P: .66). ChatGPT statistically significantly answered a higher proportion of questions correctly in the short questions group compared to the long questions group (P = .03), and in the single select multiple choice questions group compared to the multi-select multiple choice questions group (P < .001). ChatGPT has been successful in the MSE, a challenging exam for doctors in our country. However, it is a fact that ChatGPT is still behind the expert in the field for now, and what will happen with program developments in the future is a matter of curiosity for all of us.In our study, we aimed to evaluate the success of ChatGPT by determining its performance in the last 5 medical specialty exams (MSE) conducted and its ranking among the candidates of that year, and to determine its potential use in healthcare services. Publicly available MSE questions and answer keys from the last 5 years were scanned, a total of 1177 questions were included in the study, all questions were asked to the ChatGPT  GPT-3.5 series, which is the March 23, 2023 version. The average score and rank that ChatGPT would receive if it had entered the exam that year were determined. Questions were categorized as short question group, long question group, single select multiple-choice questions, and multi-select multiple-choice questions. The lowest success proportion was determined as 54.3%, and the highest success proportion was 70.9% correct answer percentage. It achieved a sufficient result as 1787th out of 22,214 people in its most successful exam, and 4428th out of 21,476 participants in its least successful one. No statistically significant difference was found between the correct answers it gave to clinical and basic science questions ( Launched in 2022, ChatGPT is a large language model AI product aiming to give human-like answers to asked questions.,3 Being free and providing support in multiple languages, ChatGPT has led to a rapid increase in its popularity and usage in many fields of science, including medicine. This situation has raised questions about the reliability of ChatGPT.,5With the increase in internet usage, artificial intelligence (AI) applications have emerged as an important element that facilitates our lives. The most popular of these artificial intelligence programs is seen as ChatGPT, a product of OpenAI. In addition, whether ChatGPT reaches the correct conclusion after given short pieces of information has also been tested in the literature before. Terwiesch C. asked ChatGPT undergraduate exam questions in a business course and published that it successfully passed the exam. Similarly, Choi JH et al subjected ChatGPT to a law school entrance exam and demonstrated that it passed the exam. Giannos P. et al evaluated ChatGPT’s performance in 4 different UK Standardized Admission Tests and found it successful in reading comprehension, problem-solving, and critical thinking. Duong et al compared the performance of ChatGPT and human participants with 85 questions asked about genetics and found no difference between them. In the field of medicine, Kung TH et al have proven that ChatGPT performed adequately in the United States Medical Licensing Exam (USMLE).ChatGPT has been previously used to write scientific articles based on given preliminary information, and although it has limitations, its potential in this field has been observed.Medical education lasts 6 years in our country. Students who graduate from medical school gain the right to become a medical doctor and can work as such. To specialize in a field, they need to take the medical specialty exam (MSE), prepared by the Student Selection and Placement Center, which is held twice a year. Depending on the score they get in this exam, they can choose to work as a resident doctor in the city and department they want. We thought that using MSE questions, which are objective, standardized, have clear answers, and are applied to almost all physicians in our country, would be appropriate to question the reliability of ChatGPT in the field of medicine.In this study, we aimed to evaluate the success of ChatGPT by determining its performance in the last 5 MSE and its ranking among the candidates of that year, and to determine its potential use in healthcare services.The MSE questions consist of a total of 240 questions, 120 basic sciences, and 120 clinical sciences in each exam. Basic sciences include questions on Anatomy, Physiology–Histology–Embryology, Biochemistry, Microbiology, Pathology, and Pharmacology; Clinical sciences consist of questions on Internal Medicine, Pediatrics, General Surgery, Obstetrics and Gynecology, and Minor Rotations. Minor rotations include questions related to Neurology, Neurosurgery, Psychiatry, Public Health, Dermatology, Radiology, Nuclear Medicine, Otolaryngology, Ophthalmology, Orthopedics, Physical Medicine and Rehabilitation, Urology, Pediatric Surgery, Cardiovascular Surgery, Thoracic Surgery, Plastic Surgery, Anesthesiology and Reanimation, and Emergency Medicine. Ethical approval was not needed for the study because this is not a study about humans or animals, and only consists of questions asked to AI.The MSE questions and answer keys of the last 5 years were scanned, questions containing visual elements were excluded. Then, questions marked as faulty in the answer key were also excluded. A total of 1177 questions were included in the study, with 232 questions from the 2021-1st term, 237 questions from the 2021-2nd term, 234 questions from the 2022-1st term, 240 questions from the 2022-2nd term, and 234 questions from the 2023-1st term.https://tuskocu.com/tus-puan-ve-siralama-hesaplama/. While calculating the score, the excluded questions of the study were considered as ChatGPT left the question blank.All questions were asked to ChatGPT , the GPT-3.5 series version of March 23, 2023. To reduce recall bias, a new chat session was started for each question in ChatGPT. The questions were manually typed into ChatGPT’s new chat bar, and it was awaited to generate an answer. All answers were recorded as correct or incorrect. Since the scoring per question changes according to the number of people taking the MSE and the proportion of correct answers, the average score and ranking that would have been achieved if it had taken the exam that year was calculated using the website A total of 1177 questions were categorized into 2 groups by recording the number of letters in each question. Questions with <45 letters were taken into the short question group, questions with 45 or more letters were taken into the long question group. The 1177 questions were also categorized as single select multiple choice questions (SS-MCQ), and multi-select multiple choice questions (MS-MCQ):SS-MCQ: These are the best choice questions in situations where there can only be one answer, where one selects the most appropriate one out of 5 options.MS-MCQ: This is the type of question where one or more of the given 3–4 or 5 options may be the correct answer Figs.  and 2.P < .05 was considered statistically significant.All statistical analyses were performed using the JAMOVI program . In summarizing the data obtained from the study, descriptive statistics were used as mean ± standard deviation for continuous variables, and frequency (percentages) for categorical variables. The Chi-square test was used to compare independent two-group proportions. A A total of 1177 questions were included in the study, and the correct answers provided by ChatGPT and their ratios are given in Table P > .05).When all years are evaluated together, of the total 1177 questions answered by ChatGPT, 586 were basic sciences questions and 591 were clinical sciences questions. ChatGPT correctly answered 376 (64.2%) of the basic sciences questions and 372 (62.9%) of the clinical sciences questions. No statistically significant difference was found between the proportion of correct answers given to clinical and basic sciences questions (P = .03) , and in the long questions group, it correctly answered 294 out of 489 (60.1%). It was statistically significant that it gave correct answers at a higher proportion in the short questions group than in the long questions group (P < .001)  out of 1071 SS-MCQ and 38 (35.8%) out of 106 MS-MCQ. It was statistically significant that it gave correct answers at a higher proportion in the SS-MCQ group than in the MS_MCQ group (1) Table .The most significant finding of our study is the degree of success achieved by ChatGPT in one of the toughest exams in our country, the MSE, that physicians are required to pass to step into specialization which consists of multiple-choice questions with definitive answers. ChatGPT has achieved a satisfactory result, ranking 1787th among 22,214 individuals in the most successful exam and 4428th among 21,476 individuals in the least successful. Our study revealed that ChatGPT achieved success in a good percentile when the exam results were compared to the doctor examinees who took the same exam that year. With these results, it has been shown that ChatGPT can easily apply to major specialities such as Orthopedics and Traumatology, Anesthesiology and Reanimation, Neurosurgery, Pediatrics, Internal Medicine, Cardiology, Neurology, Gynecology and Obstetrics, and Urology in big hospitals of metropolises in our country.When compared to previous research,In our study, when basic science and clinical science questions were evaluated separately, no statistical difference was found in the answers given by ChatGPT. This suggests that, considering its text information integration ability, it can support physicians in real-world decision making. Furthermore, it is anticipated that ChatGPT can also be used for basic science education, potentially being beneficial for both students and instructors. When categorized by the number of letters in the questions, it was seen that ChatGPT was more successful in short questions. This suggests that it is relatively unsuccessful in human skills such as data processing and analyzing, causing the success rate in long and complex questions to decrease compared to short and simple questions. This result indicates the need for improvement in this aspect of its algorithm. Finally, when the questions were categorized as SS-MCQ and MS-MCQ, it was largely seen that ChatGPT was more successful in the SS-MCQ group. Considering that multi-select questions are also more confusing for examinees, ChatGPT has struggled more in these confusing questions, similarly to human performance. A significant portion of USMLE questions consist of open-ended questions, which are more subjective to evaluate their answers compared to multiple choice questions. All the questions included in our study consist of objective questions with only one answer, making it a more valuable study in this context. In addition, the exam they tested ChatGPT on consists of a total of 350 questions, many of which are open-ended. In our study, by asking a total of 1177 single-answer questions included in 5 exams, we believe we have evaluated ChatGPT more comprehensively. While questions with clear answers may provide more objective evaluations of ChatGPT, it is true that real-world medical situations often involve ambiguous or open-ended questions that require interpretation and subjective judgment. ChatGPT can still be a valuable tool in assisting with information and generating potential insights, but ultimately, human judgment and expertise play a vital role in addressing complex medical scenarios.In the literature, the most recent study similar to our study is the one in which Kung TH et al asked USMLE questions to ChatGPT.–14 However, the fact that ChatGPT has correct data at hand and as revealed in our study, unlike other internet sites where unproven people can easily generate content, it reaches correct data at a higher rate and even achieves success in exams that doctors enter, suggests it can be used as a more reliable source of consultation. In addition, the ability to provide quick and ready answers to the question asked is another significant advantage over known web sites. MSE questions are formed by bringing together questions from different branches to a certain extent. In our study, ChatGPT, which reached a 70.9% accuracy proportion in the exam in which it was most successful in MSE, still has an error proportion around 30%. Given that a specialist would be more successful in questions related to their own field, and a 1% error can even lead to fatal situations in human life, the need to consult a specialist continues. It has been suggested in the literature that the warning regarding ChatGPT can provide different guidance from a specialist in matters requiring expertise, should be added to its website. While ChatGPT needs to go a longer way to replace humans in the health field, we can say that it is a suitable artificial intelligence model that can be used to reduce the workload of doctors and other health workers and to help with the effective use of the workforce.It has been proven many times in the literature that search engines on YouTube and the internet give incorrect results in various medical conditions to a large extent and can mislead people at a high rate.Study limitations: The most important limitation in our study is that the questions asked are in the Turkish language. Problems stemming from translation issues could have led to incorrect answers to the questions. Additionally, we tested ChatGPT on MSE that is only valid in Türkiye and may not represent tests used in schools of medicine located in other countries with different academic programs. Another limitation is that we only used ChatGPT as an artificial intelligence program in our study; there are many developing artificial intelligence programs, different studies evaluating their results may yield different results. Lastly, ChatGPT is an artificial intelligence program that is continuously updated with new versions, and the version we used in our study may not represent the latest version at the time this article was published.ChatGPT, with its successful performance in the challenging MSE for doctors in our country, has the potential to serve both as a tool for clinical decision making and for providing contributions to basic science education, thereby alleviating the workload of health professionals. The fact that ChatGPT performs more successfully in shorter questions suggests the need for improvement in this aspect of its algorithm. It’s also significant to note that ChatGPT has a lower success rate with multi-select multiple-choice questions, it struggles as humans do with questions that require nuanced reflection and inference. At this stage, it is also a fact that ChatGPT still lags behind the expertise of professionals. What future developments in the program will bring is a matter of curiosity for all of us.Conceptualization: Ayse Dilara Oztermeli, Ahmet Oztermeli.Data curation: Ayse Dilara Oztermeli.Methodology: Ayse Dilara Oztermeli, Ahmet Oztermeli.Supervision: Ahmet Oztermeli.Writing – original draft: Ayse Dilara Oztermeli, Ahmet Oztermeli.Writing – review & editing: Ayse Dilara Oztermeli, Ahmet Oztermeli.


Cytoplasmic aggregation and concomitant nuclear clearance of the RNA-binding protein TDP-43 are found in ~ 90% of cases of amyotrophic lateral sclerosis and ~ 45% of patients living with frontotemporal lobar degeneration, but no disease-modifying therapy is available. Antibody therapy targeting other aggregating proteins associated with neurodegenerative disorders has shown beneficial effects in animal models and clinical trials. The most effective epitopes for safe antibody therapy targeting TDP-43 are unknown. Here, we identified safe and effective epitopes in TDP-43 for active and potential future passive immunotherapy. We prescreened 15 peptide antigens covering all regions of TDP-43 to identify the most immunogenic epitopes and to raise novel monoclonal antibodies in wild-type mice. Most peptides induced a considerable antibody response and no antigen triggered obvious side effects. Thus, we immunized mice with rapidly progressing TDP-43 proteinopathy (“rNLS8” model) with the nine most immunogenic peptides in five pools prior to TDP-43ΔNLS transgene induction. Strikingly, combined administration of two N-terminal peptides induced genetic background-specific sudden lethality in several mice and was therefore discontinued. Despite a strong antibody response, no TDP-43 peptide prevented the rapid body weight loss or reduced phospho-TDP-43 levels as well as the profound astrogliosis and microgliosis in rNLS8 mice. However, immunization with a C-terminal peptide containing the disease-associated phospho-serines 409/410 significantly lowered serum neurofilament light chain levels, indicative of reduced neuroaxonal damage. Transcriptomic profiling showed a pronounced neuroinflammatory signature  in rNLS8 mice and suggested modest benefits of immunization targeting the glycine-rich region. Several novel monoclonal antibodies targeting the glycine-rich domain potently reduced phase separation and aggregation of TDP-43 in vitro and prevented cellular uptake of preformed aggregates. Our unbiased screen suggests that targeting the RRM2 domain and the C-terminal region of TDP-43 by active or passive immunization may be beneficial in TDP-43 proteinopathies by inhibiting cardinal processes of disease progression.The online version contains supplementary material available at 10.1186/s40478-023-01592-z. Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) are related, incurable neurodegenerative disorders with overlapping genetics and neuropathology. Most cases show neuronal and glial cytoplasmic inclusions of the multifunctional RNA-binding protein TDP-43 . NormallOver the past years, active and passive antibody approaches targeting different extracellular (Aβ) and intracellular  aggregating proteins have emerged as promising options for the treatment of neurodegenerative diseases . DespiteC9orf72 repeat expansions  signals are represented and statistically analyzed.1 µM TDP-43-MBP-His6 preparation was exchanged to Hepes buffer . 7 µM Alexa 488-coupled TDP-43-MBP-His6 was mixed with 3.5 µM DyLight 650-labeled monoclonal antibody  in low binding tubes and 100 µg/ml His6-TEV protease was added to the reaction. Samples were transferred to µ-Slide 18 Well—Flat chambers and imaged using a Zeiss LSM710 confocal microscope after 30–60 min. For confocal microscopy, a Plan-Apochromat 63x/1.40 Oil DIC M27 objective was used and images were captured with two-line averaging at 2048 × 2048 pixel resolution.10% Pluronics F-127 solution was used to treat uncoated µ-Slide 18 Well—Flat chambers  for 1 h at RT. Afterwards, wells were washed five times with MilliQ water. Prior to setting up the condensate formation reaction, the buffer of the purified Alexa 488-labeled TDP-43-MBP-His6, 10 µM of unlabeled monoclonal antibodies  and 100 µg/ml His6-TEV protease in Hepes buffer. After an incubation step of 10 min at RT, condensates were imaged in 5 s intervals within a time window of up to 45 min after cleavage. An inverted spinning disc microscope , built on a Nikon Ti2 stand equipped with a confocal spinning disc  with 50 µm pinhole diameter and a 60x/1.2 NA water immersion objective was used for acquisition. Imaging was performed using the 488 nm laser line for excitation and 525/50 bandpass filter (Chroma) for collecting the fluorescence emission. Images were captured using sCMOS camera  and a frame of 1033 × 1033 pixels in 12-bit acquisition mode.To analyze fusion events of antibody-treated and TEV protease-cleaved TDP-43, condensates were formed in 18-well ibiTreat slides  using 20 µM of Alexa 488-labeled TDP-43-MBP-His4 cells/well). The following day, recombinant pHrodo Green-labeled TDP-43-MBP-His6 was used to prepare 10 µM of aggregates during an incubation step of 2 h (see Aggregation assay). Subsequently, 10 µM of single mAbs  or equivalent amounts of PBS were added individually to the TDP-43 aggregates and incubated for 30 min at RT. Afterwards, media of SH-SY5Y cells were aspirated completely and 500 μl of fresh media containing 500 nM TDP-43 aggregate/mAb mix or 500 nM monomeric TDP-43-MBP-His6 were added on cells for ~ 20–24 h at 37 °C and 5% CO2. The day after, cells were washed twice and harvested for flow cytometry analysis. Flow cytometry experiments were performed on a BD FACSVerse™ instrument  and analyzed using FlowJo software . In brief, singlets were detected using FSC-A versus FSC-H parameters. Live cells were identified as SYTOX™ Blue− (negative) (Thermo Fisher) cells out of the single cell population. Lastly, the relative fluorescence of FITC in live single cells was analyzed. For each condition tested, at least 1800 live cells were recorded.SH-SY5Y cells were cultured in DMEM/F12 (1:1) medium + L-Glutamine + 15 mM Hepes (Gibco) supplemented with 10% fetal bovine serum, until they reached a confluency of ~ 80%. Cells were then harvested and seeded in a 24-well plate  were carried out in random order. Data were visualized and statistically analyzed using R (version 3.6.1) with RStudio. Assumptions for parametric tests were assessed by Shapiro–Wilk test/Q-Q plots and Levene’s test. Longitudinal changes of body weights were analyzed by two- or three-way repeated measures ANOVA. One-way ANOVA followed by pairwise t-test with Benjamini–Hochberg correction was used as parametric test to statistically compare more than two groups. Kruskal–Wallis test and subsequent pairwise Wilcoxon Rank Sum Tests with Benjamini–Hochberg correction were used as non-parametric tests. Individual datapoints are generally displayed as scatterplots plus bar graphs [mean + standard deviation (SD)] or box plots . To determine which epitopes of TDP-43 induce sufficient antibody titers to slow disease progression in a TDP-43 mouse model, we selected 14 peptide antigens, collectively covering all domains and 2/3 of sequence of human TDP-43  mediated inducible Taken together, antigens derived from the glycine-rich domain (peptides 10 + 12 and 15) reliably induced high-titer antibodies and detected disease-associated cytoplasmic TDP-43 inclusions.Since disease-relevant phenotypes progress rapidly in aged rNLS8 mice , we sacrNeurofilament levels in blood and CSF are emerging as a powerful biomarker for detecting neuroaxonal damage in mouse models and patients affected by ALS . Thus, wTo further investigate the potential benefits of TDP-43 peptide vaccination in rNLS8 mice, we analyzed disease-associated pS409/410 TDP-43 levels and neuroinflammation in treated animals. Using a previously established immunoassay, we quantified pTDP-43 in the cerebral cortex  and rNLS8 mice (TDP-PBS) as well as all rNLS8 animals vaccinated with promising peptides from the C-terminal domain (TDP10 + 12 and TDP15). Transgene induction in rNLS8 mice dramatically altered the transcriptome, dominated by a strong inflammatory signature, which is in line with our immunofluorescence stainings of GFAP and Iba1 Fig. A. Top upups Fig. B. Comparups Fig. C. The rNups Fig. C. The toups Fig. D revealeCxcl10, Ccl2, Ccl3 and Tnf were strongly upregulated at the transcriptomic level as well . The chemokine profile in rNLS8 mice is likely driven by IL-1β and TNF-α and is consistent with strong monocyte/microglia activation , we identified IL-1β, IP-10 (CXCL10), KC/GRO (CXCL1), MCP-1 (CCL2), MIP-1α (CCL3), and TNF-α to be significantly upregulated in rNLS8 mice compared to monogenic control animals Fig. E. KC/GROion Fig. A.To investigate the effects of antibodies induced by active vaccination in vitro, we generated a panel of monoclonal antibodies (mAbs) recognizing specific domains of TDP-43 by hybridoma generation using the spleens from immunized C57BL/6J mice see Fig. B/C. Sinc6 fusion protein, followed by agitation in an aggregation-promoting buffer , fluorescently-labeled TDP-43 readily formed aggregates that we visualized after 48 h  from a TDP-43-MBP-Hisfer Fig. B [14, 168 h Fig. C.As TDP-43 aggregates are believed to arise from TDP-43 condensates that are formed by liquid–liquid phase separation (LLPS) and then undergo a liquid-to-solid state transition , 57, we Next, we visualized TDP-43 condensates and assessed co-partitioning of mAbs into these condensates, by performing the condensation assay with fluorescently labeled TDP-43 and mAbs, and imaged condensates by confocal microscopy Fig. E. As expSince inhibition of cell-to-cell transmission could slow the progression in TDP-43 proteinopathies, we tested how the mAbs affect cellular uptake of TDP-43 aggregates.6 using TEV cleavage (for 30 min) and then incubated the samples with individual mAbs at equimolar ratios for additional 30 min before adding them to SH-SY5Y cells  carry a loss-of-function mutation in C9orf72 ALS model affected microglia activation more than poly-GA levels [To investigate the efficacy of anti-TDP-43 vaccination, we analyzed body weight, pTDP-43 levels, and serum NfL levels, a widely used clinical biomarker for neuroaxonal damage. While we did not observe beneficial effects on weight loss and pTDP-43 levels, immunization with the disease-associated C-terminal phospho-TDP-43 epitope significantly reduced NfL levels. These findings support the disease-promoting role of TDP-43 phosphorylation at serines 409/410, although the effects of C-terminal phosphorylation on TDP-43’s biophysical properties are controversially debated based on in vitro data , 16, 43.A levels . While aOne postulated mode of action for antibody-based therapy is the prevention of cell-to-cell propagation of transmittable species of aggregation-prone proteins. Transmission has been extensively shown for proteins implicated in various neurodegenerative diseases, including Tau, α-synuclein, and for TDP-43 , 39, 43.As passive antibody therapy may overcome weak immunogenicity of crucial epitopes and avoid acute toxicity, we generated a large panel of monoclonal TDP-43 antibodies targeting several of our selected epitopes. Four out of nine novel high-affinity antibodies targeting RRM2 , the C-terminal glycine-rich region  or the linking region  strongly inhibited aggregation and altered phase separation of recombinant TDP-43 in different assays, while antibodies binding TDP-43 further N-terminal were less effective. These findings are consistent with the dominant role of the C-terminal glycine-rich domain in driving phase separation and aggregation of TDP-43 , 17. A rInterestingly, antibodies targeting the glycine-rich region either resulted in the formation of much smaller and fewer TDP-43 condensates (31E9) or caused the formation of larger liquid-like droplets enriched in anti-TDP-43 antibodies (36C5 and 36C10), reminiscent of the phase separation behavior of a TDP-43 mutant containing multiple phospho-mimetic substitutions in the C-terminal domain . CollectMoreover, we show that aggregated TDP-43 is preferentially taken up by neuron-like cells compared to monomeric TDP-43, which can be blocked efficiently by two mAbs targeting the C-terminal domain (31E9 and 36C10), but not by 30D3 targeting the RRM2 domain. Interestingly, Afroz et al. show that a mAb targeting the C-terminal domain can inhibit seeding from patient material, promote phagocytic clearance by microglia and inhibit aggregate formation in vitro [Taken together, we addressed efficacy and safety risks of TDP-43 immunotherapy for ALS/FTD by unbiased epitope screening in vitro and in vivo. We identified several highly immunogenic peptides in TDP-43 and raised a panel of novel mAbs potentially useful for therapy. Despite concerns about on-target toxicity of anti-TDP-43 antibodies due to the essential role of TDP-43 for cellular survival, we only detected unexpected safety risks of highly immunogenic N-terminal peptides, presumably due to anaphylaxis in one of four genetic backgrounds tested. In a fast-progressing TDP-43 mouse model, targeting the glycine-rich domain was most beneficial and partially rescued the disease signature detected by transcriptomics. While none of the antigens reduced the effect on neuropathology and the characteristic body weight loss, vaccination with a peptide including the disease-associated pS409/410 site significantly reduced NfL levels, the gold standard biomarker for ALS progression. Moreover, we identified mAbs targeting the glycine-rich domain that efficiently block disease-related phase separation and aggregation of TDP-43 in vitro, and suppress aggregate uptake in cells. Spreading and seeding of TDP-43 are crucial for the clinical progression of ALS after focal onset, but we assume it may be less relevant in the fast-progressing rNLS8 mice with widespread neuronal protein expression. Our finding that mAbs can alter the phase separation properties of a disease-linked protein, establishes a new mode of action for antibody therapy. Our study suggests that targeting the glycine-rich domain with antibodies ameliorates key pathomechanisms of TDP-43 proteinopathies.Additional file 1: Figure S1. TDP-43 epitopes and preimmune control serum. A: Overview of peptide antigens conjugated with ovalbumin through the N-terminal cysteine residue. Note that OVA conjugates of peptides 7, 8, 10, and 11 showed some aggregation in vitro. Peptides in bold were highly immunogenic in C57BL/6J wild-type mice and were thus used for the treatment study in rNLS8 mice. Novel monoclonal antibodies were raised in C57BL/6J mice, except of 26A2 and 26F12, which were generated from immunized Balb/c mice. B: Preimmune serum contains no detectable antibodies against human recombinant TDP-43 or pTDP-43 peptide as determined by ELISA. Background-corrected mean optical density at 450 nm was measured in duplicates per animal. All sera were diluted 1:100. Figure S2. Combined TDP-43 peptides are equally immunogenic and safe in three inbred mouse strains. Antibody response plateaus already after two immunizations. A: Timeline of immunization with pooled TDP-43 peptides, blood sampling, and body weight measurement of C57BL/6J, C3H/HeOuJ, and CD-1 mice. For each strain, six animals per treatment group were used. B: A dilution series of pooled sera was analyzed by ELISA against recombinant full-length TDP-43 or pTDP-43 peptide. Background-corrected mean OD450 was measured in duplicates. Triangular shapes indicate CD-1, circles represent C57BL/6J, and squares depict C3H/HeOuJ animals. Open shapes correspond to first blood sampling, while b2 is illustrated by filled shapes. C: Kaplan–Meier plot indicating survival probability of mice from different strains. No antigen combination caused statistically significant lethality in any of the strains investigated. D: Development of body weight over 14-week study period. Mean values are plotted group-wise. Only age, but not treatment or the interaction between treatment and age, had a statistically significant effect on the body weight as revealed by a two-way repeated measures ANOVA. C57BL/6J: p = 1.74*10–24; C3H/HeOuJ: p = 2.56*10–33; CD-1: p = 1.37*10–29. E: Dilution series of pooled rNLS8 sera from different time points were analyzed by ELISA against recombinant full-length TDP-43 or pTDP-43 peptide. Background-corrected mean OD450 was measured in duplicates. Note that the antibody response plateaued after two immunizations. Age of animals is annotated for each timepoint. Figure S3. Additional immunofluorescence stainings of pooled antisera confirm specificity towards nuclear and mislocalized TDP-43. Double immunofluorescence stainings of pooled antisera and with either phosphorylated TDP-43 or pan-TDP-43 were performed on frontal cortex sections of an FTLD case and a healthy control. A: Antisera from TDP1 + 3# and TDP10 + 12 animals reliably stained nuclear TDP-43 but no cytoplasmic inclusions in a control case negative for pTDP-43. B: Double immunofluorescence with a commercial TDP-43 antibody validates specificity of pooled antisera in patients. Antisera from TDP1 + 3# and TDP10 + 12 mice detected both nuclear and cytoplasmic TDP-43, while TDP15 sera showed strong preference for cytoplasmic TDP-43 with little affinity to nuclear TDP-43. Representative images are shown. All scale bars = 10 µm. Figure S4. Additional comparisons of gene expression profiles and cytokine assays in rNLS8 mice. A: Expression patterns of genes characterizing early, late, and recovery stage in isolated microglia [p = 0.004), but not ctrl VAX mice as indicated by pairwise t-test with Benjamini–Hochberg correction. D: RNAseq-based gene expression of all detected cytokines from the cytokine/chemokine panel in rNLS8 mice and ALS patients compared to controls as in Fig. Figure S5. Characterization of TDP-43 mAbs on droplet formation, affinity, and patient inclusions. A: Representative time-lapse images of Alexa 488-labeled TDP-43 condensates obtained by spinning disc confocal microscopy. TDP-43 treated with isotype control antibody does not fuse, while mAbs targeting the C-terminal glycine-rich region increase droplet mobility, leading to slow fusion events and enlarged TDP-43 droplets. Scale bar = 2.5 μm. B: Novel TDP-43 mAbs were analyzed by ELISA against recombinant TDP-43 in dilution series. Background-corrected OD450 are shown. C: mAbs which most potently inhibited TDP-43 aggregation and LLPS detected pTDP-43-positive inclusions in a FTLD patient and additionally stained nuclear TDP-43 by immunofluorescence.icroglia  in rNLS8Additional file 2: Table S1. Differential expression analysis of control PBS, TDP-PBS, TDP10+12, and TDP15 neocortex using DESeq2 as described in the methods. Samples are compared to either ctrl PBS or TDP-PBS. In column D “−1” and “+1” indicate significant genes and the direction of change.Additional file 3: Table S2. Full Gene Ontology enrichment analysis for differentially expressed genes from TargetALS patients in spinal cord and genes expressed in rNLS8 mice, each both compared to non-diseased controls. Selected data are shown in Fig.


Human hindmilk contains higher concentrations of fat than foremilk and is more desirable for growth in preterm infants who can tolerate limited volumes of breastmilk. There is currently no clear demarcation between foremilk and hindmilk. This study characterized the change in breastmilk’s fat content from the start to end of milk flow and defined this demarcation.≥ 14 days after childbirth in a University hospital in Bangkok, Thailand between July, 2011, and April, 2012 were included in this cross-sectional study. Breastmilk samples were sequentially collected from the start to end of milk flow in 5-mL aliquots using breast pumps. The fat content of each aliquot from each breast was determined through creamatocrit. The average creamatocrit of foremilk and hindmilk were compared in predefined foremilk to hindmilk ratios of 20:80, 25:75, 33:67, and 50:50. Creamatocrit of the first and last aliquots were compared for mothers who expressed low- (≤ 25-mL per breast) and high-volumes (> 25-mL per breast) of breastmilk.Mothers of infants born at ≤ 32 weeks gestational age and p < 0.001). Mean creamatocrit in hindmilk portions  was significantly higher than foremilk portions  at all predefined ratios. Creamatocrit increased by 1% for every 10% incremental increase in expressed breastmilk volume until the breast was emptied. Low-volume mothers had a significantly higher creamatocrit in the first aliquot compared with high-volume mothers . No significant difference in breastmilk volume was observed between mothers with and without breastfeeding experience.Of the 25 mothers enrolled, one was excluded due to unsuccessful creamatocrit measurement. The last aliquot of breastmilk had a significantly higher creamatocrit than the first from the same breast (median [interquartile range] of 12.7%  vs. 5.6% ; test statistic 1128, Fat content in breastmilk increased on an incremental basis. More fluid definitions of foremilk and hindmilk should be adopted. Mothers should prepare their breastmilk into aliquots based on the required feeding volume of their infant. Hindmilk aliquots can be prioritized over foremilk aliquots to ensure infants obtain optimal caloric intake.The online version contains supplementary material available at 10.1186/s13006-023-00599-5. Human breastmilk is regarded as the best source of nutrition for newborns. Enteral feeding with breastmilk is associated with reduced necrotizing enterocolitis, late-onset sepsis, retinopathy of prematurity, bronchopulmonary dysplasia, and neurodevelopmental impairment in preterm very low birth weight (VLBW) infants –3. HowevFat constitutes approximately 3–5% of human breastmilk and serves as the primary source of energy for infants . During When VLBW infants are critically ill and hospitalized, the amount of breastmilk that infants can tolerate is limited. As foremilk is expressed first, this inadvertently results in infants consuming little hindmilk – leading to inadequate weight gain. Several neonatal intensive care units (NICUs) reported that hindmilk feeding improved growth in premature infants –14. PrevCreamatocrit measurements are one means of assessing fat content in breastmilk, which is determined by centrifuging breastmilk in capillary tubes – akin to hematocrit measurements. The length of the fat and total milk columns can be measured using Vernier Calipers or a hematocrit reader, and the proportion of fat to total volume (creamatocrit) expressed in percentage , 21, 22.The primary objective of the study was to characterize the progressive changes in breastmilk’s fat content from the start to end of milk flow through creamatocrit. The secondary objective was to define a demarcation between foremilk and hindmilk.This cross-sectional study was conducted at the Division of Neonatology, Department of Pediatrics, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand between July 1, 2011 and April 30, 2012. Siriraj Hospital is the largest tertiary care center in Thailand, with approximately 8,000 childbirths per year. Mothers of infants who were born at ≤ 32 weeks gestational age and ≥ 14 days after childbirth were included in the study. This was to ensure that expressed breastmilk consisted of mature milk. Mothers who used domperidone as a galactogogue or with an expressed breastmilk volume < 5-mL were excluded. Eligibility assessments were performed by W.B. and a practical nurse responsible for overseeing the neonatal ward’s lactation room. Mothers who stayed overnight in the neonatal ward were invited and informed of the study by W.B. All provided written informed consent prior to undergoing any study procedures.Breastmilk from the start to end of milk flow from each breast was collected during the same breast pumping session. Breastmilk samples were collected in the lactation room of the neonatal ward where infants were admitted. Mothers were taught how to practice proper hygiene; this entailed thorough handwashing techniques with soap and water as well as how to clean their breasts with sterile water. All mothers used a Medela® electric breast pump  to collect their breastmilk in 5-mL aliquots into sequentially-labelled, sterile 5-mL bottles from the start to end of milk flow. Samples were separately collected from left and right breasts. Bottles collected towards the end of milk flow that had a volume < 5-mL were discarded. Breastmilk samples were kept at room temperature and creamatocrit measurements performed within 30 min of collection.The proportion (in percentage) of breastmilk in each bottle was calculated based on the total expressed volume from each breast. The cumulative percentage of total milk volume (CP-MV) was calculated as shown in Fig. Creamatocrit measurements were performed by W.B. using Creamatocrit Plus® . Collection bottles were shaken to ensure thorough mixing before 0.2-mL of breastmilk was transferred from each bottle to two 75-µL creamatocrit tubes. These tubes were sealed with sealant before centrifugation in Creamatocrit Plus® for 3 min at 5,670-5,930 rounds per minute. Creamatocrit values were read thereafter using the machine-equipped reader tray and CP-MV calculated for each aliquot of breastmilk  and IBM® SPSS® Statistics 28 . Descriptive statistics were used to present mothers’ demographic data. Creamatocrit values between the first and last sample aliquots from the same breast were compared using either paired t-tests or Wilcoxon signed-rank tests, depending on the data distribution.Four random-coefficient mixed models  were fit to examine the relationship between CP-MV of a breast (represented by X) and its corresponding creamatocrit (represented by Y). The model with the lowest Akaike information criteria (AIC) and Bayesian information criteria (BIC) was selected as the final model . RegressThe researchers hypothesized that hindmilk started at the point where the fat content rose significantly. To define this point of demarcation in the breastmilk stream, the researchers arbitrarily determined the proportion of foremilk as 20%, 25%, 33% and 50% of the milk stream. This constituted 20:80, 25:75, 33:67, and 50:50 ratios of foremilk:hindmilk, respectively Many mothers (87.5%) exhibited a disparity in expressed breastmilk between their left and right breasts Table . No signp < 0.001])The last aliquot of breastmilk had a significantly higher creamatocrit compared with the first aliquot from the same breast . The results of fitting the four mixed models are summarized in Table Upon comparing the creamatocrit of different foremilk:hindmilk ratios, the mean creamatocrit in hindmilk portions  was significantly higher than foremilk portions  at all predefined ratios   and low-volume (≤ 25-mL) groups (using the median 25-mL observed in the study as a reference volume). Low-volume mothers had a significantly higher creamatocrit in the first aliquot of breastmilk compared with high-volume mothers . However, caution should still be exercised when prioritizing hindmilk over foremilk. For example, retinol and alpha-tocopherol were found in higher concentrations in hindmilk than foremilk . In addiPrevious literature found that mothers with previous breastfeeding experience expressed a greater volume of breastmilk than mothers with no experience , 29. ThiThis study had some limitations. First, the lack of data on maternal nutrition that may affect the nutritional compositions of breastmilk. Second, creamatocrit measurements were performed by a single researcher. While there is a risk of systematic measurement errors, this was highly unlikely due to the extensive training undergone by the researcher before measurements were performed. Third, the sample size was limited by resources and calculations were performed post hoc. Fourth, this was a single-center study that included mothers of very preterm infants. The findings of this study may not be generalizable to late preterm or full-term infants. Fifth, the disparity in infants’ age. However, this disparity was not a primary concern of this study, as it focused on the change in fat concentration from the start to end of milk flow. Hence, any variability in fat content found across a range of ages was welcomed. Additionally, mothers were recruited at least two weeks after childbirth in order to obtain mature milk, which already has less variation in fat concentration compared with the first two weeks after childbirth .The fat content in breastmilk increases from the start to end of milk flow. To ensure optimal nutrition for an infant, practitioners should recommend individualized feeding regimens and adopt more fluid definitions of foremilk and hindmilk. Mothers may also prepare their breastmilk in aliquots based on the required feeding volume of their infant, prioritizing aliquots of hindmilk before foremilk to ensure optimal caloric intake. Hindmilk feeding regimens warrant further nutritional considerationsBelow is the link to the electronic supplementary material.Supplementary Material 1


The aim of this manuscript is to provide a comprehensive review of the etiology, measurement, and treatment of common pulmonary symptoms associated with sarcoidosis. The assessment of symptoms associated with pulmonary sarcoidosis is an important component of disease management. Some symptoms of pulmonary sarcoidosis are sensitive but nonspecific markers of disease activity, and the absence of such symptoms provides evidence that the disease is quiescent. Although quantifiable objective measurements of pulmonary physiology and chest imaging are important in the assessment of pulmonary sarcoidosis, they correlate poorly with the patient’s quality of life. Because the symptoms of pulmonary sarcoidosis directly relate to how the patient feels, they are reasonable endpoints in terms of clinical research and individual patient care. Recently, the symptoms of pulmonary sarcoidosis are capable of being quantified via patient-reported outcome measures and electronic devices. We conclude that a thorough assessment of the symptoms associated with pulmonary sarcoidosis improves patient care because it is a useful screen for manifestations of the disease, provides insight into the pathophysiology of manifestations of sarcoidosis, and may assist in optimizing treatment. Sarcoidosis is a multisystem granulomatous disease of unknown cause. The lung is overwhelmingly the most common organ involved, with pulmonary involvement occurring in approximately 90 percent of patients ,2,3. TheAs is the case for most diseases, the presence of pulmonary sarcoidosis symptoms is a more sensitive than specific marker of disease activity. The presence of pulmonary sarcoidosis symptoms may also have prognostic significance. This manuscript will review the major symptoms of pulmonary sarcoidosis and will describe their causes, treatment implications, and impacts on clinical outcomes.Cough is an extremely common symptom of pulmonary sarcoidosis. In comparison to controls, an unselected group of pulmonary sarcoidosis patients was shown to have a markedly increased cough frequency and severity . The repThe characteristics of cough are variable in sarcoidosis depending depending on its etiology (vide infra). The cough may be non-productive or productive, and productive cough is more common in those with a high cough frequency . PulmonaPulmonary sarcoidosis-associated cough is a common reason for patients to seek medical attention . Using tThere are numerous potential causes of cough in pulmonary sarcoidosis. Probably the most common mechanisms causing pulmonary sarcoidosis-associated cough are airway irritation and mechanical airway damage caused directly by granulomatous inflammation. Sarcoid granulomas have a predilection for depositing in the airways  and the The measurement of cough is problematic because it is a multidimensional assessment involving both subjective and objective input. Cough frequency can be determined subjectively by the patient, but this method is not very accurate. Cough frequency monitors have been used in clinical trials ,33. ReceThe treatment of pulmonary sarcoidosis-associated cough depends on its etiology. If cough is a symptom related to active pulmonary sarcoidosis, it usually responds to therapy for that condition, particularly with corticosteroid doses of 20 mg/day of prednisone or less . Very frWheezing is a very common symptom of pulmonary sarcoidosis. Wheezing was second only to cough as a symptom of acute pulmonary exacerbations of sarcoidosis . AlthougA major mechanism for airflow obstruction in sarcoidosis is from endobronchial sarcoid granulomas that may narrow, distort, or rarely completely obstruct the airway . Airway Wheezing is problematic to quantify. Although smartphone-based artificial intelligence (AI) monitoring apps have been developed for cough , we are Wheezing is usually not specifically treated in pulmonary sarcoidosis. As wheezing is a manifestation of acute pulmonary exacerbations of sarcoidosis , systemiDyspnea is an extremely common symptom in pulmonary sarcoidosis patients. Dyspnea is more common in pulmonary sarcoidosis patients than in healthy matched controls, with moderate to severe dyspnea being more than 10 times more common (56% versus 4%) in the pulmonary sarcoidosis group . DyspneaThere are numerous causes of dyspnea in pulmonary sarcoidosis patients. These causes include the deposition of sarcoidosis granulomas in the lung, manifestations of fibrotic sarcoidosis, multiple mechanisms that can cause pulmonary hypertension, complications of treatment, manifestations of extrapulmonary sarcoidosis, psychosocial/functional issues, and conditions completely unrelated to sarcoidosis. These mechanisms of dyspnea are listed in Dyspnea is a sensation that may affect or be affected by emotional, psychological, and social states. For this reason, these four conditions cannot be assessed in isolation, and changes in any one of them may affect all the others. The interdependence of these states has been specifically confirmed in sarcoidosis ,52,53, aAs dyspnea is a subjective measure, it is typically quantified via a PROM or VAS . It has As there are innumerable causes of dyspnea, its treatment is dependent on its specific cause. Although exercise training regimens in sarcoidosis patients have been shown to improve the 6-min walk distance and lessen fatigue, they have no significant effect on dyspnea as measured using the Borg Dyspnea Scale . InspiraChest pain is a common symptom of pulmonary sarcoidosis. Chest pain has been reported as a presenting complaint in 9 percent of sarcoidosis patients , in 12 pPulmonary sarcoidosis-associated chest pain is usually pleuritic in character and is most common in the substernal and infrascapular areas . The cheThere are other causes of chest pain associated with sarcoidosis and pulmonary sarcoidosis. As mentioned, chest pain occurs in approximately 10 percent of patients with an exacerbation of sarcoidosis . PatientThe treatment of pulmonary sarcoidosis-associated chest pain depends on its etiology. If chest pain develops with an acute pulmonary exacerbation of sarcoidosis, it usually responds to therapy for this condition . The afoThere are innumerable alternative causes of chest pain in pulmonary sarcoidosis patients that are unrelated to the disease. The clinician should diligently explore these possibilities and not reflexively assume that the chest pain in pulmonary sarcoidosis is caused by sarcoidosis.Hemoptysis is a rare symptom of pulmonary sarcoidosis. The most comprehensive review of hemoptysis in pulmonary sarcoidosis was published more than 35 years ago, and found that six percent of 433 sarcoidosis patients developed hemoptysis over the course of their disease . A literHemoptysis may rarely be an initial manifestation of sarcoidosis, with only four percent of 433 patients presenting with this symptom . ScatterHemoptysis may occur in patients with Scadding stage 4 fibrocystic sarcoidosis via a number of mechanisms. Sarcoidosis-associated bronchiectasis occurs in up to 50 percent of patients with fibrotic sarcoidosis ,31. ThisOther causes of hemoptysis in sarcoidosis include necrotizing sarcoid granulomatosis (NSG), a condition where the granulomas are typically confluent and necrotic ,81. The Several medical conditions are associated with sarcoidosis that may lead to hemoptysis. Sarcoidosis patients appear to be at a higher risk of pulmonary embolism ,85,86 anIt has been estimated that 50 ,89 to 85Patients with asymptomatic pulmonary sarcoidosis most commonly have no evidence of parenchymal lung disease on a chest radiograph, with either a normal chest radiograph (Scadding stage 0) or bilateral hilar/mediastinal lymphadenopathy without parenchymal opacities (Scadding stage 1) ,97. The The prognosis of asymptomatic pulmonary sarcoidosis is better than that of symptomatic patients. In one series of 660 sarcoidosis patients where 175 (27%) were asymptomatic and at least 145 (83%) of those had pulmonary sarcoidosis, asymptomatic patients less frequently required treatment, developed less organ involvement, and had improved health-related quality of life .Although the symptoms associated with pulmonary sarcoidosis are not specific for the disease, they provide important clinical insights. Cough is the most frequent symptom of active pulmonary sarcoidosis, and the lack of cough greatly lowers the probability of an exacerbation of pulmonary sarcoidosis. Wheezing is underappreciated in pulmonary sarcoidosis. It is a very common symptom in acute pulmonary sarcoidosis from the granulomatous involvement of the airways and in chronic sarcoidosis from airway distortion from fibrosis. Chest pain is also an underappreciated symptom of pulmonary sarcoidosis. It is typically pleuritic and usually not associated with the severity of the disease. Hemoptysis is a relatively uncommon initial symptom of pulmonary sarcoidosis that usually responds well to corticosteroids. However, hemoptysis in chronic fibrotic pulmonary sarcoidosis may suggest bronchiectasis, aspergillus infection, or pulmonary hypertension; all of which are serious and potentially life-threatening disease complications. Asymptomatic pulmonary sarcoidosis patients tend to have minimal disease on chest imaging, less often require treatment, and have a better long-term quality of life than symptomatic patients.The assessment of the symptoms of pulmonary sarcoidosis is easy to obtain and has no cost. We believe that comprehensive knowledge of these symptoms will aid the clinician in identifying and managing various manifestations of the disease. Advances in artificial intelligence may allow for more accurate monitoring of these symptoms that may significantly improve patient management.


Large hydropower projects continue to be built in developing nations, despite their known negative impacts. Large-scale energy projects strain local infrastructure and reduce access to infrastructure for households that live near them. Here we investigate the link between large-scale hydropower projects and stress. Our results suggest that these projects create stress through two mechanisms: strains on community resources and through the process of displacement. We also ask how compensation and resettlement programs condition these relationships. Using data from the Madeira river basin in the Brazilian Amazon, we find that hydropower projects increase stress by reducing access to energy, water, sanitation and land. Compensation provided was not sufficient to moderate this effect. Over the past 50 years, developing nations have turned to hydropower to provide a stable energy source for their growing populations, facilitate economic development, and improve their position geo-politically by not relying upon expensive imported energy . This hath century due to dams . Our study allowed us to go one step further, since we selected communities that are located both upstream and downstream from the dam and are geographically diffuse see . The preCompensation programs have the potential to redress the losses experienced by communities that are impacted by hydropower projects. To date, the literature has mostly been critical of these programs, arguing that they are often implemented with little consultation with communities, are generally insufficient, and often exclude damages and communities that are nevertheless impacted , 28. In Each of the communities in this study suffered a variety of broken promises that may explain why compensation alone did not mitigate the stress felt by the communities. Compensation practices are typically neither transparent nor sufficient , and resDespite the efforts of activists, scientists, and other concerned groups, the Brazilian government has plans to continue to expand hydropower in coming years. Other nations, like China, will likely build additional hydropower capacity to satisfy the energy needs of their growing economies . Moving S1 Table(DOCX)Click here for additional data file.S1 Dataset(XLSX)Click here for additional data file.


The supermoiré lattice, built by stacking two moiré patterns, provides a platform for creating flat mini-bands and studying electron correlations. An ultimate challenge in assembling a graphene supermoiré lattice is in the deterministic control of its rotational alignment, which is made highly aleatory due to the random nature of the edge chirality and crystal symmetry. Employing the so-called “golden rule of three”, here we present an experimental strategy to overcome this challenge and realize the controlled alignment of double-aligned hBN/graphene/hBN supermoiré lattice, where the twist angles between graphene and top/bottom hBN are both close to zero. Remarkably, we find that the crystallographic edge of neighboring graphite can be used to better guide the stacking alignment, as demonstrated by the controlled production of 20 moiré samples with an accuracy better than ~ 0.2°. Finally, we extend our technique to low-angle twisted bilayer graphene and ABC-stacked trilayer graphene, providing a strategy for flat-band engineering in these moiré materials. The reliable fabrication of 2D heterostructures with controllable moiré patterns is important for the investigation of their emergent physical properties. Here, the authors report an alignment technique enabling the fabrication of double-aligned hBN/graphene/hBN supermoiré lattice structures with a yield close to 100%. Particularly, when two moiré superlattices contact and interface together, the overlay of double moiré will create a new structure called supermoiré lattice, strongly modifying the lattice symmetry and electronic band structure22. Compared with the single moiré potential, the double moiré potential will further break the lattice symmetry and create isolated flat moiré minibands, providing a strategy for the band flattening effect23. Recently, evidence of possible correlated states was observed in the double-aligned graphene supermoiré lattice24, which triggered further effort to search for other correlated phenomena, such as superconductivity and ferromagnetic states, as observed in twisted graphene systems25. However, due to the sophisticated stacking and the lack of control of rotational alignment, searching for these correlated phenomena in double-aligned supermoiré lattice remains elusive.The moiré superlattice, created by stacking van der Waals (vdW) heterostructures with a controlled twist angle21. However, these techniques are restricted by either specialized equipment or complicated sample preparation steps. Consequently, the optical alignment of straight edges is still the most popular and reliable technique for moiré device fabrication30. Nevertheless, the conventional optical alignment has two limitations. First, the lack of prior understanding of the crystallographic orientation of graphene or hBN, leading to only a 50% and 25% success rate for alignment of a single and double moiré structure, respectively, because the zigzag (armchair) edge of graphene can be unintentionally aligned to either the zigzag or armchair edge of hBN. Moreover, the lattice symmetry of hBN layer also obscures the inversion symmetry of the moiré heterostructure21, further decreasing the success rate to 12.5% in doublyaligned hBN/graphene/hBN heterostructures. Second, the optical alignment highly depends on the crystal edge of the graphene itself, and usually, it has a short and non-perfect straight edge. This can lead to an error in the representation of the actual principle crystallographic axes (PCA) during alignment.In previous studies of double-aligned hexagonal boron nitride/graphene/hexagonal boron nitride (hBN/G/hBN) supermoiré lattice, several techniques have been developed to control the rotation alignment, such as in situ rotation mediated by atomic force microscope (AFM) tips and polydimethylsiloxane (PDMS) hemisphere2/Vs at 2 K, we observe sharp resistive peaks at band fillings of 0, −4, −8 electrons per moiré unit cell, consistent with our calculated band structure. Second, we show that the neighboring graphite edge can be used to better guide the alignment, as demonstrated by the controlled production of 20 moiré samples with accuracy better than ~0.2°. Moreover, we have developed a so-called “Golden Rule of Three” that further guarantees the success rate and precision of our technique. Finally, we extend our alignment technique to other strongly correlated electron systems, such as low-angle twist bilayer graphene and ABC-stacked trilayer graphene, enabling us to examine moiré potential effects in these strongly correlated electron systems.In this work, we overcome the above two limitations and realize the control alignment of double-aligned graphene supermoiré lattice. First, we use a 30° rotation technique to control the alignment of top hBN and graphene, while we use a flip-over technique to control the alignment of top hBN and bottom hBN. Based on these two techniques, we can control the lattice symmetry and tune the graphene band structure. In a high-quality perfect double-aligned device with mobility of ~700,000 cm32.First, we study the controlled alignment between top hBN (T-hBN) and graphene. In the optical alignment technique, the G/hBN moiré superlattice is achieved by aligning the PCA between graphene and hBN. Based on the crystallographic structures, they can form two basic moiré patterns: 0° G/hBN and 30° G/hBN Fig. . When in−1, indicating the 0° rotation between graphene and hBN33  will lead to a significant deviation from an ideal alignment (>0.5°)  to ~70 cm−1  at the same area19  Precise representation of the PCA before alignment, and (2) Correct alignment techniques as reported in our main text. Since graphene and hBN come from two different flakes, there is a challenge to precisely represent the PCA of each flake. We have overcome this challenge and set several rules, the so-called “Golden Rule of Three”, that have to be strictly followed to guarantee the alignment precision and success rate. The rules are as follows:Golden Rule 1: Do not use the edge of graphene itself. Instead, use the straight edge of the neighboring graphite. Moreover, the length of the straight edge should be no less than 100 Golden Rule 2: Do not use a graphite flake with a single straight edge. Instead, use a graphite flake with multiple straight edges that are offset by integer multiples of 30 degrees to each other  semiconductors3/O2 plasma. The edge contact electrodes and top electrodes (3 nm Cr/65 nm Au) are deposited by standard electron-beam evaporation. The carrier mobility of our device is calculated from the slopes of conductivity σ(Vg) at a small concentration of n ∼1011 cm−2.For sample preparation, the general process is as follows. We use polycarbonate (PC), or poly-propylene carbonate (PPC) film mounted on a thick PDMS stamp to move and orientate the flakes. The stamp is used to pick up the first top hBN layer. The hBN is then positioned and aligned to a graphene edge or neighboring graphite edge before the two are brought into contact. We quickly lift the stamp once the hBN is fully passed across the graphene. After this, we invert the stamp and perform various characterizations. We then pick up a bottom hBN layer and repeat our characterizations. Last, the stack of crystals is positioned and brought into contact with a silicon wafer at the PC melting point of 180 °C. The membrane is then removed slowly so that all the stacks are left on the silicon wafer. The standard Hall bar geometry of the devices is shaped by electron-beam lithography and etched by CHFThe flip-over technique includes three steps. First, we used a thin film of polycarbonate  and polydimethyl-siloxane (PDMS) stack on a glass slide to pick up the first piece of hexagonal boron nitride flake (BN1) at 60 °C. Then we used the van der Waals force between BN1 and monolayer graphene to tear and pick up half of the graphene flake. The remaining graphene flake on the silicon was rotated by 30° and picked up at 40 °C. Second, the polypropylene carbonate (PPC) layer was spun on top of a bare silicon wafer, released with a hollow-shaped Scotch tape, and then transferred on top of the second PDMS stack on a glass slide. The PPC  is made of propylene carbonate and anisole with a mass fraction of 5%. To enhance the interaction between PPC and PDMS, the PDMS is treated with oxygen plasma for 10 mins before being covered with PPC film. Then the PPC/PDMS stamp is used to pick up the second piece of hexagonal boron nitride flake (BN2) at 60 °C. Third, in order to expose the bottom surface of BN2, we flip over the PDMS/PPC/BN2 and make the bottom surface of BN2 to be exposed to the bottom surface of BN1. Finally, we use PC/BN1/G to pick up BN2 from PPC at 80–100 °C. The success of this procedure relies on the stronger adhesion between PC and BN1 compared to BN2 and PPC.ns using both the gate capacitance and Hall measurements. We then calculate the twist angle from which the full filling corresponds to four electrons per moiré unit cell so the moiré unit cell area A = 4/ns. Second, we study Hofstadter’s butterfly features under magnetic fields. Here we use carrier-density-independent oscillations, also called BZ oscillations, of the longitudinal resistance Rxx fields, with the minimum of Rxx under magnetic moiré unit cell of a fraction of the flux quantum, BA = φ0/N, where B is magnetic field, φ0 is the magnetic flux. The Hofstadter spectrum will exhibit fractal signature  corresponding to φ = φ0/3, φ = φ0/4, φ = φ0/5.For transport measurement, the twist angles are estimated from two independent methods. First, we measure the gate voltages of full-filling gaps and convert these voltages to full-filling density 2D and the moiré wavelength for twist angles below 2°, −1 when the moiré wavelength is 14 nm at a perfect alignment of 0°.Raman spectroscopy offers a simple and fast way to determine the twist angle. The laser wavelength is 633 nm with a power of 1 mW through a ×100 objective. High-resolution Raman maps are used to characterize the spatially unfirm twist angles, which are acquired with a scan parameter of eight points per micrometer over a 10–20 μm area. We use the full width at half maximum (FWHM) of the Raman 2D-band to estimate the twist angle, where the FWHM is analyzed by Gaussian fitting. According to the previous study [33], there is a linear dependence between FWHM2 wafer at 80 °C. After this, the Au/Cr electrode is evaporated for the electrical contact using a standard lithographic technique. Before inserting into the STM chamber, the G/hBN device is annealed at 300 °C for 3–5 h in ultrahigh vacuum to remove the surface contaminations.For STM measurement, the graphene surface should be on the top so that it can be reached by STM tip. Therefore, we use a modified pick-up method for STM. First, we use a PC and PDMS stack on a glass slide to pick up a 20–30 nm-thick hBN flake. We then use the van der Waals force between hBN and graphene to pick up graphene. In order to expose the graphene surface at the top, the resulting stack with PC is transferred and released onto a second PDMS stamp. After dissolving the PC film in DCM solution, the inverted stack with PDMS is released on a SiO−11 mbar. Before the measurement, we check the tip by performing differential conductance (dI/dV) measurements on a clean Au (111) surface both before and after graphene measurement. dI/dV spectra are measured using a lock-in technique with a 20 mV (r.m.s.) and 963 Hz modulation applied to the sample voltage.Experiments are conducted with an Omicron LT-STM at low temperature (T = 77 K) with a base pressure better than 1 × 10Supplementary InformationPeer Review File


This atomic rearrangement underthe graphene layer results in aperiodic patterning of the two-dimensional(2D) material. The electronic structure of the resulting nanostructuredgraphene is dominated by a linear dispersion of the Dirac quasiparticles,characteristic of its free-standing state but with a p-doping character. The local effects of the underlying missing rowson the interfacial chemistry and on the quasiparticle scattering processesin graphene are studied using atomically resolved STM images. Thepossibilities offered by this nanostructuring approach, which consistsin inducing surface reconstructions under graphene, could providea novel tuning strategy for this 2D material.Artificial nanostructuringof graphene has served as a platformto induce variations in its structural and electronic properties,fostering the experimental observation of a wide and fascinating phenomenology.Here, we present an approach to graphene tuning, based on Rh(110)surface reconstruction induced by oxygen atoms intercalation. Theresulting nanostructured graphene has been characterized by scanningtunneling microscopy (STM) complemented by low-energy electron microscopy(LEEM), micro low-energy electron diffraction (μ-LEED), microangle-resolved photoemission spectroscopy (μ-ARPES), and microX-ray photoelectron spectroscopy (μ-XPS) measurements underultrahigh vacuum (UHV) conditions at room temperature (RT). It isfound that by fine-tuning the O Initially,the work focused on pristine graphene layers, where phenomena suchas the massless quasiparticle behavior,6 resulting in a linear band structure, or quantum hall effect,8 have been observed. Interestingly, the two-dimensional nature ofthis material makes it more sensitive to the presence of defects,as well as to other elements placed in the local environment. Thisfact has opened up new research opportunities, providing a platformfor the controlled tuning of graphene properties to pursue the explorationof novel phenomena.Morethan 15 years after the first isolation of graphene,9 or graphene bubbles10 has beenshown to induce an interesting phenomenology. While for the former,the existence of localized states, characterized by a certain magneticmoment,9 has been reported, for the latter,the existence of giant pseudomagnetic fields has been established.10 From the point of view offered by the possibilityof tuning the properties of graphene by interacting with other elements,the deposition of atoms,11 molecules,12 and clusters13 ontop of graphene has been shown to induce, for example, magnetism,11 doping,12 bandgapopening,13 or asymmetries in the groupvelocities of quasiparticles13 among others.Accordingly, the choice of support has also provided a versatile toolto modify the properties of graphene. These effects range from onlyslight changes in the electronic properties, such as doping14 or the opening of minigaps15 and the formation of band replicas,15 due to the potential felt by the quasiparticles, associatedwith the superstructures resulting from the lattice mismatch betweengraphene and the support, known as moiré patterns, to the completeabsence of Dirac cones near the Fermi level.17 Both the metal element forming the support and the symmetry of itsatomic arrangement have been shown to be key parameters to controlthe properties of graphene by inducing all this phenomenology.18 Taking these ideas a step further, other approaches,such as adding different elements beneath graphene as another layerof this material or an intercalation layer at its interface with thesubstrate, have also been explored. Interestingly, the addition ofa second layer led to the observation of unconventional Cooper pairingin twisted bilayers, where both graphene sheets are rotated underthe so-called magic angle.19 Likewise,the incorporation of an intercalation layer has induced effects rangingfrom doping, which can be tuned by the electronegativity of the intercalant,21 to the recovery of Dirac cones in cases in which they had been suppressedby the interaction of graphene with the support.23The presence of various defects such asC vacancies25 Reconstruction of thesurface atomic arrangement is very common in semiconductor materialsand less so in metals due to the absence of dangling bonds that increasethe surface free energy.25 However, anunreconstructed bare metal can still experience a surface reconstructiondue to the adsorption of specific atoms and molecules.26 In the present work, this circumstance is exploited to modify grapheneby inducing a surface reconstruction underneath through intercalation,thus taking a step forward in the development of approaches to tunethis 2D material. In particular, a nanostructured graphene systemis obtained by O intercalation between this 2D material and a Rh(110)surface. This metal support was chosen because the chemisorption ofoxygen atoms on it induces a surface reconstruction in the form ofmissing rows to reduce the stress caused by the repulsive adatom–adatominteraction.30 The combination of different characterization methods has revealedthe structural and electronic properties of the resulting nanostructuredgraphene, as well as the local effects of the missing rows underneath,both in the interfacial chemistry and in quasiparticle scattering.This work may pave the way for future investigations, related to thetuning of the properties of graphene or other 2D materials on metals,by taking advantage of the reconstruction of the metal surface uponintercalation of different elements.Surface reconstruction is a phenomenon where a few atoms oftheoutermost layers suffer lateral displacements with respect to theiroriginal positions or undergo a complete transformation of their periodicitywith respect to the bulk.22.12H4) as the precursor molecule. To this end, the Rh(110)single crystal was cleaned through cycles of Ar+ sputteringat 1 keV, followed by annealing at 950 °C at an oxygen partialpressure of 2 × 10–6 Torr, and finished witha flash-annealing in UHV at 950 °C and 1050 °C. Ethyleneexposure was then performed by opening a leak valve, connected toa high-purity source of this gas, at a pressure of 3 × 10–7 Torr for 150 s, while maintaining the Rh(110) substrateat 900 °C.Monolayersof graphene on Rh(110) were grown in situ by chemical vapor depositionunder ultrahigh vacuum (UHV) conditions, using ethylene , microlow-energy electron diffraction (μ-LEED), micro angle-resolvedphotoemission spectroscopy (μ-ARPES), and micro X-ray photoelectronspectroscopy (μ-XPS) experiments, a different approach to graphenegrowth was used to avoid the contribution of different rotationaldomains of graphene on Rh(110) to the total signal.Sample temperature was measured in both experimentalsystems byusing an infrared digital pyrometer.2.22) at a partial pressure of 1 × 10–6 Torr. In scanning tunneling microscopy (STM) experiments, wherea monolayer of graphene covered the entire Rh(110) surface, 3.6 ×103 L was typically required to achieve an intercalatedcoverage of ≈0.4 ML, while during the experiments at the Nanospectroscopybeamline, the dosage used was much lower (900 L) since the isolatedgraphene islands coexist with nearby bare metal regions. This differencein the procedure can be understood as follows: the presence of a fractionof bare Rh(110) surface directly supports the dissociation of molecularoxygen, allowing the intercalation process. On the contrary, thermallyactivated etching31 of the graphene layeris required to create bare Rh(110) regions, where O2 moleculescan dissociate, fostering the intercalation of the resulting O adatomsat the interface between graphene and Rh in neighboring regions.Oxygen intercalationat the graphene/Rh(110) interface was achieved by maintaining thesample at 320 °C during its exposure to molecular oxygen (O2.333 placed in a UHV chamber (basepressure below 10–10 Torr) connected to the adjacentpreparation chamber and delimited by a gate valve. All experimentaldata were measured at room temperature (RT) in the constant currentmode, using electrochemically etched W tips and with the bias voltageapplied to the sample. Data were acquired and post-analyzed usingthe WSxM software.34STM experimentswere performed using a home-built variable temperature scanning tunnelingmicroscope (VT-STM),2.436 at the Nanospectroscopy beamline of the Elettra Sincrotrone TriesteLaboratory. This instrument combines imaging, diffraction, and spectroscopyoperation modes under UHV conditions, using either electrons or photonsas probe sources. The kinetic energy of the electrons emitted or scatteredelectrons by the sample was controlled by the bias voltage appliedto the sample, known as the start voltage. For LEEM measurements,only the zero-order diffraction beam was used for imaging (BrightField). For μ-LEED, ARPES, and XPS data, the imaging area waslimited to a diameter of less than 2 μm using illumination ora field-limiting aperture. The monochromated soft X-ray beam was incidenton the sample at a grazing angle of 16° from the surface plane.36 The energy resolution of Nanospectroscopy beamtime for spectroscopicdata is below 100 meV. ARPES data were collected by acquiring constant-energysurfaces below the Fermi level, with an energy step-size of 0.025eV, and subsequently stacked upon each other. Then, the band structurecan be obtained by cutting along the desired high symmetry directions.All measurements were performed at RT.These techniques were executed using the spectroscopicphotoemission and low-energy electron microscope (SPELEEM),3c(2 ×2) structure with respect to Rh(110). Meanwhile, p2mg, c(2 × 6), c(2 × 8), and c(2 × 10) O/Rh(110) superstructures.30 These types of (1 × n) surface reconstructionsimply that one surface closed-packed row along the [11̅0] directionis missing out of n atomic spacings along the [001]one, and no oxygen atoms are located along the resulting stripes.Considering this, the superstructure observed in c(2 × 2) phase on Rh(110) under the graphenecover, which does not involve substrate reconstruction. In contrast,the stripe patterns in c(2 × 8) and O-c(2 ×16) structures, respectively. Moreover, by using the oxygen arrangementas a fingerprint of the Rh(110) crystallographic direction, informationabout the graphene rotation angle was also derived. 18 It should also be noted that the continuityof the graphene monolayer is not interrupted by the underlying missingrow reconstruction of the substrate (see S1), althoughthe sandwiched superstructure shows a lower apparent height in themissing row areas . Inthis sense, the graphene in these regions seems to behave as a continuousrippling sheet across the surface.To understand the features observedin 29 Furthermore, two distinct areas of graphene can befound when oxygen induces substrate reconstruction: the intercalatedarea (IA) or the missing row (MRA) area, as labeled in To illustrate the above situations,a plausible schematic representationof the atomic arrangement is proposed in p2mg arrangement reported on the bare metal,where the oxygen atoms induce a (1 × 2) missing row reconstructionof the Rh(110) substrate, here the O-c(2 × 2)does not involve any Rh(110) surface reconstruction under graphene.Likewise, O-c(2 × 16) with (1 × 8) surfaceRh(110) reconstruction also differs from the available literaturefor the case of O/Rh(110), since the maximum spacing in the missingrow reconstruction of Rh(110) was reported to be (1 × 5). Therefore,it seems that the graphene layer induces changes in the chemisorptionof oxygen on Rh(110). Such an idea was also conceived in the Gr/O/Ir(111)system,37 where new oxygen phases undergraphene, which do not exist on the bare Ir(111) metal, were reported.Apossible explanation for the observed experimental evidences is givenin the following lines: O adatoms chemisorbed on Rh(110) are negativelycharged due to electron transfer from Rh atoms to the more electronegativeO ones. As a result, there is a repulsive adatom–adatom interaction,which induces stress in the Rh(110) surface, given the fact the Oatoms are bounded to Rh ones in the topmost surface layer. This stressseems to be relaxed via the formation of missing row reconstructionsin the substrate. Nevertheless, when a graphene cover is over theO adatoms, the atomic lattice of this 2D material could receive someof the stress, which otherwise would be completely induced in theunderlying Rh(110) support. It could explain, for instance, why amissing row reconstruction is not needed at low amounts of O intercalationin the case of the O-c(2 × 2) superstructure.The above comparisonwith the case of O on Rh(110) inevitably raisesthe question of whether or not the graphene cover plays a role inthe underlying atomic arrangement. Compared to the O-(2 × 2)The sandwich superstructures involving substrate reconstructionwere those found predominantly under the intercalation parametersused in the present work. A broader perspective can be gained by lookingmore closely at the regions where these oxygen-induced atomic rearrangementsof Rh(110) occur. 39 as well as after the adsorption of various species on other metalsurfaces, such as acetate molecules on Au(110)26 or Si on Ag(110).40 Furthermore,in the present case, all these processes must take place at the interfacebetween graphene and Rh(110) since experimental STM data showed thatthe graphene monolayer was not disrupted between the planar regionsand the new islands . Thisalso proves the strength and stability of graphene to handle suchcomplex atomic rearrangements below. The above description is illustratedby the scheme shown in The measured apparent height ofthe islands is in agreement withthe single-step height of Rh(110), suggesting that they are formedby Rh atoms in the same configuration as in the (110) plane of bulkRh. Moreover, the presence of missing rows also indicates the chemisorptionof oxygen atoms onto Rh(110) within the islands. These two observationscould be rationalized in terms of a rearrangement of Rh atoms ejectedfrom the missing row areas after the adsorption of oxygen, forcingthe former to aggregate together to form new terraces on the Rh(110)facet with the same rectangular configuration. Analogous phenomenahave been observed for O on bare Rh(110),ands see S2. Thisn) periodicity of the missing row reconstruction of Rh(110)seemsto follow a random distribution over the entire surface, ranging from n = 4 to n = 12. Therefore, the O-c(2 × 8) and O-c(2 × 16) phasesdescribed in c(2 × 2n),with n = [4–12], are found only locally, andthe surface is characterized by a long-range aperiodicity. In otherwords, in this case, graphene could be considered as a one-atom-thicksheet of carbon atoms that ripples aperiodically over the entire surface.Based on the above results, it could be pointed out that the formationof the missing rows across the sandwiched superstructures would havea certain stochastic character due to the fact that the different(1 × n) missing rows, with 12 > n > 4, are thermodynamically allowed. Nevertheless, it seems thatthere must be some interaction between the missing rows since no (1× n) rearrangements for n <4 are found. Furthermore, it cannot be excluded that the distributionof the missing rows along the [001] direction of Rh could be influencedby some defects that could be created during the growth of grapheneor the intercalation of oxygen. In addition, no experimental evidencewas found for a possible effect of the rotational domains of grapheneon the periodicity of the missing row reconstruction.As already can be seen in Once thestructural properties of the sandwiched Gr/O/Rh(110) nanostructureswere established, the electronic properties of the system were alsoinvestigated. Such studies have been focused on regions where oxygeninduces substrate reconstruction under graphene since these are themost commonly found. 42 Thus, the features observed in 42 could also indicate a higher level of graphene interactionin the present case with O/Rh(110) underneath. Furthermore, despitethe rippling character of graphene induced by the presence of bothIA and MRA below, the fact that such patterns can be imaged in thesetwo regions  suggeststhat the graphene monolayer does not exhibit dissimilar electronicproperties in these regions. This could be tentatively explained bythe fact that the intercalation of oxygen atoms on the IA might besufficient to allow the decoupling of the entire graphene sheet fromthe Rh(110) surface in this specific region and also in the MRA. Itis noteworthy to mention that intervalley scattering patterns, similarto those observed in 43 It appears that although the graphene layer is not interrupted acrossthese boundaries, they induce quasiparticle scattering processes thatare not produced by the MRA.A direct explanation for the originof these patterns, observedin the STM images of see also S3 suggesFor a more comprehensive characterizationof the Gr/O/Rh(110) nanostructures,the results presented and discussed above are complemented by LEEM,μ-LEED, μ-ARPES, and μ-XPS data. 18 This suggests that the darker regions in theLEEM image are related to oxygen intercalation, while the other regionsare not intercalated. Furthermore, the appearance of a new featurein the LEED pattern shown in 18 reinforces thishypothesis. The diffuse path, highlighted by the dashed magenta rectangle,is aligned with the Rh[001] direction in the reciprocal space andshows a stronger intensity near the  spot. Such a feature isconsistent with the coexistence of different (1 × n) substrate reconstructions of Rh(110) under graphene, after theoxygen intercalation, in agreement with the STM measurements summarizedand discussed in First, as shown in K of the graphene first Brillouin zone and centered on K (see the blue line in vF) and the Diracpoint (ED) can be determined. Such a fittedresult is represented by the two superimposed red lines, yieldingan approximate value of vF = 1.0 ×106 ± 2 × 105 m/s and ED – EF = 0.4 ±0.1 eV. Details about the employed fitting procedure can be foundin Figure S4. For completeness, the grapheneband structure projected along other high symmetry directions of thegraphene first Brillouin zone is shown in Figure S5. Similarly, using a photon energy of 400 eV, the C 1s corelevel spectrum of graphene is shown in On the same island, wherethe μ-LEED shown in 46 However, in the caseof Gr/O/Rh(110), the Dirac point is above the Fermi level. In fact,the graphene layer shows a hole doping 22 system. However, it differs from the STS data obtained for oxygenintercalated on Gr/Rh(111)23 or Gr/Ru(0001).47 The absence of crossings of Rh(110) states withthe graphene π band, which is present in as-grown graphene onRh(110)18 suggests that, upon oxygen intercalation,the graphene layer is efficiently decoupled from Rh(110) and no furtherhybridization with the metal 4d states is expected, consistent withthe analysis of graphene quasiparticle scattering results shown in The estimated value for the Fermivelocity of graphene quasiparticlesis in agreement with the theoretical and experimental values of afree-standing graphene monolayer.18 wherethe C 1s spectrum is described by two components with a binding energyof 284.9 and 284.4 eV, implying a significant interaction of graphenewith the metal, the C 1s core level of graphene after oxygen intercalationbelow is characterized by a general shift toward lower binding energy,with a dominant peak at 284.0 eV and another one, with a more modestcontribution to the C 1s line shape, at 284.4 eV. Such an energy shiftis in qualitative agreement with the p-doping levelof graphene, as demonstrated by the μ-ARPES data shown in 48 and it is also consistent with other previouslyreported experimental results.49 Thus,the core level shift of the main component of the C 1s signal observedin the present case provides a solid fingerprint of oxygen intercalationunder graphene. As shown in S6). However, a contribution to the componentat 284.4 eV coming from MRA whose C atoms do not have oxygen atomsunderneath them but are still lifted with respect to the metal substratecannot be excluded.Comparedto graphene on Rh(110),4c(2 × 2), which does not involve any reconstructionof Rh(110), and a mixture of O-c(2 × 2n) phases with (1 × n) Rh(110) substratereconstruction as missing rows, where n ranges from4 to 12. The latter structure was the predominant one found in thisoxygen-sandwiched system, and it is characterized by an aperiodicmodulation of graphene over the surface due to the coexistence ofdifferent (1 × n) reconstructions within a narrowzone. The resulting aperiodic nanostructured graphene consists oftwo distinct regions, namely, the intercalated area (IA) and missingrow area (MRA). Joint STM-based quasiparticle interference analysisand μ-ARPES and μ-XPS data obtained in the missing rowreconstructed regions show that the graphene is efficiently decoupledfrom the Rh(110) substrate and exhibits the characteristic lineardispersion of graphene, albeit with p-doping. Furthermore,the experimental data do not support a significantly different chemicaland electronic landscape on IA and MRA. These results could open aninnovative method of graphene nanostructuring via the intercalationof species that induce surface reconstruction.By combiningdifferent imaging, spectroscopy, and diffraction techniquesunder UHV conditions, the structural and electronic properties ofaperiodically modulated graphene monolayers have been investigated.Such a nanostructured system was realized by the intercalation ofoxygen at the interface of graphene grown on Rh(110). Oxygen atomsunder graphene form two types of superstructures with respect to Rh(110):the O-