Contractile activity is a fundamental property of skeletal muscles. We describe the establishment of a “feeder-supported in vitro exercise model” using human-origin primary satellite cells, allowing highly-developed contractile myotubes to readily be generated by applying electrical pulse stimulation (EPS). The use of murine fibroblasts as the feeder cells allows biological responses to EPS in contractile human myotubes to be selectively evaluated with species-specific analyses such as RT-PCR. We successfully applied this feeder-supported co-culture system to myotubes derived from primary satellite cells obtained from sporadic inclusion body myositis (sIBM) patients who are incapable of strenuous exercise testing. Our results demonstrated that sIBM myotubes possess essentially normal muscle functions, including contractility development, de novo sarcomere formation, and contraction-dependent myokine upregulation, upon EPS treatment. However, we found that some of sIBM myotubes, but not healthy control myotubes, often exhibit abnormal cytoplasmic TDP-43 accumulation upon EPS-evoked contraction, suggesting potential pathogenic involvement of the contraction-inducible TDP-43 distribution peculiar to sIBM. Thus, our “feeder-supported in vitro exercise model” enables us to obtain contractile human-origin myotubes, potentially utilizable for evaluating exercise-dependent intrinsic and pathogenic properties of patient muscle cells. Our approach, using feeder layers, further expands the usefulness of the “in vitro exercise model”. However, this widely used model has limitations, especially as regards human myotubes derived from primary satellite cells. For example, given that human myotubes generally display a very flat and spread-out morphology, attaching firmly to the culture substratum, human myotubes usually have much poorer contractile activity than murine myotubes originating from the C2C12 muscle cell line under conventional culture conditions even with EPS treatment8. Moreover, while inoculation with a very high cell-density of human-origin myoblasts (e.g. more than five-fold higher than murine C2C12 cells) slightly improves their contractility development and myokine upregulations upon EPS7, it is usually difficult to obtain sufficient numbers of human satellite cells from biopsy samples, particularly from those of patients suffering from rare muscle diseases such as sporadic inclusion body myositis (sIBM)9. Thus, a useful and simple method allowing the generation of human myotubes capable of vigorously contracting in the culture system, which would markedly boost not only basic muscle research but also the diagnostic use of muscle cells obtained from patient biopsy samples, a very limited resource, is eagerly anticipated.Myotubes obtained under conventional culture conditions normally exhibit little or no contractile activity due to a lack of excitation stimuli from motor neurons. Recently, “in vitro exercise models” with contractile myotubes generated by applying electric pulse stimulation (EPS) in culture11 and is pathologically characterized by sarcoplasmic rimmed vacuole formation along with protein aggregates of p6212 and mis-localized cytoplasmic TAR DNA-binding protein of 43 kDa (TDP-43)13 as well as mitochondrial abnormalities in affected muscle fibers15. This disease results in slowly progressive and often asymmetric weakness with finger flexors and knee extensors being predominantly affected16. To date, the basic cellular functions of muscles from sIBM patients have not been well investigated, though one study demonstrated that primary sIBM myoblasts show no apparent morphological abnormalities and are fully capable of forming normal myotubes that can be properly innervated with rat embryo spinal cord in an in vitro co-culture system17. Since the outcome of this degenerative muscle disease is progressive loss of muscle contractile activity, analysis of sIBM muscle cells, ideally under contractile (exercising) conditions, is necessary for gaining meaningful insights into the pathogenic mechanisms underlying this disease.sIBM is an acquired progressive muscle disease that typically affects patients older than 50 years of ageWe herein succeeded in establishing a new “in vitro exercise model” applicable to human-origin muscle cells, which is based on a co-culture system with species-different (murine) fibroblasts as feeder cells efficiently supporting the contractility development of human myotubes upon appropriate EPS treatment. We applied this “feeder-supported in vitro exercise model” to sIBM myotubes derived from the primary satellite cells of sIBM patients and examined their functional muscle properties in response to EPS-evoked contraction by comparing them with those of myotubes derived from healthy human subjects. Our results indicate sIBM myotubes to have basically the same muscular properties upon EPS-evoked contraction as normal myotubes, though some of sIBM myotubes often exhibited cytoplasmic TDP-43 accumulations in response to EPS-evoked contraction, which was not detected in healthy controls. While such an exercise-loading test would not be feasible for patients suffering from severe diseases of muscular dysfunction, such as sIBM, the culture system developed in this study should allow us to better understand a patient’s muscle cell condition, particularly responses to actual contraction loading, potentially providing important diagnostic information allowing for customized therapies.7, myotubes derived from human skeletal muscle myoblasts (HSMM) displayed a flattened myotubular morphology reflecting their firm adhesion to the substratum. In the absence of 3T3L1 feeder cells, though human myotubes appeared morphologically well-differentiated, they exhibited minimal contraction in response to EPS . Their contraction activity, as assessed by the movement index during the final ~ 15 min. of the EPS treatment, was dependent on an EPS intensity of at least until 20 V  confirmed that nuclei of the myotubes were all positive for anti-HNA in the co-presence of murine 3T3L1 fibroblast feeders in the same culture Fig. A. This rity Fig. B.Figure Taking these data together, we can reasonably assume that although human myotubes alone without feeders exhibited poor or little contractile activity even when subjected to EPS treatment, feeder layers of fibroblasts provide a favorable substratum for human myotubes, resulting in better development of their contractile activity and de novo sarcomere structure formation upon EPS treatment.panels e–h) on feeder cells was equivalent to that of control satellite cells (panels a–d), but no spontaneous contractile activity was detected in either sIBM (panels e and f) or control myotubes (panels a and b) when EPS treatment was lacking. However, EPS treatment efficiently endowed vigorous contractile ability in both control (panels c and d) (Supplementary Movie panels g and h) (Supplementary Movie panels c and d). These results indicate that the sIBM myotubes fully retained EPS-dependent de novo sarcomere formation and the consequent vigorous contractile activity lasted for at least the duration of the 24-h EPS treatment.Taking advantage of the in vitro exercise model co-cultured with feeder fibroblasts (feeder-supported in vitro exercise model), we next examined the contractile activity of myotubes derived from primary satellite cells obtained from biopsy samples of healthy subjects or sIBM patients. As shown in Fig. 2 and in vitro7. We therefore examined CXCL1 mRNA expression levels in human myotubes of the feeder-supported in vitro exercise model . Hence, consistent with the movement index results, sIBM myotubes on the feeders have similar responsiveness to EPS-evoked contraction in terms of contraction-inducible CXCL1 upregulation.CXCL1 has been recognized as a myokine that is upregulated in response to muscle contractile activity in vivodel Fig. C. Specie20, and immunostaining analysis using anti-phospho-TDP-43 and anti-p62 antibodies confirmed that biopsy samples of all three of our sIBM patients possessed myofibers containing pTDP-43-positive and p62-positive granular structures . Unlike healthy control myotubes, some of the sIBM myotubes exhibited, especially after EPS-dependent contraction, obvious TDP-43 accumulations in cytoplasm , though such accumulation of TDP-43 was not evident in sIBM myotubes without EPS treatment. It should be noted that nuclear TDP-43 localization was also observed in 3T3L1 fibroblasts. In this set of experiments, we further examined a total of 6 individual human myotubes originating from 3 sIBM patients and 3 healthy control subjects and evaluated the TDP-43 deposits by counting numbers of myotubes possessing intracellular TDP-43 accumulations. All three healthy control myotubes only rarely displayed TDP-43 deposits, regardless of EPS-evoked contraction. In contrast, some populations of sIBM myotubes had TDP-43 deposits after EPS-evoked contraction, especially those derived from sIBM patients #4 and #6, while there were fewer deposits in those from patient #5  or healthy control subjects , and the total p62-positive areas in myotubes also did not differ among the experimental conditions .In contrast to EPS-induced alterations in TDP-43 localization in sIBM myotubes, the p62 localization pattern was not influenced by EPS-evoked contraction in any of the 6 myotubes studied, i.e., regardless of whether they were derived from sIBM patients . It should be also noted that all three sIBM patients similarly exhibited the typical sIBM phenotype of rimmed vacuoles. Thus, future studies are warranted to elucidate potential pathogenic contributions of abnormal TDP-43 accumulation in sIBM myotubes as well as to clarify the mechanism underlying the abnormal TDP-43 accumulation triggered by EPS-evoked contraction in some, but not all, sIBM myotubes. Nevertheless, our data strongly suggest the existence of intrinsic vulnerability, affecting the sIBM myotube itself, that emerges only upon actual contractile activity but not under routine culture conditions17.Taking our present in vitro data and the aforementioned clinical in vivo data together, abnormal extracellular TDP-43 accumulations are indeed sporadic and such TDP-43 abnormalities might occasionally be induced even under cell culture conditions wherein there are no other cell types such as immune cells that may directly and/or indirectly be related to the pathogenesis of sIBM40 and abnormal p62 accumulation has thus been implicated in the pathogenesis of sIBM41, due to a mechanism involving impaired autophagic clearance of aggregation-prone proteins including amyloid precursor protein43. However, unlike the very distinctly different TDP-43 localizations in sIBM and healthy control myotubes, the p62-positive small granule-like (puncta) structures were similar, being scattered throughout the cytoplasm in both sIBM and healthy control myotubes regardless of whether or not of EPS treatment had been applied  culture conditions45, we can only speculate that our culture conditions during EPS treatment likely influenced the autophagic responses in which p62 might participate. In addition, we examined only one EPS treatment condition because this EPS protocol consistently provides highly reliable and reproducible outcomes from in vitro exercise models7. Nonetheless, the current results do not allow us to assert the significance of the aforementioned TDP-43 abnormality observed in some sIBM myotubes in this study. Moreover, the underlying mechanism of abnormal TDP-43 accumulation triggered by EPS-evoked contraction in some, but not all, sIBM myotubes, remains unclear. These crucial issues thus need to be addressed in future studies by examining large numbers of myotubes originated from sIBM patients. As the limitations of this study, it should also be noted that control and sIBM samples were taken from different muscle tissues, i.e., the biceps brachii or rectus femoris muscles from two male and one female sIBM patient, all 69–86 years of age, while the subscapularis muscles were sampled in three healthy male subjects 60–68 years of age. Thus, while their ages are relatively well matched, with all being categorized as elderly, background features, i.e., sex and tissue type differences, might have influenced the phenotypes of myotubes observed in the present study. This issue needs to be carefully evaluated in a future study. Accordingly, further improvements of the feeder-supported in vitro exercise model, aimed at appropriately mimicking the pathogenic processes of myopathic diseases, are essential. Prospectively eliciting such processes in response to a contractile burden under certain experimental culture conditions such as various nutritional states, different exercise intensities/modalities, in the presence of other cell types, and so on, would likely provide valuable clinical and research insights.p62 serves as a cargo receptor in the degradation of ubiquitinated proteins and organelles during autophagic responsesied Fig. A,B. In bied Fig. C, though27 as it can regenerate and mimic muscle tissue-like environments that contain numerous fibroblastic cells. In this case, co-culture with fibroblastic cells from the same individual with sIBM, instead of mouse fibroblasts, as a feeder, may establish situations more pathologically similar to the disease state by allowing cells to communicate with each other as observed in the samples derived from patient muscle biopsy specimens.In the present study, we focused on contractility development in human-origin myotubes, and its impacts, in response to actual contraction, on intrinsic muscle properties. For sIBM myotubes particularly, this co-culture system may also be useful for elucidating higher-order functional interplays between contracting muscle cells and surrounding fibroblastic cellsIn summary, we established a “feeder-supported in vitro exercise model” applicable to sIBM myotubes derived from the primary satellite cells of sIBM patients. This newly established “feeder-supported in vitro exercise model” enables us to readily produce contractile human-origin myotubes that can be subjected to further experimental and diagnostic analyses to gain details regarding exercise-evoked biological responses in vitro. Thus, our approach, using feeder cells, further expands the usefulness of the “in vitro exercise model”.Dulbecco’s modified Eagle’s medium (DMEM), and Ham’s F-10 medium were purchased from Fujifilm Wako Pure Chemical Corp. . Penicillin/streptomycin, and trypsin–EDTA were obtained from Thermo Fischer Scientific . Cell culture equipment and rectangular 8-well plates were obtained from BD Biosciences  and Thermo Fisher Scientific, respectively. SUMILON Cell-disk LF1 was from Sumitomo Bakelite Co. LTD . Calf serum (CS) and fetal bovine serum (FBS) were obtained from BioWest . Matrigel was obtained from Corning . Unless otherwise noted, all chemicals were of the purest grade available from Sigma Chemical  or Fujifilm Wako Pure Chemical Corp.2 as previously reported46. Primary satellite cells were cultured in a growth medium containing DMEM/Ham’s F10 mixture supplemented with 20% FBS, 1% penicillin–streptomycin, 1% chicken embryonic extract , and 2.5-ng/ml basic fibroblast growth factor  at 37 °C under a 5% CO2 atmosphere. Mouse 3T3L1 fibroblasts were maintained in DMEM containing 4.5 g/l glucose supplemented with 10% CS, 4 mM l-glutamine, 30 μg/ml penicillin, and 100 μg/ml streptomycin (growth medium) at 37 °C under a 5% CO2 atmosphere.Human skeletal muscle myoblasts (HSMM) (Cat.# CC-2580) were purchased from Lonza  and were cultured following the vendor’s instructions. Primary human satellite cells were obtained by fluorescent-activated cell sorting (FACS) from muscle biopsy tissues at Tohoku University Hospital under the approval of the Ethics Committee of Tohoku University, and written informed consent was obtained from all subjects (see below). The primary human satellite cells were amplified and stocked in liquid N5 cells onto an 8-well plate. Three hours after seeding, human myoblasts (activated satellite cells) were seeded onto the feeder layer of 3T3L1 fibroblasts at a density of 3 × 105 cells in each well of an 8-well plate in 3 ml of growth medium. For immunofluorescent analysis, 3T3L1 fibroblasts were seeded onto a Cell-Disc LF in a 24-well plate at a density of 1 × 104 cells. Three hours after seeding, human myoblasts (activated satellite cells) were seeded onto the feeder 3T3L1 fibroblasts on the Cell-Disc LF in 1 ml of growth medium at a density of ~ 6 × 104 cells. Two days after plating, differentiation was induced by switching the growth medium to DMEM supplemented with 5% horse serum, 30 μg/ml penicillin, and 100 μg/ml streptomycin (differentiation medium). The differentiation medium was changed every 24–48 h during the 7–8 days of differentiation.For the feeder layer, 3T3L1 fibroblasts were seeded at a density of 0.5 × 102. In the present study, 24 h in total of EPS  were applied and the cells were then harvested for analyses.The differentiated human myotubes on feeder 3T3L1 fibroblasts in 8-well plates or on Cell-Disc LF being transferred into an 8-well plate, were placed in a chamber for EPS . EPS  was applied to the cells in the C-Dish using a C-Pace 100 pulse generator (IonOptix). DMEM containing 2% CS supplemented with 200% amino acids (Sigma) was used during the EPS treatments, as previously reported7. Briefly, high-quality images of cells were taken sequentially with a CCD camera  and a dissection microscope (Olympus CKX41) equipped with a 20× objective lens during EPS. We obtained the movies at completion  of the total 24 h of EPS treatment applied for the contractile activity evaluations. The differential images were obtained by subtracting the relaxation image from the contraction image. Given that the difference in pixel intensity between the first (contraction) and successive (relaxation) images is due to changes in the movement of scattering objects, the overlaid images of these subtracted images indicate moving parts. The calculated average intensity of the differential image indicates the amount of myotube movement (movement index). Three different fields under each culture condition were used for evaluation of the movement index.The index of movement was calculated by the differential image subtraction method with a slight modification, as we reported previouslyAfter the experimental treatments, cells were washed with PBS and fixed for 20 min with 2% paraformaldehyde in PBS containing 0.1% Triton X-100, then washed and blocked in PBS containing 5% CS and 1% BSA at room temperature. For immunofluorescence analysis, we used anti-human nuclear antigen (HNA) antibody , anti-caveolin 3 antibody , anti-sarcomeric α-actinin , anti-TDP-43 , and anti-p62/SQSTM1  as the first antibodies, and Alexa Fluor 488-conjugated anti-mouse IgG and Alexa Flour 594-conjugated anti-rabbit IgG as the secondary antibodies (Thermo Fischer Scientific) at 1:100 and 1:1000 dilutions, respectively, in a solution of 1% BSA in PBS. The samples were mounted on glass slides with Vectashield  and observed with a confocal fluorescence microscope  with an oil-immersion objective lens  and ASW v.1.3 software (Olympus). The fluorescence of DAPI, Alexa 488, and Alexa 555 was excited at 405, 488, and 543 nm laser wavelengths and detected through BA430-470, BA505-525, and BA560-600 nm bandpass filters, respectively. The pinhole diameters are set automatically according to the selected dyeing method, and imaging was performed at 8.0–10 μs/pixel (Scan speed) for 1024 × 768 (4:3) or 1024 × 1024 (1:1) pixels applying a sequential scan mode with Kalman filtering. The number of myotubes displaying cytoplasmic TDP-43 aggregations and the total number of myotubes, as assessed by the sarcomeric α-actinin staining profile for discriminating feeder 3T3L1 fibroblasts, were counted in each field, and the ratio of aggregated TDP-43-positive myotubes vs. the total number of myotubes was evaluated. Images were imported into Adobe Photoshop 6.0  for processing. Three independent experiments were performed under each condition.For evaluating TDP-43-positive nuclei and intracellular TDP-43 accumulations, we used Fiji ImageJ software . Briefly, we first defined the myotube boundary according to the anti-sarcomeric-α-actinin immunofluorescent signal, and then obtained all the nuclei (by DAPI) and TDP-43-positive nuclei according to their respective immunofluorescent signals. The number of all nuclei in the myotube was used as the denominator, and the number of all TDP-43-positive nuclei was used as the numerator to obtain the ratio of TDP-43-positive nuclei in the myotube. For evaluating cytoplasmic TDP-43 accumulation, we used the Image calculator function of Fiji ImageJ software. Briefly, we first determined the entire TDP-43-positive area and the TDP-43-positive nuclear area of the myotubes by delineating sarcomeric-α-actinin staining and DAPI staining, respectively. Then, we subtracted the TDP-positive nuclear area from the total TDP-43 area to obtain the fluorescent intensity of the TDP-43 accumulated in cytoplasmic regions. Finally, we counted numbers of total myotubes and the myotubes exhibiting cytoplasmic TDP-43 accumulation to determine the ratio of cytoplasmic TDP-43-positive myotubes. For the p62 area, we calculated the p62-positive puncta area to the total myotube area using Fiji ImageJ software.−∆CT method with reference genes. The following primers for qRT-PCR analyses were employed; for human CXCL1, 5ʹ-GCT TGC CTC AAT CCT GCA TC-3ʹ and 5ʹ-GGT CAG TTG GAT TTG TCA CTG T-3ʹ; for human IL6, 5ʹ-ATC TGG ATT CAA TGA GGA GAC T-3ʹ and 5ʹ-TGT TCC TCA CTA CTC TCA AAT CTG-3ʹ; for human TDP-43, 5ʹ-GAG AAG TTC TTA TGG TGC AGG TC-3ʹ; 5ʹ-GCT CAT CTT GGC TTT GCT TAG-3ʹ; for human p62/SQSTM1, 5ʹ-GCG TAG AAT TGC AGG TCT CTG T-3ʹ and 5ʹ-TCA CTT GTT TTG CTG CCC TAA ATG-3ʹ; for human RPLP0, 5ʹ-GGA AAC TCT GCA TTC TCG CT-3ʹ and 5ʹ-GCA AGT GGG AAG GTG TAA TCC-3ʹ.Total RNA was extracted from cells employing TRI reagent  and cDNA was then synthesized using a Transcriptor First Strand cDNA synthesis kit with oligo-dT primers . Next, qRT-PCR was performed with SsoAdvanced Universal SYBR Green Supermix, and detected with a Bio-Rad CFX Connect Systems . The relative expression levels of the target genes were calculated using the 215. As the healthy controls, we used intact subscapularis muscle specimens from three male patients, ranging in age from 60 to 68 years, who had agreed to undergo biopsy during surgical repair of arthroscopic rotator cuff tears46.The three patients, who had been clinically diagnosed with sIBM at Tohoku University Hospital, ranged in age from 69 to 86 years and had no family history of muscle diseases. The basis of the sIBM diagnosis was the presence of rimmed vacuoles in muscle biopsy tissue. The distribution of muscle weakness, creatin kinase activity, and the results of electrophysiological studies were evaluated during the disease course. Clinical data obtained from all sIBM patients, two males and one female, who had been hospitalized between 2015 and 2016 in the Department of Neurology of Tohoku University hospital, were evaluated47, with mouse monoclonal antibodies (diluted to 1:200) raised against p62  and mouse monoclonal antibodies  raised against phosphor-TDP-43 .Immunohistochemical staining was carried out using the avidin–biotin complex immunostaining method as previously described46. This study was approved by the Tohoku University Hospital Institutional Review Board , and written informed consent was obtained from all participants. All experiments were performed in accordance with relevant guidelines and regulations.Human satellite cells of both the three sIBM patients and the three healthy subjects were isolated from muscle tissue biopsy specimens, as previously reportedα (clone 16A1). The negative set included blood markers CD11b and CD45, and endothelial markers CD31 and CD34. While CD34 is known to be expressed by the majority of mouse satellite cells48, human muscle-derived CD34+ cells are myogenic and adipogenic, whereas CD34− cells are myogenic but not adipogenic49. We therefore used CD34 as a negative selection marker. Human satellite cells were defined as single live mononuclear CD11b−CD31−CD34−CD45−CD56+ cells. FACS was performed on a FACS ARIA II flow cytometer (BD Biosciences). Cells were cultured in a growth medium and cultured at 37 °C in a 5% CO2 atmosphere. When cells reached 60–80% confluence, adherent cells were split onto a new Matrigel-coated 15-cm dish to expand the activated satellite cells. Activated satellite cells (myoblasts) were suspended in Cell Banker  and stored in liquid nitrogen.Briefly, the muscle tissue was minced and digested with collagenase, filtered through a 70-μm cell strainer  and then subjected to immunostaining for FACS. Cells were incubated with an Fc receptor blocking solution and then labeled with fluorescein isothiocyanate (FITC)-conjugated anti-CD45 (clone HI30), FITC-conjugated anti-CD11b (clone ICRF444), FITC-conjugated anti-CD31 (clone WM59), phycoerythrin (PE)/Cy7-conjugated anti-CD34 (clone 581), allophycocyanin (APC)-conjugated anti-CD56 (clone MEM-188), and PE-conjugated anti-PDGFRt-test or ANOVA with Tukey’s multiple comparison test, and p values < 0.05 were considered to indicate a statistically significant difference unless otherwise specified. Data are expressed as means ± SE unless otherwise specified.Statistical analyses were performed using Student’s Supplementary Figures.Supplementary Video 1.Supplementary Video 2.


As COVID-19 took the world by storm, primary care clinicians (PCCs) played a critical role in identification and management of this disease. Yet, knowledge around COVID-19 is constantly evolving, leaving clinicians with many unanswered questions. We sought to examine what questions PCCs had about COVID-19 and whether there were any trends over time. We sought to examine what questions PCCs had about COVID-19 and whether there were any trends over time. We collected questions from PCCs during 4 live virtual 60-minute continuing medical education (CME) panel discussions on COVID-19 led by infectious disease experts from November 2020 to February 2021. Questions were independently sorted and analyzed by 2 MDs using constant-comparison and tie-break methodology. A total of 600 questions pertaining to COVID-19 were collected across 4 sessions. Top questions asked by PCCs ranked in descending order related to the following topics, with most common themes listed in parentheses: 1. Vaccines  2. Medication-Specific Treatment  3. Testing  4. Other Management  5. Personal Protective Equipment . [Table 1] The percentage of questions around vaccination increased from 5% of total questions in October 2020 to 67% in February 2021. Questions related to Treatment declined from 20% to 6%, Testing declined from 21% to 3%, Other Management declined from 6% to 1% and PPE increased from 3% to 8% during this period. Table 1. Top 5 topics of questions listed in descending order of frequency across all 4 COVID-19 panel sessions.Table 2. Percentage of questions in the top 5 topics for each of the 4 COVID-19 panel sessions, with associated trendline.PCCs nationally have gaps in knowledge around COVID-19 which can impact clinical decision-making. Based on our analysis of questions submitted by PCCs to infectious disease experts in a CME setting, the greatest gaps in knowledge were around vaccination, treatment, and testing with vaccination showing the greatest shift in interest over time.All Authors: No reported disclosures


Background: Recurrent or persistently active sarcoidosis is a risk factor for permanent organ damage. Whether this damage is due to accumulated focal injuries or progressive disease extent is not known, as the natural history of chronic inflammation in sarcoidosis is poorly characterized. The objective of this study is to determine the pattern of disease in recurrently active sarcoidosis.Methods: We identified patients with recurrent cardiac sarcoidosis (N = 21) retrospectively from an imaging database, and with recurrent cutaneous sarcoidosis (N = 17) from a prospective registry. The longitudinal patterns of cardiac sarcoidosis were established by findings on cardiac positron emission tomography scans, and of cutaneous sarcoidosis by the validated Cutaneous Sarcoidosis Activity and Morphology Instrument clinical scoring system. Patterns of recurrent disease were compared to baseline findings.Results: Recurrent sarcoidosis occurred in a nearly identical pattern and distribution as baseline disease, and spread of disease was rarely observed for both cardiac and cutaneous sarcoidosis: 97% of heart segments positive on recurrence scans were positive on baseline scans, and only one new region of facial disease was observed. In some cases, recurrence followed years of apparent remission.Discussion: Across phenotypes, and across a long period of follow-up, the extent of sarcoidosis was stable in spite of fluctuations in disease activity. For patients with a demonstrated history of recurrent disease affecting critical organs, our findings support the need for long-term follow-up. Sarcoidosis is an inflammatory disease of unknown etiology, marked by the formation of granulomas in affected tissues. The diagnosis is established by compatible clinical and histopathology findings, following the exclusion of other causes of granulomatous inflammation . While tKey immunopathologic events in sarcoidosis include an influx of CD4+ T cells, the release of inflammatory cytokines, and the formation of epithelioid granulomas at sites of disease. The oligoclonal expansion of CD4+ T cells and granuloma features support the fundamental assumption that sarcoidosis is antigen driven , 7. Yet,Treatment with immunosuppression can improve symptoms and the function of organs with active sarcoid inflammation. However, for patients with chronically active disease, improvements are often tenuous and several studies have demonstrated a high rate of recurrence following withdrawal of immunosuppression –12. Whet18F]fluorodeoxyglucose positron emission tomography (FDG-PET) imaging and expert visual inspection, respectively.With the hypothesis that the extent of recurrently active sarcoidosis is stable, we serially examined patients with chronic cardiac sarcoidosis and characterized longitudinal disease patterns. To test whether our observations are restricted to a single organ phenotype, we also measured the pattern of recurrently active lymph node disease and, in a separate cohort with cutaneous involvement, of recurrently active cutaneous sarcoidosis. These phenotypes were chosen as they are amenable to anatomical disease tracking via serial assessments of disease activity with [18F]FDG-PET studies for suspected active myocardial disease from 2006-2018 . Subjects were accrued from two sources: (1) a clinical database of patients with sarcoidosis who underwent three or more thoracic FDG, a glucose analog  at each visit, with disease activity mapped via the cutaneous sarcoidosis activity and morphology instrument (CSAMI) . For thiCalculations were performed using Graphpad Prism 8.0 . Where indicated, data were expressed as means +/-SD.From the 39 subjects with cardiac sarcoidosis who had undergone at least three valid cardiac PET scans, 21 had evidence of recurrent disease. Nearly all subjects (18/21) were White, and 71% (15/21) were male . The diaAcross the 21 subjects with recurrent cardiac sarcoidosis, a combined 163 diagnostic PET scans were available for review. At baseline, cardiac sarcoidosis was highly prevalent throughout the left ventricle (LV). On average, 9.6 segments  on baseline scans demonstrated increased FDG uptake. Certain regions of the LV were more likely to be affected than others . IncreasThe first negative PET scan following a positive baseline or recurrent PET scan defined a resolution event. For over half of these events , resolution was evident on the first scan following the initiation of treatment. However, 18 resolution events followed multiple sequential positive PET scans. For most (16/18) of these events of delayed resolution, the presence of persistently positive PET scans was associated with failure to initiate treatment, or initiation of an insufficient regimen as clearance was eventually achieved following an increase in treatment. Subject 13 , was an For the majority of subjects (17/21), the distribution of recurrent sarcoid inflammation occurred at anatomical locations that were grossly identical to the location of original lesions . Of the Of the 104 patients in the cutaneous sarcoidosis database, 38 had three or more visits. Of those, 17 had a CSAMI score decrease of >5 at a follow-up visit, followed by recurrent disease involving the face. Facial sub-compartments assessed on serial skin exams included the nose, cheeks, perioral regions, and ears. Patients had on average 2 regions affected by sarcoidosis. Peri-nasal disease  was most common, followed by disease involving the cheek . Perioral disease was observed in 9/17 subjects at baseline, 7 of whom had disease recurrence at this site. Ear involvement was less common (3/17 at baseline). For 94% (16/17) of cases, recurrently active lesions occurred in the same region as initial disease activity . Direct A total of 16 subjects had PET scan findings consistent with recurrent lymph node inflammation; 12 were from the recurrent cardiac sarcoidosis cohort and an additional four subjects were identified from the 37 subjects with cardiac sarcoidosis screened for recurrent disease . While rWe found that recurrent cardiac sarcoidosis has a strong predilection to recur in a distribution similar to previously active disease. The same finding was observed for patients with recurrently active cutaneous sarcoidosis. The finding that the extent of disease remains remarkably stable over time was observed over many years of follow-up. Our data suggest that, for a given organ affected by sarcoidosis, persistent or recurrent inflammation, with the potential transition to irreversible fibrosis, are more concerning risks than local expansion of disease. For approximately half of our cardiac sarcoidosis cohort, recurrence events either did not trigger initiation of treatment or were followed by an insufficient regimen such that multiple serial cardiac PET scans were positive, in some cases resulting in active disease spanning several years. For these cases, disease extent still remained stable. Therefore, even in the setting of persistently active inflammation, cardiac sarcoidosis does not appear to have a high propensity to progress beyond the initial extent of disease. This novel finding has profound implications for understanding and treating chronic sarcoidosis.Lymph node involvement is common in sarcoidosis, and many of our patients had their diagnosis established by lymph node biopsy. However, the specificity of PET imaging for nodal sarcoidosis activity is unknown, and increased nodal FDG uptake may be seen in a variety of inflammatory conditions. In contrast to the findings for cardiac and cutaneous sarcoidosis, the distribution of thoracic lymph node activity was more variable over time, with a higher rate of new FDG avid lymph node stations observed on recurrently positive PET scans. This finding may be due to a lower specificity of PET imaging for nodal sarcoidosis activity, with the possibility of false positive findings on follow-up scans. Alternatively, the lymph node network truly may be more prone to a progressive extent of disease in patients with chronic sarcoidosis.The stability of lesional boundaries in cardiac and cutaneous sarcoidosis contrasts with the possibility that the systemic burden of sarcoidosis may increase over time, which has been observed in previous studies of patients followed longitudinally . An incrWhile immunosuppression therapy is often successful in suppressing inflammation, disease recurrence following cessation is a vexing outcome in both cardiac and cutaneous sarcoidosis . The optThe mechanism of recurrent sarcoidosis is unknown. In a study on recurrent Lofgren's disease, repeat exposure to antigen was invoked to account for episodes of recurrence occurring remote from the time of initial disease . In contOne cautious interpretation of our findings is that the containment of antigen is not compromised over time. Altered immunity has been implicated in antigenic spread in tuberculosis, another granulomatous disease , 29. YetThere are several important limitations of this study. The ability to anatomically localize inflammatory lesions by imaging techniques, such as PET, is inherently imprecise. We cannot rule out that minor or microscopic changes in the extent of disease occurred. We found that the average extent of disease was lower at disease recurrence compared to baseline scans. However, treatment was often started or increased upon disease recurrence, which may chemically constrain the extent of inflammation. This single site study has a modest cohort size; larger studies are needed to more robustly establish the patterns of chronically active disease. For two subjects with cardiac sarcoidosis, biopsy was deferred after a careful risk-benefit assessment, and the diagnosis was established on other clinical grounds. While a common situation, the diagnosis is less certain in such cases. Finally, we recognize the stark differences in demographics between our cohorts of cardiac and cutaneous sarcoidosis. These differences raise concern that identification of cardiac sarcoidosis, which is often done through screening studies initiated by health care providers, may be influenced by demographics. Differences in access to health care may also play a role.We focused on the evaluation of organs which tend to have countable and localizable lesions, and our findings may not be generalizable to all phenotypes of sarcoidosis. An especially important question un-answered by our study is the nature of recurrent lung sarcoidosis. Tracking individual lung lesions, which are often tiny, clustered, or indistinct, is inherently less precise than tracking the more distinct cardiac and cutaneous lesions. Yet, a better understanding of how pulmonary sarcoidosis progresses is critically important as chronic lung disease, along with cardiac involvement, accounts for the majority of fatal outcomes in sarcoidosis . While wIn conclusion, the evolution of sarcoidosis has been a key unknown of the disease process, and a challenge for clinical management. In our cohorts of cardiac and cutaneous sarcoidosis, the extent of inflammation in recurrently active disease was strikingly stable. The anatomical fidelity of recurrent disease activity raises the possibility that the sarcoid antigen is contained but not cleared. A paradigm shift away from sole reliance on long-term immunosuppression to the development of therapeutics which target immune clearance or halt fibrotic transformation is needed. Our findings also indicate that, for patients with demonstrated disease recurrence or with high-stakes organ involvement, long-term follow-up is warranted even for those who appear to be in clinical remission soon after stopping treatment.Upon request, the raw data supporting the conclusions of this article will be available by the authors.The studies involving human participants were reviewed and approved by University of Pennsylvania Institutional Review Board. Written informed consent for participation was obtained for patients in the cutaneous sarcoidosis cohort, but was not required for the cardiac sarcoidosis cohort in accordance with institutional requirements. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images included in this article.KP had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors have made substantial contributions to the work reported in this manuscript, including study design, data collection and analysis, and writing and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Graphene nanoribbons are a type of graphene characterized by remarkable electrical and mechanical properties. This review considers the prospects for the application of graphene ribbons in biomedicine, taking into account safety aspects. According to the analysis of the recent studies, the topical areas of using graphene nanoribbons include mechanical, chemical, photo- and acoustic sensors, devices for the direct sequencing of biological macromolecules, including DNA, gene and drug delivery vehicles, and tissue engineering. There is evidence of good biocompatibility of graphene nanoribbons with human cell lines, but a number of researchers have revealed toxic effects, including cytotoxicity and genotoxicity. Moreover, the damaging effects of nanoribbons are often higher than those of chemical analogs, for instance, graphene oxide nanoplates. The possible mechanism of toxicity is the ability of graphene nanoribbons to damage the cell membrane mechanically, stimulate reactive oxidative stress (ROS) production, autophagy, and inhibition of proliferation, as well as apoptosis induction, DNA fragmentation, and the formation of chromosomal aberrations. At the same time, the biodegradability of graphene nanoribbons under the environmental factors has been proven. In general, this review allows us to conclude that graphene nanoribbons, as components of high-precision nanodevices and therapeutic agents, have significant potential for biomedical applications; however, additional studies of their safety are needed. Particular emphasis should be placed on the lack of information about the effect of graphene nanoribbons on the organism as a whole obtained from in vivo experiments, as well as about their ecological toxicity, accumulation, migration, and destruction within ecosystems. Graphene nanoribbons (GNRs) are narrow strips of graphene composed of repeating hexagonal carbon cells, up to 50 nm wide and up to several dozens of micrometers long, depending on the synthesis method . GraphenMoreover, the structure and physical properties of GNRs vary significantly, depending on the synthesis method. In fact, GNRs obtained via different methods are very different and have little in common. Today, three main approaches to obtaining GNRs exist :1. Electron-beam lithography and photolithography: This method makes it possible to obtain single-layer GNRs on the substrate surface; however, the fields of application of lithographically obtained GNRs are very limited. In addition, the ribbons obtained by the lithographic method have jagged edges ,13,14.2. Bottom-up synthesis from polycyclic molecules: This method includes multistage organic synthesis based on the cyclization of previously synthesized polymer chains. This method provides very narrow ribbons with an atomically precise edge configuration ,16,17,183. Unwrapping carbon nanotubes (CNTs): The third approach is based on longitudinal opening or cutting of multiwalled carbon nanotubes (MCNTs) ,20. ThisTypical characteristics of GNRs are determined by transmission electron microscopy (TEM), atomic force microscopy (AFM), Raman-scattering spectroscopy, and X-ray photoelectron spectroscopy (XPS).As previously noted, GNRs can be considered as quasi-one-dimensional graphene strips. This makes them similar to a large class of conjugated polymers, for which the synthesis conditions and the method of film formation determine the performance characteristics ,23. The GNRs with armchair-type and zigzag-type edges are the most common ones. AGNRs are characterized by a wide band gap that varies with width, while ZGNRs are predicted to have smaller bandgaps with localized edge states that are magnetic and show great potential for spintronic applications ,28,29,30Thus, to take full advantage of the exceptional characteristics of GNRs for practical applications, it is necessary to take into account the fabrication method and the type of spatial structure.Remarkable electrical and mechanical properties of GNRs make them a promising material for biological and medical applications ,34,35,36According to the available information, GNRs in biomedicine are mainly used to create various ultra-small devices such as molecular sensors, photo-, thermo-, and acoustic detectors, sequencers, drug and gene delivery vehicles, and tissue engineering constructions .The research works aimed at the integration of GNRs into devices are going in several directions, but all of these devices require connections and heterojunctions .8 V/m at the heterojunction, these materials are applicable for the photovoltaic industry and electronics 85], the The analysis of the cytotoxicity of films consisting of chitosan, alginate, and 2.5 wt % GNRs (CHI/f–GNR–ALG) obtained by unzipping MWCNTs on the mouse fibroblasts (L929) showed the sample cytocompatibility. In particular, L929 is able to proliferate on the surface of CHI/f–GNR–ALG (200 µL) . On thisForeman et al.  analyzed50 values for chemo-photothermal therapy toward U87 glioma cells than the IC50 values in traditional chemotherapy. They found that PL–PEG–O-GNRs was excreted from the body via the renal pathway in the urine, and the hematological analysis showed that this nanomaterial was not toxic. The authors consider PL-PEG-O-GNRs as potential nanocarriers of drugs to develop an effective cancer treatment strategy that will not only increase the effectiveness of therapy, but also reduce the risk of side effects of the nanocarrier in the body.The authors of reference  investigThus, there is evidence that the cytotoxicity of GNRs can be significantly reduced by their edge or surface chemical modification. For example, doping with nitrogen, oxidation, coating with biocompatible polymers such as PEG or chitosan give an obvious increase in biocompatibility. The hydrophilization of carbon nanomaterials is a versatile strategy to provide their bioavailability and applicability in biomedicine. This is especially important to take into account when developing bioengineering constructs based on GNR, which should have minimal cytotoxicity.According to Mbeh et al. , O-GNRs The results of the study  showed tThe same authors showed that O-GNR–PEG-DSPE at concentrations of 20, 80, and 160 μg/mL caused a low concentration-dependent deformation of erythrocytes, which did not lead to hemolysis . There wIn the study , the cytChowdhury et al.  demonstrA comparative analysis of the toxicity of single-layer reduced O-GNRs (rO-GNRs) and reduced graphene oxide sheets (rGOS) in relation to human mesenchymal stem cells (hMSCs) showed significant cytotoxic effects of rO-GNR at a concentration of 10 μg/mL of after 1 h of exposure, while rOGS showed the same degree of toxicity at a concentration of 100 μg/mL after 96 h. The main mechanism of rGOS action was assumed to be oxidative stress, which causes minor damage to the cell membrane. While the outflow of RNA from hMSCs showed that neither the formation of reactive oxygen nor significant damage of the membranes cells can explain the destruction of cells caused by rO-GNRs. The results also showed that rO-GNRs could penetrate cells and cause DNA fragmentation and chromosomal aberrations even at a low concentration of 1.0 μg/mL, after a short exposure time of 1 h ,95.Talukdar et al.  investigFor the imaging and phototherapy of human glioblastoma (U87MG), Akhavan et al. functionalized rO-GNRs with polyethylene glycol (rO-GNR–PEG). Cytotoxic and genotoxic effects on the cells depended on the concentration of rO-GNR–PEG. When the cells were incubated in the dark with 100 μg/mL of rO-GNR–PEG for 24 h, more than 72% of cell death and more than 29% of DNA fragmentation were observed. At a lower concentration (1 μg/mL), cell death and DNA fragmentation decreased to about 11% and 7%, respectively ,98.According to the considered researches on cyto- and genotoxicity, the possible mechanisms of the toxic action of GNRs can be suggested mechanical damage to cell membranes; ROS production, which leads to the inhibition of proliferation, induction of apoptosis, autophagy, and DNA fragmentation; chromosomal aberrations .It should be noted that the studies of the toxicity of GNRs for the whole organism have not yet been carried out, excluding the work of Lu et al. . HoweverSimilar to other carbon nanomaterials, GNRs can enter the environment not only as part of the nanoindustry products, but also during natural processes, for example, during the combustion of organic matter. There is no information on the environmental toxicity of GNRs for ecosystems in the analyzed researches. An exception is the work of Lalwani et al., which investigates the oxidative biodegradation of O-GNRs and rO-GNRs by lignin peroxidase (LiP) . LiP is The studies that are concerned with the closest chemical and structural analogues of GNRs–CNTs and graphene indicate their significant effects on various types of living organisms. A large number of studies demonstrate the toxic effects of carbon nanomaterials in relation to bacteria ,107,108,Based on this, it can be assumed that GNRs can also have a significant impact on components of ecosystems, especially microorganisms, and it can be bioaccumulated and migrate within ecosystems. These issues certainly warrant further study.GNRs demonstrate the greatest prospects in the field of biomedicine, particularly in creating nanodevices for the biomolecules detection and single-molecular techniques. In addition, GNRs are of interest as gene and drug delivery vehicles. There are researches that prove the possibility of using GNRs in tissue engineering.Practical applications of GNRs in direct contact with the human body are limited by their potential toxicity. A number of researchers have noted the damaging effects of GNRs, including cytotoxicity and genotoxicity. Moreover, the toxicity of nanoribbons is higher than those of such chemical analogs as O-GNPs. According to several studies, the possible mechanisms of toxicity are as follows: the ability to induce ROS production and autophagy; inhibition of proliferation; induction of apoptosis; DNA fragmentation; chromosomal aberrations. However, other authors have not confirmed any toxic effects of GNRs in experiments with human cell lines. The discrepancy in the observed results can be explained by differences in the methods of synthesis and structure of GNRs, their functionalization by different groups, and the use of different concentrations. In addition, different cell lines exhibit individual responses to exposure to nanomaterials. Furthermore, in order to obtain consistent results, the unification of the nanotoxicological experimental conditions is needed.The chemical modification of the surface of GNRs with hydrophilic groups can significantly increase their bioavailability and biocompatibility.There is an evidence of the biodegradability of GNRs in the environment. By analogy with other carbon nanomaterials, GNRs can be toxic to living organisms of various species. They can be bioaccumulated and migrate through ecosystems, but there are currently no data on this.In general, this review allows concluding that GNRs, as components of high-precision nanodevices and therapeutic agents, have good prospects for application in biomedicine, but the limiting factors for the use of GNRs are their high hydrophobicity and insufficiently studied toxicity. There is a lack of information on the effect of GNRs on the whole organism using in vivo experiments, as well as on environmental toxicity, accumulation, migration, and destruction within ecosystems.


Microaggressions, a concept originally introduced for ethnic minorities, represent subtle day‐to‐day discrimination, damaging the psychological health and well‐being of lesbian, gay, bisexual, and transgender individuals as well. This study aimed to assess whether microaggressions occur in psychotherapists’ assessments of clients who identify as either lesbian or transgender woman when compared with those identifying as heterosexual woman.N = 5) and microaggressive (N = 5), used to determine a clinical impression of the client. A repeated measure analysis of variance was conducted to understand the likelihood of clients of different gender identity and sexual orientation receiving microaggressions.The study included a sample of 135 licensed psychotherapists (110 cisgender women and 25 cisgender men). Participants were presented with an audio file of a woman introducing herself during her first therapy session. Three versions were presented: a transgender, a lesbian, and a heterosexual client. Participants were asked to assess the clinical relevance of 10 questions defined as neutral (Participants were more prone to consider microaggressive questions relevant where the client identified as either lesbian or transgender.The findings highlighted a bias against lesbian and trans women in evaluating the relevance and usefulness of clinical information while making a psychological assessment of a case. In an eTransgender people also experience situations in which they necessarily have to come out , which can expose them to an increased risk of discrimination and its consequences  link of the patient's gender identity or sexual orientation to the problematic or symptomatic aspects presented. We also expect that the self‐assessed emotional arousal generated by a patient belonging to a marginalized group will moderate the relevance attributed to microaggressive questions for the transgender and lesbian condition. Moreover, starting from the evidence of the relation between emotional arousal and automatic responses, we hypothesized highly activated clinicians to be more prone to use microaggressions toward sexual and gender minorities than clinicians at moderate levels of arousal.2To test whether psychotherapists could enact microaggressions in the context of the first consultation with a client, we had to create ad hoc questions that were relevant to the presented case. For this purpose, we asked 84 early career psychologists to listen to the audio files, that were then used in the main study, and produce up to 10 questions they would have asked the client to have a better understanding of the clinical case.Participants generated 575 questions in total, with an average of 6.8 questions each. Three different raters (blind to the study's hypotheses) independently classified each of the items as nonmicroaggressive, possibly microaggressive, or microaggressive. The raters were trained to recognize microaggressions; they were provided with the definition of microaggressions several examples of microaggressions targeting sexual and gender minorities. Approximately 30.9% of the sample produced at least one microaggressive question (rated as microaggression by two out of three raters). Only the questions from the original pool of items on which all three raters agreed upon were used to construct the questions for the following study.33.13.1.1η2partial = 0.25) suggested a sample size of N = 120 for a 3 between‐participants × 2 within‐participants analysis of variance (ANOVA). In total, 135 licensed psychotherapists took part in the study, all of whom self‐identified as cisgender, White Italian citizens, with 110 (81.5%) identifying as female. Other demographic characteristics of the sample are summarized in Table An a priori power analysis was conducted to estimate the required sample size using G*Power  introducing herself and describing her problems (related to panic) during the first session with the participant  to 7 (extremely relevant). The questions reflected both microaggressive and neutral themes. The questions generated from the pilot study were used to build five microaggressive (Cronbach's α = 0.90) and five neutral questions (Cronbach's α = 0.71) for the current study  Hayes, . Finally44.1To compare the relevance of microaggressive and neutral questions depending on the sexual identity of the client, we computed a 3  × 2  ANOVA with the first‐factor as between‐participants and the second‐factor as within‐participants.F  = 119.36, p < 0.001, ηp2 = 0.47. Independent from the experimental condition, neutral questions were considered significantly more relevant  than microaggressive questions . The ANOVA did not yield a significant main effect of condition: F  = 1.76, p = 0.18, ηp2 = 0.03. However, in line with the hypothesis, a significant interaction effect between condition and type of questions emerged: F  = 14.56, p < 0.001, ηp2 = 0.18. As displayed in Figure M = 5.70, SD = 0.75), than in the transgender client condition , t(86) = 2.22, p = 0.03, d = 0.48, 95% confidence interval [CI] [0.05–0.90]. The analysis revealed no differences between cisgender lesbian  and transgender conditions, t(95) = 0.92, p = 0.36, and between cisgender straight and lesbian conditions, t(83) = 1.36, p = 0.18. The post hoc comparisons of the relevance of microaggressive questions showed no difference between the cisgender lesbian  and transgender conditions , t(95) = 1.68, p = 0.09. Crucially, microaggressive questions were rated as significantly less relevant for the cisgender straight  than for the cisgender lesbian client, t(83) = 2.26, p = 0.03, d = 0.49, 95% CI [0.06–0.93], and the transgender client, t(86) = 3.94, p < 0.001, d = 0.85, 95% CI [0.41–1.29].The analysis suggested a main effect for the type of questions: F , 132 = 14.2α = 0.78), suggesting that both positive and negative items of the scale measured the same construct. A more theoretical reason is based on the argument that the positive affect and negative affect subscales of the PANAS both measure the latent construct of emotional arousal . At a high level of arousal ( = 2.55), therapists rated microaggressive questions more relevant for cisgender lesbian and transgender women than for cisgender straight women . At low levels of arousal ( = 1.85), the difference between cisgender straight and cisgender lesbian/transgender conditions was not significant .To ascertain whether emotional arousal influenced the relevance attributed to microaggressive questions, a moderation model was carried out through PROCESS  Hayes, . In this4.3F  = 0.001, p = 0.97, or any interaction effects with gender . The same results were obtained introducing sexual orientation as an independent variable . We also controlled for the effect of the therapists’ theoretical background  and years of practice on relevance evaluations. No interaction or main effects of theoretical background were identified , or for years of practice .To ascertain whether the therapists’ individual differences influenced their responses, we controlled for the effect of the therapists’ gender and sexual orientation. The first ANOVA yielded no significant main effect for gender, 5The aim of this study was to investigate the presence of microaggressive responses when psychotherapists work with clients of a marginalized sexual orientation or gender identity . The findings highlighted a bias in evaluating the relevance and usefulness of information while making a psychological assessment of a case. Data showed the effect of the client's gender identity and sexual orientation in rating the relevance of microaggressive questions by the therapists. Such questions were considered less important for the normative straight cisgender client and more important for the less conforming clients (cisgender lesbian and transgender). Conversely, asking general questions about symptoms, life events, and mental health history was considered less relevant for the assessment in the transgender client condition compared to the cisgender straight client condition.Results indicated that both cisgender lesbian and transgender women were equally targets of microaggressions. In addition, neutral questions were considered significantly less relevant only in the transgender condition compared with the cisgender straight condition. This result could be explained by considering the long history of pathologization of transgender identities. Once a client is labeled as transgender, they are immediately “diagnosed” by some of the psychotherapists that probably still rely on a pathologizing understanding of transgender identities. Thus, the main issues and complaints they bring to the consultation fade into the background as the therapist no longer needs additional information. Such bias relies on specific assumptions based on cisgenderism and heteronormativity .6In spite of these limitations, the study presents numerous strengths, including investigating microaggressions directed toward gender/sexual minorities “in vivo,” with a sample of licensed and experienced psychotherapists. This approach, rather than relying on the reported experiences of LGBT patients in therapy, allows observing directly the therapists' response to the client's disclosure of information. The use of an audio file in which the client introduces herself identifying as lesbian, trans, or heterosexual, allows the understanding of the intrinsic and implicit functioning of microaggressions through the approach of the therapist. Furthermore, the inclusion of emotional arousal among the study variables allows spreading lights on other dimensions that can potentially be targeted in the training of mental health professionals on LGBT issues.https://publons.com/publon/10.1002/jclp.23140The peer review history for this article is available at


The relative free volume fraction rf gives a good explanation of the variation for surface resistivity, melting temperature, and thermal stability, and the variation of tensile strength and thermal conductivity agree well with the results of interfacial interaction intensity β. The results showed that rf and β have a significant effect on the properties of the obtained graphene/PE composites, and the effect on the properties was revealed.The quantitative characterization of microstructure is most desirable for the establishment of structure-property relationships in polymer nanocomposites. In this work, the effects of graphene on the microstructure, mechanical, electrical, and thermal properties of the obtained graphene/polyethylene (PE) composites were investigated. In order to reveal the structure-performance relationship of graphene/PE composites, especially for the effects of the relative free volume fraction ( Polyethylene (PE) is one of the most important commercial polymer materials. In the PE family, low density PE has a wide application in our daily life due to its low cost and good flexibility ,2. When rf) of the obtained composites, and the interfacial interaction (β) between graphene and the polymer matrix. As β determines the dispersion state of graphene, it has a dramatic influence on graphene’s stress dispersion and phonon scattering. In addition, the rf determines the transport performance, such as electrical conductivity, thermal stability, and gas/liquid barrier properties [rf and β), and the correlation between these parameters and properties for graphene/PE nanocomposites.The key factor that influences the properties of graphene/polymer composites is the microstructure, including the relative free volume fraction . Positrons are preferentially localized in the cavity of the free volume hole. According to the free volume hole model, the pick-off annihilation lifetime τ3 is related to the cavity size. When the cavity size of the free volume hole increases, the opportunity of electron seen by o-Ps decreases, leading the lifetime of o-Ps to become longer [Positron annihilation lifetime spectroscopy (PALS) is a useful tool in determining the atomic scale defects and free volume hole for a wide variety of materials ,29. The e longer .τ2 is assigned to the positron annihilation in various vacancies [I2) is related to the interfacial interaction in the polymer composites. According to the simple mixture rule, if there is no interfacial interaction between polymer matrix and filler, I2 only comes from the annihilation in the matrix and filler, respectively. In this case, I2 is the linear correlation with the content of filler. Actually, the interfacial interaction always exists in polymer composites, thus deviations occurred between the theoretical results and the experimental results [β was introduced to investigate the interface between the polymer matrix and the filler [Recently, the PALS technique can not only be used for the probe of the free volume hole, but also for the characterization of the interfacial interaction. The second lifetime acancies . The cha results ,32. Conse filler ,34. Thisrf and β had a significant effect on the mechanical, electrical, and thermal properties of the obtained graphene/PE composites, and their effect on the properties was revealed.In this work, the PE incorporated with different contents of graphene were prepared by premixing followed by hot pressing. Firstly, the interfacial interaction of the obtained graphene/PE composites was qualitatively characterized with traditional infrared spectroscopy. In order to give a deep insight into the microstructure, PALS was used to determine the free volume hole characteristics and interfacial interaction intensity of graphene/PE composites. The results showed that the Graphene was purchased from Suzhou TanFeng Graphene Tech Co., Ltd. . Low density polyethylene  was purchased from Aladdin Chemical Reagent Co., Ltd. . n-Hexane was obtained from Sinopharm Chemical Reagent Co., Ltd.  without purification.Graphene/PE composites with different graphene concentration (0~2.0 wt%) were prepared by premixing followed by hot pressing. Following typical procedure, the graphene was dispersed into 40 mL n-Hexane using a bath sonicator for 1 h. Then, 8 g of PE powder was added into the dispersion with vigorous stirring for 2 h and the mixture was sonicated for 1 h. The obtained mixture was centrifuged at 4000 r/min for 0.5 h and the precipitate was dried at 60 °C under vacuum to obtain a constant weight. Finally, the obtained mixture was pressed at 140 °C, 20 MPa for 10 min with a d = 0.5 mm steel mould.Fourier transform infrared (FTIR) spectra were recorded on an Avatar 360 Nicolet instrument  by measuring the powder mixture. The mechanical properties of the graphene/PE composites were measured by a tensile testing machine . Five pieces of each sample were tested to obtain average values. The surface resistance of the obtained composites was measured with an ultra-high resistance micro-current tester . The thermal conductivity of the composites was measured using thermal conductivity test equipment . The Differential scanning calorimeter (DSC) curves were recorded with a DSC-200-F3  from 30 to 160 °C, with a heating rate of 10 °C/min. Thermogravimeter (TG) analysis was conducted with a TG-209-F3  under nitrogen atmosphere with a heating rate of 10 °C/min.−1, the C-H bending vibration signal at 1471 cm−1, and the C-H rocking vibration signal at 721 cm−1—are present in the spectrum of PE [−1 observed in the composite, and this change in the C-H bending vibration signal is evidence supporting the occurrence of CH–π interactions between PE and graphene sheets [To demonstrate the chemical structure of pure PE and reveal the interfacial interaction between PE and graphene, the FTIR spectra of PE and 1 wt% graphene/PE composites were shown in um of PE . All sete sheets ,35. Acco22Na (15 μCi) was sealed in a 7 μm thick Kapton foil and sandwiched between two identical pieces of sample for measurement. One million counts were recorded for each spectrum, which was resolved into three components by a PATFIT fitting program. The long lifetime component τ3 was used to calculate the volume of free volume hole (fV) according to the following equations:To quantitatively reveal the effects of graphene on the microstructure of graphene/PE composites, the PALS was recorded. The PALS was carried out by a conventional fast–fast coincidence spectrometer at room temperature. The positron source R represents the radius of the cavity, and ΔR = 1.656 Å [o-Ps (I3) is related to the concentration of the free volume hole. The fraction (f) of free volume hole was calculated as: 1.656 Å ,33. The C can be approximately regarded as a constant [−3; the relative free volume fraction (rf) is defined as:constant ,36, withτ3), free volume hole size (fV), the formation probability of o-Ps (I3) and the relative fraction (rf) of the free volume hole as a function of graphene content were shown in τ3 than that of pure PE, suggesting that the obtained composites have a lower mean size of free volume hole, which is consistent with the results of PVA/rGO composites [τ3 and fV for the composites were observed containing 0.25 wt% graphene. This can be ascribed to the highly exfoliated and well dispersed graphene at low content, where the large surface area provided by the graphene is beneficial to the formation of CH-π interaction  is opposite to the o-Ps lifetime, which has also been observed in the PVA/rGO composites reported by Wang and co-workers [I3. However, with a further increase of graphene content, this disruption of graphene gets weak due to the agglomeration of graphene, which results in the decrease of free volume hole concentration and I3 [As show in -workers . This can and I3 ,33.rf) is present in rf decreases with the increase of graphene content (31.63% to 28.38%), meaning that graphene sheets limit the moving space of PE chains [Based on the analysis of the free volume hole size and the free volume hole concentration, the relative free volume fraction (E chains . These rI2) change of the second lifetime can be used to analyse the interfacial interaction intensity in the composites. The interfacial interaction determines the dispersion state of graphene; thus, it has a dramatic influence on its stress dispersion. For the description of the interfacial interaction, PALS is more useful than FTIR spectra. The FTIR can only determine the type of interfacial interaction , while PI2 only originated from positron annihilation in graphene and PE, which should be a linear correlation to the content of graphene. Actually, interfacial interaction exists in most polymer composites, and variation with the change of nanofiller content. Consequently, the interfacial interaction parameter (β) was introduced to reveal the interaction intensity between graphene and the PE matrix, and the β can be calculated according the following equation [According to the simple mixture rule, if no interfacial interaction exists between the graphene and the PE matrix, equation ,38,39,40G and P represent the graphene and PE, respectively; W is the graphene weight fraction; the I2 of graphene here is 94.78% [β as a function of the graphene content is shown in β (1.99), indicating the strongest interfacial interaction. With the increase of graphene content, the β decreased gradually, which is due to the aggregation and poor dispersion of graphene sheets at high loading [fV, which will have a significant effect on the mechanical property and thermal conductivity of the obtained composites.The superscripts s 94.78% . The int loading ,37, Thisβ against graphene content  of the obtained composites [rf can produce a high-density interfacial region, restricting the mobility and relaxation of PE chains, which is of benefit for the formation of a conductive network, thus lower surface resistivity composites were obtained with a lower rf. Similar phenomena have also been observed in the PC/rGO composites [According to electrical percolation theory, graphene sheets provide percolated pathways for electron transfer, which impart electrical conductivity to the composites. However, the improved efficiency of graphene sheets dramatically depends on the relative free volume fraction  characterization of PE and graphene/PE composites . The results of FTIR absorption spectra revealed that the CH-π interaction between PE and graphene can be formed in the processing. The CH-π interaction can serve as a sacrificial bond to dissipate stress, leading to the interaction intensity β having the same trend as the tensile strength. The highest values of β (1.99) and tensile strength (11.35 MPa) were found for the composites containing 0.25 wt% graphene. The decreased rf is beneficial for the formation of a conductive network, thus the lowest log(surface resistivity) (9.65) was obtained for those composites possessing the lowest rf (28.38%). The strong interaction intensity β for the 0.25 wt% graphene/PE composites can scatter phonons and damp the vibration amplitude, inducing a higher thermal resistance. As the content of graphene increased from 0 to 2.0 wt%, the rf decreased from 31.63% to 28.38%, making movement of the polymer chain more difficult. This leads to an increase of mT from 109 °C to 112 °C. As the low rf can prevent the heat transfer and the diffusion of pyrolysis products, the greatest thermal stability was obtained for the composites possessing the lowest rf (2.0 wt% graphene/PE). These results give deep insight into the relationship between structure and performance for polymer composites; future work will focus on the establishment of quantitative relationships.In summary, the graphene/PE composites were prepared by premixing followed by hot pressing; the microstructure of the obtained composites was determined simultaneously by using the qualitative (FTIR) and quantitative (PALS) methods. Enhanced properties were obtained for the graphene/PE composites, and the structure–performance relationship was established through the quantitative characterization of microstructure (


Bioengineered have retracted the following article:We, the authors, Editors and Publisher of the journal Bioengineered, Volume 11, 2020, Pages 958-971.“Upregulation of oxidative stress-responsive 1(OXSR1) predicts poor prognosis and promotes hepatocellular carcinoma progression,” Jianhui Chen, Jiangfan Zhou, Haotian Fu, Xiaofeng Ni & Yufeng Shan. Since publication, the authors raised the following concerns:Recently, in order to further explore the molecular mechanism of oxidative stress-responsive1(OXSR1) promoting the malignant progression of hepatocellular carcinoma (HCC), we repeated the previous research results. However, when we repeated the proliferation experiments, we found that compared with the result of the control group, there was no statistically significant difference in proliferation after knocking down OXSR1. Therefore, we continued to repeat the proliferation experiments several times. Unfortunately, the results of our repeated experiments did not show statistical differences. This indicates that the OXSR1 that the previous study proved to promote the proliferation of HCC is an unreliable result. Actually, OXSR1 does not promote the proliferation of HCC. Considering that in our previous study, OXSR1 promotes the proliferation of HCC is a very important conclusion, we decided to retract this article.The authors alerted the issue to the Editor and Publisher and all have agreed to retract the article to ensure the integrity of the scholarly record.We have been informed in our decision-making by our policy on publishing ethics and integrity and the COPE guidelines on retractions.The retracted article will remain online to maintain the scholarly record, but it will be digitally watermarked on each page as ‘Retracted’.


The development of polyaniline (PANI)/biomaterial composites as humidity sensor materials represents an emerging area of advanced materials with promising applications. The increasing attention to biopolymer materials as desiccants for humidity sensor components can be explained by their sustainability and propensity to absorb water. This review represents a literature survey, covering the last decade, which is focused on the interrelationship between the core properties and moisture responsiveness of multicomponent polymer/biomaterial composites. This contribution provides an overview of humidity-sensing materials and the corresponding sensors that emphasize the resistive (impedance) type of PANI devices. The key physicochemical properties that affect moisture sensitivity include the following: swelling, water vapor adsorption capacity, porosity, electrical conductivity, and enthalpies of adsorption and vaporization. Some key features of humidity-sensing materials involve the response time, recovery time, and hysteresis error. This work presents a discussion on various types of humidity-responsive composite materials that contain PANI and biopolymers, such as cellulose, chitosan and structurally related systems, along with a brief overview of carbonaceous and ceramic materials. The effect of additive components, such as polyvinyl alcohol (PVA), for film fabrication and their adsorption properties are also discussed. The mechanisms of hydration and proton transfer, as well as the relationship with conductivity is discussed. The literature survey on hydration reveals that the textural properties (surface area and pore structure) of a material, along with the hydrophile–lipophile balance (HLB) play a crucial role. The role of HLB is important in PANI/biopolymer materials for understanding hydration phenomena and hydrophobic effects. Fundamental aspects of hydration studies that are relevant to humidity sensor materials are reviewed. The experimental design of humidity sensor materials is described, and their relevant physicochemical characterization methods are covered, along with some perspectives on future directions in research on PANI-based humidity sensors. To provide a literature overview of PANI/biopolymer sensor materials;To provide insight on the moisture-sensing mechanism of PANI and its hybrid composites that contain biopolymer systems;To develop a rational approach for the design of PANI-based materials that are suitable for humidity-sensing applications.To advance the field of multicomponent polyaniline (PANI)/biomaterial composites for humidity-sensing devices, a review of past studies on related developments enables the identification of knowledge gaps in the field. This contribution provides an overview of recent studies that highlight humidity-sensing PANI materials and their key structural and physicochemical properties. The mechanism of polyaniline-water vapor interaction will be presented along with physicochemical properties of composites related to pore structure, flexibility, hydrophilicity, surface area, conductivity and others. Suitable synthetic methodologies will enable the design of new (bio)polymer systems from known, accessible and naturally abundant components. This information can assist in advancing practical moisture-sensitive systems via a bottom-up approach, by incorporating tunable properties to yield improved materials for use in moisture-measurement systems. This review will address several objectives, as follows:g-CHT” and will be used interchangeably herein. The relevance of “chitaline” materials relates to the uncertainty in the structure of such PANI/CHT composites, which will be discussed later. Some authors refer to PANI/CHT composites to as “chitaline” or “PANI-The regulation of humidity plays a significant role in the maintenance of human health and providing comfortable conditions in many aspects of daily life. Relative humidity (RH) is related to the ratio of the partial pressure of water vapor to the total pressure of the saturated vapor at a given temperature and is often expressed in %. Humidity control within indoor environments, such as commercial and residential buildings, plays a role in maintaining the quality of infrastructure and consumable goods. For example, moisture can affect the stability of paintings, computers, plants, food, fruits, vegetables, egg incubation, coatings, chemicals, etc. Moisture-responsive materials have been tested and designed over the decades to monitor humidity in order to prevent the degradation of various items. They range from a simple human hair to sophisticated ceramics ,6,7,8,9 A humidity sensor is synonymous with a hygrometer, where early hygrometers were based on human hair due to its ability to stretch and shrink according to the changes in the humidity . About hAs shown in +) or hydroxide (OH−) ions  ; PAAs seen from Carbon nanomaterials are represented by the various types of carbon allotropes, such as nanotubes and nanofibers, as well as graphene and carbon black. These materials stand out due to the superior properties that make them perfect candidates for various electronic devices, including humidity sensors. Carbon nanomaterials can acquire various structural hierarchies: one-dimensional (fibers), two-dimensional (films) and three-dimensional (monoliths) shapes that can be used for humidity sensing. Among their various properties that support their utility in the design of sensor materials are their large specific surface area, which allows for the design of sensor materials with high sensitivity and quick response times .In a similar manner, ceramic-, carbon-, and biopolymer-based sensors can work according to resistive or capacitive transduction principles ,148. Sim2S sensor based on carbon aerogel (CA) and PANI ..I versus−1. This occurred due to the change from a glassy nematic state to a smectic state of starch ..(N) thatThe advantage of the Box–Behnken design is that it enables the selection of the optimal conditions of an experiment, excluding extreme cases, which results in the prevention of failed experiments. Each level of condition is coded as –1, 0 and 1, which can be interpreted as “low”, “moderate” and “high” values, respectively. All possible points for the BBD , avoiding extrema conditions, are shown in detail elsewhere (cf. Table 1 in ).The BBD was employed successfully for various types of polymer-based sensors’ design , optimiz2, WO3, V2O5) and metal chalcogenides . Some of these metals that have been developed as sensors may suffer from limitations due to material cost and the relative availability of materials, in some cases. Therefore, there is a need to find alternatives that are more cost-effective and sustainable, such as biopolymers and oligomers: chitin, chitosan, cellulose, starch, cyclodextrin, or carrageenan. Although the coverage of the scientific literature reported for various humidity-sensing materials appears to be broad in scope, there are limited reports on PANI/biopolymer composites as potential humidity sensors. Various review articles ,64 indicFor instance, the electrical conductivity of many PANI/biopolymer samples is known, whereas the PANI/cellulose samples have the highest conductivity among the various biopolymer composites, as compared with those containing chitosan and chitin. It may appear that chitin/chitosan is not suitable for humidity sensing. However, the conductivity of the entire composite depends on the component ratio, and on the presence of other (additive) components, including the degree of PANI-doping. A knowledge gap in the role of multi-factorial effects remains to be studied, which can be addressed through statistical optimization using methods such as the Box–Behnken design.There are knowledge gaps for other relevant humidity-sensing parameters. For example, the porosity for the PANI/biopolymer composites has not been widely studied, especially under relevant environmental conditions. In the case of studies other than SEM imaging, an estimate of the porosity  of such materials by this approach is prone to uncertainty, particularly for samples in an anhydrous state that are likely to undergo solvent swelling. The surface accessibility of pores and the average pore diameter can be estimated from the Brunauer-Emmett-Teller (BET) analysis of vapor adsorption isotherms. In contrast to rigid carbonaceous or inorganic solids, biopolymers can undergo incremental swelling, with greater levels of water uptake (vide supra). This is in contrast to ceramic materials that are rigid in nature, and their structure is relatively stable regardless of the moisture content or RH. Hysteresis in a sensor device is also important, as discussed in To design a good humidity sensor, the response and recovery times are key parameters that have not been widely studied. Moisture absorption, calculated as a percentage increase by mass, represents a potential topic area with limited reported research. In the case of the design of humidity sensor materials, it is a key feature that governs the sensitivity and efficiency parameters. Finally, as PANI composites are relatively new materials for use as humidity sensors, the literature reviewed herein highlights the need for further studies on humidity sensor design and their functional properties. In particular, this statement refers to the use of soft (biopolymers) versus hard , in conjunction with PANI.In conclusion, PANI is a preferred candidate for humidity sensor materials with unique hydration properties. It has an electrical conducting mechanism that relies on the role of molecular interactions between water and the hydrophilic sites of PANI. The conductivity of PANI is highly dependent on the ambient RH, which is a key factor to consider in the fabrication of such devices. To enhance the hydrophilic properties of PANI, it can be modified by the formation of composites with biopolymers, such as chitosan, cellulose, starch, cyclodextrin and other types of polysaccharides that facilitate the hydrogen-bonded composites. The formation of these composites ultimately affect the structure and surface chemical properties of the system. The moisture-responsive mechanism of PANI/biopolymer systems depend on two factors: (i) electron/proton hopping between water molecules and acidic nitrogen groups of PANI; and (ii) on the morphology of the composites, which can be altered under the influence of relative humidity (curling–uncurling of PANI/biopolymer chains). The kinetics of this process have not been fully reported to date, despite the importance of kinetic factors that affect the performance of fast-response humidity sensor devices. In practice, the hydration properties of PANI/biopolymer materials can be investigated with the aid of complementary methods that provide insight into the thermodynamics, kinetics, and structural aspects of the humidity-sensing process. Computational methods are anticipated to provide further insight on the structure of hydrated PANI-based composites, through the deployment of methods such as molecular dynamics and DFT calculations cf. .PANI-based composites are normally obtained via oxidative polymerization in acidic media. The nature of the dopant (acid) plays an important role as a template in the resulting morphology and humidity-sensing performance of the composites. A secondary component (biopolymer) results in a synergistic effect for improving the sensitivity and performance of PANI but may affect the electronic properties (conductivity) of PANI, according to structural considerations. The conductivity of PANI is an intrinsic property that is influenced by the functionalization of PANI via physical or chemical means.The configuration of a humidity sensor consists of at least two components: (i) a sensitive layer that may comprise a PANI-based composite, and (ii) a substrate layer with embedded electrodes, where the humidity response is measured as impedance (resistance) or capacitance at variable RH. To optimize the humidity-sensing performance, multifactorial design approaches have been employed, such as the Box–Behnken design. The latter is desirable and serves to guide experimental design for the optimization of multi-parameter systems.Nevertheless, the literature on PANI-based moisture-responsive materials is at an early stage, in comparison with conventional ceramic and carbonaceous sensor materials. A perspective drawn from this contribution recommends that further studies be directed at gaining an improved understanding of humidity-sensing mechanisms for PANI/biopolymer-based nanocomposites. Dedicated experimental and computational studies are anticipated to achieve this goal, through the use of complementary experimental methods. The holistic approaches and methodologies outlined herein are anticipated to provide a molecular-level understanding of the kinetics and thermodynamics of hydration for such systems. In turn, emerging materials such as PANI-based biopolymer nanocomposites are envisioned to occupy an increasingly important role in the future development of sensor technology that employs PANI-based materials for diverse applications.


Low-abundance biomolecule detection is very crucial in many biological and medical applications. In this paper, we present a novel electrolyte-gated graphene field-effect transistor (EGFET) biosensor consisting of acoustic tweezers to increase the sensitivity. The acoustic tweezers are based on a high-frequency bulk acoustic resonator with thousands of MHz, which has excellent ability to concentrate nanoparticles. The operating principle of the acoustic tweezers to concentrate biomolecules is analyzed and verified by experiments. After the actuation of acoustic tweezers for 10 min, the IgG molecules are accumulated onto the graphene. The sensitivities of the EGFET biosensor with accumulation and without accumulation are compared. As a result, the sensitivity of the graphene-based biosensor is remarkably increased using SMR as the biomolecule concentrator. Since the device has advantages such as miniaturized size, low reagent consumption, high sensitivity, and rapid detection, we expect it to be readily applied to many biological and medical applications. Mt doping , the grat doping ,17.Sensitivity is one of the key parameters of modern biosensors ,19,20. IIn this work, we proposed a novel integrated biosensor consisting of an electrolyte-gated graphene field-effect transistor (EGFET) and solid mounted resonator (SMR) as acoustic tweezers. The SMR serves as an active biomolecule concentrator, which was verified in this paper. Thus, the sensitivity of the graphene-based biosensor was greatly enhanced. Meanwhile, the device fabrication is compatible with conventional semiconductor manufacturing processes and system-level integration, which means a low cost and miniaturization. Overall, the proposed method is very promising in biological and medical applications.Acoustic tweezers are based on the interaction between acoustic waves and target particles within the fluids. One convenient way to generate acoustic waves is to use transducers made of piezoelectric materials. Piezoelectric materials can generate electrical polarization after applying mechanical stress, and the electrical polarization can also lead to mechanical deformation. Due to the piezoelectric effect, the radio frequency (RF) source applied to the SMR device can generate high-frequency longitudinal acoustic waves. When the piezoelectric material is immersed in solution, the high-frequency acoustic waves will trigger the acoustic streaming effects due to acoustic power leakage during the propagation of the acoustic waves into liquid ,30,31. HThe fluid motions excited by the SMR device are visualized in The quantity of the biomolecules absorbed nonspecifically by the EGFET has a positive correlation with the accumulation of the biomolecules. The charges of the absorbed molecules can modulate the drain current of the EGFET; therefore, the Dirac point will drift. The accumulation of the biomolecules around the EGFET can be measured by monitoring the Dirac point. Since the SMR works as a biomolecule concentrator, the accumulation of the biomolecules around the EGFET is much higher than the bulk accumulation, thus lowering the detection limit of EGFET.After graphene patterning, the chip was mounted onto an evaluation board with the TIE and TOE connected to the center pin and outer shell of a coaxial small-A-type (SMA) connector. The board was then connected to a Bias-Tee to split RF and direct-current (DC) signals from each other. The configuration allows the dual modes to operate independently and simultaneously at distinct frequencies (GHz vs. DC) with negligible interference. The two output terminals of the Bias-Tee were connected to a network analyzer  and the source meter  separately to characterize the RF and DC responses, respectively. The TOEs were grounded. A 100 μL solution reservoir was mounted onto the device, and an Ag/AgCl reference electrode was used as the gate electrode to minimize environmental effects. The gate electrode was connected to the other channel of the Agilent B1500. The schematic diagram of the measurement setup is shown in IgG labeled with green fluorescence  was used for the detection experiments. Various concentrations of IgG solution were obtained by stepwise diluting a certain volume of IgG solution (1 mM) into phosphate buffer saline (PBS). The IgG solution was added into the solution reservoir by pipette. The top-view images of the device were acquired by a microscope  with a CCD camera .2) layers were deposited onto the silicon substrate to form the Bragg reflector [2 pair were 1200/700, 1000/1300, and 1000/650 nm from bottom to top. The bottom electrode (BE) of the SMR was made of 600 nm thick molybdenum (Mo) on top of the Bragg reflector. The film was then patterned into isolated islands. A 1000 nm thick AlN film was then deposited on top of the BE. Orientation of the AlN crystal was along the c-axis. In the final step, the SMR was capped with a pair of gold (Au) top electrodes. The thicknesses of the Au electrodes and the chromium (Cr) adhesive layer were 300 and 50 nm, respectively. The TIE was intentionally shaped like a pentagon to suppress spurious resonance in the device. The electrode area was configured to be 3.0 × 104 μm2 so that the SMR has a characteristic impedance of 50 Ω to match the impedance of external circuits. The TOE was separated 15 μm away from the TIE. The area of TOE was several times larger than that of TIE. This geometrical arrangement ensures good electric field confinement within the active area under the TIE electrode. More details have been reported in the literature by us [The devices were fabricated on 100 mm undoped silicon wafers. The alternating aluminum nitride (AlN) and silicon dioxide  stamp and held in position for 2 h at room temperature for water evaporation. The fixture was then heated up to 150 °C with a hot plate and kept for 15 min. The heating step promotes van der Waals binding at the graphene–SMR interface, leaving an even and strongly-adhered graphene film after the removal of the PMMA stamp by immersing in acetone for 10 min, as shown in 2 plasma etching. We used a negative E-beam resist , which introduced much fewer surface contaminants compared to most photoresists. Finally, the biosensor was mounted onto an evaluation board, as shown in Graphene field-effect transistors gated by electrolyte were used for detecting the biomolecules. Then, monolayer graphene film was transferred to the SMR. The chemical vapor deposition (CVD) grown monolayer graphene film  first went through a thorough RCA cleaning process to remove surface contaminants. Afterward, the device wafer was pretreated with O2/(V⋅s).The electrical performance of the device was measured from the RF port of the Bias-Tee, as shown in The solution containing green fluorescence-labeled IgG molecules was added into the reservoir. An RF signal of 1.645 GHz at a power of 10 mW was applied to the SMR device to generate acoustic waves. The IgG molecules were driven by acoustic waves to the gap between the TIE and TOE on the graphene. The green fluorescence intensity of the gap was observed to be increased on the gap through a fluorescence microscope, which indicated that the biomolecules were accumulated. After 10-min actuation, the fluorescent intensity of the biomolecules became stable. The final accumulated result is shown in gs. Meanwhile, the Dirac points of the EGFETs merged into the same various concentrations of the IgG molecule without accumulation was also measured. Each concentration was measured three times. The Dirac point shifts against the concentration with and without accumulation are shown in 2 of 0.96.After accumulation at various concentrations  of the IgG molecule by the SMR device, the Dirac points were calculated by sweeping the VIn this paper, we have demonstrated a novel graphene-based transistor biosensor using acoustic tweezers to increase the sensitivity of the biosensor. Since the fabrication steps are compatible with IC processes, which means a small size, low reagent consumption, and low cost, this biosensor can be used as a disposable sensor to ensure repeatability and avoid cross contamination. The operating principle of the device has been described in detail and verified by experiment. We demonstrated that the biomolecules can be concentrated onto the graphene surface efficiently, which greatly increases the sensitivity. Overall, the device has great potential in biological and medical applications.