While the evolution of cooperation has been widely studied, little attention has been devoted to adversarial settings wherein one actor can directly harm another. Recent theoretical work addresses this issue, introducing an adversarial game in which the emergence of cooperation is heavily reliant on the presence of “Informants,” actors who defect at first-order by harming others, but who cooperate at second-order by punishing other defectors. We experimentally study this adversarial environment in the laboratory with human subjects to test whether Informants are indeed critical for the emergence of cooperation. We find in these experiments that, even more so than predicted by theory, Informants are crucial for the emergence and sustenance of a high cooperation state. A key lesson is that successfully reaching and maintaining a low defection society may require the cultivation of criminals who will also aid in the punishment of others. The punishment of defectors is vital for sustaining cooperation in social dilemmas Yet, previous game theoretic and experimental research on human cooperation has generally ignored settings with distinctly adversarial characteristics, wherein one actor is uniquely positioned to harm another. This lacuna is puzzling given that such settings better reflect the asymmetries inherent in many social interactions and given the many sociological studies of adversarial behavior within various societies. For example, consider criminal activity, a prototypical adversarial interaction. Criminological research has found that civilians play a crucial role in fostering peaceful communities Recent theoretical work availability of the Informant strategy is nevertheless a necessary condition for the evolution toward and maintenance of the Utopian state, albeit only under favorable game parameters and initial conditions.Mathematical analysis and simulations reveal that the Informant strategy  plays a critical role in the coevolution of cooperation and punishment when this adversarial interaction is repeated over the course of many periods and actors are given the opportunity to adapt their strategies over time in certain prescribed ways. Two steady states can emerge over such repeated play: high crime, Villain-dominated “Dystopia” or low crime, Paladin-dominated “Utopia”. Under an imitation strategy updating rule Although the main finding of this prior theoretical work – that Informants are crucial to keeping criminal defection low – closely mirrors anecdotal evidence gathered by law enforcement agencies, there has been no systematic testing involving human subjects to determine the exact role Informants play in promoting cooperative behavior within a society. Our current work aims to shed light on this issue by presenting the results of a series of behavioral science experiments that were designed to mimic as closely as possible the game outlined above, and that specifically address the question of whether and how Informants may foster long-term cooperation.We conducted 16 sessions under 10 different treatments of a computer-based experiment at the Experimental Social Science Laboratory at the University of California, Irvine; the experiment was approved by UC Irvine's Institutional Review Board (IRB) under protocol HS# 2011-8378, with informed consent obtained via the laboratory's website and in-lab instructions. Budgetary constraints forced us to implement a partial factorial over the treatment variables of interest, which include differing initial states, two different parameter profiles, and different combinations of allowed strategies. Students enrolled at the university were contacted via email advertisements to register to be in the laboratory's online subject pool. For each experimental session, an email was sent to all eligible students in the subject pool, and eligible students then signed-up for the session via the laboratory's web site. Each student was only allowed to participate in one of the 16 sessions; that is, a student was eligible if and only if he or she had not yet participated in one of the prior conducted sessions of the experiment. Every session started at 10:00AM and lasted about an hour. Subjects showed up to the lab, signed in, proved their identity by showing their student ID cards, and were assigned a computer terminal. See The experiment itself consists of three phases – instructions, multiple rounds of decision making, and a questionnaire – and was implemented using the z-Tree software package The instructions inform the subjects about the basic structure of the adversarial setting. They are told that they will be randomly assigned into groups of two and randomly given a role of either “potential criminal” or “potential injured party”. They are also informed about how the points that they earn during the session will be converted into real dollars that they will be payed at the end of the experiment. Finally, they are asked a number of questions to test their understanding of the game and the payoffs associated with various scenarios.The next phase involves multiple periods of decision making in the adversarial setting. Each session lasts a minimum of 25 periods, after which each successive period occurs with a 75% chance, with a maximum of 35 periods. This end-game design creates uncertainty about which period is the last, thereby reducing concerns about idiosyncratic behaviors in the last period while still guaranteeing that the session will end within the announced time.Out of the 16 total sessions, 14 were initialized to specific states for the first five periods of the experiment, in order to test how different initial conditions might alter the outcome of the experiment. To do this, the computer program randomly assigns certain specified proportions of subjects to each of the four strategies  in period one. During the first five periods, the subject's enforced choices are shown on the screen, and are not allowed to be altered by the subject. The subject is therefore aware of which strategy he or she is being forced to play for the initialization periods. The interactions and payoffs are recorded and shown to the subject during the initialization period. In the 2 remaining sessions with no initialization period, all subjects were allowed to choose their strategy in the first period. We employed three different initializations: 90% Villains - 10% Informants, 90% Villains - 10% Paladins, and 60% Villains - 40% Paladins. The first two start the system very near Dystopia to see the role of Informants in transitioning to Utopia. The third has a strong presence of Paladins but, under the deterministic imitation dynamic Starting in the first period after initialization , subjects are allowed to choose whatever strategy they would like (from the list of available strategies) at the beginning of each period, constrained only by the level Within each decision making period of the experiment, there is a decision screen and a results screen, screen shots of which are shown in After each subject has selected “OK” to move on to the results screen, each participant is randomly and anonymously paired with another participant, and the program randomly assigns the roles of “potential criminal” (player one) and “potential injured party” (player two) within each pair. The program then plays out each player's strategy based on the assigned roles. Four possible outcomes can occur. First, if player one selected to not steal , then both subjects receive their endowment of The top 5 entries in The crucial role played by Informants vis-à-vis Utopia in the experimental sessions qualitatively matches the theoretical results, yet the data contain three striking patterns that reveal discrepancies with the theoretical work. First, Informants are vital for the emergence of Utopia under experiments performed with both parameter profiles, despite starting from initial conditions in which they should play no role under the theoretical best response dynamic To help visualize the role Informants play in transitioning a population to Utopia and helping it remain there, we partition the data into early (1–15) and late (16-end) periods and calculate the number of instances in which a subject, when given the opportunity to switch strategy, either keeps his or her current strategy or chooses another. On the other hand, The dramatic effect of removing the Informant strategy is evident in We now turn to a detailed look at all 16 sessions. Each session number corresponds to a unique treatment, while sessions with an “(a)” appended represent a replicated session of the same treatment number. We first present results obtained from sessions where all four actor strategies were available, then we consider those where Apathetics or Informants were forbidden, leaving only three available strategy choices. Each of these subsections is further subdivided into two parts, which describe the results for experiments run with Profile A and Profile B, respectively. We refer throughout to the evolution of strategy types presented in Sessions 1, 1(a), 6, 6(a), 7, 9, and 10 all use Profile A, have all strategies available, and converge  to Utopia. Sessions 1/1(a), 6/6(a), and 7 vary in how they are initialized, and Sessions 9 and 10 enforce a high level of behavioral inertia with different initializations.Sessions 1 and 1(a) initialize the population at 90% Villains - 10% Informants for the first 5 periods and both exhibit a large proportion of Informants and Paladins present as soon as subjects are allowed to choose their strategies. As these sessions continue past these initial bursts, the Paladins bolster in numbers as the number of Informants declines. The trend of a rapid increase of Informants and Paladins in early periods followed by growing Paladins and dwindling Informants is seen in many sessions. Over the last five periods, Sessions 1 and 1(a) average at least 70% Paladins, thus ending in Utopia.Sessions 6 and 6(a) are unique because they have no initialization periods. These are the only sessions where, in the first period, all of the subjects were allowed to select a strategy type. In both of these sessions, the Informant is the second most commonly played strategy  for most periods. In Session 6, the number of Informants follows an upward trend from period 1 until period 9, where there are actually more Informants than Paladins . After period 9, we observe the expected behavior of Paladins growing as subjects playing the Informant strategy declines. Over the last five periods, these two sessions average at least 75% Paladins and less than 10% Villains.Session 7 initializes the subjects at 60% Villains - 40% Paladins, and behaves similarly to Sessions 6 and 6(a). Initial conditions were chosen closer to Utopia here than in Sessions 1 and 1(a), so it is no surprise that Session 7 ends in Utopia, and that there is an average of 79% Paladins over the last five periods.Sessions 9 and 10 are unique because they enforce a very high level of inertia, Sessions 2, 2(a), 8, and 8(a) all use Profile B, have all strategies available, and converge toward Utopia. Sessions 2 and 2(a) are initialized at 90% Villains - 10% Informants for the first five periods. Under Profile B, the best response dynamic suggests that this low initial level of Informants should prevent the transition to Utopia, but the evolution figures for Session 2 and 2(a) again illustrate the Informant strategy “leading” the Paladin strategy. Within the first two periods after the initialization period, the percentage of Informants is up to 44% and 50% in Session 2 and 2(a), respectively. In Session 2, the percentage of Paladins has spiked at 78% by period 14. The percentage of Paladins hovers right at, or slightly below, this number for the remainder of the experiment. Session 2(a) did not quickly adopt such a high percentage of Paladins, but we conclude that Session 2(a) was close to converging to Utopia. This is mainly because of this session's consistently high conviction rate, closer to Utopia would produce the same result. This is what we observe from Sessions 8 and 8(a). Session 8 exhibits similar behavior as Session 2(a), in that, while the system only has an average of 44% Paladins over the last five periods, the reporting rate is very high: 76% over the last five periods. If more periods had been played, it is likely that we would have observed subjects switching to non-stealing strategies, most likely Paladins. Session 8(a) displays a steady rise in Paladins and an average reporting rate of 91% over the last five periods.Sessions 8 and 8(a) are the same as Sessions 2 and 2(a), except that they are initialized for the first five periods with 60% Villains - 40% Paladins. Since we observed Sessions 2 and 2(a) converge to Utopia, we would expect that an initialization Sessions 3 and 3(a) do not allow the Informant strategy, and are of primary interest because they are the only sessions under parameter Profile A that do not converge (or are not on a clear path to converge) to Utopia. Both sessions are initialized near Dystopia  for the first five periods and average over 55% Villains over the last five periods. In comparison, the highest percentage of Villains in any of the 12 sessions that converge to Utopia is 18% (Session 9). The evolution of the strategy types gives credence to the theory that Informants play a role in stabilizing Utopia. The lack of a pro-reporting criminal type (Informant) causes instability for the players in coordinating on non-criminal behavior. This is seen in both sessions, but more clearly in Session 3, where the percentage of Paladins jumps up to 72% in periods 8 and 9 before that number plummets down to 27% in period 13 and down to 6% in period 20. Session 3(a) displays similar behavior with a jump of Paladins to 63% in period 7 and a sharp decline in the next period to 38%. The proportion of Paladins in Session 3(a) hovers around 33% for the remaining periods. Without the Informant strategy available to stabilize the initial burst of Paladins, the number of Paladins falls and the number of Villains rises.do allow the Apathetic strategy: an initial boom of Informants and Paladins, followed by a continuing upward trend of Paladins with a downward trend of Informants. Session 5 provides evidence that the Informant and Apathetic strategies are not analogous hybrid strategies to one another, and that, indeed, there is something unique about the Informant strategy.Session 5 is the only treatment that restricts the set of possible strategies while still including the Informant strategy. This treatment was used to test if the Apathetic strategy played an analogous role to that of the Informant. The Apathetic and Informant strategies are theoretically analogous, in that they are both hybrid strategies that mix defection and cooperation. This theoretical similarity is not illustrated in the experimental results. The fundamental difference between the Informant and Apathetic strategies is clearly seen when examining the frequency at which each of these strategies were chosen in the experiments. When only counting the periods where subjects were allowed to choose strategies , the Informant strategy was chosen much more often than the Apathetic strategy. In the 15 sessions when the Apathetic strategy was available , subjects only selected it 7.6% of the time. Additionally, there were 53 periods out of a possible 343 where no one in the session was playing the Apathetic strategy even though it was available. Comparatively, in the 12 sessions when the Informant strategy was available, subjects selected it 21.4% of the time, and there was only one observed period out of 270 where no one selected the Informant strategy. This happened in Session 8(a) on period 25, where there were 96% Paladins. In Session 5, we observe similar results to other treatments that Sessions 4 and 4(a) both initialize the population at 90% Villains - 10% Paladins, do not allow the Informant strategy, and converge to Dystopia. These sessions are crucial in illustrating the vital role of the Informant in leading to the emergence to cooperation. While there is an initial “burst” of Paladins in the first periods after initialization, it is not as dramatic as the burst in Sessions 3 and 3(a). In period 6, Sessions 4 and 4(a) experience a jump in Paladins to 38% and 27%, respectively. Both sessions experience a dwindling or stagnation in the percentage of Paladins after this period for the rest of the session. Session 4(a) exhibits another small burst of Paladins in periods 17 and 18 at 41%, but on average, both sessions report low numbers of Paladins toward the end of the session. Over the last five periods of Session 4, only 12% of subjects are playing the Paladin strategy compared to 73% choosing the Villain strategy. Over the last five periods of Session 4(a), only 25% of subjects are playing the Paladin strategy, and 60% are playing the Villain strategy.To summarize, we find experimental evidence that the Informant strategy, which allows actors to defect by committing crimes but cooperate by assisting in the punishment of other criminals, is critical for the emergence of overall cooperation in an adversarial setting. In our experiments, Utopia is always converged upon when the Informant strategy is available and never converged upon when it is not, regardless of parameter profiles. The Informant strategy is also found to be essential for keeping the system in Utopia once it is reached. Overall, our results constitute new evidence that transitioning from a high crime to low crime society may require successful cultivation of actors that both commit crimes and cooperate with authorities.It is instructive to compare our findings with other research on the emergence of cooperation. The robustness of our findings is supported by prior theoretical work Table S1Subject details.(TIF)Click here for additional data file.Table S2Session details and outcomes.(TIF)Click here for additional data file.


In 2006, TAR-DNA binding protein 43 kDa (TDP-43) was discovered to be in the intracellular aggregates in the degenerating cells in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two fatal neurodegenerative diseases . ALS causes motor neuron degeneration leading to paralysis . FTLD causes neuronal degeneration in the frontal and temporal cortices leading to personality changes and a loss of executive function [5]. The discovery triggered a flurry of research activity that led to the discovery of TDP-43 mutations in ALS patients and the widespread presence of TDP-43 aggregates in numerous neurodegenerative diseases. A key question regarding the role of TDP-43 is whether it causes neurotoxicity by a gain of function or a loss of function. The gain-of-function hypothesis has received much attention primarily based on the striking neurodegenerative phenotypes in numerous TDP-43-overexpression models. In this review, I will draw attention to the loss-of-function hypothesis, which postulates that mutant TDP-43 causes neurodegeneration by a loss of function, and in addition, by exerting a dominant-negative effect on the wild-type TDP-43 allele. Furthermore, I will discuss how a loss of function can cause neurodegeneration in patients where TDP-43 is not mutated, review the literature in model systems to discuss how the current data support the loss-of-function mechanism and highlight some key questions for testing this hypothesis in the future. Amyotrophic lateral sclerosis (ALS) is a disorder where progressive degeneration of large motor neurons in the spinal cord and cerebral cortex leads to paralysis and death ,4. FrontTDP-43 is a RNA binding protein containing two RNA-recognition motifs (RRM), a nuclear localization signal (NLS) and a nuclear export signal (NES) . The proBecause mutations in TDP-43 lead to ALS, a causal role of TDP-43 for neurodegeneration is firmly established ,28,29. TA gain-of-function  Gene mutations that enhance the mutant protein aggregation propensity and cause ALS-FTLD with TDP-43 aggregation. Examples in this category include VCP, optineurin, dynactin, ataxin 2 and ubiquilin 2. All the mutant proteins form aggregates and some form coaggregates with wild-type TDP-43 ,65-69. T Gene mutRecent studies have suggested that a redistribution of TDP-43 to the cytoplasm may be a precursor to TDP-43 aggregation. In ALS and FTLD patients, some neurons show an increase in cytoplasmic TDP-43 immunoreactivity with diffused or granular appearance, which may represent an early stage of TDP-43 aggregation -86. The Discussion on therapeutic implication based on the loss-of-function hypothesis may be premature since the hypothesis remains to be tested. However, such an exercise may be helpful for illustration of the critical importance for a resolution of this question. In the case of a gain of function, strategies that reduce the function should be effective. This may be achieved by lowering the protein levels through an inhibition of its synthesis or a stimulation of its degradation. If the toxic activity is known, strategies that inhibit the specific toxic activity may also be effective. In the case of a loss of function, on the other hand, strategies that increase the function should be effective. This may be achieved by increasing expression and stability of the protein, or stimulating its activity.The therapeutic strategy for the dominant negative mechanism differs from both purely gain- or loss-of-function mechanisms and will be most challenging. We cannot simply increase the level of TDP-43 because uncontrolled increase of TDP-43 may inhibit the function of TDP-43 rather than improving it. High levels of TDP-43 could also further accelerate its aggregation and produce aberrant interactions with other proteins and RNA. Moreover, we do not understand why TDP-43 stays in the cytoplasm and becomes depleted from the nucleus in the disease. Therefore, it is not clear whether a simple increase of TDP-43 will replenish its level in the nucleus. In the case of mutant TDP-43, allele-specific inhibition of the mutant TDP-43 may be helpful but may not be sufficient to compensate for the lost function of the mutant allele. If the hypothesis that TDP-43 aggregation drives nuclear depletion of the TDP-43 is correct, preventing or reversing the aggregation may be a rational and safe approach to mitigate the loss of TDP-43 function. To achieve this, we need to understand how TDP-43 aggregation is triggered and propagated. We also need to understand the TDP-43 aggregation process at molecular and structural levels. Alternatively, strategies that enhance the function of TDP-43 without resorting to increase the protein level, or retain TDP-43 in the nucleus may also be effective.TDP-43 aggregation and nuclear depletion have been observed widely in neurodegenerative diseases. The role of TDP-43 in neurodegeneration remains to be defined. Chief among the questions is whether a gain of function, a loss of function or a dominant-negative mechanism is responsible for neurotoxicity. The answer to this question is of critical importance because it guides the future direction of research and sets the foundation for therapeutic strategies. Current experimental data from model systems has been predominantly invoked to support the gain-of-function mechanism. However, a careful review of the data suggests that a loss of TDP-43 function caused by its mutations, its aggregation and nuclear depletion, and the inhibition of TDP-43 function by a dominant-negative mechanism in the overexpression models, are at least as plausible as the gain-of-function theory, if not more so. Therefore, in our future research, we need to gain a more detailed understanding of the normal function of TDP-43, particularly in the cells of the CNS. We need models of loss of TDP-43 function in the CNS, particularly in mammalian species, to understand the consequence of TDP-43 dysfunction. In such a pursuit, models with a partial loss of TDP-43 function may be especially desirable because in humans, it is unlikely that the TDP-43 function is totally lost. We need evidence from human diseases to determine whether the conditions are more in tune with a gain or a loss of TDP-43 function. Lastly, we need to design strategies to address the difficult problem of how to restore the normal levels of TDP-43 function as a therapy.The authors declared that they have no competing interests.ZX conceived the ideas and wrote the manuscript.Notes after the proof After the proof of this article, James Shen and colleagues demonstrated that deletion of TDP-43 in motor neurons led to a loss of motor neuron in mice , thus indicating that, similar to other cell types, TDP-43 function is also required for motor neuron survival.


LINE-1 (L1) retrotransposons are repetitive elements in mammalian genomes. They arecapable of synthesizing DNA on their own RNA templates by harnessing reversetranscriptase (RT) that they encode. Abundantly expressed full-length L1s and theirRT are found to globally influence gene expression profiles, differentiation state,and proliferation capacity of early embryos and many types of cancer, albeit by yetunknown mechanisms. They are essential for the progression of early development andthe establishment of a cancer-related undifferentiated state. This raises importantquestions regarding the functional significance of L1 RT in these cell systems.Massive nuclear L1-linked reverse transcription has been shown to occur in mousezygotes and two-cell embryos, and this phenomenon is purported to be DNA replicationindependent. This review argues against this claim with the goal of understanding thenature of this phenomenon and the role of L1 RT in early embryos and cancers.Available L1 data are revisited and integrated with relevant findings accumulated inthe fields of replication timing, chromatin organization, and epigenetics, bringingtogether evidence that strongly supports two new concepts. First, noncanonicalreplication of a portion of genomic full-length L1s by means of L1 RNP-driven reversetranscription is proposed to co-exist with DNA polymerase-dependent replication ofthe rest of the genome during the same round of DNA replication in embryonic andcancer cell systems. Second, the role of this mechanism is thought to be epigenetic;it might promote transcriptional competence of neighboring genes linked toundifferentiated states through the prevention of tethering of involved L1s to thenuclear periphery. From the standpoint of these concepts, several hithertoinexplicable phenomena can be explained. Testing methods for the model areproposed.This article was reviewed by Dr. Philip Zegerman (nominated by Dr. Orly Alter),Dr. I. King Jordan, and Dr. Panayiotis (Takis) Benos. For the complete reviews,see the Reviewers’ Reports section. Retrotransposition of an L1 element occurs through reversetranscription of its RNA intermediate and subsequent insertion of an L1 cDNA copy ata new location in the genome -4. AmongA human FL-L1 element contains a 5′ untranslated region (UTR), two open readingframes (ORF1 and ORF2), and a 3′ UTR followed by an A-rich tail . The L1 per se is notsufficient for retrotransposition, and the factors that allow for retrotranspositionin embryos but not in the germ cell line remain unknown.It has long been thought that a substantially increased retrotransposition rate islinked to a noticeable synthesis of FL-L1 transcripts and, therefore, occurs inpreimplantation embryos , severalSince the acknowledgement of Barbara McClintock’s discovery of mobile geneticelements , the traSome efforts have been made to understand the biological significance of theabundance of L1s in the genome in the context of functionally meaningful elements andthe abundance of L1 transcripts in particular cell types. LINEs constitute asubstantial portion of scaffold/matrix attachment regions (S/MARs) in the humangenome . S/MARs An important point in this regard is that the involvement of FL-L1 transcripts in theglobal regulation of early development and carcinogenesis seems to be mediated by L1RT . This raIn this review, an attempt is made to fathom how upregulated FL-L1s and their RTglobally influence the differentiation state and proliferation capacity of earlyembryos and many types of cancer. In this context, the most intriguing phenomenon tobe explored is the massive nuclear L1-linked reverse transcription found at the onsetof embryogenesis. It is difficult, if not impossible, to explain the globalepigenetic role of L1 RT and the nature of massive L1-linked reverse transcriptionwithin the framework of current concepts. Therefore, conceptual advance is the mainchallenge. Herein, available L1 data are revisited and examined in concert withrelevant findings from the fields of replication timing, chromatin organization, DNAtopology, and epigenetics. The broad picture that emerges from this integrativeapproach favors two novel fundamental concepts. First, noncanonical replication of aportion of genomic FL-L1s by means of L1 RNP-driven reverse transcription is likelyto co-exist with DNA polymerase-dependent origin-based replication of the rest of thegenome during the same round of DNA replication in embryonic and cancer cell systems.Second, the role of this mechanism is likely epigenetic. Moreover, endogenousretrotransposition may be associated, to a great extent, with failure of thisnoncanonical DNA replication of an L1 unit. An exploration of this hypothesis showsthat the mechanism of DNA replication is worthy of being retested for specificgenomic locations (distinct FL-L1 sequences) in mammalian early embryonic and cancercell systems. This is important to advance understanding of DNA replication, thebiology of L1s, and mechanisms of pluripotency and carcinogenesis.L1 RNAs and RT, abundantly expressed in preimplantation embryos and some cancer celllines, have been targeted in numerous experiments to investigate their potentialroles. These experiments have brought about very important but overlooked findings.Specifically, they demonstrate that the functional knockdown of L1 expression viaL1-specific RNA interference (RNAi) and the inhibition of RT both independentlyresult in the same biological outcomes ,28. This3H-thymidine incorporation in adose-dependent manner in human transformed hepatoma (Hep3B) cells . Add. AddCCNDpatients  but revein vitroretrotransposition assays when used at similar concentrations . Despite the growing interest in the G4-ORC link, no one hasattempted to estimate what portion of G4s associated with ORCs is represented byL1-derived G4s. With so many unknowns, a landmark for future investigations couldbe what we can see in the nucleus. The abundance of L1s among MARs andpreferential colocalization of origins and MARs suggest that chromatin isorganized in such a way that many L1s likely serve as MARs and ORC binding sitesat the same time.Response:  with G4s; howevering tract, the numI would urge the author to reassess the review and re-balance the description andinterpretation of the experimentation. This should be married with a considerablereduction (50%) in the length of the review to allow it to be accessible to as wide acommunity of scientists as possible.I appreciate the concerns with respect to making the manuscript moreaccessible to as wide a community as possible; however, I believe the paper willlose its value to specialists as well as the integral view if the experimental orinterpretative components are so drastically pruned.Response: This is a multidisciplinary work that integrates experimental data from a numberof fields. Therefore, some introductory information regarding L1 biology,replication timing, etc., are worth inclusion so the paper is accessible to amultidisciplinary readership. Moreover, retaining the experimental data that mightbe considered as non-key facts is important. From the perspective of theintroduced concepts, the whole picture that emerges from the integration of thekey and non-key facts is a more convincing piece of information than severalfindings standing alone. This is important because the concepts and interpretationof certain experimental data are provocative.This review is not in a narrative style. As mentioned above, this is an inductivepaper that purports a considerable interpretative component. The interpretativeportion of the manuscript is as important as the experimental with respect tointegrating the experimental material, introducing alternative explanations,pointing out issues pertinent to the current L1 paradigm, proposing conceptualchanges, and examining how the available data fit the model. The discussion ofpotential links between the phenomena that have never been thought linked opensnew avenues for research. I believe there is some value in this intellectualcontribution.In recognition of the length issue, I have deleted a few details such as thenames of genes that changed their expression levels in response to thedownregulation of L1s in A-375 melanoma cells and the concentrations of the RTinhibitors used to reprogram cancer cell lines and to assess their effects onretrotransposition of L1s. Some redrafting has also been done to make someparagraphs more concise.Quality of written English: AcceptableThe manuscript on the functional significance of  non-canonical L1expression and replication by Ekaterina Belan is a provocative mix of a reviewarticle and a hypothesis paper. The author extensively reviews current experimentalevidence on the role of L1 reverse transcription in early embryos and cancer in lightof recent findings on genome regulation, organization and epigenetics. A key tounderstanding the authors approach is the desire to explore novel functional rolesfor L1s that do not fit within the current paradigm of L1 biology, which focusesmainly on retrotransposition dynamics and host genome mechanisms for the repressionof transposition.The search for a functional role of L1s rests on the author’s notion that sincethe main role of L1s is not the introduction of genomic variation “it islogical to assume that an important function (or functions) of L1s remains to bediscovered.” While this kind of teleological thinking is tempting, one does notneed to invoke a direct function of L1s to explain their existence and abundance inthe genome . As is held by the selfishDNA theory, the existence of such elements can be explained solely by their abilityto out-replicate the genomes in which they reside.This is a very good point. I have revised the paragraph to includeconsideration of the evolutionary aspect.Response: Having said that, once having established themselves in their hosts’ genomes,it is almost certainly the case that elements of this kind can have a profound effecton genome function. Accordingly, what the author refers to as the current‘retrotransposition-centered paradigm’ of L1 biology may indeed lead tointerpretations of experimental evidence that are markedly different from thoseoffered in this manuscript. As such, the alternative hypotheses and views proposedhere do seem to cover new ground, are thought provoking, lead to testable predictions(to some extent), and are thus worthy of publication in Biology Direct.Some of the interpretations of L1 experimental data presented here are likely to becontroversial, particularly to the extent that they differ from interpretationsoffered by the authors of the studies that generated the data. Thus, the paper hasthe potential to generate a substantive response and a potentially interestingdiscussion in the field and/or the literature. To her credit, the author does providespecific experimental tests of her models as they relate to the occurrence ofnon-canonical L1 DNA replication and the role of full-length L1 expression in genomeregulation.Finally, it is worth noting that the topics covered in this review, and in particularthe experimental tests proposed, could have biomedical relevance with respect to thelink between L1 reverse transcriptase-dependent DNA synthesis and cancer and/or stemcells. A better understanding of this phenomenon could hold promise for thedevelopment of L1 related anti-cancer therapies and/or novel methods for thereprogramming of differentiated cells to pluripotent stem cells.Quality of written English: AcceptableThis reviewer provided no comments for publication.Homo sapiens) L1 elements ; L1PA2 L1PA3, L1PA4, L1PA6,L1PA7: Subfamilies of primate-specific L1 elements; Ta-1: Transpositionally activesubfamily of human L1 elements ; UTR: Untranslated region; ORF:Open reading frame; ORF1: Open reading frame 1; ORF2: Open reading frame 2; ORF1p: Openreading frame 1 protein; ORF2p: Open reading frame 2 protein; bp: Base pair(s); kb:Kilobase pairs; Mb: Megabase pairs; siRNA: Small interfering RNA; RNAi: RNAinterference; S/MAR: Scaffold/matrix attachment region; SAR: Scaffold attachment region;MAR: Matrix attachment region; ERV: Endogenous retrovirus; HERV-K: Human endogenousretrovirus family; MuERV-L: Murine endogenous retrovirus-like element; AML: Acutemyeloid leukemia; ICM: Inner cell mass; ESC: Embryonic stem cell; hESC: Human embryonicstem cell; mESC: Mouse embryonic stem cell; iPSC: Induced pluripotent stem cell; EpiSC:Stem cell derived from the epiblast; NPC: Neural precursor cell; EtoL: Replicationtiming change from early to late S; LtoE: Replication timing change from late to earlyS; Xa: Active X chromosome; Xi: Inactive X chromosome; BrdU: 5-Bromodeoxyuridine; IdU:Iododeoxyuridine; CldU: Chlorodeoxyuridine; HCG: Human chorionic gonadotropin; qPCR:Quantitative real-time polymerase chain reaction; INM: Inner nuclear membrane; ORC:Origin recognition complex; ORCA: ORC-associated protein; HP1: Heterochromatin protein1; G4: G-quadruplex; H3K9me3: Histone H3 trimethylated at lysine 9; H3K9ac: Histone H3acetylated at lysine 9; TDP: Timing decision point; 3D: Three-dimensional; wt: Wildtype; R loop: RNA•DNA displacement loop; ssDNA: Single-stranded DNA; dsDNA:Double-stranded DNA; FISH: Fluorescence in situ hybridization; ChIp-seq:Chromatin immunoprecipitation followed by high-throughput DNA sequencing; HIV: Humanimmunodeficiency virus.LINE: Long interspersed nuclear element; LINE-1(L1): Long interspersed nuclearelement-1; FL-L1: Full-length L1; RT: Reverse transcriptase; EN: Endonuclease; RNP:Ribonucleoprotein; cDNA: Complementary DNA; L1Hs: Subfamily of human-specific (fromThe author declares that she has no competing interests.Extended Discussion.Click here for file


LINE-1 (L1) retrotransposons make up a significant portion of human genomes, with an estimated 500,000 copies per genome. Like other retrotransposons, L1 retrotransposons propagate through RNA sequences that are reverse transcribed into DNA sequences, which are integrated into new genomic loci. L1 somatic insertions have the potential to disrupt the transcriptome by inserting into or nearby genes. By mutating genes and playing a role in epigenetic dysregulation, L1 transposons may contribute to tumorigenesis. Studies of the “mobilome” have lagged behind other tumor characterizations at the sequence, transcript, and epigenetic levels. Here, we consider evidence that L1 retrotransposons may sometimes drive human tumorigenesis. The only active mobile DNAs in modern-day humans are the autonomous L1 retrotransposon and non-protein-coding (nonautonomous) sequences its machinery mobilizes.Repetitive sequences collectively make up greater than half of the human genome and are subdivided into two principal types  [1], [2]antisense L1 promoter may occur concurrently. In the second step, the RNA is translated into two L1-encoded proteins: ORF1p, an RNA-binding protein, and ORF2p, a protein with reverse transcriptase and endonuclease activities. These proteins associate with the L1 transcript, and the resulting ribonucleoprotein (RNP) complexes are then transferred to the nucleus. The third step is termed target-primed reverse transcription (TPRT). In the course of TPRT, ORF2p cleaves the target DNA (often at a 5′-TTTTAA-3′ consensus sequence) and uses the 3′ hydroxyl group to prime the reverse transcription reaction. Synthesis of the second strand and resolution of the structure are poorly understood. Because the L1 life cycle generates DNA breaks, cell host proteins that mediate DNA repair are likely involved.The L1 life cycle entails three steps , red boxIn the human genome, the majority of our estimated 500,000 L1 copies are (1) present on both homologous chromosomes, (2) truncated at the 5′ end , and (3) incapable of encoding ORF1p and ORF2p and transposing. A relatively small number are potentially active, full-length L1 elements (approximately 6 kb long) with intact coding sequences for ORF1p and ORF2p In this review, we discuss inhibition of L1 retrotransposition in normal somatic cells and activation of L1 in cancer cells. We also consider possible causal roles of L1 in tumorigenesis, discussing ways in which it may influence regulation of host genes or genomic stability apart from the canonical transposition pathway.L1 is regulated by distinct pathways in different cell contexts. In the male germline, L1 is inhibited via an elaborate system involving Piwi-interacting RNAs (piRNAs) that ultimately methylates genomic L1 sequences. This depends on methylation regulator DNMT3L In addition to the host factors mentioned above, other proteins have been implicated in L1 repression in various somatic tissues. These include methyl CpG binding protein 2 (MECP2) MYC oncogene locus in breast ductal adenocarcinoma Somatic L1 insertions have the inherent potential to drive tumorigenesis by activating oncogenes or inactivating tumor suppressor genes. However, somatic L1-related rearrangements that have driven tumorigenesis have rarely been discovered in humans. Before 2010, only two such cases had been reported. The first case involved a gene-activating rearrangement at the The introduction of targeted next-generation sequencing and analysis methods in recent years has led to additional reports of somatic L1 retrotransposition in colon cancer The fact that the somatic insertions reported by Lee and colleagues were skewed toward genes that are commonly downregulated or mutated in the tumors that they studied suggests that L1 insertions may contribute to cancer formation. In order to determine whether L1 actually does so, it would help to identify specific loci that are recurrently disrupted by L1 insertions. Ongoing work to profile L1 positions in additional tumor types, metastatic samples, and samples of disease relapse after therapy may lead to the identification of such loci.Whereas normal adult tissues do not express L1 ORF1p In addition to these definitive cases of L1 expression, there have been other cases indicating L1 derepression. For example, L1 promoter hypomethylation has been reported in multiple myeloma L1 RNA expression, ORF1p expression, and L1 methylation status are more readily detected than ORF2p expression using current reagents. While it is plausible that ORF2p expression also occurs in cases exhibiting the first three features, it is possible that examples of decoupled regulation will be described. ORF2p expression is a major mechanism, in addition to retrotransposition, through which L1 may impact the genome. Cell-culture-based studies convincingly show that ORF2p expression can be mutagenic, inducing not only mutations associated with canonical retrotransposition events but also DNA breaks Several nucleoside analogues have been shown in biochemical analyses to inhibit the RT activity of ORF2p and, in cell-culture-based retrotransposition assays, to reduce the number of L1 retrotransposition events In cell-culture-based cancer models, both nucleoside RT inhibitors and NNRTIs have been shown to promote senescence and differentiation, and reduce invasive growth. For example, the nucleoside analog abacavir inhibits the proliferation and migration of a prostate cancer cell line In some cases, transcription is initiated within an L1 to form a chimeric mRNA that contains L1 sequence (sense or antisense) and downstream exons of a host gene. In fact, in normal mouse tissues, transcription is initiated frequently within L1 sequence, as Faulkner et al. determined by sequencing the 5′-most nucleotides of RNAs from both normal and neoplastic tissues, using cap-analysis gene expression tag technology (CAGE) MET proto-oncogene locus, is specifically induced in CML cells by the demethylating agents azacytidine and decitabine L1 transcription initiation can be drug induced. For example, Weber et al. showed that transcription of the L1-cMet sequence, which originates within the hypomethylated transcriptional start sites of single-copy genes in cancer cell lines L1 insertions may also influence transcription initiation from the canonical transcriptional start site of a gene locus. Estecio et al. found evidence of this by noting an increased number of L1 insertions near selectively There is growing evidence that L1 expression and even complete retrotransposition occur in selected human cancers, suggesting that L1 may drive tumorigenesis. Even if L1 does not drive tumorigenesis through new insertions or ORF2p genetic damage, L1 sequences are such prevalent genomic passengers that they are likely to contribute to gene expression during tumor development, and affect responses of tumors to treatment. New reagents, experimental approaches, and informatics tools are being developed to provide more complete pictures of L1-mediated roles in human neoplasms.


Biomphalaria glabrata, B. straminea, B. tenagophila. In the Biomphalaria genus, the myoglobin gene has three introns; these were amplified by PCR, analyzed by PCR-RFLP. Results showed that the size (number or nucleotides), the nucleotide sequence of the coding gene of the myoglobin are variable in the three species. We observed the presence of size polymorphisms in intron 2, 3; this characterizes a homozygous/heterozygous profile, it indicates the existence of two alleles which are different in size in each species of Biomphalaria. This polymorphism could be explored for specific identification of Biomphalaria individuals.Introns are common among all eukaryotes, while only a limited number of introns are found in prokaryotes. Globin, globin-like proteins are widely distributed in nature, being found even in prokaryotes, a wide range of patterns of intron-exon have been reported in several eukaryotic globin genes. Globin genes in invertebrates show considerable variation in the positions of introns; globins can be found without introns, with only one intron or with three introns in different positions. In this work we analyzed the introns in the myoglobin gene from  In 1977, several research groups published their findings about DNA coding sequences (exons) broken by non-coding sequences (introns) in eukaryotes ,3. IntroCavalier-Smith  suggesteIndependently in 1978, Doolittle , DarnellIn the evolutionary history of introns, the selective stretch which modulates their evolution, the relative contribution of its eukaryotic gene loss or insertion is not understood . In inteMany authors have attempted to interpret the distribution of intron in terms of a mixture of movement, removal of introns, but the difficulty of phase-shifting movement causes serious doubts about these interpretations. In contrast, some gene families for which phylogenies can be traced show patterns that clearly indicate intron insertions .The protein coding sequences are interrupted by introns at specific points, which could be used to investigate, for instance, the evolution of protein families. The gene encoding the ancestral globin chain is assumed to be interrupted by three introns inserted in the B, E, G helices. However, the conservation of this intron pattern, the exact insertion positions of the introns during evolution is a matter of ongoing discussion .Intron location, sequence may also shed light on the origin of some protein genes, for instance the polymeric globin genes occurring in mollusks, arthropods . In thisIdentifying individuals from the Biomphalaria genus is a difficult task because they are very similar in morphology. Classic methodology of identification is based on comparative morphology of shells, anatomy of the reproductive organs, however there is much intra-specific variation . ClassicIn this work, we analyze the introns of the myoglobin gene of mollusks from the Biomphalaria genus in order to evaluate their potential use for precise identification in systematic studies. The size (in base pairs) of the amplicons of intron 1 was analyzed by polyacrylamide gel electrophoresis. All individuals of the three Biomphalaria species showed a DNA fragment of about 1,300 bp .Amplification of intron 2 of B. straminea generated an amplicon larger than 1,300 bp, of about 1,500 bp. In B. glabrata, B. tenagophila, some individuals were observed to have a large amplicon of about 1,400 bp,, some individuals showed a smaller fragment , whereas some individuals showed the two fragments .The amplification of the intron 3 presented a similar homozygous/heterozygous pattern in B. straminea. Some individuals showed an amplicon of about 1,400 bp, other individuals showed an amplicon of about 1,200 bp,, some ones showed the two amplicons. In B. glabrata, was observed one amplicon only of about 750–850 bp,, B. tenagophila showed an amplicon of about 1,300 bp . Clear, satisfactory amplification using genomic DNA as a template is not a simple task, since nonspecific amplifications, artifacts could contaminate the true results. After obtaining the best protocol for amplification, this was repeated at least five times. The monitoring of all amplifications showed the results on the size of introns from Biomphalaria, which are described in the text; the gels presented as figures have some artifacts that do not refer to amplification of introns.The models for putative alleles from the three Biomphalaria species are shown in Figure 4. They were built based on the nucleotide sequence of the myoglobin gene from B. glabrata deposited in data bank, sequence of the cDNA myoglobins of B. tenagophila, B. straminea that were deposited in Genbank , which are very similar to B. glabrata myoglobin (>96% identity). PCR-RFLP was used to obtain rapid information about the nucleotide sequence of the myoglobin introns from Biomphalaria; for individuals who had introns of different sizes, the largest fragment was digested.In no individual from the Biomphalaria species was fragmentation of intron 1 observed by restriction enzymes. The intron 2 from B. straminea, B. tenagophila was digested by Hind III only, however it was digested in different places, generating fragments of different sizes.Intron 3 from B. straminea was cutted by Xba I, Xho I, Bgl II, Hind III,, the same intron from B. tenagophila was cutted by Xba I, Hind III. Intron 3 from B. glabrata was not cut by any enzyme .In 2004, Sokolova, Boulding  describeThe first intron of B. glabrata has 1,116 bp, the second intron has 1,008 bp, third has 582 bp . The difThe first elucidation of globin gene structure was that of the human hemoglobin α, β chains. These genes have two introns conserved exactly at positions B12.2 (α-helix B in the protein), G6.3 . AccordiThe typical gene structure of three exons/two introns are found in all vertebrate globins, in most annelids . The resThe globin genes of vertebrates, plants contain, respectively, two, three introns, their insertion positions are conserved. The three intron/four exon pattern of plants is proposed to be ancestral, all other globin gene structures would be derived mainly by intron loss . SeveralB. straminea presents a size polymorphism in the third intron of the myoglobin gene. B. tenagophila, B. glabrata contain size polymorphism in the second intron,, any of the three species showed size polymorphism in the first intron. This kind of polymorphism, the homozygous/heterozygous pattern was not shown for the myoglobin gene of B. glabrata which had been described.Analysis of unique individuals indicates that there are two alleles of different sizes which code for myoglobins in the Biomphalaria genus. The difference is about 200 bp, each Biomphalaria specie presents polymorphism in one intron only. The smaller alleles were observed in B. glabrata, the bigger ones were observed in B. straminea. By analyzing the size it is believed that the nucleotide sequence of the myoglobin gene of the B. glabrata deposited in data bank could be the minor allele of this specie.No differences were observed in size of the primary sequences (amino acids) of the myoglobins from the three Biomphalaria species such that the contribution of the exon to the size of the amplicons is the same for all three Biomphalaria species. The difference among the myoglobin introns from B. glabrata, B. straminea e B. tenagophila suggests a potential for using these as molecular markers to carry out the specific identification of these mollusks. Analysis of the distribution of the possible alleles of the gene encoding myoglobin among individuals of three species of Biomphalaria do not indicated a relation to the geographical origin of the mollusks, but this relationship cannot be ruled out, since it would be necessary to analyze a larger number of individuals.The sequencing of heterozygous genotypes with different alleles in size is a difficult task,, RFLP analysis is a way to get quick information about the nucleotide sequence . AnalysiThe Biomphalaria genus covers organisms morphologically very similar, making their identification difficult. The classical identification is based on comparative morphology of the shells, anatomy of reproductive organs, but the enormous intraspecific variation of morphological, anatomical features commits to a specific classification. Identification may also be hampered by the small size of the specimens, whose distinctive morphological characters are not very evident .Monis  highlighIn the same context, Knight et al. , using tB. glabrata, B. tenagophila, B. straminea from several Brazilian provinces  belonginGenomic DNA was purified from foot tissue of each individual of Biomphalaria using a DNA purification kit  according to recommendations from the manufacturer. The DNA quantification, purity were analyzed by spectrophotometry at 260, 280 nm using a UV-160A UV-Visible recording spectrophotometer (Shimadzu). The DNA quality was determined by 1% agarose gel electrophoresis. The DNA was not pooled at any step.Amplification of the introns were performed by PCR using three sets of primers which were drafted based on nucleotide sequence from the B. glabrata myoglobin gene available in GenBank Accession Code U89283. The primers were BIO 1F, BIO 2 , the other primers annealing in exon regions or in the UTR (untranslated region) to provide the complete sequence of the three introns of the myoglobin gene . All amplifications were reproduced at least five times.Primer pair BIO 1F (forward) (5’ TACTGTCACACAACCAGCCC 3’), BIO 1R (reverse) (5’ GCGTTCTTTTTGCCGTCAGC 3’) were used to amplify the first intron (intron 1), primers BIO 4F (5’ GGCAAAAAGAACGCTGGAAT 3’), BIO 4R (5’ GAGTCCGATTTTCAGGTTG 3’) were used to amplify the second intron (intron 2), primers BIO 2, BIO 3 (5’ GAGTTGGTCAGGATCCGTGG 3’) were used to amplify the third intron (intron 3).The PCR for intron 1 was performed for 30 cycles of 94 °C for 30 s, 65 °C for 60 s, 72 °C for 2 min, a final elongation step at 72 °C for 10 min. The amplification of the introns 2, 3 used the same reaction, except for an annealing temperature of 60 °C. The amplicons were analyzed by 6% polyacrylamide gel electrophoresis with silver stain; the base pair markers φX174/Hae III digested (Amersham Biosciences), 1 kb Plus DNA ladder (Invitrogen) were applied.PCR-RFLP was used to analyze the similarity of the nucleotide sequence from myoglobin introns. The three introns of the myoglobin gene from three Biomphalaria species were amplified as previously described, amplicons were submitted to digestion with the restriction endonucleases Xba I, Bgl II, Xho I, Hind III, Bam HI (Invitrogen). Only intronic fragments were digested, because the aim was to observe if the nucleotide sequence of the introns differed among species of Biomphalaria. The use of the full gene (whole gene amplification) in RFLP would not allow identification of the differences (cuts) belonging to the introns, exons,, in which intron. Reactions were performed according to the manufacturer, were evaluated by 6% polyacrylamide gel electrophoresis with silver staining; the base pair marker φX174/Hae III digested (Amersham Biosciences) was applied. The gene encoding myoglobin from B. glabrata, B. straminea, B. tenagophila displays size polymorphisms (base pairs) in the intronic regions, featuring a profile of homozygosity/heterozygosity in this gene. Considering the difference among the introns of myoglobin from B. glabrata, B. straminea, B. tenagophila, there is the possibility of using these introns as molecular markers for specific identification of these mollusks.


Delayed comparison tasks are widely used in the study of working memory and perception in psychology and neuroscience. It has long been known, however, that decisions in these tasks are biased. When the two stimuli in a delayed comparison trial are small in magnitude, subjects tend to report that the first stimulus is larger than the second stimulus. In contrast, subjects tend to report that the second stimulus is larger than the first when the stimuli are relatively large. Here we study the computational principles underlying this bias, also known as the contraction bias. We propose that the contraction bias results from a Bayesian computation in which a noisy representation of a magnitude is combined with a-priori information about the distribution of magnitudes to optimize performance. We test our hypothesis on choice behavior in a visual delayed comparison experiment by studying the effect of (i) changing the prior distribution and (ii) changing the uncertainty in the memorized stimulus. We show that choice behavior in both manipulations is consistent with the performance of an observer who uses a Bayesian inference in order to improve performance. Moreover, our results suggest that the contraction bias arises during memory retrieval/decision making and not during memory encoding. These results support the notion that the contraction bias illusion can be understood as resulting from optimality considerations. Comparing magnitudes of two temporally separated stimuli is one of the fundamental tools of experimental psychology and neuroscience. Interestingly, choice behavior in these experiments reveals a fundamental bias: when the first stimulus is small, subjects tend to overestimate it, whereas when it is large, they tend to underestimate it. The first account of this bias, known as the contraction bias, was published a century ago by Harry Levi Hollingworth who later became one of the pioneers of applied psychology. Hollingsworth presented subjects with square cards of various sizes for a brief period of time and asked them to memorize their sizes perceived magnitude of a stimulus is a weighted combination of its veridical magnitude and a reference magnitude, such as an average of all contextually relevant stimuli, that serves as an anchor The customary explanation for the contraction bias is that the There is a growing body of literature suggesting that the brain utilizes Bayes' rule to optimally combine information from different sources In order to test this hypothesis, we conducted an experiment in which we instructed subjects to memorize the length of a bar presented on a computer screen and then compare this memorized length to the length of a probe bar. We show that contraction bias depends on the prior distribution of bar lengths, and increasing the uncertainty in the memory of bar lengths enhances the contraction bias, both of which are consistent with the Bayesian hypothesis.after memory encoding and measuring the magnitude of the contraction bias we demonstrate that prior information is introduced during memory retrieval/decision making rather than when the first stimulus is encoded in memory.When within a trial does the Bayesian computation take place? Is the encoded memory biased or does the prior information bias the result of the length comparison? By manipulating uncertainty in the memory of bar lengths Some of the findings presented here have appeared previously in abstract form 1L) for 1 sec and were instructed to memorize its length. After a delay of 1 sec, during which screen remained blank, they viewed a probe bar (2L). The probe bar remained visible on the screen until subjects reported which of the two bars was longer by pressing dedicated keys on the keyboard. The first bar, L1, was drawn from a uniform distribution in the logarithmic scale between 150 and 600 pixels. The difference in length between 1L and 2L varied between −30% and +30%. Both bars were presented at random locations on the screen and no feedback was provided to the subjects on performance on individual trials (See L1>L2’ responses as a function of the difference between the memorized and probe stimuli. The average psychometric curve of the subjects (n = 9) is plotted in L1 and L2 increased.In the standard task , subjectials See . We quanpattern of errors made by subjects depends on the magnitude of the memorized stimulus. When the memorized stimulus is small, subjects tend to make more errors in trials in which the probe stimulus is larger than the memorized stimulus, compared to trials in which the probe is smaller than the memorized stimulus. The opposite behavior is observed when the magnitude of the memorized stimulus is large L1 and L2 in our experiments) relative to the width of the psychometric curve. We used a different approach to overcome this limitation: Unbeknownst to the subjects, we included a subset of trials in which the lengths of the two bars were identical. We term these trials “impossible trials” because there is no correct answer to the question “which bar (L1 or L2) was longer”. Impossible trials are well suited for the analysis of the contraction bias because performance on these trials is independent of the proficiency of individual subjects in distinguishing the difference in the length of the two bars.Our purpose is to quantify the contraction bias in these experiments. Previous studies have demonstrated a contraction bias in delayed comparison tasks by showing that the L1>L2’ as a function of the length of L1 (L1 = L2). Note that despite the fact that L1 and L2 were identical on these trials, subjects reported that L1 was longer than L2 on roughly 60% of the shortest trials  the control region knows that the neural representations of L1 and L2 are noisy, e.g. due to noise in the sensory pathway. Moreover the representation of L1 is noisier than that of L2 because L1 has to be stored in memory, a process that may contribute additional noise to the representation of L1; (2) the control region has information about the marginal distribution of bar lengths. This distribution can be approximated based on the history of the experiment; (3) the control region utilizes Bayes' rule and combines the noisy representations of L1 and L2 with knowledge about the prior distribution in order to construct the posterior distributions for the two bar lengths. These posteriors are then used to minimize error in judgment. A formal description of this process appears in the Our aim is to account for the contraction bias in a Bayesian framework of decision making. In order to see how the contraction bias emerges from Bayesian inference, we consider a control region in the brain, such as the prefrontal cortex L1 is unknown, L2 is known. Consider a hypothetical subject who forgets the length of L1, but has no ambiguity about the length of L2, i.e., the neural representation of L1 is infinitely noisy whereas there is no noise in the neural representation of L2. In this case, the posterior of L1 is the prior distribution. In contrast, the prior distribution makes no contribution to the posterior of L2. Therefore, the optimal strategy would be to report ‘L1>L2’ in trials where L2 is smaller than the median of the prior distribution and to report ‘L1<L2’ in trials in which L2 is larger than the median. Therefore, in the impossible trials in which L1 = L2, the subject would report ‘L1>L2’ if L1 is smaller than the median of the prior distribution and would report ‘L1<L2’ otherwise, as depicted in L1 and underestimating relatively large L1.L1 and L2 are equally uncertain. Consider a case where the estimated uncertainties in the representations of L1 and L2 are equal. This would be true if the only uncertainty in the representations of L1 and L2 results from sensory noise, and memory storage does not add any additional noise to the representation of L1. In the impossible trials in which the two bars are physically identical, the contribution of the prior distribution to the posteriors of L1 and L2 is equal. Symmetry considerations indicate that the subject would report that ‘L1>L2’ at chance level for all bar lengths, i.e., there is no contraction bias, as depicted in L1 is less certain than L2. In intermediate cases where the level of uncertainty in L1 is larger than that of L2, for example, as a result of added noise due to memory storage, we expect the resultant response curve to reside between the response curves of L1 is reported to be larger than L2 as a function of the lengths of L1 and L2 . Similar to the first experiment, all lengths were presented in logarithmic scale to satisfy the assumption of independence of noise and bar length. We compared the responses of this group to the original group who saw stimuli that were drawn from a uniform distribution in the logarithmic scale between 150 to 600 pixels. The accuracy of subjects in the trials in which L1≠L2  was indistinguishable between the two groups , supporting the assumption that the level of noise in the neural representation of the bars is independent of bar length in these ranges. Response curves for the two groups in the impossible trials are depicted in If the contraction bias results from Bayesian inference, then changing the prior distribution is expected to change the response curve. In particular, assuming that noise is independent of the length of the bars, a translational shift in the prior distribution would result in an equal translational shift in the response curve without changing its shape. To test this prediction, we asked a new group of naïve subjects to participate in the experiment of L1 is 30% higher than the uncertainty in L2 , the intermediate trials in which the difference between L1 and L2 was ±15% (t-test) and the most difficult trials in which the difference between L1 and L2 was ±7.5% (t-test).The distracting task was designed to interfere with the memory of the first bar. In the Bayesian framework, it was intended to add ‘noise’ to the representation of ary task . This dep = 0.0002, two-tailed), supporting the assertion that a decrease in performance is associated with an increase in the magnitude of the contraction bias.In order to characterize the effect of the secondary task on the contraction bias, we compared the response curve of subjects in the impossible trials with interference from the secondary task  the subjects could not know whether they would be presented with an interfering task and therefore could not know what weight to give to the prior distribution. Therefore, if the computation had taken place at the time of the encoding of L1, we would have observed no difference in the slope of the response curve between the two conditions. Therefore, the Bayesian computation necessarily took place after the interfering task, at the time of L1 retrieval or later, when L1 and L2 were compared.Within a single trial, when does information about the prior distribution combine with the sensory measurement? One possibility is that it takes place during the encoding of d trials . In otheHow do subjects learn the prior distribution? In order to address this question, we compared the level of contraction bias, as measured by the slope of the response curve, in the first 20 impossible trials to the slope in the last 20 impossible trials for subjects who completed the experiment in In this study we examined the effect of a translational shift in the prior, but we did not alter the shape of the prior distribution. Previous studies have shown that subjects are sensitive to the shape of the prior distribution in category and sensimotor learning Contraction bias in delayed comparison tasks is a common cognitive illusion observed in many different modalities and under different experimental conditions All subjects gave written informed consent using methods approved by the Massachusetts Institute of Technology Committee on the Use of Humans as Experimental Subjects.Subjects were undergraduate and graduate students from the Massachusetts Institute of Technology. All subjects had normal or corrected-to-normal vision and no subjects took part in more than one of the experiments. Each subject received $10 plus 1 cent for every correct trial in the experiment for a session lasting less than an hour.Stimuli were white horizontal bars on a black background displayed on a 17″ computer screen with a resolution of 1024×768. All bars were 3 pixels wide.L1 at a random location on the screen for 1 sec. After a delay period of 1 sec, during which screen remained blank, L2 appeared at another random location on the screen. L2 remained visible until the subjects pressed one of two keys indicating which bar was longer. The difference in length between 1L and 2L varied between −30% and +30%. Unbeknownst to the subjects, in roughly 50% of the trials, the lengths of the first and second bars were equal (L1 = L2). Subjects did not receive feedback on performance on individual trials. Each trial was followed by a 2 sec intertrial interval during which the screen remained blank. Two distinct groups of subjects completed the standard task. One group (n = 9) saw L1 bars chosen uniformly in the logarithmic scale from the  pixel interval, while the other group (n = 10) saw bars chosen from the  pixel interval.Subjects sat approximately 60 cm from a computer screen in a dimly lit room. Each subject completed 400 to 600 trials in one hour and received feedback on their overall performance after every 20 trials. No other feedback was provided. In the standard task, each trial started with the presentation of a L1 bars were chosen uniformly in the logarithmic scale from the  pixel interval; (2) subjects completed a distracting memory task between the presentation of L1 and L2 in a randomly selected 50% of the trials: 500 msec after L1 disappeared, a random sequence of four colors  were displayed on the screen for 400 msec each. 400 msec after the disappearance of the last color, a number from 1 to 4 appeared in yellow on the screen. Subjects were instructed to recall the color that corresponded to the number and press one of four dedicated keys to indicate this color. L2 appeared 500 msec after subjects made their color choice.The modified task was identical to the standard task with two exceptions: (1) According to our Bayesian hypothesis, the contraction bias emerges because subjects use Bayes' law to combine noisy information about the lengths of the bars with knowledge about the prior information in order to optimize performance. In this section we formalize this intuition.iL and iR be the logarithm of the length of bar i and its neural representation, respectively. We assume that this representation is noisy such that iz is drawn from a zero-mean Gaussian distribution with variance iR for a given representation of bar length In accordance with Weber's law, the lengths of the bars are measured in logarithmic scale. Let  uniform . Bayes' niform  such that L1 has to be stored in memory, a process that may contribute additional noise to the representation of L1. An ideal Bayesian observer, who has access to R1 and R2, would report ‘L1>L2’ in trials in which L1<L2’ in trials in which L1>L2’ in a trial in which L1 and L2 are presented is given byGiven a pair of neural representations, L1 is infinite, whereas there is no uncertainty in the representation of L2. In other words, L1>L2’ if R2 is larger than the median of L1. In trials in which L1 = L2, Eq. (3) dictates that he would report ‘L1>L2’ in trials in which L2 is larger than the median and ‘L1<L2’ otherwise.In order to construct the response curve we compute  . For furAll slopes were computed after normalizing the range of lengths to 0 and 1 in the logarithmic space.We used a pairs bootstrap resampling procedure In order to compare the response curve slopes between subjects who saw 50–200 pixel lines and those who saw 150–600 pixel lines we sampled from each group independently using the algorithm above, and then constructed a 95% confidence interval on the difference between the bootstrap slopes of the two groups.In order to compare trials with and without the interference task we calculated the difference in the bootstrap slope of each subjects' standard and interfered trials, and found the 95% confidence interval of this difference. The same method was also used to compare the slope of the response curve in the first 20 impossible trials of the experiment to the slope of the response curve in the last 20 impossible trials of the experiment.In order to compare behavioral performance to that predicted by the model, we used the model presented above to generate a set of response curves of ideal observers characterized by different values of L2>L1’ in the 50–200 and 150–600 standard experiments. Subjects reported that ‘L1>L2’ in the impossible trials in 41% and 46% respectively. This tendency has been reported previously p = 0.58, two tailed t-test). More importantly, this explanation is circular because a bias in the opposite direction could equally well have been explained by arguing that the prior distribution is biased in favor of large magnitudes. Therefore we did not attempt to account for the global bias and subtracted it before fitting, assuming that it is generated by a different mechanism. Thus, for the purpose of finding the parameters we added a constant to each of the response curves to normalize them such that mean(Pr[‘L1>L2’]) = 0.5. For purposes of comparison, the range of the logarithm of bar lengths was normalized to lie between 0 and 1 and we used a least square fit to find the parameters that best fit the population-average experimental data. We found that the best fit model parameters for the groups who saw 50–200 and 150–600 pixel-long bars were given by Note that subjects exhibited a small bias in favor of reporting ‘


L1 is a cell adhesion molecule associated with a spectrum of human neurological diseases, the most well-known being X-linked hydrocephalus. L1 knockout (L1-KO) mice have revealed a variety of functions of L1 that were crucial in brain development in different brain regions. However; the function of L1 in neuronal migration during cortical histogenesis remains to be clarified. We therefore investigated the corticogenesis of mouse embryos in which L1 molecules were knocked down in selected neurons, by employing in utero electroporation with shRNAs targeting L1 (L1 shRNA). Although more than 50% of the cells transfected with no small hairpin RNA  vector at embryonic day 13 (E13) reached the cortical plate at E16, significantly fewer (27%) cells transfected with L1 shRNA migrated to the same extent. At E17, 22% of cells transfected with the MGFP-only vector were found in the intermediate zone, and significantly more (34%) cells transfected with L1 shRNA remained in the same zone. Furthermore, the directions of the leading process of neurons transfected with L1 shRNA became more dispersed compared with cells with the MGFP-only vector. In addition, two transcription factors expressed in the neurons, Satb2 and Tbr1, were shown to be reduced or aberrantly expressed in neurons transfected with L1 shRNA. These observations suggest that L1 plays an important role in regulating the locomotion and orientation of migrating neurons and the expression of transcription factors during neocortical development that might partially be responsible for the abnormal tract formation seen in L1-KO mice. © 2012 Wiley Periodicals, Inc. The neural cell adhesion molecule L1 is one of the most intensively studied adhesion molecules expressed in the developing central and peripheral nervous systems , agenesis/dysgenesis of the corpus callosum, and X-linked spastic paraplegia and CRASH syndrome  strategy and in utero electroporation  was used in this study. The small interfering RNA (siRNA) sequence targeting L1 was designed and provided by SuperArray Bioscience Corporation . We prepared four shRNAs targeting L1 cloned in pGeneClip hMGFP vector and a negative control shRNA (shNC) showing no homology to any of the known mammalian genes , as well as hMGFP-labeled mock plasmid (MGFP only plasmid). The siRNA sequences are shown in 6 dissociated cells were suspended in 100 μl of transfection reagent, and the cells were then transfected with 10 μg of shRNA using the Amaxa Nucleofector , according to the manufacturer's instructions, using program No. O-005. We incubated the shRNA-transfected cells in 5 ml Neurobasal medium (Invitrogen Japan), supplemented with 2% B27 , 0.5 mM L-glutamine , and 1% penicillin/streptomycin solution . We added 4 ml of 1 × 106/ml untransfected cells into the 1 ml shRNA-transfected cells (at a density of 1 × 106/ml) and plated mixed cells at a final density of 1 × 106 cells/ml in 24-well plates, which had been precoated with 0.1 mg/ml poly-D-lysine (PDL) and 50 μg/ml laminin (Invitrogen Japan) or 0.1 mg/ml PDL, 40 μg/ml anti-Fc antibody, and 1 μg/ml L1-Fc. All of the cultures were maintained at 37°C in a 95% air, 5% CO2 (v/v) humidified atmosphere. All animal studies were approved by the Institutional Review Board for Biomedical Research using Laboratory Animals at Kyoto Prefectural University of Medicine, and the animals were handled according to the institutional guidelines and regulations.Dissociated cultures of cortical neurons were prepared from embryonic day 13.5 murine fetus . Briefly, the dorsal forebrain was dissected and treated in 0.125% trypsin/0.5 mM EDTA for 15 min at 37°C and then dissociated by gentle trituration (in a Pasteur pipette). In total 5 × 10Gapdh, Rplp1, and Rsp18.Total RNA was extracted from the transfected cultured cells using an RNeasy Micro Kit (Qiagen Japan), and cDNAs were synthesized and amplified as described previously. Briefly, first-strand cDNAs were synthesized using an iScript cDNA Synthesis Kit (Bio-Rad Japan) with oligo(dT) primer. The relative gene expression levels of L1 were measured by qRT-PCR on an ABI Prism 7000 sequence detection system (Applied Biosystems Japan) using SYBR Premix Ex Taq  according to the manufacturer's protocol. The optimized number for thermal cycling was set at 40. The primers for L1 were designed as follows: L1cam_2: 5′-gctttgcctccgagggctgg-3′ (forward), 5′-ccagggacctgtactcgccga-3′ (reverse) and L1cam_6: 5′-ccaagtggagttccgctggacg-3′ (forward) and 5′-cttcggccacttgggggcac-3′ (reverse). TaqMan rodent control reagent (Applied Biosystems Japan) was employed for housekeeping genes, such as P < 0.05 was designated as statistically significant.To evaluate the functional loss of L1 by shRNA targeting L1, at 48 or 96 hr after transfection, the primary cortical neurons were fixed with 4% paraformaldehyde for 5 min at 4°C. After fixation, we observed electroporated neurons expressing MGFP under a confocal microscope . The length of the longest neurite, the number of branches, and the total length of the neurites were measured in NeuronJ, which is an ImageJ plugin allowing the tracing and quantification of elongated structures. Because the shRNA2-transfected cells showed a unique histogram with regard to the longest neurite extension at 48 hr after transfection, we classified the cells into three types, according to the longest neurite length: 1) neuron type 1, longest neurite less than 80 μm; 2) neuron type 2, longest neurite between 80 and 220 μm; and 3) neuron type 3, longest neurite more than 220 μm. All values are expressed as means ± SEM. The SEM values indicate the variation between mean values obtained from at least three independent experiments. Statistical comparisons were conducted by using Dunnett's multiple comparison test. C57BL/6J pregnant mice were purchased from CLEA Japan Inc. . The day on which the vaginal plug was found was designated as embryonic day 0 (E0). In utero electroporation was performed as described previously . After blocking with 10% goat serum, 1% bovine serum albumin (BSA), 0.01% Triton X-100 (Sigma-Aldrich Japan) in 0.1 M PBS, the sections were incubated in primary antibody at 4°C overnight; doublecortin , Satb2 , Ctip2 , or Tbr1 , diluted in 0.1% Triton X-100 and PBS. The sections were rinsed several times with PBS and then incubated in the secondary antibody . The sections were then coverslipped and observed under a confocal microscope .t-test was performed to detect the differences in the dispersion of the “cell process angle.”Images acquired with the Olympus FluoView FV1000 confocal microscope were processed with an Olympus FV10-ASW 1.7 viewer to adjust color and contrast. To conduct a quantitative analyses of the transfected cells, which were clearly detectable with MGFP fluorescence, at least three independent brains were analyzed at each embryonic stage: transfected at E13 and analyzed at E16 or transfected at E13 and analyzed at E17. The wall of the embryonic dorsal brain was divided into six layers, the ventricular/subventricular zone (VZ); the upper and lower intermediate zone (IZ); and the upper, middle, and lower cortical plate CP; D. The waP < 0.05 by one-way ANOVA; The expression level of L1 mRNA was significantly reduced in transfected cortical cells with shRNA2 plasmids at 48 hr after transfection compared with the negative control plasmid shRNA (shNC), no shRNA (MGFP only), as well as with no treatment , and MGFP-only plasmid by in utero electroporation at embryonic day 13 (E13). In utero electroporation allowed us to introduce shRNA into the fetal VZ to knock-down the targeted gene so that neuronal migration from the VZ toward the CP could be traced. We examined the distribution of MGFP-labeled cells in the presumptive somatosensory cortical area of the dorsolateral forebrain, which was located at the level of the medial and lateral ganglionic eminences by coronal section . MGFP-onWhen we observed the migration pattern of the GFP-expressing cells in the fetal telencephalon, we found that the cells transfected with MGFP-only plasmid showed a fine neurite fasciculation and radial migration to the marginal zone, similar to that found in the physiological situation. However, the neurons transfected with shRNA2 showed a wide variation in the orientation of neurites during migration. To confirm the difference in terms of the direction of cellular migration, we measured the angle of the leading neurite relative to the orientation of the CP, as described in Materials and Methods A,B. StatTo investigate whether the process of neuronal differentiation is affected by L1 knockdown, we studied the expression patterns of the transcription factors that are known to regulate the differentiation of cortical neurons, such as Satb2, Ctip2, and Tbr1. These factors are all expressed specifically and associated with the cortical layering. Satb2  belongs to the family of DNA-binding proteins, which regulates gene expression through chromatin modification and interacts with other proteins. It is expressed in corticocortical projection neurons. Ctip2, belonging to the family of zinc finger transcription factors, is expressed in subcortical projection neurons (Leid et al.,Nontransfected neurons observed in both MGFP-only plasmid- and shRNA2-transfected dorsal telencephalon at E16 or E17 showed a similar immunoreactivity for these transcription factors. Satb2 expression was found from the upper to the middle portion of the CP and was expressed more weakly in the IZ. Many MGFP-only plasmid-transfected neurons migrated into the upper and middle CP, showing intense immunoreactivity for Satb2 at E16 as well as at E17, whereas shRNA2-tranfected neurons showed immunoreactivity for Satb2 mainly in the subplate and IZ at E16 and E17 arrow, . Ctip2 iWe found that the downregulation of L1 using in utero electroporation of shRNA affected the radial migration of cortical neurons, with aberrant expression of the transcription factors that are important for cortical layer formation. L1 plays a variety of functional roles required for normal brain development in different brain regions (Lemmon et al.,Our observations suggested that downregulation of L1 induced either a delay in the exit of neurons from the VZ to the IZ or an inability to sense a molecular cue for neurons to migrate into the CP. Our study showed an increased ratio of multipolar shRNA2-tranfected neurons stuck in the IZ at E17, which partially resembled the migration disorder observed in doublecortin loss-of-function mice (LoTurco and Bai,We have demonstrated that downregulation of L1 perturbed not only the locomotion of neurons but also the orientation of the leading process. It is known that the intracellular domain of L1 contains an ankyrin-bindng region and a neuron-specific sequence, RSLE, which is critical for sorting L1 to axonal growth cones (Kamiguchi and Lemmon,To assess this phenomenon followed by downregulation of L1 and disturbed neuronal migration, we examined how the expression of transcription factors known to be involved in cortical development is affected. Satb2 is expressed in corticocortical projection neurons and plays essential roles in the axonal pathfinding of projection neurons in brain development (Britanova et al.,In conclusion, we have demonstrated that downregulation of L1 by using in utero electroporation of shRNA perturbed the locomotion and orientation of radially migrating neurons in neocortical histogenesis, together with alterations in the expression of transcription factors involved in cortical development. These results first indicate that L1 plays an important role not only in axonal guidance but also in neuronal migration during neocortical development. The methodological advantages of the in utero electroporation method we employed allowed us to downregulate L1 in only a subset of cortical cells, so that the novel functions of L1 in cortical development could be elucidated.


It is found that graphene layers can be joined by different domains, with a relative misorientation of 30°. These graphene layers can be decoupled from Cu(110) through low temperature thermal cycling.Revealing the graphene growth mechanism at the atomic-scale is of great importance for achieving high quality graphene. However, the lack of direct experimental observation and density functional theory (DFT) verification hinders a comprehensive understanding of the structure of the carbon clusters and evolution of the graphene growth on surface. Here, we report an in-situ low-temperature scanning tunneling microscopy (LT-STM) study of the elementary process of chemical vapor deposition (CVD) graphene growth via thermal decomposition of methane on Cu(110), including the formation of monodispersed carbon clusters at the initial stage, the graphene nucleation and the ripening of graphene islands to form continuous graphene film. STM measurement, supported by DFT calculations, suggests that the carbon clusters on the surface are C Ever since its mechanical exfoliation from small mesas of highly oriented pyrolytic graphite, graphene has spurred a tremendous of interest because of its exceptional electronic and mechanical properties, such as anomalous quantum Hall Effect (QHE), long-range ballistic transport, high carrier mobility, tunable band gap, high elasticity and intrinsic strength1231291011141516171819202122232425The graphene growth on TM surfaces is based on high-temperature pyrolysis of hydrocarbons and different growth mechanisms can be involved according to the carbon solubility limit in the metal. For the growth on TM where carbon is soluble, the graphene forms when the sample is cooled and carbon segregates on the surface (surface segregation)2930312H2 can be easily formed on a Cu(111) surface, which represents a more favorable reaction path compared to CH dissociationN on Ni(111), Gao et al. indicated a ground state structure transition from a one-dimensional (1D) carbon chain to a two-dimensional (2D) sp2 carbon network at N ~ 10–1221 is a very stable magic carbon cluster on Rh(111), Ru(0001), Ni(111) and Cu(111) surfaces1516Graphene growth has been demonstrated on a variety of TMs. For example, Li and colleagues reported a CVD method that used copper-foil to produce single crystal graphene with dimensions of up to 0.5 mm14333435363738394041424) on Cu(110) using low-temperature scanning tunneling microscopy (LT-STM), including the formation of monodispersed carbon clusters at low temperature, nucleation and ripening of graphene islands at high temperature. Combined with first principles calculations, the monodispersed carbon clusters are identified as C2H5. Different domains stitch together to form a graphene layer, with a preference angle of 30° at the grain boundaries. These graphene layers can be decoupled from Cu(110) through low temperature thermal cycling.Here, we report an atomic scale characterization of the elementary process of CVD graphene growth via thermal decomposition of methane (CH4 at room temperature (RT) and subsequent annealing at 480°C in CH4 at a pressure of 2 × 10−5 mbar for 50 min, the Cu(110) surface was almost decorated with carbon clusters of monodispersed size. Each carbon cluster appears as a bright spot with an identical size of 0.4 nm. Careful inspection of the STM image reveals that the surface is decorated by isolated but well-defined superstructures, where the carbon clusters are adsorbed in an epitaxial relationship with the underling Cu(110). As indicated by the dashed lines in 0 of 0.512 nm; while it is 2b0 of 0.723 nm between two columns (a0 and b0 are the unit cell dimensions of Cu(110)). It can also be revealed that the carbon cluster arrays are aligned precisely with the crystal orientation of the underlying Cu(110). The carbon cluster at this low coverage was referred to as “cluster 1” with a density around 2.70 × 1014/cm2. Previous theoretical studies proposed that carbon dimmers are energetically favorable on the Cu surface37382Hx).As shown by the high magnification STM image in 14/cm2, referred to as “cluster 2”. In this regime, the arrangement is supposed to be cluster-cluster interaction dominated. Some brighter lines can be frequently observed, induced by the stress relaxation at high cluster coverage with increased lateral inter-cluster interaction.Further increasing the coverage of the carbon clusters can result in the formation of a hexagonally close packed structure, as shown in 4 at a pressure of 2 × 10−5 mbar for 130 min can promote the nucleation of small graphene flakes. As shown in Annealing the Cu(110) surface at high temperature at 550°C in CH1Hx (0 ~ 4) and C2Hx (0 ~ 6) clusters on Cu (110) were studied. We define the formation energy in tot is the total energy of the adsorbed system, Esub is the energy of clean Cu (110) substrate, μi and ni  represent chemical potential and the number of atoms in the cluster, respectively. Considering the equilibrium of CH4 and H2, the relationship of μH and μC in unit of electron volt can be obtained as 4 and H2.To obtain the atomic structure of the carbon clusters, the adsorption of various carbon clusters on Cu(110) were simulated using DFT. First, the stability of Clong site), bridge-short site (Bshort site) and Top site (T-site)2). The χ here was set to be 20:1; we also tested χ = 1:20, which gave similar results.For each carbon cluster species, the most stable adsorption configuration was found by checking different adsorption sites on Cu(110) surface, including the hollow site (H-site), bridge-long site  at high temperature. The average lifetime of C2H6 and C2H5 can be estimated by their adsorption energy Ea via 2H6 and C2H5 on Cu(110) surface are 0.41 and 2.85 eV, respectively. υ0 is about 1013 s−1. Therefore, their average lifetime on the surface at 480°C is 5.5 × 10−11 and 1.2 × 106 s, respectively. Such a short lifetime makes C2H6 not be able to be observed by STM. Therefore, C2H5 could be the most possible abundant species from the thermodynamic point of view.From 2, C2H4, C2H5 and C2H6. The optimized unit cell of the carbon cluster is 2a0 = 0.504 nm, 2b0 = 0.713 nm. Among these carbon clusters, the simulated STM image of C2H5 is in good agreement with the experimental results. All other stable species cannot reproduce the experimental circular shape. Hence, the basic structures of the carbon clusters are elucidated by the STM images in combination with DFT calculations as C2H5.STM images of several partially dehydrogenated carbon dimer species were also simulated using the Tersoff and Hamann approximationLarge graphene flakes can be achieved through low temperature thermal cycling process as described in the 2519As described in the 4 decomposition, similar to the previously reported hydrogenThe formation of such physically decoupled graphene can arise from the intercalation at the graphene/Cu(110) interface by hydrogen atoms released from CH4. Low temperature annealing (>480°C) in CH4 results in the formation of carbon clusters at the initial stage; further high temperature annealing (>550°C) activates the graphene nucleation; prolonged annealing in the absence of CH4 propels the diffusing and ripening of these graphene island to form continuous graphene films extended over the surface. Low temperature thermal cycling induced decoupling of graphene from Cu(110) has also been demonstrated. Our systematic investigations identify the fundamental carbidic building blocks by STM measurement, and further elucidate their atomic structures through DFT calculations. Our work could lay the foundation for providing rational design rules for synthesis of large area single crystalline graphene films.Through the combination of the LT-STM and DFT calculations, we reveal the elementary process of graphene growth on Cu(110) surface via thermal decomposition of CH4. Prior to the deposition of CH4, Cu(110) substrate was cleaned by a few cycles of Ar+ ion bombardment and subsequent annealing at 530°C. The CH4 gas was introduced into the growth chamber through a leak valve, and the pressure was monitored by a cold cathode gauge. A typical growth procedure is as follows: the Cu(110) substrate was exposed to CH4 at a pressure of 2 × 10−5 mbar for 20 min; annealing the sample at 480°C in CH4 at a pressure of 2 × 10−5 mbar resulted in the formation of carbon clusters; further annealing the sample in the absence of CH4 at 550°C initiated the graphene nucleation; prolonged annealing without CH4 at higher temperature up to 720°C propelled the ripening of graphene islands.Graphene was grown on a single crystal Cu(110) via thermal decomposition of CH−10 mbar, housing an omicron LT-STM interfaced to a Nanonis controller. All STM imaging were performed at 77 K using constant current mode with an electrochemically etched tungsten tip. All the bias voltage was applied to the tipThe LT-STM experiments were carried out in a custom-built multichamber ultra-high-vacuum (UHV) system with base pressure better than 1.0 × 101Hx (0 ~ 4) or C2Hx (0 ~ 6) clusters on Cu (110) surface were studied using DFT calculations. A 5-layer slab with a 20 Å vacuum layer was used as the substrate. The bottom layer was fixed to its bulk configuration and all other atoms were fully relaxed. A (3 × 4) supercell was chosen to make sure that clusters were separated to their neighboring clusters by more than 10 Å. In STM simulation, a (2 × 2) supercell was chosen according to the experimental coverage.Stability of C−5 eV and 0.02 eV/Å. For the (3 × 4) and (2 × 2) models, (7 × 7 × 1) and (10 × 14 × 1) k-point grids were used, respectively. STM images were simulated using the Tersoff and Hamann approximationAll the calculations were performed using DFT implemented in the Vienna Ab Initio Simulation Package (VASP) within the generalized gradient approximation55W.C. conceived and designed the experiments; J.L.Z. and T.C.N. performed the experiments; Z.Z.W., S.N.W. and Z.Y.L. performed theoretical calculations; W.C. and J.L.Z. wrote the manuscript. All authors contributed to writing and revising the manuscript.Supplementary Information