Twisted and coiled actuators (TCAs), fabricated by twisting cheap nylon sewing threads, have attracted a great deal of attention for their use as artificial muscles or soft actuators. Since the dynamic behavior of a thermally-operated TCA is governed by its thermal properties, graphene and silver nanoflowers (AgNFs) were spray-coated onto the surface of an actuator to achieve enhanced heat transfer. Addition of AgNFs improves interfacial thermal contacts between graphene flakes, while pristine graphene flakes have extremely high in-plane thermal conductivity. Thus, the synergistic effect of graphene and AgNFs reduced the total cycle time of the TCA by up to 38%. Furthermore, when a pulsed current with a 40% duty cycle was applied to the TCA, the graphene/AgNF-coated TCA exhibited a threefold larger peak-to-peak amplitude of the displacement oscillation of the actuator, as compared to that of the non-coated TCA, which demonstrates that the combination of graphene and AgNFs effectively reduced a cooling time of the TCA. This work shows great potential for a simple coating of graphene and AgNFs to produce high-performance thermally-operated soft actuators. Therefore, a great deal of effort has been invested in developing muscle-like actuators with high mechanical compliance and high specific power, including shape memory alloys (SMAs)5, pneumatic elastomeric actuators6, electro-active polymers (EAPs)8, twisted and coiled actuators (TCAs)10, and others. Among these soft actuators, TCAs fabricated by twisting and coiling sewing threads made of inexpensive polymers demonstrated an extremely high specific power of 5.3 kW/kg along with additional advantages including a large operating strain of over 20%, small hysteresis, high flexibility, low weight, and low fabrication cost9, which makes them a promising candidate for practical artificial muscles.Muscle-like actuators or soft actuators are a key component of advanced applications such as robotics, wearables, and medical devices because conventional actuators are bulky, heavy, rigid, and noisy9. However, this requires additional bulky components for temperature control of the TCA. Thus, conductive silver-coated polymer fibers were used to fabricate TCAs that enable temperature control by application of an electric current through the actuator for Joule heating11. This is a simple and efficient method for operating the TCAs with controlled actuation.When making TCAs, extreme twisting of polymer fibers forms spring-like coils and the gaps between coils are controlled by temperature change of the actuator; when the actuator is heated, the gaps between coils are closed and the actuator contracts. Repeated heating and cooling are typically achieved by applying hot air or flowing hot water through a tube surrounding the TCA12. However, twist-spun CNT yarns require a complicated fabrication process involving spinnable CNT forests, resulting in high fabrication cost and technical difficulties. To take advantage of TCAs made from cheap polymer fibers, a simple coating of graphene-based materials was recently introduced to enhance dynamic performance13. High-quality monolayer graphene with minimal defects has shown an extremely high thermal conductivity of over 2000 W/mK16. Thus, TCAs fabricated from nylon fibers were coated with various forms of graphene, such as monolayer large-area graphene, three-dimensional (3D) graphene foam, and graphene flakes, which enabled a reduction in the cycle time of the TCA; in particular, coating of graphene flakes demonstrated a dramatic decrease in the cycle time by 30.9%13. However, it has long been known that the out-of-plane thermal conductivity of graphitic materials is two orders of magnitude lower than the in-plane thermal conductivity due to the weak van der Waals interactions between individual graphene layers17 and the weak interaction between graphene flakes increases the interfacial thermal resistance. This limits the enhancement of the thermal properties of the graphene-based coating layer. In this respect, there is still a demand for coating layers comprised of graphene flakes with improved thermal properties that would allow for creation of high-performance TCAs. Metallic nanoparticles have been used to improve the thermal conductivity by bridging two-dimensional (2D) materials18. Recently, flower-shaped silver nanoparticles  have been developed for conductive electrodes and composites20. AgNFs have single-crystal petals that are around 20 nm thick. Due to their hierarchical structure with ultrathin petals, they provide better dispersion without agglomeration and allow for coalescence of petals at low temperatures of 80–120 °C19. Therefore, they have great potential for enhancing the interfacial thermal conductance between 2D materials by melting the silver petals and generating improved physical contacts to 2D materials.The dynamic behaviors of a TCA are dependent on its thermal properties. Since polymer fibers have relatively low thermal conductivity, other materials, such as carbon nanotubes (CNTs), have been utilized to fabricate super-coiled actuators. A coiled yarn made from sheets of CNTs demonstrated fast tensile actuation with large forces and strokes because of the high thermal and electrical properties of CNTsIn this study, AgNFs were embedded into graphene flakes to enhance thermal contacts between graphene flakes and thus improve the dynamic performance of TCAs. Since low-temperature curing can induce the active coalescence of silver petals, the interfaces between graphene flakes can be bridged by the AgNFs, ensuring better thermal contacts. AgNFs mixed with graphene flakes were spray-coated on the surface of the TCAs. The enhanced dynamic performance of the TCA coated with the graphene/AgNF hybrids was investigated by cyclic operations under repeated heating and cooling.21, a 2–4/TCA was used to maximize the stroke under an applied load . TCA performance is dependent on fabrication method. Following previous works22. A dispersion of graphene flakes (5 mg/mL) was prepared in a water/ethanol mixture with a volume ratio of 1:913. After AgNFs were synthesized using a previously published method19, AgNF powder was poured into the graphene dispersion. The fabricated TCAs were spray-coated with the mixture of graphene and AgNFs using a spray gun and dried in air  by sonicationScanning electron microscopy  was used to observe graphene flakes, AgNFs, and the surface of the fabricated TCAs. The surface morphology and the cross-sectional view of graphene flakes were observed by transmission electron microscopy . Raman spectroscopy  with a 532 nm wavelength laser was employed to characterize the graphene flakes.21. Briefly, the temperature of the TCAs was monitored with a micro thermistor placed on the end of the TCAs, which enabled closed-loop temperature control. A linear magnetic encoder was used to measure the displacement of the TCAs under a temperature range between 35 °C and 100 °C, while a 500 g payload was attached to the end of the vertically-positioned TCAs.As the TCAs were fabricated with electrically conductive silver-coated nylon fibers, the dynamic performance of the fabricated actuators was evaluated using Joule heating. A DC current was applied to the two ends of the TCAs to control the temperature for actuation. A detailed experimental set-up was described in previous papers−1 and the 2D band at 2700 cm−1  of the normalized displacement. When the current is shut off, the TCA starts to naturally cool from exposure to air and extends to its maximum length  at the lowest operating temperature. Figure TCAs coated with graphene/AgNF hybrids were tested with repeated heating and cooling. Displacements of each TCA were normalized to the maximum displacement in order to compare the cycle performance of different TCAs27:0 is the ambient temperature, P is the input electric power, and Cth and λ are the thermal mass and thermal conductivity of the thermally-operated actuator, respectively. The dynamic performance of the TCA can be determined by the time constant, τ = Cth/λ. Therefore, increasing the thermal conductivity of the TCA is one way to improve its cycle performance. As the thermal conductivity of pristine graphene is extremely high, a simple coating of graphene flakes on the surface of the TCA enhances the thermal response of the actuator13. However, the out-of-plane thermal conductivity of multi-layer graphene flakes is two orders of magnitude lower than the in-plane thermal conductivity17 and the weak interaction between graphene flakes induces thermal resistances at interfaces.A first-order linear model can be used to understand the thermal behavior of a TCA with the following equation28 and CNTs29 due to their high-aspect-ratio geometries and outstanding physical properties. However, our study (not shown here) revealed that the use of AgNWs could not grant the TCAs better dynamic behavior. This may be because long AgNWs reinforced the coating layer, interfering with TCA actuation. In addition, silver nanoparticles (AgNPs) have been utilized synergistically with other nanomaterials30. However, AgNPs tend to easily agglomerate31, which can increase stiffness of the TCAs. Due to their better dispersibility, high thermal and electrical conductivities, and ability to coalesce at very low temperatures, AgNFs offer thermally robust and mechanically flexible bridges between graphene flakes in the TCA coating.To obtain better thermal interfaces between graphene flakes, AgNFs were embedded into the graphene flakes. Silver nanomaterials with different morphologies have been used to bridge other electrically and thermally conductive nanomaterials. For instance, silver nanowires (AgNWs) provided robust electrical and thermal connections for graphene13. In contrast, the addition of AgNFs significantly improved the cycle time by 38% at a mass ratio of AgNFs to graphene of 1 (Fig. The synergistic effect of graphene/AgNF hybrids was investigated by evaluating the cycle performance of the coated TCAs. Heating, cooling, and total cycle times were evaluated from the dynamic responses shown in Fig. f 1 Fig. b; the im13.The effect of AgNF concentration on the performance of the TCAs was investigated by varying the mass ratio of AgNFs to graphene from 1 to 10. As shown in Fig. In addition, the dynamic behaviors of the TCAs were characterized by monitoring the displacement responses as a function of the frequency of the heating and cooling cycles Fig. . A pulseIn summary, the dynamic behaviors of a TCA, a promising artificial muscle material, were improved by treating the surface of the actuator with a graphene/AgNF hybrid coating. A simple spray-coating method was used to obtain thin, uniform layers on the fabricated TCAs. As the nylon fibers were coated with silver, the electrically conductive TCA was actuated by Joule heating and natural cooling in air. AgNFs had ultrathin petals with tens of nanometers in thickness, which enabled coalescence of the AgNFs by heat treatment at a low temperature. Therefore, the graphene/AgNF hybrid coating dramatically reduced the total actuation time in repeated heating and cooling cycles; the maximum reduction in cycle time was 38% at a mass ratio of AgNFs to graphene of 1. This is attributable to improved interfacial thermal contacts between graphene flakes caused by coalescence of AgNFs as well as high-quality graphene flakes with extremely high in-plane thermal conductivity. Furthermore, when a pulsed current with frequencies ranging from 0.1 to 0.4 Hz was applied to the TCAs, the graphene/AgNF-coated TCA showed a threefold greater peak-to-peak amplitude in the displacement response as compared to the neat TCA without any coating. This work demonstrates the great potential of the graphene/AgNF hybrid coating for development of high-performance thermally-operated soft actuators.


Cellular microenvironments are known as key factors controlling various cell functions, including adhesion, growth, migration, differentiation, and apoptosis. Many materials, including proteins, polymers, and metal hybrid composites, are reportedly effective in regulating cellular microenvironments, mostly via reshaping and manipulating cell morphologies, which ultimately affect cytoskeletal dynamics and related genetic behaviors. Recently, graphene and its derivatives have emerged as promising materials in biomedical research owing to their biocompatible properties as well as unique physicochemical characteristics. In this review, we will highlight and discuss recent studies reporting the regulation of the cellular microenvironment, with particular focus on the use of graphene derivatives or graphene hybrid materials to effectively control stem cell differentiation and cancer cell functions and behaviors. We hope that this review will accelerate research on the use of graphene derivatives to regulate various cellular microenvironments, which will ultimately be useful for both cancer therapy and stem cell-based regenerative medicine. Cells experience growth within the cell microenvironment ,2. SpeciECMs, which are part of the cellular microenvironment, are primarily composed of proteoglycan and fibrous proteins. In particular, fibrous protein contributes to cell adhesion mechanisms and tissue development and plays a pivotal role in the aforementioned control of the cellular microenvironment ,21,22,23Graphene is one of the most popular nanomaterials used in various studies mostly due to its unique physicochemical properties . GeneralFor these reasons, graphene and GO are considered to have sufficient properties to control cellular microenvironments. In fact, graphene and GO can be used in more diverse forms compared to fibrous proteins because they can form various artificial structures with other materials or by themselves relatively easily ,46,47,48To fully understand the use of graphene-based materials for controlling cellular behaviors, the control of cellular microenvironments using conventional protein-based materials should be overviewed. Standard protein-based materials are fibrous proteins, such as collagen and fibronectin, which possess excellent cell adhesion properties. They are used in many types of cell culture and assist in the proliferation of cells or induce differentiation ,51,52,53In the study, Teixeira et al. induced the osteogenic differentiation of MSCs by treating them with collagen on the PLA scaffold structure; several similar studies have been reported ,60,61,62Research on stem cell differentiation using graphene hybrid composites has been very prolific since 2010. Considering that graphene is less cytotoxic and allows stem cells to attach and proliferate ,68, grapLiu et al. developed conductive hydrogel containing carbon nanotubes and graphene oxide for nerve cell differentiation e–g 73,7,75,76,77Recently, Kim et al. induced differentiation of C2C12 mouse myoblast cells using graphene. Kim et al. produced a “crumpled grapheme” structure for cell differentiation  84]. Cr. Cr84]. Graphene patterns create a more deliberate physical impact beyond the control of microenvironments that can be expected from random arrangements or the electrical properties of graphene itself. An advantage is that graphene can be patterned into a specific shape or a specific arrangement to construct the cell culture type desired by researchers.Kang et al. reported a new form of GO patterning technology  91,92].,92.91,92If a graphene hybrid pattern is applied to stem cell culture, it can serve as a platform to induce their differentiation. Lee et al. produced nanoscale graphene–Au hybrid nanoarrays to induce differentiation of human mesenchymal stem cells (hMSCs)  97,98].,98.97,98This study is one NSC differentiation platform using LIL, and there are many cases where LIL is applied to NSC differentiation ,106,107.Controlling the cellular environment using graphene is not limited to stem cells. This control can also be applied to tumor cells, as demonstrated in recent studies . As prevCancer cell microenvironmental control using graphene has also been applied to a 3D cell culture method. Kim et al. produced a graphene oxide-based platform and reported HepG2 cell spheroid formation through cell proliferation  109]. T. T109]. Hu et al. devised a slightly different approach to control 3D cellular microenvironments  115]. T. T115]. Recently, Wan et al. also reported that GO exerts a positive effect on the growth of cancer cells when incorporated with the scaffold structure . In thisThrough the studies discussed above, we divided cellular microenvironmental control using graphene into two main categories. One is ‘cellular microenvironments of stem cells’, and the other is ‘cancer cell microenvironments’. These cellular microenvironments were controlled using various types of graphene-based platforms. In the case of stem cells, differentiation was induced or promoted. In the case of cancer cells, proliferation was adjusted to enable the formation of 3D spheroids of cancer cells and long-term cell culture. Graphene derivatives regulate the microenvironments of target cells in various ways. Specifically, the induction of differentiation of hNSCs has been demonstrated using graphene–polymer scaffolds, and the control of HeLa cell attachment and growth using graphene platforms such as crumpled graphene, and graphene patterning are notable. In addition, the GO vertical patterning method in which graphene is three-dimensionally patterned on the side wall of micro-well arrays, has provided a new platform for an effective method for 3D culture of cancer cells. All of these “controlling cellular microenvironments” approaches are enabled due to distinct physical and electrical properties of graphene including excellent cell adhesion, flexibility in physical transformations, and stability against physical/chemical stimuli. Such characteristics of graphene and GO are known to affect various biological phenomena including (i) the absorption or repulsion of growth/differentiation factors on the surface, (ii) ECM protein alignment, and (iii) anchoring of integrin receptors on the cell membrane to the artificial surface. These physicochemical properties of GO allow a limitation or enhancement of cell adhesion and spreading, which ultimately lead to the changes in various cell functions  via alterations in the upstream cytoskeletal dynamics and downstream gene expression. Broadening our horizons, we were able to determine the positive changes that graphene-based materials and platforms will have on regenerative and clinical medicine in the future. In particular, graphene-modified hydrogels and graphene patterns can be used to achieve cells of interest from various stem cell sources with high conversion efficiency, which will ultimately accelerate the use of stem cells for regenerative therapies. In the case of cancer research, graphene hybrid materials, including graphene-coated micro-wells, can be used to restrict cell adhesion and growth through generation of 3D tumor spheroid arrays and further rapid anticancer drug screening. Taken together, we can conclude that controlling cellular microenvironments using graphene-based materials is highly promising for both stem cell-based regenerative medicine and cancer research.


We have previously identified a delay in general practitioner (GP) referrals for patients with degenerative cervical myelopathy (DCM). The aim of this study was to evaluate whether an education gap existed for DCM along the GP training pathway by quantitatively assessing training in, and knowledge of, this condition.Gap analysis: comparison of DCM to other conditions. Comparators selected on the basis of similar presentation/epidemiology (multiple sclerosis), an important spinal emergency (cauda equina syndrome) and a common disease (diabetes mellitus).Medical students, foundation doctors and GP trainees.(1) Assessment of training: quantitative comparison of references to DCM in curricula (undergraduate/postgraduate) and commonly used textbooks (Oxford Handbook Series), to other conditions using modal ranks. (2) Assessment of knowledge: using standardised questions placed in an online question-bank (Passmedicine). Results were presented relative to the question-bank mean (+/−).DCM had the lowest modal rank of references to the condition in curricula analysis and second lowest modal rank in textbook analysis. In knowledge analysis questions were attempted 127 457 times. Performance for DCM questions in themes of presentation (+6.1%), workup (+0.1%) and management (+1.8%) were all greater than the question-bank mean and within one SD. For students and junior trainees, there was a serial decrease in performance from presentation and workup (−0.7% to +10.4% relative to question-bank mean) and management (−0.6% to −3.9% relative to question-bank mean).Although infrequently cited in curricula and learning resources, knowledge relating to DCM was above average. However, knowledge relating to its management was relatively poor. Search terms relating to degenerative cervical myelopathy (DCM) were queried from three UK specific medical school curricula and relevant postgraduate curricula.A large number of responses were obtained by placing questions in an online question-bank, relating to DCM.A limited number of learning resources were searched to assess references to DCM.Degenerative cervical myelopathy (DCM) is a common and insidious condition that can lead to severe disability.The diagnostic pathway for DCM almost exclusively starts with assessment and triage by a community physician, termed in the UK a general practitioner (GP).This period of the diagnostic pathway is difficult to examine in detail, and while delayed patient presentation is likely to contribute, delayed detection measured by multiple consultations and patient perspective, is certainly a relevant component.Our objective therefore was to evaluate whether an education gap exists for DCM among GPs, by quantitatively assessing their training in, and knowledge of, DCM.DCM patients were surveyed online and this confirmed that the diagnosis of DCM is frequently delayed.A gap analysis is a process to identify gaps in existing systems such as education curricula. It involves an assessment of existing knowledge or standards against predetermined standards that define core competency requirements.The study objective was therefore approached in two separate gap analyses .Assessment oftraining: quantification of DCM in curricula and commonly used learning resources, including assessment of relative importance through comparison to other conditions.Assessment ofknowledge: formal assessment of trainees’ knowledge using questions placed in an online question-bank.UK medical graduates complete the UK Foundation Programme to enter higher specialty training as formal GP trainees. There are several assessments along this route including: medical school final examinations to gain registration to practice in the UK, or the equivalent Professional and Linguistic Assessments Board (PLAB) test for international medical graduates; the Specialty Recruitment Assessment (SRA)—a written assessment taken towards the start of the second foundation year prior to higher specialty applications; and the Membership of The Royal College of General Practitioners (MRCGP), an exit examination for GP trainees. While alternative entry routes to general practice exist, this pathway represents the most common training route for GPs today.DCM was chosen as an inclusive term for a variety of diseases resulting in compressive myelopathy.Direct comparator to myelopathy: a disease that is a differential diagnosis for DCM with equivalent or greater incidence.Degenerative spine comparator: an alternative degenerative spine disease that is widely taught.Generic non-neuroscience comparator: a common disease that all clinicians would have some knowledge about and interaction with.The a priori hypothesis was, if an education gap existed within DCM, we would expect metrics of training and knowledge of DCM to rank inferior to controls. Controls were identified through a consensus author meeting and finalised after confirmation of epidemiological profiles through literature review.Oxford Handbook of Clinical Medicine, the Oxford Handbook for the Foundation Programme and the Oxford Handbook of General Practice.We selected learning resources that were in common usage by trainees, available in electronic format (to be amenable for searching) and stratifiable by training stage. Specifically, training curricula, the Oxford Handbook seriesSearch terms for analysing learning resources were created for DCM and each comparator using relevant terms from the search syntax of Cochrane Reviews, or if absent, recent systematic reviews. Curricula were searched for the number of references per disease, text books for the number of words per disease section and question-banks for the number of questions.The author panel includes GPs with an interest in education (JW), university appointed educationalists (JG) and neurosurgeons . The authors devised a set of questions to be included in an online question-bank, composed both multiple choice questions (MCQs) and extended matching questions (EMQs), designed to cover the different components of medical assessment; presentation, investigation and management. The final set consisted of 19 questions (13 MCQs and six EMQs). The questions together with answers are included in 10.1136/bmjopen-2018-028455.supp1Supplementary dataOnline question-banks including subsections devoted to each stage of the GP training pathway were contacted with details of the study. Only one question-bank—Passmedicine,Statistical analysis was performed using SPSS V.22. For assessment of training, frequencies of search terms were described and the mode and modal ranks determined. For assessment of knowledge, histograms were constructed with either a condition or question-bank along the x-axis and user performance relative to question-bank mean along the y-axis. The user performance relative to question-bank mean corresponded to the raw difference between the mean value for each theme and the mean value for a particular question-bank.Multiple sclerosis (MS), cauda equina syndrome (CES) and diabetes mellitus were selected as the three disease comparators and list of search terms compiled accordingly .16–19 MSDCM and CES had the lowest modal rank in curricula search analysis . This waOverall, DCM had the second lowest modal rank but above that of CES . The relPassmedicine was approached, on the basis it did not have any DCM questions previously which could influence analysis. Questions were introduced between the period June 2017 and October 2017. Section editors for each question-bank ultimately selected which of the 19 questions were relevant, such that finals/PLAB contained 14, SRA—19 and MRCGP—19.Overall, questions were attempted 127 457 times; finals/PLAB—36 706; SRA—47 530; MRCGP—43 221 . Data on10.1136/bmjopen-2018-028455.supp2Supplementary dataThere were differences in user performance in the three DCM question themes—presentation, workup and management, though average scores were all within one SD of the mean . PerformUser performance by disease showed large variation between question-banks . The meaIn this study, we found that DCM is relatively infrequently cited in curricula and commonly used textbooks, even compared with diseases such as MS, with similar epidemiology. Despite this however, user performance in some of the DCM questions remained consistently above question-bank averages. There was a sequential decrease in user performance across the themes of DCM presentation, workup and management for early years’ trainees, whereas for senior trainees, performance did not vary by theme of question. For other neuroscience themes such as MS and CES, the user performance was below average. This was in contrast to user performance in questions relating to diabetes mellitus, which was consistently above question-bank mean.We observed a below average performance in questions grouped under the neurology theme, which included all questions relating to the central and peripheral nervous system. Reduced knowledge pertaining to neurosciences has previously been linked to a term called neurophobia, though this is by no means a universally accepted concept.Second, our study supports the observation of poorer performance in management themes by clinicians at an earlier training stage. Prior studies on medical students that have evaluated knowledge formally have shown similar results in specialties such as ophthalmology.Our original hypothesis attempting to correlate delays in diagnosis for DCM with a deficiency in training and knowledge, was not conclusively demonstrated in the present study. Earlier stage trainee doctors demonstrated comparably higher levels of knowledge with regards to the presentation and work up of DCM and a potential gap with regards to management-related questions. These differences disappeared in individuals studying for the GP exit examinations. Nevertheless, the clinical argument for an education gap is strong—our previous analysis of the referral pathway for DCM identified a time to initial referral by the GP as representing 51% of the overall diagnostic delay.This study demonstrated DCM received relatively less training than other conditions. In the west, the prevalence of DCM is underestimated but at least 60 per 100 000, compared with around 100 per 100 000 for MS.One potential way of reconciling the absence of a gap in formal knowledge with the deficiencies in the diagnosis and management of DCM, is that factual knowledge may not translate into recognition of the disease in the clinical context. Causal association between training and clinical practice have not been clearly demonstrated, although prior studies have found good correlation between examination performance and clinical performance in a variety of medical and surgical specialties.There are several limitations to be considered in the context of these findings. First, only select learning resources were used in this study and questions were inserted into a single online question-bank. Second, question-bank data were not extractable per user, nor per first time answer. While this was not a comprehensive strategy, this is unlikely to have limited our results for at least two reasons: questions were attempted more than 100 000 times and analysis was relative within learning resources, and therefore limitations were applicable to each comparator. The native question-bank data extraction technique also did not provide data on the number of attempts on question themes other than DCM.The methodology employed in the present study may not have been sufficiently sensitive to detect a knowledge gap for DCM, for several reasons. First, our questions may not have been challenging enough given that they tested core principles all clinicians would be expected to achieve. Although we did not employ pilot testing of the questions in our target population, questions were designed by an experienced author panel including educationalists. Furthermore, our questions were subject to additional scrutiny by the question-bank editors, such that only those questions deemed appropriate were included in a particular question-bank. Second, there was no first-time answer data available for analysis. This means that ‘random effect answering’, users selecting an answer to read the explanation, was not accounted for, though this was the case for both DCM and controls. Third, our controls may not have been appropriate and certainly, educational comparisons between specialties is not always the best method of assessing knowledge adequacy.In this study, we set out to evaluate whether there is a deficiency in training and knowledge regarding DCM in the GP training pathway. Although DCM was infrequently referenced in learning resources, trainees performed above average for DCM questions in an assessment of knowledge using an online question-bank. The clinical suggestion of an education gap is strong, and these contrasting findings do not conclusively allay this concern. Future studies are required to better understand these observations.


Sarcoidosis is a systemic disease of unknown etiology defined by the presence of noncaseating granulomatous inflammation that can cause organ damage and diminished quality of life. Treatment is indicated to protect organ function and decrease symptomatic burden. Current treatment options focus on interruption of granuloma formation and propagation. Clinical trials guiding evidence for treatment are lacking due to the rarity of disease, heterogeneous clinical course, and lack of prognostic biomarkers, all of which contribute to difficulty in clinical trial design and implementation. In this review, a multidisciplinary treatment approach is summarized, addressing immunuosuppressive drugs, managing complications of chronic granulomatous inflammation, and assessing treatment toxicity. Discovery of new therapies will depend on research into pathogenesis of antigen presentation and granulomatous inflammation. Future treatment approaches may also include personalized decisions based on pharmacogenomics and sarcoidosis phenotype, as well as patient-centered approaches to manage immunosuppression, symptom control, and treatment of comorbid conditions. Sarcoidosis is a systemic disease of unknown etiology that can cause organ dysfunction and diminished quality of life. The disease is diagnosed by a constellation of radiographic, clinical and histopathologic findings; it is most often defined by the presence of noncaseating granulomatous inflammation that occurs in the absence of infection, exposures, malignancy, or alternative immune-related disease. Lung and thoracic lymph nodes are most often involved, but any organ can be affected, with multi-system involvement having a worse prognosis. Treatment is directed at alleviating organ dysfunction, preventing irreversible scarring, and improving quality of life. Herein, we review the indications for treatment, pharmacotherapy, treatment duration, side effects, adjunct non-pharmacologic therapies, and outcomes for patients.Propionibacterium acnes has also been proposed as an etiologic agent given the higher frequency of genetic material found in sarcoidosis granulomas compared to controls, as well as similar exaggerated immune response to Priopionibacteria in sarcoidosis T cells compared to normals  and to prevent eventual scarring and fibrosis as seen in the lungs. Current standard of care focuses on suppressing highly activated macrophages and T cells and their production of cytokines such as TNF-α, IL-1, IFN-γ, and IL-6. More recent study has also suggested a possible role of anti-B cell therapy given the presence of B cells in the granuloma and increased B cell activating factor (BAFF) in sarcoidosis patients . AdditioTo date, treatment of sarcoidosis is largely guided by small, uncontrolled trials and expert consensus –19. A feOver half of patients with sarcoidosis will incur spontaneous resolution or never have clinical manifestations of the disease, whereas the remaining half will experience a more chronic course, often requiring treatment. Mortality appears to be increasing over time, and burden of disease can be substantial due to complications of the disease or its treatments . It is uCorticosteroids are considered first-line treatment by consensus of sarcoidosis providers , 29. Mulvia the adenosine a2a receptors  to methotrexate in sarcoidosis. Its safety and efficacy are supported by a retrospective study of 76 patients who had progressive sarcoidosis or had failed alternative immunosuppressive medications. A small, but significant improvement was seen in forced vital capacity (FVC) for pulmonary involvement, and a partial or good response was seen in 83% of the patients with extrapulmonary sarcoidosis . A steroAzathioprine, an inhibitor of purine metabolism, can also be considered as a steroid-sparing agent in patients with sarcoidosis, with similar efficacy compared to methotrexate to improve FVC and diffusing capacity (DLCO), and to taper corticosteroids . AzathioMycophenolate mofetil (MMF) is another second-line option that acts by inhibiting purine nucleotide synthesis specifically in lymphocytes and decreases production of autoantibodies by B cells; therefore, the drug is used commonly for immunosuppression in a variety of rheumatologic diseases and interstitial lung diseases . The larChloroquine and hydroxychloroquine have been frequently used in treatment of sarcoidosis based on early randomized trials that showed a long-term benefit with chloroquine . HoweverInhaled corticosteroids have not been shown to have clear benefit in patients with sarcoidosis in a RCT , but givTNF-α is a predominant cytokine, consistently elevated in active sarcoidosis as a product of macrophage activation, particularly at sites of granuloma formation . It is aIncreasing data for adalumimab would suggest a role for this TNF-α antagonist in sarcoidosis , 76. In On the other hand, etanercept, a fusion protein that antagonizes the TNF receptor, has not shown clear efficacy in treatment of sarcoidosis. Therefore, this drug is not recommended for use in sarcoidosis. An open-label Phase II study in stages 2 and 3 pulmonary sarcoidosis was stopped early due to excessive treatment failures in patients treated with etanercept 25 mg subcutaneously twice weekly . AlthougSimilarly, ustekinumab and golimumab were tested in a three-arm double-blind RCT in chronic pulmonary sarcoidosis patients with a primary endpoint of FVC change . GolimumThe advent of biosimilars has provided another, potentially less expensive, option for clinicians who desire an anti-TNF therapy. The biosimilar to infliximab has been tested in rheumatoid arthritis and found to have equivalent efficacy to its original form  . A retroSarcoidosis has been associated with altered B cell homeostasis , high BAAntifibrotics, now approved for treatment of idiopathic pulmonary fibrosis and progressive fibrotic interstitial lung diseases (ILDs), are also an enticing possibility for fibrotic sarcoidosis. The INBUILD trial, a positive RCT of nintadenib in various progressive fibrotic interstitial lung diseases included a few patients with fibrotic sarcoidosis . In the A few other immunosuppressive drugs have been reported as potential options in sarcoidosis, but lack a significant body of evidence in the form of RCTs or larger series. For example, cyclophosphamide has been reported as an effective treatment in corticosteroid-resistant disease in both neurosarcoidosis and cardiac sarcoidosis, and has been associated with a lower relapse rate for patients with neurosarcoidosis –99. HoweAdditional therapies targeting a variety of pathogenic mechanisms are also undergoing further study, but do not have enough evidence yet to be incorporated into treatment recommendations. For example, nicotine acts upon NFκB in macrophages to decrease cytokine production and acts to decrease the Th17/T-reg ratio by its effects on CD4+ lymphocytes, leading to evaluation of this compound as a treatment for sarcoidosis. A small RCT of 13 patients with sarcoidosis treated with transdermal nicotine versus standard treatment alone showed that those treated with nicotine had normalization of their TLR-2 and TLR-9 responsiveness and increased the T-reg response . Based oAnother trial, entitled the CLEAR trial , is targeting the hypothesis that mycobacteria are the elusive antigen in some cases of sarcoidosis. A pilot study of thirty patients with cutaneous sarcoidosis showed decrease in size of skin lesions and granulomatous burden with the CLEAR regimen . In a sivia modulation of neuropilin-2, is also ongoing in pulmonary sarcoidosis . Apremilast, a phosphodiesterase-4 inhibitor approved for psoriasis that decreases production of TNF-α, interferon γ, IL-2, IL-12, and IL-23 was recently investigated in 15 patients with cutaneous sarcoidosis. In this study, skin lesions improved significantly after 12 weeks of therapy . A Phasecoidosis . ResultsAdditionally, evolving research in granuloma formation and propagation has suggested new potential therapeutic targets. For example, mTORC1 activation of macrophages has been associated with disease progression, and alveolar macrophages from sarcoidosis patients have upregulation of interleukin-1 receptor associated kinases (IRAK1 and IRAK-M) and receptor interacting protein 2 (Rip2) , 110. Inin vitro to inhibit proliferation of Th1 lymphocytes, as well as suppress antigen presentation and activation of macrophages, all of which could potentially diminish granuloma formation and propagation  and angiotensin converting enzyme levels, suggesting an effect on granulomatous inflammation , familiarity of therapies by the clinician, side effect profiles, and patient preferences. The choice can be influenced by a patient’s age, alcohol intake, likelihood of pregnancy, concurrent medications, or comorbidities such as diabetes, liver or kidney dysfunction. Given the polypharmacy often involved in the patient regimen, drug interactions should be considered in medication choice. Corticosteroids are most often first line therapy for any type of sarcoidosis, but second or third-line therapies can be considered in cases where corticosteroids are risky  or these drugs can be added in the more aggressive treatment of life-threatening or severe organ derangement such as can be seen in neurosarcoidosis, ophthalmic injury, or severe infiltrative heart disease. On a more chronic, outpatient regimen, steroid-sparing agents are often used when corticosteroids cannot be reduced to reasonable doses (less than 10–15 mg per day), there is corticosteroid toxicity, or the anticipated course of treatment is lengthy.Duration of therapy, whether with corticosteroids or other immunosuppressive treatment, is generally considered to be approximately 1 year, based on data suggesting an increased risk of relapse with shorter courses , 116–118The question of whether there are organ systems which benefit from a particular steroid sparing agent is not wholly clear, and generally, extrapulmonary sarcoidosis is treated with the same algorithms as pulmonary sarcoidosis . HoweverAlthough some demographic and clinical characteristics have been associated with worse outcomes and increased disease severity in the broader sarcoidosis population, there are no patient-specific biomarkers used in clinical practice that can directly predict who will progress and necessitate treatment, nor are there clear biomarkers that will predict relapse in any one individual patient. However, increasing data shows promise for potential biomarkers. Multiple differing HLA haplotypes seem to track with both disease onset and differing presentations, implying that the genetic profile of the immune system can account for some of the varied clinical manifestations . For insFor treatment response, genetic polymorphisms may account for some of the variability in treatment response by therapy. For example, a study of 111 refractory sarcoidosis patients who were started on infliximab or adalimumab showed that patients without the TNF-alpha-308A variant allele (GG genotype) had a three -fold higher response to the TNF antagonists compared to those with the allele . Higher Although immunosuppression dominates the forefront of sarcoidosis therapy, in reality, holistic treatment of patients with sarcoidosis often involves addressing the comorbid conditions associated with the disease and mitigating side effects of immunosuppression. Sarcoidosis can cause a number of “danger situations”, including fibrocystic sarcoidosis, sarcoidosis-associated pulmonary hypertension (SAPH), bronchiectasis, and mycetomas . AdditioStrategies to circumvent detrimental toxicities of both corticosteroids and alternative immunosuppression are important to incorporate into the multidisciplinary management of patients. For example, maintaining routine recommended vaccinations for infections such as influenza or pneumococcus should be considered , 137. AdAspergillus within lung cavities, can cause hemoptysis or lead to invasive infection. Similarly, bronchiectasis resultant of fibrotic sarcoidosis can also complicate treatment. In these cases, use of antibiotics and pulmonary therapies including bronchodilators, mucolytics, and chest physiotherapy is often most effective , the treatment of pulmonary hypertension in sarcoidosis can be complex. Corticosteroid treatment may result in the improvement of pulmonary pressures in some, but not all cases, reflecting the differing mechanisms of pulmonary hypertension. Case series suggest benefit of prostacyclin analogues in some patients and endothelin receptor antagonists (ERAs) have conflicting data reports –148. RetIn case of progressive organ failure, transplantation can be considered in patients with sarcoidosis. Approximately 3% of lung transplants done in the United States are due to sarcoidosis . SuccessSmall Fiber Neuropathy in sarcoidosis causes chronic pain, autonomic dysfunction, and altered sensation . TreatmeFatigue in sarcoidosis is extremely common. Its presence is an interplay of inflammation, musculoskeletal disease, mental health, treatment side effects, and sleep issues , 163. GiPhysical training studies in sarcoidosis have suggested that a structured physical activity program can improve exercise capacity, muscle strength, and fatigue in sarcoidosis , 169. AdTreatment of sarcoidosis is complex and non-standardized for clinicians and patients. Further research is necessary to inform clinical guidelines and provide higher quality evidence for treatment regimens. Drug development is challenging due to the lack of animal model and rudimentary understanding of pathogenesis. Additionally, research should involve clinical trial design and prognostic biomarkers to appropriately select and evaluate those patients who will require therapy. Advancing the knowledge regarding etiology and pathophysiology may one day lead to prevention or cure. Future treatments will likely involve prevention of exposure, treatment of an antigen, mitigation of granulomatous inflammation, and interrupting fibrotic pathways. Management decisions should evolve to include personalized medicine based on pharmacogenomics and sarcoidosis phenotype, as well as patient-centered approaches to incorporate immunosuppression, symptom control, and treatment of comorbid conditions.The author confirms being the sole contributor of this work and has approved it for publication.AG is funded, in part, by the Federal Drug Administration Grant#FD-R-0005993 to study the natural history of sarcoidosis.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The synthesis of metal-free but electrochemically active electrode materials, which could be an important contributor to environmental protection, is the key motivation for this research approach. The progress of graphene material science in recent decades has contributed to the further development of nanotechnology and material engineering. Due to the unique properties of graphene materials, they have found many practical applications: among others, as catalysts in metal-air batteries, supercapacitors, or fuel cells. In order to create an economical and efficient material for energy production and storage applications, researchers focused on the introduction of additional heteroatoms to the graphene structure. As solutions for functionalizing pristine graphene structures are very difficult to implement, this article presents a facile method of preparing nitrogen-doped graphene foam in a microwave reactor. The influence of solvent type and microwave reactor holding time was investigated. To characterize the elemental content and structural properties of the obtained N-doped graphene materials, methods such as elemental analysis, high-resolution transmission electron microscopy, scanning electron microscopy, and Raman spectroscopy were used. Electrochemical activity in ORR of the obtained materials was tested using cyclic voltamperometry (CV) and linear sweep voltamperometry (LSV). The tests proved the materials’ high activity towards ORR, with the number of electrons reaching 3.46 for tested non-Pt materials, while the analogous value for the C-Pt (20 wt% loading) reference was 4. Therefore, over the past decades, graphene’s potential uses have been examined within many fields5: fields emission (FE) devices6, sensors and biosensors7, optoelectronic devices (transparent electrodes for solar cells), liquid–crystal displays (LCD)9, and electrode materials in batteries and supercapacitors11. Graphene can exist in the form of zero-dimensional (0D) dots , one-dimensional (1D) fibers (ribbons)14, two-dimensional (2D) sheets15, and three-dimensional (3D) foams16. 3D graphene foam is a network of connected graphene sheets and, just as 2D graphene, can be widely used to improve the electrical, mechanical, or thermal properties of electrochemical devices. The size, quality, and type of graphene sheet synthesis determines the properties and potential application of the 3D materials17. The synthesis of a three-dimensional graphene structure is based on either the assembly of two-dimensional graphene flakes or on a direct synthesis. Many papers describe 3D structure synthesis based on chemical vapor deposition (CVD) of graphene on a nickel foam matrix21. A relatively new synthesis method is the 3D printing of graphene materials24. Well-known methods of synthesis produce three-dimensional graphene materials effectively, but they are too expensive and require specialized equipment. The assembly of graphene flakes26 by the hydrothermal method27 is also popular, although not without disadvantages; self-organization of graphene sheets is the most common problem that researchers want to eliminate28. The microwave method30 supports the synthesis of graphene, combining many advantages: it is fast, simple, does not require specialized equipment, and the starting material for foam production can be expanded graphite31 or sucrose32.Graphene possesses excellent mechanical and thermal properties34. Hence, to further improve their properties, increase electrode capacity, and power or energy density, many researchers try to dope graphene materials with metal compounds or metal oxides, or to create composites with other active materials38. These solutions involve increased production and disposal costs. The fact that these substances are not environmentally friendly leads researchers to try to introduce heteroatoms (mainly nitrogen) into the carbon structure using natural reagents40, thus increasing the range of potential applications of 3D graphene materials.Graphene foams, due to their excellent porous structure, are a light material that combines the properties of two-dimensional graphene sheets and the three-dimensional system they form when joined together. The large pore volume and subsequent high specific surface area of the material, its thermal stability, high electronic conductivity, and high ion transfer rate all contribute to graphene foams’ functionality in energy storage devicesChlorella vulgaris as an N-precursor. Different solvents  were applied in the microwave reactor study. After the microwave process, the samples were carbonized. The study investigated the influence that the microwave (duration time) and carbonization processes had on graphene structure and nitrogen content.The synthesis of any metal-free but electrochemically active electrode material is an important contribution to protecting the environment and the key motivation for engaging this research approach. To this end, we present a fast and facile method of producing N-doped graphene foam using a microwave process. In the proposed process, expanded graphite was used as a starting material and green algae The proposed method to obtain N-doped graphene foam using a pressure microwave reactor was confirmed to be effective using instrumental methods. Figure Chlorella vulgaris. Heteroatoms were successfully introduced into the graphene structure using the natural reagent. For samples synthesized in different solvents with N-reagent, the percentage by mass of nitrogen was high, at 2.44 wt% for the 1A-10 sample. Along with extending the reaction time for the 1A-T series produced in the ethyl alcohol solvent, a significant decrease of the nitrogen content, down to 0.65 wt%, was noted. In the case of the 1B-T series, where DMF was the solvent, an increase in the samples’ nitrogen content was observed when holding time was increased. The highest nitrogen content for the 1B-T series was recorded for a sample maintained for 90 min, at 0.89 wt%.A basic premise of this method of enriching the final material with nitrogen is the use of green algae. After carbonization, it can join graphene flakes  into a durable, porous 3D structure resembling a foam or sponge. Aside from providing amorphous carbon matter adhering graphene flakes, green algae is a source of nitrogen. It was selected due to its useful features, such as ease of mixing with graphene flakes in EtOH or DMF and thermal degradation to a non-porous nitrogen-rich matrix. A low carbon matrix yield from the starting green algae mass makes it possible to avoid soaking the graphene flakes and retains the graphene flakes’ surface accessibility to chemical reagents, e.g., electrolytes. The green algae-originated carbon phase is assumed to be present in the form N-rich bridges between graphene flakes which enable electron transfer throughout the whole mass of material. The effective exfoliation of materials obtained with a holding time of 10 min in the reactor led to the investigation of the effect that extending holding time had on the structure and elemental composition of graphene foams. The influence on the nitrogen content was examined in two different electrolytes, while the carbonization process was used to change the structure of graphene by creating exfoliated graphene foam with embedded nitrogen functional groups. The elemental analysis was used to determine the composition of the N-doped graphene foams. The influence of the elemental composition for materials in series 0A/B-T and 1A/B-T obtained in different solvents is shown in Table 41. On the Raman spectra for graphene, three characteristic bands can be observed, D, G, and 2D, for which the shift at the 532 nm laser line is 1347 cm−1, 1577 cm−1, and 2698 cm−142 respectively. Using Raman spectroscopy analysis, it is possible to estimate the number of graphene sheet layers that have been laid, as well as determine defects in the material obtained. Raman spectra were compiled for the N-doped graphene foams carried out in two selected solvents, EtOH , C–C (sp3), C–O–C, C=O, and O–C=O, respectively. The total content of carbon for all samples, determined by XPS measurement, was in the range from 90.2 to 93.2 at.% , C–C sp, C–O–C, 43. Modified graphene structures subjected to microwave heating facilitated nitrogen substitution in the graphite plane. The effect of high carbonization temperatures on doped structures was the formation of mainly quaternary-N functional groups, responsible for the materials’ effective catalytic activity in the oxygen reduction reaction44. Due to access to an electron pair in the graphene structure, the pyrolytic N-group is active in response to the oxygen reduction reaction, which leads to an increase in the catalytic activity of N-doped materials45. The overall content of nitrogen atoms for all samples in the 1A-T series was between 0.6 and 2.7 at.%. The XPS results show that nitrogen doping in graphene is successful; there were two characteristic types of nitrogen functional groups that improved catalytic properties in response to ORR results, the highest content belonging to the pyrrolic-N groups. Deconvolution of the high-resolution O1s spectrum fit into three bands at energy values of 531.6 eV, 533.2 eV, and 536.0 eV which correspond to C–O–C, O–C–O, and absorbed H2O bonds, respectively46.After deconvolution of the high-resolution spectrum of N1s, two characteristic types of bonds were determined for nitrogen in the form of pyrrolic-N and quaternary-N in binding energies of 399.1 eV and 400.8 eV, respectively48. In this case, graphene structures served as a matrix for the introduction of nitrogen, using the natural substrate Chlorella vurgalis as the source. As the literature reports, N-precursor is transformed into conductive carbon forms under the influence of high carbonization temperatures, 800–900 °C, which increase electrocatalytic properties50. The reference material in electrochemical tests was a carbon material based on platinum (20 wt%). All measurements were made in an oxygen-saturated 0.1 M KOH electrolyte for the materials from the 1A-T and 1B-T series, however the plots are presented for the 1A-T series only  at one rotation speed, 1600 rpm. Sample 1A_60 exhibits the highest current density among the samples obtained, but it is still lower than a commercial material based on platinum. This proves that, despite sample 1A-60 having the lowest nitrogen content at 0.65 wt%, the material shows good catalytic properties in ORR. The value of the onset potential for the 1A-60 sample, shown in Fig. 51. ORR performance parameters for the 1A-T series and commercial platinum-based carbon are presented in Table 52. The LSV results show that the resulting materials have good catalytic properties. This is proof that the electrocatalysts doped with heteroatomic nitrogen, and mainly nitrogen functional groups in the graphene structure, are responsible for catalyzing the ORR reaction. The products are completely free of any metals and can potentially achieve wide application in the field of electrochemistry. Remarkably, the 1A-60 and 1A-90 exhibited prominent ORR activity with the most positive onset potential (Eonset) at 0.91 V and 0.92 V. The half-way potential (E1/2) was also highest for 1A-60 and 1A-90 compared to other samples. The prominent ORR activities of 1A-30 and 1A-60 in an alkaline medium may be benefitting from the synergistic effect of the structure and elemental composition. First, the unique few-layer architecture of graphene is conducive to exposing more of the active sites and facilitates the accessibility of oxygen. Second, the nitrogen heteroatoms can induce charge redistribution and facilitate the adsorption of oxygen and the consequent reduction reaction on carbon. The proposed materials’ composition is their advantage, because generally structures described for ORR application tend to be decorated with expensive, precious, or environmentally-unfriendly metals or metal oxides, e.g., Pd, Ru53, perovskie oxides54, Fe(II)55, Fe7C356, or Au57.In order to check the catalytic activity of the obtained materials in the oxygen reduction reaction, cyclic voltammetry and linear voltammetry measurements were taken. The ORR reaction is distinctive in metal-air batteries and fuel cells, therefore examining the catalytic activity of the produced nitrogen-doped materials was an important aspect of the work. In recent years, heteroatom-doped graphene has become an alternative to platinum-based materials owing to the heteroatoms’ improvement of electrochemical propertiesnly Fig. . Figure Chlorella vurgalis. Research indicates that the use of a natural N-reagent during synthesis in a microwave reactor in various solvents can contribute to the incorporation of nitrogen atoms into the graphene structure at a level of several percent. It can be concluded that ethyl alcohol and dimethylformamide are good solvents which contribute to the exfoliation of graphene layers and the production of pure graphene material. The high quality of the obtained material, without defects, shows that the method is a promising way of producing heteroatom-doped graphene foams. The optimal time for synthesis in the microwave reactor was 30 min and introduced mainly pyrrolic-N groups descend from N-precursor Chlorella vurgalis. The best electrochemical results were obtained for materials using ethanol as a reaction medium. The solvent also influenced the introduction of nitrogen functional groups into the graphene structure. The products showed good catalytic activity owing to the nitrogen functional groups introduced into the graphene structure, which are responsible for the catalytic properties in the oxygen reduction reaction. The tests proved their high activity towards ORR, i.e., the number of electrons n reached 3.46 for tested materials (no Pt added), while the analogous value for the C-Pt (20 wt% loading) reference was only slightly higher and equal 4. For pristine, i.e., non-N-doped graphene, the n value was only 2. It has to be underlined that the progress in electrochemical performance was achieved in an environment-friendly way, not only without the use of noble metals, but also with a reduction in energy consumption. Heteroatom doping of graphene materials is an alternative to more expensive materials based on metals, an alternative that can have promising properties for electrochemical applications and a huge impact on the potential use the composite materials in supercapacitors, metal-air batteries, or fuel cells.In summary, an effective method of obtaining N-doped graphene in a microwave reactor was developed, one which uses expanded graphite and Chlorella vulgaris. The compounds were mixed in a solvent with a 1:4 mass ratio of graphene to Chlorella vulgaris, then sonicated to obtain a better dispersion. The samples were loaded into a microwave reactor (Microwave 400) and heated under pressure to 180 °C, with the power of about 300–600 W. The microwaving duration was either 10, 30, 60, or 90 min; after this time, the samples were stirred. The obtained mass was then washed with distilled water to remove the solvent and dried at 120 °C overnight. Finally, samples were carbonized at 900 °C with a heating rate of 3 °C min−1 and then heated for 4 h under the flow of nitrogen (to provide an inert environment during carbonization). Reference samples were made using the same procedure, but without the addition of an N-precursor nor the carbonization process. For all samples without the addition of Chlorella vulgaris, the process in the reactor was maintained for 10 min. The reference samples obtained without carbonization, only the microwave process, are designated as 0A/B-T, where A and B describe the solvent and denote EtOH or DMF, respectively, while T is the time in minutes that the sample was held in the microwave reactor. Samples obtained with the addition of the N-precursor and using carbonization are designated as 1A/B-T, where A and B describe the solvent and mark EtOH or DMF, respectively, and T is the duration of time spent in the microwave.The production of N-doped graphene foams was based on a one-step process using microwave radiation in two dispersed solvents: ethyl alcohol (EtOH), and dimethylformamide (DMF). The starting materials used were expanded commercial graphite  and the natural green algae N-precursor, The atomic structure of the obtained materials was analyzed using a high-resolution transmission electron microscope  at an accelerating voltage of 200 kV. A scanning electron microscope operating at 30 kV  was used to determine the structure of the samples. The elemental composition of the materials was analyzed by means of a combustion elemental analyzer . Raman-microscopic spectroscopy analysis was performed using Renishaw InVia , excitation wavelength 532 nm at an ambient temperature. Nitrogen sorption isotherms were determined through nitrogen physisorption at 77 K in a volumetric apparatus ASAP 2010 . The attachment, bonding configuration, and compositional analysis of the material was carried out by X-ray photoelectron spectroscopy measurements . A monochromatic Al-Kα X-ray (1486.6 eV) was used as the operating excitation source. Atomic Force Microscopy (AFM) investigations were conducted using a Scanning Probe Microscope (SPM) apparatus produced by Veeco , working under ambient conditions.−2. Before electrochemical testing, the alkaline solution was saturated with O2/N2 for 20 min. The activity of the samples in an oxygen reduction reaction was evaluated by measuring cyclic voltammetry (CV), carried out at a scanning speed of 10 mV s−1, and linear voltammetry (LSV), at a scanning speed of 5 mV s−1 and rotation speed of 800–2800 rpm, in a solution of 0.1 M KOH at room temperature. The number of electrons (n) directly participating in ORR was calculated using the Koutecky–Levich equation (K–L):L is the current density limiting diffusion, and JK is the kinetic current density; ω is the angular velocity of the electrode; n is the number of electron transfers in the reaction; F is the Faraday constant ; C0 is the concentration of dissolved oxygen (1.2 × 10–6 mol L−1 in 0.1 M KOH); D0 is the diffusion coefficient of dissolved oxygen (1.9 × 10–5 cm2 s−1 in 0.1 M KOH); ν is the kinetic viscosity of the electrolyte for 0.1 M KOH, 0.01 cm2 s−1. The slope of the K-L graph can be obtained using Koutecky–Levich Eqs. . The standard measuring setup was made up of three electrodes—an Ag/AgCl electrode in 3 M KCl was the reference, a Pt electrode the counter, while the working electrode was glassy carbon (GC rotating disk electrode with a 5.0 mm diameter) onto which ink, with the catalyst, was applied. The catalyst ink was prepared basing on the dispersion of 2.5 mg of the test sample in a mixture of distilled water and ethanol (ratio 1:4) and Nafion (0.5 wt% Nafion). As a point of comparison, electrochemical studies were also performed on a commercial catalyst from Sigma Aldrich, platinum on graphitized Pt/C carbon (20 wt% Platinum). The prepared ink was pipetted onto glassy carbon in an amount of 15.63 µl and the electrode was dried afterwards. Catalyst content was about 0.4 mg cm


Correction to: J Foot Ankle Res (2020) 13: 44https://doi.org/10.1186/s13047-020-00411-1Following publication of the original article  the authIncorrect values: 20.3 ± 5.1(4–15) 11.0 ± 2.6(16–43)Correct values: 20.3 ± 5.1(16–43) 11.0 ± 2.6(4–15)


Here, we examined the KCa3.1 channel expression and its role in rat bone marrow‐derived MSC (BMSC) proliferation in response to mechanical stretch. Application of mechanical stretch stimulated BMSC proliferation via promoting cell cycle progression. Such mechanical stimulation up‐regulated the KCa3.1 channel expression and pharmacological or genetic inhibition of the KCa3.1 channel strongly suppressed stretch‐induced increase in cell proliferation and cell cycle progression. These results support that the KCa3.1 channel plays an important role in transducing mechanical forces to MSC proliferation. Our finding provides new mechanistic insights into how mechanical stimuli regulate MSC proliferation and also a viable bioengineering approach to improve MSC proliferation.Mechanical stimulation is an important factor regulating mesenchymal stem cell (MSC) functions such as proliferation. The Ca Here, we showed that application of mechanical stretch to rat bone marrow‐derived MSCs (BMSCs) significantly increased cell proliferation, mainly via altering cell cycle progression. Such mechanical stretch also up‐regulated the KCa3.1 expression at mRNA, protein and functional levels. Importantly, mechanical stretch‐induced stimulation of BMSC proliferation and alteration in cell cycle progression were prevented by pharmacological inhibition of the KCa3.1 channel or siRNA‐mediated knockdown of the KCa3.1 expression. Taken together, our results provide compelling evidence to support an important role of the KCa3.1 channel in mechanical stimuli‐induced stimulation of MSC proliferation, thus revealing a new molecular mechanism in transducing mechanical forces to regulate MSC proliferation and identifying a viable bioengineering approach to improve MSC proliferation.Mesenchymal stem cells (MSCs) have many promising applications in regenerative medicine.22.1Ca3.1‐specific siRNA (siKCa3.1) or control siRNA (siCTL) was described previously,Isolation of BMSCs from 30‐day‐old male Sprague‐Dawley rats, BMSC characterization and culture were previously described.2.25 on silicone chambers pre‐coated with collagen I (Becton Dickinson). When cells reached 80% confluence, the silicone chambers were mounted to a stretch device  and were exposed to stretch by 2.5%, 5%, 10% and 15% for 6, 12 and 24 hours. Cells cultured without stretch were used as static control (SC). To block the KCa3.1 channel, 100 nmol/L TRAM‐34 (Alomone) was added into the culture medium.Cells were seeded at density of 1 × 102.3Total RNA extraction and RT‐PCR were described in the Supplementary File. The primers used were listed in Table 2.4Ca3.1 protein expression was determined using fluorescence‐activated cell sorting (FACS) as previously described.Ca3.1 (Alomone). Isotype control IgG was used as control. The fluorescence intensity was determined by FACSCalibur (Becton Dickinson) and analysed using CellQuest software.The cell surface K2.5Cell proliferation was examined by determining the number of living cells using CCK‐8 kits according to the manufacturer's instructions (Dojindo). The absorbance at 450 nm was measured using a microplate reader (Thermo Scientific).2.6Cells, after being synchronized for 24 hours in serum‐free medium, were subjected to mechanical stretch for further 24 hours in media containing 10% foetal bovine serum. The cell cycle distribution was determined by FACSCalibur (Becton Dickinson) and analysed by ModFit software as previously described.2.7+ currents was performed using a HEKA amplifier (Lambrecht) as described previouslyCa3.1 channel currents were derived from TRAM‐34‐sensitive current components.Whole‐cell patch‐clamp recording of the K2.8post hoc Fisher's test as indicated. P < .05 was considered statistically significant.Data are presented as mean ± standard deviation, where appropriate. Statistical analysis was conducted using Student's test or one‐way ANOVA and 3We started with examining the effects of mechanical stretch on BMSC proliferation. We chose mechanical stretch from 2.5% to 15%, the most commonly used range in study of mechanical stretch‐induced regulation of MSC functions.Ca3.1 channel is critically engaged in MSC proliferation.Ca3.1 channel expression. Exposure to mechanical stretch of 5%‐15%, but not 2.5%, for 24 hours increased the expression of KCa3.1 at the mRNA level shown by RT‐PCR (Figure + currents was enhanced by membrane stretch induced using hypotonic solution (Figure Ca3.1 channel expression and activity. We finally investigated whether the KCa3.1 channel plays a role in mechanical stretch‐induced stimulation of BMSC proliferation. Treatment with TRAM34, a KCa3.1 channel‐specific inhibitor, prevented mechanical stretch‐induced increase in cell proliferation, without effect on cell proliferation under normal control condition (Figure Ca3.1 expression (Figure Ca3.1 channel or genetic depletion of the KCa3.1 expression prohibited mechanical stretch‐induced arrest of cell cycle in the G0/G1 phase (Figure Ca3.1 channel in mediating mechanical stretch‐induced stimulation of BMSC proliferation.As introduced above, the Kn Figure  suppress4Ca3.1 expression in BMSC (Figure Ca3.1 channel activity (Figure Ca3.1 channel with TRAM‐34 (Figure Ca3.1 expression (Figure Ca3.1 channel inhibited mechanical stretch‐induced alteration in cell cycle (Figure Ca3.1 channel in cell proliferation.Ca3.1 channel in rat BMSC had no effect on cell proliferation (Figure 2+‐activated K+ channels, including KCa3.1, in BMSCs is strongly species‐dependent,2+ increase and cell proliferation in BMSCs.[Ca3.1 channel activity (Figure Ca3.1 channel plays a critical role in coupling mechanical stimulus‐induced Ca2+ signal to activation of the downstream signalling pathways to stimulate MSC proliferation. It has been also shown that the KCa3.1 channel can regulates extracellular Ca2+ entry and thereby MSC proliferation.Ca3.1 channel is critically engaged in the transduction of mechanical forces to intracellular Ca2+ signalling and cell proliferation in MSCs.We here show that exposure to mechanical stretch stimulates BMSC proliferation Figure A, in supC Figure A‐D and, y Figure E. More i4 Figure F or siRNn Figure G strongle Figure H‐K. Prevn Figure F. It is in BMSCs., 20, 21 Ca3.1 channel in mediating mechanical stretch‐induced stimulation of BMSC proliferation, thus revealing a new molecular mechanism in the transduction of mechanical forces to MSC proliferation. Such a finding should be helpful in developing new strategies to maintain and stimulate the capacity of MSC proliferation and increase the cell number required for MSC‐based applications in regenerative medicine.In summary, this study shows a critical role of the KThe authors confirm that there are no conflicts of interest.YF developed and organized this paper. XJ, HS, XC, YH and YZ carried out experimental work. XJ and HS mainly drafted the paper and created the figures. PJ, CG, XG and YH assisted in data analysis and interpretation. LH J critically reviewed the manuscript. This manuscript is not under review elsewhere, and all authors read and approved the final manuscript. Click here for additional data file.


Lipomyces, the most 5' terminal intron position is occupied by one of three complex intervening sequences consistent of differently nested U2 intron units, as demonstrated in L. lipofer, L. suomiensis, and L. starkeyi. In L. starkeyi, the donor elements of the constituent introns are abutting and the complex intervening sequence can be excised alternatively either with one standard splicing reaction or, as a [D] stwintron, by two consecutive reactions. Our work suggests how [D] stwintrons could emerge by the appearance of new functional splice sites within an extant intron. The stepwise stwintronisation mechanism may involve duplication of the functional intron donor element of the ancestor intron.In primary transcripts of eukaryotic nuclear genes, coding sequences are often interrupted by U2-type introns. Such intervening sequences can constitute complex introns excised by consecutive splicing reactions. The origin of spliceosomal introns is a vexing problem. Sequence variation existent across fungal taxa provides means to study their structure and evolution. In one class of complex introns called [D] stwintrons, an  U2 intron is nested within the 5'-donor element of another  U2 intron. In the gene for a reticulon-like protein in species of the ascomycete yeast genus  Few U12-type introns are excised by the minor spliceosome). Differently from spliceosomal introns, group I and group II introns are ribozymes, which may or not need accessory proteins for self splicing. In eukaryotes, group I and group II introns are primarily restricted to mitochondrial and chloroplast DNAs.In the eukaryote nuclear genome, coding sequences are often interspersed with intervening sequences (introns). Introns have to be precisely excised from the primary transcript to generate the open reading frame that translates into the appropriate peptide. Excision of U2-type introns and splicing of the bordering exons is catalysed by the major spliceosome6. Previously, we have described a particular class of nested U2 introns, the stwintron, for which the excision of the internal intron is required to accomplish the subsequent excision of the external intron because the former interrupts one of the three conserved intron splicing elements of the latter, the 5'-donor, the sequence element around the lariat branch point adenosine, or the 3'-acceptor11 . Correct stwintron excision requires intron definition whereby the nearest 5’- and 3'-splice sites are paired across the intron prior to splicing16. This results in excision of the smallest possible U2 intron; in the case of a stwintron, this is thus the internal intron. In fungi, spliceosomal introns are generally quite small – often <100 nucleotide (nt). Nevertheless, one of the fungal stwintrons we have described is crucial to an authentic alternative splicing event11.Intervening sequences of any of the three main classes – group I, group II and spliceosomal – can be made up of more than one functional intron unitPezizomycotina taxa8. One possible mechanism by which such a stwintron could emerge involves the stepwise formation of new 5'- and 3' splice sites within an extant intron of sufficient size to evolve into two U2 introns 18 could occur during repair of an asymmetric DNA double-strand break (DSB) which would leave a 3' overhang. In an independent event, mutations in the central region of the host intron could result in functional 3'-splice sites. The new internal splice sites may pair to define the new U2 unit, whose subsequent excision is necessary for the formation of a functional donor for the second U2 definition and standard splicing reaction to properly remove all intervening sequences.Two [D] stwintrons conserved across whole classes of filamentous fungi are located at exactly the same intron position as a canonical U2 intron present in early divergent 11, we identified a  stwintron in the gene encoding a reticulon-like protein in the Pezizomycotina, one of the three Ascomycota subphyla. The gene product is a “protein of unknown function which associates with the endoplasmic reticulum” (protein family PF02453). While assessing the ancestry of this  stwintron, we encountered different complex intervening sequences (CIS) consistent of nested U2 introns in the orthologous gene in seven species of Lipomyces, a taxon of the Saccharomycotina, another subphylum of the Ascomycota. The parallel evolution of these three different CIS at the very same intron position in species of the same fungal genus supports the stwintronisation mechanism of stwintron evolution.In a previous paperTaphrinomycotina and in the family of the Lipomycetaceae (Saccharomycotina) is consistent with their reticulon-like genes being genuine orthologues of the one in Aspergillus nidulans (rtnA) and in other Pezizomycotina11. Figure Lipomyces genus (nine species) drawn from an extensive maximum likelihood tree of the reticulon-like protein in all Ascomycota (see Methods section for details). Lipomycetaceae occupy a unique phylogenetic position at the basis of the Saccharomycotina clade19. The species Lipomyces starkeyi harbours many more ORF-interrupting introns than ascomycete yeasts outside the Lipomycetaceae20. The intron density of L. starkeyi resembles that seen in Pezizomycotina rather than that apparent in other Saccharomycotina.The conservation of intron positions in multiple species of Saccharomycotina, the primary transcript of the gene for the reticulon-like protein harbours multiple introns in eight genome-sequenced Lipomyces. However, the intron content varies considerably from the maximum of five introns in L. starkeyi and three closely related species, as well as in L. lipofer , or species of Taphrinomycotina (position 3), or species from both these other subphyla (positions 4 and 5). The observed simplification of the intron-exon structure of the gene encoding the reticulon-like protein suggests that some early divergent Lipomyces species have lost introns rather than that new introns have been gained in the other taxa, completely independent of the massive intron loss observed in all other families of Saccharomycotina21.Exceptionally within the fer Fig. . L. japoLipomyces species. The second exon is either 162 or 165 nt (L. japonicus & L. lipofer) long. The extra codon is located upstream of the codon of the absolutely conserved Trp of the PF02453 domain  is disrupting the 5'-donor element of an 162-nt long external intron (5' GUGA | GU – 137-nt – ACUAAC – 10-nt – UAG) between A4 and G5. The splicing scheme is shown in Fig. L. lipofer stwintron is the first donor-disrupted stwintron of the  type as well as the first ever stwintron (sensu stricto) described outside the Pezizomycotina subphylum.In L. suomiensis and L. japonicus, we detected two potential canonical 5' donor sequences separated by one nt at the 5' extremity of the predicted CIS. Furthermore, a fully canonical 3'-acceptor and associated lariat branch point element could be identified within the CIS in both species. Sequence analysis of the mature messenger in L. suomiensis . Similarly, in L. japonicus, we predict that an 162-nt long canonical U2 intron (5' GUAAGU – 135-nt – ACUAAC – 12-nt – UAG) is disrupting an 124-nt long canonical U2 intron (5' GUAAGUG | A – 97-nt – AUUAAU – 10-nt – UAG) between G7 and A8. The whole CIS is 286 nt long and the two functional donor elements are identical in sequence (5'-GUAAGU) . A difference between the two species is that the constituting U2 introns in L. suomiensis are half the size of those in L. japonicus.In two species, sensu stricto as previously defined7, since the internal intron does not interrupt the donor element of the external intron; the two canonical donor elements are actually one nt apart from each other. In the primary transcript of the gene for the reticulon-like protein in L. suomiensis, the primary selected 5'- and 3'-splice sites are clearly those that define the smallest possible internal intron (intron definition). We can therefore consider such CIS of nested introns examples of a new class of fungal stwintron, the “stwintron sensu lato”.To excise these  CIS, two standard splicing reactions are necessary. This intron nesting does not constitute a [D] stwintron in L. starkeyi and the three closely allied species  whether the intervening sequence could be alternatively excised using either the one or the other donor element. Amongst the cDNA clones of matured mRNA generated, we indeed found two different ORFs, one of them containing six nt (GTACGT) more than the other (GenBank MN689086 & MN689087), demonstrating alternative splicing. Under our experimental conditions, fewer cDNA clones contained the GTACGT sequence. Subsequent inspection of the intervening sequence suggested the presence of a coupled lariat branch point element and 3'-acceptor roughly in its centre, of which the lariat branch point element could be the imperfect 5'-AUUCAU or the equally imperfect 5'-UUUAAU, associated with a 5'- CAG shortly downstream. If the internal splice signals were recognised by the spliceosome, one would expect a complex  intervening sequence of 252 nt, where an 120-nt long internal U2 intron (5' GUAAGU – 75-nt – AUUCAU – 14-nt – UUUAAU – 10-nt – CAG) is disrupting an 132-nt long external intron (5' GUACGU | A – 109-nt – GCUGAC – 7-nt – UAG) between U6 and A7 . However, if the spliceosome would ignore the imperfect internal 3' splice site halfway the intervening sequence but not the fully canonical proximal donor, one would expect that a 246-nt long canonical U2 intron (5' GUAAGU – 224-nt – GCUGAC – 7-nt – UAG) is excised in one standard splicing reaction , which is poorly conserved both in length and in sequence in ascomycete reticulon-like proteins.In the genes for the reticulon-like protein of genome-sequenced species of the 22. Some modes of autonomous intron gain could involve repair of double-strand breaks (DSB) by an end-joining mechanism26. The formation of a new stwintron from the sequences of an existing canonical intron can occur similar to the intronisation mechanism17, by the formation of new 5'- and 3'-splice sites within the parental intron at the chromosomal locus  could represent an intermediate stage in which the CIS can be removed by one- as well as by two subsequent U2 splicing reactions, as the internal imperfect 3' lariat branch point element is apparently functionally weak.The origin of spliceosomal introns is a vexing problem. Some commonly purported mechanisms involve mobile elements like transposons or group II introns of mitochondrial or bacterial origin, while other proposed mechanisms  are autonomous and have a strictly endogenous originpomyces D,5-,  stwintron in the gene for the fungal reticulon-like protein, we searched the DNA databases at the National Center of Biotechnology Information (NCBI) for homologous genes in other fungi. Additional homologues were found in the Saccharomycotina and Taphrinomycotina subphyla of the Ascomycota, the Blastocladiomycota, the Glomeromycota, the Mucoromycotina and the Mortierellomycotina , to verify the status of the Lipomycetaceae as the earliest divergent family of Saccharomycotina20. Most of these analyses yielded monophyletic clades for the nine sequenced Lipomyces: A representative maximum likelihood subtree is shown in Fig. Lipomyces genome sequence resources are: L. starkeyi (GenBank Whole Genome Shotgun Master accession: LSGR00000000.120), L. mesembrius (PPJS00000000.219), L. arxii (PPJT00000000.219), L. kononenkoae (PPJW00000000.119), L. lipofer (PPJX00000000.119), L. doorenjongii (PPJU00000000.119), L. suomiensis (PPJQ00000000.219), L. oligophaga (PPJR00000000.219) and L. japonicus (PPJV00000000.119).Species of Lipomyces clade shown in Fig. 28, applying the L-INS-i iterative refinement method trained on recognising one conserved domain with long extensions – appropriate for the PF02453 domain – with the BLOSUM80 scoring matrix. This MAFFT alignment was trimmed to 136 informative residues with Block Mapping and Gathering with Entropy software (BMGE version 1.12)29, using the BLOSUM40 similarity matrix with the block size set at 4. A maximum likelihood tree was then calculated from the trimmed alignment by PhyML (version 3.0) with automatic substitution model selection30 in default mode. The Smart Model Selection (SMS) software selected LG + G + I + F as the substitution model31 using the Akaike Information Criterion. Branch support was estimated by approximate likelihood-ratio tests32 implemented by the PhyML webserver. The inferred tree was drawn from the PhyML Newick output with FigTree (version 1.4.3: http://tree.bio.ed.ac.uk/software/figtree). The Lipomyces subtree was selected in FigTree (select clade option) and saved in a separate file, then exported as an interactive pdf. Final annotation was done in Adobe Illustrator.The phylogenetic tree at the basis of the monophyletic Lipomyces lipofer CBS 944 , Lipomyces suomiensis CBS 7251 , and Lipomyces starkeyi CCY 33-1-1  were used to verify the existence of a particular CIS in the primary transcript of the orthologous genes encoding a reticulon-like protein. Lipomyces kononenkoae CBS 2514 was also assessed in support of results obtained for L. starkeyi. All yeasts were maintained and cultivated on rich growth media listed in Supplementary Table –1 (rpm) for 16 h. The biomass was harvested by centrifugation at 10,000 rpm for 5 min then transferred to tubes with ceramic beads  and homogenised at 4000 rpm for 30 s with the help of a MagNA Lyser Instrument (Roche). Macherey-Nagel NucleoSpin kits were used for the extraction of genomic DNA (NucleoSpin Plant II) and total RNA (NucleoSpin RNA Plant) from the homogenate. Nucleic acids were quantified using NanoDrop technology (Thermo Scientific).For isolation of nucleic acids, yeast cultures were grown in 500-mL Erlenmeyer flasks with 100 mL medium in an orbital shaker (Infors HT Multitron) at 200 revolutions minReverse transcription was primed off 1 μg of total RNA with Oligo(dT) as the primer using the First Strand cDNA Synthesis Kit (Thermo Scientific). PCR reactions were performed in a 25 μL volume containing 4 μL of single strand cDNA, using gene-specific oligonucleotides  and cloned . Plasmid DNA was isolated using the NucleoSpin Plasmid EasyPure kit (Macherey-Nagel). Independent clones were sequenced over both strands using universal primers hybridising to the vector . All sequences determined were deposited at GenBank. For convenience, determined sequences of RNA stwintron splicing intermediates are also given in the Supplementary Information.Accession numbers for sequences determined during this study: MN689081–MN689090.Supplementary Information.


While control cells displayed robust puromycilation, TDP-43-ΔNLS transfected cells exhibited reduced ongoing protein synthesis. Next, by using a transgenic mouse overexpressing cytoplasmic TDP-43 in the forebrain (TDP-43-ΔNLS mice) we assessed whether cytoplasmic TDP-43 regulates global translation in vivo. Polysome profiling of brain cortices from transgenic mice showed a shift toward non-polysomal fractions as compared to wild-type littermates, indicating a decrease in global translation. Lastly, cellular level translational assessment by SUNSET was performed in TDP-43-ΔNLS mice brain slices. Control mice slices incubated with PURO exhibited robust cytoplasmic PURO signal in layer 5 neurons from motor cortex, and normal nuclear TDP-43 staining. Neurons in TDP-43-ΔNLS mice slices incubated with PURO exhibited high cytoplasmic expression of TDP-43 and reduced puromycilation respect to control mice. These in vitro and in vivo results indicate that cytoplasmic TDP-43 decreases global translation and potentially cause functional/cytotoxic effects as observed in ALS/FTD. Our study provide in vivo evidence (by two independent and complementary methods) for a role of mislocalized TDP-43 in the regulation of global mRNA translation, with implications for TDP-43 proteinopathies.TDP-43 is a major component of cytoplasmic inclusions observed in neurodegenerative diseases like frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). To further understand the role of TDP-43 in mRNA/protein metabolism and proteostasis, we used a combined approach with cellular and animal models overexpressing a cytoplasmic form of human TDP-43 (TDP-43-ΔNLS), recapitulating ALS/FTD features. We applied in HEK293 cells a method for labeling  TAR DNA-binding protein 43 (TDP-43) is a ubiquitously expressed, predominantly nuclear protein with multiple roles in RNA processing  recapitulates multiple features of TDP-43 proteinopathies of the ALS/FTD spectrum, including progressive neurodegeneration, gliosis, global gene expression changes and behavioral abnormalities encompassing the motor, cognitive and social domains  are virtually unknown. In this work, we investigated both in vitro and in vivo if cytoplasmic expression of TDP-43 modifies global protein synthesis, using two different approaches, polysome profiling and surface sensing of translation (SUnSET) technique.While most studies have focused on the regulation of global or specific translation using cell lines or primary neurons in culture, the consequences of 2. Cells (5 × 105 per well) were plated on poly-D-lysine-coated 12 mm glass coverslips in a 6-well plate, and grown for 24 h before transfection. Lipofectamine 2000 reagent (Invitrogen) was used for transfection of constructs (empty vector as control or hTDP-43-ΔNLS), according to the manufacturer's instructions. After 48 h, medium was replaced with fresh medium with or without puromycin  for 20 min and washed with PBS.Myc epitope-tagged hTDP-43-ΔNLS construct was generated in pcDNA 5/To plasmid (Invitrogen) using restriction sites BamHI and XbaI, as previously described  for 10 min, permeabilized with 0.2% Triton X-100 (Sigma) in PBS for 10 min, blocked with 5% powdered milk in PBS for 2 h, and incubated overnight with primary antibody at 4°C. Primary antibodies used in this study (diluted in 5% powdered milk in PBS) were as follows: anti-total-TDP-43  at a ratio of 500 μl buffer /10 mg tissue. Homogenates were then placed on ice for 10 min and centrifuged at 5,000 rpm for 10 min at 4°C. Supernatant, consisting in cytosolic extract containing polysomes, was collected and protein concentration was determined by bicinchoninic acid (BCA) method (Pierce). Sucrose gradient, ranging from 15 to 50%, were prepared in gradient buffer . Briefly, 2.4 ml 50% sucrose solution was added to a tube and chilled for 30 min at −80°C. The procedure was repeated with layers of sucrose solution at lower concentration  and stored at 4°C if used the next day. 0.5 to 1 mg of cytosolic protein extract were added to each gradient, and centrifuged in a SW40 rotor at 35,000 rpm for 2–4 h, at 4°C. Five hundred microliters aliquots were separated and absorbance at 260 nm was measured . A peak area ratio between polysomal (P) and non-polysomal  fractions was determined for each group.Cortices of control (non-transgenic) or hTDP-43-ΔNLS animals were homogenized in cold lysis buffer [15 mM Tris-HCl, pH 8.0, 300 mM NaCl, 15 mM MgClFive hundred microliters of sample from each fraction was subjected to methanol/chloroform protein extraction method and resuspended in sample buffer. Equal amounts of samples were resolved on 10% sodium dodecyl Sulfate polyacrilamyde gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes . After transfer, membranes were stained with Ponceau S for total protein load assessment, then blocked in 5% powdered milk in 0.1% Tween-TBS, and incubated in anti-total TDP-43 primary antibody overnight . Detection of primary antibody was performed with horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch), and ECL was used for blot development. Digital images were acquired using a G-BOX Imager (Syngene). Ponceau S staining was used as a control for equal protein loading in our immunoblot analysis of polysomal fractions, since typical housekeeping genes are barely detectable in polysome fractions . The brain was next glued onto a vibratome stage and acute sagittal slices (400 μm) were cut. Then, the slices were recovered at room temperature for an hour in ACSF . After recovery, slices were placed on a fine grid mesh in a petri dish and incubated in ACSF (C- group) or ACSF supplemented with 5 μg/μl puromycin  at a final volume of 30 ml. Incubation time was 45 min under continuous aeration with carbogen (95% O2/ 5% CO2). Slices were then rinsed in ACSF and fixated in 4% paraformaldehyde (Sigma-Aldrich Inc.) in 0.1 M PBS for 24 h at 4°C. Next day, slices were washed three times in 0.01 M PBS for 10 min each under continuous agitation in an orbital shaker, incubated with 10% sucrose in 0.01 M PBS for 2 h at 4°C and stored in 30% sucrose-0.01 M PBS at −20°C until use.SUnSET labeling in brain slices was adapted from Cook et al. , with soBrain slices containing the motor cortex [approximately bregma 1.18 mm, . In non-transgenic  mice incubated with puromycin (C+), each cellular area was defined in the green channel corresponding to puromycin signal, as well as background areas between cells; the same approach was employed in hTDP-43-ΔNLS animals incubated with puromycin (ΔNLS+) but in the red channel . For non-transgenic mice incubated in ACSF (C-), a circular area centered in each red nucleus and background areas between cells were quantified. Background signal was normalized to individual cell areas, and a ratio between raw cell intensity signal and normalized background signal was calculated. The signal from individual cell populations and averaged signal per animal were compared between experimental groups.t-test when comparing only two groups on one measure. A one-way ANOVA followed by Tukey's multiple-comparison post hoc test was used when comparing three groups. Prism Version 8 (GraphPad Software) was used for statistical analysis and differences were considered to be statistically significant when the probability value was < 0.05.Data are expressed as means ± SEM. Statistical analysis was performed using a standard unpaired two-tailed student's +) show a decrease in ongoing protein synthesis  in ALS/FTD pathogenesis.Protein synthesis is an essential process for all cellular types, but neurons are particularly sensitive to proper control of this metabolic process due to their high polarization and the demands of plasticity mechanisms  as demonstrated by immunoprecipitation, and that TDP-43-ΔNLS co-localizes in the cytoplasm with RACK1 and rpS6, suggesting that TDP-43 associates to translational machinery  leading to translational modulation events such as eIF2α phosphorylation .in vitro and in vivo SUnSET studies, image processing, and data analysis. MB provided key reagents, funding, and helped design and analyze the in vitro experiments. SC and LI wrote the manuscript. LI conceived and supervised all aspects of the project and obtained study funding. All authors read and approved the final manuscript.SC, LL, and LI designed the study. SC and AV performed the cell culture experiments. LL performed the polysome profiling and immunoblot experiments. SC performed 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.