (ECM) deposition. Disruption of alveolar type II (ATII) epithelial cell homeostasis and the characteristics of mesenchymal cell populations in IPF have received particular attention in recent years. Emerging data from single cell RNA sequencing (scRNAseq) analysis shed novel light on alterations in ATII cell progenitor dysfunction and the diversity of mesenchymal cells within the fibrotic lung. Within this minireview, we summarize the data from most recent human scRNAseq studies. We aim to collate the current knowledge on cellular plasticity and heterogeneity in the development and progression of IPF, effects of drug treatment on transcriptional changes. Finally, we provide a brief outlook on future challenges and promises for large scale sequencing studies in the development of novel therapeutics for IPF. Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible, and usually fatal respiratory disease with limited therapeutic options . The curDisruption of ATII cell homeostasis and the characteristics of mesenchymal cell populations in IPF have therefore received particular attention in recent years. Apart from ATII cells and alveolar fibroblasts, recent studies also suggested that other cell types may have an important role in IPF initiation. This entails further, recently identified, sub-populations of specific epithelial and/or mesenchymal cells and possible contributions of other cells in the distal lung, most notably macrophages –15.Overall, detailed cellular contributions to the onset and progression of IPF are still not fully elucidated. The advent of scRNA technology has increased our understanding of lung cell heterogeneity and cellular interplay in healthy and diseased lung tissue. Initial efforts were mainly conducted in mice, which also offer cell-type specific lineage tracing to study disease onset and progression , 16–18. Inadequate alveolar epithelial repair is believed to constitute the initial trigger for fibrotic remodeling in IPF. Within the alveolar epithelium, constituted of alveolar type I epithelial (ATI) and ATII cells, ATII cells are considered self-renewing stem cells that also regenerate ATI cells after injury . The conAxin2 , and transdifferentiation towards a KRT5+ airway basal cell-like state, whereas Wnt activity prior to Krt5 activation favors differentiation toward ATII cells, leading to normal alveolar epithelial repair , were also reported in explant tissue of patients undergoing transplant for end-stage IPF  cells are Wnt-active , and in Axin2 , 18. A rfibrosis . The stufibrosis . Similarl repair . Atypicatage IPF . Identiftage IPF  31). Ep. EpAxin2tage IPF .CD66+ secretory cell primed basal (SPB) cells, that share a molecular signature with basal cells recovered from fibrotic lung tissue . Two scRNAseq studies published this year, further added to the notion of aberrant basaloid cells, expansion of airway epithelial cells and reduction in alveolar epithelial cells in IPF lungs , whereas alveolar epithelial cells were recovered to a lower extend  and pericytes, a derivative of fibroblasts with myofibroblast characteristics, that is closely associated with capillaries . Recent COL1A1, COL1A2), two clusters enriched for ACTA2, a canonical marker of myofibroblasts, and a fourth cluster representing lipofibroblasts , a cell that's presence in the human lung has long been controversial  and was termed “fibromyocyte”. Both expressed a rich set of genes associated with TGF-β signaling   .ACTA2+ myofibroblasts and a population of lipofibroblasts , and an EBF1+ sub-population. Concordantly, myofibroblasts expressed contractile genes (MYH11). This was increased in IPF. These authors also found that all mesenchymal subtypes contributed to the excessive ECM production in fibrosis and that there is little evidence for trans-differentiation of fibroblast subtypes and that fibroblast fate is determined early in lung development , and one highly expressing SPP1 and MERTK (SPP1hi). Proliferation of SPP1hi macrophages was strikingly increased in IPF lungs. Colocalization and causal modeling supported the role for these highly proliferative SPP1hi macrophages in activation of IPF myofibroblasts in lung fibrosis  macrophages could contribute to progression of fibrosis through overreactive responses to alveolar injury , 43. Rec samples . A subsefibrosis . In liner stages .COL15A1 was found in the IPF lung. This is transcriptomically indistinguishable from systemically supplied bronchial vascular endothelial cells detected in control lungs. However, in IPF lungs, these cells can readily be found in affected regions in the distal IPF parenchyma whereas these cells are restricted to the peri-bronchial vasculature in the normal lung and are never seen in the lung parenchyma. The authors speculate that the ectopic presence of these cells provides the cellular molecular correlate to an old notion that the bronchial vascular network is expanded throughout the IPF lung (In addition, this recent study also identified changes in vascular endothelial cells in IPF lungs. A vascular endothelial cell population that expresses IPF lung .scRNAseq studies have vastly increased our understanding of cell types resident in the distal lung and also highlighted differences in cell populations/states between healthy and IPF tissue , 44, 45.CEMIP, linked to cell migration and ECM production, was downregulated in fibroblasts and lung homogenates (DDX11, E2F1, and PLXNA4, genes linked to fibroblast proliferation and reduction of angiogenesis (To date, Nintedanib and Pirfenidone are the only approved drugs for treatment of IPF , 48. Twoogenates . Nintedaogenesis . Furtherogenesis  and mighogenesis . scRNAsescRNAseq methods enable unbiased, high-throughput, and high-resolution transcriptomic analysis of individual cells. This provides an additional dimension to transcriptomic information relative to traditional methods that profile bulk populations of cells and are uniquely suited to look for heterogeneity within cell populations that might emerge during disease . HoweverNevertheless, new tools enabling data analysis, visualization, and interpretation are being developed at a rapid pace and are readily available as “open source” tools . Within The rising number of sequenced cells and the number of patients per study are strengthening accuracy and resolution . The redJN, AS, and MF wrote the manuscript. All authors agree to be accountable for the content of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The obtained results reveal that the electronic properties of graphene and MoTe2 layers are well preserved in heterostructures due to the weak van der Waals interlayer interaction, and the Fermi level moves toward the conduction band minimum of MoTe2 layer thus forming an n type Schottky contact at the interface. More interestingly, the Schottky barrier height and contact types in the graphene-MoTe2 heterostructure can be effectively tuned by biaxial strain and external electric field, which can transform the heterostructure from an n type Schottky contact to a p type one or to Ohmic contact. This work provides a deeper insight look for tuning the contact types and effective strategies to design high performance MoTe2-based Schottky electronic nanodevices.Two-dimensional (2D) transition metal dichalcogenides with intrinsically passivated surfaces are promising candidates for ultrathin optoelectronic devices that their performance is strongly affected by the contact with the metallic electrodes. Herein, first-principle calculations are used to construct and investigate the electronic and interfacial properties of 2D MoTe Two-dimensional 2D) layered crystals have attracted increasing interest due to their novel physical properties and potential applications in various fields since the discovery of graphene . UnconveD layered2 includes three interesting phases: hexagonal  phase [d, type II Weyl semimetal) phase [2 has an indirect band gap of about 1.0 eV for bulk and a direct band gap of about 1.1 eV for monolayer [2 is simply referred as MoTe2. Compared with other TMDs, MoTe2 has many advantages, for example, the conductivity is lower [2 and graphene, fabricating a type of heterostructure by graphene and MoTe2 for device applications could be considered. Actually, recently vertical heterostructures based on 2D-layered-structure materials have been attracted increasing interests [2, some experiments have investigated the graphene-MoTe2 heterostructure [2. It was reported [2 vertical heterostructure is as high as ~(0.5 − 1) × 10−5, and the photo responsivity can reach 20 mAW−1, which are comparable to the corresponding values of graphene-MoS2 device. Later, based on graphene-MoTe2-graphene vertical vdW heterostructure, a near-infrared photodetector was fabricated [2 vdW vertical transistor which exhibits suitable V-shaped ambipolar characteristics [2 heterostructures are promising candidates for optoelectronic nanodevices with high responsivity, high-speed, and flexible. In this sense, it is essential to carry out a theoretical investigation on graphene-MoTe2 vertical heterostructure which has not been reported yet.As a member of TMDs, bulk MoTeg) phase , monoclig) phase , and octl) phase , 21, in onolayer , 23, whiis lower , Seebeckis lower , and theis lower , 25. Comnterests –33 due tnterests –38, as ctructure –43 due treported  that thebricated , 42 witheristics  was repoFor the metal-semiconductor heterostructure, the contact type (Schottky contact or Ohmic contact) has to be considered, because it determines the existence of rectifying characteristics or not for the heterostructure. For the Schottky contact, the Schottky barrier height (SBH) would play a key role on the behaviors of the corresponding devices  and has 2 heterostructure have been investigated. The calculated results demonstrate that the electronic properties of graphene and MoTe2 monolayer are preserved quite well after being vertically stacked up as a heterostructure. The Schottky barrier of the heterostructure can be changed between p type and n type by applying an external electric field or strain, and the heterostructure can even reach the Ohmic contact when the external electric field or strain is strong enough.In this paper, based on first-principles calculations, electronic structure, and external electronic field and strain dependence of the SBH of graphene-MoTe−6 eV for energy and 0.01 eV/Å for force. The Brillouin zone k-point mesh is set as 9 × 9 × 1 within the Monkhost-Pack scheme. A vacuum space of 25 Å along the z direction is adopted to avoid the interaction between the neighboring layers. As it was revealed that the spin-orbit coupling effect on band structures of 2H-MoTe2 is very weak [First-principle calculations have been carried out using the Vienna Ab-initio Simulation Package (VASP) –47 basedery weak , all of 2 heterostructure is constructed by graphene and MoTe2 monolayer via stacking the two 2D materials along the vertical direction. Both graphene and MoTe2 adopt the hexagonal lattice and their lattice parameters are 2.46 Å [2 monolayer, here, three typical stacking modes are considered: HS-1, HS-2, and HS-3, which are shown in Fig. The graphene-MoTee 2.46 Å  and 3.52e 2.46 Å , respectWhen the strain dependence of the SBH is investigated, strain is applied equally along the zigzag and armchair directions of graphene, respectively.2 monolayer and three typical stacking modes  of the graphene-MoTe2 heterostructure have all been fully optimized. The obtained binding energies of the three typical stacking modes are all nearly the same, i.e., −0.85 eV, while the equilibrium interlayer distances of the three modes are all approximately equal to 3.53 Å. Hence, we solely focus on the HS-1 graphene-MoTe2 heterostructure for the following discussion and omit “HS-1” for simplicity in the following text. The optimized geometry structures of MoTe2 monolayer and graphene-MoTe2 heterostructure are shown in Fig. 2 monolayer adopts the hexagonal lattice and the optimized lattice constant is 3.52 Å, which is consistent with the experiment results [2 monolayer, as it is shown in Fig. 2 monolayer is a semiconductor with a band gap of 1.14 eV, which is also consistent with the experiment results [2 monolayer are vertically stacked up as a heterostructure, the equilibrium interlayer distance is 3.53 Å, which is comparable to the value of the Sb-MoTe2 heterostructure (about 3.94 Å) [2 layer and graphene layer in the graphene-MoTe2 heterostructure almost remain the same as the original structures of MoTe2 monolayer and graphene, which indicates the interaction between these two layers is weak. The binding energy of equilibrium structures −0.85 eV is lower than that of the Sb-MoTe2 heterostructure (about −0.37 eV) [2 and graphene is weak vdW type.The lattice crystal structures for MoTe results . It can  results , 23. Whe 3.94 Å) . It coul0.37 eV) , so the 0.37 eV) , graphen0.37 eV) , graphen0.37 eV) , and gra0.37 eV) , indicat2 monolayer are stacked up to form the heterostructure. The 3D charge density difference in the graphene-MoTe2 heterostructure defined as Δρ = ρH − ρG − ρMT has been calculated, where ρH, ρG, and ρMT are the charge densities of heterostructure, isolated graphene, and MoTe2 monolayer, respectively. The result is shown in Fig. 2 layer, which indicates that the electrons accumulate around the MoTe2 layer; while graphene layer is surrounded by the dark pink area, implying that the holes accumulate around the graphene layer. To see the property of the charge transfer more clearly, the planar average 〈∆ρ〉, which is defined as the average value of the 3D charge density difference Δρ in planes with z = const. that are parallel to the graphene layer, is shown as a blue line in Fig. 2 layer, and there are oscillations in 〈∆ρ〉 in both the graphene and MoTe2 layer. The electron localization function (ELF) is also plotted in Fig. Actually, the charge redistribution and transfer would inevitably occur when graphene and MoTe2 heterostructure are shown in Fig. 2 layer demonstrate that the semiconductor characteristics of MoTe2 layer with a direct band gap are retained. The band gap of MoTe2 layer is 0.85 eV in the heterostructure, which is changed compared with the result of 1.14 eV for the isolated MoTe2 monolayer. One striking feature in Fig. 2 layer is insufficient to modify the characteristics of the band structure of each component in the heterostructure, so the interlayer interaction effect on the band structure is very weak. This further indicates that the vdW interaction dominates between MoTe2 layer and graphene layer in the heterostructure, and thus preserving the intrinsic key properties.Many physical properties are determined by the band structures and density of states (DOS), and the calculated band structures and DOS of the graphene-MoTe2 heterojunction-based transistor has been designed, and the schematic diagram is shown in Fig. 2 monolayer is used as the channel material and graphene as both source or drain and gate electrodes. Due to the difference in work functions of the metal and semiconductor, there is a band bending at the interface, which can be estimated by the Fermi level difference (ΔEF), defined by ΔEF = WG − MT − WMT, where WG − MT and WMT are the work functions of the heterostructures and the corresponding MoTe2 monolayer, respectively. The calculated WG − MT and WMT are 4.36 eV and 4.84 eV, respectively, as is shown in Fig. EF) is about 0.48 eV in the heterostructure, which is comparable to the result of graphene-hydrogenated phosphorus carbide heterostructure [The contact properties of heterostructures are of importance for device applications. A graphene-MoTetructure .Fig. 6a2 layer), which determines the current flow across the interface of heterostructures thus playing a significant role in the corresponding device performance. In general, according to the types of the semiconductors in heterostructures, SBH is divided into n type and p type, respectively. The n type SBH (ΦBn) is defined as the energy difference between the conduction band minimum (CBM) of the semiconductor (EC) and the Fermi level of the metal (EF), i.e., ΦBn = EC − EF. The p type SBH (ΦBp) is defined as the energy difference between the Fermi level of the metal and the valence band maximum (VBM) of the semiconductor (EV), i.e., ΦBp = EF − EV. The SBH results of the graphene-MoTe2 heterostructure is shown in Fig. 2 monolayer to conduction band side of MoTe2 layer in the heterostructure, which denotes that the SBH of the heterostructure is n type with the value of about 0.33 eV at the interface. That is to say, the charge conduction of the heterostructure will be mainly through electrons.One of the most important contact properties of metal-semiconductor heterostructures is Schottky barrier at the vertical interface  of the MoTe2 layer almost remains constant under the external electric field, which denotes that the external field has little effect on the pristine electronic properties. This can be understood as follows: although the external electric field can change the energy eigenvalues of the valence electron such as CBM and VBM, their relative values are unchanged, resulting in the band gap remaining constant. In other words, the external electric field could not change the band structure except the band bending. It can be also seen clearly from Fig. 2 without any barrier, indicating that MoTe2 possesses a metallic conductivity, and thus realizing a Schottky-to-Ohmic contact transition. For the negative electric field when the intensity exceeds 1.0 V/nm, the heterostructure could also be tuned to the Ohmic contact. All these results demonstrate that applying an external electric field is an effective strategy to modulate the SBH and contact type for the graphene-MoTe2 heterostructure.To improve the performance of heterostructure-based transistors, it would be desirable to tune the SBH. It is demonstrated that the SBH can be tuned via applying an external electric field and in-plane strain , 30, 58.z coordinate of the Te atoms are relaxed while the positions of other atoms remain fixed after changing the size of the unit cell. It is shown that strain can also tune the SBH of the heterostructure between n type and p type and drive the heterostructure to approach the Ohmic contact. The behaviors of strain dependence of SBH are very different with that of the electric field dependence. The situation becomes much more complex. For a wide strain range, ΦBn is smaller than ΦBp, while only in a narrow tensile strain range ΦBp maintains smaller than ΦBn. That is to say, the strain range of n-type SBH (the strain is about −10 ~ 4%) is much wider than that of the p type (about 4 ~ 7%). When the tensile strain reaches 7% and the compressive strain reaches 10%, the Ohmic contact for the heterostructure also appears. It is worth noting that the band gap of the MoTe2 layer in the heterostructure would change strongly with the variation of the strain in the Schottky contact region, which is strongly different with the results of the electric field case. When the lattices are under strain, they would deviate from the equilibrium state, thus causing the change of the band structure. In fact, not only the value of band gap but also the type of band gap (direct or indirect) would be change by strain. For small strain, MoTe2 layer remains a direct band gap while it changes to indirect band gap for large strain. Here, it should be pointed out that for a real transistor the actual conditions to realize the Schottky-to-Ohmic contact transition may be somewhat different with the calculated results due to the actual situations.The SBH as a function of the in-plane biaxial strain is also calculated and the results are displayed in Fig. 2 heterostructure, which is indispensable to design 2D vdW heterostructure based field-effect transistors. Furthermore, the graphene-MoTe2 heterostructure can be applied for tunable Schottky diodes in nanoelectronic and optoelectronic devices.The above results suggest that both applying an external electric field and in-plane biaxial strain are effective methods to control SBH and the type of contact of the graphene-MoTe2 heterostructure under different electric fields and biaxial strains have been systematically investigated based on first-principle calculations. The electronic structures of graphene and MoTe2 are well preserved after being stacked up together along the vertical direction, which suggests that the interlayer interaction of the heterostructure belongs to the vdW type. However, the Fermi level moves toward CBM of the MoTe2 layer after the formation of the heterostructure, i.e., the Schottky contacts are n type with a 0.33 eV SBH. The SBH and the type of contacts at the heterostructure interface can be effectively modulated by applying an external electric field or strain. When an electric field is applied, in the Schottky contact region, the n type SBH exhibits an approximately upward linear relationship with the electric field, and p type SBH behaves reversely. The heterostructure can be tuned to the Ohmic contact for an electric field which is greater than 1.0 V/nm at both positive and negative sides. For the case of applying biaxial strain, the situation is more complex than the electric field case. The strain range of n type SBH is much wider than that of the p type. When the tensile strain reaches 7% or the compressive strain reaches 10%, the Ohmic contact also appears. All the results demonstrate that applying an electric field or strain is a good way to control of the SBH as well as the type of contact of the heterostructure, even drive the system into the Ohmic contact. These features are quite significant for designing high performance nanoelectronic and optoelectronic devices.In summary, the band structures of the graphene-MoTe


Since its discovery, graphene and its multiple derivatives have been extensively used in many fields and with different applications, even in biomedicine. Numerous efforts have been made to elucidate the potential toxicity derived from their use, giving rise to an adequate number of publications with varied results. On this basis, the study of the reproductive function constitutes a good tool to evaluate not only the toxic effects derived from the use of these materials directly on the individual, but also the potential toxicity passed on to the offspring. By providing a detailed scientometric analysis, the present review provides an updated overview gathering all the research studies focused on the use of graphene and graphene-based materials in the reproductive field, highlighting the consequences and effects reported to date from experiments performed in vivo and in vitro and in different animal species . Special attention is given to the oxidized form of graphene, graphene oxide, which has been recently investigated for its ability to increase the in vitro fertilization outcomes. Thus, the potential use of graphene oxide against infertility is hypothesized here, probably by engineering the spermatozoa and thus manipulating them in a safer and more efficient way. Leafing through the newspapers or magazines, listening to the radio, looking at the television or surfing the Web it is impossible to do not stumble on news regarding the use of graphene in everyday life, from the formulation of new batteries  and the Graphene is chemically defined as a two-dimensional material or, more impressively, a one-atom thick species formed exclusively of carbon atoms packed in a honeycomb crystal lattice ,6,7. DesThe spreading of graphene and, overall, its oxidized derivatives into the biomedical research world  has led The present review is aimed to outline all the research studies focused on the use of graphene and graphene-based materials in the reproductive field, highlighting the consequences and effects reported by the scientific community during the last years. As a first step, a detailed scientometric analysis will be presented, created after analysing the published literature regarding the interaction between graphene and Graphene-Derived Materials (GDMs) and the reproductive function in animal models and humans, with the aim of evidencing the research efforts made on that issue.Then, particular attention will be directed to the recognition of the various derivatives belonging to the family of graphene-based materials investigated in these researches, with special attention to the oxidized form of graphene, GO. The idea is to follow the classification created by Wick et al. in order to understand structure–activity relationships in the context of health science and safety and to avoid generalizations of the term graphene . In addiA detailed bibliographic search was carried out to create a data set for the scientometric analysis, made up with the Advanced Search Function of Web of Science Database (WoS) .For the present analysis the topic “graphene” was used in combination with “sperm”, “spermatozoa”, “human spermatozoa”, “oocytes”, “reproduction”, “fertility” and “human fertility”. As a result, 29 documents  were found published from 2012 to 2019 see . Since dIt is evident from the analysis performed that the number of research products is recently increasing , as well as the number of citations , even ifTo explore the most relevant issues related to the scientific production in the selected products, the International Scientific Indexing (ISI) keywords were analysed. The words most commonly used inferred from the analysis were: “toxicity” (11), “in-vitro” (8), “nanoparticles” (8), “carbon nanotubes” (7) and “graphene oxide” (6). Moreover, the thematic map showed that “graphene oxide” and “in vitro” are of great importance in the research field studied. Regarding the most relevant source, the analysis showed that the majority of the manuscripts were published in journals included in the Web of Sciences Category (WC-Subject Area) “toxicology”, while only one article was published in a journal within the category “reproduction”.The analysis of the contribution from each country is an interesting tool to evaluate the scientific production. Researchers from China, Italy and Korea represent the most active countries in these topics, representing the 60% of the total of publications. On the other hand, the analysis of the collaborations evidences the absence of strong collaborations among researchers from different countries, with no multiple interactions. The analysis of the network with the dynamics of authors and co-authorships establishes that there are many sub-networks, with all the components characterized by the tendency to form highly clustered structures that do not communicate with each other. Taken together, these data suggest that the scientific community is highly fragmented, highlighting the lack of communication among scientists. Cytoscape Software  was used to integrate and analyse the networks . SupplemMany methods of preparation have been developed in order to obtain graphene, but they differ in the final size, quality and production costs. In general terms, graphene can be obtained following two different strategies: the top down and the bottom-up. Most mass scale graphene is produced by a top-down approach via graphite exfoliation. This process consists of the peeling off of graphene from graphite, requiring large amounts of solvent and mechanical mixing, sonication or electrochemical treatment ,32,33,34On the other hand stands the bottom-up synthesis of high quality graphene, exploiting chemical vapor deposition or advanced organic synthetic methods that allows to recover very small amounts of the final material and at very high costs ,40.In the present analysis of research studies evidencing the effects of graphene and graphene derivatives on reproductive functions, the biggest problem that came to light, as mentioned above, was the nature and features of the investigated materials. As a matter of fact, the term graphene was often used as a general term to indicate carbon materials spanning from zero-dimensional carbon materials such as carbon black (CB)  and nanoIn order to shed light and clarify the different materials investigated and generically referred to as graphene or carbon materials, the following is a list of the most representative materials that have been encountered during the present bibliographic analysis.D/IG, index of occurred oxidation, is 1.55.Pristine and acid purified SWCNT, differing for their metal (iron) content  and oxidGraphene nanoplatelets or graphene nanoparticles (GNP) in their pristine version ,49 or fuDespite the reduced molecular weight of such material, only a few studies reported detailed properties and dimensions of GQDs, although bottom up and top-down approaches of synthesis could entail completely different products. For example, whereas a commercial sample was described only in terms of round shape of 40–45 nm size and X-ray diffraction pattern , other sThese particles are a variant of pure carbon quantum dots that contain also nitrogen and oxygen. They are prepared by different methods: solvothermal approach in DMF using GO as a precursor  or pyrol4/NaNO3 and H2SO4 [2sp carbon atoms and oxygen-functionalized regions containing moieties such as hydroxyl (–OH) and epoxide (–O–) functional groups on the two accessible sides, and carboxylic acid (–COOH) or carbonylic (C=O) groups at the edges. It is a hydrophilic derivative of graphene and possesses different degrees of oxidation depending on the precise protocol of preparation [w/w, that may vary from 0.25 [Graphene Oxide (GO) is typically produced, as reported above, by a Hummer’s method  or slighnd H2SO4 . The proparation . The graparation . Researcparation ,56,57,58paration , oxygen rom 0.25  to 0.39 rom 0.25  or as firom 0.25  or >36% rom 0.25 ), dimensrom 0.25  to 3–5 µrom 0.25 ,47), surrom 0.25  to −56.7rom 0.25 ). In a from 0.25 ,63 or arrom 0.25  sheets wrom 0.25  or harshAll materials obtained by reaction of GO with different agents can be inserted in this class. For instance, carboxylic GO (GO-COOH) can be obtained through oxidization on GO surface of epoxy and hydroxyl groups to carboxyl groups by performing chemical modification with sodium chloroacetate . Within 3sp carbon atoms, missing in pure graphene. rGO can be obtained following different approaches. The widely used reductive agent is hydrazine [This compound is a reduced version of graphene oxide, often named as pristine graphene due to the fact that, theoretically and as reported above, the reduction in oxygenated moieties of GO should bring graphene oxygen to its original progenitor, graphene. Obviously, this is not perfectly true and rGO keeps numerous defects and ydrazine ,57,66 buIn recent years, the spread of graphene-derived materials has caused many researchers concern regarding the environmental and health impacts derived from their use, giving rise to different manuscript publications.As has been inferred from the scientometric analysis, the majority of the studies conducted to date regarding graphene and GDMs and reproduction have aimed to analyse the toxicological events involving the reproductive function, a powerful approach that allows examining not only the detrimental effects caused to the single individual but also the effects left in bequest to the offspring. In that way and from 2014 see , some reOther researchers, by contrast, have carried out their experiments in different animal species and either in vivo or in vitro by analysing the health of the offspring after exposing the forefathers with different graphene derivatives and concentrations, as it will be further reviewed  to mammals .Caenorhabditis elegans and paracentrotus lividusCaenorhabditis elegans (C. elegans) is one of the most common species used to study the toxicological phenomena derived from the use of non-physiological compounds, thanks to its relatively short life, low cost, well-known genetic background and easy handling. In this regard, various graphene-derived compounds at specific concentrations have been added by some research groups to the soil living-medium, showing low or no toxicity [The soil nematode hermaphrodite toxicity ,49,52 ortoxicity ,71. For toxicity . It is wZhao et al. investigated a completely different material, nitrogen-doped GQDs characterized by the presence of both nitrogen and oxygen moieties and by a really small size . They were used to evaluate the potential toxic effects at different concentrations . Authors demonstrated the absence of toxicity for the nematodes, targeted organs and embryos .2 (amide) and COOH (carboxylate) at various concentrations  for 72 h. In this case, GO and GNP were precisely characterized, being essentially micrometric flakes, not necessarily single layer. Their findings, based on the number of offspring at all stages beyond the egg, showed some toxic effects varying with the graphene type, surface functionalization, number of layers, dose and time exposure, with no toxicity for the lowest concentrations (5 and 10 µg/mL). In a later work, they further investigated and compared the effects of few-layered nano-sized GO and rGO at doses of 20, 50 and 100 µg/mL for 72 h, demonstrating a higher reproductive toxicity of GO than rGO, likely due to the activation of a non-canonical Wnt-MAPK signaling cascade as the possible underlying mechanism causing the GO reduced reproductive capability [On the other hand, Chatterjee and co-workers have found some negative effects in the reproductive potential of the worms influenced by the surface functionalization and layer number of the different graphene materials utilized . Concretpability .The toxicity caused directly to the reproductive organs (concretely the gonads) has been also evaluated. Zhao and collaborators added GO at different doses  to the worms’ living medium until their young adults stage. In this case, GO was synthesized from graphite and had a size of 40–50 nm, prevailingly monolayered. They found that concentrations of GO 10–100 µg/mL induced the production of more germ cell corpses (thus inducing germ line apoptosis) as well as a slightly decreased brood size and number of oocytes, an inhibition of the egg injection rate and an increased embryo mortality. Moreover, they suggested an epigenetic protection mechanism probably activated by GO, hypothesizing a novel self-protection mechanism against toxicity .After long-term GO exposure  of young adult nematode, GO-characteristic Raman spectral bands measured throughout the bodies of C. elegans revealed the accumulation of GO in the reproductive organs, a reduced sperm number and a damage of the spermatogenesis . AdditioParacentrotus lividus) has been also used by researchers to study the sperm toxicity after the exposure to commercial graphene materials, such as carbon black (CB) and GO. As far as GO is concerned, it was prevailingly mono-layered with a mean size ranging between 0.5–5.0 µm and containing 20% by weight of oxygen. Their findings showed that, after 1 h of sperm exposure to different concentrations , egg fertilization was significantly affected with a reduction of around 50% in the case of CB or had almost no effect in the case of GO [The Mediterranean purple sea urchin  to evaluate the effects of commercial GO and rGO at concentrations of 250 and 1000 mg/Kg of dry soil. By evaluating the life cycle of the soil, the Authors confirmed reduced hatching, survival and reproduction rates after the treatment with the highest concentrations and in a dose-dependent way [Spodoptera frugiperda (S. frugiperda) by Martins et al. at different doses . Both monodimensional and bidimensional carbon nanomaterials affected the reproductive performance when they were used at the highest concentrations, which could entail ecologic and economic consequences for the agriculture sciences [Similar experiments were performed with the soil invertebrate dent way  for the sciences .Mouse2+, to analyse GO biodistribution. Male mice were exposed to this compound by oral gavage  or IV injection  to study the short and long-term reproductive ability. Their results illustrated that the exposed mouse possessed normal sexual behaviours with no affectation of the testes and epididymis, normal pregnancy rates and litters, and a normal development and growth of the offspring. GQD-exposed mouse sperm retained high quality, suggesting that GODs avoided to penetrate the blood-brain and blood-testis barriers [Mouse species is commonly used for the study of reproduction, since mice are cheap, easy to maintain and straightforward to breed in captivity . Additiobarriers . On the barriers . NeverthAkhavan et al. performed a work in which they confirmed the uptake of NGO sheets by the testis of male mice after IV injecting the compound to both female and male mice in vivo every week for 8 weeks at concentrations of 2, 20, 200 and 2000 µg/mL (last one corresponds to 4 mg/kg mouse). They used GO nanosheets  obtained by oxidation and sonication from GO. They analysed the spermatozoa, finding a reduced viability, motility and progressive motility and a decrease in some kinetic parameters, as well as a higher reactive oxygen species (ROS) production and DNA fragmentation when exposed to the highest NGO concentrations. In addition, they reported for the female mice a decreased fertility, gestation rates and hormone concentrations  when using the highest concentrations of this graphene material .125I, whose stability is known to be not very good for long-term tracking [Similar nanoGO samples were used by Liang and co-workers to perform their experiments, but results were completely different from the previous study. Concretely, they used two sizes of NGO previously used for cancer phototherapy and in vivo tumour targeting ,75  intraperitoneally injected at doses of 0.4, 2 and 10 mg/kg for 7, 15 or 30 days . Their findings demonstrated a dose-dependent reduction in the number of spermatozoa, spermatogonia and spermatids, a decreased sperm motility, and some morphological abnormalities in the groups that were treated with the highest NGO concentrations. Moreover, some oxidative stress was confirmed with the increased activity of antioxidant enzymes, without causing damage to the testis or reducing the fertility potential, data confirmed with the healthy offspring derived from the matching with female rats .The study of the toxicological effects in vivo provides an interesting amount of information about the general and systemic effects, as well as the evaluation of many vital functions, organs and tissues as a whole. However, this model poses an important problem, since it acts as a black box: it is possible to know the details about the input  but not the output . Moreover, it is of great importance to consider the neurohormonal control of reproduction, involving a network of central and peripheral signals in the hypothalamic-pituitary-gonadal axis . Due to To our knowledge, only one research work has been published to date regarding the toxicity of graphene on the female germ cell, the oocyte. Lin et al. recently incubated in vitro GDQs with mouse oocytes at doses of 0.5, 1 and 1.5 µg/mL. By evaluating the oocyte maturation at different times, they found an interference in the maturation process, with no appreciable toxicity regarding the development of the offspring in vivo . FurtherRegarding the toxicity of the graphene materials in vitro caused to the male germ cells, a modest number of articles have been published to date. Asghar and co-workers functionalized carbon nanotubes (SWCNT-COOH) and rGO to evaluate their influence on human spermatozoa when incubated with different concentrations  over 30 min and 3 h. The Authors found an absence of toxicity on sperm viability and kinetic parameters, but an increase in ROS production when using SWCNTs-COOH, supporting the possibility to develop microfluidic sorting systems to select the sperm subpopulations .2H4-GO), reduced by hydrothermal reaction (HT-GO), and with green tea polyphenols (GTPs-GO). After incubating for 2 h with different doses , they found a reduced viability, motility and progressive motility whereas some kinetic parameters, for concentrations >1 µg/mL, were influenced in a dose-dependent manner. Moreover, all the compounds but the GTPs-GO increase the ROS and nitric oxide (NO) production, while decreasing the ATP and NAD+/NADH [Hashemi and co-workers found some cyto- and genotoxic effects in mice spermatozoa when exposed to GO and various rGO: GO reduced with hydrazine , most oIn order to confirm the positive results obtained in the IVF assays in a different animal model and in more physiological conditions, further experiments were carried out using a bovine in vitro model in which a somatic component, the oviductal epithelial cells, was included. This innovative model allowed us to verify the absence of toxicity when spermatozoa were capacitated in the presence of GO 1 µg/mL, to confirm the positive effects in terms of IVF outcomes, and even more important to evidence the absence of toxicity during the early embryo development (until Day 8 post-fertilization). Furthermore, the increase in the bull sperm fluidity was confirmed, giving rise to an interesting number of proteins and lipids modified after the treatment with the GO solution .Very recently, our group moved forward towards the use of the mouse model, currently used for the study of most of human diseases since mice and humans share virtually the same set of genes  . The resAs has been exposed, the vast majority of the works published to date related to graphene and reproduction are focused on the study of the toxicological events that this fusion entails. However, the first approach of what might be the first steps into the bioengineering of the male gametes to minimize the problem of infertility has also been presented, a worldwide issue that affects around the 20% of couples in the world (48.5 million) and entails psychological, social, demographic and financial problems for the society. It is known that the male factor is responsible for around the 50% of the cases, and among them, the 30% are due to unknown causes ,86,87. Thttps://www.grandviewresearch.com/press-release/global-assisted-reproductive-technology-market) “the global assisted reproductive technology (ART) market is expected to reach USD 45.4 billion by 2025, according to a new report by Grand View Research, Inc. Childbearing postponement is one of the high impact rendering drivers of the infertility treatment market. The reproductive behavior and pattern of some of the nations such as few European nations have shifted from early to late childbearing pattern. Increasing marital age, rising tobacco and alcohol consumption, increasing obesity rate are some of the other factors contributing to the market growth. Furthermore, increasing incidence rate of conditions such as poly-cystic ovarian syndrome (PCOS), tubal factors and endometriosis are other drivers of the market”.It is important to mind that, as reported by the Grand View Research Incorporation (Archea to mammals, including Human, has been provided here. What is emerging is a very interesting perspective presenting two sides of the same coin: on the one hand they could be harmful materials, with possible toxic effects on such delicate and important function; on the other hand, it is possible to speculate that the GRMs, and GO in particular, could represent a possible way to fight the problem of infertility by manipulating spermatozoa in a safer and better way.In summary, an updated overview of the scientific literature concerning graphene and graphene-related materials and reproduction in several organisms, from