A major transition to electric vehicles (EVs) is underwayglobally,as countries target reductions in greenhouse gas emissions from thetransport sector. As this rapid growth continues, significant challengesremain around how to sustainably manage the accompanying large volumesof waste from end-of-life lithium-ion batteries that contain valuablerare earth and critical materials. Here, we show that high-shear exfoliationin aqueous surfactants can upcycle spent graphite anodes recoveredfrom an EV into few-layer graphene dispersions. For the same hydrodynamicconditions, we report a process yield that is 37.5% higher when usingspent graphite anodes as the precursor material over high-purity graphiteflakes. When the surfactant concentration is increased, the averageatomic layer number reduces in a similar way to that of high-purityprecursors. We find that the electrical conductance of few-layer grapheneproduced using the graphite flake precursor is superior and identifythe limitations when using aqueous surfactant solutions as the exfoliationmedium for spent graphite anode material. Using these nontoxic solution-processablenanomaterial dispersions, functional paper-based electronic circuitboards were fabricated, illustrating the potential for end-to-end,environmentally sustainable upcycling of spent EV anodes into newtechnologies. In the past decade, the number of EVs on theroad has grown from almost negligible numbers to ∼10 millionas the urgency for climate action has been recognized by governmentsand manufacturers globally. Over the next 10 years, this is projectedto increase dramatically from ∼10 to 200 million 1. Both b1 Pyrometallurgyand hydrometallurgy recovery is restricted to high-value materialssuch as cobalt, lithium, nickel, copper, and aluminum with graphiteanodes seen as having a low recovery value. Graphite is more abundant,although natural resources are geographically concentrated. A relianceon importation has lifted it to the status of strategic importanceand on critical minerals lists for many nations and regions . Despite being placed on the critical minerals list and contributingto 20% of the total lithium-ion battery weight,2 graphite is currently burned as an energy source, usedas a reducing agent in pyrometallurgy, and ultimately disposed ofas waste. As the demand for graphite in lithium-ion battery applicationsincreases to meet the global growth of EVs, developing solutions fora circular materials system will be crucial for the environment andeconomic security.Current material recovery processes are insufficient,and thereare opportunities to improve the sustainability of the entire batterymanufacturing lifecycle.5 A complementary option that will be explored in thiswork is the upcycling of graphite anodes into graphitic nanomaterialsthat could have multiple end uses. For example, Large et al. usedsize-selected graphene nanosheets to print radio frequency antennasand thin-film electronics.6 Using meltmixing, Paton et al. dispersed small volume fractions (0.07 wt %)of shear exfoliated graphene nanosheets in poly(PET), improving the strength of PET by 40%.7 Graphene nanosheets can also be used in 2D/2D heterostructures forthe improvement of visible-light photocatalytic water treatment processesby enhancing the separation of charge carriers from the parent semiconductingphotocatalyst.8Developing effective methods that recyclegraphite anodes for reusein new batteries has recently become a focus area for addressing thiswaste problem.10 By taking advantage of synthesis methods in the field of two-dimensionalmaterials, it may be possible to develop scalable upcycling processesto handle the large waste streams from end-of-life EV batteries.Graphite is a layeredmaterial containing individual graphene layersheld together by van der Waals forces. Various chemical and nonoxidizingtop-down liquid exfoliation processes have been developed to synthesizefew-layer graphene and other graphene-related materials from graphite.12 mixed chemical–mechanical approaches,13 and entirely chemical exfoliation methods.16 However, challenges remain, particularly around the environmentalsustainability of processes that can convert lithium-ion anodes tographene, GO, and rGO materials.17Liquid-phase exfoliation techniques have recently been appliedto anode graphite recovered from lithium-ion batteries in electronicdevices to synthesize graphene, graphene oxide (GO), and reduced grapheneoxide (rGO). The techniques vary from ultrasonication-assisted,Previous approaches have required the use of either toxic solvents in the treatment/pretreatment step or methods that werebased on other chemical processes . In this work, we explore the use of high-shear exfoliation,without harsh chemicals or toxic solvents, to produce solution-processablegraphene nanomaterials that are then used to fabricate paper-basedelectronic devices. We assess this upcycling approach on anode graphiterecovered from an electric vehicle whose battery has reached its endof life. By doing so, the methods and research outcomes are directlyrelevant to this rapidly growing industrial sector.18 The vehicle batterywas considered to be at its end of life (2.5 V) and was dischargedto a 0% state of charge. The resulting anode black mass powder containedthe graphite material and a binder (poly(vinylidene fluoride), PVDF)with an estimated content of 18Spent graphite anodematerial was reclaimed from a Nissan Leaf (generation 1). An illustrationof the battery breakdown to the cell level is shown in V = 160 mL) using deionized water (5 MΩm) and sodium cholate . The binder was retainedin the starting graphite material to investigate synthesis withoutany pretreatment steps that require toxic solvents such as NMP towash PVDF from the graphite. The dispersion was subjected to high-shearexfoliation using a cylindrical stirred vessel with diameter 70 mm,height 95 mm, and a contoured base , resulting in turbulent flowinside the vessel (Re = ρωD2/μ ≈ 106) and shear rates of –1.19 This rotational speed was chosen to operate above the criticalcriterion required to exfoliate few-layer graphene from graphite flakes, –1.7Graphite recoveredfrom electric vehicle anodes was dispersed in an aqueous-surfactantsolution  were chosen because they are one ofthe most commonly used precursor materials for the production of few-layergraphene in the literature. Laser diffraction measurements were taken,and these flakes were found to have a volume-based median particlesize of 550 μm. Identical material preparation, high-shear exfoliation,and postproduction steps were followed as described for spent EV anodegraphite in the g for 45 min. The top 5 mL of thesupernatant contained few-layer graphene and was removed for analysisusing UV–vis–nIR spectroscopy (PerkinElmer 365). Theaverage atomic layer number (N) was obtained by measuringthe extinction spectra (E(λ)) of the nanomaterialdispersions and utilizing spectroscopic metrics for graphene, N = 25(E550 nm/Emax) – 4.2.20After liquid-phase exfoliation,the aqueous-surfactant dispersions were pipetted into centrifugationtubes with a capacity of 15 mL and centrifuged at a relative centrifugalforce (RCF) of 243Cgr) was determined by filtering the dispersions through 25-mm-diameterPTFE filters with a pore size of 220 nm. The mass of few-layer grapheneretained on the filters was measured and then used to calculate theextinction coefficients, ε(λ). At λ = 660 nm, theextinction coefficient is independent of the nanosheet thickness andsize. Therefore, this wavelength was chosen when measuring the concentrationof the few-layer graphene dispersions using the Lambert–Beerrelationship, Cgr = E660 nm/ε660 nmL, where L is the optical path length of the cuvette(10 mm). The extinction coefficient was measured to be ε660 nm = 1014 L g–1 m–1 for material produced using the spent EV anode precursor and ε660 nm = 1521 L g–1 m–1 for the material produced using the graphite flake precursor.The few-layer graphene concentration  is shown in Csc ≤ 1 g/L) where differences of up to 10-fold were found. For surfactant concentrations of 1 ≤ Csc ≤ 20 g/L, the concentration of few-layergraphene extracted from the spent anode precursor was comparable toand up to twofold higher than that of the graphite flake precursor.The optical extinction spectra for both materials and various surfactantconcentrations are shown in 23The upcycling performanceof high-shear exfoliation in aqueous surfactants was assessed by comparingthe production outputs of the spent graphite anode and high-puritygraphite flake precursors. The concentration of few-layer grapheneproducts for a range of surfactant concentrations (Cgr/Ci) after 15 min ofhigh-shear exfoliation(–1) were 1.87 wt % (upcycledEV anode) and 1.36 wt % (graphite flake), respectively. Although theabsolute yields are low, they are favorable when compared to otherscalable nonoxidizing liquid exfoliation techniques such as sonication,7 microfluidization,24 high-shear mixers,7 spinning disc, andTaylor–Couette-type approaches25 that produce yields of 0.1–3 wt % for longer processing timesof V = 160 mL), and repeatedthe shear exfoliation process to explore if it is possible to extractadditional graphene nanomaterial using solvent exchange. This secondexfoliation step produced 69% of the initial output over the same15 min process interval, demonstrating that graphite anode sedimentcan be reprocessed multiple times and that yields of >3% can beobtainedin only 30 min.The maximum yields  and process waste is substantially largerby ∼103. Alternatively, we can investigate the scalerequired to achieve the production rates of a batch high-shear mixer.For a similar high-shear stirred vessel in this work, only with largerprocess volumes of up to ∼1 L, Pérez-Álvarezet al. showed that few-layer graphene concentration follows the scalingrelationship 19 Applying this scaling, we estimate an upcyclingproduction rate of ∼5 g/h with a process volume of V = 3 L. This is only 1% of the volume required by high-shearmixing in batch operation, indicating that high-shear upcycling ofspent graphite anodes is a promising and environmentally sustainableapproach.We can use the process yield results to estimatethe upcyclingmaterial production rate. Using Csc = 0), and this resulted in a yield of 0.69 wt %. Considering thatresidual surfactant can adversely impact material properties and canbe challenging to remove from solution-processed functional devices, high-shear upcycling has the potential to provide the sustainablesynthesis of few-layer graphene with low additive requirements.In terms of end-use considerations, it is equallysignificant thatthe maximum yield using a spent graphite anode was obtained for asurfactant concentration that was one order of magnitude lower thanfor graphite flakes. Indeed, we also measured the concentration forhigh-shear upcycling in water only . This suggests that the same nanosheetdestabilizing mechanism at Csc ≈10 mM also applies to the solution processing of spent graphite anodematerials in aqueous surfactants.Interestingly, two different characteristics were observed foreach precursor. The upcycled product featured a bimodal concentrationprofile, whereas the few-layer graphene produced from graphite flakescontained a single peak in concentration at Csc ≤ 1 g/L),the primary (largest) peakin Cgr for the upcycling process occurs(Csc = 0.1 g/L). This contrasts with thetrend observed for shear exfoliated high-purity graphite flakes, wherethe dispersions have poor stability and the lowest few-layer grapheneconcentrations. Further investigation into this finding is necessary;however, the enhanced concentrations using spent anode precursorsmay be attributed to (1) the presence of significantly more edge siteson the sphericalized particles for intercalation and delaminationto occur; (2) graphite expansion from the intercalation of lithiumbetween layers during charge/discharge battery cycles; and (3) increasedwettability of the PVDF binder due to the presence of the surfactant.In the low-surfactant-concentrationregion  is a method known toimprove the production output;10 therefore,the presence of dilated layers in the anode graphite would also benefitthe exfoliation process. Finally, the PVDF binder is hydrophobic,which should reduce the dispersibility of graphitic particles in water.However, anionic surfactants have been shown to have an an affinityfor PVDF and increase the wettability below the critical micelle concentration(CMC).28 Increased wettability may alsoplay a role in the enhanced anode material dispersibility that isobserved here below the CMC for sodium cholate. Nevertheless, thehigh recovery performance is encouraging, and further research inthis low surfactant region would help to optimize the mechanisms whichenhance concentration using spent graphite anode material.Expanding on the previous points (1–3), the sphericalizedgraphite particles from the EV anode are much smaller than the flakegraphite particles, with a D50 of 21 μm compared to 550 μm.As part of the sphericalization process, these particles are jet milledto create a morphology which also contains more sites for the intercalationand delamination of layers than the graphite flakes. Also, duringthe operational lifetime of the battery, lithium ions intercalateinto the graphitic layers in the anode, weakening the van der Waalsattractive force. It has been suggested that this process can expandthe layer spacing by an average of 3.5% (0.352 nm) and up to 14.7%(0.39 nm).20 The number ofatomic layers is plotted against surfactant concentration in Csc ≈1 g/L and then decreases further as the surfactant concentration increases.This corresponds to the threshold of Csc ≈ 10 mM and aligns with the previous observations for Cgr noted above and for other 2D materials synthesizedfrom high-purity precursors.27 This demonstratesthat the atomic layer number of few-layer graphene exfoliated fromspent graphite anodes recovered from EVs can also be tuned using surfactants.Variationsin surfactant concentration modified the extinctionspectra for both graphene materials in the UV wavelength region 4. For feCsc was found to be lower for the upcycled material.At thehighest Csc = 20 g/L, the differencesin the layer number increased from ΔN ≈2 to 3. Overall, it appears that few-layer graphene dispersions producedfrom graphite flakes have a lower average number of layers than theupcycled product. This suggests that for the same hydrodynamic conditionsand aqueous-surfactant solutions, the quantity of few-layer nanosheetsproduced from spent EV anodes is less than for the high-purity graphiteflakes. Shear exfoliation using turbulent flows erodes the outer surfacesof graphite flakes to produce few-layer graphene.25 The resulting few-layer sheets continue to decrease inthickness by rate-controlling processes that span the entire rangeof turbulent energy-containing flow structures down to the Kolmogorovlength. The binder layer that coats the surfaces of graphite anodeparticles and smaller exfoliated platelets potentially acts as a barrierfor slip and peel mechanisms of delamination to proceed at the nanoscale.The rate of change in the layer number with N isbased on an empirical correlation that predicts the average layernumber to within 15% for a number of different graphite precursors.20 The shaded regions in The spectroscopic metric used to calculate Maintaining the themeof environmentally compatible processes, we explored the applicationof upcycled graphene materials for fabricating paper printed circuitboards. The electronic properties were investigated by solution-processinggraphene dispersions to create paper-based conductive thin films.Graphene inks were prepared using shear exfoliation in deionized water/sodiumcholate followed by centrifugation and spray deposition onto papersubstrates . Anillustration of the spray deposition process is shown in N ≤ 10.29 This suggests that the graphite mass is dominatedby a small number of poorly exfoliated particles. To remove these,the as-prepared dispersions were centrifuged at RCF = 115g for 20 min, following a similar approach recently described forbiocompatible few-layer graphene inks.30 As these formulations contain thicker nanoplatelets, we refer tothe inks as graphene-related material (Grm) to distinguish them fromthe few-layer graphene dispersions formulated at RCF = 243g.With few-layer graphene yields on the order of Ci = 50 g/L and using a surfactant concentration, Ci/Csc = 100,corresponding to the peak Cgr shown in CGrm = 1.66 g/L.To obtain an equivalent ink concentration from the high-purity graphiteflake precursor, exfoliation was first performed using Ci = 100 g/L and Ci/Csc = 20, resultingin CGrm = 2.8 g/L. This “flakeGrm” product was then diluted to match the concentration ofthe upcycled “EV Grm” product before spraying the papersubstrate.The ink concentration was increased to above 1g/L by exfoliatingspent graphite anode material at 2 per spray pass. Multiple spray passes were used to createflexible paper substrates that were electrically conducting on thecoated side and insulating on the other. The coatings were physicallyrobust with strong adherence to the paper backing and the abilityto withstand bending and folding. To accelerate the drying process,air at a temperature of 200 °C was blown over the paper usinga heat gun, evaporating the water solvent after each spray pass. Anexample of a paper substrate with 100 passes of a shear exfoliatedgraphite anode material is shown in 31 This is confirmed by the low-magnitude error bars in A 90 mm × 40 mm area was airbrushed with a supplypressureof 20 psi, resulting in a graphene material deposit of 18.4 g/m6 Copper tape electrodes  were positioned in parallel and bonded to the Grm coatedpaper (W = 40 mm) using low contact resistance adhesion(R = 0.001Ω). The electrical resistance wasmeasured between electrodes spaced various distances, L, apart (Keithley model 2000). The sheet resistance was then determinedfrom the slope of the linear trend between L/W and paper resistance, shown in 31The sheet resistancesof various spray-coated paper substrateswere measured in accordance with the transmission line method.N ≈ 2 to 3). To explore this further,we isolated the fraction of few-layer graphene contained in the shearexfoliated Grm inks by performing an additional centrifugation stepat RCF = 243g and 45 min. We measured the extinctionspectra for both RCF = 115g and RCF = 243g and compared the spectroscopic ratio ε550 nm/ε267 nm as a proxy to illustrate the changesin the atomic layer number (N ≈ 10) in the EV Grm ink was found to be ∼64 wt %.In contrast, the graphitic material within the flake Grm ink is dominatedby few-layer graphene nanosheets (N < 10) includinga significant fraction with N = 8 (∼64 wt%). This difference in composition impacts the electronic propertiesof the deposited film and results in a higher sheet resistance forthe upcycled spent EV anode material.Using the flake Grm as the reference,the sheet resistance wasfound to be 10-fold higher for the EV Grm coating. This is due toa combination of factors including thicker and smaller nanosheets,the presence of a residual anode binder in the nanosheet network,and the potential presence of nanosheet defects. Under the same shearexfoliation conditions, r number 6D. The f6 Similar approachesmay also work with liquid exfoliated spent graphitic anode materials,maximizing the conductivity of upcycled functional inks where graphiticnanoparticles, additives, and surfactants are present.The conductance of thethin film produced from EV Grm increasesalmost fourfold when the number of spray passes is doubled to 200.This highlights that further reductions in sheet resistance toward Althoughthe electrical conductivity of the paper coatings wassuperior using high-purity graphite flakes, contemporary electroniccircuits rely on a suite of different components and materials . We exploited the differences in electronicproperties between EV Grm and flake Grm solution-processed thin filmsto fabricate a simple paper circuit board with a white light-emittingdiode 7. PaperCsc < 10 mM. At concentrations above this, the surfactant influencesthe upcycled product concentration and layer number similarly to few-layergraphene exfoliated from graphite flakes. Finally, we investigatedthe use of upcycled graphene-related material for fabricating paperelectronics. The sheet resistance of thin films formed from upcycledmaterial is an order of magnitude higher, which may be addressableusing alternative solvents to remove the binder and any additives.However, the environmental sustainability of the proposed water-basedliquid processing techniques is advantageous over chemical treatmentsor traditional toxic solvent use. Furthermore, lithium-ion batteriesalso utilize different binders that are dissolvable in water  and graphite sources which provide opportunities for improvements without sacrificing thesustainability of the high-shear upcycling process.This work demonstrates that high-shearexfoliation in aqueous surfactantsis a viable approach for upcycling spent graphite anodes from electricvehicles into solution-processable graphene. The nanomaterial yieldwas found to be comparable to and in most cases higher than that obtainedusing high-purity graphite flakes as a precursor. The maximum concentrationrecovered using this upcycling approach was 37.5% higher than thepeak concentration of few-layer graphene obtained from graphite flakes.By demonstrating the feasibility of shear exfoliation, this work showsthat other shear exfoliation techniques that have achieved high yieldsusing natural graphite precursors  may alsoprocess spent electric vehicle anodes with similarly high yields.However, it is noted that significant improvements to this techniquewill be necessary to achieve economical upcycling of spent anodesfrom electric vehicles. Spectroscopic measurements suggest that theaverage atomic layer number in aqueous-surfactant dispersions is 2to 3 atomic layers less when using the high-purity graphite flakeprecursor. The sphericalized graphite morphology together with thepresence of the PVDF binder impacts production output, particularlyat low surfactant concentrations of


Idiopathic pulmonary fibrosis is a poorly prognosed form of progressive interstitial pneumonia. Patients with IPF have a significantly increased risk of developing lung cancer, which further worsens the course of the disease. The most common histological types of LC among patients with IPF are squamous cell carcinoma and adenocarcinoma. Furthermore, all LC treatment modalities can lead to developing an acute IPF exacerbation. In this report, we present a rare case of coexistence of IPF and small cell lung cancer in a 76-year-old patient with chronic obstructive pulmonary disease, and a former smoker. For over 2 years, the patient was treated with an anti-fibrotic drug-pirfenidone, which slowed down the progression of IPF. Unfortunately, after being diagnosed with an active SCLC, the patient was excluded from further participation in the pirfenidone drug program. SCLC is characterized by high aggressiveness, rapid growth and high metastatic potential; therefore, it is necessary to apply antitumor treatment as soon as possible. The described patient was treated with carboplatin–etoposide chemotherapy. Early treatment tolerance was good and after two cycles of cytotoxic treatment, a partial response was present in CT. The presented case emphasizes the need for further research to determine the treatment regimens in patients with coexisting IPF and LC and the appropriateness of antifibrotic treatment in them. In addition, it can help to choose the treatment method for similar patients, indicating a combination of carboplatin and etoposide as an effective and, at the same time, relatively safes method in terms of the risk of IPF’s exacerbation. Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic, progressive, fibrosing interstitial pneumonia of unknown cause. It is a rare disease with an estimated prevalence ranging from 10–60 cases per 100,000 people, which occurs primarily in older adults. IPF is defined by the histopathologic and/or radiologic pattern of usual interstitial pneumonia (UIP). It leads to a progressive worsening of dyspnea and an irreversible decline of lung function. The clinical course of IPF varies and is difficult to predict, nevertheless, the prognosis is poor and the mean survival is 3–5 years from diagnosis. However, there are two anti-fibrotic drugs—pirfenidone and nintedani—which decrease physiological progression and likely improve progression-free survival .There are several pulmonary and extrapulmonary comorbid conditions that affect the course and prognosis of IPF, such as chronic obstructive pulmonary disease, pulmonary hypertension, congestive heart failure and lung cancer (LC). It is worth noting that among them, lung cancer has the biggest impact on mortality in the setting of IPF, and LC survival is significantly worse than in patients with IPF without LC  . The mosSCLC is an aggressive cancer of neuroendocrine origin, which is strongly associated with cigarette smoking. It is characterized by rapid doubling time and early dissemination in lymph nodes and distant organs. Despite its high sensitivity to chemotherapy and radiotherapy, survival rate is low due to its high relapse rate. Considering the fact that the prognosis is poor even with IPF alone, when IPF coexists with SCLC, the patient has a very low chance of survival .The aim of this study is to present a rare case of coexistence of IPF and SCLC and to analyze the available therapeutic options, which are very limited due to the fact that in patients with IPF, all LC treatment modalities can lead to lung function deterioration .2 44 mmHg, and pO2 48 mmHg. During hospitalization, a positive COVID-19 antigen test result was obtained (patient was not vaccinated against COVID-19). A 76-year-old man was admitted to the Department of Pneumonology, Oncology and Allergology in November 2019 due to symptoms that had been intensifying for several months: dry cough, exercise dyspnea, exercise intolerance and general weakness. He had been suffering from chronic obstructive pulmonary disease since 2013; for this reason, he was being treated with home oxygen therapy. The patient is a former smoker of 40 pack-years. His past medical history includes arterial hypertension and ischemic heart disease. The computer tomography examination revealed the features of massive honeycomb pulmonary fibrosis, with a predominance distribution in subpleural and basal areas. Moreover, reticular abnormalities such as thickening of intra-and interlobular septa, increased lung density, bronchial tree dilatation and the presence of emphysema were visualized, and in the HRCT option, small air traps during the expiratory phase A. In pulIn a chest CT performed in March 2022, polycyclic confluent tissue masses were visualized, infiltrative in the left lung occupying a significant part of the lower lobe and the dorsal parts of the upper lobe with radiological features of the neoplastic lesion. Compared to the CT performed in February, progression of nodal lesions in the mediastinum and hilars was visible A. Nodal Lung cancer has been shown to significantly worsen the course of the disease and the prognosis of patients with IPF. Despite many studies on the cause of frequent LC development in the course of IPF, the underlying pathomechanism has not yet been clearly confirmed . Proven The guidelines for the treatment of lung cancer for the general population are unambiguous, but there are still no commonly accepted recommendations for the group of patients with IPF, especially for the rare co-occurrence of IPF with SCLC. Various treatment methods for lung cancer, including surgical intervention, chemotherapy and radiation therapy, carry the risk of inducing an acute exacerbation of IPF, which can be fatal ,15. In oRecent studies show that pirfenidone combined with carboplatin-based regimens or immune checkpoint inhibitors might be a safe first-line chemotherapy for patients with IPF and non-small cell lung cancer (NSCLC), which accounts for the vast majority of LC in the course of IPF . SCLC ocNintedanib, another antifibrotic agent, is frequently used in combination with docetaxel for treatment of NSCLC . NintedaFor over 30 years, platinum-based chemotherapy has remained the primary therapeutic option for patients with SCLC. The recent CASPIAN and IMpower133 studies on the effectiveness of the combination of chemotherapy and immunotherapy in this group of patients have opened a completely new chapter in SCLC treatment. They proved that the addition of an antibody blocking the PD-L1 molecule  to first-line chemotherapy (based on platinum and etoposide) has a more favorable effect on patients’ survival than chemotherapy alone. In both studies, the median overall survival of previously untreated patients with extensive stage SCLC was significantly longer in the group receiving immunotherapy in combination with chemotherapy compared to the group receiving chemotherapy alone . The safety profile was acceptable. These two immune checkpoint inhibitors proved to be the first agents in the last decades to determine an improvement in outcomes of patients with extensive stage SCLC patients, so it led to a new standard of care . UnfortuSince the coexistence of both diseases is rare, so far not enough clinical trials have been conducted to determine the optimal chemotherapy regimen for patients with IPF and SCLC that would not adversely affect the course of IPF. Therefore, when choosing a chemotherapy method, we can only use similar cases described in the literature. Among these few cases are two SCLC and IPF patients with poor lung function described by Zhang et al. They also received chemotherapy based on a derivative of platinum and etoposide. At the same time, anti-fibrotic treatment was continued. After treatment, both patients’ clinical condition and lung function improved .In conclusion, an individualized approach is essential when choosing a treatment method for patients with IPF and SCLC. Chemotherapy seems to be the most effective method against SCLC, and at the same time, relatively the safest method in terms of the risk of exacerbation of IPF. The case of the patient described by us emphasizes the need for research into the development of new therapeutic agents that would enable effective treatment of both SCLC and IPF.


The substituent effect is characterized by the substituent effect stabilization energy (SESE) and substituent Hammett constant σ. For adenine, SESE is obtained with purine as the reference system. Additionally, for both adenine and purine, SESE characteristics are estimated with benzene, imidazole and amino-pyrimidine as reference systems, when possible, taking into account substitution in topologically equivalent positions. The role of a C6–NH2 group in adenine in modifying the substitution effect is observed and discussed. Additionally, the proximity effect for some asymmetric substituents  is recognized and meticulously analyzed.Substituent effects at the C2-, C8- and N-positions of adenine and purine in their four the most stable tautomers are studied by means of B97D3/aug-cc-pvdz computation applying substituents of varying electronic properties: NO 2, CN, CHO, Cl, F, H, Me, OMe, OH and NH2.Substituent effects at the C2-, C8- and N-positions of adenine and purine in their four the most stable tautomers are examined using substituents of varying electronic properties: NO The low energetic prototropic abilities of adenine result from the presence of four endocyclic nitrogen atoms, one of which is bonded to a hydrogen atom. The proton may change its position, which leads to a formation of four low-energy tautomers. Among them, the 9H tautomer  level of theory.14 It was found that the stability of tautomers of substituted adenine molecules strongly depends on solvation. It was also found15 that, for 20 different substituents in the position 8 of adenine, electrostatic potentials at the bonding atoms of monomers correlate nicely with interaction energies of adenine–thymine pairs.ADF computational package was used to study substituent effects on nanoswitches based on DNA base pairs16All of the above-mentioned papers present research results indicating that many important properties of nucleic acid bases significantly depend on the substituent attached to their molecules. In addition, it should be mentioned that in most cases observed changes of the studied systems were not related to the electron properties of substituents. It is well known that substituents can exert either electron withdrawing or electron donating properties. Thus, they may influence substituted systems in a different way and have a fundamental impact on the nature and consequences of the substituent effect.σ  are linearly related to the substituent constants σ(X), as shown in eqn (1)ρ is termed reaction constant and characterizes sensitivity of the property P(X) on influence of the substituent X. For exhaustive reviews see Classically, the quantitative characteristics of the substituent effect is described by the Hammett substituent constants σ  or their24,25 has resulted in the introduction of several models based on quantum chemistry describing substituents effects. One of them is the use of homodesmotic reactions26–28 which for disubstituted species is presented by eqn (2)Dynamic development of applications of quantum chemistry in chemical research29 describes the substituent effect in energetic scale. A positive SESE value means that the intramolecular interactions between the substituents X and Y stabilize the X–R–Y system. This descriptor of the substituent effect takes into account all interactions in X–R–Y systems. Another quantum chemistry concept of the substituent effect is based on atomic charges. It is known that due to polarity of X–C bonds in substituted carbocyclic systems, atomic charges of substituents (in e.g. benzene derivatives) do not correlate with substituent constants.30 However, when instead of the charge of the X substituent, q(X), the sum of charges in the substituent X and the ipso carbon atom are taken into account, the correlations with the constant substituents are very good.31,32 This descriptor is named cSAR(X) (charge of the substituent active region) and independently of the atomic charge assessment method, it correlates well with substituent constants.33 Recently, it has been shown that in the case of series of disubstituted benzene, cyclohexadiene and bicyclooctane derivatives, both descriptors, SESE and cSAR(X), modeled theoretically correlate well with traditional substituent constants.29,34–36 It is important to note that cSAR(X) not only describes electron attracting/donating properties of the substituent, but also informs how these properties depend on its position in the substituted molecule. Another fruitful theoretical tool to study the substituent effect is the analysis of molecular electrostatic potential (MESP), which was recently published by Remya and Suresh37 and references therein.The difference between energy of products and substrates, named SESE (substituent effect stabilization energy),Adenine and purine are π-electron systems. As mentioned above, four endocyclic nitrogen atoms with the possibility of prototropic rearrangements give four tautomers. There are several questions that we should ask here: (i) how substituent effect descriptors based on quantum chemistry work in such complex molecules and whether they differ from benzene systems; (ii) how the substituent effect works in four different tautomers; (iii) whether the substituent can change the stability of the tautomer; (iv) how far the amino group in adenine may differentiate the substituent effect comparing to purine.2, CN, CHO, Cl, F, H, Me, OMe, OH and NH2) of the four most stable adenine and purine tautomers  are presented and discussed.The research objects were C2–X, C8–X and N–X substituted derivatives  in purine and adenine are presented in The energy changes due to the presence of the most electron-accepting and electron-donating substituents . Moreover, significant differences can be noticed when looking at the nitro and amino derivatives substituted at the C2 and C8 positions. In the case of the C2- and C8-nitro purine derivatives tautomer energies increase monotonically, while for adenine derivatives the lines are irregular and intersect between 7H and 3H tautomers. For amino derivatives of purine and adenine energy changes are similar. In both cases substitution at C2 leads to lower stability of 3H and 1H tautomers than in the case of C8 substitution. This means that the amino group affects energy of substituted tautomer derivatives strongly enough to modify the sequence of decreased stability. For the N–X systems shown in Erel, (with respect to the 9H tautomer 9H), for studied C8–X, C2–X and N–X substituted derivatives of adenine and purine tautomers can be found in Tables S1–S3.2-purine, more stable than 9-NO2-purine by ca. 1.7 kcal mol−1, and the reason is a formation of a weak C–H⋯O intramolecular hydrogen bond in the former. The mean values of average relative stability in substituted adenines is ∼10 kcal mol−1, whereas for purine series is only ∼7.3 kcal mol−1. Undoubtedly, this difference results from the presence of the NH2 group at the 6-position in adenine, which additionally leads to greater variability of relative energies in adenine series. Secondly, we noted that for both adenine and purine derivatives, in both C8 and C2 substituent positions, the Erel values for tautomers 7H have the smallest range of variation . The direct neighborhood of the C8 substituent in 9H and 7H tautomers is almost identical: one nitrogen atom with a lone pair in the molecular plane (2pxy) and one electron on the perpendicular 2pz orbital and second nitrogen (in N–H group) with 2pz electron pair. Thus, even if there are some proximity effects, they are similar, and the resulting Erel differences are small. For C2–X substitution the ranges are the smallest, since there are no proximity effects that appear in the vicinity of C8. No such situations are found in other cases, with much larger Erel ranges, generally between 8.07 kcal mol−1  and 13.21 kcal mol−1 . In addition, it is worth noting that the substituents, except for the N–X cases with X = NO2, do not change the stability of purine tautomers  differences with respect to the unsubstituted systems (X = H) allow for easier analysis of the SE. For 7H tautomers, this difference can be written as the energy of the homodesmotic reaction, named SESErel:The use of relative energy  on SESErel(purine) are analyzed  of N–X substituted tautomers do not lead to unequivocal conclusions. However, when some of the most deviating points are omitted (empty circles in 2 and OH) and finally 0.673  for 3H substituted tautomers.Relationships between SESErel model, in adenine with that present in purine, allows to conclude that more significant effect is observed in adenine when substituent is attached to the 5-membered ring.Thus, the comparison of the substituent effect, based on SESEσ constants are the most commonly used to describe the electron donating/accepting properties of the substituents. Their application to the data in relvs. σ regression (see Table S5R2 lower than 0.3 (for 3H C8–X purine series). These results provide important information that the model of interactions which describes well the substituent effect in benzene derivatives fails completely in some cases of adenine and purine derivatives. This problem will be discussed in more details later.Hammett EC8–X and EC2–X are the energies of the mono-substituted molecule at positions C8–X and C2–X, respectively.Differences between the energies of pairs of the same molecule with different substitution positions allow to compare the substituent effect from these positions. For all adenine and purine tautomer series with substituents at the C8–X and C2–X positionsE8−2 values for all adenine and purine tautomers are given in It should be noticed that the close vicinity of C2 and C8 substitution positions in adenine and purine is topologically identical. The obtained ΔE8−2 averaged for all four tautomers of adenine and purine derivatives do not correlate with each other (R2 = 0.421), while the range values do very well (R2 = 0.954), as shown in Fig. S1.Some interesting points resulting from the comparison of the data presented in E8−2 values for adenine and purine – in other words – shows how the amino group in adenine influences interactions in the molecules. As mentioned above, the closest vicinity of positions C2 and C8  and slopes variety (between 0.868 and 1.256) could indicate dependence of the systems sensibility on the position of the substituent. Undoubtedly, the difference should be related to the presence of NH2 group in adenine.Despite the similarity of the closest vicinity for substituents in the C2–X and C8–X positions and the fact that their energy difference is considered, differences appear in Δe series . It is iEnergies of substituted purine (PU) and adenine (AD) can be applied for homodesmotic reactions:For C2–X substituted systems of the 9H tautomer we can write:PU approach are gathered in PU values indicate greater stability of substrates than reaction products.Analogous equations can be written for the remaining substituted tautomers. The results of the SESEPU values are usually negative (i.e. destabilizing interactions) for electron-donating substituents and positive for the electron-attracting ones. In the case of strongly interacting substituents, for the C8–NH2 and C2–NH2 systems the average SESEPU values  are −2.47 and −0.46, whereas 2.37 and 1.72 for NO2 derivatives, respectively. These differences in the energy values of the substituent effects in adenine, in comparison with purine, can be connected to the presence of the amine group at the 6-position in adenine.Observed SESEPU values with the traditional substituent effect descriptor, i.e. Hammett constants. Its mutual correlations for C8–X and C2–X adenine series are shown in σp and σm relations).It is interesting to compare the SESEPU correlates better with σp than with σm, (ii) the same applies to C2–X series of 3H and 1H tautomers, when X = CHO systems are excluded, whereas (iii) for C2–X derivatives of 9H and 7H tautomers, a better correlation of SESEPU with σm than with σp can be found.A verification of the obtained interrelationships shows: (i) for C8–X systems SESEPU values are based on differences in the energy values of the analogically substituted adenine and purine derivatives, obtained values inform about changes related to the amino group present in adenine tautomers. In the 7H tautomer the 2pz electron pair residing at N7 atom hinders charge transfer between electron-attracting substituents at the C8 position and the amino group at C6  interacts with a lone pair and the CO part with the NH, while in the second conformation, when CHO is rotated by 180°, strong repulsive interactions appear: CO is interacting with the lone pair and CH is interacting with NH. The observed difference in energy between these two rotamers is 6.59, 5.50, 9.53 and 5.92 kcal mol−1, respectively. Similar effects are observed for other nonsymmetric substituents, such as X = OH and OMe  and the NH with the 2p Table S7. These a Table S7 predominDifferent interactions can be noticed in N-substituted adenine derivatives. Here, the proximity effect is mainly observed for N7- and N1- substituted species, where amino group in the 6-position is neighboring to substituents. Good examples of proximity effects are intramolecular hydrogen bonds presented in 2 group at C6, have been compared. One can ask how different are substituent effects observed in adenine and purine derivatives compared to those in benzene (BEN) and anilines (AN), which are considered classical objects in the SE research.So far, substituted adenine and purine series, differing only in the NHThe corresponding equations are as follows:BEN and SESEAN values are presented in PU.Analogous equations can be written for the remaining substituted tautomers of adenine and purines. The obtained SESEBEN and SESEAN are negative for electron-attracting substituents and positive for electron-donating ones. In addition, their absolute values are higher for purine systems than for adenine ones. This can be depicted by the slope of a linear relation of the SESE descriptors (SESEBEN and SESEAN) between the values for adenine and purine systems as shown in R2 > 0.95).In almost all cases the obtained values for both SESEBEN and SESEAN are based on homodesmotic reactions with benzene derivatives as references. Thus, these descriptors, by definition, include a comparison of the substituent effect in heterocyclic systems (adenine or purine) with that realized in traditional benzene series (monosubstituted benzene or aniline derivatives). In the case of para-substituted aniline derivatives, we can characterize the SE using their homodesmotic reaction with benzene, where energy, SESEAN–BEN, can be calculated as:Both SESE34 are also given. These values are congruous with the results calculated at B97D3/aug-cc-pvdz level of theory.The obtained values are presented in Table S9.PU, whereas benzene for SESEAN–BEN. The obtained results of linear relation SESEPU(adenine) vs. SESEAN–BEN(aniline), shown in The use of the SESE concept, with appropriately selected reactions, allows the comparison of the influence of a transmission moiety on the substituent effect. Purine is a reference for SESEIn conclusion, a subsequent study confirmed that the more significant effect is observed in adenine when the substituent is attached to the 5-membered ring (substitution at C8–X position).2–pyrimidine) and imidazole rings. Therefore, it can be of interest how the substituent effects observed in adenine and purine derivatives differ from those in imidazole (IM) and amino-pyrimidine (PY), substituted in a similar topological environment as in the purine (adenine) molecules. For this purpose, appropriate homodesmotic reactions, for the 9H tautomer shown in IM and SESEPY, both for adenine and purine C8–X and C2–X substituted series, respectively; for 9H adenine C8–X and C2–X derivatives, according to the following equations:Purine (adenine) can be formed by the fusion of pyrimidine  for electron-donating substituents and usually positive for the electron-attracting ones. The opposite changes apply to SESEIM, that is for C8–X series both adenine and purine. Furthermore, the slopes of a linear relation of the SESE descriptors (SESEIM and SESEPY) between the values for adenine and purine (both 9H and 7H) systems (R2 > 0.92).For particular substituents, calculated SESE systems  documentBEN, SESEAN, SESEIM and SESEPY on σp for C8–X and C2–X adenine and purine derivatives are presented in Table S10.†Statistical details of the mutual dependences of SESEσp should be understood as the generally accepted characteristic of the substituent effect in cases where contributions from inductive/field and resonance effects are equal.47–49 The greater R2 of the regression SESE vs. σp, the interactions between substituents in adenine/purine series are more similar to those in benzene. This could mean that in the studied systems the classical resonance and inductive/field effects are realized nearly alike. For almost all cases in the adenine series R2 values are worse than the ones in purines  as a reference for the SE description in adenine systems is clearly worse than in purines. However, if aniline (i.e. 4-X–aniline) is a reference system, the determination coefficients improve, and SESEAN becomes a good SE characteristic in both adenine and purine series. The same applies to SESEIM, but this descriptor can only be used for the C8–X series. In addition, better correlations of SESEAN with σp for C8–X series than for C2–X indicate a greater role of the resonance effect in the first case.It should be added that Table S10. This caConsidering the slope absolute values and benzene (aniline) as a reference, for the purine derivatives the strength of the SE increases with the stability of tautomers for C2–X series, while opposite trends can be seen for C8–X systems. In the case of adenine derivatives, the picture is not so clear. In addition, again, lower absolute slope values in the second series may be related to the presence of the amino group in adenine.2) and SESEPUR (as the SE descriptor) for all studied adenine tautomers are shown in 2) values are gathered in Table S11BEN and SESEAN are used as the SE descriptor are presented in Table S12.†The classical or traditional SE is the most common studied type of interaction – the properties of the “reaction site” Y (the fixed group in the series) are related to the properties of the X substituent in the disubstituted X–R–Y system. In the case of studied adenine derivatives, the C6-amino group is the fixed group (Y), purine moiety can be defined as R and substituents (X) can be introduced at the C2 or C8 positions . The rel2) on SESEPUR indicate an interesting difference between regressions for the C8–X and C2–X series. It is important to note the high values of the determination coefficient , which entitle us to a deeper analysis. The variability of cSAR(NH2) sensitivity to the SE in the C8–X series is significantly greater (slopes varies from 0.012 to 0.027) than in the C2–X series (the slope ranges from 0.025 to 0.029). This discrepancy can be easily explained by resonance structures for these two systems, which resemble the interactions with electron-attracting groups observed in the case of para and meta positions in the substituted aniline34 (or phenolate).50 Adenine C8–X derivatives resemble para-substitution in benzene, where the intramolecular charge transfer between the electron donating and electron accepting substituents can be described by canonical forms with charge separation, shown in meta-nitrophenolate). In general, when the number of bonds between carbon atoms to which electron attracting and electron donating substituents are attached is odd, then a charge transfer occurs (as in para substituted benzene), if the number is even, then interactions are like those for meta substituted benzene.51Obtained dependences of cSAR(NH2 groups or nitrogen atom in the CN group). In this case, if the number of all bonds between donating and accepting atoms in D and A groups, respectively, is even, then the charge transfer is privileged and well visible.52An another equivalent way of counting the bonds is the number of bonds between the electron-donating atom  and the electron-accepting atom  relation to SESEBEN and SESEAN (obtained with benzene derivatives as a reference). In the case of the C8–X series, they are higher than 0.857 , while the 1H tautomer systems are the least stable. In the case of 7H and 3H tautomers, the order of energy changes for substituted adenine derivatives differs from the sequences in purines, which can be explained by the presence (interaction) of the NH2 group. The amino group in adenine induces about 1.6 times greater energy difference between the most and least stable tautomers comparing to purine. In addition, substituents, except for the N–NO2 systems, do not change stability of purine tautomers, which decreases in the order 9H, 7H, 3H and 1H.For all substituted adenine and purine series, the 9H tautomeric systems are the most stable  analysis indicate the stronger substituent effect in adenine than purine derivatives for substituents attached adjacent to the NH group of the ring. In addition, a more significant effect is observed in adenine systems when the substituent is attached to the 5-membered ring. This observation is also confirmed by the results of analysis of the differences between the energies of pairs of the same molecule with the substituent at the C8–X and C2–X positions, for all tautomers.The results of SESEPU (purine as reference) approach informs about changes in the substituent effect due to the presence of the amino group in adenine tautomers. Like in the substituted aniline derivatives, the obtained values show stabilizing and destabilizing interactions of the amino group with electron-attracting and electron-donating substituents, respectively. Moreover, these interactions are more pronounced in C8–X than in the C2–X series.The SESEPY, suggest stabilizing interactions in the case of electron-donating substituents.The description of the substituent effect in adenines and purines based on their similarity to monocyclic reference systems  allows to conclude that the amino group of adenine reduces the effect of substituent when compared to purine. This weakening is more pronounced for the C8–X substitution, independently of the tautomer. However, it should be emphasized that these SESE values, with the exception of SESEPU reveal the similarity of the substituent effect in C8–X to the para, while in C2–X to the meta positions in benzene . As in the case of aniline derivatives,34 the sensitivity of the amino group to the substituent effect, along with much better correlations, exhibit C8–X systems when compared to C2–X ones. In addition, weaker transmission of the substituent effect by the purine moiety than by benzene is observed.Dependences of SESE descriptors on the Hammett substituent constants allow to compare the similarity of the substituent effect in adenine (and purine) to that observed in benzene. The obtained relationships for SESEFurthermore, analysis of the classical substituent effect in adenine confirms the more pronounced substituent effect from the C8-position what can be explained by the stronger resonance effect in such arrangements.Specific interactions between substituents and neighboring N or NH are the reason for deviations and lower similarity of the substituent effect when compared with systems without these kind of interactions. Particularly strong deviations are observed for CHO group.The authors declare no competing financial interest.RA-009-C9RA04615A-s001


The drawbacks of conventional model (no coating) have been resolved by presenting this one at blue spectrum specifically. Finally, mfERG clinical and experimental results show that this model can stimulate the electrochemical voltages and currents in photoreceptor cells.The absorption of light is very important task for retina photoreceptors. Graphene is an energy harvesting material and one of the best models for the electromagnetic wave absorption and its conversion into signals. In this paper, an electromagnetic modeling of human retinal photoreceptors has been presented based on graphene coated material as a receiver antenna. The proposed electromagnetic model based on dielectric resonator antenna (DRA) is being analyzed for retina photoreceptors of human eye (cones and rods) by Finite Integral Method (FIM) collaborated with CST MWS. The results show that the model is good for vision spectrum with a proper field enhancement in cone photoreceptor due to its sensitivity to light. The results indicate proper S Dielectric resonator antenna; Electromagnetic model; Graphene; Photoreceptor; Retina In recent years, blind people are searching for a proper device to restore their sights. That is why one of the electromagnetic challenges is analysis and modeling of retina and its optic nerve in order to replace a prosthesis as a bionic eye. There have been many good endeavors for realizing commercial prosthesis such as Argus I, Argus II (the second sight), Alpha-IMS, fractal prosthesis (FracRet AIM), and etc. Actually, there is no existing model (fabricated as mass production) as the view point of antenna concept. The graphene coated model is a unique and novel model. This is the first time that photoreceptors model is being introduced as a viewpoint of antenna concept.The incoming light waves are converted to electrical signals that stimulate neurons in retina and then convey information to other structures in the central nervous system (CNS) by optic nerve. The typical human trichromatic color visual system combines the response of three types of cone cells in retina; the three cone types-labeled for short, medium, and long wavelengths are sensitive primarily to light with central wavelengths of 437 nm (Blue), 533 nm (Green), and 610 nm (Red), respectively. The rod cells are also receiving the signal with a center wavelength of 498 nm for lower luminance of light. A block diagram of retina photoreceptor system has been shown in In this paper, the proposed electromagnetic model on graphene coated dielectric resonator antenna (DRA) is being analyzed for retina photoreceptors of human eye (within its interior medium) by Finite Integral Method (FIM) collaborated with CST MWS. A comparison is done between two models i.e., graphene coated and no coating ones.2The human tissue can act like either an absorber or a reflector at high frequencies; Blocking, attenuating, or redirecting any signal that propagates in its direction. Model of two photoreceptors is illustrated in 2 and is responsible for different wavelength detection (color detection) but the rods are achromatic, one type of pigment; Rods dominate in scotopic vision by less than 0.003 cd/m2 and are responsible for intensity detection.The conical resonator is a chromatic one, three types of pigment ; it dominates in photopic vision by more than 3 cd/mA human rod cell is about 2 microns in diameter and 100 microns long and the cone cells are typically 40-50 μm long, and their diameter varies from 0.5 to 4 μm 3Computer Simulation Technology (CST) MWS is a special tool for the 3D electromagnetic simulation of high frequency components. It enables the fast and accurate analysis. In this broadband structure, it helps to simulate accurately.There are several stages for defining the graphene material in CST MWS as shown in 1- Click on Macros (Top right button).2- Click on Materials.3- Choose the “Create Graphene Material for Optical Applications” Option.4- Enter the temperature in K.5- Enter the chemical potential in eV.6- Enter the carrier relaxation time in ps.7- Enter the thickness of graphene material already created in the CST environment.The graphene is single layer of graphite. A planar monoatomic layer of carbon bonded in a hexagonal structure.ω is radian frequency, τ) that is assumed to be independent of energy, and T is temperature.The Kubo model for the surface conductivity of graphene material can be shown as following equation A code using MATLAB software has been terminated in accordance with the equation An equivalent effective permittivity for the graphene as a very thin metal can be defined as following 11 for waveguides and HEMδ11 for cavities transversal  in addition to the klongitudinal  due to its radial shape around the media and photoreceptor (dielectric part). It generates an extra applied electric field mostly in the radial  direction. This field impacts on both transversal and longitudinal fields simultaneously as they are related to each other for supporting the HEM mode.In other words, the structure (port) is supported by Hybrid Mode (HEM), both TE and TM. Hybrid electromagnetic modes (HEM) are supported by structures exhibiting certain boundary condition(s) satisfying which requires a hybrid of TE and TM mode solutions, rather than either. These modes are usually found as the higher order modes of a waveguide or cavity structure, possessing some unique characteristics. Two structures that use the hybrid modes for popular but distinct applications are the (i) Iris loaded cylindrical waveguide and the (ii) Cylindrical dielectric resonator. Usually, hybrid mode structures are operated in the dominant hybrid mode designated as the HEMThe carrier relaxation time of electrons is estimated to be in the specified range of When 4The incidence procedure is defined by absorbed, transmitted and reflected signals as shown in It indicates that the absorption of graphene coated photoreceptor is roughly acceptable in all spectrum  behavior, normal or negative-index diffraction. The optical properties can be dynamically tuned by controlling the operating frequency and the doping level of graphene The real part of epsilon for graphene at visible spectrum shown in  correspoThe wide spectrum response, transmitted, reflected and absorbed signals of graphene coated model has been illustrated in The field distribution (Electric and Magnetic) of two models along Z axis , central axis of structure as an example, indicates a familiar phenomenon nominated localized field enhancement The field distribution in graphene coated model has gotten an improvement with respect to the simple one specially at higher frequencies of blue light  are shown in the following. The far-field characteristics of graphene coated rod cell are being illustrated in The Co-polarization and Cross-polarization of graphene coated cone and rod models have been illustrated in Radiation efficiency of a single graphene coated model is not significant as its array. This parameter has been compared for both single and array Directivity is a fundamental antenna parameter that shows a measure of how well the antenna directs the received energy toward a particular direction. As a receiver antenna, the directivity is more important than the gain. This is a model for the photoreceptors of retina in a human eye. So, it is a receptor not a transmitter. The gain will be meaningful when the antenna transmits the signal. This model indicates that how much values of the received energy will reach to the end of photoreceptors as electrochemical signals; then it defines the directivity parameter not the gain.G, D are gain, radiation (antenna) efficiency and directivity of antenna respectively. The gain can be realized by the mentioned directivity and the radiation efficiency . The radiation efficiency of single graphene coated antenna is not so much (between 10-25 percent) at visible frequencies; but its array  of retinal cells is the amplitude. The mfERG waveform includes three points N1, the first negative peak, P1, the positive peak, and N2, the second negative peak. The overall mfERG amplitude is defined from N1 to P1. It shows the largest amplitude at the fovea region. The amplitude of the N1 is the average voltage of the encompassed window for N1 point; the N1 amplitude is from the baseline to the N1 point.Implicit time (the time to peak) is the required time for the electrical response to reach the maximum amplitude. It is measured from stimulation time to the corresponding wave-component peak one and returns the signal conduction rate.The array of traces in the top row (It is noteworthy that N1 wave includes contributions from photoreceptors in comparison to other cells such as bipolars that contribute to the P1 wave 611 (return loss below -10 dB) with invaluable resonances in a wide range of frequencies from 405 THz to 790 THz (vision spectrum), suitable S21 (insertion loss 3-dB bandwidth), very good field distribution for flowing the power within desired radiation characteristics.The electromagnetic model based on dielectric resonator antenna (DRA) has been analyzed for retina photoreceptors of human eye (cones and rods) by Finite Integral Method (FIM) collaborated with CST MWS. The results show that the model is good for vision spectrum with a proper field enhancement in cone photoreceptor due to its sensitivity to light. The results indicate proper SThe comparison of two models, graphene coated and simple one shows that there are significant improvements specially in blue light and beyond of spectrum. It can be expressed that many drawbacks of previous model have been covered by this graphene coated model.The received level signal (field distributions) of the proposed model regarding to the input level can stimulate the signals of retina photoreceptors in accordance with the amplitude of clinical results obtained by mfERG technique . However human visual system contains other parts beyond optics that may be a good reference for future works. In 711 (return loss below -10 dB) with invaluable resonances in a wide range of frequencies from 405 THz to 790 THz (vision spectrum), suitable S21 (insertion loss 3-dB bandwidth), very good field distribution for flowing the power within desired radiation characteristics. Finally, clinical and experimental technique, mfERG, was compared to the numeric electromagnetic fields.In this paper, an electromagnetic modeling of human retinal photoreceptors was presented based on graphene coated material as an antenna concept. The proposed electromagnetic model based on dielectric resonator antenna (DRA) was analyzed for retina photoreceptors of human eye (cones and rods) by Finite Integral Method (FIM) collaborated with CST MWS. The results showed that the model was good for vision spectrum with a proper field enhancement in cone photoreceptor due to its sensitivity to light. The results indicated proper SMahdi NoroozOliaei, Hamid Riazi Esfahani, Mohammad Abrishamian: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data included in article/supp. material/referenced in article.The authors declare no conflict of interest.No additional information is available for this paper.


The nanoscale-wide 1D contacts allow spininjection both at room and at low temperature, with the latter exhibitingefficiency comparable with 2D tunnel contacts. At low temperature,the spin signals can be enhanced by as much as an order of magnitudeby electrostatic gating, adding new functionality.Spintronicsinvolves the development of low-dimensional electronicsystems with potential use in quantum-based computation. In graphene,there has been significant progress in improving spin transport characteristicsby encapsulation and reducing impurities, but the influence of standardtwo-dimensional (2D) tunnel contacts, via pinholes and doping of thegraphene channel, remains difficult to eliminate. Here, we reportthe observation of spin injection and tunable spin signal in fullyencapsulated graphene, enabled by van der Waals heterostructures withone-dimensional (1D) contacts. This architecture prevents significantdoping from the contacts, enabling high-quality graphene channels,currently with mobilities up to 130 000 cm The useof tunnel barriers has grown out of the need to overcome the so-calledconductivity mismatch problem,20 whichleads to a drastically reduced spin injection efficiency when theresistance of the contact is lower than the spin resistance of thechannel,21Rs = ρλ/W, with W the graphene channel width, λ thespin relaxation length, and ρ the graphene sheet resistance.Nevertheless, standard 2D contacts, even with the use of a tunnelbarrier, are known to introduce strong doping across the channel leadingto inhomogeneity24 and present challenges in the growth of the barrierwithout pinholes, which lead to enhanced spin relaxation.25 On the other hand, fully encapsulated graphene with 1D contacts26 has been shown to produce exceptionally cleandevices and localize the doping within ∼100 nm near the contacts.28 1D contacts have recently enabled spin injection in graphene,29 albeit only at low temperature, with graphenechannel mobilities below 30 000 cm2 V–1 s–1 and contact polarization with multiple inversionsof polarity. Therefore, further study of this architecture is warranted.Full encapsulation by hexagonal boron nitride(hBN) protects graphenefrom direct contact with lithographic polymers, whereas the self-cleaningprocess driven by van der Waals interactions limits any contaminationpresent at the interfaces within small bubbles, ensuring atomicallyclean interfaces in the rest of the heterostructure.2 V–1 s–1, fully encapsulatedby hBN layers, where a spin current is injected via nanoscale-wide1D contacts. Spin precession measurements yield a quantitative understandingby extracting the channel spin relaxation length λ and timeτs and the 1D contacts’ spin injection efficiencyor polarization P. The fabrication process produceshomogeneous graphene channels, including 1D contacts that preventsubstantial charge doping within the channel and offer a gate-tunablecontact resistance. While the spin polarization is comparable to thatfrom standard tunnel 2D contacts, spin transport can be electrostaticallytuned into a mismatch-free spin injection regime. These elements leadto the realization of a ballistic injection process via nanoscale-wide1D contacts and spin transport in graphene with a spin relaxationlength of ∼18 μm and a long mean free path of ∼1μm, opening up possibilities for lateral spintronic elementsthat exploit quantum transport.Here, we report spin transport in high-quality graphene channelswith low-temperature mobility up to ∼130 000 cmSupporting Information, Section 1. Briefly, we use the dry-peel transfertechnique30 to prepare van der Waals heterotructureson a Si/SiO2 substrate. We then use standard electron beamlithography to pattern a hard polymer mask, which defines the channelgeometry by using reactive ion etching.31 Due to different etch rates for hBN and graphene, a ∼10 nmwide step can be seen in the profile of a hBN–graphene–hBNedge  layers of hBN, with ferromagneticcontacts deposited directly onto narrow (∼10 nm wide) stripsof graphene at the sides of the channel . In orderto characterize charge transport, we measured the four-probe resistanceof graphene as a function of its charge carrier density, n, by using a back-gate voltage applied between the highly doped Sisubstrate and the graphene channel /e, as shown in n| ∼ 1 × 1012 cm–2. For device A, the mobility is then45 000 cm2 V–1 s–1 at room temperature and 79 000 cm2 V–1 s–1 at 20 K. The mobilities of our devices, typicallywithin the range of 20 000 to 130 000 cm2 V–1 s–1 , are significantly higherthan previous graphene-based spintronic devices32 that exhibitedmobilities <20 000 cm2 V–1 s–1, the majority of which have used only a partial hBNencapsulation, whereas we ensure this full hBN encapsulation throughoutthe spin transport channel. The charge diffusion coefficient (andcorresponding mean free path) for representative devices is obtainedfrom the graphene sheet resistance via the Einstein relation, D = 1/(ρe2ν), with ν being the density of states for single layer graphene. The contacts exhibit typical Rc values within the range of 3–15 kΩ, whichimplies they should be able to inject efficiently spins into a graphenechannel with Rs < 10 kΩ. Somecontacts had electrical access via their two ends, see Supporting Information, Section 3). The magnetic 1D contacts present charge transportcharacteristics consistent with those of reported nonmagnetic ones26 . Among these are (i) an inverse scaling of Rc with the width of the contact wc , (ii) anegligible temperature dependence of Rc for high carrier density , and (iii) a sizable dependence on carrier density, with a moderateelectron–hole asymmetry and a maximum Rc near the Dirac point . The electron–hole asymmetry of the contact in Rc at n ≲ 0, indicatesthe presence of an n-doped region adjacent to the metal electrode,consistent with the difference between the work functions of the metal(Co) and graphene.33To characterize the 1D contact resistance t wc see S4a, (ii)t wc see S4a, (ii)I into graphene through contacts 1 and 2,and measure a nonlocal voltage VNL betweencontacts 3 and 4. The nonlocal resistance is defined as RNL = VNL/I. The spin valve signal is given by the difference between the twodistinct levels corresponding to the parallel and antiparallel alignmentof the injector and detector electrodes, ΔRNL = RNLP – RNLAP. Spin signals were measuredfor different separations between injector and detector, L, ranging from 2 to 15 μm . An increase of approximately 1 orderof magnitude in ΔRNL from room tolow temperature was observed, as shown in parts a and b of RNL > 1 Ω. Thisstrong temperature dependence is distinct from the weaker dependenceobserved in standard tunnel 2D contacts,5 indicating a different transport mechanism for spin injection in1D contacts.Spin transportis characterized by spin-valve and spin precession(Hanle) measurements in a standard nonlocal geometry, as shown in 34 enablesus to confirm the presence of spin transport and rule out spuriouscontributions which hindered previous efforts in devices with 1D contacts.32 Here RNLP and RNLAP are measuredwhile sweeping an external magnetic field applied perpendicular tothe device plane (Bz).This causes the injected spins, having a polarization along the x-direction (see x–y plane while moving within the channel. The resulting ΔRNL(Bz) , the spin signal is described by a balanceof spin injection and relaxation within the channel, given by34L theinjector–detectordistance and W the channel width. The channel parametersfor charge (σ ∝ D) and spin (τs) transport have a moderate 2-fold increase at low temperature,see 26 Furthermore, P has a 4-foldincrease at low temperature, as confirmed by the parameters extractedfrom Hanle measurements. These observations indicate that the strongtemperature dependence seen in ΔRNL is dominated by the polarization of the contacts, via the scalingΔRNL ∝ P2.The spin signal ΔRc, together with the observation of a more electron–holesymmetric channel resistance, as seen in 38 to the restof the channel. This doped region only exists near the 1D contact,which in our devices has a nominal width wc of 100—350 nm. The n-doped region being localized in thevicinity of the 1D edge is in stark contrast to standard 2D junctionswhich cover the full width of the channel and lead to substantialinhomogeneity.24 Here, the geometry of the 1D contacts plays a keyrole. On the other hand, for the case of the channel sheet resistance,most of the channel is undoped, except for the nanoscale region indirect vicinity of the 1D edge. Therefore, the channel resistance,probed at a length scale L, W ≥1 μm, exhibits a more electron–hole symmetric response.The identification of an n-doped graphene regionnext to our 1Dcontacts from the electron–hole asymmetric response of 38 and is hitherto unaddressed in nonmagnetic28 or magnetic32 1D contacts, is a nonmetallicincrease in resistance at low temperature. All measured 1D contacts and the doped graphene region (∼100 nm); see The descriptionof contact resistance in 2D metal–graphenejunctions involves two processes: carrier transport from the metalto the (doped) graphene region in direct contact with the metal andtransport from the doped graphene region to the rest of the (undoped)graphene channel. The transmission for those two processes gives riseto the observed contact resistance.I (cyandata) has a high electronic quality close to the upper limit of ourmobility range, with μFE = 130 000 cm2 V–1 s–1. Device A (red data) also has a high electronic quality, with μFE = 79 000 cm2 V–1 s–1, whereas device B (black data) has alower quality (μFE = 27 000 cm2 V–1 s–1). As shown in 34 .Parts f–h of s and length λ,shown in parts f and h of s andλ for device I being about twice as large as thosefor device A, while the parameters for device A are ca. three times as great as for device B. Thisobservation indicates spin relaxation dominated by the Elliot–Yafetmechanism,41 where τs increaseswith the diffusion coefficient D . It is clearthat, with our experimental accuracy, it is indistinguishable fromthe fit obtained using s are not significantlyaffected. Further indication for the role of the Elliot–Yafetmechanism is obtained by studying the carrier density dependence.For all our devices, τs and λ increase withincreasing carrier concentration, having a minimum close to the Diracpoint. This behavior, also observed in single layer graphene using2D contacts, has been attributed to the Elliot–Yafet mechanism45 and results in a linear scaling between spin relaxation time τs and momentum relaxation time τp . We have observed spin transport acrossdistances of 15 μm, while crossing several (noninvasive) 1Dcontacts, only limited by device dimensions . Overall, the demonstration of spin transport parametersreaching values up to D ∼ 0.7 m2 s–1 and λ ∼ 18 μm opens upthe exploration of lateral spintronic architectures involving long distance spin transport,42 now within high-quality and homogeneous channels.The spin relaxation time τnt D see 2d. A pris is rather short, in fact shorterthan in many devices with 2D contacts and of lower quality reportedin the literature.12 In the latter case, τs ∼ 2 ns or more have been observed, while the maximumspin lifetime in our device I with the highest mobilityis only ∼0.7 ns. We speculate that the reason is likely tobe due to the specific geometry of spin injection through 1D contacts:here spin-polarized electrons enter from the opposite sides of a graphenechannel which is followed by propagation in the perpendicular directionalong the channel. In this case, spin transport becomes essentially2D and may not be accurately described by the standard 1D Hanle model(where spins are injected uniformly across the channel and continuepropagation in the same direction).We note that, despite the clear indications that Elliot–Yafetmechanism of spin relaxation plays a dominant role in our devices,the observed τRc, to spin resistanceof the graphene channel, Rs, where a ratio Rc/Rs ≫ 1indicates a regime of efficient spin injection.11 Ingraphene spintronic devices, the ratio Rc/Rs has been tuned by using an electrostaticback-gate via the carrier density dependence of ρ and λ,whereas for tunnel junctions Rc typicallyexhibits a weak dependence.25 In our work, not onlythe channel resistance but also the 1D contacts exhibit a dependenceon carrier density (see Rc/Rs > 1 for all carrier densities,increaseswith carrier density, and it is tunable to values Rc/Rs ≫ 1 at high density.We evaluate the degree ofconductivity mismatch via the ratio ofcontact resistance, sity see 2c, whichRNL. As shown in RNL has a minimum at the Diracpoint, consistent with the minimum observed for the spin transportparameters (RNL exhibits a nonmonotonic behavior: in the hole regime,the signal increases and reaches a maximum for n ∼−0.8 × 1012 cm–2, after whichit decreases. For the electron regime, this behavior is much lesspronounced and the spin signal remains comparatively low. This overallbehavior is understood by considering two distinct regimes of conductivitymatching.25 First, at high carrier density,where Rc/Rs ∼ 10, there is a regime free of conductivity mismatch. Here,ΔRNL is insensitive to the valueof Rc, and it scales as ΔRNL ∝ ρ (see Rc/Rs ∼ 3;see Rc. In this case ΔRNL exhibits a scaling similar to that in themismatch regime46 ∝ Rc2/Rs. As indicated by the cyan line in RNL11 at low carrier density and in the electron regime.To further evaluate the spin injection efficiency and its tunability,we discuss the spin valve signal, Δ ∝ ρ see 2. This i32 and tunable spinsignal and achieves a mismatch-free regime at moderate carrier density.The spin signal shows a consistent behavior across all devices, witha minimum near the Dirac point. This observation is compatible withthe proposal of magnetic proximity at the Co–graphene interface,33 where a reversal in the polarity of the spin polarization is expectednear the Dirac point, implying no spin polarization and thus no spinsignal near the Dirac point. The fact that our devices do not showany inversion of the spin signal as a function of carrier densityis consistent with a homogeneous potential profile within the graphenechannel, where both injector and detector contacts would reverse theirpolarizations at similar carrier density. The demonstration of spintransport in graphene with a large mean free path of ∼1 μmat low temperature, comparable to device dimensions, while ensuringsizable spin relaxation lengths, is a key advance in the developmentof low-dimensional spintronic systems approaching47 the high electronic mobility of state of the art charge-baseddevices.48This device architecture demonstrates efficient spin injectionin graphene using nanoscale-wide 1D contacts, reproducibly acrossseveral devices. The ballistic spin injection process allows for observationof the Hanle effect


O,4′-C-ethylene nucleic acids (ENAs), which are modified nucleic acids with high stability, and tested the therapeutic potential of lowering TDP-43 levels using ENA-modified ASOs. We demonstrated that intracerebroventricular administration of ENA-modified ASOs into a mouse model of ALS/FTD expressing human TDP-43 results in the efficient reduction of TDP-43 levels in the brain and spinal cord. Surprisingly, a single injection of ENA-modified ASOs into TDP-43 mice led to long-lasting improvement of behavioral abnormalities and the suppression of cytoplasmic TDP-43 aggregation, even after TDP-43 levels had returned to the initial levels. Our results demonstrate that transient reduction of TDP-43 using ENA-modified ASOs leads to sustained therapeutic benefits in vivo, indicating the possibility of a disease-modifying therapy by lowering TDP-43 levels for the treatment of the TDP-43 proteinopathies, including ALS/FTD.The abnormal aggregation of TDP-43 into cytoplasmic inclusions in affected neurons is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although how TDP-43 forms cytoplasmic aggregates and causes neurodegeneration in patients with ALS/FTD remains unclear, reducing cellular TDP-43 levels is likely to prevent aggregation and to rescue neurons from TDP-43 toxicity. To address this issue, here we developed gapmer-type antisense oligonucleotides (ASOs) against human TDP-43 using 2′-  in vivo, indicating the therapeutic potential of the TDP-43-lowering approach for ALS/FTD treatment.TDP-43 plays central roles in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Takeuchi et al. demonstrate that the transient reduction of TDP-43 using gapmer-type antisense oligonucleotides suppresses cytoplasmic TDP-43 aggregation and disease phenotypes  Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease that is characterized by the selective loss of motor neurons in the brain and spinal cord, leading to paralysis and death within 5 years after clinical onset.,,,,,,Although how TDP-43 forms cytoplasmic aggregates and causes neurodegeneration in patients with ALS/FTD remains unclear, a reduction in cellular TDP-43 levels will likely prevent its aggregation and potentially rescue neurons from TDP-43 toxicity. Excess amounts of either wild-type (WT) or mutant TDP-43 was reported to cause neuronal toxicity and severe paralysis together with pathological TDP-43 aggregation in several lines of transgenic mice.SOD1,ataxin-2,FUS,C9orf72-associated repeat,,Antisense oligonucleotides (ASOs) are emerging as an attractive therapeutic strategy for neurological diseases. ASOs are designed to bind disease-associated target genes or their transcripts, leading to modulation of the expression of target genes. After the first successful approval of the molecularly targeted ASO therapy (nusinersen) for spinal muscular atrophy,O,4′-C-ethylene nucleic acids (ENAs), which are modified nucleic acids with high stability, and tested the therapeutic potential of the TDP-43-targeting approach using ENA-modified ASOs. We demonstrated that intracerebroventricular administration of ENA-modified ASOs into a mouse model of ALS/FTD expressing human TDP-43 results in the efficient reduction of TDP-43 levels in the brain and spinal cord. Surprisingly, a single injection of the ENA-modified ASO into TDP-43 mice led to long-lasting improvement of behavioral abnormalities and suppression of the mislocalization/cytoplasmic aggregation of TDP-43 even after the levels of TDP-43 had returned to the initial levels. Our results demonstrate that lowering TDP-43 levels using ENA-modified gapmer ASOs leads to the sustained improvement of disease phenotypes and TDP-43 pathology in vivo, indicating the therapeutic potential of the TDP-43-lowering approach for treatment of the TDP-43-associated diseases (TDP-43 proteinopathies), including ALS/FTD.To address this issue, in this study we developed gapmer-type ASOs against human TDP-43 using 2′-O and 4′-C positions of furanose.N-type conformation, enables ENAs to interact with their complementary RNAs with comparably high affinity as other bridged nucleic acids (BNAs), such as 2′-O,4′-C-methylene-bridged nucleic acid/locked nucleic acid , but provides substantially higher resistance against nuclease degradation than 2′,4′-BNA/LNA.,To obtain proof of concept of the potential of the TDP-43-lowering approach for ALS/FTD treatment, we utilized gapmer-type ASOs with modified nucleic acids. Gapmer ASOs are short ASOs in which a DNA segment is placed at the center and flanked on both sides by RNA segments. Gapmer ASOs bind to their target RNAs and form a DNA:RNA hybrid, which can be recognized and cleaved by ribonuclease H, leading to silencing of the target gene. For the RNA segments in gapmer ASOs we utilized ENAs, which are modified nucleic acids with an ethylene bridge between the 2′-1 and 4, decreased TDP-43 levels compared with the nontargeting control ASO C1  293 cells, and 48 h post transfection the cellular levels of endogenous TDP-43 were analyzed by western blotting. Densitometric analysis demonstrated that most of the gapmer ASOs, except for ASOs l ASO C1 B and 1C.6 to optimize the target sequence of the gapmer ASOs. For this purpose, we further synthesized the gapmer ASOs 11–21, in which the sequences were designed to be shifted by two bases each from the target sequence of ASO 6 , and the evaluation of TDP-43 knockdown was repeated using HEK293 cells   E and 1F.15 against human TDP-43, as well as its potential cytotoxicity. HEK293 cells were transfected with various concentrations of ASO 15 and incubated for 48 h. Western blotting analysis after a 48-h incubation demonstrated that the protein level of endogenous TDP-43 was markedly reduced in the tested concentration range of ASO 15 of 25–150 nM 15, or control ASO C1 into the cerebral ventricles of hTDP-43 mice at 6 weeks of age and analyzed the expression levels of hTDP-43 using a human TDP-43-specific antibody (clone 2E2-D3) at 2 weeks, 12 weeks, and 24 weeks after injection. We confirmed that the administration of both gapmer ASO 15 and control ASO C1 by intracerebroventricular (i.c.v.) injection was well tolerated and demonstrated no acute toxicity up to a dose of 200 μg of ASOs per mouse. Injection of ASO 15 at a dose of 200 μg resulted in a marked reduction in hTDP-43 levels, not only in the brain including the cortex and hippocampus, but also in the spinal cord 2 weeks after injection, whereas injection of ASO 15 at a dose of 100 μg modestly decreased hTDP-43 levels in the cortex and spinal cord but not in the hippocampus  and at 5 months   and 5 months of age   B, indica WT mice A, and sp < 0.01) F. These C2 at 6 weeks of age traveled a longer distance than ASO C2-injected WT mice at 3 months of age   C, implyi < 0.05) D and 3E, < 0.05) F. These -43 mice C and 3F.15 improved the abnormal locomotor function of hTDP-43 mice. The increased total travel distance of hTDP-43 mice due to hyperactive behavior at 3 months of age was effectively attenuated by the injection of ASO 15 at 6 weeks of age , to a level comparable with that of ASO C2-injected WT mice , which reached a level similar to that of WT mice  and at 6 months   and 6 months of age   B and 4C. < 0.01) D and 4E.15  and 6 months of age , showing values similar to those of WT mice  and 6 months of age   E, demons15 into hTDP-43 mice at 6 weeks of age appeared to improve this weakness at 5 months and 8 months of age, when the grip strength scores were normalized by body weight  and 8 months of age , but not at 3 months of age  and 8 months of age , resulting in the normalization of the body weights of hTDP-43 mice to levels similar to those of ASO C2-injected WT mice, with no statistically significant difference between the two groups . Reagents for ASO synthesis were purchased from Glen Research. The coupling of 2′-O,4′-C-ethylene nucleoside-3′-phosphoramidite was performed according to standard synthesis cycles, except for an elongation of the coupling time (15 min). After synthesis, the resins were treated with concentrated aqueous ammonia at 55°C for 8 h. The crude products were purified by C18 silica gel column chromatography with a gradient of CH3CN , treated with 10% acetic acid in H2O for 8 h, and then purified by reverse-phase high-performance liquid chromatography with a gradient of CH3CN .Modified oligonucleotides containing 2′-TDP-43 forward, 5′-GGG AAA TCT GGT GTA TGT TGT C-3′; TDP-43 reverse, 5′-TTT CAG GTC CTG TTC GGT TG-3′; GAPDH forward, 5′-AAG GTG AAG GTC GGA GTC AAC-3′; GAPDH reverse, 5′-GGG GTC ATT GAT GGC AAC AAT A-3′. For the cell viability assay, HEK293 cells were transfected with ASOs and incubated for 48 h as above. After incubation, the cells were treated with WST-1 reagent (Takara) for 2 h, and cell viability was analyzed according to the manufacturer’s instructions. All experiments were performed independently at least three times.HEK293 cells were grown and maintained in DMEM supplemented with 10% (v/v) fetal bovine serum. For western blotting analysis, HEK293 cells were transfected with gapmer ASOs using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific), incubated for 48 h, and lysed with RIPA buffer . Lysed solutions were collected and centrifuged to remove cell debris, resulting in clear cell lysate solutions. The protein content of the cell lysates was estimated using a DC Protein Assay kit (Bio-Rad). Proteins in cell lysates were separated using 5%–20% gradient SDS-PAGE gels (ATTO) and transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad). The membranes were incubated overnight with the following antibodies at 4°C: anti-TDP-43 , and anti-β-actin . As secondary antibodies, horseradish peroxidase (HRP)-conjugated immunoglobulins (IgGs) were used. The HRP signal was visualized with ImmunoStar Zeta chemiluminescence solution (Fujifilm Wako) and captured with an Amersham Imager 600 CCD imaging system (Cytiva). Acquired images were analyzed using ImageJ software. For qRT-PCR analysis, HEK293 cells were transfected with ASOs and incubated for 48 h as above, and total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized from total RNA using a QuantiTect Reverse Transcription Kit (QIAGEN), and real-time PCR was performed using the CFX96 Touch Real-Time PCR System (Bio-Rad) and TB Green Premix Ex Taq II (Takara). The primer sequences are as follows: ad libitum. For ASO administration, mice at 6 weeks of age were stereotactically injected with 5 μL of ASO in saline  into the right ventricle using a 5-μL syringe (Hamilton). The coordinates for injection were 2 mm anterior to the lambdoid suture, 1 mm lateral from the sagittal suture, and a depth of 2–3 mm.All mouse experiments were approved by the Animal Experiment Committee of Osaka University and Nagoya University, and were performed in accordance with their guidelines on animal experiments. hTDP-43 mice were generated by the Julien laboratory and have been described previously.Body weight, grip strength, and rotarod performance were assessed as described previously.Anesthetized mice were perfused with PBS followed by 4% paraformaldehyde fixative in phosphate buffer through the left cardiac ventricle. Brains were removed after perfusion, post-fixed in 10% phosphate-buffered formalin, and processed for paraffin embedding. Brain sections of 3 μm thickness were deparaffinized and blocked in PBS containing 2% BSA and 0.5% Triton X-100 for 1 h at room temperature. The sections were then incubated with an anti-TDP-43 antibody , or an anti-Iba-1 antibody  at 4°C overnight, followed by an Alexa Fluor 488-conjugated goat IgG  for 1 h at room temperature. The sections were mounted with VECTASHIELD HardSet antifade mounting medium (Vector Laboratories) and analyzed using a fluorescence microscope . The formation of cytoplasmic inclusions and the nuclear localization of hTDP-43 in the cerebral cortex were quantitatively analyzed .g for 10 min at 4°C, after which the supernatants were collected. Proteins were separated using 5%–20% gradient SDS-PAGE gels and transferred to PVDF membranes. The membranes were incubated overnight with the following primary antibodies at 4°C: anti-hTDP-43 , and anti-β-actin . As secondary antibodies, HRP-conjugated IgGs were used. The HRP signal was visualized by ImmunoStar Zeta chemiluminescence solution and captured using an Amersham Imager 600 CCD imaging system. Acquired images were analyzed using ImageJ software. For qRT-PCR analysis, total RNA was extracted from the brain cortex, hippocampus, and spinal cord using TRIzol reagent (Thermo Fisher Scientific). cDNA was synthesized from total RNA using a QuantiTect Reverse Transcription Kit (QIAGEN), and real-time PCR was performed using the CFX96 Touch Real-Time PCR System (Bio-Rad) and TB Green Premix Ex Taq II (Takara). For semi-quantitative RT-PCR analysis, cDNA was synthesized from total RNA as above, and PCR was performed using PCR Thermal Cycler Dice Touch (Takara). The resultant PCR fragments were analyzed by 5%–20% gradient PAGE gels (ATTO). The primer sequences used were as follows: mouse TDP-43 forward, 5′-ATT TGA GTC TCC AGG TGG GTG TGG-3′; mouse TDP-43 reverse, 5′-GTT TCA CTA TAC CCA GCC CAC TTT TCT TAG G-3′; Actin forward, 5′-CGT GCG TGA CAT CAA AGA GAA-3′; Actin reverse, 5′-CAA TAG TGA TGA CCT GGC CGT-3′; Sort1 forward, 5′-ATG AAT CCC GCC AGA GAA G-3′; Sort1 reverse, 5′-GAC AAG CAT CAG TCC CAC GA-3′; Dnajc5 forward, 5′-ACC TGA GGG TGA GGA GAC AG-3′; Dnajc5 reverse, 5′-GGC TGT ATG ACG ATC GGT GT-3′; Ppp3ca forward, 5′-CTC TCT GGC GGG AAA CAG AC-3′; Ppp3ca reverse, 5′-GAG GCG AGA GCC TTG TTG AT-3′; Kcnip2 forward, 5′-AAT CCC GAG ATT TGG ACG GC-3′; Kcnip2 reverse, 5′-TGG CAC ACC GTG GAT AGT TC-3′.For immunoblotting of hTDP-43 in hTDP-43 mouse tissues, the brain cortex, hippocampus, and spinal cord were homogenized in RIPA buffer containing protease inhibitor cocktail  and centrifuged at 15,000 × Western blotting and cell viability data were analyzed by one-way analysis of variance (ANOVA) followed by the Dunnett’s multiple comparison test. qRT-PCR data from the cell experiments and the knockdown efficiency and immunohistochemistry data from mice were analyzed by Student’s t test. Data from body weight, grip strength, the open-field test, and the light/dark transition test of hTDP-43 mice were analyzed either by one-way ANOVA followed by the Tukey multiple comparisons test or by two-way repeated-measures ANOVA followed by the Tukey multiple comparisons test. For all analyses, GraphPad Prism (GraphPad Software) was used. A p value of less than 0.05 was considered to indicate a statistically significant difference between groups.