This review (162 references) focuses on two-dimensional carbon materials, which include graphene as well as its allotropes varying in size, number of layers, and defects, for their application in electrochemical sensors. Many preparation methods are known to yield two-dimensional carbon materials which are often simply addressed as graphene, but which show huge variations in their physical and chemical properties and therefore on their sensing performance. The first section briefly reviews the most promising as well as the latest achievements in graphene synthesis based on growth and delamination techniques, such as chemical vapor deposition, liquid phase exfoliation via sonication or mechanical forces, as well as oxidative procedures ranging from chemical to electrochemical exfoliation. Two-dimensional carbon materials are highly attractive to be integrated in a wide field of sensing applications. Here, graphene is examined as recognition layer in electrochemical sensors like field-effect transistors, chemiresistors, impedance-based devices as well as voltammetric and amperometric sensors. The sensor performance is evaluated from the material’s perspective of view and revealed the impact of structure and defects of the 2D carbon materials in different transducing technologies. It is concluded that the performance of 2D carbon-based sensors is strongly related to the preparation method in combination with the electrical transduction technique. Future perspectives address challenges to transfer 2D carbon-based sensors from the lab to the market. Justin Gooding and Filip Braet have asked, “Should you use nanotubes or graphene as carbon nanomaterial in biosensors?” , motivatuctivity , 4. Thesuctivity –8.For sensor development, it is desired that the electrode fabrication can be performed in an easy way, allowing mass production, which is not in coincidence with the preparation of individual graphene flakes from graphite by the famous scotch tape method . Many otPreparation techniques for 2D materials are often classified either bottom-up or top-down approaches which are capable to create nanomaterials with a varying distribution of lattice defects. These include vacancies, grain boundaries, oxygen functional groups, dangling bonds, and Stone-Wales defects resulting in a certain degree of functionalization. Excellent reviews are published on the high diversity of preparation methods to yield two-dimensional materials –14. With4 and NaNO3 in concentrated sulfuric acid [2-hybridized carbon system and its conductivity. Figure The chemical synthesis is the widest distributed technique to obtain graphene, ensured by the simple instrumentation and the advantage to obtain aqueous dispersions of 2D carbon nanomaterials. Graphene oxide (GO) is synthesized by treating graphite with a strong acid and an oxidizing species, which delaminates the graphite crystal structure introducing oxygen functional groups. This material can be reduced in many ways to reduced graphene oxide (rGO), which is widely applied as sensor layer. Brodie paved the way for the graphene oxide synthesis already in 1859 by oxidizing graphite with potassium chlorate and fuming nitric acid . In 1957ric acid . This syric acid , 22. Theric acid . Oxidatiric acid . The subThe thermal treatment is the simplest way to reduce GO –28. ManyAn advantage of the aqueous dispersed rGO compound is the facile transfer mechanism for chemically synthesized 2D materials , 34–36. Chemical synthesis of 2D materials provides a large degree of freedom to adjust process parameters. So far, these options have not been fully exploited, e.g., by the design of 2D materials with a controlled number of defects. Such materials might be of great interest in sensing applications. It is observed for surface functionalization by nanoparticle (NP) deposition that the structural irregularities act as nucleation sites for the particles . Defects+ ions or quaternary ammonium salts in organic solvents [4Cl, Na2SO4, (NH4)2SO4, NaNO3, K2SO4, and NaClO4). Sulfate yields large graphene flakes with a lateral size up to 44 μm (> 85% with ≤ 3 layers) and no exfoliation was observed for ClO4−, Cl−, and NO3− [In comparison to chemical synthesis, which needs, in total, several days to proceed, the exfoliation can be speeded up to hours by applying a strong electrical potential to graphite electrodes. Upon application of an electrical voltage, solvated ions of the electrolyte intercalate within the graphite working electrode, weakening the interlayer forces and driving the individual flakes apart, which are released into the electrolyte , 40. Thesolvents –43. AnodRaman spectroscopy, a versatile tool for non-destructive optical characterization of 2D materials, revealed that the material got oxidized during the exfoliation procedure exhibiting major structural defects . The eleChemical vapor deposition (CVD) gained its popularity due to the possibility to grow large high-quality 2D carbon materials . The gro2 senor, a pulsed laser deposition of ZrO2NPs- or AgNPs on cvdG did not only decorate the carbon layer but also introduce defects [Apart from the deposition, the layer transfer to a substrate of choice is still challenging for 2D carbon materials prepared by CVD. Etching of the metallic substrate increases the cost and introduces ionic impurities , 53. A p defects .2 range or even smaller. Moreover, the preparation method still is time-consuming and compensates a lot of energy due to the high operating temperature of around 1000 °C. To circumvent the need of a metallic catalyst requiring a subsequent transfer step as well as to reduce the deposition temperature, the growth of graphene on non-conductive materials, such as SiO2, was developed [Even though, research aims for ongoing improvement of the growth mechanism to achieve large-area films of high-qualitative graphene material, mostly for optoelectronic applications such as displays, it became apparent that there is no need for the growth of large films in sensing applications, since most electrochemical sensors operate with small electrodes in the mmeveloped . Figure An alternative synthesis routine was investigated to obtain high qualitative 2D materials dispersed in liquid medium. This method can be applied to numerous bulk crystals, yielding colloidal stable dispersions Fig. , which aIn contrast to chemical synthesis, either by oxidizing graphite under harsh conditions yielding GO, the liquid phase exfoliation (LPE) takes advantage of a direct, mild exfoliation of the bulk material in liquid medium , 61. It Coleman et al. pioneered the field of LPE . The exfN,N-dimethylformamide (DMF) and N-methyl-1-pyrrolidone (NMP) were found to be versatile dispersants [The choice of the solvent is the key in terms of production. Apart from the effective delamination of the material, it is mandatory to keep the dispersion colloidal stable. This can be achieved when the solvent and delaminated material match in surface energies and possess similar Hansen solubility parameters , 68. Higpersants –71. One Liquid-phase exfoliation is gaining more interest due to the timesaving, up-scalable exfoliation, which yield materials mostly unchanged in their chemical structure ranging from dispersed monolayers to few-layers . So far,In contrast to many noble metal, 2D carbon materials have been reported as stable against electrode fouling. This was demonstrated by copper electrodes which were partly covered by cvdG. Even for inner sphere redox couples, no apparent fouling was observed .2-hybridized carbon system are key properties of 2D carbon materials to be integrated into a sensor device. Analyte interaction can take place via π-interaction of the extended aromatic carbon system or by hydrogen bonding with the oxygen residues present within and at the edge-planes of the graphene flakes. The introduction of additional recognition elements is highly desired to enhance the sensitivity and selectivity of the carbon material. Georgakilas et al. published detailed reviews on the functionalization of graphene, covalently and non-covalently [2-hybridized carbon lattice remains intact. Intermolecular interactions with materials exhibiting a π system are feasible [2-hybridized carbon lattice [The high surface-to-volume ratio and large spvalently , 76. Thefeasible , 77–79. feasible , 81. Onefeasible , 82, 83.feasible . The carfeasible , 84–87.  lattice , 88. Oxy lattice . The rea lattice . A non-d lattice , 92.The direct interaction of graphene-like materials with analytes or ease of functionalization as well as the tenability to obtain materials either of metallic, semiconducting, or insulating character is highly attractive for electrochemical sensors. Excellent reviews focus on the sensing capabilities of 2D carbon materials towards numerous analytes –97. A crIn field-effect transistors (FET), semiconducting 2D carbon materials act as channel between source and drain electrode applying a potential of the gate electrode. Two sensing types are accomplished, either back-gating or top-gating, also known as solution-gating. The interaction of the analyte with the 2D carbon material changes its charge carrier density due to electric charge distribution making this technology capable to develop rapid, miniaturized sensors . A chemiN-hydroxysuccinimide ester showing an outstanding limit of detection (LOD) of 25 aM for 24 mer target DNA [Vds = 0.1 V) the highly selective sensor showed a LOD of 100 fM for a 22-mer DNA strand [In a graphene FET (GFET), the material is accessible to surface functionalization as the channel is partially integrated into the device and the electrical double layer allows operation at low gate voltage with high conductivity. Monolayer graphene may be superior compared to multi-layered graphene as the conductance is improved. This is demonstrated by a DNA sensor, where the probe DNA was coupled to monolayer graphene via a pyrene butyric rget DNA . A FET trget DNA . To enhaA strand . These t−1 for the graphene mesh. A similar effect was reported, when it was discovered that certain graphene-based pH sensors can exceed the theoretical maximum Nernst limit of 59 mV pH −1 in sensitivity. This was also attributed to defects, which allow in addition to the electrostatic gating effect a direct transfer of the charge carriers to the 2D carbon material [On the other hand, defects are reported to enhance the sensitivity. This was demonstrated for a glucose sensor by fabricating a graphene mesh via CVD on a copper foil where certain areas were blocked by silica spheres . The edgmaterial .Disease-related small marker molecules like glucose , dopaminIn general, cvdG is the material of choice in terms of miniaturization and mass production. Even when mass production still suffers from defects, originating of polymer contamination, affecting 44.2% of all chips , this ma2-hybridization of the graphene lattice [One of the rare exceptions of using rGO in FETs was reported by the group of Wolfgang Knoll. They report on a layer-by-layer (LbL) assembly of urease and polyethyleneimine on rGO and exploit the pH dependency of liquid-gated GFETs when detecting urea . The LbL lattice . Reduced lattice .−1 for the SARS-CoV-2 antigen protein. The sensor proved to be successfully applied in clinical diagnosis as nasopharyngeal swab specimen from COVID19 patients and healthy persons were investigated [Graphene FETs have also been reported as a platform for immunosensors, e.g., to determine emerging pathogens such as coronavirus 2 (SARS-CoV-2) Fig. . Graphenstigated .Fig. 9Sc3 gas under different environmental conditions, which is enhanced by the recognition elements, cobalt meso-arylporphyrins [Chemiresistors are extremely popular in gas sensing. Here, 2D carbon materials can add additional value by their high affinity to gasses, together with excellent conductivity and catalytic effects. Figure rphyrins .Fig. 10A3O+ is formed, which acts as charge carrier. In such cases, both electrical and ionic conductivity will be enhanced and the sensor is interfered. Therefore, the choice of the 2D carbon material has an impact on sensitivity as well as on selectivity. The complexity in the surface chemistry of 2D materials decreases in the order of GO < rGO < LPE graphene < cvdG. In the same order, the conductivity of the material increases. A large variety in surface chemistry might be attributed to many binding sites, either for adsorption of the gas itself or for easy modification with metal or metal oxide nanoparticles, which further increase the surface area, number of binding sites, binding affinities, or even may introduce electrocatalytic effects. Table 2 nanorods [2O and the SnO2 surface of the particles/rods with generated radicals, which are known for a fast reaction. In comparison of silver-nanoparticle-modified 2D carbon materials, a huge difference in the range of detection is found for cvdG [The high operating temperatures (often > 200 °C) are one of the major drawbacks in gas sensing with semiconducting metal oxides, which can be overcome. Ammonia is one of the most frequently studied analytes, as it is harmful to humans already at very low concentrations. The threshold concentration is 25 ppm for an exposure time of 8 h . Besidesnanorods . This mifor cvdG  and rGO for cvdG . This cafor cvdG . The autGraphene-modified electrodes as impedimetric sensors are popular in determination of biomolecules, biomarker, proteins, or DNA. Mostly, the sensing mechanism depends on the blocking of the electrode upon analyte binding, which inhibits the interaction of the electrode with a redox marker present in the electrolyte. Table 2-hybridized structure of graphene intact, retaining the physical properties. Electrochemical impedance spectroscopy revealed an increase in the charge transfer resistance (RCT) from bare cvdG via an anti-IgG/cvdG to a BSA/anti-IgG/cvdG assembly  onto a multi-layered cvdG, grown on nickel . Edges ably Fig. . The inc2-hybridized carbon lattice, changing the electronic properties of the material, i.e., by opening a bandgap. The LOD was 0.9 pg mL−1 OVA in a linear range of 1 pg mL−1 to 100 ng mL−1.Better stability can be achieved by attaching a receptor covalently. This is difficult for cvdG as it exhibits only functionalities upon an imperfect handling. A straightforward approach was developed by electrografting of 4-aminobenzoic acid on cvdG to design a sensor for the determination of ovalbumin (OVA) . Even thCT value between redox marker and electrode surface due to electrostatic repulsion. Upon hybridization with the target DNA, the resistance increases even more. The enzyme exonuclease III recycles the sensor layer upon cleavage of the dsDNA  or rGO modified electrodes (5 aM–1 nM ssDNA). The GO-modified electrode revealed a LOD of 50 fM DNA, whereas the LOD is significantly decreased to 10 aM for rGO modified electrodes. Compared to GO which is almost an insulating material due to the enormous structural irregularities, highly conductive rGO is sensitive towards surface processes resulting in an enhanced change of Rke place .2-hybridized carbon lattice intact, maintaining high conductivity. In contrast to pyrene groups, porphyrins are able to incorporate an electrochemically active metal ion. The manganese ion served to monitor the stability of the porphyrin linker attached to the electrode during electrochemical measurements. The use of rGO as supporting material prevented the MnTPP from detaching, in contrast to a glassy carbon electrode (GCE). An increased electron transfer attributed to graphene’s high surface area as well as the short distance between graphene and porphyrin result in a sufficient sensor performance [In contrast to glassy carbon (GC), rGO provides a high affinity to aromatic molecules as demonstrated by porphyrins, which easily attach by π-stacking to rGO. A metallic porphyrin-modified rGO impedimetric sensor was prepared to capture hepatitis C-DNA by a complementary ssDNA, which is linked to manganese(III) tetraphenylporphyrin (MnTPP) . The nonformance .Table In contrast to frequency-dependent impedance spectroscopy, which monitors binding events in an equilibrium, time-dependent sensing techniques, e.g., voltammetry and amperometry, are attractive to investigate induced electrochemical reactions.Numerous exfoliation protocols strive for the preparation of graphene materials with a low number of defects to come close to the ideal graphene. For time-dependent electrochemical sensing techniques, the presence and number of defects within the graphene affects their electrochemical activity. The defect-dependent sensing capability of the 2D carbon compounds generated by sonication was investigated upon the determination of phenolic compounds. The quality of the carbon materials ranges from a low amount of defects present within the structure to graphene compounds, which were anodized to provide additional functional groups . NMP-disElectrochemical graphite exfoliation is predestined for simplification as the preparation procedure and material functionalization can be combined to one step, which was proved for a graphene-porphyrin compound. Graphite was exfoliated electrochemically with porphyrin as intercalant, simultaneously functionalizing graphene flakes by π-interaction, introducing an oxidizing agent for catechol . ApparenThe design of improved electrochemical sensors envisions the hybridization of electrocatalytic active groups with graphene substrates. Besides the functionalization of graphene with carbon allotropes by π-stacking, highly attractive became the decoration with electrocatalytic active metallic nanoparticles (NPs). They provide a suitable substitute to natural enzymes, as they are more stable and are less prone to operation and environmental conditions. Metallic nanoparticles combine easily with carbon materials upon hydrothermal route or by direct electrodeposition onto the substrate of choice , 139–143The diameter of nanoparticles is crucial towards the electrocatalytic behavior, as the potentials of redox reactions can be shifted by the nanoparticle’s size and shape. The size of the particles can be either modulated by applied deposition voltage or deposition time, as shown in Fig. 4, forming a AuNPs-modified rGO hybrid [Tables O hybrid . Gold na2O2 [3. Glassy carbon electrodes were modified by drop-casting of rGO-Nafion-AgNPs. Cyclic voltammetric studies showed that an excessive AgNPs decoration led to the deterioration of the reduction ability of the sensor, as the aggregation of AgNPs reduces the electrocatalytic active sites of the material. Amperometric sensing was performed by applying a potential of − 0.65 V revealing a LOD of 0.53 μM H2O2, with a high selectivity for H2O2 compared to the five times increased concentration of urea, glucose, dopamine, uric acid, and ascorbic acid [2O2 sensor was prepared consisting of a heterostructure, decorated by microspheres of noble metals, Au and Pt, supported by rGO [2PtCl6, and HAuCl4 were mixed in equal molar ratio adding NaBH4 to trigger the reduction reaction. A GC electrode was drop-coated with rGO/Au/Pt. To detect H2O2 amperometrically, a potential of 80 mV vs. Ag/AgCl was applied, subsequently injecting the analyte into PBS (pH 7) resulting in a low LOD of 0.008 μM [A ternary hybrid composed of graphene, AgNPs and Nafion, targets the determination of H2O2 . The combic acid . In contd by rGO . Reduced0.008 μM . Apparen2-hybridized honeycomb-lattice of carbon atoms becomes vivid, when investigating the physical and chemical properties of the material, depending on the exfoliation technique. It can be stated that the preparation method guides the performance of the sensor material due the different intrinsic characteristics.Two-dimensional carbon materials have been intensively studied regarding synthesis, fabrication, and their implementation in electrochemical sensors. The fact that this class of nanomaterial is much more than a spThe variances originate from defects distributed within the carbon lattice, layer inconsistencies, oxygen functional groups, and adatoms. The still ongoing development of fabrication methods for 2D carbon materials in the last years’ aims for a high-yield production with a reproducible quality. Overcoming the weak interlayer van-der-Waals forces results in the delamination of the bulk material, yielding robust and flexible nanomaterial flakes due to strong intralayer forces , 160.In 2018, the group of Castro-Neto has investigated the quality of graphene supplied from 60 different producers with the result that the quality of these materials is rather poor, and this is claimed as one of the key issues for the slow development of applications . Especia2-hybridized carbon system of graphene. On the other hand, the linkage of active biomolecules might become an issue as the natural structure of the biomolecule can be disrupted followed by a loss of activity [The modification of the 2D materials are easily achieved either by covalent or non-covalent functionalization to improve the selectivity of the sensor , 76. On activity .2-hybridized carbon lattice, but for most electrochemical sensors, this is not a limitation so far. It is to point out that cvdG is prone to an unintentional introduction of non-reproducible defects, i.e., smallest variations of the processing parameters, complex transfer protocols inducing ionic impurities, and structural defects, deteriorating the physical and chemical properties. Besides the rather high-quality cvdG, synthetically derived rGO is widest applied as sensor layer since it is either produced in an ordinary laboratory or purchased from the growing market of suppliers offering graphene materials up to a liter-range. A large defect density is distributed along the material’s basal planes. Even though this term has a negative connotation, those structural defects do not pose a drawback when it comes to sensing applications. Quite the contrary, the defects are resembled as active functional groups, preferably interacting with a potential analyte, when applied as sensor surface. Not any other material was used as frequently as reduced graphene oxide in sensor design, due to the simplicity of the material and its physicochemical properties. Additionally, the oxygen moieties enable the dispersibility of rGO in water.Numerous sensors were reported for an enormous variety of analytes based on 2D materials beyond the scope of this review. Different exfoliation techniques yield 2D carbon materials of a diverse structure, which consequently exhibits altered physical properties. Graphene, derived by CVD, is rather integrated in FETs or chemiresistors, as these sensors base on the change of the intrinsic electrical characteristics of the sensing layer upon analyte adsorption. The advantage of this material is claimed to be the intact spIn terms of easily derived, low-cost, liquid processable material, one has to keep in mind the LPE-process of graphite. The exfoliation via sonication or shear exfoliation is widely performed either in toxic, high-boiling point, organic solvents with a matching surface tension to the material or in aqueous solution with a stabilizing surfactant. Two-dimensional materials are obtained without a chemical change in the basal plane of their structure, but with varying flake size and number of layers from mono- to multi-layered compounds. The diverse surface chemistry often affects the physical properties in a non-reproducible way. The mechanical exfoliation process may not introduce structural irregularities within the carbon layer, yet the number of defects increase upon formation of smaller flakes dominated by edge-planes. Electrochemical exfoliation is performed in aqueous solution using inorganic salts as intercalating species upon application of a high voltage. A bonus is the possibility of direct functionalization during exfoliation. Many reports exist on how to exfoliate 2D materials, but little is derived on the sensing behavior, especially when the morphology of the materials changes upon variation of the delamination process. Nevertheless, the reported LPE-derived materials applied in impedimetric or amperometric sensors revealed promising results. Future trends envision the use of all carbon-fabricated wearable electrochemical sensors. For a direct application on the skin, deeper understanding of the toxicity of each class of 2D carbon materials is still needed. The sustainability of the preparation procedures is barely addressed even though the growing demand of production and processing procedures result in an enhanced consumption of resources.2-hybridized carbon system of cvdG was intentionally destructed by introduction of sp3-hybridized functionalities. To become an essential part in mass-produced electrochemical sensors, the system integration must be compatible to already existing complementary metal-oxide-semiconductor (CMOS) technologies [A deeper understanding of the electrochemical properties of the 2D carbon materials has to be obtained to unravel the question, whether a graphene material suits the requirements of the potential sensor. As stated before, the widest applied materials for sensor development were contrary materials, cvdG and rGO, known for either high-quality or defective graphene. Instead of implementing the materials according to their unique features, it was found that the intact spnologies . It is v


TDP-43 is a DNA and RNA binding protein involved in RNA processing and with structural resemblance to heterogeneous ribonucleoproteins (hnRNPs), whose depletion sensitizes neurons to double strand DNA breaks (DSBs). Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder, in which 97% of patients are familial and sporadic cases associated with TDP-43 proteinopathies and conditions clearing TDP-43 from the nucleus, but we know little about the molecular basis of the disease. After showing with the non-neuronal model of HeLa cells that TDP-43 depletion increases R loops and associated genome instability, we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation, R loop-dependent increased DSBs and Fanconi Anemia repair centers. These results uncover a new role of TDP-43 in the control of co-transcriptional R loops and the maintenance of genome integrity by preventing harmful R-loop accumulation. Our findings thus link TDP-43 pathology to increased R loops and R loop-mediated DNA damage opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies. Amyotrophic Lateral Sclerosis (ALS) is an adult onset, progressive neurodegenerative disease, caused by the selective loss of upper and lower motor neurons in the cerebral cortex, brainstem and spinal cord. The nuclear TDP-43 RNA binding protein, is encoded by a major gene for ALS susceptibility whose mutations are found in 3% of familial and 2% of sporadic ALS cases. Thanks to its ability to recognize DNA and RNA, TDP-43 is involved in different steps of mRNA metabolism and in several mechanisms of genome integrity. This, together with the fact that R loops or DNA-RNA hybrids are a common source of genome instability, prompted us to investigate whether TDP-43 deficiency has any role in R loop homeostasis that could explain previously described DNA damage response defects of ALS cells. We show that TDP-43 plays a role in preventing R loop-accumulation and associated genome instability in neuronal and non-neuronal cells, as well as in patient cell lines. Thus, our study opens the possibility that R loop-modulation in TDP-43-defective cells might help develop ALS therapies. TDP-43 is a nuclear RNA binding protein (RBP) with a repressor role of HIV-1 transcription. It binds to the trans-active response element DNA sequence of the viral genome . Like otMutations in TDP-43 are associated with sporadic and familial cases of Amyotrophic Lateral Sclerosis (ALS), an adult onset, progressive neurodegenerative disease, caused by the selective loss of upper and lower motor neurons in the cerebral cortex, brainstem and spinal cord , 10. TARIn addition to transcriptional autoregulation, TDP-43 can be cleaved into smaller C-terminal fragments before being enzymatically degraded to maintain its physiological levels , 19 by aEvidence indicate that a major source of spontaneous DNA damage comes from the accumulation of R-loops, consisting in DNA-RNA hybrids and a displaced single strand DNA (ssDNA) . Non-phyAll this, together with the fact that a number of neurodegenerative diseases highlight a particular sensitivity of the nervous system and motor neurons are associated with deficiencies in RNA metabolism and DDR, prompted us to investigate whether TDP-43 deficiency, as found in ALS cells, have a role in R-loop homeostasis that could explain previously described DDR defects of ALS cells. We show that TDP-43 plays a role in preventing R-loop accumulation and R loop-mediated DNA breaks in neuronal and non-neuronal cells and in patient cell lines, thus opening the possibility that R-loop modulation in TDP-43-defective cells might help develop ALS therapies.A key regulatory role of TDP-43 in essential metabolic processes was previously suggested since silencing of TDP-43 in HeLa cells lead in dysmorphic nuclear shape, misregulation of the cell cycle, apoptosis, increase in cyclin-dependent kinase 6 (Cdk6) transcript and protein levels . As a maGenomic DNA-RNA hybrids in siTDP-43 HeLa cells were first assessed by immunofluorescence microscopy (IF) using the anti-DNA-RNA hybrid S9.6 antibody, and determining the levels of the S9.6 signal in the nucleoplasm after subtracting the nucleolar contribution , 34. As Next, we determined R-loop accumulation by the more accurate method of DNA-RNA immunoprecipitation (DRIP)-qPCR, based specifically on the purification of genomic DNA-RNA hybrids of different sizes. In this case the S9.6 signal was determined for genes expressed at different levels such as APOE, RPL13A, WDR90, EGR1 and MIB2, which have been previously validated for the detection of R loops , 33, 35,Then, we investigated the functional impact of nuclear DNA-RNA hybrid enrichment on DDR, given that hybrids have been shown to enhance transcription-replication conflicts . As can Our results show that TDP43 depletion causes R loop-accumulation, R loop-dependent DNA damage and accumulation of the transcription-replication collision-associated FANCD2 repair foci similar to depletion of other mRNP processing factors that function co-transcriptionally together with RNA polymerase II (RNAPII). Indeed, genome-wide ChIP-seq data obtained in K562 erythroblastoma cells  reveals that TDP-43 colocalizes with expressed genes defined by RNA-seq . 6011 ouIn ALS patients harboring TDP-43 mutations, TDP-43 mislocalizes from the nucleus to the cytoplasm in detergent-resistant aggregated forms either full-length (43 KDa) and fragmented forms , which can be ubiquitinated and hyperphosphorylated . Protein-containing supernatants were collected and stored at −80°C. Total soluble protein lysates were loaded onto 8–16% SDS-PAGE gel (Bio-Rad). Samples were transferred to nitrocellulose membrane with Trans-Blot Turbo Transfer System (Bio-Rad). After 5% non-fat dry milk blocking, nitrocellulose membranes were incubated ON at 4°C with the following antibodies: anti-TDP-43 antibody , anti-GAPDH antibody . Densitometric analysis of the bands was performed using ImageJ software from three independent experiments.g). Cytoplasmic proteins were collected and stored at −80°C until use. The pellet was re-suspended in an ice-cold hypertonic nuclear extraction buffer  and incubated on ice with agitation. The nuclear extracts were centrifuged at the maximum speed and the nuclear proteins were collected. GFP antibody , rabbit polyclonal anti-H1  and mouse monoclonal anti-lactate dehydrogenase  were used as nuclear and cytoplasm loading controls.SH-SY5Y subcellular fractionation was performed according to Pansarasa et al. 2018. Cells were first re-suspended in an ice-cold hypotonic lysis buffer  and incubated on ice. 10% Nonidet NP-40 was added and samples were centrifuged at the maximum speed  ENCSR033VAZ entry, while DRIPc-seq, DRIP-seq and RNA-seq was gathered from Gene Expression Omnibus under accession number GSE127979 We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.Yours sincerely,Dmitry A. GordeninAssociate EditorPLOS GeneticsGregory BarshEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: In the manuscript “TDP-43 deficiency links Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated damage,” Giannini and co-authors explore the role of TDP-43 in the maintenance of genome integrity and try to dissect the mechanism of the neurodegenerative pathogenesis caused by the mutated version of the protein. They test the hypothesis that TDP-43 prevents the accumulation of DSB-inducing, deleterious R-loops and claim that an R-loop-dependent increase in DSB foci and Fanconi Anemia repair centers is caused by mis-localization of the mutated protein in transfected neuronal cells and in cell lines from ALS patients. Without a doubt, the findings reported in the manuscript are significant and worth further investigation; however, the conclusions drawn from the data are not sufficiently substantiated.Major concerns.1. The depletion of TDP-43 in HeLa cells by siRNA leads to a minor increase in S9.6 signal intensity . Even though the increase is statistically significant, the biological relevance of such an increase is unclear without comparison to the changes in intensity after depletion of the known protein involved in R-loop processing. This concern is exacerbated by the absence of an increase in DRIPqPCR product for the APOE gene in siTDP-43 treated cells . Taking into account this marginal effect on R-loop formation on the one hand and the multiple functions of TDP-43 in mRNA processing and its interaction with DNA on the other, the increase in gammaH2AX and FANCD2 foci  could reflect consequences of TDP-43 depletion that are unrelated to R-loop processing. The decrease in foci in cells expressing RNaseH could also be an indirect consequence of the protein depletion.2. The statement from the abstract, “we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation, and R loop-dependent increased DSBs and Fanconi Anemia repair centers” is misleading. Mis-localization of both proteins - mutant and wild type - leads to an increase in S9.6 intensity , and the difference in S9.6 signal intensity between the cells with mutant and wild type proteins is not significant. It is well documented that overexpression of proteins can cause their mis-localization and misfunction. So, “significantly decreased” nuclear localization of the mutant protein in SHSY-TDP382 cells (decrease of less than 15%) compared to wild type SHSY-TDP+ cells  can result from (slightly) different expression levels of each of the proteins in the corresponding cell lines. In other words, the somewhat increased deleterious consequences of expression of the mutant allele could be caused by a higher level of overexpression of the mutant protein, compared to the wild type allele, rather than the presence of a missense mutation.Unless the levels of TDP+ and TDP382 proteins are measured and found similar, the “mutation” effects could be attributed to the differential expression levels of TDP-43 alleles in the cells.3. The same critique applies to the experiments with the lymphoblastoid cells from the patients.4. The data in Figure 6 are neither quantified nor normalized. The quantity of the loading control proteins varies from lane to lane. Also, it is unclear how many times the experiment has been performed, so the discussion of the results of this experiment is mere speculation.5. In Figure 3A, the increase in S9.6 signal in the cells with overexpression of TDP+ cannot be attributed to R-loop formation because overexpression of RNaseH does not decrease the signal. Nevertheless, the authors make the following conclusion: “Therefore, we conclude that mislocalisation of TDP-43 also causes R-loop accumulation “(page 8). This statement contradicts the data. It is unclear whether the levels of overexpression of RNaseH were monitored during the experiments in different cell lines. One explanation of these data could be that the expression of RNaseH in the specific cell line was different (lower) from the others.6. Throughout the study, the only assay utilized for providing evidence for DNA damage or activation of DNA damage response is the detection of DNA repair foci. Additional, independent tests  are necessary to address physiological consequences of TDP43 malfunctioning.Minor concerns.1. There is no description of how overexpression of RNaseH is achieved in Figures 1-5. Reference #33, used for an explanation , does not provide relevant information.2. Since in Figures 3A and Supplemental Figure 1, the data presented as “signal per nucleus” and the differences in these signals are minor, it seems to be important to make sure that the area (size) of the nuclei are similar among cells transfected with different constructs.3. Flow cytometry plots in Figures 5B and C do not include the plot of S9.6 signal for the LCL-SALS cells (cells from sporadic ALS patient), making the data representation skewed.Reviewer #2: In this manuscript, Gianini et al. have investigated in detail the role played by TDP-43 in the generation of R-loops and RNA:DNA hybrids in neuronal and non-neuronal cell lines. In general, the manuscript contains interesting data for the field and several novel observations that contribute to a better understanding of the protein in disease. A few required additions and clarifications are as follows:-The title “TDP-43 deficiency links Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage” does not seem appropriate since the manuscript principally focuses on a disease-associated mutation, A382T. Therefore, it might be better to change it in “TDP-43 mutation links Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated DNA damage”.- The order followed in the abstract is not the one respected in the results  in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation and then a non-neuronal model of HeLa cells depleted of TDP-43. However, in the results they present first the depletion of TDP-43 in Hela cells and then the transfection of mutated TDP-43 in SHSY-5Y. Please be consistent in the order of the text.- In the work the authors used silenced TDP-43 in Hela cells but transfected mutated TDP-43 in SHSY-5Y. Please explain better the choice of the two cell lines and how they are connected.- In the LCLs mutated in TDP-43 the authors observe an increase of R-loops compared to CTL and SALS LCLs. RNAse H could revert partially the S9.6 increase signal. How can this partial decrease be explained?- Experiments were run in SHSY5Y and in LCL carrying only one mutation A382T. It might be interesting to carry out at least part of the reported experiments in SHSY-5Y cells overexpressing TDP-43 with another mutation. These data would confirm whether the readouts observed with this mutation are specific or not.Reviewer #3: The paper by Gianini et al focuses on understanding how deficiency of TDP-43 observed in ALS is connected with R-loop homeostasis and R-loop mediated DNA damage. Previous work from Livinsgton lab has demonstrated that TDP43 is involved in prevention/repair of transcription-associated DNA damage through R-loop-related mechanisms in neuronal cells , therefore the novelty of this paper is partially compromised by this publication. One interesting aspect which the authors were trying to pursue is to uncover if TDP-43-disease-associated mutations which result in re-localisation of usually nuclear TDP43 to the cytoplasm has any effect on R-loop-mediated DNA damage. This is certainly an interesting question worth addressing. The authors pursued these questions in HeLa cells , neuroblastoma cells  and lyphoblastoid cells . Using these model systems, the authors have shown that TDP43 deficiency results in accumulation of R-loops (by IF and DRIP-qPCR), DNA damage (γH2AX) and Fanconi Anemia repair centers. Unfortunately, the authors have not managed to take these initial findings, some of which were already reported in Hill et al PNAS 2016. (i.e. R-loop and DNA damage increase), into subsequent mechanistic investigations. For the reasons of the quality and amount of data presented in this paper, at this current stage the paper is not suitable for publication in PLoS Genetics.Major comments:1. The amount of data presented in this paper is minimal and not of a good quality which overall does not give me the confidence of the general paper conclusions. In particular the conclusions drawn from S9.6 IF analysis are rather ‘pushed’ and not visible from the images presented. For example, Fig 1A does not have a clear nuclear signal in any of the images shown . The differences between quantified signals are very small and represent only a background signal, in my opinion). The RNase H over-expression does not result in any visible change of the signal on the images. The positive controls (known factor which up-regulates R-loop level) are missing. The authors have not quantified nucleolar signal (are there any changes there?), though they have chosen to study gene encoding ribosomal protein in their DRIP analysis (see below).2. It is surprising to me that the only gene which shows statistically significant accumulation of R-loops is RPL13A, which is a ribosomal protein. I would like to see more comprehensive analysis of R-loop increase by DRIP-qPCR. Other multiple genes of different categories should be tested in different model systems.3. Experiments in Fig 3-4 are carried out with neuroblastoma cells, where TDP-43 Wt or mutant (TDP382) are expressed on the endogenous background. The authors need to characterise this system a bit better, since the imaging in Fig S1 is not sufficient. They need to present Westen blot showing the level of expression of endogenous and over-expressed proteins in different cellular fractions  in all three cell lines. What is the level of endogenous protein contribution to all results observed? The authors have not really explained why do we observe a significant decrease of nuclear signal for TDP43 WT cells compared to control cells? These data are important for further interpretation of IF and DRIP results. All these experiments should have been performed on the background of depleted endogenous TDP-43 protein to have a clear interpretation.4. The authors have carried out IF in Lymphoblast cells from patients with TDP43 and ALS mutations. Similar to my comments for Figures 1A and 3A, the IF data are not convincing and show extremely small changes between cell lines studied. These cells derived from different people and are subject to patient –to patient genetic variation. Since the data are showing rather small changes, the authors should use multiple cell lines representing each category. Possibly using alternative approaches such as slot blot may provide more quantitative data than IF.5. Figure 6 contains results of S9.6 IP experiments in lymphoblast cells. The authors propose that they observe less TDP43 interaction with R-loops when TDP43 is mutated. This is not visible from the protein gel presented, and as far as I am concern represents a wishful thinking of the authors and not the result of a given experiment. There is absolutely minimal pull-down observed with TDP-43 in the nucleus, and mainly there is a pull down of TDP-35 which should be really cytoplasmic, and not in the chromatin fraction? Clearly these TDP-35 fragments are also IPed in the WT conditions – why is this happening? This experiment lacks RNase H control to show that interaction between TDP43 and R-loops is specific. Comments regarding stronger pull-down of TDP35 in TDP43 mutant in cytoplasm are not really substantiated since it is clear that this sample is over-loaded on the gel as can be seen from H3 and GAPDH westerns.Minor comments:1. On page 9 first paragraph, the authors say that ‘TDP-43 mutation causes an increase in DNA breaks derived from R-loop accumulation that promotes transcription-replication collisions..’ . I think that this is an over-statement since it has not really been tested if accumulation of R-loops following TDP-43 depletion results in transcription-replication collisions.2. The authors need to explain why SNRPN gene does not have DRIP signal – it is not expressed?3. All the data in this paper can be grouped in 2-3 figures max (one figure for each cell model used).4. The paper would benefit from additional English editing (i.e. - ‘significant’ on pages 6 and 11 should be spelled correctly..etc)5. Concluding model demonstrating TDP43 role in disease and R-loop biology would be helpful.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1: No: The numerical data underlying graphs are not provided in spreadsheet form.Reviewer #2: YesReviewer #3: Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article  and your responses to reviewer comments. 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Gordenin, Ph.D.Associate EditorPLOS GeneticsGregory BarshEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: The revised manuscript by Gianini et al., “TDP-43 deficiency links Amyotrophic Lateral Sclerosis with R-loop homeostasis and R loop-mediated damage,” is much improved compared to the original version.Most of the issues that caused concerns in the paper’s previous version regarding data interpretation and appropriate controls are addressed and resolved in the updated manuscript.The added comparison of the consequences of TDP-43 depletion on R-loop formation (reported as changes in S9.6 fluorescence intensity) to the measurements already published in the studies of the contribution of proteins directly involved in R-loop processing , as well as DRIP-seq of additional R-loop - prone targets, allows appreciation of the biological relevance of relatively minor changes in fluorescence levels.The data from the new experiments involving treatment of the cells with RNAse III for the removal of dsRNA  eliminate the ambiguity of the results reported in the first version of the manuscript and validate the statement, “we prove that mislocalization of mutated TDP-43 (A382T) in transfected neuronal SH-SY5Y and lymphoblastoid cell lines (LCLs) from an ALS patient cause R-loop accumulation…”Measurements of the expression levels of the wild type and mutant constructs  strengthen the conclusion that indeed mislocalization, rather than an increased level of the mutant protein, causes accumulation of R-loops.Several points still require the authors’ attention.1. Even though the new genome-wide analysis of TDP-43 localization  does not provide direct evidence of the contribution of TDP-43 to R-loop processing but instead suggests that TDP-43 binds to highly expressed loci in DNA, these data offer new insights into TDP-43 functions. Nevertheless, the statement “…TDP-43 RBP controls transcription at expressed genes that are prone to accumulate R loops...” is not substantiated by the data and should be toned down.2. Since statistically significant “reduction in nuclear fraction” of endogenous TDP-43 was NOT observed either following expression of SH-TDP+, or SH-TDP382, it is formally incorrect to attribute an absence of a reduction in SH-TDP294 to any phenotype . .3. The model in Figure 6C must be adjusted to accommodate the data showing that a decrease (compared to control) in nuclear localization of overexpressed wild type TDP-43  does not cause an increase in S9.6 intensity .4. The spreadsheets with the raw data reflected in graphs and figures are not provided.Reviewer #2: The authors have provided several new data and have addressed in detail most of the queries raised by reviewers.Reviewer #3: The authors have made an effort to improve this manuscript including additional genes for DRIP-qPCR analysis (as requested in my comment 2) and some modest visual improvement for S9.6 IF.Related to previous comment 1:To address my concern related to small changes in IF signal in the nucleus associated with Pol II genes, the authors introduced genomic data from publicly available Encode and previously published data, showing a correlation between TDP-43 binding and R-loops in expressed genes under physiological conditions in K562 cells (fig 3). Based on these datasets TDP-43 is mainly enriched at the 5’end /TSS of the expressed genes, while R-loops can be found in multiple locations . However, these new genomic data in Fig 3 does not clarify what happens with this correlation when TDP-43 is depleted? Do R-loops accumulate only at the locations where TDP-43 is bound which would support the direct effects of TDP-43 on the regulation of these R-loops? The authors have not demonstrated if they see a specific enrichment of TDP-43 binding at R-loops of their tested genes by DRIP-qPCR supporting the specificity and direct effects of TDP-43 on these R-loops.Related to previous comment 4:Unfortunately the authors have failed to introduce additional patient cell lines to verify the effects of R-loop accumulation in patients with different genetic backgrounds. Therefore, in my opinion, providing a direct evidence that TDP-43 binds at the regions of R-loops affected following its depletion (as discussed above) is crucial.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the No: The data underlying the figures are not providedReviewer #1: Reviewer #2: YesReviewer #3: Yes**********what does this mean?). 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Spanish cañón “canyon” /kaɲon/ [ka.'ɲon]). With these crosslinguistic differences in mind, we ask: (1a) Do L1 English learners of L2 Spanish produce acoustically distinct Spanish /n/ and /ɲ/ and (1b) Does the distinction of /n/ and /ɲ/ vary by proficiency? In the case that learners distinguish /n/ and /ɲ/, the second question investigates the acoustic quality of /ɲ/ to determine (2a) if learners' L2 representation patterns with that of an L1 Spanish representation or if learners rely on an L1 representation  and (2b) if the acoustic quality of L2 Spanish /ɲ/ varies as a function of proficiency. Beginner (n = 9) and advanced (n = 8) L1 English/L2 Spanish speakers and a comparison group of 10 L1 Spanish/L2 English speakers completed delayed repetition tasks in which disyllabic nonce words were produced in a carrier phrase. English critical items contained an intervocalic heterosyllabic /nj/ sequence ; Spanish critical items consisted of items with either intervocalic onset /ɲ/  or /n/ ['xa.na]. We measured duration and formant contours of the following vocalic portion as acoustic indices of the /n/~/ɲ/ and /ɲ/ ~/nj/ distinctions. Results show that, while L2 Spanish learners produce an acoustically distinct /n/ ~ /ɲ/ contrast even at a low level of proficiency, the beginners produce an intermediate /ɲ/ that falls acoustically between their English /nj/ and the L1 Spanish /ɲ/ while the advanced learners' Spanish /ɲ/ and English /nj/ appear to be in the process of equivalence classification. We discuss these outcomes as they relate to the robustness of L1 phonological constraints in late L2 acquisition coupled with the role of perceptual cues, functional load, and questions of intelligibility.This study examines whether L1 English/L2 Spanish learners at different proficiency levels acquire a novel L2 phoneme, the Spanish palatal nasal /ɲ/. While alveolar /n/ is part of the Spanish and English inventories, /ɲ/, which consists of a tautosyllabic palatal nasal+glide element, is not. This crosslinguistic disparity presents potential difficulty for L1 English speakers due to L1 segmental and phonotactic constraints; the closest English approximation is the heterosyllabic sequence /nj/ (e.g., “canyon” /kænjn/ ['k A lasting question that has occupied a central role in the study of second language (L2) phonology across several decades asks which factors modulate the acquisition of L2 contrastive sounds that are not part of the first language (L1) grammar. A look at the collective body of research reveals that, while there is robust evidence that novel L2 sounds are acquirable , which is restricted to heterosyllabic position. With these crosslinguistic differences in mind, to converge on the L2 Spanish target, the learner's grammar must come to allow a single alveopalatal nasal segment. The question, then, that follows, is whether these constraints can be overcome in the L2. While there are no L2 Spanish /ɲ/ acoustic or perception data to inform our predictions for the current studyIn the present study, we examine L1 American English speakers' acquisition of the alveopalatal nasal /ɲ/ in L2 Spanish. This sound is a challenge for L1 American English speakers for a number of reasons. First, this is a scenario in which the L2 sound does not exist in the L1. Second, it is the least frequent phoneme in the Spanish inventory  includes an alveopalatal nasal that contrasts with the other nasals in word-medial onset positionju:zik]) . Together, these L1 constraints yield a learning task in which the grammar must come to allow a single nasal segment alveolar and palatal places of articulation in syllable onset position.According to Martínez Celdrán and Fernández Planas , the alvTo determine whether L2 Spanish learners produce a single segment (/ɲ/) or a sequence (nj), we follow Bongiovanni  by acousj/~/C/ contrasts , with Diehm also reporting production data of within-language and between-language contrasts.As mentioned, although this is the first study to our knowledge to examine L1 English speakers' acquisition of the L2 Spanish palatal nasal, we can look to a small body of research that has examined L1 English speakers' acquisition of L2 Russian palatalized consonants to inform predictions for the current study. Similarly to Spanish /ɲ/, and unlike the English /nj/ sequence, Russian palatalized consonants are single complex segments with dual places of articulation that contrast phonemically with non-palatalized counterparts. Therefore, the L2 learning task for L1 English speakers is similar. In onset position/. Diehm  and Luky/. Diehm  examinedj/ ~ /Cjj/ contrast, Lukyanchenko and Gor's data from an ABX task reflected above-chance accuracy in low-proficiency (~73%)j/ and /Cjj/ more accurately than low-experience learners when followed by a low vowel  and that the increase in /Cj/ accuracy corresponded with a decrease in inaccurate identification of /Cj/ as /Cj/. While the learners in these two studies are neither at ceiling nor in line with L1 Russian accuracy, the data suggest that learners can develop perceptual acuity that at least partially circumvents the L1 phonological constraints that yield /Cj/ perception. Production data from Diehm, the only study to our knowledge to report a within-subjects comparison of L1 English and L2 Russian production data, points to partial acquisition in the oral modality. She found that even advanced L2 Russian learners' productions of /CjV/ and /CjjV/ syllables did not differ in F2 trajectory or duration from the point of consonantal release to the offset of the palatal element. This result lends support to the observation that L1 English learners decompose L2 /Cj/ into an L1-like /Cj/ sequence. Interestingly, a crosslinguistic comparison of a subgroup (n = 4) of advanced learners' L1 English and L2 Russian productions revealed that, while one learner produced L1 /Cj/ and L2 /Cj/ as (L1-like) /Cj/, the other three produced a distinct sequence in Russian that fell between their L1 English /Cj/ and the L1 Russian /Cj/ comparison. That is, although the L2 Russian /CjV/ F2 trajectories did not approximate those of the L1 Russian comparison group, they were shorter and had a more negative slope  than their English /CjV/. Diehm posited that this intermediate representation was indicative of partial L2 acquisition.Regarding perception of the /CjV/ ~ /CV/ contrast is higher than the functional load of the Spanish /ɲ/~/n/ contrast. Russian, which has 42 consonantal phonemes, has 15 pairs of consonants that are phonemically distinguished by palatalization; palatalized consonant phonemes range in frequency ranking from 14 to 42  overcoming the relevant L1 English constraints is possible. However, we recognize that the learning scenario is different in L2 Russian vs. L2 Spanish. Specifically, the functional load  of the Russian /CThere are two research questions that drive the current study. The first regards whether learners acquire the relevant contrast in the L2, independent of how their /ɲ/ productions compare with the L1 Spanish /ɲ/.(1a) Do L1 English learners of L2 Spanish produce acoustically distinct Spanish /n/ and /ɲ/, as measured by duration and formant trajectories of the following vocalic portion?(1b) Does the distinction of /n/ and /ɲ/ vary by proficiency?Following the acoustic description of the nasal segments in section Nasal consonants in English and Spanish, distinct segments are predicted to take the form of a longer vocalic portion following /ɲ/ than following /n/; /ɲ/ is expected to present a higher F2 and a lower F1 than /n/, with an overall flatter shape for /n/ vs. /ɲ/.In the case that learners distinguish /n/ and /ɲ/, the second question concerns the acoustic quality of /ɲ/.(2a) If learners distinguish /n/ and /ɲ/, (i) do they rely on an L1 representation to produce /ɲ/ or (ii) have they overcome L1 constraints to establish a novel L2 representation? In this latter case, does the acoustic quality of the L2 representation pattern with that of an L1 Spanish representation?(2b) Does the acoustic quality of the L2 Spanish /ɲ/ vary as a function of proficiency?There are two logical outcomes. The first is that we will encounter evidence that learners have mapped Spanish /ɲ/ in the input onto their representation of English /nj/. In the Speech Learning Model . The Spanish/English bilinguals were divided into three groups based on order of acquisition and level of proficiency: (1) L2 Beginner (n = 9), 2) L2 Advanced (n = 8) and (3) L1 Spanish (n = 10). The L2 Beginner and L2 Advanced groups are comprised of L1 English speakers who learned Spanish as an L2. The L1 Spanish baseline comparison group mirror the L2 groups and are L1 Spanish speakers who learned English as an L2. The use of a Spanish baseline from a mirror-image bilingual group avoids the problematic comparison of bilinguals to monolinguals and acknowledges that a bilingual's systems do not act in isolation  and Modern Language Association (MLA) that was first used in Slabakova and Montrul CV1.ɲV2 and (C)CV1.nV2 structures, respectively; critical and control items in English consisted of (C)CV1n.jV2 and (C)CV1.nV2 structures, respectively. Across conditions, V1 was a mid or low vowel  and V2 was /a/ in Spanish and /ɲ/ in English. Distractors followed the same general (C)CV.CV structure as the control and critical stimuli. English and Spanish stimuli were recorded by phonetically trained female native speakers of Midwest American English and Northern Peninsular Spanish, respectively. The experiment consisted of Delayed Repetitions Tasks ; audio stimuli were presented over Sennheiser HD-280 PRO headphones through a MOTU Ultralite mk3 interface. Recordings were made in a sound-attenuated booth using a head-mounted Shure SM 10A dynamic microphone and a Marantz PMD 661 solid-state recorder at a 44.1 kHz sampling rate.The experiment was conducted in a single session divided into Spanish mode and English mode segments, with the language mode order counterbalanced across participants. After providing informed consent, participants started the first segment with a 10-min interview in order to establish the first language mode, followed by the delayed repetition task in that language. The English mode segment ended with completion of the BLP and the Spanish mode segment ended with the Spanish written proficiency assessment. The L1 Spanish comparison group only participated in the Spanish mode segment and completed the interview, repetition task, and English written proficiency assessment, in that order.Following the research questions presented in section Research questions and predictions, this study examines the duration and formant trajectories of the following vocalic portion as acoustic indices to differentiate between nasal segments. To that end, we used Praat [6.1.16]  the visual presence of an abrupt change in formant structure and frequencies (onset) and 2) a breaking up of the formant structure and a loss of energy and periodicity in the waveform (offset). Following Bongiovanni  perceptual mapping of /ɲ/ to English /nj/ or (ii) development of a novel L2 representation. We first compared the advanced learners' /ɲ/ to their English /nj/ and to the L1 English baseline  and a between-groups comparison (L2 Spanish /ɲ/ vs. L1 Spanish /ɲ/). Recall that, in addition to non-overlapping confidence intervals, differences between English /nj/ and Spanish /ɲ/ are expected to present in the form of a higher F2 peak and lower F1 valley for English /nj/ vs. Spanish /ɲ/. Keeping that in mind, For the Beginner L2 group, the within-group comparison (Beginner /ɲ/ vs. Beginner /nj/) revealed no overlap in confidence intervals for F1 (maximum difference =0.47 Bark), with the exception of the point where the two curves cross at 35–45%. The same pattern presents for F2, with no overlap in confidence intervals between 0–35 and 70–100% (maximum difference =0.51 Bark) and an overlap between 35 and 70% where the formant curves cross. The formant trajectories reflect the expected differences between /nj/ and /ɲ/, i.e., a lower F1 valley and a higher F2 peak for /nj/ vs. /ɲ/. The between-groups comparison (Beginner /ɲ/ vs. L1 Spanish /ɲ/) revealed no overlap in F1 confidence intervals between 0–55% and 85–100% (maximum difference =0.32 Bark), with an overlap between 55 and 85% where the curves cross. Further, there was no overlap in F2 confidence intervals between 0 and 65%, with a maximum difference of 0.51 Bark.For the advanced L2 group, the within-groups comparison (Advanced /ɲ/ vs. Advanced /nj/) revealed that the F1 confidence intervals overlap between 0 and 50% and then run adjacent to one another from 50 to 100%. The F2 confidence intervals overlap at the beginning of the formant trajectory (0–20%) and then again when the curves cross around 65%. There is no overlap between 20 and 55% nor between 70 and 100% (maximum difference =0.21 Bark). Visually, the formant trajectories illustrate a higher F2 peak for English /nj/ vs. Spanish /ɲ/ but a lower F1 valley for Spanish /ɲ/ vs. English /nj/. However, the confidence intervals are overlapping at both of these points, thus rendering this distinction non-significant. The between-groups comparison (Advanced /ɲ/ vs. L1 Spanish /ɲ/) revealed zero overlap in the F1 confidence intervals with a difference of 0.64 Bark units at their most different. The F2 confidence intervals do not overlap at the beginning and the end of the trajectory (between 0 and 20% and between 75 and 100%) with a maximum difference of 0.22 Bark units). Visually, the Advanced /ɲ/ demonstrates a higher F2 peak  when compared with the L1 control /ɲ/.To summarize the results for RQ 2: As with RQ1, there were no differences in duration within learner groups or between learner groups and the L1 Spanish group. In terms of the beginners' formant trajectories, there was a significant three-way distinction between the beginners' Spanish /ɲ/, their English /nj/, and the L1 Spanish /ɲ/. In contrast, the advanced data's considerable overlap between /nj/ and /ɲ/ formant contours suggests a lack of difference. However, comparably limited overlap between the advanced /ɲ/ and L1 /ɲ/ contours indicate a significant difference in formant trajectories .This study examined the speech production of beginner and advanced groups of L1 English/L2 Spanish learners and a comparison group of L1 Spanish/L2 English speakers to determine whether their production evidences a phonemic distinction between Spanish /n/ and /ɲ/, and, if so, whether learners establish the contrast via L1 English /nj/ or creation of a novel L2 representation. Between-segment patterns were established via two acoustic indices: Z-score transformed durations of the vocalic portion that follows the nasal segment and formant trajectories of the same vocalic portion. Durational differences were predicted to take the form of longer duration for /nj/ than /ɲ/ and for /ɲ/ than /n/. The formant trajectory for /nj/ was expected to consist of an F1 with a lower valley and an F2 with a higher peak compared with /ɲ/; /n/was predicted to evidence an earlier and higher F1 peak and an overall lower F2 contour.Before we turn to the discussion of the results, there are two notes regarding our analysis: First, because this is the first study to measure these sounds in L1 English/L2 Spanish bilinguals and there are no data to inform relative cue strength for these contrasts, we avoid the arbitrary assignment of relative weights to the indices of duration, vowel height (F1), and vowel frontedness (F2). Instead, we treat them as three separate strategies that speakers may use to distinguish between these nasal segments. Second, we eschew an arbitrary quantification of how little overlap in the confidence intervals of the spline curves constitutes a meaningful difference between the nasal segments and focus our qualitative interpretation on the first half of the formant trajectories (0–50%). In doing so, we home in on the nature of the transitions in /nj/ vs. /ɲ/ rather than differences in the vocalic portion, which is expected to differ due to cross-linguistic differences in the following vowel ([ə] in English vs. [a] in Spanish).Our first research question asked whether L1 English learners' L2 Spanish production reflects a distinction between /n/ and /ɲ/ in Spanish and if that distinction is subject to differences in proficiency. Beginning with the duration data, our beginner group did not evidence a significant difference in duration between /n/ and /ɲ/ but the advanced group did, producing longer vocalic portions following /ɲ/ than /n/ with a medium effect size. This difference between the learner groups suggests that the durational difference increases as a function of L2 proficiency. In comparison to the advanced group, however, the L1 Spanish group distinguishes via duration to a greater degree. Thus, while the advanced L2 Spanish and L1 Spanish both distinguish the segments via duration, the degree to which the L2 group does so does not approximate the L1 Spanish comparison.If we were to use duration as the only acoustic index of the /n/~/ɲ/ distinction, we would conclude that beginner learners have not acquired the /ɲ/ phoneme in Spanish since there is no durational difference between Spanish /n/ and /ɲ/. However, the formant trajectory data indicate that the beginners and advanced learners alike utilize height (F1) and frontedness (F2) of the vocalic portion to distinguish /n/ and /ɲ/. Given that the Spanish /n/~/ɲ/ contrast varies by proficiency, the question that follows is: Why do beginner learners use vowel height and frontedness to make a distinction, but not duration? There are two points to consider. First, it could be the case that duration is not the primary cue that learners attend to in the input to distinguish the /n/~/ɲ/ contrast, but rather that it is a later-acquired cue that learners have available to them at advanced proficiencies . Second, we remind the reader of the large standard deviation values for the duration results , which ir(17) = 0.08, p = 0.760]. Thus, the individual data are suggestive of individual differences in cue weighting, which have been documented in L2 acquisition  = −0.30, p =0.237], the weakness is likely due to the variation in the beginners' duration differences (proficiency scores < 30).Our second research question concerned the quality of the learners' Spanish /ɲ/. That is, (a) do they rely on their L1 English /nj/ to approximate the novel Spanish contrast, or (b) have they overcome L1 constraints and established a single segment? We begin with the duration results, which are limited here to the advanced group since the beginner group did not use duration to contrast Spanish /n/ vs. /ɲ/. Since the learners' Spanish /ɲ/ was not different from their English /nj/, we posit that they do not use duration to differentiate them. Solely based on this outcome, we might conclude that the learners rely on English /nj/ to approximate the L2 Spanish /ɲ/ target. However, neither of these was different from the L1 Spanish /ɲ/. What might explain a scenario in which a learner's L1 and L2 sounds do not differ from each other and also do not differ from the L2 target? One possibility is that the learners' L2 Spanish /ɲ/ has affected their L1 English /nj/. L2 influence on the L1 aligns with a scenario of equivalence classification in which /ɲ/ is initially mapped onto /nj/ and, over time, the representation shifts in the direction of the L2 sound. Nevertheless, the advanced learners' English /nj/ did not differ from the English baseline . These inconclusive findings cast doubt on the reliability of duration as an acoustic correlate in this case, at least at the group level. A look at the individual-level duration difference between the advanced learners' English /nj/ and Spanish /ɲ/  and frontedness (F2) as illustrated by non-overlapping formant contours in the first half of the vocalic portion that follows the nasal segment see , 8. The Based on these comparisons of duration and formant contours, our tentative response to RQ 2a is that (i) the advanced group relies on their L1 /nj/ representation when producing Spanish /ɲ/ while the beginner group does not, and (ii) neither group approximates the L2 Spanish target as measured by an L1 Spanish baselineThe finding that beginner and advanced L2 learners differ from one another as well as from the L1 Spanish baseline is not unexpected; intermediate representations have been commonly documented in L2 production research Zampini, . In factWhat we find is that the F1 contour is indeed different, but in the opposite direction of what would be predicted , and there is no difference in F2. Thus, there is no evidence of a shift toward Spanish. Going forward, longitudinal data will best inform the following outstanding questions regarding the relationship between /ɲ/ and /nj/ via within-group developmental observations: (1) Do learners who first establish a three-way crosslinguistic distinction (/n/~/ɲ/~/nj/) eventually develop a single representation that is used for perception of English /nj/ and Spanish /ɲ/? (2) In the case of a shared representation, does the representation trend in the direction of the L1 or L2? And finally, as a complement to the variables we have examined here: (3) Is the representation that learners use to produce /ɲ/ tautosyllabic or heterosyllabic? That is, is there evidence that learners can overcome the L1 English constraint that militates against /nj/ in onset position? To address this question, future analyses will (a) examine production data for syllabic breaks via identification of glottalization  consonants that have been shown to influence outcomes in L2 phonology—the functional load of a contrast (e.g., Best and Tyler, j/ representation that differed from their English /Cj/ representation, thus outperforming the advanced L2 learners in our study. We could reasonably hypothesize that the difference between the L2 Russian and L2 Spanish learners can be attributed to the comparatively higher functional load of the contrast in Russian. However, the L2 Russian learners' new representation did not converge on the L2 target, which suggests that high functional load might be necessary but not sufficient for L2 convergence.First, recall Diehm's  finding Instead, a lack of L2 convergence might be at least partially attributed to cue robustness. Archibald , 2009 prcañon” (Diversity Style Guide, Returning to the question of intelligibility, if an L2 speaker's message is not at risk of being lost, what are the practical implications of these findings? Pronunciation pedagogy objectives have shifted away from adherence to native-speaker norms and toward intelligibility (see e.g., Levis, The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of Illinois Office for Protection of Research Subjects. The patients/participants provided their written informed consent to participate in this study.SS and JC contributed equally toward conceptualization, methodology, and validation of the project, as well as preparation of the manuscript. SS was responsible for data collection and data analysis. JC was responsible for the theoretical framework. All authors contributed to the article and approved the submitted version.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.


All these devices have one point in common: they have been built using graphene-based materials like graphene, graphene oxide, reduced graphene oxide, inkject printing graphene, graphene-based composite sponges, graphene screen-printed electrodes or graphene quantum dots.This Special Issue compiles a set of innovative developments on the use of graphene-based materials in the fabrication of sensors. In particular, these contributions report original studies on a wide variety of sensors, such as gas sensors for NO Therefore, sensing is one of the many fields that can benefit from the use of this exciting material.Graphene has become the most explored material since Novoselov and Geim (Nobel Prize winners for Physics in 2010) achieved its isolation in 2004. The exceptional properties of graphene have attracted the attention of the scientific community from different research fields. Graphene is a monolayer of hexagonally arrayed spHowever, the development of a method for the production of high-quality graphene in large quantities at a competitive cost is essential to further exploit its full potential. For that reason, the use of graphene-based materials as graphene oxide (GO) and reduced graphene oxide (rGO), among others, has gained widespread consideration, as a compromise between the interesting properties of graphene and the synthesis price and complexity. Consequently, not only graphene but also other graphene-based materials are considered very good substitutes of graphene in many applications. In particular, graphene-based materials have been widely used for sensing applications in the last few years due to their high specific surface area, high electronic mobility and low electrical noise. A wide range of chemical sensors, biosensors and gas sensors have been developed using graphene materials.This Special Issue includes seven works focused on sensors based on diverse technologies for different applications with the common particularity that all of them use graphene materials.2-Decorated rGO as NO2 Gas Sensor” [2 commercial powders into mono-to-few-layer flakes of WS2, and dispersed them with rGO flakes to obtain a WS2-decorated rGO. They deposited it on Si3N4 substrates with platinum finger-type electrodes to build a chemo-resistive NO2 sensor that operates at near room temperature conditions and achieves a detection limit of 400 ppb NO2 and reproducible gas responses. Cross-sensitivity tests with humid air showed a very low influence of water vapor on the NO2 response.In “Near Room Temperature Light-Activated WS Sensor” , Paolucc2 and NH3 Sensing Characteristics of Inkjet Printing Graphene Gas Sensors” [2 and NO3. Here, the sensitivity of graphene-based chemiresistor gas sensors, fabricated through inkjet printing, is investigated using different concentrations of graphene in the inks. The response of these sensors towards humidity, nitrogen dioxide and ammonia has been characterized, showing that when the sensing layer is not homogeneous and does not cover all the electrode area, the reaction of the sensor towards gases is slower. Another important parameter is the thickness of the graphene film. This work shows that when the layer is too thick, the current flows through independent parallel paths, which brings it to a lower sensitivity. Conversely, if the film is too thin, thermal noise degrades the signal to noise ratio at the output. Moreover, thinner graphene films show a higher sensitivity and faster recovery time.In “NO−22 g. The benefit of stress immunity indicates the great robustness of the proposed sensor against external disturbances in real conditions.In “Stress-Insensitive Resonant Graphene Mass Sensing via Frequency Ratio” , the appThe addition of nanomaterials such as nanoparticles, carbon nanotubes or graphene-based materials may enhance the sensitivity of biosensors. In particular, “Graphene-Based Glycan Biosensor for Electrochemical Label-Free Detection of a Tumor-Associated Antibody”  describeAlthough graphene has been widely used as a nano-filler to enhance the conductivity of porous materials, it is still a requirement to prepare graphene-based sponge porous materials by simple and low-cost methods to enhance their mechanical properties and make them have good sensing and capacitive properties. In “Multifunctional Graphene-Based Composite Sponge” , a multi“Mechanical, Electrical, and Piezoresistive Sensing Characteristics of Epoxy-Based Composites Incorporating Hybridized Networks of Carbon Nanotubes, Graphene, Carbon Nanofibers, or Graphite Nanoplatelets”  comparesTo finish, “Development of Graphene Quantum Dots-Based Optical Sensor for Toxic Metal Ion Detection”  exhausti