Informed by the poststructuralist theory, this study investigates the case of Ming, a Chinese professor of English, about the impacts of his first language (L1) and second language (L2) learning experience, and the changes of social contexts on his L1 and L2 identities construction. It was found that being a learner of English as a Foreign Language (EFL), Ming’s identities development aligned with the poststructuralist theory, in which it is considered dynamic, fluid and conflicting. Ming negotiated and renegotiated his identities in various social contexts in China and the United States and finally gained acceptance into the L2 academic community. This study not only analyzes Ming’s experience with his language learning and identities, but also unravels that conflicts may be part of the process of identities construction, and encourages learners to be persistent and emotionally resilient, while using certain strategies to retain a stable L1 identity so that they can navigate through the negative encounters during the second language acquisition (SLA) process to sustain the development of their identities and L2 abilities. The relationships between social contexts, language and identities, and how they affect the learners’ second language acquisition (SLA) have been well documented in the literature. For instance, Traditional frameworks such as the acculturation model embraced the monolingual model, which unrealistically oversimplifies the process of L2 learning and disregarded the learners’ heritage language and culture. However, the exponential increase in mobility and globalization in the last two or three decades has resulted in greater diversity in various communities. English as a lingua franca also seems to gain its momentum, and many people from non-English speaking countries began to learn English. The monolingual model can no longer reflect the realities of the world today. As such, more recently, several researchers argued against these frameworks with the poststructuralist model, which captures the complexity of L2 learning and renders the learning outcome unpredictable. For example, Based on the previous literature, there seems to be a need for more research on the complexity of how L1 and L2 learning affect the learner’s identities construction. In addition, there is a paucity of studies on the development of the learner’s identities associated with changing social contexts, which can be operationally defined in this study as the different residence countries, school settings, communities, circles of friends and family of an English as a Foreign Language (EFL) learner.In order to better understand L1 and L2 identities construction along this line of research, the current study examines the case of Ming, a Chinese professor of English, about his L1 and L2 learning experience, L1 and L2 identities construction, and how language related experience and social contexts positively or negatively impact both identities.The theory of poststructuralism has been widely accepted in the academic community. Previous empirical studies substantiated the poststructuralist framework in learner identity and learner identity construction. Although English learners’ L2 identity construction may be independent of that of their official language in their home country, the process of their L2 identity construction may be difficult and complex. Therefore, a number of researchers conducted case studies investigating how L2 students negotiate their identities in an attempt to gain access to their L2 community. In more recent literature, scholars continue conducting similar studies, or updating their theories to gain a deeper insight into English learners’ L2 identity construction, especially how L2 learners negotiate their identities, the techniques they use and how they may mitigate such struggles. The research conducted on learner identity and its development supported the advocacy of multi-competencies and multilingualism. Furthermore, While previous studies unfolded the L2 learners’ dynamic negotiation of identities, L2 competence, and power positions in diverse contexts to gain legitimate membership in the L2 and international communities, the complexity of how L1 and L2 learning experience impact the construction of L1 and L2 identities, and how both identities are affected by social contexts, are yet to be fully explored. The current study aims to shed lights on these issues by conducting a case study on a highly proficient Chinese professor of English, Ming, who was born and raised in China but went to the United States for his MA and Ph.D. studies. The case study seeks answers to the following research questions:1)To what extent did Ming’s L1 and L2 learning experiences affect his L1 and L2 identities construction in different social contexts of China and the United States?2)How had Ming’s different social contexts in China and the United States impacted his L1 and L2 identities development?This study took a qualitative case study approach, with Ming as our focal participant. The case study approach was used in this research because of its many advantages. For example, Ming was born and raised in a southern province of China and excelled in his Chinese and English, from a young age. After graduating with a BA in English in China, he prepared for the TOEFL and the GRE on his own and achieved nearly perfect scores, with 110 on TOEFL (its scores range from 0 to 120) and 98th percentile on the GRE. He went to the United States and stayed there for 10 years to pursue his MA and Ph.D. degrees in English. Upon graduating from his Ph.D. program, he started teaching and conducting research at a university in China. His L2 learning experience and identities of L1 and L2 might have been impacted because of a change in social contexts from China to the United States in general. More specifically, his social contexts in this study encompass different countries, school settings, communities, the circle of friends and family, etc. Therefore, he was chosen for this case study. A summary of Ming’s profile is presented below.Two different data sources, namely, Ming’s written autobiographical narrative and a semi-structured interview, were employed in this study. Both were done in English. Five guiding questions see  were genIn order to investigate Ming’s L1 and L2 learning experiences and identities construction, his narrative data were analyzed chronologically, based on three critical time periods. Each of the time periods is of significance to his L1 and/or L2 identities development. The first period occurred in the context of a high school and a university in China, where his L1 and L2 learning experiences as well as L1 and L2 identities construction began. The second period impacted his L1 and L2 identities development as his social contexts changed, when he first arrived in a mid-west city in the United States for his MA study. This is the period when he experienced the most struggles with his L1 and L2 identities. In contrast, the third period witnessed how Ming mitigated such conflicts and how he further developed both of his identities during his Ph.D. study in a large metropolitan city on the east coast of the United States. To deepen the analysis and triangulate the data, follow-up interview data were transcribed, coded, analyzed and presented based on the three time periods in the findings as follows: (1) L1 and L2 learning experiences and related language identities construction in China; (2) Initial L2 learning experiences in a mid-west city of the United States and language identities struggles; and (3) Reconciling L1 and L2 identities during the Ph.D. program in a large city of the east coast of America. Moreover, the poststructuralist theory was used to provide a deeper analysis of this case study. Member check was conducted with the participant to ensure accuracy and validity of data analysis.It is necessary to analyze Ming’s English learning experiences from three different time periods chronologically in order to capture his changing L1 and L2 identities. Ming’s profile and English learning are summarized in As Ming grew up in a multilingual environment with exposure to different dialects, he considered himself a multilingual Chinese. It seemed that at that time, his L2 identity was much weaker as compared to his L1 identity, since it was only “a foreign language” to him. Nevertheless, his L2 identity developed from a beginner of English in junior high school to an advanced learner at university:Excerpt 1 from narrativeAt the age of 11, I began to learn English as a foreign language in junior high school…I became an English major in college and studied the language, culture, and pedagogy of English. Apparently, I am a native speaker of Chinese… English is just a foreign language to me.”“…As an avid reader and skillful writer in his L1, Chinese, Ming believed that some of his L1 reading and writing skills were positively transferred to his acquisition of L2 English, which boosted his confidence in reading and writing in English and subsequently reinforced his L2 identity.Excerpt 2 from narrative“My solid foundation in Chinese literacy contributed a lot to my acquisition of English literacy later on which increased my confidence in the English language immensely.”When Ming arrived in a mid-west city of the United States for his MA study, he still considered himself an English learner:Excerpt 3 from narrative“I considered myself a lifetime English learner. In the MA program, I read on average 300 pages English materials per week, and delivered six to eight presentations and wrote four term papers  every semester, plus a thesis in the 4th semester. Meanwhile, I was watching English TV and interacted with Americans on a daily basis. the language environment changed to English. most of my professors and classmates were Americans.”Excerpt 4 from interviewmy English improved a lot. In the second semester of my MA program, I wrote a critical and insightful book review in English and published it in a refereed journal. Then I published another paper in my 3rd semester. At that time, none of the American students in my MA cohort published any papers.”“…He was developing his L2 identity in the United States. Meanwhile, he reached out to various Chinese and American discourse communities, and retained his L1 identity.Excerpt 5 from narrativeHowever, my roommate was a Chinese graduate student and I called my family once a week. In addition, I regularly interacted with people in the Chinese community. I was able to retain my L1 self in the United States.”“… However, such developments of his identity were not without struggles and conflicts.Even though he was able to gain acceptance into both the American and the Chinese communities in the United States, the communities themselves were separated from each other. He felt as if two people, namely, his L1 self and his L2 self, were trapped living within one body. Moreover, Ming also felt that his L1 identity threatened his L2 identity at times, and vice versa:Excerpt 6 from interview“When Americans were hostile toward my L1 self, it impeded my L2 learning, because I felt like I was not eligible to speak English. One day, I was doing grocery shopping at a local supermarket in that mid-west city. I was talking to my Korean friend about some random stuffs. All of a sudden, an American guy walked by and said to me “You are Chinese, why do you speak English? That’s my language!” I did not confront him or argue with him. Somehow I felt that he owned the English language and I did not have the right to use it. It was a horrible feeling.”Another hurdle that Ming needed to overcome was the unfamiliarity of American culture, for instance, the background knowledge of jokes. A lot of the time, when the professors said something funny in class, he did not know what was going on when everyone else was already laughing out loud.Excerpt 7 from interview“Sometimes, when talking to my American classmates, they were also joking too , but I did not get the joke and had no clue what they were talking about. Those were embarrassing moments.”He also encountered problems when interacting with Americans.Excerpt 8 from interview“Chinese people may say “have you eaten?” as a way to say “hello” to each other. Of course, I was not stupid enough to say that to greet my American friends. However, I was not used to saying “how is it going?” or “what’s up?” yet when I bumped into my friends in the street or on campus. Instead, I greeted them in the Chinese way by saying “where are you going?”. Guess what? Some Americans responded “none of your business.””Apparently they were offended by what he said. Americans seem to have a strong sense of boundaries between each other. They value privacy and try to mind their own business. He stumbled over cultural issues like that quite a bit.On the other hand, after living in the United States for about a year, Ming began to encounter issues with some friends back in China. After he went to the United States for graduate study, he barely hand wrote Chinese anymore. As a result, his ability in Chinese writing decreased. Even though his everyday Chinese was not much affected, he sometimes had trouble expressing academic stuffs in Chinese.Excerpt 9 from narrative“One day, I was talking on the phone in Chinese to a friend in China and he asked me about the format of a research paper. I could not think of the Chinese words for “double spaced,” so I just said the English words, which he did not understand. He was offended and screamed at me: “Why do you have to show off your English? Do you think you are all that?”. Things like that happened to me a few times.”Even though it was not his intention to say English words to his friends in China, his L2 identity still posed a threat to his L1 self and caused miscommunication with his Chinese friends.After the master’s program, Ming moved to a large city on the east coast for his Ph.D. degree. There, his academic English further improved. He was serving as a research assistant and later a teaching assistant at university where he interacted on a daily basis with students and colleagues. During his doctoral study, he published quite a few papers in international refereed journals.He was also able to maintain his L1 identity, especially when his American undergraduate students and his American colleagues highly respected him and his L1 identity, and welcomed him into the academic community. Perhaps it was because the university was located in a metropolitan city with a quite diverse population, people tended to embrace diversity more, and incidents like the one in the supermarket did not happen to Ming again.Excerpt 10 from narrative“For some reason, some of my American undergraduate students and professors in the doctoral program preferred to call me by my Chinese name Ming rather than my English name Tom. I felt that they respected me for who I am and valued my Chinese cultural background.”His confidence in his L2 continued to grow as he published more journal articles and excelled in his doctoral study. He finally found a way to accept both his L1 and L2 identities, and became a multilingual, multicultural individual.Excerpt 11 from interview“In the Ph.D. program, I got straight A’s. I remembered a professor of the Ph.D. program was a well-known tough grader. He rarely gave A’s to his students and his tough feedback even made some doctoral students cry. I survived his class and got an A for active participation in class discussions and writing compelling term papers.”When people doubted his L2 identity, he seemed to be able to fall back on his L1 identity, which was more stable and comforting to him. His knowledge about World Englishes also helped.Excerpt 12 from interview“Sometimes people in my neighborhood picked on my Chinese accent and said that You don’t have a perfect American accent. How can you teach English then?”Ming was bothered by those negative comments at the beginning, but then as he read more literature on World Englishes, he knew well enough to tell those people that the monolingual model of English learning was problematic and unrealistic. In addition, as a lingua franca, English is being used world wide, instead of by people from English speaking countries only. Different varieties of Englishes are being redefined by researchers. His Chinese accent just shows that he was speaking a variety of English, which he was feeling comfortable with.Furthermore, Ming was inspired by some well-established scholars in his field, who originally came from Asia but were working in the United States. They shared similar cultural backgrounds and probably went through similar struggles to fight prejudice as well.Excerpt 13 from interview“In addition…there were quite a few Asian researchers…(who) were leaders in the field. I looked up to them as my role models and thought that my Chinese accent did not matter as long as I kept publishing papers and that should not be an obstacle to my career. Instead, my L1 self and culture were an asset in my research and teaching.”Finally, Ming no longer thought his L1 and L2 identities were conflicting. Instead, both were assets.Below is a summary table of Ming’s L1 and L2 identities construction in different social contexts, reflecting the intricate relationships among his language learning, social contexts and identities construction.Ming considered himself a life time English learner, and kept expanding his L2 identity in these three critical periods of his L1 and L2 learning. In the beginning, Ming’s L1 and L2 identities developed separately in China. Although his identity became a struggle when Ming first arrived in the mid-west of the United States for his MA study, during his Ph.D. study, his L1 and L2 identities converged and he was finally feeling comfortable with his identity as a multilingual. Ming was an additive bilingual. He moved to the United States after he graduated from university in China. By that time, his L1 identity was already fully developed. He managed to retain his L1 identity throughout his stay in the United States. Thus, his L2 identity was additive to his L1 self.The construction of Ming’s L1 and L2 identities can be explained by Norton’s poststructuralist theory. His identities were fluid, dynamic and changed depending on the contexts. For example, during Ming’s MA study, his L1 and L2 identities were dynamic and fluid. When he was in school, he may present his L2 self to his professors and peers. However, when he was at home or talking to his family in China over the phone, his L1 self dominated. The struggles and challenges Ming went through were not only due to cultural gaps between the east and the west, it was also because of the power relations. For instance, when Ming could not think of the Chinese word for “double spaced,” he spoke English to his friend in China (who didn’t speak English), which made his friend angry. This is mainly because English is at the top of the language hierarchy, and the fact that Ming spoke English unintentionally put his friend at a disadvantage. It was after going through different changes in contexts and difficulties that Ming successfully developed his L2 identity.Other findings show Ming had much positive L1 transfer to his L2 literacy, hence he looked at his L1 identity positively. Such may be due to his social context (a high school in China) at the time that allowed him to construct his L1 identity with ease. Furthermore, it seemed that Ming’s L1 identity conflicted with his L2 self after he went to the United States for his MA study. This was due to his social contexts at the time. He was studying in an MA program in a mid-west city in the United States. The culture in the mid-west is relatively conservative, being less liberal than that in the west and east coasts. Even though Ming was well accepted by both the Chinese and the English communities at that mid-west university [this is consistent with Ming’s struggles in his L2 identity are consistent with the previous studies e.g., . SpecifiThis is similar to the research conducted by Moreover, Ming’s Ph.D. study and continued publications  helped him gain confidence in his English and L2 identity. The fact that his professors, and peers valued his diverse background helped to nurture his identity as “a multiple and a dynamic social construct” , p 56. Hheld dear” in his “heart.” He also expressed that his “favorite food is still Chinese dishes.” While existing studies promote multilingualism, and advocate against acculturation and monolingualism, it is essential to point out that although Ming was not fully acculturated, he became more inter-culturally sensitive, and embraced diversity. This implies that although English learners do not have to fully accept the L2 culture and lifestyle, it may be beneficial for them to have an open mind and to be more inter-culturally sensitive, so that they may interact with their L2 communities easily.Furthermore, Ming was raised in a multilingual environment with exposure to different dialects. Consequently, he thought of himself as a multilingual, which may have enabled him to maintain such an identity in the United States, even when his L1 and L2 identities conflicted with each other. Contrary to Ming’s autobiographical narrative and interview data seemed to align with the conceptual framework of the poststructuralist theory, described by Furthermore, it is equally important to recognize that not all discourse communities augment Ming’s struggles with his identities. Some  may nurture and fast-track his L2 identity construction and encourage both of his L1 and L2 identities to co-exist and thrive.Ming’s experience revealed important implications on English learners’ identity construction and sustainable development. First, there may be a relationship between students’ L1 and L2 identities and L2 learning. Various researchers studied the socio-contextual framework of L2 learning  of the learner identities construction. There may be others who may have different experiences in their English learning and their identities construction. For example, some may have moved to an English speaking country at a younger age, at an older age, or may be born in that country. Some learn L2 in an L2 speaking context whereas others may learn it in an L1 environment. Nevertheless, they are also multilinguals/bilinguals who may have a unique point of view on various pertaining topics. Therefore, it may be important for one to acknowledge, respect and embrace such diversity, instead of giving preference to one “type” of multilinguals/bilinguals. Findings of various studies  may offer empirical data to substantiate or challenge existing theories, or develop new theories and techniques to alleviate the challenges in students’ L2 identity construction, and contribute to the sustainable development of students’ L2 skills.For the above reasons, more empirical research (case studies and survey plus interviews) should be conducted to provide stakeholders with a more realistic and panoramic picture that reflects the complexities of L2 learners identities construction, the potential opportunities and the role of learner agency as a result of sustaining the development of their L2 identity and creating their multilingual identity.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Foreign Studies College, Hunan Normal University. The patients/participants provided their written informed consent to participate in this study.YX was responsible for the conceptualization, most of the methodology and editing. AZ contributed to the first draft of the manuscript. Both authors contributed to the data collection, revising, 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.


We employ graphene microelectrodes fabricated on PET substrate in chronic in vivo lifetime. We demonstrate stable chronic recordings of cortical potentials in multimodal imaging/recording experiments using transparent graphene microelectrodes fabricated on PET substrate. On the other hand, graphene microelectrode arrays built on SU-8 substrate exhibit extensive crack formation across microelectrode sites and wires after one to two weeks, resulting in total failure of recording capability for chronic studies.Our results show that graphene microelectrodes fabricated on PET substrate work reliably after 30 days accelerated aging test performed at 87 °C, equivalent to 960 days in vivo experiments.PET shows superior reliability as a substrate for graphene microelectrode arrays for chronic Graphene is a unique neural interface material enabling cross-talk free integration of electrical and optical recording and stimulation techniques in the same experiment. To date, graphene-based microelectrode arrays have been demonstrated in various multi-modal acute experiments involving electrophysiological sensing or stimulation, optical imaging and optogenetics stimulation. Understanding chronic reliability of graphene-based transparent interfaces is very important to expand the use of this technology for long-term behavioral studies with animal models. I.in vivo experiments [Electrophysiology has been one of the major tools for basic neuroscience research and clinical practice for decades. There have been vast technological advances in electrode technologies, allowing for high-temporal resolution recording of neuronal spikes and local field potentials generated by neuronal ensembles. Optical techniques such as multi-photon imaging has opened new avenues for high-spatial resolution mapping of neural activity from hundreds of neurons , 2. Comberiments –8. Grapheriments , high coeriments  and bioceriments , 12, makeriments .in vivo chronic animal experiments on mice with simultaneous 2-photon imaging and electrical recordings using the PET graphene electrode array and evaluate the stability of the electrode impedance and the recording noise over time.Transparent graphene arrays can be fabricated on different types of flexible polymer substrates, which exhibit optical transparency in wavelength ranges from 450 nm to 850 nm, commonly used in optical imaging and stimulation experiments. Different types of polymers including polyimide , SU-8 1, PET 5,, ParylenII.A.We fabricated 16-channel graphene microelectrode arrays with a 100 μm electrode size and 400 μm spacing on SU-8 and PET substrates –d. BesidB.s mimics the series resistance of the solution, the constant phase element (CPE) represents the Helmholtz double-layer capacitance (Cdl), the bounded Warburg element (Wb) simulates any diffusion processes occurring, and Rct is the charge-transfer resistance, which simulates any faradaic reactions [Both graphene microelectrodes fabricated on a PET substrate and SU-8 substrate were characterized using electrochemical impedance spectroscopy (EIS) in 0.01 M phosphate buffered saline (PBS) to study the graphene/electrolyte interface –d. Impedeactions . Represeq [dl. Computational values of Cdl (around 10 μF cm−2) are larger than computational values of Cq (around 1–4 μF cm−2) [dl is experimentally measured using Au electrodes [q is computed by fitting EIS results to the equivalent circuit model shown in ct and Wb values are both large, so the electrode behaves capacitively, which is consistent with well-known characteristics of graphene.Graphene gives rise to an additional quantum capacitance component represented by Cq , 21 in sμF cm−2) . Cdl is ectrodes , and Cq C.in vivo lifetime for implantable devices [ref). For our study, Tref is set at 37 °C, for that is the average body temperature of mice.Accelerated aging tests have been used to simulate  devices –27. The  devices . ΔT is ein vivo environments, we suspended the microelectrode arrays in a glass beaker and immersed them in 0.01 M PBS at pH 7.4, a buffer solution commonly used in accelerated aging tests due to the constant pH level [in vivo. Four graphene microelectrode arrays fabricated on a PET substrate and four graphene microelectrode arrays fabricated on an SU-8 substrate were used for a 30-day accelerated aging test. The impedance of working channels was tracked every three days for PET and every day for the SU-8 arrays which showed early failure.To simulate pH level . The setpH level , and theD.2) was made over the left hemisphere and the graphene microelectrode array was implanted to cover visual cortices. A glass imaging window was placed on top of the electrode array and further secured with vetbond (3 M) and dental acrylic. To protect the array while the mice were in the home cage, a custom 3D-printed case was employed. Electrophysiological data were recorded by the RHD2000 amplifier board and RHD2000 evaluation system (Intan Technologies), which has a large input impedance which allows us to achieve high SNR with our relatively high impedance electrodes. It is important to note that relatively high impedance of graphene electrodes necessitates recording systems with large input impedance to prevent signal attenuation. The data was processed using custom Matlab code.All procedures were in accordance with protocols approved by the UCSD Institutional Animal Care and Use Committee and the guidelines of the National Institutes of Health. Adult mice (cross between CaMKIIa-tTA (JAX 003010) and tetO-GCaMP6s (JAX 024742), 2 months old) were anesthetized with isoflurance (3% for induction and 1% for maintenance) and a circular piece of scalp was removed. A custom-built head-post was implanted to the exposed skull (1 mm posterior to lambda) with cyanoacrylate glue and cemented with dental acrylic . A stainless-steel screw  was implanted over right olfactory bulb as reference. A square craniotomy  exhibited visual PBS permeation between the PET substrate and SU-8 encapsulation layer . Liquid B.All four graphene microelectrode arrays fabricated on SU-8 substrate succumbed to crack failures prior to the conclusion of the 30-day accelerated aging test. The average impedance of SU-8 arrays over the 30-days is plotted along with the standard deviation in A summary of all the arrays detailing equivalent lifetime age and failure mechanisms is listed in Some studies in literature have suggested SU-8 as a reliable material for neural applications , 35, 36 C.in vivo studies. Therefore, for chronic in vivo experiments, we only used transparent graphene microelectrodes fabricated on PET substrates. To further evaluate the reliability of the transparent graphene microelectrode arrays in chronic imaging and recording experiments, we implanted the graphene microelectrodes over the visual cortex of transgenic mice. The transgenic mice express GCaMP6s [Based on the results of the accelerated aging tests, transparent graphene microelectrode arrays fabricated on a PET substrate were determined to be more reliable for chronic  GCaMP6s , which i GCaMP6s . Similar2) positioned 15 cm away from the right eye using Psychtoolbox [2. The neuron cell bodies are clearly identified due to the high transparency of the graphene electrodes and PET substrate (in vivo experiments combining electrophysiological recordings with 2-photon microscopy.In addition, we observed stimulus-induced responses in multiple ECoG channels, showing delayed on-responses and elevated signal power across wide frequency bands , 6e. Durhtoolbox . One of ubstrate . Our resIV.in vivo lifetime. On the other hand, graphene microelectrodes fabricated on SU-8 substrate exhibited failures due to different forms of crack formation across the electrodes or wires. We further demonstrated the reliability of graphene microelectrode arrays fabricated on PET in chronic 45-day in vivo studies in mice. After 45 days, graphene electrodes still exhibit low noise and impedance and can reliably record ECoG during calcium imaging. Our studies conclude that compared to other substrate materials, PET shows superior reliability and is therefore an ideal substrate of the graphene microelectrodes arrays for chronic in vivo studies, combining simultaneous electrophysiology and optical imaging.We performed systematic accelerated aging tests to investigate reliability of transparent graphene microelectrode arrays fabricated on PET and SU-8 substrates. Graphene microelectrodes fabricated on PET substrate show high reliability with all functioning channels after 30 days of accelerated aging or 2.63 years equivalent supp1-2979475


The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C‐terminal fragments of the protein TDP‐43. Here, we tested the hypothesis that highly sensitive mass spectrometry with parallel reaction monitoring (MS‐PRM) can generate a high‐resolution map of pathological TDP‐43 peptide ratios to form the basis for quantitation of abnormal C‐terminal TDP‐43 fragment enrichment. Human cortex and spinal cord, microscopically staged for the presence of p‐TDP‐43, p‐tau, alpha‐synuclein, and beta‐amyloid pathology, were biochemically fractionated and analyzed by immunoblot and MS for the detection of full‐length and truncated (disease‐specific) TDP‐43 peptides. This informed the synthesis of heavy isotope‐labeled peptides for absolute quantification of TDP‐43 by MS‐PRM across 16 ALS, 8 Parkinson’s, 8 Alzheimer’s disease, and 8 aged control cases. We confirmed by immunoblot the previously described enrichment of pathological C‐terminal fragments in ALS‐TDP urea fractions. Subsequent MS analysis resolved specific TDP‐43 N‐ and C‐terminal peptides, including a novel N‐terminal truncation site‐specific peptide. Absolute quantification of peptides by MS‐PRM showed an increased C:N‐terminal TDP‐43 peptide ratio in ALS‐TDP brain compared to normal and disease controls. A C:N‐terminal ratio >1.5 discriminated ALS from controls with a sensitivity of 100% (CI 79.6–100) and specificity of 100% (CI 68–100), and from Parkinson’s and Alzheimer’s disease with a sensitivity of 93% (CI 70–100) and specificity of 100% (CI 68–100). N‐terminal truncation site‐specific peptides were increased in ALS in line with C‐terminal fragment enrichment, but were also found in a proportion of Alzheimer cases with normal C:N‐terminal ratio but coexistent limbic TDP‐43 neuropathological changes. In conclusion this is a novel, sensitive, and specific method to quantify the enrichment of pathological TDP‐43 fragments in human brain, which could form the basis for an antibody‐free assay. Our methodology has the potential to help clarify if specific pathological TDP‐43 peptide signatures are associated with primary or secondary TDP‐43 proteinopathies. The pathological hallmark of amyotrophic lateral sclerosis (ALS) is the presence of cytoplasmic inclusions, containing C‐terminal fragments of the protein TDP‐43. Here, we used a high‐resolution map of TDP‐43 peptides to form the basis for absolute quantitation of abnormal C‐terminal TDP‐43 fragment enrichment. The C:N‐terminal peptide ratio is increased in ALS brains and N‐terminal truncation‐specific peptides confirm the pathological cleavage of TDP‐43 in ALS and surprisingly AD. TDP‐ as CTFs , 5. Alth as CTFs , 7, biociofluids . Studiesiofluids . Limitatiofluids , 12, 13.post mortem tissue. The detection of additional N‐terminal truncation site‐specific peptides was used to quantify the process of pathological cleavage of TDP‐43 into N‐terminal truncated CTFs. This method specifically discriminated ALS from other neurodegenerative diseases such as Parkinson’s disease (PD) and AD. Interestingly, by this method we were able to identify some cases of AD with coexistent LATE neuropathological change (LATE‐NC); however, further work is required to establish whether our method is suitable for the identification of disease‐specific “TDP‐43 strain signatures” associated with the spectrum of primary and secondary TDP‐43 proteinopathies.We aimed to develop an antibody‐independent method to biochemically detect CTFs by mass spectrometry to enable the absolute quantification of pathology. Detecting specific C‐ and N‐terminal TDP‐43 peptides allowed us to measure the pathological accumulation of C‐terminal TDP‐43 relative to its N‐terminal end in urea fractions extracted from TDP‐43 pathology positive ALS 22.1Primary motor cortex (BA4) or, if unavailable, dorsolateral prefrontal cortex (BA9/46) and spinal cords from individuals diagnosed with ALS, PD or AD, and from normal controls (CTL) were obtained from the Oxford Brain Bank. Donors had provided written informed consent for brain donation and the use of the material and clinical information for research purposes under Research Ethics Committee approval .2.2Post mortem delay was calculated in hours from time of death to fixation and the age at death was recorded. Genetic testing was only conducted in the presence of a clear family history.For several ALS cases severity of physical symptoms was classified as per the revised ALS Functional Rating Scale (ALSFRS‐R) with a maximum of 48 points. Here, lower values represent a more severe disease stage  and the 2.3All cases were formally assessed for microscopically visible protein aggregates using standard criteria used in international brain banking (PMC3266529). In brief, formalin‐fixed paraffin‐embedded sections (six microns) were stained with the following primary antibodies : phosphorylated TDP‐43 (pTDP‐43) that targets pS409/410 ; Anti‐ß‐Amyloid ; AT8 ; and Purified Mouse Anti‐alpha‐Synuclein . All were developed with the DAKO Envision Kit and 3,3‐diaminobenzidine tetrahydrochloride (DAB) as chromogen. Sections were examined using an Olympus BX50 microscope with an attached Olympus digital camera (for photography).2.4Fractionation of tissue was performed as described by Neumann and colleagues , with th2.5The urea fractions were diluted in LDS sample buffer (Thermo Fisher) supplemented with reducing agents (Thermo Fisher) and boiled for 5 min. Proteins were separated on 10% NuPAGE pre‐cast Bis‐Tris gels (Thermo Fisher), transferred using an iBlot2 apparatus (Thermo Fisher). Membranes were Ponceau‐stained, then blocked using protein free Blocking buffer (Thermo Fisher), and probed overnight at 4°C with a polyclonal TDP‐43 C‐terminus antibody recognizing TDP‐43 at epitopes from amino acid 260 onwards  or phosphorylated TDP‐43 at S409/410  and 2 h at room temperature with secondary antibody rabbit HRP. Antibody signal was detected with ECL reagent  using a ChemiDoc MP Imaging System (Bio‐Rad). For interpretation of the immunoblots the ratios of the intensities of the CTFs over full‐length TDP‐43 were compared.2.6Total protein amounts were visualized by silver staining using a ProteoSilver Silver Stain kit (Merck) as per the manufacturer’s protocol and using solutions provided in the kit. Briefly, after electrophoresis, NuPAGE Bis‐tris gels were fixed overnight at room temperature in fixing solution (10% acetic acid (Millipore) and 50% Ethanol (VWR) in MilliQ water). The gels were consecutively washed for 10 min in 30% Ethanol, MilliQ water, sensitizer solution, and silver solution. Staining was visualized by incubating with developer solution for 30–70 s, the reaction was stopped by addition of ProteoSilver Stop solution and washing with MilliQ water for 15 min.2.7Urea fractions were thawed on ice and used for digestion. For in‐gel digestion gel bands were excised with a scalpel after electrophoresis and gel pieces rinsed in 200 μl wash solution (50% methanol and 5% acetic acid in MilliQ water). Two‐hundred microliters of acetonitrile was added to dehydrate the gel pieces prior to reduction with 5 mM DTT and alkylation with 20 mM IAA. After dehydration samples were first rehydrated in 200 μl of 100 mM ammonium bicarbonate and then, in 30 µl of 200 ng/µl chymotrypsin (Promega) in 50 mM ammonium bicarbonate and incubated overnight. Fifty microliters of 50 mM ammonium bicarbonate buffer was added to the samples, 50 µl of extraction buffer 1 (50% acetonitrile and 5% formic acid in MilliQ water) and 50 µl of extraction buffer 2 (85% acetonitrile and 5% formic acid in MilliQ water) were added and supernatant with eluted peptides collected. For in‐solution digestion samples were reduced with 5 mM of dithiothreitol (Alfa Alsar) followed by alkylation with 20 mM IAA. Samples were subsequently precipitated by methanol–chloroform extraction. Pellets were fully resuspended by addition of 6 M urea buffer (Merck) and brief sonication. Samples were reduced to a final concentration of 1 M Urea by adding 50 mM TEAB buffer. The samples were incubated with 2 μg trypsin/chymotrypsin (Promega) at 37°C/25°C on a shaker for 14–16 h. The reaction was stopped by addition of formic acid (Millipore). Samples were desalted using SOLA SPE columns (Thermo Fisher). All in‐solution and in‐gel digested samples were dried in a speed‐vac (Thermo Scientific) and pellets were resuspended in 2% acetonitrile with 0.1% formic acid diluted in MilliQ water.2.8Peptides from in‐solution and in‐gel digestion were analyzed by nano ultra‐high‐performance liquid chromatography (nano‐UPLC) tandem mass spectrometry (MS/MS) using a Dionex Ultimate 3000 nano‐UPLC, (Thermo Fisher) coupled to an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher). Data were acquired in data‐dependent mode with a resolution of 120,000 full width half maximum at m/z 200 in the survey scan (375–1500 m/z) and with EASY‐IC using the reagent ion source (202 m/z) for internal calibration. MS/MS spectra were acquired after precursor isolation in the quadrupole with an isolation window of 1.2 Th, dynamic precursor selection (top speed mode) with a 3‐s fixed duty cycle time, and 60‐s dynamic precursor exclusion. Isolated precursor ions were fragmented by CID with a Normalized Collision Energy of 35%. Parallelization was enabled and MS/MS spectra were acquired in the linear ion trap for up to 250 ms with an ion target of 4000 in rapid scan mode. Raw MS data were analyzed using Progenesis QI for Proteomics v3.0 (Nonlinear Dynamics). MS/MS spectra were searched against the UniProt Homo Sapiens Reference proteome (retrieved 15/11/2016) using PEAKS with precursor mass tolerance of 10 ppm and a fragment ion tolerance of 0.5 Da. Carbamidomethylation of Cysteines was defined as a fixed modification, deamidation of Asparagine and Glutamine, and oxidation of Methionine as variable modifications. Peptides scoring ≥20 and FDR <1% were imported into Progenesis QIP.2.9As previously described, in‐gel digestion of low molecular weight bands (20–28 kDa) of ALS urea fractions were used to identify the truncation site‐specific TDP‐43 peptides . These p2.10For the measurement of endogenous (light) TDP‐43 peptides by mass spectrometry with parallel reaction monitoring (MS‐PRM), C‐ and N‐terminal and truncation site‐specific peptides detected from the discovery experiments and a previously reported sequence  were use2.1110 ratio (light:heavy peptide)) were compared across diagnostic groups. The C:N‐terminal peptide ratio was calculated by dividing non‐log absolute abundances of the light peptides. All significant differences were tested comparing ALS to all other diagnostic groups using one‐way ANOVA with Dunnett’s multiple comparison test. p‐values <0.05 were considered as statistically significant. Standard measures of diagnostic test validity were performed on non‐log abundances of the light peptides. Sensitivity, specificity, and predictive values accompanied by their confidence interval (CI) stated at the 95% level were calculated for varying peptide cut‐off levels. The optimal cut‐off level for dichotomizing values was selected as the situation maximizing the Youden index (For interpretation of the PRM results absolute abundances of the light peptides (logen index . The rec33.1Post mortem time from death to fixation was the same between groups (p = 0.6). Clinical characteristics of patients are given in Table All clinical diagnoses were neuropathologically confirmed and the characteristic neuropathology of each diagnostic group used for the final MS‐PRM analysis is shown in Figure 3.2First, we used LC–MS/MS analysis to detect unique peptides for the absolute quantification of TDP‐43 Figure . In‐soluFor absolute quantification of endogenous (light) peptides by MS‐PRM the corresponding heavy isotope‐labeled analogs of a list of tryptic and chymotryptic TDP‐43 peptides were generated Table  and test3.3p = 0.04) and an increased level of CTF‐25 in ALS compared to CTL, PD, and AD  in accordance with the enrichment of CTFs in ALS brain tissue   protein fractions from the same tissue cohort of 16 ALS, 8 PD, 8 AD, and 8 CTL used for PRM were performed at equal volumes to verify the recovery of protein prior to digestion and to confirm depletion of abundant proteins after sequential fractionation Figure . The uren tissue . It is n) Figure .3.410 ratio (light peptide:heavy peptide), Figure p = 0.001)  . To investigate if peptide abundances reflect the pathological increase of CTFs in insoluble protein fractions from ALS motor and prefrontal cortex tissue, we calculated the C:N‐terminal peptide ratio. The C:N‐terminal peptide ratio was increased in ALS compared to all other diagnostic groups   Figure . In cont) Figure , but una) Figure . This suTo determine the potential of using C‐ and N‐terminal peptide quantification as a diagnostic tool to discriminate ALS from other diseases, receiver operating characteristic (ROC) analysis was used Figure . A N‐ter3.5p = 0.0008 and p < 0.0001, respectively) . Only when calculating the Truncation 1: N‐terminal peptide ratio an increase in ALS compared to AD was observed (p = 0.04) caused by the respective higher N‐terminal peptide abundance in AD.To specifically quantify N‐terminal truncation of TDP‐43, to further proof the pathological truncation of TDP‐43 in smaller CTFs, the truncation site‐specific peptides were measured within the insoluble protein fraction from motor and prefrontal cortex tissue. Quantification of truncation site‐specific peptide 1 (Truncation 1) showed an increase in ALS motor and prefrontal cortex urea fractions compared to PD and CTL  Figure . SurprisROC analysis showed that a Truncation 1 peptide abundance >0.005 yielded the optimal discrimination between ALS and PD, with a sensitivity of 100% (CI 67.56%–100.0%) and 100% specificity (CI 56.55%–100.0%) Figure . Applyinp = 0.001 and p = 0.01, respectively) , PD, and CTL (p < 0.0001 and p = 0.0001). No significant difference between ALS and AD was observed when calculating the Truncation 2: N‐terminal peptide ratio (p = 0.79). According to this, ROC analysis demonstrated a Truncation 2 peptide abundance >0.004 yielded the optimal discrimination between ALS and PD with a sensitivity of 100% (CI 70–100) and 100% specificity (CI 18–100)  showed a significant increase in ALS motor and prefrontal cortex urea fractions compared to PD and CTL  Figure , but Tru) Figure . ApplyinIn summary, quantification of the TDP‐43 peptide signature in motor and prefrontal cortex urea fractions showed that in ALS the C:N‐terminal peptide ratio and truncation site‐specific peptides were increased in the presence of TDP‐43 neuropathology. In contrast, in AD motor cortex where TDP‐43 neuropathology is microscopically absent the C:N‐terminal ratio was normal, but truncation site‐specific peptides were similarly increased Figure .3.6To investigate, if an increase of C:N‐terminal peptide ratio and truncation peptides also represent pTDP‐43 pathology we immunoblotted motor cortex urea fractions of ALS and AD cases with the highest MS‐PRM values compared to PD and CTL cases. We observed the highest immunoreaction for pTDP‐43 at 45 and 25 kDa in ALS followed by AD with LATE‐NC, while pTDP‐43 was absent in PD and CTLs with low MS‐PRM values Figure . As expe3.7Due to the biochemical evidence of pathological TDP‐43 truncation in AD motor cortex samples using highly sensitive PRM, we sought to investigate if this finding represents the coexistence of LATE‐NC in significant cases of AD. Therefore, we re‐examined our cohort for microscopic evidence of pTDP‐43 pathology. We found evidence of LATE‐NC in six out of eight cases of AD, but not in PD, ALS, or controls confirming the sensitivity of our assay. The principal anatomical distribution of pTDP‐43 in LATE‐NC was different from that typically found in ALS, mainly involving the amygdala, entorhinal cortex, and hippocampus, but not the motor cortex Figure .3.8p = 0.0001) , PD, or AD (both p = 0.9)   Figure . This su) Figure . When ca) Figure . Truncat4Post mortem validation of TDP‐43 pathology currently relies on qualitative rather than absolute quantitative data . Professor Benedikt Kessler, Dr. Mattéa Finelli, and Mr Connor Scott have nothing to report.Conception and design of the study: RF, EG, BMK, KT, and MRT; Acquisition and analysis of the data: EF, PDC, MJF, CS, OA, and RF; Drafting a significant portion of the manuscript or figures: EF, PDC, MJF, CS, BMK, RF, EG, KT, and MRT; Review and editing: RF, BMK, EG, KT, and MRT; Funding acquisition: MRT, EF, BMK, and PDC; Resources: RF, BMK, OA, and MRT; Supervision: RF, EG, KT, and MRT. All authors critically reviewed the manuscript and approved its submission.Supplementary MaterialFIGURE S1 Neuropathology of the study cohort. (A) ALS‐TDP with granular‐, skein‐ and neuritic pTDP‐43 aggregates in the motor cortex (layer three) and nucleus hypoglossus (inset). (B) PD with alpha‐synuclein‐positive Lewy bodies, granular deposits and Lewy neurites inthe substantia nigra (inset: pigmented dopaminergic neuron with Lewy body H&E). (C and D) classical AD with a neuritic beta‐amyloid‐positive plaque (C) and neurofibrillary tangles in the granule cells of the hippocampus (D) . Scale bar 20 μmFIGURE S2 Silver stains of insoluble and soluble protein fractions. Representative silver stains demonstrate the total protein amount of (A) 0.5 μl of the soluble (TX) protein fractions. (B) 1 μl of the insoluble (urea) protein fractions extracted from cortex and spinal cord post mortem tissue of CTL, ALS, PD and AD patients. Depletion of abundant proteins is demonstrated in urea fractionsFIGURE S3 Skyline export of light and heavy peptides for PRM analysis. Representative Skyline exports shown with Savitzky‐Golay  retrieved from a pooled ALS cortex urea fraction sample. The top row represents light peptides and the bottom row heavy isotope‐labelled peptides, where different coloured graphs represent fragment ions. (A) N‐terminal chymotryptic light and heavy peptide. (B) C‐terminal chymotryptic light and heavy peptide. (C) Truncation 1 chymotryptic light and heavy peptide. (D) Truncation 2 chymotryptic light and heavy peptideTABLE S1 Detailed characteristics of post mortem tissueTABLE S2 Detection of TDP‐43 peptides from in‐solution trypsin digestions of cortex urea fractions from ALS and CTL casesTABLE S3 Detection of semi‐specific truncation site‐specific TDP‐43 peptides from chymotrypsin in‐gel digestion of cortex urea fractionsTABLE S4 List of TDP‐43 peptides for heavy isotope‐labelled peptide productionClick here for additional data file.


Using Rhodamine 6G (R6G) as a probe, SERS spectra were compared on MoS2 N-donuts/graphene nanohybrids substrates. A remarkably high R6G SERS sensitivity up to 2 × 10−12 M has been obtained, which can be attributed to the more robust LSPR effect than in other TMD nanostructures such as nanodiscs as suggested by the finite-difference time-domain simulation. This result demonstrates that non-metallic TMD/graphene nanohybrids substrates can have SERS sensitivity up to one order of magnitude higher than that reported on the plasmonic metal nanostructures/2D materials SERS substrates, providing a promising scheme for high-sensitivity, low-cost applications for biosensing.Nanohybrids of graphene and two-dimensional (2D) layered transition metal dichalcogenides (TMD) nanostructures can provide a promising substrate for extraordinary surface-enhanced Raman spectroscopy (SERS) due to the combined electromagnetic enhancement on TMD nanostructures via localized surface plasmonic resonance (LSPR) and chemical enhancement on graphene. In these nanohybrid SERS substrates, the LSPR on TMD nanostructures is affected by the TMD morphology. Herein, we report the first successful growth of MoS Tancement ,6,7 In amentally ,10. 2D amentally ,12 are ementally  in nanohmentally ,16,17,182 and MoS2) of lateral dimension ~500 nm and height ~3–5 nm grown on the monolayer of graphene (TMD N-discs/graphene), [−12 M on the TMDs N-discs/graphene nanohybrids [−1 on different substrates, the EM enhancement factors could be estimated using graphene only SERS substrate as reference. Specifically, the enhancement factors of 10.3 and 8.7, respectively, are obtained by dividing the intensities of the 613 cm−1 peak on TMDs (WS2) N-discs/graphene and gold nanoparticles (AuNPs) on graphene (or AuNPs/graphene) SERS substrates by that on graphene [While metallic plasmonic nanostructures have been the major focus to induce EM enhancement, nonmetallic ones have recently emerged as a promising alternative to address the drawbacks of the metallic plasmonic nanostructures including strong spectra noise background due to contamination on the metal surface, the deformation and distortion of the probe molecules due to the strong metal-adsorbate interactions, the metal-catalyzed side reactions of the probe molecules and substantial joule dissipation ,10,19,20aphene),  a strongohybrids . It shouohybrids ,28. Usingraphene . While tgraphene ,30.Quantitatively, SERS enhancement relies on many factors such as the size and the shape of plasmonic nanostructures and substrate morphology ,32,33,342 nanodonuts (N-donuts) by controlling the size of the (NH4)2MoS4 precursor nuclei via variation of its concentration in the range of 0.06−0.40 wt%. The hypothesis is that MoS2 N-donuts could be achieved on large-size precursor nuclei due to the nonuniform conversion to MoS2 along the radial direction while MoS2 N-discs form on smaller nuclei when such nonuniformity is negligible. In addition, the solution-based deposition may lead to nonuniform distribution as the solvent is evaporated, which could also lead to donut-shaped structures. With increasing precursor concentrations, the precursor nuclei dimension increases monotonically and MoS2 N-donuts become predominant at precursor concentration of 0.26 wt% or higher. Interestingly, a resonant (at 532 nm excitation) R6G SERS sensitivity ~2 × 10−12 M has been achieved on these MoS2 N-donuts/graphene SERS substrates, in contrast to 5 × 10−12 M for the 0.06 wt% MoS2 where MoS2 N-discs are more prevalent [In this work, we explore the growth of MoSrevalent ,43,44,452 (7 sccm) and CH4 (40 sccm). For graphene transfer on SiO2/Si substrates via a wet transfer procedure [3 solution) for about 4 h, followed by rinsing the PMMA/graphene samples at least five times with deionized water. The PMMA/graphene samples were then placed onto the SiO2/Si and left overnight to dry at room temperature in air. Afterward, the PMMA/graphene/SiO2/Si stack was soaked in acetone four times to remove PMMA. Finally, the samples were rinsed with isopropanol (IPA) and thermally annealed at 400 °C for 30 min in forming gas (Ar (500 sccm)/H2 (300 sccm)) to remove PMMA residues on graphene.Graphene was synthesized on polycrystalline Cu foils  using chemical vapor deposition (CVD) and the details of the growth procedure have been described in detail in our earlier works ,47,48. Brocedure ,47, 3% P2 was grown on graphene using a vapor transport process established in our previous works [2 N-donuts, the (NH4)2MoS4 precursor solution of variable concentrations was prepared by dissolving (NH4)2MoS4 powder in  with different concentrations of 0.06 wt%, 0.10 wt%, 0.20 wt%, 0.26 wt% and 0.40 wt%. It should be noted that continuous layers of MoS2 were achieved at ~0.10 wt% with multiple precursor dips [2 N-donuts could be obtained on graphene with a single dip of the precursor of low (0.06 wt%) to high (0.40 wt%) precursor concentrations. However, at the lower end of precursor concentrations in this range, MoS2 N-discs seemed preferable due to possibly the small precursor nuclei dimension. The nuclei dimension increases with the precursor concentration, MoS2 N-donuts became predominant at the higher end of precursor concentrations. After the graphene/SiO2/Si samples were dipped in the precursor solution, spinning at 3000 rpm for 30 s was followed to achieve a uniform coating of the precursor on graphene. The samples were annealed afterward in a tube furnace at 450 °C for 30 min in sulfur vapor transported by a mixed gas of H2 (10 sccm) and Ar (50 sccm) from the sulfur powder placed upstream from the sample in a quartz tube at about 200 °C.MoSus works ,49. To o2 N-donuts were measured using the Nanoscope software . Specifically, densities were measured using the particle analysis feature, and the diameters and heights were measured by taking cross-sections through at least 20 MoS2 N-donuts for each sample.The sample phase and morphology were characterized using Raman spectroscopy and microscopy , and atomic force microscopy (AFM). For Raman characterization of graphene, 488 nm excitation laser was employed using a 20× microscope objective. The integration time was 3 s and the laser power was ~1–5 mW. For the Raman spectroscopy and maps of TMDs, a 100× microscope objective was used with either 532 or 488 nm laser excitation. For consistency, Raman maps were collected at a minimum of 4 different locations on each sample. AFM images were collected using a Digital Instruments Multimode Nanoscope IIIA AFM system in contact mode using standard silicon nitride cantilevers . Images were collected in at least three locations on the sample surface at multiple scan sizes. The average diameter, height, and density of the MoS−5 M to 10−13 M) was placed on the substrate surface and left to dry for about one hour on a hotplate set at 70 °C. The diameter of the R6G samples on the substrate after it dried is around 4–5 mm. R6G SERS characterization was carried out using laser excitation of 532 nm on the same WiTec alpha 300 confocal Raman system. The laser beam spot was about tens of µm that is further reduced through a 20× microscope objective to allow multiple scan spots near the center of the droplet for consistency. An integration time of 3 s and a low intensity (~1–5 mW) were used to avoid the possible damage of the R6G probe molecules. For an improved signal-to-noise ratio, each presented Raman spectrum is an average of 5–10 spectra gathered at the same spot of the sample. It should be noticed that every Raman spectrum demonstrated in this work is representative because it is based on an average of ~10 or more Raman spectra gathered at different spots arbitrarily chosen on a sample and typically 2–3 samples were tested for reproducibility. To prevent the coffee-ring effect, the tested spots were chosen not too close to the R6G sample’s edge.For the investigation of SERS, R6G was used as a probe molecule. A droplet of R6G  was used. Our simulation model is mostly used to highlight how these two structures support different plasmonic field enhancement factors, defined as the ratios of the field intensities in the presence of the N-donuts and N-discs to those in their absence. Simulations were carried out considering metallic N-donuts and N-discs with different sizes, placed on SiO2 substrates.A finite-difference time-domain simulation (FDTD) study of the characteristic plasmonic effect was carried out to reveal the differences between MoS2 N-donuts/graphene nanohybrids (see Method for details). Controlling the (NH4)2MoS4 precursor concentration in a DMF solution was found critical to obtaining the MoS2 N-donuts. N-donuts nanostructures can be obtained on graphene with precursor concentrations as low as 0.06 wt% while precursor nuclei lateral dimension increases with the precursor concentration. At lower precursor concentrations, MoS2 N-discs seem to be dominant on smaller precursor nuclei  and with 0.26 wt% precursor concentration  (−1 due to the primary in-plane vibration mode and 2D peak at ~2707 cm−1 due to secondary in-plane vibration of zone-boundary phonons [−1 (associated with the defect in graphene8) are clearly observable on these samples. On the pristine graphene, the intensity ratio of 2D to G peak (I2D/IG) is around 1.8 while ID/IG is negligible, indicating that the graphene is a monolayer with high quality. On the MoS2/graphene nanohybrid made with 0.10 wt% precursor concentration, the graphene’s Raman peak intensities are notably enhanced most probably due to the LSPR effect by the MoS2 nanostructures [2/graphene nanohybrid sample made with 0.26 wt% precursor concentration, indicating a stronger LSPR effect. It should be noted that such an enhancement in the Raman signatures of graphene by metallic plasmonic nanostructures has been reported on metal-nanostructures/graphene nanohybrid samples [2/graphene nanohybrids indicates that the LSPR can be effectively generated on nonmetallic TMD nanostructures. 2 taken on the MoS2/graphene nanohybrids made with 0.10 wt% (red) and 0.26 wt% (blue) precursor concentrations, respectively, using Raman excitation at 488 nm. The Raman peaks in both spectra can be indexed to the −1 (due to S and Mo atoms in-plane displacement mode) and −1 (due to the S atoms out of displacement mode) of MoS2 [2 was grown on graphene in the MoS2/graphene nanohybrid samples.ominant) . The gra phonons , and D p samples ,51,52. A of MoS2 , confirm2 Ag1 peak taken on the MoS2/graphene nanohybrid samples made with different precursor solution concentrations of (a) 0.06 wt %, (b) 0.10 wt %, (c) 0.2 wt %, (d) 0.26 wt %, (e) 0.32 wt%, and (f) 0.40 wt%. The Raman maps show that the MoS2 formed is not in a continuous layer, rather in the morphology of NPs of approximately round shape in the selected (NH4)2MoS4 precursor concentration range. Quantitatively, the (NH4)2MoS4 precursor concentration exhibits an effect on both the MoS2 NP density and shape. At the lowest precursor concentration of 0.06 wt%, the round shape of MoS2 NPs grows in two different sizes: smaller size with an average diameter of ~200 nm (majority) and larger size with an average diameter of ~1.0 µm with low density. With increasing precursor concentrations in the range of 0.20–0.26 wt%, the size of the MoS2 NPs changes moderately in the range of 300–600 nm, while the NP density increases. Above the precursor concentration of 0.26 wt%, the lateral dimension of the MoS2 NPs increases considerably and the NP density decreases.2 NPs is shown in 2 NPs have been confirmed in the entire range of the precursor concentration 2MoS4 precursor concentration from ~200 nm at 0.06 wt% precursor concentration to 600 nm at 0.26 wt% concentration. However, the average diameter seems to saturate at higher precursor concentrations.AFM images of 5 µm × 5 µm at selected concentrations of 0.06 wt%, 0.26 wt%, and 0.40 wt% are shown in −5 M) probe molecules on MoS2/graphene nanohybride substrates that were synthesized with different precursor solution concentrations from 0.06 wt% to 0.32 wt%, measured using the same excitation of 532 nm wavelength. Most of the R6G Raman peaks are visible on all spectra. Especially, the spectra include the R6G fundamental peaks at 613 cm−1 allocated to the C–C–C ring in-plane vibration mode, 773 cm−1 assigned to aromatic C–H bending mode, 1190 cm−1 and 1648 cm−1 allocated to C–O–C stretching mode and the C–C stretching mode, respectively [2 N-discs are predominant in this sample. In contrast, the highest SERS enhancement is observed on the spectrum for MoS2/graphene nanohybride made with 0.26 wt% precursor concentration (black) on which MoS2 N-donuts are predominant. Quantitatively, the enhancement factor can be estimated using the ratio of the intensity of the R6G Raman peaks on the MoS2/graphene samples made using different precursor concentrations at 613 cm−1 and 773 cm−1 to the ones on the MoS2 (0.06 wt%)/graphene). 2/graphene sample at 0.26 wt% precursor concentration, suggesting that the SERS enhancement is a compromise of the shape, dimension, and density of MoS2 NPs. Interestingly, MoS2 N-donuts are predominant in MoS2/graphene nanohybrides when the precursor concentration is exceeding ~0.20 wt%. The higher SERS sensitivity in this precursor concentration range, especially the peak SERS sensitivity in MoS2/graphene nanohybride made from 0.26 wt% precursor concentration, suggests that MoS2 N-donuts may support LSPR in a more preferable way than other N-discs or other shapes of MoS2 nanostructures.ectively ,54. Amon−5 M to 2 × 10−12 M were cast and dried on the substrate and then Raman spectra were gathered with a 532 nm laser. The R6G Raman spectra are compared at the R6G concentrations of 5 × 10−5 M to 5 × 10−9 M in −10 M to 2 × 10−12 M in −10 M. With further reduction of the R6G concentration to 2 × 10−12 M, most of the R6G Raman signature peaks are still detectable, particularly the vibrational modes with larger polarizability, such as 613 cm−1, 773 cm−1, 1191 cm−1, and 1648 cm−1. Therefore, the R6G sensitivity of 2 × 10−12 M on the MoS2 N-donuts/graphene nanohybrid is about half an order of magnitude higher than that obtained on MoS2 N-discs/graphene [The R6G molecule droplets of varying concentrations ranging from 5 × 10graphene  and abougraphene .−1 peak enhancement factor  on different SERS substrates of graphene only, MoS2 N-discs/graphene [2 N-discs (0.10 wt%)/graphene [2 N-discs+MoS2 N-discs (0.10 wt%)/graphene [2 N-donuts/graphene (made from 0.26 wt% precursor concentration in this work). The result shows that the best enhancement was obtained on the MoS2 N-donuts/graphene.graphene , AuNPs+Mgraphene , WS2 N-dgraphene  and MoS2−1 and 773 cm−1 as a function of the R6G concentration are depicted in 2 (0.26 wt%)/graphene. The R6G Raman peak intensities at 613 cm2 material studied in this paper. However, since LSPRs are ubiquitous optical signatures of charge carriers, as shown below, our model can explain the impact of the shapes of MoS2 structures studied in this paper on the SERS enhancement. The simulations were done considering a normal planar polarized light reaching an NDs or N-donut placed on a SiO2 substrate  of 500, 360, and 240 nm a. The thThe bi-resonance nature of N-donuts allows them to present a robust field enhancement over a wide range of wavelengths. To see this in In the case of N-discs c, howeve2 N-donuts/graphene nanohybrids SERS substrates. The MoS2 N-donuts growth process consists of two major steps: coating of (NH4)2MoS4 precursor at room temperature in air, followed by vapor transport anneal in sulfur vapor transfer process at 450 °C for 30 min. We have found that the predominant MoS2 N-donuts of the larger lateral dimension of 300–700 nm and height of 5–10 nm can be obtained by varying the precursor concentration to exceeding 0.20 wt% to promote nonuniform MoS2 conversion on larger precursor nuclei. This is in contrast to the formation of predominantly MoS2 N-discs of smaller lateral dimension at lower precursor concentrations down to 0.06 wt%. A SERS study using R6G probe molecules has shown that higher R6G SERS sensitivity has been observed on MoS2 N-donuts/graphene nanohybrid substrates than on the MoS2 N-discs/graphene ones. Specifically, the highest SERS sensitivity up to 2 × 10−12 M has been achieved on the MoS2 N-donuts/graphene nanohybrid SERS substrates made using 0.26 wt% precursor concentration. This is a half order of magnitude higher than 5 × 10−12 M on the MoS2 N-discs/graphene SERS substrates. FDTD simulation indicates that MoS2 N-donuts can better support LSPR than their MoS2 N-discs counterparts. This result illustrates the feasibility of tuning TMD nanostructure morphology for high-performance nonmetallic plasmonic nanohybrids for low-cost, large-scale applications in biosensing and other optoelectronics.In summary, we have developed a layer-by-layer growth process for the fabrication of MoS


The in situ investigation of the dynamic growth process and novel assembly phenomena of graphene on liquid copper (Cu) is of great significance to deeply understand the special behavior of graphene and self‐assembly mechanism. Here, the direct observation of the graphene growth and motion behavior on liquid Cu via in situ imaging is reported. Evidence of graphene movement on liquid Cu is offered and it is demonstrated that the translation and rotation behaviors of graphene are affected by the surface condition of liquid Cu. The self‐assembly process of graphene array is also revealed by capturing the dynamic changes of graphene in real‐time. Further analysis highlights the importance of surface energy of liquid Cu and the interaction between graphene building blocks during the self‐assembling process. The growth parameters are also investigated to flexibly control the assembly configuration of graphene arrays. This work provides an insight into the mechanism of graphene motion and assembly behavior that can be used to guide the controllable manipulation of 2D materials and on‐demand fabrication assembly structures with desired properties. This work develops an in situ investigation system to study the growth and self‐assembly of graphene on liquid copper in real‐time. The translation and rotation behavior of graphene are captured for the first time, which is attributed to the fluidity of liquid copper surface. The self‐assembly mechanism is further revealed, enabling the precise synthesis of various assembly structures. Currently, liquid metal catalysts  with a quasi‐atomically flat surface, show great potential in synthesizing uniform graphene with significantly higher quality and faster growth speed. Meanwhile, the intrinsic fluidity of liquid metal provides a platform for precise manipulation of graphene to achieve complex assembly behaviors.The controllable synthesis of large‐size, defect‐free graphene is a main challenge toward its practical application. We discovered that graphene could assemble spontaneously into a super‐ordered structure on liquid Cu surface. Research on this self‐assembly behavior of graphene is of great significance to scalable and cost‐effective fabrication of integrated devices. However, the current exploration of the growth and self‐assembly mechanism only relies on ex situ experiments and theoretical calculations. The experimental setups of CVD are similar to “black box” in most cases. There are some scientific issues still unclear, such as the dynamic growth and self‐assembly process, and the thermodynamics and kinetics mechanism of self‐assembly behavior on liquid Cu. In situ research enables direct visualization of the dynamic process during the crystal growth and evolution, which is expected to help understand the special behavior of graphene on liquid Cu and provide guidance for precisely synthesizing various assembly structures.In our previous works, we have achieved the controllable synthesis of a series of 2D atomic crystal arrays based on liquid metals.Here, we investigate the growth and motion behavior of graphene on liquid Cu via in situ imaging for the first time. We offer explicit evidence to the movability of graphene on liquid Cu at high temperature. The translational and rotational motion of graphene are captured during the growth and assembly, which are related to the condition of liquid Cu surface. We show the dynamic process of graphene self‐assembly behavior and further reveal the self‐assembly mechanism. We demonstrate that both the minimum surface energy and interaction between graphene are crucial to the formation of aligned graphene arrays via self‐assembling. We further explore the influence of the growth parameters on the growth and self‐assembly of graphene. Our work highlights the unique potential of an in situ technique that enables improved understanding of mechanisms in the CVD process. We believe that our in situ investigation could guide the precise assembly of 2D atomic crystals and promote applications in integrated systems.2Figure The schematic of in situ growth of graphene in a CVD reactor is shown in Figure 4. Based on different emissivity between graphene and Cu, they could be distinguished at high temperature. The typical Rad‐OM image of as‐grown graphene is shown in Figure For studying growth and motion behavior of graphene on liquid Cu, we built an in situ investigation system  and an objective . The quantum efficiency of camera was 72% over 525 to 580 nm and decreased rapidly to less than 5% at 1000 nm (on the website of the company). A 20× long working distance microscopy objective was applied to focus the samples and prevent the thermal radiation damage due to the high temperature.−1. In order to prevent the oxidation of Cu, 200 sccm Ar and 10–30 sccm H2 were introduced into the custom‐made CVD reactor during the whole heating process.The melting process of Cu under high temperature was investigated using the in situ system. After electrochemically polished, the 0.05 mm Cu foil  was put on the W  substrate, which was located in the center of the heating zone. The Cu foil was heated to the melting point and the maximum of the heating rate was less than 30 °C min−1. When the temperature reached 1100 °C, 1–2 sccm CH4 was introduced into the custom‐made CVD reactor for growing graphene. The oxide nanoparticles introduced from the ceramic heater tended to be the nucleation sites, serving as good tracers. After 2–5 min, the CH4 was turned off, and the temperature was decreased to room temperature. The whole process was conducted under 200 sccm Ar and 12 sccm H2.First, the Cu foil was put on the W substrate, which was located in the center of the heating zone. After that, the Cu foil was heated to 1100 °C and the maximum of the heating rate was less than 30 °C minOptical images and videos were taken with a radiation‐mode optical microscopy. Raman spectroscopy was performed with WITec alpha 300R system (488 nm excitation wavelength). The low‐resolution transmission electron microscopy (TEM) images were obtained by a high‐resolution TEM system (JEM‐2100) and the operating voltage was 200 kV. Atomic‐resolution scanning transmission electron microscopy (STEM) image was obtained with JEOL ARM‐200F operated at 80 kV.All the images were conducted with image manipulation software developed by the authors. The image was subtracted after dulling edges and denoising from all the images for background deduction and the minimum value was added at each pixel to avoid the negative value. Then, all the pixels were stretched equally to form the processed image. The contrast and brightness of images were adjusted in image manipulation software. The adjusted gray‐scale images were converted into RGB images. The final images were achieved via RGB color analysis to distinguish the nuclei, graphene, and liquid Cu more clearly.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.Supplemental Video 1Click here for additional data file.Supplemental Video 2Click here for additional data file.Supplemental Video 3Click here for additional data file.Supplemental Video 4Click here for additional data file.Supplemental Video 5Click here for additional data file.


Poly Adenylate Binding Protein Interacting protein 1 (PAIP1) plays a critical role in translation initiation and is associated with the several cancer types. However, its function and clinical significance have not yet been described in oral squamous cell carcinoma (OSCC) and its associated features like lymph node metastasis (LNM). Here, we used the data available from Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), and Clinical Proteomic Tumor Analysis Consortium (CPTAC) to analyze PAIP1 expression in oral cancer. The publicly available data suggests that PAIP1 mRNA and protein levels were increased in OSCC. The high PAIP1 expression was more evident in samples with advanced stage, LNM, and worse pattern of invasion. Moreover, the in vitro experiments revealed that PAIP1 knockdown attenuated colony forming, the aggressiveness of OSCC cell lines, decreasing MMP9 activity and SRC phosphorylation. Importantly, we found a correlation between PAIP1 and pSRC through the analysis of the IHC scores and CPTAC data in patient samples. Our findings suggest that PAIP1 could be an independent prognostic factor in OSCC with LNM and a suitable therapeutic target to improve OSCC patient outcomes. Like most epithelial cancers, OSCC is a common cancer type of the mucosal linings of the oral cavity. LNM is one of the deterministic prognostic indicators in OSCC2. Despite the multimodal approach, including surgery or radiotherapy as treatment approaches in patients with OSCC, the low LNM-associated survival rate of less than 50% remains a major concern4. Thus, there is crucial to identify novel LNM-related biomarkers to improve the clinical outcomes in patients with OSCC.Tumor metastasis is critical for tumor progression, depending on motility and invasion, cancer cell plasticity, the modulation of the microenvironment, and cancer cell colonization9. PAIP1 displays a PAM1 and PAM2 domain, of which PAM1 interacts with the RRM 1and 2 domains of the Poly Adenylate Binding Protein (PABP) and PAM2 with the C-terminal domain of PABP5. Its interaction with eIF3 is regulated via the S6 kinase10. These interactions help the circularization of the RNA and translation initiation8.The coding gene of PAIP1is located at 5p12 chromosomal location, encoding a protein of 479 amino acids sharing homology with the central domain of human eukaryotic initiation factor 4G (eIF4G), consequently interacting with the eIF4A and eIF3 complexes to facilitate translation initiation11. Similarly, it reportedly regulates different aspects of oncogenesis, such as cellular proliferation, cell death, and metastasis in several cancers15. Although these findings suggest that PAIP1 plays a part in tumor progression and metastasis as a potential prognostic factor, the correlation between PAIP1 expression and OSCC metastasis has not yet been described.Since its identification by Sonenberg et al. in 1998, it has been described to be frequently upregulated in several types of cancer. In breast cancer, the high PAIP1 expression was related to late clinical stage, high histologic grade, and a low survival rateIn this study, we tried to examine whether PAIP1 is frequently overexpressed in human OSCC tissues and cell lines and to investigate the correlation between PAIP1 expression and the clinicopathological parameters of patients with OSCC, just as well as to evaluate the prognostic value of PAIP. Furthermore, to explore the function of PAIP1 and its underlying molecular mechanisms, we observed the migration and invasion in PAIP1-silenced OSCC cell lines.in silico PAIP1 expression analyses. In public database, PAIP1 was frequently amplified and overexpressed in a variety of cancers including head and neck squamous cell carcinoma (HNSCC) . To further evaluate the PAIP1 expression in HNSCC, we assessed differentially expressed genes in 45 healthy and 45 cancer samples from the GSE dataset 30784 and found different, significantly upregulated PAIP1 transcripts  Fig. . We alsopts Fig. . Comparepts Fig. . Moreovepts Fig. . Consistnes Fig. . Based ones Fig. . These rin silico data . In a recent comparative study, the worst pattern of invasion (WPOI) was reportedly superior to histological grade as a prognostic indicator of OSCC16. To investigate the relevance of PAIP1 expression on WPOI, we categorized it into 5 different patterns and classified it into two categories: cohesive and non-cohesive, following their grading schemes  and ITF (invasive tumor front). The IHC scores of the PAIP1 staining in ITM or ITF are shown as a heat map. The ITF exhibited significantly higher IHC scores than ITM  using siRNA interference Fig. . First, P- and R-values of 0.000 1 and 0.50, respectively). We performed a multivariable analysis to evaluate the correlation between PAIP1, pSRC (Tyr 419), WPOI, and nodal metastasis, observing a positive correlation between these variables . These data highlighted that SRC phosphorylation is involved in the poor OSCC prognosis with a high PAIP1 expression. A summary of the working model by which PAIP1 has metastatic potential and prognostic significance in OSCC is illustrated in Fig. To examine the involvement of PAIP1 in SRC phosphorylation, closely related to tumor invasion and metastasis, we performed western blot in PAIP1 knockdown cells. The SRC (Tyr416) activation was strongly attenuated by PAIP1 inhibition in both cell lines Fig. . We thenles Fig. . Finally6. Amplification of short arm of chromosome 5 is frequently seen in cancers18. This led us to hypothesize that PAIP1 might undergo amplification in head and neck cancer. Concurrent with our hypothesis, we found that the genetic alterations seen in head and neck cancers were mostly amplifications with mutations and fusions and a strong correlation between PAIP1 copy numbers and mRNA levels  when compared to inner regions (ITM)  show molecular heterogeneity when compared to the preceding zones of the tumor, which can better predict the tumor outcomesTM) Fig. . PAIP1 eTM) Fig. 24,25. ThLNM Fig. . FurtherLNM Fig. . HoweverLNM Fig.  and S4c.15. In particular, Guan et al. reported that PAIP1 knockdown in pancreatic cancer cells markedly decreased in the expression of MMP2, which affects the invasion and migratory process and MMP2 in OSCC has also been described as a potential biomarker for metastasis33, whereas we found no change in the MMP2 levels, rather MMP-9 activity downregulation in the PAIP1 knockdown of OSCC cells 15. Against to our expectations, these findings indicate that EMT-related molecules regulation of PAIP1 was unlikely to be pertinent in our model. If the migratory and invasive abilities of OSCC cells are blocked by PAIP1 knockdown, it may still occur by an alternative mechanism.Although the recently growing pieces of evidence suggest that high PAIP expression contributes to the aggressive behavior of various tumors, its role has never been determined in OSCCls Figs.  and S61536. Wang et al. reported that PAIP regulated AKT/GSK3β pathways for migration in lung cancer14. Although we did not find any significant changes including the AKT signaling pathway (data not shown), we for the first time found that PAIP1 was associated with activation of SRC by affecting phosphorylation of tyrosine residue 416/419  were used to extract expression data and identify PAIP1 expression and its relationship with the clinicopathological features in HNSCC.Three data repositories . Reference for the codes developed was accessed from, https://labs.cd2h.org/gitforager/repository/repository.jsp?id=77901462. The results shown here are partially based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.HNSCC dataset was used to create a custom cohort, which was accessed via https://pdc.cancer.gov/pdc/ including the base of the tongue NOS (Not Otherwise Specified), cheek mucosa, lip NOS, gum NOS, tonsil NOS, the floor of the mouth NOS, head, face, or neck NOS, just as well as the overlapping lesions of the lips, oral cavity, and pharynx. The log values for reporter ion intensity for unshared PAIP1 peptides were extracted after the manual downloading of the files CPTAC3_Head_and_Neck_Carcinoma_Proteome.tmt11.tsv and CPTAC3_Head_and_Neck_Carcinoma_Phosphoproteome.phosphopeptide.tmt11.tsv. The obtained values were used to compare the physiological and cancer-affected expression levels44.We analyzed the proteomic PAIP levels of a custom cohort, accessed from 45 and staging of tumors was based according to the TNM system recommended by the American Joint Committee on Cancer46. We performed a histologic risk assessment to record the worst invasion patterns (WPOI)27. The clinicopathological and histological features of the patients with OSCC are summarized in Supplementary Tables The specimens analyzed were retrieved from 58 OSCC patients who were surgically treated at the Seoul National University Dental Hospital at Department of Oral and Maxillofacial Surgery in 2009. The clinical data were collected from the patient medical records which included age, sex, recurrence, and survival of the cases. The tumor grading was based on the World Health Organization Classification of TumorsFour μm serial sections were obtained from 58 and 36 formalin-fixed and paraffin-embedded OSCC and adjacent healthy oral mucosal tissue specimens, respectively. The specimens were serially sectioned for each case to detect PAIP1 and pSRC (Tyr419). One section per case was immunohistochemically stained using a mouse monoclonal anti-PAIP1 antibody  after heat-induced antigen retrieval in a microwave oven for 10 min in citrate buffer (pH 6.0) incubated overnight at 4 °C, followed by detection using Dako real detect HRP-conjugated system (Dako). The other section was immunohistochemically stained after heat-induced antigen retrieval in a microwave oven for 8 min in citrate buffer (pH 6.0) using anti-pSRC (Tyr419)  incubated for 1 h at room temperature, followed by detection using Dako real detect HRP-conjugated system. Slides incubated without primary antibodies were used as a negative control.21 along with the total tumor score. The percentage of positive cells was scored as follows: Score 0, less than 5% positively stained cells; Score 1, 5%–25% positively stained cells; Score 2, 26%–50% positively stained cells; and Score 3, 51%–100% positively stained cells. The intensity was also scored for positive cells as follows: Score 0, negative; Score 1, weak (light brown); Score 2, moderate (brown); and Score 3, strong (dark brown). Each intensity score was added to the percentage score to produce a combined score. The final IHC score varies between 0 and 6. IHC scores between 0 and 2, less than 4, and equal to or greater than 4 were classified as negative, low, and high, respectively  assessed immunohistochemical staining semi-quantitatively, individually following blinding. In the case of disagreement, the scores were reached to a consensus following discussion. Tumor cells that demonstrated cytoplasmic staining were considered positive. The tumor was demarcated into two different zones: inner tumor mass and invasive tumor front (most advanced layers and detached tumor cells). Both zones were scored independentlyely Fig. . For sta2 until reaching a confluency of 70%–80%.Eight OSCC cell lines were screened along with a positive and negative control for PAIP1 protein and mRNA expression. The Hokkaido University kindly provided the HSC2, HSC3, HSC4, Ca9.22, HO1-N1, and HO1-U1 cells and the Dankook University kindly provided the HN22 cells. The SCC-9 was acquired from the Korean Cell Line Bank and MDA-MB-231 cells were purchased from the American Tissue Culture Collection. The Yonsei University kindly provided the iHOK cells. The MDA-MB-231 and iHOK were used respectively as positive and negative controls. The cells were routinely maintained in DMEM-F12 or RPMI 1640 media, supplemented with 10% FBS and penicillin/streptomycin and KSF medium (for iHOK cells) supplemented with BPE/EGFR at 37 °C, 5% CO−∆∆CT method47. We used the qPCR primers as follows: PAIP1 forward, 5′-TTT GGA AGA TGC TTG GAA GG-3′, PAIP1 reverse, 5′-TGA AGT TGC ATG GAC TCT GC-3′; GAPDH forward, 5′-GTGGTCTCCTCTGACTTCAAC-3′, GAPDH reverse, 5′-CCTGTTGCTGTAGCCAAATTC-3′.1 microgram of RNA was used to reverse transcribe using the AMPIGENE cDNA Synthesis Kit  according to the manufacturer’s protocol and then, the resultant cDNA was subjected to PCR using the AMPIGENE qPCR Green Mix Hi-Rox . The real-time PCR was performed using the Applied Biosystems StepOnePlus Real-Time PCR System (Applied Biosystems) and the amplification parameters for all genes were as follows: 95 °C for 2 min, followed by 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. Fold amplification of each gene was normalized to the amount of GAPDH and calculated using the 2Protein lysates were prepared using RIPA buffer supplanted with a protease inhibitor cocktail. The concentration of protein was determined by the DC Protein Assay Kit. The equal amounts of protein samples were separated on SDS polyacrylamide gels and transferred to a polyvinylidene fluoride membrane. This membrane was blocked with a blocking solution, TBST, supplemented with 5% Difco Skim Milk (BD bioscience) for 1 h, the membrane was then incubated with PAIP1-specific antibody (Santa Cruz), anti-GAPDH antibody (Abcam), anti-α-Tubulin (Santa Cruz), anti-SRC , anti-pSRC (Tyr416) , or anti-MMP9  in TBST. Following incubation with HRP-conjugated secondary antibodies, proteins were finally detected using the SuperSignal West Pico Chemiluminescent Substrate and captured using ImageQuant LAS 500 .PAIP1 were provided by Bioneer. The PAIP1 siRNA sequences were as follows: human PAIP1 siRNA (#10605-1): sense 5′-CCAGGUGGUUGUAGCUCCU-3′, antisense 5′-AGGAGCUACAACCACCUGG-3′. The cells were seeded onto 60 mm dishes. The cells were cultured along with 25 nmol·L−1 of sicontrol or PAIP1 siRNA using Lipofectamine 2000 reagent according to the manufacturer’s instructions.Small-interfering RNAs (siRNA) targeting PAIP1 siRNA-treated cells were seeded onto six-well culture dishes (2000 cells per well) and incubated. The medium was changed every 3 days for a week. The colonies (clusters of 20 or more cells) were then stained with 1% crystal violet and taken under an inverted microscope, and the number of colonies was calculated using the Image J software.4 cells per well) were grown in the upper chamber of transwell inserts in 500 μL of serum-free DMEM/F12 medium. To assess cell migration, 8 µm Transwell membranes of chamber (Corning) were precoated on the lower surface with Collagen Type I to yield a concentration of 0.5 mg·mL−1. For the invasion assays, the transwell inserts were precoated with growth factor reduced Matrigel (1:15 dilution). A total of 700 μL of DMEM-F12 with 10% FBS was inoculated in the lower chamber and then, the assay plates were incubated at 37 °C in 5% CO2 for 24 h. The upper chamber was then wiped off with a cotton swab, following this hematoxylin and eosin were used to stain the chamber. The cells were counted under a light microscope . Finally, 10 unique microscopic fields at ×100 magnification from each chamber were randomly chosen for cellular counting.The siRNA-treated cells  at 37 °C for 72 h. The gel was then stained with Coomassie Brilliant Blue R250 (Bio-Rad Laboratories). Thereafter, de-staining of the gel was performed using a solution of 40% methanol and 10% acetic acid, until the MMP-9-degraded part of the membrane became clear. Evaluation of the density of the clear bands was done using the ImageJ software.The siRNA-treated groups were sustained in a serum-free medium for 72 h. To collect samples of the MMP protein extract, the conditioned medium was concentrated using Amicon Ultra-15 Centrifugal Filter Units (Millipore) at 4500 g and 4 °C for 80 min. To assess the MMP-9 activities, 10 and 30 µg protein samples were applied to an acrylamide gel, 8% which was prepared with 1.5 mg·mLP < 0.05 was considered statistically significant.Statistical tests were conducted using the SPSS ver. 25.0  and GraphPad Prism 8 software. All results were expressed as the means ± standard deviation (SD). Multivariable correlations between the immunohistochemical staining and clinicopathological parameters of the tumors were performed via multivariable Pearson correlation, Student’s t-test, the Chi-square test, and Fisher’s exact test. The relative risks were calculated for different variables for high PAIP1 expressions. The statistical significance of the in vitro experimental data was analyzed using the two-tailed Student’s t-test (for comparisons of two treatment groups) or one-way ANOVA with Tukey’s post hoc test (for comparisons of three or more groups). A Supplementary Figure LegendsFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Supplementary Tables