The aim of this study was to investigate the effect of icaritin (ICT) on TAR DNA-binding protein 43 (TDP-43)-induced neuroblastoma (SH-SY5Y) cell damage and to further explore its underlying mechanisms.To investigate the possible mechanism, TDP-43 was used to induce SH-SY5Y cell injury. Cell viability was evaluated by the CCK-8 assay. The mitochondrial membrane potential (MMP) was determined with JC-1. The expression levels of TDP-43 and cytochrome C (CytC) were measuring by Western blotting. Changes in adenosine 5′-triphosphate (ATP) content, total antioxidative capacity (T-AOC), glutathione peroxidase (GSH-Px) activity, superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were detected with specific kits.The results showed that ICT reduced the cell damage induced by TDP-43. ICT reduced the expression level of TDP-43; increased ATP content and the MMP; decreased CytC expression; increased T-AOC and GSH-Px, total SOD (T-SOD), copper/zinc SOD (CuZn-SOD), and manganese SOD (Mn-SOD) activity; and decreased MDA content.The results suggest that ICT has a protective effect on TDP-43-transfected SH-SY5Y cells that is related to reductions in TDP-43 expression and mitochondrial damage and alleviation of oxidative stress. TAR DNA-binding protein 43 (TDP-43) is a multifunctional nucleic acid-binding protein composed of 414 amino acids and six exons. Under physiological conditions, it can regulate RNA splicing, transport and translation in a variety of ways and is involved in the biosynthesis of microRNAs. It is an important transcriptional regulator . This prEpimedium, is rich in flavonoids and easily crosses the blood–brain barrier (Icaritin (ICT), a component of  barrier . It impr barrier . Studies barrier . TherefoTherefore, to confirm our hypothesis, the protective effect of ICT against TDP-43-induced mitochondrial damage in SH-SY5Y cells   was evalReagent-grade ICT,  was obtained from Zhongke Quality Inspection Biotechnology Co., Ltd.  and dissolved in dimethyl sulfoxide (DMSO); the final concentration of DMSO in the medium was less than 0.1% (v/v). Human TDP-43 and polybrene were purchased from Hanbio Biotechnology Co., Ltd. , a TransZol Up Plus RNA Kit and EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix were purchased from TransGen Biotechnology Co., Ltd.  and NuHi Robustic SYBR Green Mix was purchased from Nuhigh Biotechnologies Co., Ltd. . High-Sig ECL Western Blotting Substrate was purchased from Tanon Technology Co., Ltd. . RIPA buffer (high) (R0010) was purchased from Solarbio Life Science . A GAPDH antibody, a TDP-43 antibody, a CytC antibody, and HRP-conjugated Affinipure goat anti-rabbit IgG (H+L) were purchased from Proteintech Group . A goat anti-mouse IgG (H+L) secondary antibody was purchased from Thermo Fisher Scientific .2 cell culture incubator at 37 °C. When the cells covered approximately 80% of the bottom of the bottle, they were collected in a single tube and centrifuged at 1000 rpm for 5 min after trypsin digestion. After discarding the supernatant, the cells were seeded in 96-well plates, 24-well plates, or 6-well plates at a density of 5  × 104/mL in complete medium, and the medium was changed every 2 days.SH-SY5Y human neuroblastoma cells were purchased from Wuhan University Collection Center. The cells were cultured in DMEM-F12 medium containing a mixture of 15% fetal bovine serum and 1% penicillin streptomycin in a 5% CO5 cells/mL) were plated in a 96-well plate at a density of 1 ×104 cells per well and maintained at 37 °C and 5% CO2 A total of 200 µL of fresh medium containing virus at an MOI of 0, 10, 20, 30, 40, 50, or 60 (including 1.2 µL of polybrene at a working concentration of 6 µg/mL). After 12 h, the transfection was terminated, and the optimal MOI was selected by observing and photographing the cells under a fluorescence microscope.Virus transduction construct, cloning, and production were carried out by Hanbio Biotechnology Co., Ltd. The detailed steps and more information are provided in the supplementary files. SH-SY5Y cells showing good growth were chosen. The SH-SY5Y cells (1 ×105 cells/mL) were seeded in each well of a 96-well plate for 24 h. After treatment, CCK-8 solution (10 µL) was added to each well of the 96-well plate, the cells were incubated at 37 °C for 2 h. The absorbance was measured at 450 nm with a Synergy HTX microplate reader .Cell viability was assessed by the CCK-8 assay ; in this assay, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5--2H-tetrazolium monosodium salt produces water-soluble maz dye after it is formed and undergoes biological reduction in the presence of electron carriers. Briefly, SH-SY5Y cells . The proteins were then heated at 100  °C for 5 min for denaturation. Equal amounts of total protein (25 µg per lane) were loaded on 12% SDS-PAGE gels and separated. After sample loading, the voltage was set to 90 V and then to 120 V until the sample reached the bottom of the gel. The proteins were then transferred to a nitrocellulose (NC) film by the sandwich method. The membrane was washed three times with TBST and then blocked with 5% skimmed milk. The membrane was then incubated for 12 h at 4 °C with a GAPDH antibody , TDP-43 antibody , and CytC antibody . The membrane was washed with TBST 3 times and incubated with HRP-conjugated Affinipure goat anti-rabbit lgG (H+L)  for 2 h at room temperature. The membranes were developed using hydrogen peroxide and Supersignal West Pico Luminol . Finally, High-Sig ECL Western Blotting Substrate  was used to visualize the membrane.A JC-1 MMP test kit  was used according to the manufacturer’s protocol. The culture medium of each group of cells was discarded, the cells were wash once with PBS, and the same amount of culture medium and JC-1 staining working solution was added and mixed well. Afterwards, the cells were placed in a 37 °C cell incubator and incubated for 20 min. Then, the supernatant was discarded, the cells were washed twice with prepared 1 × JC-1 staining buffer, and cell culture medium was added. After this, the cells were washed with PBS and observed under an inverted fluorescence microscope .We used a commercial ATP content test kit, GSH-Px test kit, SOD typing test kit, MDA test kit  and T-AOC test kit . A BCA protein quantitative kit (Beijing Solarbio) was used to determine the protein content.To measure ATP levels, reagents and the sample to be tested were added to tubes according to the manufacturer’s protocol. A blank tube, a standard tube, experimental tubes, and a control tube were analyzed, and the experiment was performed in triplicate. The absorbance value of each tube at a wavelength of 636 nm was measured .To determine T-AOC, cells were scraped off, lysed on ice, and centrifuged at 4 °C at 12,000 rpm in a low-temperature centrifuge for 5 min, and the supernatant was taken for determination. Reagents and the sample to be tested were added to each well according to the manufacturer’s instructions, the absorbance value of each well at a wavelength of 734 nm was measured, and the T-AOC of the sample was calculated according to the standard curve.To measure GSH-Px levels, prepared cell homogenates were centrifuged in a low-temperature centrifuge at 2000 rpm for 10 min, and the supernatant was centrifuged at 10,000 rpm in a low-temperature centrifuge for 15 min. After adding 0.5 mL of PBS and mixing, the prepared detection reagents were added, and the absorbance value of each sample was determined at a wavelength of 412 nm.via the hydroxylamine method according to the manufacturer’s protocol. SH-SY5Y cells were seeded into a 6-well plate, and the absorbance of the samples was measured at 550 nm using a microplate reader.To analyze SOD levels, T-SOD, CuZn-SOD, and Mn-SOD activity in SH-SY5Y cells was measured using a SOD typing assay kit To assess MDA levels, treated cells were washed with cold PBS and homogenized in PBS in a glass homogenizer to form a suspension. The supernatant was used for analysis according to the manufacturer’s protocol, and the absorbance of each sample was measured at a wavelength of 530 nm.t-test with correction. A value of P < 0.05 was considered statistically significant.All statistical analyses were performed with SPSS 22.0 . All results are expressed as the mean ± SD. The difference between the means of more than two groups was analyzed by one-way analysis of variance (ANOVA). When ANOVA showed significant differences, pairwise comparisons of means were made by Bonferroni’s post hoc TDP-43 in the TDP-43 group was higher than that in the control group (Cell were transfected with virus an MOI of 0, 10, 20, 30, 40, 50 or 60. Cells transfected at an MOI of 0 were in good condition and showed no green fluorescence under a fluorescence microscope. Cells transfected at an MOI of 30 were in good condition, showed clearly visible dendrites, fluoresced under a fluorescence microscope, and showed high transfection efficiency. The fluorescence and transfection efficiency of cells transfected at MOIs of 10 and 20 were lower than those transfected at an MOI of 30. The cells transfected at MOIs of 40 and 50 were in a poor state; there cell bodies were crumpled and collapsed, and some of the cells were dead. Cells transfected at an MOI of 60 were blurred or absent, and a large number of cells were dead. In conclusion, the optimal MOI was determined to be 30 . The morol group . Westernol group . In summAfter modeling, ICT at concentrations of 0.0, 0.1, 1.0 and 10.0 µmol/L was added, and the cell activity of each group at 12, 24 and 48 h were determined. The results showed that optimal cell viability was obtained at a drug concentration of 1.0 µmol/L and a treatment time of 48 h. Therefore, subsequent experiments were carried out under these conditions .Changes in cell activity in the control group, control + ICT group, TDP-43 group, and TDP-43 + ICT group were assessed after treatment with 1.0 µmol/L ICT for 48 h. The results showed that compared with that of the control group, the activity of the TDP-43 group was decreased, while the cell activity of the TDP-43 + ICT group was increased after ICT treatment .The Western blot results showed that compared with that in the Con group, the expression level of TDP-43 in the TDP-43 group was increased; however, after ICT treatment, the expression level of TDP-43 decreased .JC-1, which can quickly and sensitively detect MMP changes, was used a fluorescent probe in this experiment. In normal mitochondria, JC-1 aggregates in the mitochondrial matrix to form a polymer that emits strong red fluorescence. In unhealthy mitochondria, due to a decrease in the MMP, JC-1 can only exist in the cytoplasm as monomers, which produce green fluorescence. The results showed that compared with the control group and control + ICT group, the TDP-43 group showed reduced red fluorescence, enhanced green fluorescence and a decreased MMP. Green fluorescence was weakened, red fluorescence was enhanced, and the MMP was increased in the TDP-43 + ICT group compared with the TDP-43 group  and 4B.The results showed that there was no significant difference in ATP content between the control + ICT group and control group and that ATP content in the TDP-43 group was lower than that in the control group. Compared with that in the TDP-43 group, the ATP content in the TDP-43 + ICT group was increased .The Western blot results showed that compared with that in the control group, the expression level of CytC in the TDP-43 group was increased, while the expression level of CytC decreased after ICT treatment .T-AOC , GSH-Px TDP-43 is a highly conserved nuclear RNA/DNA-binding protein involved in the regulation of RNA processing . TDP-43 Abnormal accumulation of TDP-43 in mitochondria is an important cause of mitochondrial dysfunction and neurodegenerative diseases . PreventA large amount of TDP-43 is localized in mitochondria mainly because a long-term increase in oxidative stress increases the nuclear depletion and cytoplasmic accumulation of TDP-43 and promotes the entry of a large amount of this protein into mitochondria. The interaction between TDP-43 and OXPHOS-related mt-mRNA affects the mitochondrial respiratory chain, resulting in a decrease in ATP production, a decrease in the MMP and an increase in ROS levels. This increase in ROS levels promotes the TDP-43/OXPHOS-related mt-mRNA interaction, forming a vicious cycle . Lipid pHow does ICT reduce the level of TDP-43? According to our findings and previous results, we speculated that ICT promotes the degradation of TDP-43 by increasing the levels of LonP1 and ATP. We also speculate that the mechanism is related to the antioxidant capacity of ICT based on existing experiments and related research. A large number of studies have shown that ICT is a relatively good plant-based antioxidant and it has good antioxidant capacity in different cell and animal experiments . This exIt is well known that TDP-43 gene mutations are one of the recognized genetic causes of ALS, which is closely related to an imbalance of energy homeostasis and disease susceptibility and progression . Other rThe results suggests that the protective effect of ICT against TDP-induced SH-SY5Y cell injury is related to a reduction in TDP-43 expression, alleviation of mitochondrial injury and amelioration of oxidative stress.10.7717/peerj.11978/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.11978/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.11978/supp-3Supplemental Information 3Click here for additional data file.


In faculty development, understanding each participant’s cultural context is important. However, there is scarce evidence on how to improve cultural understanding in faculty development. Cultural anthropology is a discipline that focuses on developing cultural self-awareness by understanding different cultures. Professionals from this field can be crucial to the goal of cultivating cultural awareness among medical educators. The aims of this study are to 1) develop and modify cultural anthropology sessions in faculty development and 2) evaluate the effectiveness of these sessions, including their long-term impacts.The cultural anthropology sessions were organized as part of a longitudinal faculty development program—Foundation Course for Medical Education—at Kyoto University in Japan. The study included 47 medical educators participating in faculty development and three lecturers: two cultural anthropologists and a medical educator. We developed the cultural anthropology sessions and implemented them in the longitudinal faculty development program. In these sessions, cultural anthropologists used inquiry-guided reflection. An action research methodology was employed and repeated in four cycles from 2015 to 2018. Qualitative and quantitative data were collected during the action research cycles. The qualitative data were thematically analyzed.The cultural anthropologists’ inquiries fostered learning during the sessions, and three themes—cultural relativism, attention to context, and reframing—were synthesized. As a long-term impact of the sessions, the learners reported becoming more aware of the cultural contexts in their daily educational and clinical activities.The cultural anthropology sessions in the faculty development program were shown to have enhanced the participants’ awareness of cultural contexts. The concept and format of these sessions may be used more widely in faculty development programs.The online version contains supplementary material available at 10.1186/s12909-022-03260-7. It is well known that awareness of cultural context is imperative in medical education, and the importance of considering cultural context during faculty development (FD) has been recognized and discussed more frequently in recent years –3. As FDEffective FD programs should therefore include a session focused on cultivating participants’ cultural awareness. Cultural anthropology (CA), a discipline that focuses on the study of different cultures, provides a suitable foundation for developing cultural self-awareness in individuals  and for The history of medical education recognizes that methods and theories of medical education—many of which were developed in Western culture—have been introduced to other countries. However, such methods and theories have not always been successful in the highly context-dependent field of medical education . MedicalIn this study, the authors developed a CA session to cultivate cultural awareness among medical educators and implemented the same in a longitudinal FD program. The aims of this study are to 1) develop and modify the CA session and 2) evaluate the effectiveness of the CA session, including its long-term impact.We used an action research methodology, which is appropriate for making decisions about program improvement through repetitive reflection, data collection, and analysis , 16. We The CA sessions were developed as part of a longitudinal FD program—Foundation Course for Medical Education (FCME)—at Kyoto University in Japan. FCME is a one-year part-time medical education program conducted in web-based and face-to-face sessions. The curriculum contains sessions on teaching and learning, assessments, curriculum development, informatics technology, research, philosophy, leadership and management, general, others, and CA. Twelve participants enroll in the program each year; they are physicians who teach medical students and residents at medical schools or teaching hospitals. The program was first established in April 2015 and over 70 participants have graduated from the program as of 2021.This study involved 47 participants: 12 each for the classes of 2015, 2016, and 2018, and 11 for the class of 2017. Additionally, the study involved three lecturers: one medical educator and two cultural anthropologists. One participant from the class of 2017 was excluded due to absence from the CA session. The participants were a diverse group of individuals with different ages and areas of specialty , and a cultural anthropologist (JI) initiated discussions about drafting CA sessions, establishing learning objectives, scheduling, and educational format. They have collaborated for over a decade in research and teaching settings and obtained successful results . Based oAs an educational format, they offered a case-based, inquiry-guided reflection. In this format, each participant presented a case of educational practice that they learned in one context and applied in another, with detailed descriptions of their backgrounds; for example, the size, location, or organization of the institutions they learned from and where the practices had been applied. Following the presentations, the participants reflected on how and why their practices succeeded or failed, and the cultural anthropologist lecturer asked questions about the case. By answering the anthropologist’s questions, participants were expected to gain a self-relativization perspective and view themselves differently. In addition to the cultural anthropologist, the medical educator and other participants also asked questions about the case. The CA sessions were web-based, and after the session, each presenter wrote a short reflective essay on the insights gained from the session and submitted it as a post-session assignment. We introduced three books for the participants as references –22.In this case-based, inquiry-guided reflection, participants reflected on the case, and then the cultural anthropologist asked questions to help them reflect further. The developers selected this format because guided reflection is known to help learners develop reflective thinking , and theEach year, the lecturers reflected on the CA session and, depending on the survey results, modified the contents if necessary.To assess the lecturers’ modification of the CA sessions, the main author investigated the updated syllabus of the CA session in the planning phases of 2016 to 2018.To assess the effectiveness of the CA sessions, we collected multiple data in each cycle of action research. In the observation phase of each year, all CA sessions were video recorded; and observation logs, including the qualitative data of participants’ presentation and questions asked by the lectures and other participants, were collected. The post-session assignments, post-session questionnaire, and post-program questionnaire of all participants were also collected via Google forms  with experience as an ethnographer in hospital and educational facilities joined the team. Furthermore, the 15 min for presentation and discussion per participant was often insufficient. Therefore, starting from the Class of 2016, the presentation and discussion time per participant was increased to 30 min.The final model of the CA sessions is shown in Table Based on the observation logs, the questions by the anthropologist were divided into two themes: questions about the institutions’ socio-cultural backgrounds and those about their educational practices. The anthropologist asked questions that revealed the cultural contexts in the healthcare setting that medical professionals often take for granted. After elucidating the cultural context of the participants’ cases, the cultural anthropologist asked questions that encouraged the participants’ reflection regarding their educational practices. As an example, the inquiry-guided reflection for Dr. A, an internist, is shown in Table In terms of how the cultural anthropologist’s inquiries fostered learning during CA sessions, three themes—cultural relativism, attention to context, and reframing—were synthesized. Initials were used to indicate the type of contributor (P = participant and L = lecturer), type of data source , and the year of the class or year of data collection.Although the participants tried to describe the cultural and contextual differences in detail, it was still accomplished through their lenses as medical professionals. The contextual issues, including differences in the learning environment and learners’ needs or motivation for learning between institutions, were easily shared with peers without detailed explanations, and these were often perceived as a superior-inferior relationship. However, the cultural anthropologist had difficulties understanding such “common perceptions among medical professionals,” and upon inquiry, the presenter noticed differences in their views on culture. Some participants struggled to understand the distinctive aspects of CA, but, gradually, they were able to reflect on the two different contexts as relative rather than as having a superior-inferior relationship. The phrase “cultural relativism” has several variants, including the idea that individuals’ beliefs are relative to the social contexts surrounding them , and thaWhen I was teaching learners as an attending physician, I didn’t doubt that the hospital culture I was trained in was superior to the learners’ hospital culture. I think I made many mistakes with that. Even in the same country, there are cultural differences between hospitals. If I had known that there is no superiority or inferiority between cultures, I might have avoided such mistakes. (PP 2017)A sample response:The assigned books and discussion with the cultural anthropologist allowed the participants to understand that different contexts have different practices and that educators should consider the context in which teaching methods are applied. Furthermore, they recognized which teaching method was more appropriate in individual situations and that they should objectively analyze contexts. Throughout this session, it was evident that participants began to focus more on context.I realized that I focused too much on the methodology of this journal club as a cause of failure. The book ‘Situated learning: Legitimate peripheral participation’ widened my view. Learning should not be an independent activity, and people learn things better within social contexts. This perspective is what I should have had when I introduced a new project. I should also focus on the communities of practice to which the participants belong. For example, learning objectives should be based on the learners’ needs and relevant to their practice situations. I obtained a new viewpoint, “the participants’ situation or context,” by reflecting on my educational activities. (PP 2017)A sample response:Based on the reflection, the main role played by the cultural anthropologists in the sessions was to “reframe.” Inquiry-guided reflection by the cultural anthropologist effectively fostered the participants’ self-awareness of their cultural and social contexts by reframing their ways of thinking, beliefs, or positions. Therefore, participants discovered new insights through discussion with the cultural anthropologist.The anthropologist’s questions reminded me that what I consider obvious while talking to healthcare professionals may not be obvious for people in other specialty fields. I think the anthropologist lecturers in this session were instrumental in helping me think about understanding others. Because our learners are the others. (PQ 2015).We can provide the participants with something to shift them from the frame they are using now or give them another viewpoint. Maybe we can do nothing more. I think it would be great if they are encouraged through discussions with anthropologists who specialize in thinking about cultural contexts. (LI 2018).Interestingly, we [physicians] usually did not ask questions about our educational practice backgrounds, considering the socio-cultural contexts. However, that is important when introducing a new educational activity. The anthropologist asked how many members were in the department, about the venue of the journal club, and details of the hospital’s situation. As physicians, we sometimes ignore these points of view. (LI 2018)Sample responses:Fourteen out of 47 (29.8%) participants responded to an e-questionnaire sent out in 2019; all respondents indicated that the CA sessions impacted their educational activities and practice. In terms of how the CA sessions affected their education and clinical practices, the main theme synthesized in the thematic analysis was intense awareness of context.I used to focus on the abilities and qualities of the medical students and residents, but after attending the CA session, I also pay attention to other contexts such as the learners’ background and environment. (PQ 2016)I became more aware of the cultural context of the patients in the clinical practice. (PQ 2015)I started a new pre-clinical clerkship session for medical students in collaboration with cultural anthropologists inviting the concept of fieldwork with the learning objective of enhancing cultural context-awareness. (PQ 2016)Figure Sample responses:ment. PQ 016I becaThe authors found that the anthropologist’s questions fostered the learners’ self-reflection by deepening their insight about their educational practices with contextual perspectives. In the CA sessions, unexpected questions about cultural contexts were raised during the inquiry-guided reflection. By answering such questions, the participants’ awareness of “context” developed, and they began to focus on “culture” and “context” more than before. Faculty development activities should employ strategies that stimulate participants’ reflection , 29. HowAs the theme of “cultural relativism” suggests, the perspective of the cultural anthropologist had a significant impact on the participants’ realization that there is no superiority-inferiority relationship between the cultures of training hospitals. As to why the participants unconsciously perceived a superiority-inferiority relationship among the training hospitals, the results of previous studies on resident matching programs might have influenced their perceptions. Studies showed that resident applicants placed great importance on the quality of the education provided in the hospital , as wellThe result of this study showed that being aware of one’s unconscious perceptions of the superiority-inferiority relationship between cultures of training hospitals might be useful, especially when introducing educational practices from one context to another. Cultural relativism could act as a framework based on which educators reflect on their practices when they are unable to conduct these practices as successfully as they expect.The CA sessions in FD programs enabled an awareness of conditions and circumstances that are often too mundane for the participants to notice. The cultural anthropologist’s purely explorative questions led the participants to review their fixed ideas and norms from a different viewpoint. As previous research has shown, the approach of cultural anthropology has contributed to other specialties by reframing the questions of neuroscience research  and mitiIn terms of long-term impact, our study showed that CA sessions might have a certain effect on the participants as educators as well as physicians. The participants increased their awareness of the cultural contexts of not only their students but also their patients. Furthermore, the participants became interested in the discipline of anthropology itself through learning about its theories and concepts. A participant applied the concept of fieldwork to his educational activities by collaborating with the anthropologist. These results have not been described in previous studies about outcomes of FD , 36, andConsidering the above findings, we recommend that FD organizers work with cultural anthropologists to make FD programs more culturally contextual. Collaboration between medical education and cultural anthropology has enormous potential. As the next step, inviting cultural anthropologists into teaching practice settings may be useful, as they could analyze the teaching activities from an ethnographic perspective. Hence, external consultants for FD are recommended for assisting educational change . This stThis is a single institution study involving 47 graduates. Due to curriculum modification implemented in 2016, the impact of this session on the participants in 2015 and thereon in 2016 may not be the same. In the analysis of long-term impact, we should interpret the results carefully since the response rate was low. In addition, there is a methodological limitation with collecting the graduates’ e-questionnaire once in 2019, which meant that the duration after graduation was not equal for all graduates. Ideally, the graduates’ behavioral changes should be observed in their workplace with an immersion approach after graduation.We developed and analyzed a model of FD that includes CA sessions. Our results showed that medical education could learn a lot from CA, as working with individuals from different cultural backgrounds requires self-reflection and personal awareness. Furthermore, our results implied that more collaboration between medical education and CA would enrich our medical education activities, including FD.Additional file 1. Post-session questionnaire.Additional file 2. Post-program questionnaire.


Effects of electropolishing on the quality and the Raman response of the grown graphene layers are studied by microtexture polarization analysis. The obtained data are compared with the Raman signal of graphene after transfer on glass substrate revealing the complex interaction of graphene with the Cu substrate.Graphene films were grown by chemical vapor deposition on Cu foil. The obtained samples were characterized by Raman spectroscopy, ellipsometry, X-ray photoelectron spectroscopy and electron back-scatter diffraction. We discuss the time-dependent changes in the samples, estimate the thickness of emerging Cu Since its discovery, graphene has been viewed as promising candidate for novel optoelectronic devices due to its extraordinary ballistic transport over micron lengths and large carrier mobility ,2,3 combThese optimistic prospects are, however, confronted by plenty of technological difficulties. The main problem is the challenge to achieve growth of nondefective graphene over sufficiently large areas. An important prerequisite for this goal is to have reliable backfeed by a standardized characterization of the obtained graphene directly on the metal substrate, which has to account for the whole complexity of graphene’s interactions with the metallic substrate/stabilizer, its possible oxides and gases/liquids which can come in contact with it during processing. As the commonly used CVD substrate is commercially obtained polycrystalline Cu foil, intensive research work was devoted to study the impact of Cu surface morphology and grain orientation on the grown graphene ,9. It haG) and 2D band (ω2D) of graphene is commonly used for quantifying strain and doping effects [Raman spectroscopy is a preferred characterization method for the study of graphene and the novel graphene-like 2D materials, as it is nondestructive, achieves high resolution with relatively simple commercially available setups and provides valuable information on the defect density, number of layers and their mechanical state. Most importantly, this technique reveals details of the electronic structure via the electron–phonon coupling and the rich single- and double-resonance phenomena characteristic for the Raman response of carbon-based materials. However, the versatility of this method is inherently connected to its main disadvantage—complex data interpretation—because of the need to account for the variability of many parameters. For instance, the combined plot of frequencies of the G-band , ellipsometry and electron back-scatter diffraction (EBSD). We find that immediately after CVD growth, the strong Cu-graphene coupling is still detectable in the XPS spectra in addition to its manifestation in the Raman spectra. In order to find a measure for the relaxation of this coupling with time, we use ellipsometry to estimate the thickness of the emerging Cu3PO4 solution with the polished copper foil as the anode and a copper plate as the counter electrode at polarization voltage of 1.98–2.02 V at room temperature. Similar to [Both raw (as supplied) and electropolished copper foil  was used as the growth substrate. Electropolishing was performed in 85% concentrated Hmilar to , the ano4 flow combined with 200 sccm H2 flow, were introduced for 30 min into the reaction chamber for the growth stage. Afterwards, the sample was quenched to 300 °C at 15 °C/min in hydrogen/argon atmosphere. Cooling to room temperature was done in high-purity argon atmosphere. The grown samples were kept in clean room environment prior to characterization and during aging.The graphene was grown in a cold wall Plasmalab System 100 research reactor of Oxford Instruments using low-pressure chemical vapor deposition (LPCVD). Low-pressure growth (4000 mTorr) was preferred because it yields lower graphene nucleation density  and the −1 spectral width. The 632.8 nm line of a He/Ne-laser was applied for excitation using a 600 gr./mm grating, the absolute accuracy being about 1 cm−1. The laser beam was focused on a spot of about 1–2 μm in diameter on the sample surface using microscope optics with an objective of 50× magnification. The Raman measurements were performed on the graphene-coated Cu foils and after PMMA-assisted transfer onto glass substrates. The measured frequencies were calibrated by means of the T2g vibration of silicon at 520.7 cm−1 and the lines corresponding to the stretching vibrations of oxygen and nitrogen at 1556 and 2331 cm−1, respectively, which are contained in the spectra.Raman spectra were measured on a Jobin Yvon Labram HR-800 spectrometer with a CCD detector and entrance slits set to 1.5 cm−8 Pa and a total instrumental resolution 1 eV. The binding energies (BE) were determined utilizing the C 1s line (from an adventitious carbon) as a reference with energy of 285.0 eV. The accuracy of the measured BE was 0.2 eV. The photoelectron lines of constituent element on the surface were recorded and corrected by subtracting a Shirley-type background and quantified using the peak area and Scofield’s photoionization cross-sections. The decovolution of spectra where is necessary was done with XPSPEAK41 software.The X-ray photoelectron spectroscopy (XPS) studies were performed in a VG Escalab MKII electron spectrometer using AlKα radiation with energy of 1486.6 eV under base pressure 10The ellipsometry measurements were performed using a Woollam M2000D rotating compensator spectroscopic ellipsometer with a wavelength range from 193 nm to 1000 nm in reflection mode. The involved data acquisition and analysis software was CompleteEASE 5.10 J. A. Woollam Co., Inc.  The spectroscopic ellipsometry data of Ψ and Δ were taken at room temperature at angles of incidence 50°, 60° and 70°.® 6–60) on specimens tilted at 70° with 5 kV acceleration voltage in order to find the appropriate surface. The high tilt angle and the lower acceleration voltage ensures the low penetration of the electrons, i.e., small interaction volume close to the specimen surface.The EBSD measurements were performed on a Thermo Fisher Scios 2 FIB/SEM instrument equipped with BRUKER e-FLASH FS EBSD detector with acceleration voltage 15 kV. Oxygen elemental distribution maps were measured by EDS (BRUKER XFlash2O). When freshly taken out of the CVD chamber, the entire area of the foil looks similar to the left part of the left image with corresponding typical Raman spectrum as the “blank”-marked trace in 2O bands at 149, 216, 525 and 640 cm−1 [A typical case of time evolution of a graphene-coated Cu (Gr/Cu) foil is depicted in 640 cm−1 ) while oTo obtain a more quantitative picture of the oxidation development with time we performed a detailed characterization with different experimental methods on 3 samples with different storage times after CVD growth (6 months for the second and 20 months for the third sample) and with consecutive increasing density of reddish coloring. These samples will be referred to as “6 months” and “20 months” in what follows. The first sample referred to as “fresh” was examined immediately after graphene growth.A detailed XPS examination of the three samples was performed. Microscopic images and fitted C1s spectral bands are displayed in 2 graphene at 284.2–284.4 eV. Interestingly, only for the “fresh” sample there is a peak at around 284.9 eV with much higher intensity than that of the conventional sp2 peak. This is consistent with the assumption that immediately after the growth process, the major part of the graphene layer is still strongly coupled to the copper substrate via exchange interactions between the Cu valence electrons and the carbon C1s core hole which yields a C1s sp2 signal at 284.75 eV [3 sites as well as defects in the graphene honeycomb lattice and C-H bonds whose signal is located between 285.0 and 285.5 eV. In the C1s spectra of the other samples these three contributions form a separate peak which is denoted as sp3 peak. Several other satellite peaks due to oxygen-containing groups are also detected. The peak at around 286 eV belongs to C-O/C-OH moieties, that around 288 eV to O=C-O bonds and carbonate groups, and that at ≈283 eV to sp1 type carbon bond in C2Cu. As expected, the oxygen-related peaks are more intense for longer storage time.The C1s spectra are complex and their most intense component is typically the C1s peak of sp84.75 eV . In our 2O. As expected, this oxidation-related peak has maximum intensity for the sample with the longest storage time of 20 months. There is a shoulder at the higher binding energy side which is accounted for by the third peak at around 532 eV. The appearance of this shoulder is related to formation of C-O/C-OH and C=O bonds. From the Cu 2p photoelectron spectra in 0  and Cu1+ (Cu2O) for all studied samples. The same is evidenced by the Auger CuLMM line  values from our measurements on the three samples. As can be appreciated from −1 for ωG and around 2660 cm−1 for ω2D. This indicates significant compressive strain and, on the other hand, ω2D is additionally upshifted due to the Fermi velocity reduction which is significant for our low excitation laser energy of 1.96 eV [G, ω2D) values around the zero-strain point at  cm−1 [2O bands, which are completely missing in the “fresh”-sample spectra, display considerable intensity in the spectra of the two older samples. The  values for sample “20 months” are more scattered with some points implying a transition to tensile strain imposed by local Cu2O aggregations [Comprehensive spatially resolved Raman measurements were performed on the three samples. Representative spectra from this characterization are presented in ed shift  and a shed shift . As thered shift ,29, we c 1.96 eV . As the .3) cm−1  with somegations . It shou−1 (compared to ~29 cm−1 before transfer). In From the collected Raman data, we obtained values for the peak intensity ratio of the 2D and G band I(2D)/ I(G) ranging from 2 to 2.8 which can, however, be impacted by the graphene-Cu coupling. In the 2O patches on this grain. Still, we regard oxygen etching of graphene edges and grain boundaries as the main mechanism ensuring a slowly advancing oxidation of the substrate [The oxygen content in the 3 examined samples as calculated from the XPS spectra is given in ubstrate . As was ubstrate , vacuum ubstrate  at even 2O layer by means of spectroscopic ellipsometry (SE) for the fresh and the 6-month-old sample. There are only few ellipsometric studies of as grown graphene on metals [To examine the weakening of the Cu-graphene coupling upon oxygen intercalation it is instructive to find a measure for the separation of the graphene layer from the Cu surface. We therefore estimated the thickness of the formed Cun metals , while mn metals ,33.As there is increasing evidence that the Cu oxidation rate beneath the graphene depends on the surface orientation of the copper grains , we exam2O participating in the model with optical constants taken from Palik’s Handbook of Optical Constants [2O average thicknesses were obtained: fresh sample: ≈0 nm; sample “6 months” low-oxidation region—0.5 nm; and sample “6 months” high-oxidation region—6.5 nm. From the strong variation of the oxide thickness and the obvious coincidence of the boundaries of differently colored regions with the Cu grain boundaries, as can be appreciated from the microphotographs in The ellipsometric data for Ψ and Δ were modelled within a model consisting of two consecutive layers on top of an oblique substrate which is the copper foil. The first layer is Cuonstants   orientation exhibit the lowest oxidation rate. Grains with orientation near to (111) display an intermediate affinity towards oxidation. Surprisingly, these results are the opposite to those reported in [orted in , where f near to 1 displayorted in , the parorted in  on polycElectropolishing has been frequently applied as part of the Cu foil pretreatment to lower its surface roughness and to achieve better quality of the grown graphene ,38. Here−1 after transfer onto glass as compared to ~15 cm−1 for unprocessed Cu foil). It is also found that the Cu-graphene coupling makes the intensity ratio of the 2D and G band less informative for determination of the number of layers and it becomes a reliable parameter only after the transfer on isolating substrate.From the Raman spectra in To further illustrate the effect of electropolishing, we visualize the domains in the graphene layers grown on an electropolished and on a nonprocessed Cu foil by means of the nematic liquid crystal (LC) E7. This is possible due to the interaction of two-dimensional graphene-honeycomb structure with the LC benzene rings through π–π electron stacking (binding energy of −2eV) which induces a planar alignment of the LC, thus creating LC pseudonematic domains (PNDs) at the graphene surface . As the 2O as a way for its relaxation. Correlating results were obtained from Raman and XPS characterization, ellipsometry and the EBSD technique. We find strong Cu-graphene coupling on the nonoxidized Cu grains and gradual release of this coupling along the “strain” line without significant doping effects. Our ellipsometric results provide an estimate for the typical oxide layer thickness beneath the graphene after 6 months of sample aging, which varies from fractional parts of a nanometer to 6–7 nm. We confirm that in the case of polycrystalline foil oxidation to Cu2O of graphene-coated copper is grain-selective, with (001)-oriented grains being most resilient and (011)-oriented ones most susceptible to oxidation. Correspondingly, the strong Cu-graphene coupling and the strain in the graphene lattice is released very nonuniformly and over different time scales.We investigated single-layer graphene-coated Cu foils with respect to the Cu-graphene coupling and Cu oxidation to CuFrom a visualization of graphene grains by means of coating by liquid crystal, it is found that graphene grown on electropolished Cu foil exhibits larger grains with lower defect density. It is found that the Cu-graphene coupling alters the frequency and the intensity ratio of the G and 2D band, and a determination of the number of graphene layers from Raman spectral parameters should preferably be made after transfer on isolating substrate.


TARDBP gene) is strongly linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). From the identification of TDP-43 positive aggregates in the brains and spinal cords of ALS/FTD patients, to a genetic link between TARBDP mutations and the development of TDP-43 pathology in ALS, there is strong evidence indicating that TDP-43 plays a pivotal role in the process of neuronal degeneration. What this role is, however, remains to be determined with evidence ranging from gain of toxic properties through the formation of cytotoxic aggregates, to an inability to perform its normal functions due to nuclear depletion. To add to an already complex subject, recent studies highlight a role for TDP-43 in muscle physiology and disease. We here review the biophysical, biochemical, cellular and tissue-specific properties of TDP-43 in the context of neurodegeneration and have a look at the nascent stream of evidence that positions TDP-43 in a myogenic context. By integrating the neurogenic and myogenic pathological roles of TDP-43 we provide a more comprehensive and encompassing view of the role and mechanisms associated with TDP-43 across the various cell types of the motor system, all the way from brain to limbs.TAR DNA-binding protein 43, mostly referred to as TDP-43 (encoded by the  TARDBP gene, establishing the causal involvement of this protein in disease pathogenesis. The vast majority of the ALS-causing mutations are missense mutations located in the C-terminal region , and limbic-predominant age-related TDP-43 encephalopathy (LATE) . TDP-43 l region . ALS is l region . ALS is LS cases . ClinicaLS cases . The cliLS cases . In 40–5LS cases . In the LS cases . Recentlneration .TARDBP gene are mainly associated with ALS but these mutations only contribute to pathology in a small fraction of all ALS cases. In FTD and AD, mutations in the TARDBP gene are rare although the TDP-43 pathology is present in a quite large proportion of all cases , hnRNPA1 and hnRNPA2B in multisystem proteinopathies (MSP)  . Despitemyopathy . Over thmyopathy .The aim of this review is to provide an up-to-date overview of the many different physiological and pathophysiological roles of TDP-43, with a special focus on its tissue-specific functions. Furthermore, we will highlight the role of TDP-43 in muscle tissue and the current findings relating to this less well-studied facet of TDP-43 proteinopathies.TARDBP gene , containing glycine and uncharged polar amino acids (a.a.). This PLD is an important factor in regulating many protein-protein interactions and the liquid-liquid phase separation (LLPS) behavior of TDP-43 . AlthougMislocalization of TDP-43 in the cytoplasm is typically observed in pathological conditions, hence understanding the regulation of its normal subcompartmentalization is of great importance. TDP-43 is mainly localized in the nucleus but continuously shuttles to and from the cytoplasm . Part ofAll in all, the proper regulation of the nucleocytoplasmic trafficking is important for allowing TDP-43 to exert its proper physiological functions via active re-uptake of TDP-43 protein to the nucleus, but also to maintain steady-state levels.The specific functions of TDP-43 are mainly studied in the central nervous system (CNS) as TDP-43 is a common pathological hallmark in patients suffering from ALS, FTD, and other neurodegenerative diseases. As can be expected, taking into account the complexity of the protein structure, the different protein species, and the variable subcellular localization; TDP-43 is a protein that participates in many cellular and molecular processes.Drosophila melanogaster (D. melanogaster) homolog, referred to as TBPH, results in reduced eclosion and shorter lifespan  and proteins . The facA well-described function of TDP-43 is its regulatory role in RNA splicing and this through either direct interaction with the RNA itself, or interaction with proteins involved in the splicing machinery . In geneD. melanogaster, TBPH is responsible for the regulation of the pre- and postsynaptic Discs large protein (Dlg) levels in muscles and motoneurons by binding the Dlg mRNA and in this way regulating its splicing patterns. Of note, Dlg has an important role in the formation of neuromuscular junctions and organization of the glutamate receptors on muscle membranes, suggesting an important role for TBPH in muscle tissues  and gain-of-function (GOF) of TBPH results in high upregulation of snoRNAs (NEAT1) and metastasis associated lung adenocarcinoma transcript 1 (MALAT1). NEAT1 is involved in the structural formation of paraspeckles, whereas MALAT1 acts as a splicing regulator by modulating the distribution of splicing factors in paraspeckles and the phosphorylation of splicing factors (in vitro and in vivo experiments TDP-43 interacts with the microRNA-1 (miR-1) family that promotes differentiation of striated muscle progenitors. TDP-43 negatively regulates miR-1 activity by limiting its interaction with the RNA-induced silencing complex which is required for miRNA to perform its activity. Overexpression of human TDP-43 in mice models decreases the activity of the miR-1 family which in turn increases the translation of the miR-1 targets, IGF-1 and HDAC4. HDAC4 is associated with muscle denervation and blocks innervation. This indicates that in steady-state conditions the miR-1 targets are suppressed, therefore the interaction between TDP-43 and miR-1 is required to maintain homeostasis of the miR-1 targets, and subsequently mediate muscle development and preserve balance in adult muscle tissues , small nucleolar RNA (snoRNA) and long non-coding RNAs (lncRNA). The regulation of miRNAs occurs either through direct binding of TDP-43 to the primary miRNAs in the nucleus and their precursors in the cytoplasm, or by a protein-protein interaction between TDP-43 and the nuclear Drosha and cytoplasmic Dicer complexes. The association between TDP-43 and the Drosha or Dicer complexes is mediated by several RNA species and the CTD . The put snoRNAs . In the  factors . In musc tissues . Furthermyotubes .CAMTA1 and DENND4A  (PAR)-binding motifs (PBM), PBM1 and PBM2, which interact with the PAR biopolymer, a sugar covalently linked to proteins. This binding results in the initiation of LLPS and the formation of stress granules . SecondlThe membraneless organelles can originate via LLPS in either the nucleus or the cytoplasm, resulting in varied functions and properties . TherefoTDP-43 undergoes phase separation in response to various stressors into reversible nuclear granules with cytoprotective functions such as interrupting DNA transcription and RNA processing which can be resumed when the stress has passed.NEAT1 lncRNA, whereas RRM2 suppresses LLPS by binding tRNA in vitro. During stress conditions NEAT1 lncRNA is upregulated as a defense mechanism against stress and pathological conditions in neurons (These membraneless nuclear granules are defined as nuclear bodies (NBs) as they are visible with a microscope, collect specific nuclear factors and the components shuttle between the granule and the surrounding nucleoplasm. Cell-based studies indicate the relevant, but distinct roles, of the RRMs in the formation of these NBs. The different functions can probably be attributed to the interaction with diverse RNAs, since RRM1 suppresses LLPS by indiscriminate RNA binding and conversely is primarily involved in the formation of LLPS by binding  neurons .NEAT1, a long non-coding RNA with a strong binding affinity for TDP-43. NEAT1 has a short and a long isoform, NEAT1_1 and NEAT1_2 respectively. Both transcript isoforms are elements in the structural integrity of paraspeckles, however, only NEAT1_2 is essential in the formation of the paraspeckles  are cytoplasmic membraneless organelles formed during stress conditions, e.g., oxidative stress, osmotic stress, temperature and pH changes. SGs contain mainly non-translating mRNAs and, translation and transcription factors . In thesThere are some conflicting opinions as to whether TDP-43 is a crucial component of these SGs. Several studies show the presence of endogenous TDP-43 in SGs . ColombrNEAT1 lncRNA which reduces the chance to form cytoplasmic SGs and therefore prevents a build-up of cytoplasmic TDP-43  are cytoplasmic mRNP granules consisting of non-translating mRNA, RNA-binding proteins and other proteins. P-bodies are present in physiological conditions but can be upregulated during stress conditions to regulate translation and mRNA degradation . It is kIn muscle tissue there is a specific counterpart of the stress granules, referred to as myo-granules. Skeletal muscles are required for movement and for the body’s structural integrity, but also have important endocrine and metabolic functions . The musTARDBP mutations. The key hallmarks of TDP-43 proteinopathy are cytoplasmic TDP-43 accumulations, accompanied by a nuclear depletion as cytoplasmic TDP-43 “captures” nuclear TDP-43. Three possible hypotheses giving rise to TDP-43 proteinopathy are still debated: a gain-of-function – novel gain of toxic function – or loss-of-function  which are associated in both positive and negative ways with TDP-43 pathology. The best studied PTMs are phosphorylation and ubiquitination. Phosphorylation sites on threonine (T), serine (S) and tyrosine (Y) residues, are scattered throughout the entire protein. Ubiquitination is another well-studied PTM of TDP-43 and is necessary for the clearance and degradation of soluble full-length TDP-43 via the Ubiquitin proteasome system (UPS). A blockage of this system triggers the formation of insoluble and immobile TDP-43 aggregates and the accumulation of ubiquitylated TDP-43. Full-length TDP-43 aggregates are associated with the presence of K48- and K63-linked ubiquitin chains. Proteins with K48-linked polyubiquitin chains are degraded by UPS-dependent mechanisms, while K63-linked chains target proteins to the autophagic pathway. The fact that full-length TDP-43 aggregates are labeled by both types of ubiquitin chains and inhibition of autophagy prevents the clearance of aggregated TDP-43 indicates an important role for the autophagy pathway in the removal of TDP-43 aggregates in conjunction with UPS-mediated degradation. Impairment of the UPS or autophagy in patients with ALS provokes the accumulation of misfolded and insoluble TDP-43 and prevents the clearance of these aggregates . Moreovein vitro aggregation propensity of TDP-43, hypothesized by an increased net charge due to acetylation and hence electrostatic repulsion  present in the NLS and the RRMs are all subject to oxidation, however, it is thought that only oxidation of C173 and C175 in RRM1 causes protein aggregation. This oxidation-induced aggregation could be strengthened by oxidation of the residues in the RRM2, C198 and C244 . Upon oxin vitro experiments mutations in the PBMs impair LLPS and thus cause the formation of irregular solid structures ation (PARylation), is described as modulating the LLPS behavior of TDP-43 through the two PBMs located within the NLS . PARylatructures . In NSC3toxicity .A final PTM of TDP-43 is SUMOylation. SUMOylation is a three-step enzymatic cascade in which small ubiquitin-related modifiers (SUMOs) are conjugated, via an isopeptide bond, with lysine residues in a consensus motif. In TDP-43, K136 and the SUMO-interacting motif (a.a.: 106–110), both located within RRM1, are especially susceptible to this enzymatic process  Maurel . SUMOylapost-mortem tissues derived from ALS patients  of ∼25 and ∼35 kDa are prominent species observed in human patients . Severalpatients .Overexpression of the ∼25 and ∼35 kDa fragments in HEK293T cell-lines indicates that the ∼25 kDa fragments are more susceptible to form numerous and large aggregates, mainly located in the cytoplasm . MoreoveThe same residues in the NTD important for physiological dimerization and oligomerization contribute to TDP-43 pathology as well. In pathological conditions, the first 10 residues are capable of sequestering nuclear full-length TDP-43 into cytoplasmic inclusions and therefore trigger aggregation. This implies that the first residues have both LOF and GOF capacities . Along tFinally, the NTD, together with RRM1, are able to form an ∼86 kDa dimeric form of TDP-43 through intermolecular interactions. Overexpression of this dimer in HEK293T cells triggers the production of aggregates and therefore may be involved in seeding the formation of pathological aggregates . AltogetAggregation is one of the most typical hallmarks of TDP-43 proteinopathies. The aggregate formation can be triggered by many routes, some already discussed in the previous sections like post-translation modifications, the formation of aberrant TDP-43 species, oligomerization, aberrant LLPS and dysfunction of several cellular processes . TypicalInterestingly hyperexcitability causes the formation of TDP-43 isoforms lacking the CTD. These isoforms are located within the cytoplasm where they aggregate and sequester nuclear TDP-43. Besides the formation of aggregates, these isoforms lack their physiological function such as autoregulation and splicing .However, neither the CTD nor the NTD by itself harbors the full ability to induce the formation of aggregates. The RRMs are important in the formation of these oligomers and subsequently aggregates as shown by TDP-43 constructs in which the RRMs are replaced by monomeric EGFP. These synthetic proteins display a higher ratio of monomer versus oligomer over time, showing the synergistic function of the RRMs on the aggregation induced by the CTD and NTD .Based on the above data, it appears that alterations to the protein structure sensitize the protein to form insoluble structures.in vitro and in vivo studies have already been described and are reviewed by The CTD of TDP-43 is also referred to as PLD as it contains a segment enriched in glutamine and asparagine residues. This region is used to regulate the self-interaction, aggregation and amyloidogenic propensities of TDP-43 . In prioMost of the physiological and pathophysiological processes of TDP-43 are studied in the context of neurodegenerative diseases. Interestingly, strong evidence for a pathological role of TDP-43 in skeletal muscle is provided by the observation that TDP-43 aggregates, reminiscent of the aggregates in neurons, accumulate in patients with sporadic inclusion body myositis (sIBM), IBM associated with Paget’s disease of bone and frontotemporal dementia (IBMPFB), oculopharyngeal muscular dystrophy (OPMD), distal myopathies with rimmed vacuoles (DMRV) and myofibrillar myopathies, including desminopathy and myotilinopathy .GNE gene. This hereditary form is also known as DMRV. The first clinical manifestation is an alteration in gait patterns and at a later stage the patients become wheelchair dependent. This disease affects younger patients when compared to sIBM, as the average disease onset is 26 years of age. The muscular pathological hallmarks are akin to those observed in sIBM, but a few differences are noted. First, in DMRV the tibialis anterior muscle is the most affected, while the quadriceps muscles are spared. Second, in DMRV there is no lymphocytic inflammation and there are no mitochondrial-disease markers present. The rimmed vacuoles stand as a common muscle pathological finding between sIBM and DMRV , however, muscle weakness is generally present. Typically, in all myofibrillar myopathies, myofiber degeneration starts around the Z-disc of the sarcomere . Furtherectively .The presence of TDP-43 aggregates in the above-described myopathies strongly supports a broader pathogenic and primary role of TDP-43 in muscle degeneration. Similar to the physiological processes, the pathological processes related to TDP-43 pathology are described in cell-lines and mostly interpreted and confirmed in a neuronal context. In diseased conditions, TDP-43 typically accumulates in the cytoplasm and this accumulation is accompanied by a nuclear depletion. This situation makes the understanding of the pathogenic mechanism complicated as the cytoplasmic accumulation, and hence the aggregation, point toward a gain-of-toxic-function, but in contrast the nuclear depletion may indicate a loss-of-function . This onD. melanogaster. TBPH knock-down during muscle development, larval stage and adulthood cause age-related motor abnormalities. These abnormalities involve alterations in the overall motor activity, mean speed and the total distance travelled  and abnormal myofibers. The mean size of the myofibers is smaller than that in the non-transgenic mice, especially in the aggregate-containing myofibers. In addition, the inclusions are enriched in proteins required for protein homeostasis or the UPS pathway and the muscle fibers have autophagic and mitochondrial abnormalities. These data indicate that the aggregates induced by overexpression are not completely able to induce all the pathological aspects of sIBM, since the infiltration of inflammatory cells is lacking. The ∼35 kDa fragments in these transgenic mice may indicate that the ratio of ∼35 kDa over ∼25 kDa fragments is higher in muscle tissues when compared to neuronal tissues . Interesin vivo experiments in mice that show that cytoplasmic TDP-43 is increased in injured muscle tissue where it forms amyloid-like assemblies, called myo-granules  and ALS patients alike. This hints toward a need to broaden the disease spectrum of TDP-43 proteinopathy to include, not only neurodegenerative diseases, but also myopathies, further highlighting the need for further research of TDP-43 in a myogenic context.BaD coordinated the manuscript and approved the final version of the manuscript. LV and EB wrote the initial draft. PE, NS, BoD, JD, and BaD critically reviewed and edited the manuscript. LV created the figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The nuclear RNA-binding protein TDP-43 forms abnormal cytoplasmic aggregates in the brains of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients and several molecular mechanisms promoting TDP-43 cytoplasmic mislocalization and aggregation have been proposed, including defects in nucleocytoplasmic transport, stress granules (SG) disassembly and post-translational modifications (PTM). SUMOylation is a PTM which regulates a variety of cellular processes and, similarly to ubiquitination, targets lysine residues. To investigate the possible regulatory effects of SUMOylation on TDP-43 activity and trafficking, we first assessed that TDP-43 is SUMO-conjugated in the nuclear compartment both covalently and non-covalently in the RRM1 domain at the predicted lysine 136 and SUMO-interacting motif , respectively. By using the SUMO-mutant TDP-43 K136R protein, we demonstrated that SUMOylation modifies TDP-43 splicing activity, specifically exon skipping, and influences its sub-cellular localization and recruitment to SG after oxidative stress. When promoting deSUMOylation by SENP1 enzyme over-expression or by treatment with the cell-permeable SENP1 peptide TS-1, the cytoplasmic localization of TDP-43 increased, depending on its SUMOylation. Moreover, deSUMOylation by TS-1 peptide favoured the formation of small cytoplasmic aggregates of the C-terminal TDP-43 fragment p35, still containing the SUMO lysine target 136, but had no effect on the already formed p25 aggregates. Our data suggest that TDP-43 can be post-translationally modified by SUMOylation which may regulate its splicing function and trafficking, indicating a novel and druggable mechanism to explore as its dysregulation may lead to TDP-43 pathological aggregation in ALS and FTD.The online version contains supplementary material available at 10.1007/s12035-021-02505-8. TDP-43 is an ubiquitous RNA-binding protein (RBP) localized in the nucleus where it mainly regulates splicing but, by shuttling between the nucleus and the cytoplasm, it also controls RNA metabolism at different levels, including miRNA biogenesis, mRNA transport, stability and translation . In the The PTM SUMOylation consists in the conjugation of different small ubiquitin-related modifiers  to specific lysines of the target protein through different steps which, similarly to ubiquitination, involve SUMO-E1 activating enzyme, SUMO-E2 conjugating enzyme UBC9), and SUMO-E3 ligases. SUMOylation is reversible by means of the SENP proteases that cleave SUMO from the target proteins . SUMOyla, and SUMSUMOylation was already studied in association to neurodegenerative pathologies, such as Alzheimer’s, Parkinson’s and Huntington’s diseases, where this PTM may favour or prevent abnormal protein aggregation . In ALS,TDP-43 was initially identified by proteomic analyses to be SUMO-2-modified in response to heat shock in HeLa cells  and in tIn this study, we therefore aimed to better characterize TDP-43 protein SUMOylation and to study its impact on TDP-43 splicing activity, subcellular distribution and aggregates formation in different experimental cell models. We also modulated SUMOylation using the deSUMOylating cell-permeable SENP1-derived peptide TS-1 or the SUMOylation inducer KCl and investigated if NCT could represent a potential druggable target in TDP-43 proteinopathies.www.abcepta.com), GPS-SUMO  µm2, > 1 µm2) after cell exposure to the TS-1 peptide Below is the link to the electronic supplementary material.


This paper presents the effect of deposited graphene oxide coating on fatigue life of austenitic steel 1.4541 at 20 °C, 100 °C, and 200 °C. The study showed a decrease in the fatigue life of samples with a deposited graphene oxide layer in comparison with reference samples at 20 °C and 100 °C. However, an increase in fatigue life of samples with a deposited graphene oxide layer in comparison with reference samples occurred at 200 °C. This relationship was observed for the nominal stress amplitude of 370 and 420 MPa. Measurements of temperature during the tensile failure of the sample and microfractographic analysis of fatigue fractures were performed. Tests have shown that graphene oxide deposited on the steel surface provides an insulating layer. A higher temperature of the samples with a deposited graphene oxide layer was observed during fracture compared to the reference samples. Improvements in mechanical properties after the addition of graphene or its derivatives to composites have resulted from many research projects ,2,3,4. TLi et al.  showed tNajfi et al.  conducteTaking into account the increase in fatigue life of composites containing graphene ,4,5,6,7 Currently, the most well-known method for depositing graphene on the surface of (mainly) copper is chemical vapor deposition (CVD) ,9,10. HoHowever, to date, fatigue life tests at elevated temperatures  of flat specimens made of 1.4541 steel with a deposited graphene oxide (GO) layer have not been performed. Therefore, taking into account the operation of many machine parts and structural elements at elevated temperatures, the aim of this work was to determine the effect of a deposited graphene oxide layer on mechanical properties.Static tension tests and fatigue life tests were performed on flat paddle specimens of 5 mm thickness made of 1.4541 steel according to the following designations: BM—base material (as supplied) , and BM p < 0.045; S < 0.015; T < 0.70; and Fe = bal. Test specimens were cut by the Water jet method from a 2000 × 1000 × 5 mm3 metal sheet perpendicular to the rolling direction. The inner edges of the specimens were subjected to a milling process as shown in The dumbbell specimens  were madGraphene oxide (GO) flakes dispersed in water were obtained from flake graphite by a modified Hummers method . The concentration of graphene oxide water dispersion was 10 g/L. Flake size was 3–10 µm. The detailed synthesis method was described previously in ,12,13.In the first step of graphene oxide deposition on BM + GO samples, surface activation and cleaning with RF (radio frequency) plasma was performed using a Plasma Prep III device  a. The foStatic tensile and fatigue tests were performed using an Instron 8862  electromechanically driven testing machine with a force range of ±100 kN a coupledFatigue tests were carried out for different stress levels Rm = 0.6, 0.7, 0.8, and 0.9 at maximum stress levels of 370, 420, 500, and 540 MPa respectively . The cycDuring static and fatigue measurements at 20 °C, the temperature distribution was performed using a FLIR  model SC6000 thermal imaging camera c. The caGraphene oxide deposited on screw was investigated by scanning electron microscopy (SEM) using a Quanta 250 FEG SEM, FEI, Hillsboro, OR, USA. SEM images were acquired using a backscattered detector  with an accelerating voltage of 2–5 kV for GO and 5–10 kV.The measurements of the changes in surface roughness of stainless steel 1.4541 as a result of plasma cleansing and GO deposition were made using a confocal microscope Zeiss LSM 700 . The experimental parameters were laser wavelength 405 nm, pinhole 0.5 AU (Airy Unit), and gain 416 nm. Five measurements for each sample were made.−1 with a resolution of 4 cm−1.Measurements of the spectra of GO in the infrared range were made using the FTIR technique in reflection mode. The measurements were made to confirm GO deposition on the stainless steel 1.4541 surface (BM). For FTIR measurements, a Thermo Scientific Nicolet iN10 with a DTGS detector  was used. Spectra were recorded nine times in different locations with 126 scans in a range of 675–4000 cm1) and 32.8% (Rz2) was observed . −1, 2976 cm−1, 1745 cm−1, 1636 cm−1, 1238 cm−1, and 1156 cm−1 corresponding to the vibrations of the C–O bond, C=C bond, C=O bond, aliphatic C–H, and O–H bond, respectively. This shows that the surfaces examined have oxygen-containing groups in the BM + GO sample, which proves successful deposition of the GO as compared to the BM sample spectrum.The GO samples (BM + GO) have absorption peaks at 3289 cmStatic tensile tests were performed for three temperature levels 20, 100, and 200 °C. The mean results of the five trials along with the standard deviations are listed in The maximum temperature during the static tensile test at the point of breakage was 9.4% lower for BM specimens as compared to BM + GO specimens. It was also observed that after tensile failure, the cooling process of BM + GO samples was faster . The recmax levels of 0.9, 0.8, 0.7, and 0.6 Rm . The material strength limit Rm was determined experimentally and was 600 MPa  were performed at σ 600 MPa . For eleAveraged results of fatigue life tests (from four measurements) in the temperature range of 20, 100, and 200 °C were used to prepare the Wohler diagram .No significant effect of graphene oxide deposition on fatigue life was observed at 20 °C. Test results obtained at 20 °C, presented as curves in max of 370 and 420 MPa are presented. At 20 °C and 100 °C, an increase in the fatigue life of BM specimens compared to BM + GO was observed. However, at 200 °C the opposite situation was observed. There was an increase in the fatigue life of BM + GO samples compared to BM samples. In general, the study showed that as the temperature increases, the fatigue life of BM specimens decreases, in contrast to BM + GO specimens. This may be due to the change in the structural properties of graphene oxide flakes as a result of temperature changes and variable loads.In During the fatigue life tests, a higher temperature of 3–26% of the BM + GO specimens was also observed in comparison with BM specimens during final failure. The deposited graphene oxide layer provided an insulating layer, which made the sample heat up more slowly .Microfractography of the surface of fatigue fractures of BM specimens and BM + GO specimens observed by scanning electron microscopy SEM revealed the sites of crack initiation a,e, steaMicrofractography analysis of the fatigue fractures showed a shiny zone (2) around the fracture initiation focus in the BM + GO specimens. The next stage of sample failure was the transition zone (4) separating the fatigue zone (3) from the residual zone (5), where rapid specimen damage occurred . On the The final stage of damage occurred as a consequence of the separation characteristic for static cracking d,h. TherThe deposited graphene oxide flakes formed a uniform surface on the BM + GO samples a. In theThe graphene oxide coating applied on flat samples made of 1.4541 steel had a positive effect on surface smoothing. The arithmetic mean of the profile deviation  for BM + GO was 32.8% lower than for BM samples (without GO coating) . It was As the test temperatures of 20, 100, and 200 C increased along with effects on the mechanical properties of BM and BM + GO specimens, there was a typical decrease in yield stress, short-term tensile strength, and Young’s modulus. The GO coating at elevated temperature acted as a thermal barrier causing an increase in the temperature of the samples at the break point of 9.4% .max = 540 MPa led to an 8% reduction in the life difference of BM and BM + GO specimens. On the other hand, the fatigue cracking of the specimens at the lowest fatigue cycle stress σmax = 370 MPa developed in a stable manner, and thus the difference in durability was 77%.Fatigue tests of the samples for the assumed maximum cycle stress levels confirmed insignificant differences in the durability of BM and BM + GO samples at 20 °C . A decremax = 370 and 420 MPa. Undoubtedly, this is a surprising result, but it is probably related to the change in properties of the GO layer  and concerns a more favorable system of residual stresses occurring in the GO layer.The decrease in fatigue life of the BM + GO samples tested at 20 and 100 °C occurred at all stress levels in the maximum fatigue cycle. However, at 200 °C, the fatigue life of BM + GO specimens was higher than that of BM specimens at maximum cycle stress levels σAfter analyzing the test results, it should be noted that the graphene oxide coating alone deposited on the steel did not unequivocally increase its fatigue life. Therefore, it is proposed that an additional strengthening treatment be carried out such as mechanical shot peening, which allows the deposited graphene oxide coating to be introduced into the surface layer and takes advantage of the unique properties of graphene to unequivocally increase durability, as the authors presented in .The analysis of the results of investigations into the influence of the coating of deposited graphene oxide on the fatigue life of austenitic steel 1.4541 in different temperature ranges showed the following.(1)Decrease  of tensile strength Rm by 1.53–2.56%, yield strength Re by 2.97–5.65% and Young’s modulus E by 2.93–8.89% for samples with a deposited graphene oxide layer (BM + GO) in comparison with untreated samples (BM).(2)Increase in temperature of BM + GO samples during breaking of 9.4% in static tensile tests and 3–26% in fatigue tests.(3)∘20 °C: 12% for 370 MPa, 14% for 420 MPa, 2% for 500 MPa and∘100 °C: 77% for 370 MPa, 37% for 420 MPa, 8% for 500 MPa.Decrease in fatigue life of BM + GO specimens compared to BM specimens regarding temperature stress level, at respective levels of(4)Increase in fatigue life at 200 °C of BM + GO samples by 135% and 21% at nominal stress amplitude of 370 and 420 MPa, respectively.(5)The formation of faults at the lateral edge for BM specimens, which may be due to higher surface roughness.(6)Parallel cracking of graphene flakes on the steel surface during fatigue tests.The processing of elements presented in this work consisting of plasma cleaning and surface activation, application of graphene oxide, vacuum drying, and mechanical shot peening was called hybrid graphene treatment and is presented in patent application P. 438715.


Intensive research is underway worldwide to develop new conductive materials for applications in the power industry. Such tests aim to increase the electrical conductivity of materials for conductors and cables, thus increasing the current carrying capacity of the line and reducing the loss of electricity transmission. The scientific discovery of recent years, graphene, one of the allotropic types of carbon with very high electrical and thermal conductivity and mechanical strength, creates great opportunities for designing and producing new materials with above-standard operational properties. This project concentrates on developing technology for manufacturing aluminum-graphene and copper-graphene composites intended to be used to produce a new generation of power engineering conductors. In particular, we present the results of the research on the mechanical synthesis of aluminum-graphene and copper -graphene composites, as well as the results of the electric, mechanical, and structural properties of rods obtained after the extrusion process and wires after the drawing process. Copper has high electrical conductivity (58 MS/m at 20 °C) and is widely used in electrical applications. Owing to a favorable ratio of mechanical strength to mass density and resistivity, aluminum is a conductive material competitive for copper and commonly used in power engineering, in particular in overhead power lines as ACSR (Aluminium Conductor Steel Reinforced) cables. Continuously growing demand for higher transmission capacity of power lines is a driving force of the development of conductive materials engineering. Operating conditions of conductors impose additional requirements on conductive materials concerning strength and fatigue characteristics, rheology, and excellent corrosion resistance. It limits the application of pure metals, i.e., pure copper and pure aluminum, to produce power conductors and fosters the use of alloys that demonstrate high strength and are resistant to operating conditions. Consequently, electrical conductivity is reduced. 2 (Tc ≈ 40 K), 2010-SrFFeAs (Tc ≈60 K), 2010-FeS (Tc ≈ 100 K) and 2015-H2S @ 155 GPa (Tc ≈ 200 K). One of the latest discoveries is a H2S superconducting material that obtains superconducting properties at a temperature of 200 K but at a high pressure of 155 GPa. Extremely low temperatures are no longer a requirement to generate superconductivity, and the discovery of superconducting materials operating at close to room temperature could find application in, for example, current transmission technology, where losses due to resistance in transmission lines could be almost completely avoided [One of the dynamically developing directions of improving electric current transmission is superconductivity. Actually, the zero resistance, and consequently the lack of dissipation of electrical energy, mean superconductors are commonly used in the windings of superconducting electromagnets . The race to discover materials with an increasingly higher critical temperature, exceeding 100 K, is still ongoing, e.g., 1988-YBaCuO (Tc ≈ 100 K), 1995-HGBaCaCuO @ 30 GPa Chemical synthesis—Covetic types of an atomic blend  ,21.(b)Electrochemical deposition of graphene on elements of copper or another material .(c)Casting a mixture of liquid metal with graphene to a form facilitating further plastic processing to make wires with required characteristics .(d)Deposition of monoatomic carbon coats (graphene) using the CVD method on the surface of Al or Cu wires and their further plastic processing to form wires with required characteristics.(e)Mechanical synthesis of Al powders coated with graphene or Cu powders coated with graphene to a form facilitating their further plastic processing to make wires with required characteristics ,28,29,30Synthesis of graphene with aluminum or copper creates an opportunity to obtain new materials with previously unknown electrical and functional characteristics. There are different approaches and works on using different methods to develop such materials are in progress and include the following: There is a lot of research in the literature on the synthesis of graphene from metals. Authors of papers ,21 preseLiterature analysis shows that there are many methods of synthesizing aluminum and copper with graphene. It is difficult to say which one guarantees composites with the properties required for electrical applications, i.e., high electrical conductivity and high tensile strength. This is due to the problems associated with the poor wettability of graphene and copper or graphite and aluminum, the large difference in the density of copper and graphene, and the unstable quality of graphene currently available on the market ,36,37,38The purpose of the study was to evaluate the synthesis of copper and graphene as well as aluminum and graphene and the formability of composites during the extrusion process. The tests involved the implementation of a powder synthesis of copper and aluminum powders with graphene. −1, spectral range (Raman shift): 50 cm−1–6000 cm−1, experimental temperature: ambient.Mixtures of appropriate ratios of copper and aluminum powders and graphene were used to synthesize the powders. Graphene was produced in the Institute of Electronic Materials Technology in Warsaw within a project called “New wires made of metal-graphene composites with improved conductivity, intended for power conductors”, implemented as a part of the Applied Research Programme by METGRAF Consortium. The Raman spectrum studies were performed with Renishaw inVia Confocal Raman Microscope – parameters: excitation (cw): lasers 532 nm, 633 nm, 1064 nm, 325 nm, configuration: back-scattering, spatial resolution: 0.5 microns, resolution: about 1.5 cmThe chemical composition of Al and Cu powders selected for synthesis was tested on the JXA 8230 microanalyzer by Jeol . An accelerating voltage of 15 kV was used, the electron beam current was 30 nA. The measurements were carried out using waveguide spectrometers . The wavelength dispersion method enables a more accurate measurement of light elements. It is also characterized by a better detectability of elements and a higher resolution of spectral lines. The synthesis involved mixing and compaction copper powders and aluminum powders with graphene, pressing them into compacts, and their further consolidation using the conventional extrusion method. The powder mixtures forming the Al-C and Cu-C composites were made according to the proportion of graphene addition: 0.5% and 1% by weight based on the weight of pure aluminum/copper powder. The mixing process was carried out using industrial and laboratory ball mills for 30 to 60 min. The diagram of the composites and wires production process is shown in The criterion for selecting the mixing time of the powders was related to obtaining a homogeneous distribution of graphene in the volume of the metal matrix. In the case of ALGRAF composites, the increase in graphene content led to a deterioration of the compacting effect, which resulted in the production of moldings and presses with poor surface quality see . Composites thus made were extruded using a special stand horizontal press with a rotating die. In the next step, extruded rods with a diameter of 4 mm were strained in 98% (strain hardening) by the wire drawing process, which helped obtain wires with a diameter of 1 mm. Detailed parameters of the extrusion and wires drawing process are presented in Wires after the drawing process and rods after the extrusion process were tested for their mechanical, electrical, and structural properties. The tests of mechanical properties were carried out using the uniaxial tensile test. The mechanical properties of the samples were measured on a Zwick/Roell testing machine  with a maximum force range of 20 kN. The tests of electric properties were performed using the Thomson bridge. The maximum measuring resolution was 1 nΩ, with measuring currents from 100 μ to 10 A. The accuracy class was 0.01%. Microstructure tests were performed using a Scanning Electron Microscopy  and Jeol JXA 8230 microanalyzer . An accelerating voltage of 15 kV was used, the electron beam current was 30 nA. The main objective of the research was to answer whether the Al-Graphene and Cu-Graphene composites produced a guaranteed homogeneous structure and chemical composition throughout the entire volume of the composite. Based on the research, it was found that the composites obtained both as a result of plastic consolidation (extrusion process) of ALGRAF and CUGRAF showed a homogeneous distribution of graphene in the entire volume, which guarantees a homogeneous distribution of properties along the length of the wires produced.One can observe that the higher content of graphene in CUGRAF and ALGRAF composites significantly reduced the plasticity of the material. Nevertheless, no significant differentiation of the tensile strength was observed, and the values of 200–250 MP (for Cu-C) and 140–150 MPa (Al-C) for the majority of materials were reached. An analysis of the impact of graphene content on electrical properties of the studied composites at different processing stages revealed slightly higher resistivity values as compared to the base material, i.e., pure copper and pure aluminum. This was the evidence of the probable presence of graphene material in a form not linked with the copper or aluminum matrix. Additionally, the lack of reduction in resistivity could result from imperfect consolidation and processing of the composites.After the tensile tests, wire fracture was analyzed by SEM. Photos of fractures of wires made of ALGRAF and CUGRAF composite are presented in Based on the relations presented in On the other hand, in the case of extruded rods, it has been observed that the addition of graphene to the Al matrix and Cu matrix leads to an increase in resistivity . Interestingly, in the case of aluminum-graphene rods and wires, the differentiation of the resistivity value was concluded for various graphene content. Higher resistivity values of wires compared to rods are an effect of dislocation density increasing. While similar Al-C wires resistivity values—regardless of the graphene content—results from the increase in their density, an additional effect was compacting due to cold deformation . In the case of copper-graphene composites, this situation does not exist. The differentiation of the resistivity value for Cu-C rods after extrusion and for wires after the drawing process is similar.The wires obtained by the laboratory drawing process did not show any lower than standard resistivity at room temperature compared to the reference materials , Al-C and Cu-C composites with homogeneous carbon distribution were obtained. The results of the drawing process showed good deformability of composite wires in the drawing process. At the same time, a limited ductility was found, which is expressed by a lower total deformability of these materials compared to traditional materials. The above fact results from the significantly lower plasticity of composite wires.The addition of graphene to aluminum and copper results in an increase in resistivity and strengthThe addition of graphene to copper and aluminum results in a different type of strain hardening during the wiredrawing process. The addition of graphene to aluminum leads to greater strain hardening . On the other hand, in the case of Cu-C composites, the addition of graphene resulted in a lower strain hardening in the drawing process . The different types of strain hardening result from a different mechanism of graphene interaction on Cu and Al.The obtained results showed that it is possible to produce wires from composites based on aluminum/copper powders with the addition of graphene in the core of the wires, which can be a component of overhead power cables.Based on the results of experimental research, it was found that: