Screen-printed graphene layers on flexible substrates are one of the most advanced printed electronics developments of recent years. Obtaining thin, flexible, highly conductive components, whose applications are increasingly directed towards biomedical engineering and even medicine, requires an in-depth understanding of the correct choice of materials and procedures. Our work was aimed at investigating the influence of homogenisation in the triple rolling process over pastes dedicated to the screen printing technology, on their rheological parameters and the properties of the prints. The effect of selecting a suitable polymer matrix and different packing of graphene flakes was evaluated. Several studies were carried out, which can provide an excellent knowledge base in the context of graphene screen-printing pastes. Paste rheology, printability, path thickness, sheet resistance and adhesion to the substrate were investigated. Selected layers were also subjected to SEM imaging. One of the fastest-growing industries in recent times is printed electronics. The possibility of obtaining components with high mechanical resistance while maintaining flexibility has led to an increase in research in this area . PrintedThe most versatile and extensively employed technology for printed electronics is screen printing. This technology is not only characterised by its high versatility as well as a good resolution but also enables printing on a large variety of substrates ,7. ScreeScreen printing technology imposes specific requirements on the materials involved. A paste form is required, i.e., a stable suspension of nano- to micro-sized particles in a polymer matrix. The properties of the pastes are adjusted to the dedicated application. However, it is not only the characteristics of the final printed product that are important but also the ease and repeatability of the printing process .−5 Ω∙m [One of the breakthroughs, in terms of the expansion of prospective screen-printing technology applications, was the implementation of printable graphene-based composites. Based on these materials, these include innovative electrodes , capacit−5 Ω∙m . A graph−5 Ω∙m . Nevertheless, it should be highlighted here that for a value-enhanced application, it is not only the material of the functional phase that is crucial but also the carrier used. The right polymer can significantly improve the uniformity of the paste and provide the right properties for the composite to be used in the printing process. The polymer serves as a matrix in the final print. It is intended to provide the best possible mechanical performance, such as surface hardness, high abrasion and scratch resistance, as well as chemical resistance. Considering printing on a flexible substrate, it is also crucial to ensure adequate flexibility. Popular and widely used polymers as matrices are plastics  or thermosetting and chemically curing resins ,18. ExamThe increasing pressure to produce increasingly efficient layers, often both highly conductive and flexible, enabling ease of printing and proper adhesion to the substrate, often entails complex and multi-step paste manufacturing processes. One of the often-mentioned paste production steps is its homogenization in the rolling process ,22. ThisFurthermore, very often engineers and scientists are faced with the dilemma of the necessity to compromise between printability and mechanical and electrical properties. Therefore, investigating the impact of the triple-rolling process on various parameters can provide precious information for many researchers and manufacturers. In this paper, we present a study on the effects of the rolling process of graphene pastes based on different carriers on their rheology, printability and electrical and adhesion parameters. Flexible substrates, which are among the most researched at the moment due to the rapid growth of flexible electronics, were investigated in this study. The investigation was dedicated to graphene pastes, which represent a strong trend in the development of screen-printing technology.®  were used. According to the datasheet, they range from 1 to 2 μm in width and 8–15 nm in thickness.In this work, graphene nanoplatelets from CheapTube5 u, which dissolved in 2-(2-butoxyethoxy) ethyl acetate to form a carrier referred to in this paper as PMMA. Another polymer tested was Laroflex M35 with a density of 1.24 · 103 kg/m3, which was dissolved in a mixture of 2-(2-butoxyethoxy) ethyl acetate and 2-butoxyethanol in a ratio of (97:3); referred to as LARO carrier. Two types of thermoplastic polyurethane (TPU) were also tested, in the form of Elastollan® soft 35AP, with a density of 1.18 · 103 kg/m3, and Elastollan® hard C80A, with a density of 1.19 · 103 kg/m3. Both were dissolved in N, N-Dimethylformamide; their designations in the paper are TPU soft and TPU hard.In this study, four different polymers were used. The solvents were selected concerning the particular polymers tested, ensuring good solubility and with consideration of their use in screen-printing technology, i.e., paying attention to an appropriately high boiling point. The first polymer used was poly(methyl methacrylate) with an average molecular weight of 3.5 · 10® , Laroflex M35, Elastollan soft 35AP and Elastollan C 80A were purchased from BASF® .Poly(methyl methacrylate) and all solvents were purchased from Sigma AldrichThe printing substrate was a 36 µm PET film purchased from MICEL Sp z o.o .® ECI 1010 ink purchased from Tekra LLC .Silver contacts were applied with LOCTITEThe carriers were obtained by mixing the weighed components for 48 h at a temperature adapted to the individual polymers comprising the carriers, 50 °C for LARO and PMMA and 30 °C for soft and hard TPU, respectively.In the next step, the carriers were combined with nanoplatelets of graphene, representing the conductive functional phase. An appropriate amount of graphene and carrier was weighed and ground for at least five minutes in an agate mortar. Each series was prepared in five different degrees of packing of the functional phase   with a sThe paths were printed using an Aurel c920 screen printer , Aurel A® R/S-CPS+ rheometer equipped with an RCT-50-2 spindle , dedicated to the viscosity ranging between 0.006–50,900 Pa·s. A shear rate from 0 to 400 s−1 for was tested for 100 s. The viscosities were analysed using the dedicated Rheo 3000 software (version 1.2.2009.1). The rheology of the pastes was measured with a Brookfield® . The values were recalculated into sheet resistance (Ohms per square).The resistance of the printed paths was measured with a UT804 multimeter manufactured by Uni-Trend Technology® profilometer , with the force of the stylus set to 3 mg.Path thickness was measured using Bruker’s DektakXT® disc knife included in the BG VF 1842 C kit. Scotch® tape  was applied to the cut sections and ripped off after approximately three minutes by uniform movement. The crumbled material was removed with a brush and the results were compared according to the ISO 2409 standard, which defines the level of adhesion on a scale of 0–5, where 0 means no material detachment at all, 1 indicates from 0–5% of the total material, 2 from 5–15%, while 3 is between 15–35% and 4 is between 35% and 65% of the total pattern. Larger material detachment is qualified as 5. Adhesion was tested using a 2 mm ANTICORRScanning electron microscopy (SEM) of three layers of best conductivity was conducted on a Hitachi SU8230 instrument  with an accelerating voltage of 5.0 kV and an upper secondary electron detector.For the obtained pathways with markedly different conductivity capabilities, their structure was additionally compared under the Keyence VHX-900F optical microscope .Firstly, the dependence of the pastes’ viscosity on the shear rate was analysed. This comparison was carried out for all the tested carriers and graphene concentrations, in all cases for both triple-rolled and non-rolled materials. For all four carriers tested, higher dynamic viscosity was observed with increasing graphene packing for both triple-rolled and non-rolled materials. A tripling of the graphene concentration increased the dynamic viscosity registered for the minimal shear rate by up to several hundred times. This is understandable, as graphene nanoplatelets are a powder additive with a highly developed specific surface area and low bulk density. Furthermore, for all pastes, irrespective of the carrier used, as the packing increased, a greater curve slope angle was recorded for shear rates below 150 1/s. For higher speeds, the correlation between viscosity and the aforementioned parameter decreased dramatically, i.e., a further increase in shear rate resulted in a slight change in viscosity.Considering the effect of the rolling process on the rheological properties, various dependencies can be observed. For PMMA pastes, an improvement in rheological properties and a decrease in dynamic viscosity were noted for all pastes. For LARO pastes, the rolling process resulted in a decrease in viscosity for the packing of graphene by 15% and an increase for the other variants. For pastes containing soft TPU, the rolling process caused a decrease in the viscosity for pastes with higher packing (12.5% and 15%). Such a result indicates that for those carriers only for larger packing, the rolling process introduces a significant deagglomeration of the functional phase and contributes to a reduction of internal friction in the material which results in lower viscosity. The hard TPU-based pastes showed worse rheological properties after the rolling process. For shear rates above 100 1/s, the dynamic viscosity values decreased rapidly and were close to zero. Comparing the results obtained for various carriers, the highest dynamic viscosity was observed for the hard TPU paste d). AnalyIn screen printing technology, the standard dynamic viscosity is between 0.5–50 Pa∙s for the shear rate achieved during squeegee work (This speed is 150–180 1/s). Therefore, despite significant differences and sometimes extremely high viscosities at the zero shear rates, almost all of the pastes examined showed adequate viscosity for the speeds relevant for use in screen printing. The exceptions are rolled pastes based on hard TPU, where viscosity was too low.Next, the electrical properties of the tested materials were compared. For this purpose, the sheet resistance of a dozen rectangular paths of different thicknesses printed on dedicated silver contacts was measured. During the process of preparing the paths, significant difficulty in the printing process was noted for pastes containing hard TPU. Their high viscosity, combined with strong adhesion to the squeegee and screen, inhibited proper deposition of the paste on the substrate, resulting in frayed and patchy paths . Such a result qualifies the TPU hard carrier as not screen-printable. For this reason, further research on this material was discontinued. In addition, non-rolled pastes containing soft TPU showed limited printability. At higher graphene packing (12.5% and 15%), the material failed to settle well on the substrate despite several passes of the squeegee. Since only part of the material was squeezed through the screen in a single printing cycle, which would not be optimal for the screen-printing process, it was decided to set the maximum graphene packing of this carrier at 10% by weight without a triple-rolling process. Adding the rolling step enabled printing and the higher tested graphene packing. The results obtained for the pastes, found to be highly screen-printable, are presented in For both rolled and unrolled pastes, with all three carriers considered, a decrease in the layer resistance of the paths was observed as the graphene packing increased. This is a predictable result because graphene is the conductive functional phase of these materials. For LARO-based pastes, with a threefold increase in graphene packing, the sheet resistance decreased by up to two orders of magnitude. When comparing the paths printed from pastes subjected to the unification process on the triple-roller with those not subjected to this process, it is worth noting that in most cases a benefit in terms of a reduction in sheet resistance is evident. The greatest changes were observed for the low graphene packing. For LARO pastes with the lowest graphene content, rolling resulted in a more than 100-fold decrease in sheet resistance. For LARO pastes containing the highest tested packing, there was a decrease in the layer resistance value of 0.12 kΩ/□. For the pastes on soft TPU, rolling not only improved the bonding of the functional phase to the polymer phase and unified the material enough to make it printable, but an improvement in electrical properties is also apparent. For PMMA pastes, the rolling process only had a positive effect on pastes with up to 10% packing. In contrast, the 12.5% and 15% pastes had a slightly higher electrical resistance after the rolling process.The reason for the diminishing effect of improving electrical parameters with increasing packing can be attributed to the fact that, with excessive amounts of graphene, the rolling process can lead not only to deagglomeration but also to fragmentation of the graphene flakes, which in turn can result in more difficult carrier tunnelling and thus poorer electrical properties.Comparing all of the carriers, it is interesting to observe that, at low graphene packing, significant differences in the obtained values of sheet resistance can be noted, while for maximum graphene packing the resistance results are very similar for all the carriers. The interactions between the filler particles and the matrix polymer seem to be more crucial for low functional phase contents. For these packings, an easy path for electron tunnelling between flakes is important, so polymer density and particle size may also be significant. At higher packing levels, the role of the polymer is more directed towards maintaining the print on the substrate and enabling the material to maintain homogeneity.An important parameter that is very often neglected in investigations, but which may vary depending on the packing of the functional phase, and also on the carrier used, is the path thickness. This parameter has great significance in the context of the obtained resistance of the layers. We examined the path thicknesses of all the paths obtained . The thiThe results obtained show that, as the packing increases, the printed paths have a higher thickness. That may be directly related to the viscosity of the pastes, as pastes with lower viscosity spread over the substrate to a greater extent. It is also noticeable that LARO pastes allow for thicker paths to be printed, which may be attributable to the amount of polymer required to produce a paste with suitable rheology, but also to the higher intermolecular interactions within this carrier. However, the influence of the carrier itself is considerably lower than the packing of graphene flakes, which is understandable, given that the amount of the added functional phase is the main determinant of paste viscosity.Non-rolled pastes have been omitted from this comparison due to their very similar outcomes, indicating that the path thickness is not significantly affected by the triple roll process.In the final stage of the study, the adhesion properties of the printed paths to the flexible PET substrate used were checked for both rolled (R) and not rolled (NR) pastes. In this work, we used flexible substrates on account of the strong development of flexible electronics. It is on difficult, flexible substrates that additional material processing, in the form of three-roll milling, can allow graphene pastes to be printed with adequate adhesion. Tests were carried out for at least three 20 mm × 20 mm square prints each time and the median of the adhesion values obtained is listed .The definite effect on adhesion to the substrate for both the polymer matrix used and the concentration of the functional phase is evident. The more graphene in the layer, the better the adhesion for all three polymer matrices tested.The poorest adhesion properties among the tested polymers were noted for PMMA-based prints. For the 5% graphene packing, 35–65% detachment was observed. However, for higher concentrations of graphene, the detached material did not exceed a value of 5% of the print. Prints on both PMMA and TPU matrixes showed similar adhesion. In the most severe cases, this resulted in the detachment of no more than 15% of the print Such dependencies can originate from several factors; from the properties of the polymers themselves, such as their elasticity and density, the interaction of the used solvent with the PET substrate and the interaction of the carrier with the functional phase, finishing with the viscosity of the paste modulated by both the carrier and the graphene packing.In addition, the effect of the rolling process of the pastes on the adhesion of the layers printed with their use is also evident. This is probably related to the homogenisation of the paste itself. Since the agglomerates of the functional phase, which are present in greater quantity in non-rolled pastes, can be the link that detaches the layer from the substrate, the deagglomeration, being part of homogenisation, leads to better adhesion.With the highest packing of graphene being 15%, for pastes based on all three carriers subjected to the unification process on a three-roller machine, the results of layer resistance, layer thickness and adhesion were found to be very similar. These layers were subjected to SEM imaging to verify to what extent the structures of these prints are alike .The analysis of the SEM images shows that, despite the differences in the polymer matrix, with sufficiently high packing of the functional phase, highly similar layers are obtained. The homogeneous and dense distribution of graphene flakes is evident. The visible bulges are the result of the screen-printing technology used.The role of both the composition and the selection of the process for producing graphene-based screen-printing pastes was outlined. The importance of the selection of the polymer and the degree of packing of the functional phase was discussed, as well as the influence of the triple-rolling process concerning rheological parameters, screen-printing potential, and properties of the obtained paths concerning sheet resistance, path thickness, and adhesion to the flexible substrate.Depending on the carriers used, different rheological, electrical and adhesion properties of the composites produced were obtained. It was shown that the results can be significantly different even within a single polymer family, as in the case of the two thermoplastic polyurethanes tested, both of which were based on polyethers, and differing in hardness and the presence of a plasticiser (in soft TPU).We have shown that graphene pastes with a higher packing of the functional phase, possessing higher dynamic viscosity values, not only exhibit better electrical performance but also have better adhesion properties to PET films.Above all, we demonstrated the significance of a correct paste preparation process. The triple-rolling process revealed the influence on the rheology and printability of the pastes, as well as the sheet resistance and adhesion of the acquired paths. The rolled materials showed lower electrical resistivity, with the greatest impact at lower graphene packing. A clear improvement in the adhesion of the rolled composites was noted for all carriers.


Since the first commercial biosensor device for blood glucose measurement was introduced in the 1970s, many “biosensor types” have been developed, and this research area remains popular worldwide. In parallel with some global biosensor research reports published in the last decade, including a great deal of literature and industry statistics, it is predicted that biosensor design technologies, including handheld or wearable devices, will be preferred and highly valuable in many areas in the near future. Biosensors using nanoparticles still maintain their very important place in science and technology and are the subject of innovative research projects. Among the nanomaterials, carbon-based ones are considered to be one of the most valuable nanoparticles, especially in the field of electrochemical biosensors. In this context, graphene oxide, which has been used in recent years to increase the electrochemical analysis performance in biosensor designs, has been the subject of this review. In fact, graphene is already foreseen not only for biosensors but also as the nanomaterial of the future in many fields and is therefore drawing research attention. In this review, recent and prominent developments in biosensor technologies using graphene oxide (GO)-based nanomaterials in the field of cancer diagnosis are briefly summarized.  Graphene, called the material of the future, consists of a single layer of carbon atoms arranged in a regular hexagonal lattice appearance , is prodSince the award of the above-mentioned Nobel Prize, many researchers have been conducting research on graphene in a wide variety of disciplines, including medicine, physics, and chemistry. As of 2022, nearly 296,500 articles based on graphene have been produced and many companies have also started to work in this field. A great deal of effort has been made by researchers on the synthesis of graphene to date, and when these studies are examined in general, it is seen that mechanical exfoliation and chemical vapor deposition (CVD) methods still maintain their popularity. High quality graphene sheets can be produced by both methods . Since g2 g−1) 3−/4−, which is the parameter especially focused on in impedimetric studies, is strongly affected by electrode modification. In line with this basic information, a Nyquist curve with a very small semi-circle was obtained in the presence of a redox probe with the glassy carbon electrode (GCE) electrode used, and then an increase in the diameter of the semi-circle was observed when the experiment was repeated with the GO modified electrode. This has been interpreted as the modification of GO, whose sp2 networks are disrupted during its synthesis, and that electron transfer to the electrode interface is prevented, and this layer acts as an insulating layer. Then, a single-stranded DNA was covalently immobilized on the GO modified electrode surface and it was observed that the Ret value increased further. It is stated that this situation occurs due to the electrostatic repulsion of the negatively charged phosphate backbone in DNA against ferrocyanide. Then, a single-stranded DNA was covalently immobilized on the GO modified electrode surface and it was observed that the Ret value increased further. This situation is evaluated as both the DNA binding to the surface and the negatively charged phosphate backbone of DNA exerting electrostatic repulsion against ferrocyanide. In the continuation of the experiment, when the electrode surface was subjected to electrochemical reduction (when scanning from 0.0 to −1.7 V) after DNA modification, it was determined that the Ret value decreased dramatically, that is, the conductivity increased clearly. It is an indication that the GO on the surface decreases as a result of reduction and this situation occurs with the formation of rGO. To summarize, the use of rGO in biosensor platforms is due to its contribution to the acceleration of electron transfer. It has been proven by many studies that it improves EIS responses in DNA biosensor designs. When hybridization occurs, the Ret value increases again because this means that [Fe(CN)6]3−/4− cannot penetrate the double helix bundles with more negatively charged phosphate backbones on the surface. Similarly, in CV experiments performed with step-by-step surface modifications and with the same redox probe, the findings obtained from the electrode-electrolyte interface are compatible with the EIS results. Electrochemical impedance spectroscopy (EIS) is a technique that allows the analysis of both the resistive and capacitive properties of physical and/or biomolecular modifications of electrode surfaces, including nanomaterials, nucleic acids, proteins structures etc. In this concept, EIS is one of the basic electrochemical methods used to determine the fundamental redox events at the electrode–electrolyte interface. In fact, it is the most widely used method in studies of this type of research. However, evaluations are usually made on the basis of complementary results obtained with both the EIS and CV method. For example, in a study carried out by Gong et al. to investigate the properties changing on the surface with graphene oxide modification, Nyquist curves obtained from the solution interface with the glacial carbon electrode using the conventional redox probe ferri/ferrocyanide were evaluated . The eleIn fact, many of the GO production methods are known to damage this structure. As a result of this damage, it was determined that some functional groups were formed in the substance or on its surface, and carbonaceous or metallic impurities were formed. However, it is seen in many reports that this situation is often turned into an advantage by electrochemical or chemical means. For example, covalent bonding procedures,  which ar6]3−/4− is used for label-free analyses as given in the following sections. CV, DPV, and SWV techniques are also used in both label-free and label-based biosensors to monitor the redox events and mechanisms occurring on the electrode surface. Of these, cyclic voltammetry (CV) is the most widely used voltammetric method to explain basic electrochemical events, such as the thermodynamics of oxidation-reduction reactions and electron-transfer kinetics occurring at the electrode-solution interface, as well as mass transport towards the electrode surface ,68. In tDifferential pulse voltammetry (DPV) is another basic electrochemical technique and in a cell containing a triple electrode system, measurements are performed at the moment when the faradaic current is highest and capacitive current is lowest. Thus, the signal-to-noise ratio increases and hence sensitivity increases. In the method, the difference in current per applied pulse (∆Ip) is recorded as a function of linearly increasing potential. The resulting differential curve is in the form of a peak and its height is proportional to the concentration .Square wave voltammetry (SWV) is also a pulse voltammetry technique that has the advantage of being extremely fast and at the same time sensitive. In this method, a potential is applied to the working electrode in the form of a symmetrical square wave. In each square wave cycle, the current is measured twice, at the end of the applied forward and reverse pulses. The difference of these currents gives the net current value. The square wave voltammogram is obtained when the net current obtained is plotted as a function of the applied potential. The SWV measurement is extremely fast, it is possible to increase the precision of the analysis by averaging several voltammetric measurements .As a result, the modification and redox events occurring on the electrode surface can be elucidated by evaluating these techniques individually or together.Cancer is among the diseases that carry a serious risk of death, and it is becoming more and more common around the world. There are about 200 different forms of cancer detected so far . AccordiSince early diagnosis in cancer is life-saving, new diagnostic technologies to be developed must provide fast and accurate analysis. In this context, cancer biomarkers are becoming increasingly attractive targets for electrochemical biosensor designs alongside classical detection methods, as they are useful tools for understanding the developmental processes of cancer disease. Thanks to advances in nanotechnology, various cancer biomarkers, i.e., cancer cells, metabolites, miRNA, surface antigens/proteins, etc., biodetection is possible . Some ofHowever, there are some difficulties in developing a biodevice with features that will allow early detection of cancer. For example, the biosensor should be able to perform sensitive and selective analysis with a small sample. If an evaluation is made in terms of the number of cancerous cells, it is expected that a biosensor with clinical validity will accurately detect a tumor in the range of 100–1000 cell counts . DespiteIn this section, prominent examples of graphene-containing electrochemical biosensors developed for cancer diagnosis will be given. It was determined that real samples  analyses were also performed in all of the studies in the review. Some other interesting designs and the basic contents of them are collected in two different tables at the end of the review  and square wave anodic stripping voltammetry (SWASV). It is understood that this methodology, which was discovered by Professor Wang and his team in 2002 and is based on the dissolution of metal ions from QDs with acidic solution, is still a popular and powerful alternative electrochemical biosensor design technique despite many years since its discovery 3−/4− redox couple was used in measurements to monitor both surface modifications and antibody-antigen binding via CV or SWV methodologies. In addition, the electrochemical signal of gold nanoparticle label was also evaluated in the determination. In this system, p53 antibodies could be detected with a detection limit of 0.088 pg/mL with a linear range of 0.1 pg/mL to 10 ng/mL.An innovative and remarkable biosensor platform developed for cancer prognosis has been reported by Elshafey et al., where gold nanoparticles provide large surface area for p53 protein modification and reduced graphene oxide is used for signal enrichment . In the Dou et al.  demonstrOn the other hand, in paper-based biosensors, another sensor platform, graphene oxide nanomaterial and various labels are used for signal amplification and enrichment of the sensing surface area in the diagnosis. In fact, biosensors based on GO-modified paper have been produced since the 2010s. Electrochemical biosensor designs in cancer diagnosis have also been a prominent research area in recent years due to paper-based electrodes. Nevertheless, it is still considered that there are some issues that need to be overcome in order to reveal point-of-care systems . A few i2) to further increase the sensor performance. The paper-based part in the microfluidic paper-based analytical device (μPAD) consists of two layers of selectively patterned square filter paper of the same size (35.0 mm × 35.0 mm), and the specially patterned parts, where the analysis will be carried out, were designed with the AutoCAD 2012 program. Eight carbon-based circular working electrode regions are printed on the paper surface using screen-printed technology  sandwich assay protocol, and (iv) a microfluidic system 3−/[Fe(CN)6]4− containing redox probe solution using CV and SWV techniques. As expected, a decrease in electrochemical response was obtained after the target analyte was bound. The detection limit of the developed LSG-AuNS-based aptasensor was found to be 0.008 ng/mL. Thanks to the 3D gold nanostructures deposited on LSG, high electron transfer rate, low detection limit, and reproducible results were achieved. Another outstanding aspect of this study is the development of an aptasensor that can accurately and sensitively analyze the amount of HER-2 added to undiluted human serum. In addition, a special software has been developed to use the presented system as a POC biosensor device, and the conversion from a laboratory aptasensor to a hand-held aptasensor containing a mobile phone has been achieved. However, with the handheld device, analysis could be performed with a higher detection limit (100 ng/mL) than the aptasensor, which was developed in this study for use only in the laboratory environment.As another interesting example of graphene-containing biosensors in cancer research, Rauf et al. reported  a laser-Analysis of cancer cells or their markers using modified graphene oxide with different metallic structures such as tungsten oxide-graphene composite  and reduBiosensor designs with 3D materials have also attracted attention in recent years. To give an example of a prominent work, Jang et al.  developeFor more information on electrochemical biosensors developed for cancer diagnosis, both containing types of graphene and with other nanomaterial design contents, some valuable review studies reported recently are available in the literature ,131,132.Graphene oxide-based nanomaterials on immunosensors or biosensors offer a range of possibilities in regard to cancer diagnosis. Two tables containing some of the other recently reported studies and the basic biosensor design elements related to these studies is presented below .In this review, we tried to present prominent graphene oxide-based electrochemical studies that have been reported in recent years and have a suitable infrastructure for clinical use for cancer diagnosis. Although significant progress has been made in the synthesis of graphene and its derivatives and their use in drug carriers/therapeutics in vitro, more progress is needed in the field of in vivo analysis. On the other hand, if an evaluation is made in terms of electrochemical studies, first of all, it can be said that while promising studies continue, there are also discussions on whether graphene and its derivatives increase the electroactivity/conductivity sufficiently. For this purpose, many elements, including N, S, P, B, etc., are integrated into graphene structures by doping . We beliAs is known, zero-dimensional graphene quantum dots GQDs, a member of the latest-generation graphene family, have also attracted great interest from researchers and industries in recent years . In lineApart from all these, comprehensive studies including extensive efforts in the field of theranostics, which combine diagnosis and treatment in a single system/platform in the medical field, are also being carried out ,173. HavIt is necessary to develop at least stable surface sensors and diagnostic kit for “open–use–dispose”-type electrochemical sensors . Much moFinally, we would like to point out that we think that, among the electrochemical biosensors examined in this review, paper-based sensors or systems that can measure with an application able to be downloaded to a mobile phone are more suitable for clinical practice. However, electrochemical biosensors  are also very promising for wearable health monitoring, thanks to their important advantages such as selectivity, sensitivity, rapidity, portability . It is c


The present paper describes the effect of the concentration of two graphene oxides (with different oxygen content) in the modifier layer on the electrochemical and structural properties of noble metal disk electrodes used as working electrodes in voltammetry. The chemistry of graphene oxides was tested using EDS, FTIR, UV–Vis spectroscopy, and combustion analysis. The structural properties of the obtained modifier layers were examined by means of scanning electron and atomic force microscopy. Cyclic voltammetry was employed for comparative electrochemical studies. Electrochemical methods are based on measurement of the current associated with the molecular properties and interfacial processes of chemical species. The recorded response results from direct transformation of the desired chemical information  into a current signal , according to the selected method. Voltammetry is considered as one of the most sensitive electroanalytical methods, suitable for the determination of trace amounts of many metals and compounds in clinical, industrial, and environmental samples ,2,3,4. V2O. Seven years later, Ruess [2SO4 along with: (1) sodium nitrate for in situ production of nitric acid in the presence of KMnO4 (Hummers’ method); (2) fuming nitric acid and a KClO3 oxidant (Staudenmaier method); (3) concentrated phosphoric acid with KMnO4 (Tour method); or (4) concentrated nitric acid and a KClO3 oxidant (Hoffmann method) [3 as an oxidant (Staudenmaier’s and Hofmann’s methods) exhibit a higher C/O ratio, whereas methods employing KMnO4 (Hummers and Tour) yield a larger proportion of oxygen-containing groups. Oxidative methods employing KClO3 result in oxides containing mostly CO groups , whereas methods using KMnO4 lead to oxides containing large amounts of carbonyl (Hummers) or both carbonyl and carboxyl groups (Tours) [Graphene, which consists of a one-atom-thick planar sheet containing an sp2-bonded carbon structure with exceptionally high crystalline and electronic quality, is a novel material that has emerged as a rapidly rising star in the field of material science ,12. Everr, Ruess  suggester, Ruess  proposedr, Ruess  presenter, Ruess , GO consr, Ruess  proposedr, Ruess ,27,28. Ir, Ruess ,32,33,34 method) ,36,37,38 (Tours) . The aim of this study was to compare the properties of noble metal electrodes modified with graphene oxide and to determine how graphene oxide chemical composition and concentration affect their electrochemical and structural  properties.−1 with a resolution of 4 cm−1. Electroanalytical measurements were carried out using a µAutolab instrument  controlled by GPES 4.9 electrochemical software. The three-electrode electrochemical cell employed in the study consisted of a reference electrode, an auxiliary electrode (platinum wire), and a working disk electrode .The surface topography of the studied electrodes was investigated using an atomic force microscope . The AFM measurements with the scan size of 5 µm × 5 µm were performed in the tapping mode using silicon scanning probe  with a nominal spring constant of 42 N/m and resonance frequency of 320 kHz. The roughness parameters Ra and Rq were defined on the basis of AFM topography images . The surface morphology and elemental composition of electrodes was investigated with high-resolution scanning electron microscopy  equipped in a Schottky field emission electron emitter. The surface morphology measurements were performed using CBS detector, enabling the observation of surface using signal of backscattered electrons (BSE). The images were recorded at 3 kV with electron beam deceleration 4 kV, spot size 2.5, and working distance 6.7 mm. The chemical elemental analysis of GO I- and GO II-modified gold electrodes was determined with EDAX Roentgen spectrometer (EDS) with Octane Pro Silicon Drift Detector (SDD) . Attenuated total reflection Fourier transform infrared (ATR–FTIR) spectra were recorded on a Nicolet iS50 spectrometer equipped with an MCT detector and the GATR accessory with Ge crystal over the spectral region from 600 to 4000 cmThe potential of the working electrode was measured vs. an Ag/AgCl electrode. Double-distilled water was used throughout the experiments. All the chemicals, including graphene oxide (GO I), reduced graphene oxide (GO II), and hexacyanoferrate system were purchased from Sigma-Aldrich and used as received.The GO I/GO II suspension was prepared weekly by dispersing an appropriate amount of GO I/GO II powder in DMF (dimethylformamide) in a 5 mL volumetric flask. The resultant solution was kept in a refrigerator at 4 °C in the dark. Spectrophotometric measurements were made using a Cary 100 Bio UV–Vis spectrophotometer .The general procedure used to obtain voltammograms was as follows: Ten mL of supporting electrolyte was placed in the voltammetric cell and the solution was purged with argon for 10 min (if necessary). After recording the initial blank, the required volumes of the analyte were added by means of a micropipette. Then, the solution was deoxygenated for 10 s (if necessary), and a voltammogram was recorded. All electrochemical measurements were carried out at ambient temperature. Each measurement was repeated three times and a mean was calculated.−1 were prepared. Afterwards, 20 µL of each solution was deposited on alumina foil by sessile drop technique and, subsequently, the samples were allowed to evaporate the solvent. Final spectra were received by the addition of 64 scans at a resolution of 4 cm−1.In order to obtain FTIR spectra of both graphene derivatives, DMF solutions of GO I and GO II at a concentration of 20.0 g L−1 were prepared for each oxide. Prior to modification with GO I or GO II, the working electrode surface was sonicated in ethanol for 60 s. Next, working electrode surface was mechanically polished with 0.05 μm Al2O3 slurry on a polishing cloth to a mirror finish. Then, it was ultrasonically treated in ethanol for 180 s and washed with double-distilled water. The modifier suspension (3 µL) was dropped onto the surface of the cleaned electrode and dried at ambient temperature. A new modifier film was prepared before each series of measurements.Modifying solutions containing graphene oxides were prepared with dimethylformamide. Suspensions at concentrations ranging from 1.0 to 50.0 g L63−/Fe(CN)64− redox couple and suspensions of graphene oxides (GO I and GO II) in a concentration range from 1.0 to 50.0 g L−1. 63−/Fe(CN)64− redox couple recorded on bare and modified gold (A) and platinum (B) electrodes. Both working electrodes were modified with graphene oxide suspensions at a concentration of 1.0 g L−1.Voltammetric measurements provide a wealth of information concerning the properties and characteristics of electrochemical processes. Cyclic voltammetry is an important and widely used technique determining analyte electrochemical behavior, including the formal redox potential, thermodynamic and transport properties, electron transfer kinetics, and adsorption processes. The hexacyanoferrate(II)/(III) redox couple undergoes a nearly reversible electrode reaction without any complications of preceding or post-chemical reactions; so, it has been a popular choice as a redox standard in CV. Electrochemical measurements were performed at modified working electrodes using the Fe(CN)−1 and lower. The relationships between hexacyanoferrate anodic peak current and the surface concentration of GO I recorded at both Au and Pt electrodes are shown in −1 GO I suspension. An analogous study using GO II suspensions also revealed the strongest signals of the Fe(CN)63−/Fe(CN)64− redox couple for the 20.0 g L−1 concentration at both Pt and Au electrodes. 63−/Fe(CN)64− voltammograms recorded at the GO II-modified platinum electrode.The strongest signals at both electrodes were observed for the electrode surface-modified with GO I. Significant difference may be observed for GO II suspension, which influence tested working electrodes in the opposite way. As can be seen in −1 GO II  were much lower than those obtained at electrodes modified with 20.0 g L−1 GO I . Since GO II was purchased as reduced graphene oxide and RGO has fewer oxygen groups (than GO), which improves its electrical conductivity, the opposite results were expected (oxygen content in both GOs will be thoroughly discussed in It is worth noting that the highest currents recorded at electrodes modified with 20.0 g L63−/Fe(CN)64− is a chemically reversible system, in which the oxidized form of the solution species can be regenerated from the reduced form (and vice versa), and both forms are stable on the time scale of the voltammetric experiment. However, after preliminary studies, a negative impact of modifications on electrochemical reversibility could not be excluded. If this was the case, the electron transfer would be so slow that the peak potential would not reflect the equilibrium activity of the redox couple at the electrode surface. To determine whether GO modification had a positive or negative effect, cathodic and anodic signal separation  was analyzed. In the theoretical model, the difference between the anodic and cathodic peak potentials  should be equal to 59 mV/n for fully electrochemically reversible systems. However, the majority of redox systems used are quasi-reversible, with anodic/cathodic separation being much higher than 59 mV, even for fast quasi-reversible redox pairs. The separations measured in the present study are shown in The model redox pair Fe(CN)−1 concentration) was only one of several factors influencing peak separation. At the platinum electrode, the lowest separation values were observed for the lowest modifier concentrations, while at the gold electrode, the highest concentrations afforded the best reversibility. This shows again that GO I and GO II act like two totally different compounds. Another important diagnostic characterizing an electrode reaction is peak potential (pE). At fast electron transfer rates, Ep is independent of the scan rate, indicating a reversible electrode reaction. The influence of the scan rate was tested in the range of 10–500 mV s−1. It was found that the scan rate did not have any effect on peak potential , indicating a quasi-reversible electrode reaction. The next very important diagnostic tool consists of the relationships log Ip vs. log v and Ip vs. v1/2. For both graphene oxides, linear plots of log Ip vs. log v were obtained with slopes of approx. 0.5, indicating an electrode reaction with the rate governed by diffusion of the electroactive species to an electrode surface. A linear relationship (for both electrodes with both graphene oxides) was also observed for Ip vs. υ1/2, which confirmed that the mass transport rate of the electroactive species to the electrode surface occurred across the concentration gradient. In such a case, the peak current Ip is governed by the Randles–Sevcik equation: Ip = k n3/2 A D1/2 C* υ1/2, where the constant k = 2.72 × 105; n is the number of moles of electrons transferred per mole of the electroactive species; A is electrode area; D is the diffusion coefficient; C* is solution concentration; and v is the scan rate. The above formula was used to calculate the electroactive area of the Au and Pt electrodes modified with both graphene oxides  are shown in It is well known that the properties of a broad range of materials and the performance of a large variety of devices depend strongly on their surface characteristics . TherefoAs mentioned above, the modified electrodes were also characterized by atomic force microscopy (AFM) to give insight into their surface topography. Three-dimensional (3D) views of the graphene-oxide-modified gold electrodes are given in −1 and a broad peak of very low intensity at 3210 cm−1 can be assigned to the stretching mode of the O–H bond, which reveal the presence of hydroxyl groups in graphene oxide [−1 can be designated to the stretching and bending vibration of water molecules adsorbed on graphene oxide [−1  [−1 (epoxide C-O-C or phenolic C-O-H stretch) [−1 confirms the presence of the C=O from carboxyl and/or carbonyl groups in the GO II structure [With the aim of determining whether the chemical structure affects the electrochemical properties of the prepared graphene-based electrodes, spectroscopic measurements were conducted. ne oxide ,48. Morene oxide . The FTIoxy C-O) ,46,49,50stretch) ,46,49,50tructure . The FTIThis was also confirmed by UV–Vis spectroscopy . UV–Vis In the last step, modified electrodes were examined with Energy Dispersive X-ray Spectrometry. The EDS measurements revealed changes in the carbon-to-oxygen ratio in GO I and GO II samples . The quaIn this paper, the electrochemical and structural characteristics of two graphene oxides used as electrodes modifiers were analyzed. AFM and SEM results showed that the topographies of the investigated electrodes/modifications do not significantly differ. However, the EDS elemental analysis revealed changes in the oxygen content. This was also confirmed by combustion analysis, FTIR, and UV–Vis spectroscopy. Unexpectedly, more oxygen content was found in the case of GO II (declared as RGO) compared with GO I (declared as GO). From the electrochemical point of view, the presence of RGO  should improve the electrochemical conductivity; however, the opposite results were obtained. The detailed electrochemical studies with model redox system showed that purchased graphene oxides act as utterly different modifiers. Although basic analytical characteristics are similar—for example, the diffusional nature of registered signals remained unchanged—other more important properties are completely different . As a result, we may obtain two completely different sensors that could, but should not, be called uniformly “graphene-oxide-modified”.Based on the obtained results, we can generally conclude that GO I is more reduced than GO II, which is the exact opposite of the general conception of graphene oxide structure. Hence, the following question arises: is there a chance to achieve good reproducibility among the scientific community using the not well-established uniform structure of GO? Undoubtedly, we may find some positive aspects of this graphene oxide case because by using graphene oxides of slightly changed chemical composition we can develop sensors with tailored properties towards a given analyte. On the other hand, we have to be aware of the negative aspect, i.e., the lack of reproducibility between “graphene-oxide-modified sensors” when being developed from diverse research teams. In conclusion, the authors believe that there is an urgent need to standardize such popular nanomaterials by assigning a CAS Registry Number to them. The orderliness in graphene oxides nomenclature will greatly shorten the search for appropriate graphene oxide and will contribute to improvements in electrochemical analysis.


The long interspersed nuclear elements 1 (LINE-1/L1s) are the only active autonomous retrotransposons found in humans which can integrate anywhere in the human genome. They can expand the genome and thus bring good or bad effects to the host cells which really depends on their integration site and associated polymorphism. LINE-1 retrotransposition has been found participating in various neurological disorders such as autism spectrum disorder, Alzheimer’s disease, major depression disorder, post-traumatic stress disorder and schizophrenia. Despite the recent progress, the roles and pathological mechanism of LINE-1 retrotransposition in schizophrenia and its heritable risks, particularly, contribution to “missing heritability” are yet to be determined. Therefore, this review focuses on the potentially etiological roles of L1s in the development of schizophrenia, possible therapeutic choices and unaddressed questions in order to shed lights on the future research. Retrotransposons are a group of “jumping genes” which constitute ∼17% of the human genome. In fact, nearly half of the human genome (∼45%) is derived from insertions of transposable elements . The mecvia engineered L1 retrotransposition (Retrotransposable elements (RTEs) are only known to be de-repressed in the brain during human life. Accumulation of approximately 13.7 novel somatic L1 insertions have been noticed in human hippocampus . Althougposition . Furtherposition .L1s’ mobilization occurs more frequently during differentiation of neurons than non-neuronal cell types . HoweverMost L1s integrate into non-exonic regions and won’t cause any recognized phenotypes. So far, no hotspots for L1 insertion have been discovered in the genome. The question of whether L1 insertion is random or guided by environmental factors, hormone influence, or inherited genetic print remains unanswered.The insertion of L1 has long been proposed to predispose people with the risk of schizophrenia . The sigIt is worth noting that the retrotransposition itself can cause many by-products which may have detrimental consequences. For example, because of its endonuclease activity, the ORF2p product of L1 might generate mutations and instability. The expression of faulty protein, RNA or DNA in the cytosol may also trigger immune response, inflammation and neuron degeneration . DysreguIn vitro study also suggested that endogenous IFN signaling limits the propagation of L1 retrotransposon. Collectively it is suggested that IFN may play a protective role against L1 retrotransposon activation and propagation. The activation of L1 possibly activates the expression of low levels of IFN, which in turn antagonize the subsequent L1 retrotransposition. This hypothesis is supported by a correlation between L1 and IFN-β mRNA expression and the capacity of exogenous L1 to induce the expression of IFN-β and downstream substrates in vitro , PCNA and other regulatory elements have been extensively elaborated somewhere else , which include epimutations (changes in histone modifications and promoter methylation that affect gene expression but not the DNA sequence), usually result in somatic mosaicism. Compared to other tissues, the PZMs are comparatively common in mammalian brains especially in schizophrenia patients . MoreoveThe promoter region of DNA regulates gene transcription and its methylation shields the binding of transcription factors, ultimately silencing the gene expression. Hypomethylation exposes promoter to transcription factors and allows the subsequent transcription or protein expression. In mammalian genomes, L1 is the active autonomous retrotransposon, and hypomethylation of L1 is associated with higher retrotransposon activity. Analysis of peripheral blood samples revealed significant hypomethylation of L1 in schizophrenia patients  both in Paradoxically, hypermethylation of L1 in brain tissue of schizophrenia patients has also been reported , which ivia CpG DNA methylation and histones modification is a key repressive mechanism preventing mutagenic events from accumulation  is a protein that plays a role in global DNA methylation as well as neurodevelopmental disorders. MeCP2 appears to be crucial for normal functioning of nerve cells and serve as an inhibitory factor for L1 retrotransposition . L1 retrde novo L1 insertions selectively target CpG-poor promoters of NPC-specific genes because L1 endonuclease identifies an A+T-rich sequence motif. In fact, insertions into A+T-rich introns of housekeeping genes are equally possible  binding and activates several specific retrotransposons: L1, SINEs, and LTRs) but had dramatically increased L1 expression in NAc of the brain . CocaineIt is reported that SIRT6 is a potent suppressor of L1 retrotransposon activity . SIRT6 bIn neurodevelopmental disorder cohorts, mutations in genes which encode chromatin remodelers are overrepresented . ChromatA study in cancer cells has identified 142 genes capable of activating or repressing L1 retrotransposition . These gAs opposed to more common disorders such as anxiety or depression , schizopIt has been proposed that transposable elements contribute in genome expansion and modification not just through transposition but also through the generation of tandem repeats  and tandvia altered expression of synaptic or other schizophrenia-related genes in neurons   which isFurthermore, only ∼ 100 retrotransposition-competent (RC) different classes of L1s are found in individual human  whereas,in-vitro/in-vivo may uphold the promise for cell-type specific investigation, although it’s a time consuming, labor-demanding and technique-challenging task.Although the detailed methodology to study the L1 retrotransposition rate in neurons have been reviewed , but limin-vivo genome correction. However, the challenge of identifying off-target editing and control still remains and further technological improvement is required. The CRISPR-based genome editing has been tested for neurological diseases like autism spectrum disorder, Alzheimer’s, epilepsy, Parkinson’s and Huntington’s diseases (Accumulating evidence suggests that L1s prefer to re-insert in genes responsible for synapse formation in schizophrenia patients. It is warranted to explore further how L1 retrotransposition affects the synapse health chemically or physically? Does it cause hinderance in production, intracellular packaging and synaptic transmission of neurotransmitters? Moreover, currently available antipsychotics have no effects on L1 retrotransposition , therefodiseases  which poSchizophrenia symptoms usually appear in adolescence and dysfunctional GABAergic signaling is the common phenotype consistently found in schizophrenia pathophysiology. As the functional maturation of GABAergic interneurons can prolongs up to post-adolescence period in primate prefrontal cortex, it is highly likely that L1s may also preferentially expressed in GABAergic interneurons. Brain has wide diversity of neuronal cells in terms of function and location, and L1’ behavior in specific cell-types needs to be explored to identify cell specific effect. Moreover, it also remains unaddressed whether the sex hormones help to trigger chromatin remodeling, L1 activation and then schizophrenia. Genome sequencing of families with schizophrenia would possibly provide a clue about the heritable pattern of L1.Analysis of post-mortem brain of patients with schizophrenia and mouse models for schizophrenia exposed to various environmental insults at different times and exposure durations, will probably help to strengthen the loop in understanding. Maternal care in early life can modulate L1 activity, but how does its lacking translate into L1s escape and molecular pathology of schizophrenia is uncertain. There is a need to develop classification system in order to determine whether schizophrenia can be caused by L1 retrotransposition or other mechanisms? Categorization of L1s into “good or bad” and development of improved molecular reporter system to trace L1s in the genome are imperative. Thus, evidences regarding L1 retrotransposons being the cause of schizophrenia are insufficient. Further efforts are required for the development of techniques and skills to investigate the mechanism and epigenetic regulation of L1s leading to schizophrenia.


Long interspersed nuclear element 1  is a retrotransposon comprising ~17% of the human genome. A subset of L1s maintains the potential to mobilize and alter the genomic landscape, consequently contributing to the change in genome integrity and gene expression. L1 retrotransposition occurs in the human brain regardless of disease status. However, in the brain of patients with various brain diseases, the expression level and copy number of L1 are significantly increased. In this review, we briefly introduce the methodologies applied to measure L1 mobility and identify genomic loci where new insertion of L1 occurs in the brain. Then, we present a list of genes disrupted by L1 transposition in the genome of patients with brain disorders. Finally, we discuss the association between genes disrupted by L1 and relative brain disorders. Approximately 45% of the human genome is constitutive of transposable elements referred to as the ‘jumping genes’ . TranspoIt has been estimated that more than 7000 copies of L1 contain a complete structure of ~6 kb in length, even though most L1 sequences in the human genome are remnants of L1 fragments . Full-leRetrotransposition of L1 begins with full-length transcription via a 5′UTR having internal promoter activity . RNA polOccasionally, L1 retrotransposition occurs with the flanking sequence of L1 3′UTR or 5′UTR, which are called 3′ transduction and 5′ transduction, respectively. The weak transcriptional termination signal in L1 causes RNA polymerase to utilize the other termination signal in the downstream regions of L1 3′UTR and results in elongated L1 transcripts . If a stThe retrotransposition of L1 leads to genomic variations and alterations in gene expression. L1s in the intergenic region can influence the transcription of peripheral genes because the 5′UTR of L1 has sense and antisense promoter activities. Alternatively, intergenic L1 can function as regulatory motifs, such as enhancers ,26. L1 iA reporter system using active L1 was utilized to monitor the activity of L1 retrotransposition in a given context . The L1 Monitoring the retrotransposition of endogenous L1 is technically challenging because it is hard to distinguish active L1 transcripts among L1 transcripts produced from several loci and to accurately measure the change in L1 copy numbers in the human genome. Like protein-coding genes, the total amount of L1 transcripts and copy number of L1s can be measured by applying conventional experimental methods, such as real-time PCR, nucleic acid blotting, high-throughput sequencing, etc. However, unlike protein-coding genes, thousands of L1s are spread out in the genome as a repetitive sequence. Slight sequence variations between L1s are indistinguishable when detected by conventional experimental techniques and analysis tools.The sequence similarities in L1s have led to novel approaches for detecting specific L1 transcripts. The long-read RNA sequencing technique may be the best to identify specific L1. However, short-read sequencing platforms have been routinely used for most biological studies, including L1 investigation. The short sequence reads derived from L1 can be mapped to multiple regions of the genome. To analyze the sequence of L1 transcripts originating from specific loci, algorithms that statistically reassign multiple mapped reads have been developed. The expectation–maximization (EM) algorithm is widely adopted to reassign multiple mapped reads . The EM In order to measure the insertion rate of endogenous L1 and to identify the genomic locus where L1 is newly integrated, several methods based on the sequencing techniques have been developed. The copy numbers of newly inserted L1 are remarkably lower than those of pre-existing L1 in the genome, raising the signal-to-noise problem in detecting new insertion. Therefore, the flanking sequence of L1 is amplified together with L1 to obtain sufficient read counts of L1 with a specific sequence. For amplifying L1 with the flanking region, one primer is designed to have a complementary sequence to the 3′ UTR or 5′ UTR. The other primer is designed to have a random sequence. If there is a target region to examine whether L1 insertion occurs, primers can be designed using the reference sequence. The flanking sequence of L1 contains a specific sequence that informs the genomic locus of L1. PCR amplification provides sufficient read counts of the target sequence. In the process of sequence mapping, machine learning technology is now applied to improve the detection accuracy for nucleotide variants and indels.Dux, a master transcription activator, and to activate the rDNA expression required for the transition from the two-cell stage to the four-cell stage [DUX4, a human homolog of mouse Dux [L1 has been intensively studied in early embryogenesis and germ cells. In the mouse study, it is shown that L1 is expressed in fertilized eggs before implantation. The expression of L1 rises to the peak at the two-cell stage and gradually decreases until embryos reach the blastocyst stage ,38. It ill stage . Perhapsouse Dux .The effects of L1 on cellular functions and regulation mechanisms of L1 have been demonstrated more in germ cells. The expression of L1 dynamically changes during germ cell development. In the early stage of spermatogenesis, L1 DNAs are demethylated, resulting in the retrotransposition of L1. New insertion of L1 in germ cells will increase genetic diversity among the human population. However, hyperactive L1 will cause genome instability, resulting in cell death . The malThe retrotransposition of L1 in somatic tissues was first uncovered in the brain. In 2005, Fred H. Gage’s group showed L1 mobility in neural progenitor cells (NPCs) utilizing the L1 reporter system . In the The pathological significance of L1 insertion was first demonstrated by Kazazian in 1988. Kazazian found de novo insertions of L1 into an exon of the factor VIII gene in patients with hemophilia A . L1 retrCCND1 duplication) [CDKN2A deletion) [After the first discovery of abnormally high expression of L1 in cancers in the late 1980s , tremendication) . On the eletion) . These fMeCP2 mutations that result in hypomethylation of L1. The increase in L1 in the brain of AT patients can be speculated to be caused by the destabilization of P53. AT is caused by mutations in ATM. ATM is necessary for stabilizing P53, which is known to suppress L1 transposition [Abnormal expression and copy numbers of L1 have been reported in the brain of patients with various brain disorders, including Rett syndrome (RTT), ataxia–telangiectasia (AT), autism spectrum disorders (ASD), schizophrenia (SZ), and tuberous sclerosis (TSC) ,59 Figu. The molposition ,63.Blood cells of patients have also been utilized to investigate the association of L1 activity with brain disorders. Epigenetic analyses showed that the methylation status of L1 in blood samples is linked to the disease status of brain disorders. The hypomethylation of L1 has been observed in the lymphoblastoid cells of ASD patients with severe language deficits  and the The causality between L1 retrotransposition and diseases has not been revealed in most brain disorders. However, several disease-associated genes disrupted by L1 insertion have been identified in patients’ brains, suggesting that L1 insertion may cause disease by altering the expression of the genes. By analyzing the genome sequence of patients with ASD, RTT, SZ, and AT, specific genic regions were identified where de novo insertion of L1 occurs ,69,70,71FAM126A and OPHN were found and validated. Mutations in FAM126A cause hypomyelination in the central and peripheral nervous system [OPHN1 encodes a Rho-GTPase-activating protein (Rho-GAP) that regulates cell migration and axonal outgrowth by promoting GTP hydrolysis in Rho-GTPase. OPHN1 mutations cause epilepsy and cerebella hypoplasia [OPHN1 mutations are also known to be associated with ASD and SZ [Newly inserted L1s are more frequently found in intronic regions than in exonic regions of genes . Only tws system . The myepoplasia ,75, whicD and SZ .OPHN1, many genes possessing newly inserted L1 are listed as ASD risk factors in Simons Foundation Autism Research Initiative . SCN1A, SCN2A, CTNNA3, and CNTNAP2 found in ASD patients; RELN and DLG2 found in AT patients; GPHN found in TSC patients; and GRID2 KHDRBS2, and SYNE1 found in SZ patients are revealed to be associated with ASD.In addition to SCN1A, SCN2A, and RELN are ranked as high confidence genes associated with ASD. SCN1A and SCN2A are voltage-gated sodium channel genes that were identified as genes perturbed by L1 insertion in the brain of ASD and AT patients. Mutations in SCN1A and SCN2A cause epilepsy [SCN1A and SCN2A have been identified in patients with familial ASD [SCN1A and SCN2A genes are disrupted by L1 transposition at a critical time period during development.Particularly, epilepsy ,79,80,81lial ASD . This inRELN were found in the brain of AT patients. The Reelin signaling pathway is essential for neuronal development and migration [RELN and DAB1, the key molecules in the Reelin signaling pathway, can cause various brain disorders such as ASD and SZ [L1 insertions into the intronic region of igration ,84. The igration . The Reeigration . This imD and SZ .DLG2 in AT, GPHN in TSC, and NRG3 in SZ patients were identified as genes where L1 transposes in intronic regions. Genetic variations in these genes are associated with SZ patients. DLG2, GPHN, and NRG3 are involved in synaptic function. DLG2 encodes a postsynaptic scaffolding protein that interacts with NMDA receptor signaling [ignaling ,87. GPHNignaling ,89. NRG3ignaling .TGM6 in RTT patients’ brains and SYNE1 and GRID2 in SZ patients’ brains were listed as genes containing L1, transposed de novo. Mutations in TGM6, SYNE1, and GRID2 are related to spinocerebellar ataxias (SCA) [as (SCA) ,94,95,96as (SCA) ,98,99.Genes containing a new insertion of L1 cannot account for all the molecular mechanisms underlying brain disorders. Nonetheless, the list of genes disrupted by L1 shows that L1 can mobilize to the genes associated with human brain disorders.Due to the L1-induced gene disruption found in hemophilia A, L1 has attracted attention through its association with human diseases . Then, t


Staphylococcus epidermidis (one that was forming biofilm and one that was not) on new or used teatcups made of silicone for use in milking parlours for sheep, was assessed for 24 h after the application by smearing on the surface of the teatcup. Staphylococci were applied by smearing on an area of 0.0003142 (3.142 × 10−4) m2 on material obtained from the teatcups and their growth and expansion further on were monitored for 24 h at varying ambient conditions: temperature 21 °C or 31 °C and humidity 60% or 80%. No differences were evident between the two isolates in the frequency of recoveries in any of the conditions tested . Recovery rates were higher in humidity 80% compared to humidity 60%: 1678/2016 (83.2%) versus 1282/2016 (63.6%) (p < 0.0001), and in temperature 31 °C compared to temperature 21 °C: 1525/2016 (75.6%) versus 1435/2016 (71.2%) (p = 0.001). Recovery rates were also higher from new teatcups compared to used ones only in humidity 60%: 744/1008 (73.8%) versus 538/1008 (53.4%) (p < 0.0001). Humidity 80% was associated with higher speed of linear dissemination of the isolates on teatcup surface compared to humidity 60%: 0.000000640 (6.40 × 10−7) m s−1 versus 0.000000322 (3.22 × 10−7) m s−1 (+98.8%) (p < 0.0001); no such association was seen with higher temperature: 0.000000509 (5.09 × 10−7) m s−1 versus 0.000000453 (4.53 × 10−7) m s−1 for temperature 31 °C and 21 °C (+12.4%) (p = 0.29). As part of precision livestock farming, differing approaches can be instituted in accord with varying climatic conditions in different farms, as well as within the same farm with the change of seasons.The growth of two isolates of  Staphylococcus aureus, which is a frequent and important causal agent of clinical mastitis in sheep, various coagulase-negative species  cause subclinical mammary infection. In dairy sheep flocks, the infections of the mammary gland have a significant financial importance [Additionally to portance , becauseportance .Pathogen transmission can occur during machine-milking of animals . StaphylA hypothesis was developed that varying conditions in the environment of a milking parlour  may affect the growth of bacteria and their spread on the teatcups. However, there is clear lack of relevant studies and information in the international literature; in a search in the Web of Science platform no relevant publications were found. In general, previous relevant research has focused on the potential to reduce staphylococcal populations on teatcups through correct post-milking cleaning  and the Staphylococcus epidermidis were tested in the study. Details on the source of the organisms, multi-locus sequence typing and biofilm production have been presented by Katsarou et al. [S. epidermidis, as found by carrying out multi-locus sequence typing (MLST). One isolate was forming biofilm (isolate A), whilst the other was not (isolate B) [Two isolates of u et al. . In brieu et al. . The twoolate B) .New and used teatcups manufactured for use in milking parlours for sheep (silicone) were used as test material. Brand-new teatcups  were purchased from an agricultural merchant. Used teatcups, of the same brand as the new ones, were obtained, through the same agricultural merchant, from a local sheep farm, after these had been replaced in the milking parlour at the end of a milking season, subsequently to a use of approximately 4500 milkings.Out of the tubular shaft part of each teatcup , a piece of material was cut. The piece was square, with dimensions 8 cm × 8 cm.The pieces of teatcup material were pinned on a wooden board, thus remaining stretched and facilitating all technical work. On the middle of each piece of teatcup material, a complex like a polar chart-type (‘complex’) was designed. The complex included four concentric circles, with diametres 1 cm, 2 cm, 3 cm, 4 cm, and thus referred to an inner circle and three outer zones , as descS. epidermidis) were cultured on Columbia blood agar and then seeded into Tryptic Soy broth  for aerobic incubation at 37 °C for 6 h. After mixing the bacterial suspension in a Vortex equipment , a swab   was dipped into the broth for 3 s; excess fluid was drained and the swab was withdrawn. The swab was smeared on the piece of teatcup material as described in minute detail by Katsarou et al. [9 to 8.93 × 1010 c.f.u. mL−1; the established technique of Miles and Misra [The two bacterial isolates  were performed every 3 h up to 18 h and then at 24 h [Staphylococcus selective medium (mannitol agar) in duplicate. All the media were incubated at 37 °C in aerobic environment for a period of 48 h; if no growth was seen at that time, they were reincubated for a further 24 h. Bacterial isolation and initial identification were carried out using established techniques [S. epidermidis) from at least one of the two swab samples obtained from a quadrant on each sampling occasion  was deemed to confirm presence of the bacteria on the test material, specifically on the quadrant of the complex from where the swab sample was collected.The swab samples obtained during the above procedure were cultured on 5% sheep blood agar and chniques ,13. DeteAfter bacterial application by smearing, the board with the pieces of test material was maintained at temperature 21.0 °C or 31.0 °C and humidity 60% or 80%. Ambient temperature and humidity were maintained by means of an air-conditioning unit and a humidifier, functioning throughout each experiment.For each isolate, triplicates were performed on each environmental condition and for each type of material (new or old teatcup) assessed. Uninoculated complexes, with no bacteria smeared thereon, were also included in each assessment as controls.−4) m2, 0.0015708 (15.708 × 10−4) m2 and 0.0021990 (21.990 × 10−4) m2, respectively. Thus, the area of each of the quadrants in the respective zones were 0.0002356 (2.356 × 10−4) m2, 0.0003927 (3.927 × 10−4) m2 and 0.0005498 (5.498 × 10−4) m2. Thereafter, the length of time required for the full coverage of each quadrant in circular zone 1 and circular zone 2 was calculated as detailed previously [As the model employed had been validated by Katsarou et al. , similarp < 0.05.Data were entered into Microsoft Excel and analyzed using SPSS v. 21 . Basic descriptive analysis was performed. Frequencies were evaluated by use of Pearson chi-square test or Fisher exact test, as appropriate. Results of the speed of linear dissemination and surface covered were compared between material (new or old teatcups) and between conditions  by using the Kruskal–Wallis test. Statistical significance was defined at No bacteria were recovered from any of the swab samples obtained at the start of each trial, before applying (smearing) the staphylococcal isolates on the teat material.p > 0.95 for all comparisons between triplicate measurements in the various assessments). In contrast, we did not recover staphylococci at any sampling point from any complex, on which no staphylococci had been applied . Differences in overall recovery rates between successive sampling points were significant for up to 18 h after application by smearing .In all trials, after application by smearing, we recovered staphylococci consistently from the swab samples collected from the inner circle of the complex on all sampling occasions (3 h to 24 h post-application) . This wap = 0.83). Also, we did not find significant differences in recovery rates between the two isolates, when assessing separately the three circular zones for recoveries between the two isolates   for isolate A and 1477/2016 (73.3%) for isolate B (arisons) .p < 0.0001). We found this significant difference also in the recovery rates from the two types of teatcups, when these were considered separately for recoveries in each of the three circular zones (p < 0.0001). However, when we assessed recoveries separately in the environmental conditions created, this difference was evident in humidity 60% , but not in humidity 80%  , but not in humidity 80% .We found clear evidence that recoveries were more frequent from new teatcups compared to used ones: 1593/2016 (79.0%) versus 1367/2016 (67.8%) (arisons) . When wep < 0.0001) (p < 0.0001).In general, we found that recovery rates were higher in humidity 80% compared to humidity 60%: 1678/2016 (83.2%) versus 1282/2016 (63.6%) ( 0.0001) . The samp = 0.001)  versus 1435/2016 (71.2%) (= 0.001) . Howeverp > 0.95).Finally, we did not find any differences in the frequencies of recovery of staphylococci among the triplicate assays during the evaluation of the same isolate tested on the same material (−7) m s−1 and 0.000000471 (4.71 × 10−7) m s−1, respectively (p = 0.71). The speed of dissemination was clearly higher in new teatcups: 0.000000373 (3.73 × 10−7) m s−1 versus 0.000000271 (2.71 × 10−7) m s−1 in used ones (p < 0.0001) in 60% humidity, whilst no such difference was seen in 80% humidity: 0.000000664 (6.64 × 10−7) m s−1 versus 0.000000616 m (6.16 × 10−7) s−1, respectively (p = 0.23).There was no difference in the speed of dissemination between the two isolates overall: 0.000000491 (4.91 × 10−7) m s−1 versus 0.000000322 (3.22 × 10−7) m s−1 (+98.8%), respectively (p < 0.0001). In contrast, no association of temperature was seen with significantly higher speed of dissemination: 0.000000509 (5.09 × 10−7) m s−1 versus 0.000000453 (4.53 × 10−7) m s−1 (+12.4%), for temperature 31 °C and 21 °C respectively (p = 0.29).Higher humidity (80%) was associated with higher speed compared to 60% humidity: 0.000000640  and disseminate quickly ,19. IncrThe reduced growth of the bacteria on the used teatcups was not expected. A possible explanation for this might be that the fissures and cracks present on used teatcups , increaS. epidermidis occurs [Formation of biofilm by one of the isolates evaluated did not seem to provide an advantage in dissemination of the staphylococci on the surface of the teatcup. Through formation of biofilm, bacteria attach on surfaces and produce extracellular polymers, which enhance their attachment thereon and the formation of matrices . In the s occurs ,23; duris occurs , but evis occurs .The findings suggest that, in field conditions, differing climatic conditions can pose varying challenges in the management of milking parlours in farms. As part of precision livestock farming, differing approaches can be instituted in accord with varying climatic conditions in different farms, as well as within the same farm with change of seasons. Within a wider viewpoint, it is noted that climatic changes may lead to effects in micro-environments, e.g., within a milking parlour, and have relevant effects in pathogen dissemination. In a similar context, it is noted that humidity has been recognized as a risk factor for staphylococcal infections in orthopaedic prosthetic devices in the tropical part of Australia , which fThe study presents for the first time internationally that climatic factors may affect the bacterial numbers and their speed of dissemination on teatcups from milking parlours. Increased number of bacterial on teatcups increase the risk for mammary infections and consequently mastitis to animals milked in those parlours . PreviouThe results the study can be used to scientifically update the relevant knowledge about conditions in milking parlours, because indeed conditions change as the results of different dynamics or interactions prevailing in there change, according to time of year, technological changes and bacterial evolution.


A great wealth of studies has investigated the capacity of motivationally relevant stimuli to bias attention, suggesting that reward predicting cues are prioritized even when reward is no longer delivered and when attending to such stimuli is detrimental to reward achievement. Despite multiple procedures have been adopted to unveil the mechanisms whereby reward cues gain attentional salience, some open questions remain. Indeed, mechanisms different from motivation can be responsible for the capture of attention triggered by the reward cue. In addition, we note that at present only a few studies have sought to address whether the cue attractiveness dynamically follows changes in the associated reward value. Investigating how and to what extent the salience of the reward cue is updated when motivation changes, could help shedding light on how reward‐cues attain and maintain their capacity to attract attention, and therefore on apparent irrational attentive behaviors. Motivationally relevant objects attract attention , the question is whether the reward‐cue attentional salience does also change accordingly. Intuitively this should be the case, but the study of De Tommaso et al.  has showHowever, concerns have been raised that some of the reported evidence of the reward‐cue attentional bias may in fact result from factors other than motivation, like for instance strategic attentional allocation or selection history , followed by a test phase, when the reward cue is presented in extinction, and its ability to capture attention is measured with different tasks. An example of the two‐phase paradigm is the Value‐Driven Attentional Capture (VDAC) paradigm, originally developed by Anderson et al. . In the Different versions of the two‐phase paradigm have been adopted in a great wealth of studies showing the persistence of reward‐based attentional capture also with more complex stimuli and with a variety of tasks in different modalities  transfer to the reward cue (CS), thus altering the corresponding attentional salience /reinforcement system , so the presence of another singular element  will likely attract attention. In other words, the attentional templates of the relevant and the irrelevant information overlap, being both tuned on singularity. However, if attention is tuned to a more specific feature of a target (feature‐search mode), the strategy of searching for a singleton becomes inefficient, and the distractor singleton can be ignored with practice  could help in disentangling the learning mechanisms whereby the reward‐cues attentional bias is attained. Recent studies confirm that the reward‐cues attentional salience adapts, although with some inertia, to changes in cue‐reward mapping when reward is a desirable outcome (De Tommaso & Turatto, It is also worth noting that individual differences in susceptibility to attentional capture by motivational stimuli such as personality traits (Albertella et al., In sum, different procedures investigating reward‐cues attentional salience have been devised, highlighting several factors that need to be controlled in order to infer that attention is biased by the motivational effects of reward. Among these factors, past orienting episodes of selection or avoidance, reinforcement of the attentive behaviour and information seeking have shown to be particularly relevant and not easy to disentangle. However, several strategies have been implemented to control for the role of these factors, such as for example to test whether the attentional bias is context specific, or whether the presence of the attentional effect persists despite the removal of reward from the learning procedure. Furthermore, studying the role of state‐dependency in modulating the reward‐cue salience, as a consequence of either dynamic change in motivation or of an altered arousal level, could also offer new insights on how reward‐cues attain and maintain their capacity to attract attention.The authors declare that they have no conflict of interest.Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.


GRN) gene have been found in up to 50% of patients with familial FTLD. Abnormal deposits of post-translationally-modified TAR DNA-binding protein of 43 kDa (TDP-43) represent one of the main hallmarks of the brain pathology. To investigate in peripheral cells the presence of the different TDP-43 forms, especially the toxic 25 kDa fragments, we analyzed lymphoblastoid cell lines (LCLs) and the derived extracellular vesicles (EVs) from patients carrying a GRN mutation, together with wild-type (WT) healthy controls. After characterizing EV sizes and concentrations by nanoparticle tracking analysis, we investigated the levels of different forms of the TDP-43 protein in LCLs and respective EVs by Western blot. Our results showed a trend of concentration decreasing in EVs derived from GRN-mutated LCLs, although not reaching statistical significance. A general increase in p-TDP-43 levels in GRN-mutated LCLs and EVs was observed. In particular, the toxic 25 kDa fragments of p-TDP-43 were only present in GRN-mutated LCLs and were absent in the WT controls. Furthermore, these fragments appeared to be more concentrated in EVs than in LCLs, suggesting a relevant role of EVs in spreading pathological molecules between cells.Frontotemporal lobar degeneration (FTLD) is a complex disease, characterized by progressive degeneration of frontal and temporal lobes. Mutations in progranulin ( MAPT), chromosome 9 open reading frame 72 (C9ORF72), and progranulin (GRN), with many other genes being less frequent [Frontotemporal lobar degeneration (FTLD) is a complex disease, characterized by progressive degeneration of frontal and temporal lobes and extensive neuroinflammation, which manifests with a range of clinical disorders and inevitably leads to death. FTLD is the second most common cause of dementia after Alzheimer’s disease (AD) under the age of 65 ,2. AboutGRN mutations have been found in about 10% of patients with sporadic FTLD and in up to 50% of patients with familial FTLD [GRN mutations areloss of function, leading to mutated mRNA degradation and to a following condition of haploinsufficiency [GRN mutations cause FTLD, homozygous GRN mutations cause neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disorder [ial FTLD ,4. Progrial FTLD . In the ial FTLD ,8,9,10. ficiency ,12. Whildisorder , althougdisorder .GRN-associated FTLD include neuronal loss, glia hyperproliferation and inflammation, lysosomal dysfunction, and, in particular, abnormal deposits of TAR DNA-binding protein of 43 kDa (TDP-43) in neuronal cells [The main neuropathological hallmarks of al cells ,16,17. Nal cells . TDP-43 al cells ,22,23,24In cellular models of TDP-43 proteinopathies and in human brain samples from FTLD, FL TDP-43 and its CTFs are released via exosomes ,26. An eGRN mutation and wild-type (WT) healthy control subjects. We analyzed both lymphoblastoid cell lines (LCLs) and the derived EVs, searching for pathological hallmarks. We observed the presence of phosphorylated 25 kDa CTFs of TDP-43 in cells originated from subjects carrying a GRN mutation, while these fragments were absent in WT control cell lines. Furthermore, we confirmed the same findings in the EVs, suggesting that EVs actually reflect the pathological status of their originating cells.In this work, we investigated the presence of TDP-43 and its phosphorylated/pathological forms in peripheral cells from FTLD and NCL patients carrying a GRN mutations result in null alleles, leading to a reduction of PGRN (haploinsufficiency). PGRN expression was evaluated in LCLs from three healthy WT subjects and nine patients carrying GRN null mutations . As expected, PGRN levels were significantly reduced in heterozygous GRN mutation carriers (p = 0.0011), while the protein was absent in homozygous carriers (p = 0.0002)  .GRN mutation carriers. Western blots were performed with antibodies recognizing TDP-43 or TDP-43 phosphorylated at residues Ser409/Ser410 (p-TDP-43).As it is known that pathological TDP-43 is abnormally phosphorylated at Ser409/Ser410 residues and cleaved to generate CTFs migrating at ~25 kDa, which are more prone to phosphorylation than FL protein, we examined all these forms of TDP-43 in LCLs from heterozygous and homozygous GRN-mutated LCLs  were exclusively detected in GRN-mutated LCLs (GRN heterozygous LCLs compared to GRN homozygous LCLs (p = 0.0272).We observed an overall increase in p-TDP-43 levels in ted LCLs : while Fted LCLs C, althouGRN-mutated LCLs in order to assess the presence of pathological forms of TDP-43. We isolated the EVs from conditioned media of control and GRN-mutated LCLs and firstly analyzed them by nanoparticle tracking analysis (NTA) to evaluate EV concentrations (EVs/mL) and average sizes (nm). Only a trend toward a decrease in EV concentration in GRN-mutated LCLs compared to controls was observed, even if not reaching statistical significance . No statistically significant differences in EV size were observed among the investigated groups .As it has been demonstrated that the EVs carry different kinds of biological molecules, including proteins, which can either accomplish physiological communication between cells or transmit toxicity if they are pathologically altered, we examined the contents of EVs from ificance A,B , although this did not reach statistical significance [n = 10) or GRN mutations (n = 5) compared with sporadic FTLD subjects (n = 51) and controls (n = 22) [We have a collection of LCLs derived from patients carrying a ificance . In anot(n = 22) . An addi(n = 22) .GRN homozygous LCLs than in WT LCLs, while the situation was more variable in GRN heterozygous LCLs, as some of them did not display FL p-TDP-43. However, concerning the CTFs p-TDP-43 of about 25 kDa, the differences between WT and mutated LCLs were striking. In fact, CTFs p-TDP-43 were not detected in WT LCLs, whereas in all but one GRN heterozygous LCLs and one GRN homozygous LCL they were present at high expression level. Interestingly, in the cases where CTFs p-TDP-43 were absent, FL p-TDP-43 was present, indicating that at least one abnormal form of TDP-43 is present anyway, perhaps not (yet) truncated to CTFs.In our LCLs, TDP-43 levels were similar among all genotypes, including WT, heterozygous and homozygous, both in LCLs and EVs. This suggests that protein transcription levels and stability do not differ significantly, even in presence of the mutation. Similar levels of TDP-43 were also reported in LCLs from ALS and AD cases compared to healthy controls ,36. On tGRN patients, a significant concentration decrease of EVs was described [GRN-mutated LCLs only a trend towards concentration decrease was observed, not reaching statistical significance. In EVs, the level of FL p-TDP-43 did not show significant difference among genotypes, but the level of 25 kDa CTFs p-TDP-43 confirmed the situation of the respective LCLs: CTFs p-TDP-43 were absent in WT EVs, very abundant in GRN heterozygous EVs and also present, at lower levels, in GRN homozygous EVs. Comparing the ratio CTFs p-TDP-43/TDP-43 in LCLs to the ratio in EVs, the amounts of CTFs p-TDP-43 resulted increased in heterozygous EVs, that is the EVs appeared to concentrate these toxic fragments. This finding makes the EVs particularly relevant and critical in their role of communication between cells, as previous studies have demonstrated that TDP-43 aggregates can be transferred from cell to cell via exosomes [As for EVs, while in plasma and in human primary fibroblasts from escribed ,38, in oGRN homozygous LCLs and EVs the CTFs p-TDP-43 were at lower levels than in GRN heterozygous is intriguing. Homozygous GRN mutations are associated with NCL, a disease that shares some pathological features with FTLD but that is also very different. In a recent post-mortem analysis of a human brain carrying a homozygous GRN mutation, only a very mild TDP-43 pathology was described, without any positive detection of aggregated forms, while lysosomal storage disorder was overwhelming [GRN heterozygous FTLD context.The finding that in whelming . Our datAs EVs are released into the extracellular space and can be isolated from a number of body fluids, including blood, CSF and urine, detecting molecules associated with these vesicles may have diagnostic, prognostic, and disease monitoring potential. We think that the toxic 25 kDa CTFs p-TDP-43 detected in LCLs and their EVs may have this potential, as they are derived from peripheral cells. As a future direction, detection of these fragments in plasma or plasma EVs may be planned.GRN mutation. After acquiring informed consent for research purposes from these patients and control subjects, we obtained peripheral blood lymphocytes, from which LCLs were produced using the Epstein–Barr virus infection, according to standard procedures.Following genetic analysis for diagnostic purposes in the dementia field, we disclosed some patients carrying a GRN (RefSeq NM_002087.4) mutation  were analyzed in this study.LCLs from three WT healthy age-matched control subjects and nine patients carrying a LCLs were cultured in suspension in T25 flasks in Optimem medium  containing 10% fetal bovine serum  and 2% penicillin/streptomycin .6 cells/mL) were seeded in an Optimem medium without FBS for 24 h. The conditioned medium was then collected by centrifugation and used to isolate EVs (see below), while the cells were washed in PBS and lysed in an ice-cold RIPA lysis buffer  containing Protease Inhibitor Cocktail  and Phosphatase Inhibitor Cocktail . The cell lysates were kept in ice for 15 min, sonicated in ice, kept for an additional 15 min in ice, centrifuged at 13,000× g for 15 min at 4 °C, and then the supernatant was recovered. Protein concentration of extracts was determined by a BCA assay kit .LCLs  for nanoparticle tracking analysis (NTA) or lysed in an ice-cold RIPA lysis buffer containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail for Western blot analyses. Protein concentration of extracts was determined by BCA assay kit.EVs were isolated from conditioned media of LCLs after 24 h of starvation. EVs were prepared with a Total Exosome Isolation reagent  following the manufacturer’s instructions. Briefly, the conditioned cell culture media were centrifuged at 2000× EVs resuspended in PBS were analyzed with the NanoSight NS300 Instrument . Samples were diluted with filtered PBS to obtain an optimal range of 20–150 particles/frame. For each sample, 5 videos of 60 s were recorded and data were processed using NanoSight NTA Software 3.2. Optimized post-acquisition settings were kept constant during the analysis of all samples. The data obtained included particle concentration (particles/mL) and average size (nm). Raw concentration data (particles/mL) from NTA were normalized to obtain EV concentrations in the LCL-conditioned media.Western blot analysis was performed using 25 µg cell lysates and 15 µg EVs lysates. Samples were subjected to polyacrylamide gel electrophoresis using Bolt 4–12% Bis-Tris gels  and transferred onto PVDF membranes . The membranes were incubated overnight at 4 °C with the following primary antibodies: anti-hPGRN ; anti-TSG101 ; anti-Alix ; anti-TDP43 ; anti-p-TDP43  and anti-Actin . TSG101 and Actin were used as loading controls for EVs and cellular extracts, respectively. Horseradish peroxidase-conjugated secondary antibodies  were incubated for 1 h at room temperature. Immuno-positive bands were detected by chemiluminescence  according to the manufacturer’s instructions. Densitometric analysis was performed with QuantityOne software (BIO-RAD).t-test or Mann–Whitney test was used for comparison between two groups of normally or non-normally distributed variables, respectively. One-way ANOVAs, with Bonferroni post-hoc tests, were used for comparisons within the groups of normally distributed variables. Means with standard deviations or medians with interquartile ranges (25–75%) were graphically represented for normally or non-normally distributed variables, respectively. Significance was set at p < 0.05.All statistical analyses were performed using GraphPad Prism Software . Data distribution was assessed by D’Agostino and Pearson omnibus normality test. Student’s