Studies investigating the relationship between working memory (WM) and approximate number system (ANS) acuity in the area of arithmetic strategy utilization are scarce. The choice/no choice method paradigm was used in the present study to determine whether and how ANS acuity and WM components affected strategy utilization. The results showed that the central executive (CE) mediated the relationship between ANS acuity and strategy utilization. Furthermore, quantile regression analyses revealed that the association between CE and strategy choice was robust from the first to highest quantile. Notably, the relationship between ANS acuity and strategy choice was significant at the median and higher quantiles , but not significant at lower quantiles . These results suggest that domain-general skills play a crucial role in the relationship between children’s ANS acuity and mathematical ability. The impact of ANS acuity and CE on strategy choice was dependent on the distribution of the strategy utilization level. These results provide a further understanding of the utilization of cognitive strategies. Mathematics plays an important role in our daily and professional lives . A long Previous studies have revealed that children’s poor performance on mathematics is related to inefficient strategy utilization e.g., , making Working memory has been consistently shown to be an important predictor of children’s math performance. In the last few decades, numerous studies have shown the relationship between WM and strategy utilization. Many studies have tested the relationship between the ANS and mathematical ability e.g., . ANS refThe studies outlined above concentrated on domain-general factors or domain-specific factors that affect mathematics and strategy utilization separately. However, studies investigating the effect of the interaction between domain-general and domain-specific factors on math abilities are scarce. Additionally, although studies have shown that ANS acuity is a predictor of mathematics ability, this relationship explained only a small proportion of the total variance. ANS acuity is not the only or the strongest determinant of a child’s math achievement. A theory of early foundations for mathematics learning assumes that the first step toward learning mathematics in children is based on the ANS. The first abstract math symbols are number words and Arabic numerals, which, in turn, obtain their meaning when mapped onto the ANS, during which attention control plays an important role . As IranGiven WM and ANS acuity contributions to mathematical ability, we assumed that (1) CE predicts strategy utilization beyond measures of the PL and VS and (2) ANS acuity may play a mediating role in the relationship between CE and strategy utilization.To date, most studies have focused on the average effect of ANS acuity, PL, and CE on average strategy utilization performance using the ordinary least squares (OLS) regression technique, which can hide important features of the underlying relationship. The regression curve provides a grand summary for the averages of the distribution corresponding to the set of X’s. Thus, ordinary regression often gives a rather incomplete picture, just as the mean gives an incomplete picture of a single distribution . ChildreM = 11.09 years, SD = 0.65; female = 88). All participants gave written informed consent and were paid for their participation. This study was approved by the regional ethics committee for biomedical research.In this study, 179 fourth-grade children were selected from public schools in China. Fourth graders were selected because computational estimation problems are frequently introduced in the curriculum at this time. Two children who enrolled in the study were excluded due to the absence of Automated Working Memory Assessment (AWMA) task results, and 17 children were excluded due to an invalid Arithmetic Computational Strategy Test. A total of 160 children were included as study participants . The test–retest reliability coefficients are presented in Table 1.The AWMA  was utilIn Digit Recall, the examinee hears a sequence of digits and attempts to recall each sequence in the correct order. The test begins with a block of one number and increases to a block of nine numbers. Backward Digit is almost identical to Digit Recall in structure and administration; the child hears a sequence of digits and attempts to recall each sequence in backward order. The test begins with a block of two numbers and increases to a block of seven numbers. As the name implies, Word Recall is a series of words that the participants must recall in the correct order. The test begins with a block of one word and increases to a block of seven words.In Dot Matrix, a red dot in a 4 × 4 matrix is exposed for 2 seconds (s), and then the participants recall the positions by tapping the appropriate squares on a computer screen. The test begins with a block of one dot and increases to a block of nine dots. In Odd One Out, the examinee views three shapes presented in a row of boxes and identifies the Odd One Out shape. After a series of presentations, the examinee must recall the location of each Odd One Out in the correct order. The test begins with a block of one set of shapes and increases to a block of seven sets of shapes. In Maze Memory, the participants view a maze with a red path drawn through it. The task is to use a finger to trace the same path on a blank maze presented 3 s later on the computer screen. The test begins with small mazes and increases to large mazes.In Counting Recall, the child counts the number of red circles in an array of circles and triangles and attempts to recall the tally of numbers in sequence. The test begins with a block of one array and increases to a block of seven arrays of circles and triangles. In Mr. X, the child views a picture of two Mr. X figures and identifies whether the Mr. X with the blue hat is holding the ball in the same hand as the Mr. X with the yellow hat. The Mr. X with the blue hat may also be rotated. At the end of each trial, the child attempts to recall the location of each ball in the correct order by pointing to a picture with six possible marks. The test begins with a block of one set of Mr. X and increases to a block of seven sets of Mr. X.The Non-symbolic Number Comparison Task is a commonly used measure of ANS acuity. The Weber fraction was used as an index of ANS acuity, as the ANS is assumed to follow Weber’s law . In thisThe ACST was classified as a measure of an individuals’ strategy utilization, including strategy choice and strategy execution. It consists of 180 two-digit multiplication problems . The accuracy of each condition, calculated based on the number of correct answers, was considered as he index of the level of strategy choice or strategy execution. Higher accuracy indicates a greater level of individual strategy choice and execution. There were three conditions in this test: the no choice/rounding-down condition, in which all problems must be answered using a rounding-down strategy ; the no choice/rounding-up condition, in which all problems must be answered using the rounding-up strategy ; the choice condition, in which one strategy must be selected from rounding-up and rounding-down strategies for each problem to estimate the correct answer as closely as possible. Cronbach’s alpha coefficients for the ACST were 0.919, 0.759, and 0.613 for the conditions of choice, no choice/rounding-up, and no choice/rounding-down subtests, respectively.Following previous findings . We scheduled a short break during the testing session.Participants were tested in a quiet room. There were two sessions. The first session was a group test, in which participants were asked to complete the ACST. It lasted approximately 15 minutes (min). Before starting the ACST, the experimenter showed each participant how to use the rounding-up and rounding-down strategies. The ACST did not start until every participant could use the strategies proficiently. Participants solved problems according to the instructions. The second session was the individual test, and participants completed the AWMA task first (about 45 min). The AWMA is a computer-based assessment of WM skills. All tests were presented on a laptop computer. The experimenter recorded the participant’s response using the right arrow key on the keyboard (→) for a correct response and the left arrow key on the keyboard (←) for an incorrect response. Each memory test began with a series of practice trials. Immediately following the practice trials were the test trials. The test trials were presented as a series of blocks; each block consisted of six trials. In each case, the child had to remember a piece of information and then recall it back immediately. The participants repeated the information verbally, and the experimenter recorded the participant’s responses to the PL tests . All tests operated on the following rules.If an individual responded correctly to the first four trials within a block of trials, the program automatically proceeded to the next block. A score of 6 was given for the block that has just been completed.If an individual responded correctly to four out of five trials in a block, the program immediately started the next block. A score of 5 was given for the block that has just been completed.If three or more errors were made within a block of trials, the program stopped the test and automatically returned to the main menu. The score for that test reflected the number of correct responses up to the point at which the test was ended.Structural equation modeling (SEM) and quantile regression techniques were used to analyze the data with AMOS 17.0  and SAS 9.4 , respectively.In this section, we present our results that (a) compare the predictions of PL, VS, and CE for children’s strategy utilization, (b) demonstrate the role CE played in the relationship between children’s ANS acuity and strategy utilization, and (c) resolve the different effects of WM and ANS acuity on lower-, middle-, and higher-level strategy utilization performance.2 = 18.56, df = 17, p = 0.36, RMSEA = 0.02, GFI = 0.97, TLI = 0.99 . All structural path coefficients were significant .A first-order confirmatory factor analysis was used to determine whether the data acquired by the AWMA fit with the WM theory model and SEM using maximum likelihood estimation in AMOS 17.0. The model demonstrated a good fit to the data: χTable 2. Consistent with previous research on children of this age (w) was 0.215. The Weber fraction as an index of ANS acuity is inversely proportional to ANS acuity. Thus, to facilitate the description, w was multiplied by -1. As expected, ANS acuity was significantly related to PL, VS, and CE, with a particularly strong relationship between ANS acuity and CE (r = 0.39). Children’s performance on ANS acuity was also significantly associated with their strategy utilization performance. Consistent with a prior study, children’s PL, VS, and CE were significantly correlated with strategy utilization, with a particularly strong relationship between CE and strategy use (r = 0.50).Means, standard deviations, and correlations for the variables are shown in this age , this stt-test was used to test the gender differences in the variables. No significant gender differences were found for different components of WM , ANS acuity , or strategy utilization ; therefore, we built only one SEM without differentiating gender.The independent sample Table 3).Three SEM analyses using a maximum likelihood estimation were performed. First, two direct effects models were prepared, where the direct effects of PL, VS, and CE on strategy choice (Model 1) and strategy execution (Model 2) were tested separately. These two models indicated a good fit to our data  beyond the effect of strategy execution . PL also positively influenced strategy executive , but negatively affected strategy choice . Moreover, although they were not statistically significant, VS negatively predicted children’s performance on strategy execution  and strategy choice .Models 1 and 2 show the direct effects of PL, VS, and CE on strategy execution and strategy choice, respectively. The results indicated that CE highly predicted individual differences in strategy choice , with the exception of the non-significant path for the relationship between ANS acuity and strategy utilization . With this model structure, 30.8 and 36.5% of the variance of children’s strategy execution (2R = 0.31) and strategy choice (2R = 0.37) were explained, respectively.Given that the effect of CE on strategy utilization was much stronger than that of PL and VS, we tested the complete mediation model, which assumed that CE completely mediated the effects of ANS acuity on children’s strategy utilization, specifically in strategy choice and strategy execution. This model demonstrated good fit, χSE = 0.11, p = 0.001), but it was not significant after controlling for CE . Similarly, the strategy choice results showed that ANS acuity significantly predicted children’s performance on strategy choice , but ANS was not a significant predictor when controlling for CE . These results suggested that the associations between ANS acuity and strategy execution and ANS acuity and strategy choice were completely mediated by CE; the mediating effects accounted for 56.3 and 70.5%, respectively, of the variance in children’s performance on strategy execution and strategy choice separately.As expected, the strategy execution results revealed that ANS acuity significantly predicted children’s performance on strategy execution .It was clear that the OLS estimates do not tell the whole story. However, the quantile regression curves show that the value of the estimated coefficient on ANS acuity, PL, and CE varied over the conditional growth rate distribution, which was consistent with our prediction. In particular, at the 0.15 and 0.25 quantiles, the estimate of ANS acuity was insignificant, that is, ANS acuity did not affect the low-level strategy choice. ANS acuity was significantly associated with strategy choice at the 0.5, 0.75, and 0.85 quantiles. Namely, ANS acuity was of crucial importance for median and higher than median strategy choice performance. Moreover, the CE estimate was significant in all quantiles. In contrast, PL was not significantly associated with strategy choice. Note that when the quantile regression solution was evaluated at the lower-level of strategy choice performance , CE appeared to have a large effect on children’s strategy choice performance. However, the CE coefficient decreased sharply for higher-level strategy choice performances at the upper quantiles, suggesting that the effect of CE decreased as strategy choice performance increased  through CE. Our results showed that CE completely mediated the relationship between ANS acuity and strategy utilization. This was in line with Another significant finding in the present study was that we first detected the effects of WM components and ANS acuity on strategy choice performance in the tail distribution using the quantile regression technique. The results revealed that the association between CE and strategy choice was robust from the first quantile to the highest quantile. That is, CE contributed not only to low-level strategy choice performance but also to high-level performance. Note that the relationship between ANS acuity and strategy choice was significant at the median and higher quantiles , but not at lower quantiles , meaning that children with low-level strategy choice performance were not affected by ANS acuity, but when children’s strategy choice performance was at the median or far above the median performance, their strategy choice was remarkably affected by ANS acuity. These findings cannot be observed using traditional OLS regression. One possible explanation for this phenomenon is the two-factor theory . RobinsoAn unexpected additional interesting finding that contrasts with previous studies was the insignificant relationship between VS WM and strategy utilization. As discussed by On a theoretical level, the present findings have significant implications regarding our understanding of strategy selection. The strategy choice and discovery simulation model (SCADS) assumes that there is a WM trace of each strategy’s execution, which allows individuals to provide accurate information immediately after verbal reports concerning their strategy use . ConsistAlthough our results support our hypotheses, there were at least two additional limitations of this study that suggest directions for future studies. First, we recruited typically developing children in this study. As discussed above, the effect of ANS acuity and WM components on strategy choice varies from the 0.15 to 0.85 quantiles. Accordingly, future research should pay more attention to children who perform in the tail of the distribution. Second, we “left out critical co-requisite sources of contribution, something that happens all too often in both psychology and education” . AccordiAll procedures performed in studies involving human participants were in accordance with the ethical standards of the ethics committee on human experimentation of Shandong Normal University and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.Conception and design of the work were done by HL and JS. Data collection was done by HL, MZ, XW, and XD. Analysis of data was done by HL, JS, and all other authors interpreted the data. The original draft of the manuscript was wrote by HL, and it was edited and co-wrote by MZ, XW, XD, and JS.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer FB and handling Editor declared their shared affiliation.


Compared to RAID v2.0, new features in RNAInter include (i) 8-fold more interaction data and 94 additional species; (ii) more definite annotations organized, including RNA editing/localization/modification/structure and homology interaction; (iii) advanced functions including fuzzy/batch search, interaction network and RNA dynamic expression and (iv) four embedded RNA interactome tools: RIscoper, IntaRNA, PRIdictor and DeepBind. Consequently, RNAInter contains >41 million RNA-associated interaction entries, involving more than 450 thousand unique molecules, including RNA, protein, DNA and compound. Overall, RNAInter provides a comprehensive RNA interactome resource for researchers and paves the way to investigate the regulatory landscape of cellular RNAs.Research on RNA-associated interactions has exploded in recent years, and increasing numbers of studies are not limited to RNA–RNA and RNA–protein interactions but also include RNA–DNA/compound interactions. To facilitate the development of the interactome and promote understanding of the biological functions and molecular mechanisms of RNA, we updated RAID v2.0 to RNAInter (RNA Interactome Database), a repository for RNA-associated interactions that is freely accessible at  RNA-associated interactions involve many physiological and pathological processes, such as cell growth and development, cell differentiation and inflammation . With thhttp://www.rna-society.org/rnainter/ or http://www.rna-society.org/raid/) to address these challenges. RNAInter establishes a repository of integrated experimentally validated and computationally predicted RNA-associated interactions through manual curation of the literature, along with another 35 resources under one common framework  miRNA symbols from the miRBase database, (ii) DNA, RNA and protein symbols from the NCBI Gene or Ensembl  databaseIn particular, we collected and processed four single-cell RNA-seq (scRNA-seq) data sets from the Gene Expression Omnibus (GEO)  to visuaHomo sapiens and Mus musculus interactions took up the main part of the vertebrata , have also been documented in RNAInter.In summary, RNAInter contains 41 322 577 RNA-associated interactions, including 34 106 998 RPIs, 6 007 974 RRIs, 1 060 684 RHIs, 138 068 RDIs and 8853 RNA-compound interactions (RCIs) Figure , Table 1a Figure . Other mThen, we expanded the RNA-compound, RNA–DNA and RNA–histone modification interactions in RNAInter. Apart from basic annotation, support evidence, RNA-binding sites and references, we focused on the multifaceted supplementation of the details of RNA editing/localization/modification/structure/dynamic expression, the interaction network, the target region and the homology interaction in detail. ‘RNA editing’ provides editing position, editing type and genetic region. ‘RNA localization’ includes subcellular localization and the tissue or cell line. ‘RNA modification’ involves the modification position, modification type and genetic region. Moreover, ‘Homology interaction’ shows the conservative interactions across organisms documented in RNAInter. ‘Target region’ shows the target locus in RHI/RPI/RRI and data accession from the literature or high-throughput sequencing with their sample resources. All this information links to their corresponding databases.RNAInter provides a user-friendly platform for searching, browsing, visualizing and profiling RNA interactome data. To improve the search capability, RNAInter enables an optimized query with a new function of fuzzy and batch search. Fuzzy Search can help users to search interactions using unstandardized or uncertained interactor name under selected molecular category, then the result of interactions will be presented by selecting interactors in candidate list. Meanwhile, Batch Search supports for inputting a list of official symbols/IDs or uploading a file with text format to obtain multiple molecular categories associated interactions. Thus, users can select ‘Exact Search’ to filter the search results, or ‘Fuzzy Search’ to further focus on interactors of interest, or ‘Batch Search’ to customize their query content in batch Figure . Taking In response to the diverse needs of users, RNAInter embeds four interactome tools: RIscoper, IntaRNA, PRIdictor and DeepBind. RIscoper is a tool for RNA–RNA interaction extraction from the literature. IntaRNA is a program for the fast and accurate prediction of interactions between two RNA molecules. PRIdictor is a protein–RNA interaction predictor. DeepBind predicted the sequence specificities of DNA- and RNA-binding proteins by deep learning Figure .RNAInter is an update of RAID v2.0, a comprehensive resource for RNA interactome data obtained from the literature and other databases, containing over 41 million RNA-associated interactions of RCI, RDI, RHI, RPI and RRI. With detailed interactome information, visualized interaction network and RNA dynamic expression, enhanced search functions, and embedded RNA interactome tools, RNAInter depicts a system-level RNA interactome landscape with guides and help researchers to perform further studies. We expect RNAInter to update the manual curation of RNA interactome data and expand the available information about RNAs and other molecules in the future. Continuously integrating high-throughput data, including scRNA-seq, to provide more precise depiction of the dynamic expression pattern of RNAs illuminates the role of RNA across organisms. We may optimize the confidence score strategy with the emergence of new mass sequencing technologies, experimental methods and prediction algorithms. At the same time, more RNA-associated applications are docking with our database. Eventually, RNAInter will present the most comprehensive map of the RNA interactome to satisfy different requirements.gkz804_Supplemental_FileClick here for additional data file.


In eukaryotic cells, gene expression is highly regulated at many layers. Nascent RNA molecules are assembled into ribonucleoprotein complexes that are then released into the nucleoplasmic milieu and transferred to the nuclear pore complex for nuclear export. RNAs are then either translated or transported to the cellular periphery. Emerging evidence indicates that RNA-binding proteins play an essential role throughout RNA biogenesis, from the gene to polyribosomes. However, the sorting mechanisms that regulate whether an RNA molecule is immediately translated or sent to specialized locations for translation are unclear. This question is highly relevant during development and differentiation when cells acquire a specific identity. Here, we focus on the RNA-binding properties of heterogeneous nuclear ribonucleoproteins (hnRNPs) and how these mechanisms are believed to play an essential role in RNA trafficking in polarized cells. Further, by focusing on the specific hnRNP protein CBF-A/hnRNPab and its naturally occurring isoforms, we propose a model on how hnRNP proteins are capable of regulating gene expression both spatially and temporally throughout the RNA biogenesis pathway, impacting both healthy and diseased cells. A fascinating question in gene expression regulation is to understand how from the onset of transcription, cells regulate mRNA molecules into degradation, localization, storage, and/or translation. Several decades of mRNA biology have shown that regulation primarily happens at the level of ribonucleoprotein (RNP) particles, composed of RNA molecules and RNA-Binding Proteins (RBPs) . Within In this mini-review, we concentrate on two key steps in the mRNA regulation by focusing on a representative of a large family of RBPs, the heterogeneous nuclear ribonucleoprotein ab (hnRNPab) also referred to as CBF-A (CArG box-binding factor A). After a brief review of the different stages of mRNA biogenesis, we will address the role of hnRNPab in the formation and integrity of RNP particles, and in the regulation of the translatability of the carried mRNA. Finally, we will discuss the relevance of those mechanisms in cell specification and development.Chironomus tentans has shown in situ evidence of RNP particles decorating chromosome loops and being released after the maturation in the interchromatin space  or A2 Response Element (A2RE), in order to regulate cytoplasmic mRNA transport (in vitro and in living cells (∗RNA Binding Domain (RBDs) and Glycine-rich auxiliary domain” (2∗RBD-Gly) proteins  and a conserved C-terminal Glycine-rich region n] at telomeres  quadruplexes. However, while hnRNPA2 efficiently disrupts such a structure, CBF-A/hnRNPab has the opposite effect and stabilizes the RNA G4-quadruplexes  of hnRNP A1 in complex with single-stranded guanine-rich telomeric DNA. Guanine-rich DNA and RNA sequences have a tendency to form tetrahelical G4-quadruplex structures olecules . Althougelomeres . These bruplexes . While truplexes . Not onlruplexes , but morruplexes .cis-acting elements within nascent RNA and stabilize the formation of RNA G4 quadruplexes to sort transcripts that are not translationally active and can therefore be transported to the cellular periphery. Indeed, CBF-A/hnRNPab binds to the RTS located in the 3′ UTR of several transcripts, including the Myelin Basic Protein (MBP), β-actin, Arc, BDNF, CAMKIIα, and Protamine 2 mRNAs (∗RBD-Gly-rich family such as hnRNPA2 (in vitro (With this in mind, CBF-A/hnRNPab may cotranscriptionally target  2 mRNAs . RTS bin 2 mRNAs . The RTS hnRNPA2  and hnRN hnRNPA2 . CBF-A/hin vitro . Given tAs mentioned above, upon nuclear export, translationally repressed RNPs are further assembled into larger granules to be transported to specific cellular locations for storage or for translation. Although poorly understood, assembly of RNP particles into transport granules has been proposed to be mediated by homo-dimerization of RNA-bound hnRNPs and actin polymerization from within individual RNP particles . The homin vitro p37 shows a higher affinity than p42 and hnRNPA2 for the same RTS target (How RNA transcripts become available to the translation machinery remains a major question for future studies. Insights recently came from evidence of different roles performed by the CBF-A/hnRNPab isoforms in regulating translatability of the Protamine 2 mRNA Figure . While bS target . p42 andS target . FurtherS target  but onlyS target . AltogetS target . A similS target . In addiS target . FurtherOne of the open questions is how differential RTS binding of the two isoforms is achieved. Recently, hnRNPA2 has been shown to be involved in alternative splicing of miRNAs by recognizing methylation on adenosine residues . Since bXenopus laevis, depletion of CBF-A/hnRNPab orthologs led to a decrease in eye size due to a general increase in apoptosis, as well as a decrease in proliferative neural tissues, with cranial neurons not being properly formed, motor neurons missing and defects in migration. Depleted neurons also show a thinner and disorganized tubulin network (−/− knock-out mice have reduced expression in the stem cell marker Nestin and an increase in the differentiated marker dcx. This suggests that CBF-A/hnRNPab is involved in the regulation of stem cell maintenance and neuronal precursor differentiation. Furthermore, CBF-A/hnRNPab−/− neurons in vivo have neurites length increased by 40% while their longest neurite is 32% longer than the wild-type condition (−/− is involved in neuronal development is not known. In both neuronal cells and spermatogenic cells CBF-A/hnRNPab was found to interact with the 5′ Cap-binding complex, facilitating translation (The mechanisms of RNA trafficking are important to ensure spatial and temporal regulation of gene expression, which is, in turn, required during development and differentiation. Understanding how the CBF-A/hnRNPab isoforms promote efficient mRNA trafficking might, therefore, provide an interesting paradigm to study cell specification and neuronal development. Indeed, high levels of CBF-A/hnRNPab expression can be found in neuronal cells, in the developing neural tissues and neurogenic regions of the brains  In Xenop network . In miceondition . Finallyondition , 2000. Hnslation . An intein vivo excitotoxicity, a phenomenon that is a direct consequence of over stimulation of glutamatergic neurons that can lead to cell stress and neuronal cell death (∗RBD-Gly family mimic Alzheimer’s disease phenotypes at the cellular level when the proteins are depleted in vitro (∗RBD-Gly family members and the onset of neurodegenerative disorders.The above mechanisms are likely to also occur in the adult brain since CBF-A/hnRNPab is expressed in mature neurons, oligodendrocytes, and astrocytes . Lack ofll death . Interesll death . Consistin vitro . In lighWe suggest that the mechanisms described above based on the ubiquitously expressed CBF-A/hnRNPab isoforms overall contribute to the general translatability of mRNA transcripts. This is initially achieved in the cell nucleus; we propose that at this stage nascent transcripts are translationally repressed and consequently sorted for cytoplasmic transport and localization Figure . The conFuture work will need to proceed toward controlled cell differentiation systems in combination with genome-wide analyses to understand how cell development is controlled by CBF-A/hnRNPab. As more molecular mechanisms are being revealed that regulate mRNA biogenesis from the gene to polysomes, systems biology will provide a powerful approach to understand the importance of specific hnRNP proteins in tissue development and differentiation of complex multicellular organisms and how these mechanisms are potentially impaired in human diseases.All authors wrote the paper, read, and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The majority of breast cancer patients die of metastasis rather than primary tumors, whereas the molecular mechanisms orchestrating cancer metastasis remains poorly understood. Long noncoding RNAs (lncRNA) have been shown to regulate cancer occurrence and progression. However, the lncRNAs that drive metastasis in cancer patients and their underlying mechanisms are still largely unknown.lncRNAs highly expressed in metastatic lymph nodes were identified by microarray. Survival analysis were made by Kaplan-Meier method. Cell proliferation, migration, and invasion assay was performed to confirm the phenotype of LINC02273. Tail vein model and mammary fat pad model were used for in vivo study. RNA pull-down and RIP assay were used to confirm the interaction of hnRNPL and LINC02273. Chromatin isolation by RNA purification followed by sequencing (ChIRP-seq), RNA-seq, ChIP-seq, and luciferase reporter assay reveal hnRNPL-LINC02273 regulates AGR2. Antisense oligonucleotides were used for in vivo treatment.We identified a novel long noncoding RNA LINC02273, whose expression was significantly elevated in metastatic lesions compared to the primary tumors, by genetic screen of matched tumor samples. Increased LINC02273 promoted breast cancer metastasis in vitro and in vivo. We further showed that LINC02273 was stabilized by hnRNPL, a protein increased in metastatic lesions, in breast cancer cells. Mechanistically, hnRNPL-LINC02273 formed a complex which activated AGR2 transcription and promoted cancer metastasis. The recruitment of hnRNPL-LINC02273 complex to AGR2 promoter region epigenetically upregulated AGR2 by augmenting local H3K4me3 and H3K27ac levels. Combination of AGR2 and LINC02273 was an independent prognostic factor for predicting breast cancer patient survival. Moreover, our data revealed that LINC02273-targeting antisense oligonucleotides (ASO) substantially inhibited breast cancer metastasis in vivo.Our findings uncover a key role of LINC02273-hnRNPL-AGR2 axis in breast cancer metastasis and provide potential novel therapeutic targets for metastatic breast cancer intervention. Although the overall survival and prognosis of breast cancer patients have been improved in recent years , metastaIn human genome, there is only up to 2% of protein-coding genes that are stably transcribed, whereas the vast majority are non-coding RNAs (ncRNAs) . EmerginHere, we identified that lncRNA LINC02273 was significantly upregulated in metastatic LNs compared with primary tumors and could predict poor recurrence free survival in breast cancer patients. Knockout of LINC02273 could curb breast cancer metastasis in vitro and in vivo. Mechanistically, RNA-binding protein hnRNPL, which was also increased in metastatic tissues, bound with CA-repeats in the 3′ region of LINC02273 through its RRM1&2 motif and increased the stability of LINC02273. We further revealed that the hnRNPL-LINC02273 RNP complex could activate oncogene AGR2 transcription through enhancing H3K4me3 and H3K27ac levels around its promoter region. We also verified the potential clinical use of ASOs targeting LINC02273 to inhibit breast cancer metastasis. Altogether, our results demonstrated that LINC02273 played a pivotal role in enhancing breast cancer metastatic potential and could serve as a novel therapeutic target for mitigating breast cancer metastasis.Metastatic lymph node samples and paired primary tumors were collected at Fudan University Shanghai Cancer Center. All lymph node metastatic loci and primary tumors were confirmed by H&E staining. Lymph node metastatic loci and primary tumor sites were dissected and subjected to RNA extraction. A series of fresh frozen tissue and clinical data from breast cancer patients was collected at Fudan University Shanghai Cancer Center. Tumor tissue was obtained from patients undergoing surgery and immediately stored at − 80 °C. All the protocols were reviewed and approved by an independent institutional review board at Fudan University, and all patients gave their written informed consent before inclusion in this study. Post-operative chemotherapy and radiation therapy were suggested according to breast cancer guidelines of Fudan University Shanghai Cancer Center.bc-GenExMiner 3.0 database was used to explore the predictive role of LINC02273 for metastatic events in breast cancer . Five GE6 cells were seeded in 6-well plate. After 24 h, GapmeR ASOs were added directly to cell cultures. RNA was extracted 48 h after transfection. For actinomycin D RNA stability assay, 1 × 106 cells were seeded in 6-well plate. After 24 h, 5 μg/ml actinomycin D (Apexbio) were added. Cells were treated at the time point as indicated. Sequences of siRNAs and ASOs were listed in Additional file The HEK293T, MDA-MB-231 and BT549 cell lines were obtained from American Type Culture Collection (ATCC) . LM2 was a subline of MDA-MB-231 with high lung metastasis tendency was described previously . All celTotal RNAs from cultured cells were extracted with TRIzol (Life technologies) according to the manufacturer’s protocol. cDNAs were reverse transcribed with Hiscript III Reverse Transcriptase (Vazyme) with oligo (dT) and random hexamers followed by qRT-PCR analysis and applied for PCR/qPCR analysis. Real time quantitative PCR was performed with ChamQ SYBR qPCR Master Mix (Vazyme) and 7900HT Fast Real-Time PCR System or (Applied Biosystems). The relative expression of different sets of genes was quantified to GAPDH mRNA. Primer sequences for RT-qPCR and RT-PCR used were listed in Additional file 6) were counted and plated in 6-well plate. After 24 to 48 h, cells were rinsed with PBS. Then, 80 μl 1 × SDS Protein Loading Buffer containing protease and phosphatase inhibitors (Bimake) were added to each well of the plate. Cell lysates were scraped down and sonicated with Bioruptor Plus for 3 × 30s with low power. After denaturation at 100 °C for 10 min, the samples were resolved by SDS-PAGE, transferred to PVDF membranes (Millipore). The membranes were blocked with 5% (w/v) skimmed milk in TBS-T for 1 h, then probing with antibodies at 4 °C overnight. Antibodies used in immunoblot includes hnRNPL ; AGR2 ; GAPDH (HRP-60004). HRP-conjugated secondary antibodies were used for detection.Cells . The lower chambers contained DMEM with 20% FBS. After incubation for 8 h, the inserts were fixed and stained with crystal violet. Cells in the upper chamber were removed with cotton swabs. The average confluence of migrated cells was analyzed by ImageJ according to three random fields captured by 100× microscope. Each experiment was conducted in triplicate. Matrigel invasion assays were performed using Matrigel-coated Transwell inserts with the procedure as described above.For proliferation assay, cells were seeded in 96-well flat-bottomed plates with each well containing 3000 cells in 200 μl of culture medium and cultured in ambient environment described above. Plates were imaged by using IncuCyte ZOOM System (Essen Bioscience) at 12-h interval. The growth rate was measured according to confluence change analyzed by IncuCyte software. For wound healing assay, 30,000 cells were seeded in 96-well IncuCyte® ImageLock Plates (Essen Bioscience). After 24 h, a wound was made in each well with WoundMaker™. Plates were imaged as described above with 6-h interval. Wound width was measured and analyzed. For transwell migration assay, a total of 1 × 10The 3′ and 5′ RACE (Rapid amplification of cloned cDNA ends) was performed using the SMARTer RACE kit (Clontech) following the manufacturer’s instruction. RNA was extracted from MDA-MB-231 cells. Primers used for 3′ and 5′ RACE were listed in Additional file LINC02273 1653 nt full length cDNA were synthesized (Genewiz) according to 5′ and 3′ RACE result and cloned into pCDH-CMV-Puro and pcDNA3.1-myc vector. The ORF of hnRNPL, AGR2, ETS1, and STAT3 were cloned from MDA-MB-231 cDNA and inserted into pCDH-CMV-Puro with C-terminus 3 × FLAG tag. Serial mutations of hnRNPL were subcloned and constructed with C-terminus 3 × FLAG tag. The shRNA oligos were annealed and cloned into pLKO.1-Puro vector. Lentivirus was produced by co-transfecting HEK293T cells with desired plasmids together with psPAX2 and pMD2.G. After 72 h, virus was harvested by passing through a 0.45 mm filter. Collected lentivirus was used directly to infect cells with the addition of 8 mg/ml polybrene (Sigma-Aldrich) or stored in − 80 °C. Infected cells were selected with puromycin (Invivogen) at 1 μg/ml.The gRNA oligos were annealed and cloned into the lentiGuide-Puro . To delete LINC02273, MDA-MB-231 and BT549 cells were transiently transfected with lentiGuide-Puro plasmid carrying two gRNAs and pLentiCas9-Blast  and selected with 1 μg/ml puromycin and 2 μg/ml blasticidin for 5 days. Cells were then seeded in 96 well plates in low density (1 cell per well) to form single colonies. Cell clones were genotyped by PCR and sequencing to detect the deletions. PCR primers were listed in Additional file LINC02273 RNA FISH was performed using Stellaris FISH probes as listed in Additional file 7 cells were collected and used for nuclear and cytoplasmic protein extraction using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Life Technologies). For nuclear and cytoplasmic RNA separation, 1 × 106 cells were collected and extracted using PARIS™ kit (Life Technologies).1 × 107 cells were harvested, resuspended in 1 mL lysis buffer followed by 3 × 10 s sonication with high power with an interval of 30 s. After centrifuging at 12,000 rpm for 15 min at 4 °C, the supernatant was pre-cleared with 30 μl Dynabeads Protein G (Invitrogen). The precleared lysates were incubated with 4 μg anti-hnRNPL antibody (Santa Cruz Biotechnology) for 3 h at 4 °C. Then 40 μl Dynabeads Protein G beads (blocked with 1% BSA and 20 mg/ml yeast tRNA for 1 h at 4 °C) were added to the mixture and incubated for another 1 h at 4 °C followed by washing with wash buffer. The RNA-protein complex was eluted with 200 μl TRIzol at room temperature for 10 min. RNA was purified with Direct-zol RNA Microprep (Zymo Research). For qRT-PCR, reverse transcription was performed with Hiscript III Reverse Transcriptase (Vazyme) with oligo (dT) and random hexamers followed by qRT-PCR analysis. Primers were listed in Additional file 1 × 10Each probe was designed using LGC Biosearch’s web-based designer, biotin-TEG-labeled at its 3′ terminus and divided into odd or even groups. Probes targeted LacZ were used as negative control. For each sample, 4 × 107 MDA-MB-231 cells were used and crosslinked in 1% glutaraldehyde at room temperature for 10 min, and quenched with 125 mM glycine for 5 min. The cells were lysed by Bioruptor plus in ChIRP lysis buffer and sheared to 100–500 bp fragments at 4 °C. Cleared lysates were hybridized with probes in 37 °C in a hybridization oven for 4 h. After washes and elution, RNA and DNA were purified accordingly. qPCR and RT-qPCR were performed as described above. DNA for library construction were performed as described in the library preparation section. The probes used in the ChIRP assay are listed in Additional file PCR products were verified by gel electrophoresis and purified by phenol: chloroform extraction. Biotin-labelled RNAs was in vitro transcribed with HiScribe™ T7 Quick High Yield RNA Synthesis Kit (NEB) with Biotin-16-UTP (Roche), treated with RNase-free DNase I on column during RNA purification with RNA Clean & Concentrator-25 (Zymo Research). For each sample, 5 μg RNA was mixed with 1 × 107 cell extract and incubated at 4 °C for 1 h, followed by incubating with Dynabeads M-280 Streptavidin (Invitrogen) at 4 °C overnight. After washes, the pull-down complexes were eluted by denaturation in 1× protein loading buffer for 10 min at 100 °C. The samples were detected by Western blot or proceed to mass spectrometry.After RNA pull-down, equal amounts of samples pulled down by sense and anti-sense LINC02273 were loaded on SDS-PAGE gel. Then the gel was stained with Fast Silver Stain Kit (Beyotime) according to the manufacturer’s instructions. Specific bands were cut and analyzed by LC-MS/MS . Protein identification was retrieved in the human RefSeq protein database , using Mascot version 2.4.01 .4 cells per well in 2.5 ml of completed MammoCult™ Medium (Stemcell) in 6-well ultralow attachment plates (Corning). After 5–10 days, mammospheres larger than 100 μm were imaged and counted under microscope.Cells were trypsinized, and 3 × 106 cells per assay) were cross-linked with 1% formaldehyde at RT for 10 min and quenched in 125 mM glycine for 5 min. The cross-linked chromatin was sonicated to generate DNA fragments averaging 200–500 bp in length by Bioruptor plus. Chromatin fragments were immunoprecipitated with antibodies against mouse or rabbit normal IgG , hnRNPL , H3K27ac (1:200 CST), H3K4me3 (1:200 CST). The precipitated DNA were purified using the ChIP DNA Clean & Concentrator Kits (Zymo Research).MDA-MB-231 cell lines . For LINC02273 stability assay, 500 ng reporter vector and 20 pmol siRNA were co-transfected with Lipofectamine 3000 (Invitrogen). After 48 h, cells were lysed in 100 μl of passive lysis buffer. The firefly luciferase activity and the Renilla activity were determined by Dual-Luciferase® Reporter Assay System. For each experiment, the firefly luciferase activity was normalized to Renilla activity. The results were expressed as fold induction compared to negative control indicated.LINC02273 promoter , AGR2 promoter region, and serial truncations were cloned into pGL3-basic vector. Full length LINC02273 and LINC02273 without CA-repeat were cloned into pMIRGLO vector at the 3′ UTR of luciferase ORF. 1 × 10Five pairs of primary tumor and LN metastatic loci were dissected and verified by H&E staining, tumor percentage > 70%. Followed by RNA extraction with TRIzol reagent, samples were analyzed by Shanghai Qiming Biotechnology Co. Ltd. with Affymetrix HTA 2.0.The total RNA samples (1 μg) of MDA-MB-231 WT and LINC02273 KO cells were treated with VAHTS mRNA Capture Beads (Vazyme) to enrich polyA+ RNA before constructing the RNA-seq libraries. RNA-seq libraries were prepared using VAHTS mRNA-seq v2 Library Prep Kit for Illumina (Vazyme) following the manufacturer’s instructions. Briefly, polyA+ RNA samples were fragmented and then used for first- and second-strand cDNA synthesis with random hexamer primers. The cDNA fragments were treated with DNA End Repair Kit to repair the ends, then modified with Klenow to add an A at the 3′ end of the DNA fragments, and finally ligated to adapters. Purified dsDNA was subjected to 12 cycles of PCR amplification, and the libraries were sequenced by Illumina sequencing platform on a 150 bp paired-end run. Sequencing reads from RNA-seq data were aligned using the spliced read aligner HISAT2, which was supplied with the Ensembl human genome assembly (Genome Reference Consortium GRCh38) as the reference genome. Gene expression levels were calculated by the FPKM (fragments per kilobase of transcript per million mapped reads). Gene Set Enrichment Analysis (GSEA) pre-ranked was run on the ranked list using the Molecular Signatures Database (MSigDB) as the gene sets.Total RNA was isolated with TRIzol reagent. Next, strand-specific RNA-seq libraries were prepared using the NEBNext Ultra Directional RNA Library Prep kit , according to the manufacturer’s instructions. For the data processing, the raw sequencing reads were aligned to human reference genome (hg38) using the splice-aware aligner HISAT246. Read counts for each gene were normalized into FPKM (Fragments per Kilobase of transcript per Million mapped reads) values. The cutoff of differential gene expression was FDR < 0.05, normalized by the wild type cells. The FPKM fold change of the overlap genes of the two subgroups independent KO cells were used for further analysis.ChIP and ChIRP DNA libraries were prepared using the NEBNext DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer’s instructions. The raw reads were aligned by Bowtie2 (v2.2.9) with a human reference genome (hg38). The uniquely mapped reads were subjected to the peak calling algorithm, MACS (v1.4.2) with default parameters.Four-week-old female NOD/SCID mice or female athymic BALB/c nude mice were purchased from Shanghai Slack Laboratory animal Co., LTD and housed under SPF conditions at the animal care facility of the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine.6 LM2 cells were orthotopically injected directly into the inguinal mammary fat pads of mice in 50 μl of sterile PBS (n = 8 in each group).For xenograft models, 1 × 106 LM2 cells were orthotopically injected directly into the mammary fat pads of mice in 100 μl of sterile PBS. Two weeks after injection, mice were randomized into three groups (n = 8 in each group). ASOs were delivered according to groups, through tail vein injection, 10 nmol  each mouse, and twice a week. Tumor were measured by caliper twice a week. The xenografts were fixed in polyformaldehyde for paraffin embedding or frozen for RNA. For tail vein injection model, 1 × 105 LM2 cells were diluted in 100 μl of sterile PBS and injected through tail vein.For ASO in vivo treatment experiment, 1 × 10To investigate tumor metastasis, 8 weeks after injection, each mouse was intraperitoneally injected 200 mg/kg D-Luciferin. Bioluminescence were analyzed by IVIS system. All experiments were performed in accordance with relevant institutional and national guidelines and regulations of Shanghai Medical Experimental Animal Care Commission.P values. For bioluminescence, Mann-Whitney tests were used to calculate the P values. Spearman correlation analysis was performed to assess the relationship between different factors. Survival curves were plotted using the Kaplan–Meier method and compared using log-rank tests. All statistical analyses were performed using two-tailed P values. The experiments, with the representative data shown in the figures, were repeated three times independently with similar results obtained. Statistical details and methods used are indicated in the figure legends, text or methods.Statistical analyses were performed using SPSS v.23.0 (SPSS) or Prism GraphPad 8.0. For most of the experiments, independent sample t-tests were used to calculate the Gene microarray were used to define the transcriptome of metastatic LNs and their matched primary tumors from breast cancer patients . Compared to the primary tumors, LINC02273 was significantly increased in the metastatic loci within LNs  revealed that LINC02273 expression was significantly higher in tumors with positivie lymph nodes (P = 0.048). In addition, patients with pN3 lymph nodes metastasis have higher LINC02273 in primary tumors compared with pN1 (P = 0.026) or pN0 patients (P = 0.026)  in a breast cancer cohort with lymph node metastasis  are prognostic predictor for poor RFS  correlated with poor metastasis/recurrence free (MR-free) survival  or two clones of LM2 LINC02273-KO cells (KO-1 and KO-2) were used. We found that the growth rate of xenograft tumors was inhibited substantially, and the tumor volumes were significantly reduced when LINC02273 was depleted , indicating hnRNPL stabilizes LINC02273   drugs has gained increasing attention owing to their ability targeting diverse RNAs, which has been validated both in vitro and in vivo , 37. To Through profiling transcriptome of metastatic LNs and matched primary cancer tissues, we identified LINC02273 as a lncRNA significantly enriched in LN metastatic lesions, compared to the primary tumors. We further showed that downregulation of LINC02273 impaired the migration and invasion ability of breast cancer cells while its overexpression significantly promoted breast cancer cell mobility. Moreover, LINC02273 knock-down remarkably reduced lung metastasis in breast cancer xenograft models. In breast cancer patients, increased LINC02273 was associated with high burden of LN metastasis. Patients with high LINC02273 level had shorter RFS and worse clinical outcome than those with low LINC02273 expression. Therefore, LINC02273 could serve as an independent prognostic factor in breast cancer.Recent studies highlighted the critical roles that RNA binding proteins (RBP) may play in modulating cancer initiation and progression through binding to RNAs and regulating their processing, stability, localization, modification or translation , 10. Ourin cis or genes on different chromosomes in trans. Transcriptional regulation by lncRNAs may depend on their roles in modulating signaling transduction, regulating transcription factor recruitment and histone modification, as well as serving as a scaffold for assembly of multiple regulatory molecules at single locus. Since LINC02273 mainly localized in the nucleus, RNA-seq and ChIRP-seq were carried out to identify genes regulated by LINC02273 and respective DNA loci that LINC02273 bound. A number of LINC02273-regulated gene, such as CHRNA7, ERP27, GABRG1, FOXN4, ZNF831, AGR2, EBF1 and GUCY2C, have been reported to be involved in cancer progression and metastasis. CHRNA7 promoted pancreatic and lung cancer metastasis [The molecular mechanisms by which lncRNAs exert their biological functions are diverse and complex. Accumulating evidence suggests that lncRNAs can affect neighboring intra-chromosomal genes tastasis , 39, whitastasis . As a metastasis . Overexptastasis . In breatastasis . Upregultastasis . Furthertastasis . Althougtastasis , 23, 31,In addition, several studies have reported the role of hnRNPL in regulating gene transcription. For instance, hnRNPL plays an important role in gene regulation in epithelial cells . Recent Consistent with our in vitro findings, a positive correlation between LINC02273 and AGR2 was confirmed in both primary tumors and metastatic LNs from breast cancer patients. In addition, shorter RFS was significantly associated with high expression of both AGR2 and LINC02273, suggesting combined expression levels of AGR2 and LINC02273 may serve as promising biomarkers to determine prognosis in breast cancer.. Our data indicated that ASO could effectively suppress LINC02273 in cells and xenograft tumors. Moreover, LINC02273-targeting ASOs substantially reduced primary tumor growth and lung metastasis in our xenograft model. The observation strongly supports that suppressing LINC02273 with specific ASOs may serve as an effective therapeutic approach to mitigate LINC02273-promoted breast cancer progression.Overall, hnRNPL mRNA expression was relative stable in breast cancer samples . Indeed,Our study revealed that LINC02273, as a novel metastasis-associated onco-lncRNA, promoted tumor invasion and correlated with poor prognosis in breast cancer. Mechanistically, LINC02273 was stabilized by binding of hnRNPL to the CA-repeats within its S3–2 region. The hnRNPL-LINC02273 RNP complex could be recruited to the promoter region of AGR2 where it enhanced AGR2 transcription by increasing local H3K4me3 and H3K27ac modification (Fig. Additional file 1. Figure S1. LINC02273 correlates with poor prognosis in breast cancer. Figure S2. qPCR analysis of LINC02273 expression and genes closed to LINC02273 gene locus in LINC02273 KO cells. Figure S3. LINC02273 promotes breast cancer proliferation and metastasis but not cancer stemness. Figure S4. LINC02273 interacts with hnRNPL. Figure S5. LINC02273 transcriptionally activates AGR2 expression which was revealed by ChIRP. Figure S6. hnRNPL-LINC02273 complex promotes cancer metastasis through upregulating AGR2. Figure S7. Epigenetic regulation of modification at AGR2 promoter by hnRNPL was dependent on LINC02273. Figure S8. Targeting LINC02273 with ASO showed potential in mitigating breast cancer metastasis. Table S1. Patient characteristics of RNA microarray. Table S2. CPAT prediction of LINC02273. Table S3. CPC prediction of LINC02273. Table S4. Baseline clinicopathological characteristics of patients according to LINC02273 expression. Table S5. Univariant COX regression analyses of RFS in BC patients. Table S6. Multivariant COX regression analyses of RFS in BC patients. Table S7. LINC02273 knockout neckties. Table S8. MS result of LINC02273 RNA pull-down. Table S9. Baseline clinicopathological characteristics of patients according to AGR2 expression. Table S10. Multivariant COX regression analyses of RFS in BC patients. Table S11. Probes used in this study. Table S12. Primers used in this study. Table S13. shRNA, siRNA, CRISPR, and ASOs.


Decision making often requires weighing costs and benefits of different options that vary in terms of reward magnitude and uncertainty. Previous studies using pharmacological inactivations have shown that the basolateral amygdala (BLA) to nucleus accumbens (NAc) pathway promotes choice towards larger/riskier rewards. Neural activity in BLA and NAc shows distinct, phasic changes in firing prior to choice and following action outcomes, yet, how these temporally-discrete patterns of activity within BLA→NAc circuitry influence choice is unclear. We assessed how optogenetic silencing of BLA terminals in the NAc altered action selection during probabilistic decision making. Rats received intra-BLA infusions of viruses encoding the inhibitory opsin eArchT and were well trained on a probabilistic discounting task, where they chose between smaller/certain rewards and larger rewards delivered in a probabilistic manner, with the odds of obtaining the larger reward changing over a session (50–12.5%). During testing, activity of BLA→NAc inputs were suppressed with 4- to 7-s pulses of light delivered via optic fibers into the NAc during discrete task events: prior to choice or after choice outcomes. Inhibition prior to choice reduced selection of the preferred option, suggesting that during deliberation, BLA→NAc activity biases choice towards preferred rewards. Inhibition during reward omissions increased risky choice during the low-probability block, indicating that activity after non-rewarded actions serves to modify subsequent choice. In contrast, silencing during rewarded outcomes did not reliably affect choice. These data demonstrate how patterns of activity in BLA→NAc circuitry convey different types of information that guide action selection in situations involving reward uncertainty. Amygdala projections to the nucleus accumbens (NAc) form a neural circuit that has been implicated in guiding risk-related decision making, and dysfunction in this circuitry is thought to underlie aberrant decision making observed in a variety of psychiatric illnesses. The present study used temporally-discrete optogenetic inhibition to identify how activity in amygdala inputs to the accumbens, during different phases of the decision process, promotes optimal decision making. These findings provide novel insight into the mechanisms through which these circuits contribute to normal and abnormal decisions involving reward uncertainty.Optimal reward-seeking often requires evaluations of the relative costs and benefits associated with different actions. Among the various costs that may be associated with different rewards , decisioStudies in humans and animals have implicated various nodes within cortical, limbic and striatal circuitry in mediating different aspects of risk/reward decision making . Among tRisk/reward decision making is a dynamic process that may be partitioned into different phases, including deliberation before action selection and subsequent evaluation of action outcomes that may influence subsequent choice biases. Neurophysiological studies suggest that these different phases of the decision process are likely mediated by temporally discrete patterns of activity within different nodes of cortico-limbic-striatal circuitry. For example, in vivo electrophysiological recordings.The present study sought to address this issue by using temporally-specific optogenetic inhibition of BLA projection terminals in the NAc, to ascertain how suppressing activity in this pathway during different task events may alter risk/reward decision making. Rats received intra-BLA infusions of an adeno-associated virus (AAV) encoding for the inhibitory opsin eArchT and were well trained on a probabilistic discounting task where they chose between small/certain and large/risky rewards. During testing, light was delivered into the NAc to suppress input from the BLA to this nucleus during distinct phases of the decision process. In a separate experiment, we confirmed the effectiveness of this procedure in suppressing BLA-evoked firing of NAc neurons, using Male, Long Evans rats (Charles River Laboratories) weighing ∼275–300 g on arrival were group housed and provided food ad libitum for one week. Following daily handling and acclimatization to the colony, rats underwent stereotaxic viral infusion surgery into the BLA and were subsequently single housed for the remainder of the experiment. Rats were food restricted to ∼85% of their free-feeding weight before beginning operant behavioral training. Their weights were monitored daily and food was adjusted to maintain a weight gain of ∼5 g per week. All animal procedures were performed in accordance with the University of British Columbia animal care committee’s regulations.Forty-eight hours before surgery, rats were given food ad libitum. Rats were given a subanesthetic dose of ketamine (50 mg/kg) and xylazine (5 mg/kg) intraperitoneally and maintained on isoflurane (5%) for the procedure. They were placed into a stereotaxic frame and secured with earbars (flat skull) and analgesia was administered subcutaneously . Over the course of this experiment, rats received two surgeries, the first involving infusions of virus into the BLA before behavioral training, and the second entailing implantation of optical fibers into the NAc after initial training. During these surgeries, burr holes were drilled into the skull above the BLA or NAc. Over the course of these experiments, animals were subjected to two surgical procedures at different time points.Before any behavioral training, separate groups of rats received 1.0-µl infusions of a solution containing a virus encoding the inhibitory opsin rAAV5-CaMKIIα-eArchT3.0-eYFP (to selectively promote protein expression in glutamatergic neurons) or rAAV5-CaMKIIα-eYFP as a control vector (University of North Carolina Vector Core). Infusions were made bilaterally into the BLA via microinfusion pumps  at a flow rate of 0.1 µl/min. Injectors were left in place for an additional 10 min following infusion to allow for virus diffusion in tissue. Incisions were sutured, and rats were given approximately two weeks to recover and then commenced the initial behavioral training, which lasted approximately five weeks.After completion of their initial behavioral training, rats received their second surgery. Here, optic fibers consisting of 400-µm cores (Thorlabs) threaded through 2.5 mm-wide metal ferrules (Precision Fiber Products) were implanted over the NAc at a 12° angle . Ferrules were secured to the skull using four screws and dental cement to secure the assembly, leaving about half of the ferrules uncovered. Rats were permitted to recover from this surgery for one week before retraining and optogenetic testing.Behavioral testing was conducted in operant conditioning chambers  enclosed in sound-attenuating boxes. Each box was equipped with a fan with the purpose of providing ventilation and limiting extraneous noise. The chamber was fitted with a central food receptacle where sucrose food reward pellets  were dispensed. Two retractable levers were located on either side of the food receptacle. The chamber was illuminated by a 100-mA house light located on the top center of the box opposite the food receptacle. Four infrared photobeams located just above the grid floors monitored locomotor activity (assessed by number of beam breaks). All data were recorded on a personal computer connected to the operant conditioning chambers. Lasers were controlled by Med PC hardware and software, which delivered a TTL+ pulse to lasers to initiate light delivery and a TTL– pulse to terminate it.After ∼5 d of food restriction, rats were given ∼30 pellets in their cage on the day before beginning operant training. On the first day of training, two pellets were placed in the food receptacle, the right or left lever was extended, and crushed reward pellets sprinkled on the extended lever. Animals were trained to lever press for pellets under a fixed ratio-1 schedule until a criterion of 60 presses in 30 min for one lever, and on the next day, the other lever. Rats were subsequently trained on a simplified version of the full task, consisting of 90 trials. On each trial, rats were presented with one of the levers that, if pressed within 10 s, would deliver one pellet with a 50% probability. If the lever was not pressed within this time, it was retracted and the trial was scored as an omission. Trials occurred every 40 s. Rats trained until a criterion of <10 omissions for a minimum of two consecutive days (∼4 d of training). During the next phase of training, rats learned to choose between one lever associated with a larger, four-pellet reward (delivered with a 50% probability) and another lever that always delivered a one-pellet reward. Assignment of the large-reward lever was counterbalanced across animals. Sessions consisted of 72 trials portioned into four blocks of 18 trials. The first eight trials of each block were forced choice, where only one lever was inserted (randomized in pairs), and the remaining 10 trials were free choice, where both levers were inserted. Rats were trained until they chose the large reward lever on >60% of the free choices (∼3 d).The task was similar to that described by p > 0.10). Once stable patterns of choice were displayed, rats were given food ad libitum for 3 d before undergoing fiber optic implantation, as described in the preceding section. Following recovery from surgery, rats were retrained on the task. During retraining, ferrules were connected to dummy fiber optic patch cables, encased in stainless-steel spring coils and tethered to a rotary joint that permitted free movement through the chamber. This procedure habituated animals to the procedures they would experience during testing. Once stable behavior was re-established (∼15 d), optogenetic test sessions commenced.Rats were trained until the group demonstrated stable choice behavior (∼30 d), evaluated by analyzing data from three consecutive days using a two-way repeated measures ANOVA, with day and trial block as the two factors. Behavior was deemed stable when there was no main effect of day and no day × block interaction  even after many weeks of training . Thus, aTesting commenced approximately two months post viral infusion. This time frame permitted the opsins that were initially expressed in the BLA to be trafficked down axons and expressed in terminals in the NAc where they could be activated by laser light A,B. BefoFs < 3.50, n.s.). Animals completed all tests for a particular manipulation before moving on to the next type of manipulation. The order with which the different types of tests were administered was counterbalanced across rats. Some experiments had considerable attrition rates as a result of damage to headcaps. Ultimately, some rats did not receive all silencing tests, resulting in a differing number of subjects in each analysis. Two rats displayed abnormal discounting behavior  and their data were not included in the final analyses. Rats infused with the control virus not containing the inhibitory opsin eArchT were only tested on manipulations that induced significant changes in choice in the eArchT virus group.Each optogenetic test consisted of a 3-d sequence similar to that used in other studies using temporally-discrete manipulations of neural activity during different phases of decision making . The firIn one experiment, BLA inputs to the NAc were suppressed before animals made a choice. Laser light was initiated 4 s before lever extension  and terminated either when a choice was made or 10 s elapsed after lever extension (omission). Under these conditions, light was delivered for 4–10 s each trial depending on response latency (on average ∼5 s). Previous studies have shown that when rats choose between risky and certain rewards, NAc neural firing begins to increase during a deliberation period a few seconds prior to initiation of a choice . As suchIn separate series of experiments, we inhibited BLA inputs to the NAc after different outcomes of choices. In all of these experiments, laser light was delivered during the outcome of interest on both forced and free-choice trials, since regardless of trial type, information about probability gathered from these outcomes influenced subsequent choice biases.In another experiment, we inhibited BLA inputs to the NAc only on trials where animals chose the risky option and did not receive a reward (“risky loss”). Here, lasers were illuminated on all free- and forced-choice trials when a rat selected the large/risky lever and did not receive the larger reward. Laser light was initiated immediately after these choices and was terminated 7 s post-lever press.Another experiment inhibited activity in the BLA→NAc pathway after rewarded risky choices (“risky wins”). On these test days, light was delivered on all free- and forced-choice trials after a rat selected the large/risky lever and received the larger reward. Lasers were left on for the 7 s post lever press, which would have overlapped with pellet delivery and consumption.This experiment inhibited activity in BLA→NAc pathway after a small/certain choice . During these tests, lasers were illuminated on for all free- and forced-choice trials immediately after a rat selected the small/certain lever. Lasers were left on for the 7 s post lever press, which included the time it took for pellet delivery and consumption.To ascertain that the effects of outcome-associated silencing of the BLA→NAc pathway was attributable to inhibiting neural activity that was temporally linked to these events, a control experiment tested the effects of inhibiting this pathway for 7 s during the 40-s ITI. For all free- and forced- choice trials, light pulses were delivered during the ITI, on a random interval starting 6–14 s after the start of the ITI .A separate cohort of rats underwent viral infusion surgery and were allowed to recover for approximately two months before being used for electrophysiological experiments. Some rats received infusions of the virus encoding for eArchT3.0-eYFP, and others with a control virus that only encoded for eYFP. All rats were given food ad libitum post-viral infusion before physiologic recordings. Rats were anesthetized with urethane (1.5 mg/kg) and inserted into a stereotaxic frame. The rat’s scalp was incised, and burr holes were drilled in the skull at coordinate above the BLA and the NAc; stimulating and recording optical microelectrodes (optrodes) were lowered at the coordinates described in the subsequent section.The electrophysiological cell-searching and recording procedures were adapted from A stimulating electrode connected to an Iso-Flex optically-isolated stimulator (AMPI) that received programmed pulses from a Master-8 pulse generator (AMPI) was lowered into the BLA . Afterward, a cell-searching procedure began, wherein the optrode was lowered into the NAc with a hydraulic microdrive , while single-pulse electrical stimulation was delivered to the BLA every 8 s. Searching continued until a NAc neuron that exhibited a reliable, monosynaptic action potential in response to BLA stimulation was isolated. Stimulation currents were then titrated to evoke a baseline firing probability of ∼50% . Once a stable baseline was established (40 sweeps over ∼5 min), a second set of stimulations were administered. Here, the laser was turned on for 4 s (with 5 or 10 mW power), starting 2 s before electrical stimulation of the BLA (40 sweeps). This was to confirm whether local light application could suppress BLA-evoked firing of NAc neurons. Immediately after test sweeps, another set of 40 sweeps were tested without laser delivery, to assess recovery of evoked neural firing. We typically obtained between one to three cells per rat tested. At the end of data collection, rats were killed via transcardial perfusion and brains were obtained for histologic analysis.Rats were killed via transcardial perfusion with 4% paraformaldehyde. Brains were fixed in 4% paraformaldehyde for 24 h and then stored in PBS with sodium azide. Each brain was sliced in 50-μm sections using a vibratome (Leica). NAc sections were treated with citric acid at 95°C for 10 min, incubated for 3 d in PBS with 10% Triton X-100, 3% horse serum and chicken anti-GFP . Visualization was performed with anti-chicken 488 secondary antibody (Jackson Laboratory) diluted 1:250 in PBS with 10% Triton X-100, 3% horse serum for 60 min at room temperature. BLA and NAc sections were mounted onto slides, counterstained, and coverslipped using Fluoromount-G with DAPI (eBioscience). Viral expression was verified in the BLA using a 10× objective and terminal expression in the NAc was localized using a 63× objective B. Placemp < 0.001) and will not be mentioned further. Likewise, the main effect of sub-block was also significant (p < 0.05), indicating that within each probability block, rats tended to choose the risky option less as the block progressed. In these analyses, a treatment × probability block interaction indicates that optogenetic inhibition differentially altered choice over the 20 trials within each probability block relative to baseline. Moreover, a treatment × probability block interaction, combined with a lack of a three-way interaction would indicate that the effects of inhibition on choice within a particularly block did not differ significantly over the first versus last 10 trials of that block relative to baseline. For these ANOVAs, sphericity was not a concern, as all factors were comprised of only two levels. Additionally, while repeated measures ANOVAs are robust to violations of the assumption of normality, checks for normality were run and the distributions did not deviate from normal .For each behavioral manipulation, the primary dependent variable was the proportion of choices of the large/risky option, controlling for trial omissions. This was calculated by dividing the number of large/risky choices made in a block by the total number of choices made in that block. Choice data from the two probability blocks were further partitioned into sub-blocks of 10 trials to assess whether inhibition altered discounting within a probability block. These data were analyzed with three-way repeated measures ANOVAs, with treatment (optogenetic inhibition vs baseline), probability block (50% and 12.5%) and sub-block as three within-subjects factors. For these experiments, the main effect of probability block was always significant  and negative feedback sensitivity (lose-shift behavior). The analyses compared each free-choice to the outcome of a previous choice of the risky lever. A win-stay ratio was calculated based on the proportion of times rats chose the risky lever following previous receipt of the large reward (a risky win) over the total number of large rewards obtained, and was used as an index of reward sensitivity. The lose-shift ratio was calculated based on the proportion of times rats chose the safe lever following previous non-rewarded choice (a risky loss) over the total number of non-rewarded choices, and was used as an index of negative feedback sensitivity. Both these values were analyzed together in a two-way ANOVA with treatment and feedback sensitivity (win-stay/lose-shift) as the two within-subject factors. For all multi-factor ANOVAs, when a significant statistical interaction was obtained, simple main effects analyses were conducted using one-way ANOVAs where appropriate. Additionally, the total number of trial omissions, response latencies (the time between lever insertion and lever press), and locomotion were analyzed with one-way repeated measures ANOVAs.Evoked-firing probabilities were calculated by dividing the number of action potentials observed by the number of stimuli administered. Changes in spike probabilities were used as an index of the effect of optogenetic inhibition on the magnitude of change in NAc neuronal activity produced by subsequent BLA stimulation. This was analyzed with one-way ANOVAs with phase (baseline vs laser application) as the within-subjects factor. Only cells that showed evoked responses during the post-laser recovery phase were included in the analysis to ensure that reductions in firing were not attributable to electrode drift.Confirmation of eYFP fluorescent protein in the BLA revealed robust expression in cell bodies encompassing the entire anterior–posterior range B, top. TF = 16.44, p < 0.001; F = 0.27, p = 0.38). The three-way interaction was not significant  = 2.08, p = 0.17). Simple main effects analyses on the treatment × probability block interaction revealed this was driven by a significant (p < 0.05) reduction in risky choice across the 20 trials of the 50% block on test days. In contrast, in the 12.5% block, optogenetic inhibition increased in risky choice compared to baseline.In 16 rats, we tested the effects of optogenetic inhibition of BLA terminals in the NAc before all free-choice trials A. Under F = 8.48, p = 0.01). Simple main effect analyses comparing baseline versus test days further revealed that inhibition of BLA inputs to the NAc before choice did not affect win-stay behavior (p > 0.17), but did significantly enhance lose-shift behavior  = 11.91, p < 0.004) but not in the 12.5% block  = 1.52, p > 0.24). Thus, the reduction in risky choice during the higher probability block appeared to be driven by an increased tendency to shift to the small/certain option after a non-rewarded risky choice.To further dissect whether these changes in risky choice were related to alterations in sensitivity to preceding rewarded or non-rewarded outcomes, we analyzed each choice as a function of the outcome of the previous trial. This analysis revealed a significant treatment × feedback sensitivity (win-stay/lose-shift) interaction  = 4.90, p = 0.04; F = 5.51, p = 0.03; F = 3.82, p = 0.07; With respect to other performance measures, inhibition before choices caused slight, but statistically significant increases in choice latencies  = 2.27, p = 0.16), but did reveal a significant treatment × probability block interaction  = 5.74, p = 0.03; F = 1.69, p > 0.21). This effect was driven by an increase in choice of the large/risky lever during the low (12.5%) probability block compared to baseline  = 11.13, p = 0.005), whereas there was no change in choice behavior during the 50% block (p > 0.50).The BLA→NAc pathway was optogenetically inhibited in 15 rats during a period coinciding with reward omissions following selection of the large/risky lever A. AnalysThe individual choice data from this experiment is displayed in F = 0.38, p > 0.50); treatment × response type interaction  = 0.41, p > 0.50; Fs < 1.20, all ps > 0.30; Choice-by-choice analyses showed that this increase in risky choice was not driven by changes in win-stay or lose-shift behavior [main effect of treatment  = 1.67, p > 0.20; treatment × block interaction: F = 0.25, p > 0.60; three-way interaction: F = 4.65, p = 0.05; F values < 1.78, all ps > 0.20; In 14 rats, the BLA→NAc pathway was inhibited from the time rats chose the large/risky lever and received the large reward A. As oppn = 14) did not affect choice  = 3.50, p = 0.08; treatment × probability block interaction: F = 0.96, p > 0.35; three-way interaction: F = 0.39, p > 0.54; F values < 1.67, all ps > 0.20; Similar to the lack of effect of silencing after risky wins, optogenetic inhibition of BLA→NAc inputs after rats chose the small/certain lever and received the smaller reward  = 0.83, p > 0.38; treatment × block interaction  = 0.11, p > 0.74); three-way interaction; F = 0.49, p > 0.49). Furthermore, there were no significant effects for the other performance measures  = 1.21, p = 0.31); treatment × block interaction  = 0.002, p = 0.96). Similarly, inhibition after risky losses also did not alter choice relative to baseline  = 0.12, p = 0.74); treatment × block interaction  = 1.13, p = 0.33; In a separate cohort of rats infused with a virus that only encoded for eYFP, delivery of laser light did not affect choice when administered before choice (in vivo single-unit recordings of NAc neurons to confirm that 532-nm light application within the NAc could attenuate firing of neurons driven by the BLA (n = 4) obtained from two rats that received intra-BLA infusions of a virus encoding for eArchT, application of 10-mW light pulses for 4 s around the time of BLA stimulation caused a 76 ± 7% reduction in BLA-evoked firing compared to baseline  = 35.00, p = 0.001; n = 6) recorded from four rats infused with eArchT, application of 5-mW light during BLA stimulation caused a 67 ± 14% reduction in evoked firing of NAc neurons relative to baseline  = 9.92, p = 0.03; F = 0.002, p > 0.90; To confirm that our optogenetic manipulations could suppress neural activity of in the BLA→NAc pathway, another experiment used  the BLA A. In oneIt is well established that BLA→NAc circuitry plays an integral role in facilitating a variety of reward-related behaviors. Studies using conventional lesion/inactivation and disconnection procedures have implicated this pathway in mediating approach to reward-associated stimuli , sensitiInhibition of BLA→NAc inputs when rats were deliberating about their choices shifted choice biases away from their preferred option. When reward probabilities were comparatively high (50%) choice was biased toward the risky option during baseline, yet pre-choice inhibition of BLA inputs to the NAc reduced preference of this option. This was associated with enhanced sensitivity to reward omissions , similar to that observed after pre-choice silencing of the BLA in rats choosing between larger/punished rewards and smaller/safe ones . The preThese effects of pre-choice suppression of BLA→NAc activity on risk/reward decision making complements previous neurophysiological studies describing choice-related activity of NAc neurons during risk/reward decision making. 1 receptor (1 receptors reduces choice of larger/risky rewards and increases lose-shift behavior (1 receptors) may work cooperatively to enhance phasic firing of NAc neurons that in turn increases the likelihood that actions are directed toward more preferred options.The ability of BLA→NAc signals to promote selection of reward of greater subjective value may be facilitated by dopaminergic modulation of activity in this pathway. Activation of the BLA can promote mesoaccumbens dopamine release via local, glutamate-receptor dependent mechanisms . In turnreceptor . In thisbehavior , similarIn another experiment, suppressing activity in the BLA→NAc pathway immediately after a non-rewarded action increased risky choice during the low probability block. These findings complement the observation that reward omissions after a risky choice increase phasic firing in a subpopulation of NAc neurons , and sugClassical views of amygdala function posit that this region mediates responding to aversive stimuli such as punishment or reward omissions . The degIn contrast to the above-mentioned findings, inhibition of BLA→NAc activity during receipt of larger or smaller rewards did not alter choice. This lack of effect was interesting, considering both BLA and NAc neurons increase firing to rewards and encode differences in reward magnitude . NotablyIn a separate experiment, we confirmed that light application markedly attenuated BLA-evoked firing of NAc neurons, but only in rats that expressed the inhibitory opsin. Notably, this did not induce a complete “silencing” of evoked firing, which may explain why the magnitude of our behavioral effects were somewhat smaller compared to those induced by conventional inactivations . NeverthCollectively, the present data provide novel insight into the temporal dynamics of activity in BLA→NAc circuitry during deliberative and evaluative components of cost/benefit decision making and how this may guide action selection in situations involving reward uncertainty. These findings complement studies on the neural basis of decision making in humans, implicating the amygdala and NAc in guiding neuroeconomic choices and appraisals of their outcomes . Moreove


Cell nutrition, detoxification, signalling, homeostasis and response to drugs, processes related to cell growth, differentiation and survival are all mediated by plasma membrane (PM) proteins called transporters. Despite their distinct fine structures, mechanism of function, energetic requirements, kinetics and substrate specificities, all transporters are characterized by a main hydrophobic body embedded in the PM as a series of tightly packed, often intertwined, α-helices that traverse the lipid bilayer in a zigzag mode, connected with intracellular or extracellular loops and hydrophilic N- and C-termini. Whereas longstanding genetic, biochemical and biophysical evidence suggests that specific transmembrane segments, and also their connecting loops, are responsible for substrate recognition and transport dynamics, emerging evidence also reveals the functional importance of transporter N- and C-termini, in respect to transport catalysis, substrate specificity, subcellular expression, stability and signalling. This review highlights selected prototypic examples of transporters in which their termini play important roles in their functioning. Their activity is essential for cell survival, division and differentiation and consequently for the life of all organisms. This is reflected in the high number of corresponding genes in all genomes (approx. 5–15%) and in several associated genetic or other diseases caused by transporter malfunction . The great majority of solute transporters are either facilitators, not requiring energy for downhill transport, or secondary active transporters, which need energy coupling provided by the co-transport (symport) or exchange (antiport) of ions or other solutes down their electrochemical gradients. Two other types of transmembrane proteins involved in transport, ATP-dependent primary active transporters and ion channels, are significantly distinct in structure, function and evolution from secondary active transporters and facilitators. This review will discuss aspects concerning the roles of cytosolic tails specifically in secondary active transporters.outward- to an inward-facing topology . I. IA. nidelements  necessaridulans  in the N-terminus and a di-leucine motif (L-L or R-T-P-S-L-L-E-Q) in the C-terminus, which function autonomously as internalization motifs, and so presumably interact with AP-2 at the PM [T-E-L-E-Y-L-G-P (or L-X-X-L-X-P-D-E-X-D), 8 residues downstream from the L-L motif in the C-terminus, is critical for sorting, via the endosome, to the TGN [D-R-S-G-K-D-G-V-M-E-M-N is an example of autonomous apical targeting [One extensively studied case of transporter recycling is the insulin-stimulated glucose transporter GLUT4 in mammalian cells. GLUT4 traffics through several distinct intracellular compartments, including early endosomes, intermediate transport vesicles, recycling endosomes and/or the TGN, and insulin-responsive vesicles (IRVs) . Initially, two distinct motifs have been identified, a phenylalanine-based motif  of the Naproteins –117.(L-K-V), which seems to be important for DAT PM stability, but also for exit from the retromer complex and recycling back to the PM [Reuptake of synaptically released neurotransmitters by NSS transporters is the primary mechanism to control duration and strength of neurotransmission. In most cases, differential internalization rates and post-endocytic sorting of NSS transporters are controlled by different elements in their termini. For example, the norepinephrine (NET) and DAT transporters have distinct trafficking properties, and exchange of domains revealed that this difference was determined by non-conserved structural elements in the N-terminus, while the C-terminus had no effect . In tablo the PM –121. Simo the PM . In conto the PM . Differeo the PM . An apico the PM .5.One of the most interesting novel concepts concerning transporter tails is their role in regulating transport function and substrate selection, apparently via complex allosteric interactions with each other and cytosolic loops of transporters. Emerging evidence supports that the N- and/or C-tails of LeuT-like transporters are crucial in the rocking-bundle mechanism of transport ,126,127.hDAT–Asp436hDAT and interactions with the cytoplasmic end of TMS6, stabilized by a cation-π interaction between Arg60hDAT and Tyr335hDAT, and with Val259 (L4) and Glu428 (TMS8) [Drosophila dDAT (Protein Data Bank (PDB) 4m48) residue Tyr334dDAT (TMS6) forms hydrogen bonds with five other residues, namely Glu427 and Thr431 in TMS8, and Arg27, Glu28 and Thr29 in the N-tail. Additionally, two other salt bridges are formed between the N-tail and L2, namely Glu26–Arg92 and Asp25–Lys93 (b). These interactions reveal the possible role of the N-terminal cytosolic segment as a barrier between the substrate and the intracellular environment [In the outward conformation of LeuT and DAT an important salt bridge is formed between the N-terminal tail and TMS8, which is well conserved in the NSS family . It conc8 (TMS8) ,130. Ind25–Lys93 b. These ironment . In the ironment . Anotherironment –136. Oveironment ,137. Appironment .Figure A crucial role in the mechanism of transport of the C-tail segment was found in members of the APC family, namely the AdiC and GadC amino acid antiporters, which conform to the LeuT fold . AdiC ; however no salt bridge, similar to that shown in NSS members, is formed. Interestingly, another network of hydrogen bonds is also formed between Thr20–Glu233(TMS6)–Lys232(TMS6)–Tyr324 (TMS8), linking the N-terminus, TMS6 and TMS8, as was observed with DAT. All the above interactions are disrupted in the inward-facing conformation following the rearrangement of the hash motif of TMS3, 4, 8 and 9, although the N-terminal fragment is shown to be quite rigid with a relatively small-scale reorientation of the segment between Pro15 and Glu8. This suggests that although important differences are observed between Mhp1 and LeuT in the conformational changes related to the mechanism of substrate translocation it appears that the interruption of the contacts between the N-terminus and L8 in the inward-facing structure is similar in both cases, supporting the concept of the thin inner gate.In the case of the benzyl-hydantoin–sodium symporter Mhp1 three structures, representing the conformational changes from outward to outward-occluded and to inward, have been crystallized. Based on these, Cameron, Henderson and co-workers ,143,144 figure 3c; ,143,144)6.A. nidulans it became apparent that terminal segments host elements necessary not only for endocytic turnover and subcellular sorting, but surprisingly also for substrate specificity [N-X-D-Φ-D-P (residues 24–29). The ones that restricted specificity were deletions either of the most distal part of the N-tail (residues 1–21) or of the C-tail (last 29 residues), as well as Ala substitutions of N-tail residues 15–17.In the course of analysing the roles of the cytosolic C- and N-termini of members of the Fur group of the NCS1 family in cificity ,17. In pThe similarity of phenotypes obtained by mutations in the N- and C-termini (i.e. block in endocytosis and changes in specificity) was suggestive of an involvement in a common mechanism controlling FurE function. Indeed, intramolecular BiFC analysis showed that the FurE cytoplasmic N- and C-termini interact dynamically, and this interaction is transport activity dependent. It was thus proposed that in the absence of substrates FurE is found in a rather stable outward-facing conformation, which brings the cytoplasmic N- and C-termini into close contact, and this permits the reconstitution and detection of a fluorescence signal. In the presence of substrates, the transporter is ‘forced’ to continuously alternate from the outward- to inward-facing conformation, and thus the termini become dynamic and do not reconstitute a stable fluorescence signal . The int7.in vivo and is required for function and specificity [It is now well established that many transporters form dimers or oligomers that are important for effective trafficking to the membrane, function and/or regulation of function ,14,145. cificity ,14,38,40cificity –151, othcificity , cross-lcificity , pull-docificity , co-immucificity  and sizecificity  have beecificity ,14,145. cificity –161. Othcificity ,14 and rcificity .+/H+ antiporters from Thermus thermophilus, an additional N-terminal helix, not present in the orthologous Escherichia coli NhaA transporter, strengthens interface interactions, removing the requirement for lipids to stabilize dimer formation. This N-terminal helix seems to interact with a subset of annular lipids as further structural support to facilitate large-scale conformational changes within the membrane [in vivo [per se. A computational model of the N-terminus of hDAT obtained by an ab initio structure prediction, in combination with MD simulations in the context of a lipid bilayer, revealed that the N-tail is a highly dynamic domain, but also contains secondary structure elements that remain stable in the long MD trajectories of interactions with the bilayer. These stable elements include specific residues that interact with charged PIP2  or PS (phosphatidylserine) lipids and thus seems to control the function, and possibly the stability, of the DAT oligomers.Are transporter tails critical for oligomerization or is this information encoded solely in the main transmembrane body of transporters? Recently, mass spectrometry was used to relate the presence of interfacial lipids and oligomeric stability, and discover how lipids act as key regulators of membrane protein association . This apmembrane . The trimembrane ,166. Ano[in vivo ,167. SpeStill other reports have shown that lipids other than phospholipids in particular sterols seem to play important roles in the functioning of DAT and other NSS transporters –170. Rec8.Neurospora crassa and Ustilago maydis), plants , protozoa (Leishmania mexicana) and in human cells [Transceptors are functional transporters that possess both solute transport and direct receptor-like signalling activities ,174. Thean cells . The conWhat distinguishes a transporter that acts solely in transport from a transporter that also acts as a signal receptor? Are the cytosolic termini of transceptors, which in some cases are particularly long , criticA. thaliana homologous ammonium transporter (Amt-1) is allosterically regulated by phosphorylation, but in an opposite way [Archaeoglobus fulgidus) ammonium transporter further suggests that the C-terminus interacts physically, in a phosphorylation-dependent manner, with cytosolic loops of neighbouring subunits [A well-studied case concerning the role of termini in transceptors is that of the yeast Mep2 ammonium transporters ,181. Undsite way . Findingsubunits . Such coTruncation of the last 14 or 26 amino acids from the C-terminus creates a constitutively active allele of the Gap1 transceptor that resides permanently in the PM and causes constitutive over-activation of the PKA pathway, provided it is expressed in a mutant strain that allows such truncated alleles to be secreted to the PM. However, the specific Gap1 conformation that elicits signalling remains unknown ,185. Fur9.per se, have been identified in several eukaryotic transporters. One of the most critical emerging issues is the interaction of tails with membrane lipids and other cytosolic domains of the transporter main body. The case of the FurE transporter serves as an instructive paradigm on how specific transporter tail interactions with internal loops, and most probably with phospholipids, can allosterically finely regulate the gating process, and thus substrate specificity, while distinct tail interactions can also regulate folding, trafficking and turnover. Evolution of functional tails may represent a naturally selected adaptation required for the integration of new functions in eukaryotic transporters, which face increasingly complex cellular challenges of regulation in response to differentiation and metabolic needs.Current findings are consistent with the idea that the size of eukaryotic transporter termini increased during evolution, providing additional flexible elements that enabled the generation of novel transporter functions and specificities, as well as specific mechanisms for controlled trafficking, turnover and/or oligomerization elements not shared with their bacterial homologues. Distinct but interacting roles for distal and proximal segments of the larger N- and C-termini in folding, turnover, ER exit and trafficking to the PM, or in transport function