Long noncoding RNAs (lncRNAs) have been wildly explored in various cellular processes and their aberrant expression could lead to tumorigenesis, development and progression. Differentiation antagonizing non-protein coding RNA (DANCR), a well-known lncRNA that is aberrant expression in various tumors, including hepatocellular carcinoma, gastric cancer, colorectal cancer, breast cancer, lung cancer and glioma and so on, in which it functions as oncogene mainly, contributing to cancer development and progression. High expressed DANCR is correlated with poor prognosis. In the present review, we summarize recent progression concerning the role, potential clinical utilities and underlying molecular mechanisms of DANCR related to occurrence and development of multiple cancers. Cancer is fundamentally caused by genetic alterations that result in the deregulation of the cellular information flow to alter cellular homeostasis and promote growth Long noncoding RNAs (lncRNAs) are defined as transcripts of more than 200 nucleotides without obvious protein coding potential LncRNA differentiation antagonizing non-protein coding RNA (DANCR) also termed as anti-differentiation non-coding RNA (ANCR), which is originally identified a regulator of the suppression of progenitor differentiation Tables In 2011, Kretz et al. discovered an uncharacterized, predicted lncRNA, NR_024031, termed ANCR, was suppressed upon terminal differentiation of multiple cell types Table  And the potential as biomarkers for diagnosis and prognosis of DANCR in cancers are presented in Table  The functions and underlying molecular mechanisms of DANCR in various cancers are summarized in Table 3 and detailed in the rest of this review.Accumulating evidences have demonstrated the abnormal expression of DANCR in multiple cancers, including hepatocellular carcinoma, gastric cancer, colorectal cancer, pancreatic cancer, breast cancer, lung cancer, glioma, osteosarcoma, cervical cancer, bladder cancer, prostate cancer, nasopharyngeal carcinoma, esophageal squamous cell carcinoma, ovarian cancer and retinoblastoma. The associations between DANCR expression and the relevant clinicopathological features of various cancers are summarized in Yuan et al. found that DANCR was overexpressed in stem-like hepatocellular carcinoma cells and associated with CTNNB1, which could block the repressing effect of miR-214, miR-320a and miR-199a Hao et al. confirmed that DANCR was dramatically upregulated in gastric cancer tissues and DANCR overexpression was significantly associated with worse overall survival in gastric cancer patients. Their experiments revealed that overexpression of DANCR notably increased gastric cancer cell proliferation by influencing the gene expression programs in cell metabolic and cycle process In 2015, Liu et al. reported DANCR expression was increased in colorectal cancer tissues compared with that in adjacent normal tissues. And high DANCR expression was correlated with TNM stage, histologic grade, and lymph node metastasis. They found that DANCR expression was an independent poor prognostic factor for both overall survival (OS) and disease-free survival (DFS) Chen et al. revealed that DANCR could promote pancreatic ductal adenocarcinoma progression, with relatively higher expression levels in pancreatic ductal adenocarcinoma cell lines and tissues. Correlation analysis of clinicopathological features and DANCR expression found that high DANCR expression was statistically correlated with vascular invasion, advanced T stage, lymph node metastasis and advanced TNM stage. High DANCR expression was identified as an independent risk factor for poor OS and progression-free survival (PFS) of pancreatic ductal adenocarcinoma. DANCR could promote the proliferation and metastasis of pancreatic ductal adenocarcinoma cells. Besides, miR-33a-5p/AXL signaling pathway may be involved in mediating the function of DANCR in vitro and in vivo. In addition, ectopic expression of ANCR partly attenuated the TGF-β1-induced EMT. Therefore, as a new TGF-β downstream molecular, ANCR is essential for TGF-β1-induced EMT by decreasing RUNX2 expression Li et al. initially identified that ANCR could potentiate the CDK1-EZH2 interaction, which then increase the intensity of phosphorylation at Thr-345 and Thr-487 sites of EZH2, facilitating EZH2 ubiquitination and hence its degradation. They uncovered that ANCR level was lower in breast cancer tissues and breast cancer cell lines, in contrast to their normal counterparts. ANCR is an important player in breast cancer progression and metastasis mainly through decreasing EZH2 stability in vitro and contributed to tumor growth in vivo. Sha et al. revealed that DANCR knockdown was associated with increased binding of EZH2 on the promoters of CD44 and ABCG2, and concomitant reduction of expression of these genes, suggesting that they may be DANCR targets in triple negative breast cancer While, Sha, Tao and Tang et al. identified that DANCR was significantly overregulated in triple negative breast cancer tissues and cell lines in vitro, and DANCR interference effectively suppressed lung cancer progression both in vitro and in vivo. They found that the miR-216a level in cancer cells was negatively correlated with DANCR expression Zhen et al. saw DANCR upregulation in lung cancer, particularly in high-grade lung cancer tissues and aggressive cancer cells. The DANCR overexpression promoted cell proliferation and colony formation The study of Wang revealed that DANCR was markedly upregulated in non-small-cell lung cancer tumor tissues and cell lines compared with related normal controls. DANCR could function as a tumor promoter and promote cell proliferation, invasion and migration through directly targeting the tumor suppressor miR-758-3p in non-small-cell lung cancer in vitro, xenograft growth suppression in vivo and cisplatin-induced cell apoptosis in vitro and in vivo. DANCR upregulated AXL via competitively binding miR-33a-5p, miR-33b-5p, miR-1-3p, miR-206 and miR-613. Through upregulating AXL, DANCR activated PI3K/Akt/NF-κB signaling pathway in glioma cells. DANCR could promote cisplatin resistance via activating AXL/PI3K/Akt/ NF-κB signaling pathway in glioma Yang et al. explored that DANCR was increased in glioma tissues and cells compared with normal brain tissues and cells. DANCR expression was positively correlated with the malignancy and poor prognosis of glioma patients. DANCR contained a binding site of miR-33a-5p and DANCR expression was negatively correlated with the expression of miR-33a-5p in glioma tissues. DANCR could facilitate cancer cell proliferation, invasion and metastasis and inhibit apoptosis through competitively binding to miR-33a-5p in glioma in vitro, as well as promote xenograft tumor growth and lung metastasis in vivo. DANCR upregulated expression of the receptor tyrosine kinase AXL by competitively binding to miR-33a-5p, which targets AXL mRNA for degradation, and this function was sequentially performed through the PI3K-AKT signaling pathway The present study of Jiang et al. showed that DANCR was significantly increased in osteosarcoma tissues, and its expression was positively correlated with tumor size and metastasis as an independent poor prognostic factor. The overexpression of DANCR could increase osteosarcoma cell proliferation, migration, and invasion Li et al. found that ANCR was highly expressed in human osteosarcoma cell lines. And their data indicated that ANCR might be an oncogenic lncRNA that promoted proliferation of osteosarcoma in vitro, indicating that DANCR functioned as an oncogene in cervical cancer. They verified that DANCR could regulate ROCK1 expression by competitively binding to miR-335-5p In Liang's study, they discovered that DANCR was significantly elevated in cervical cancer tissues and cells, and was closely correlated with poor prognosis of cervical cancer patients. In addition, knockdown of DANCR inhibited cervical cancer cell proliferation, migration, and invasion in vitro and enhanced tumor lymph node metastasis and growth in vivo. DANCR could activate IL-11-STAT3 signaling and increase cyclin D1 and PLAU expression via guiding leucine-rich pentatricopeptide repeat containing (LRPPRC) to stabilize mRNA Zhao et al. found that DANCR was significantly up-regulated in bladder cancer, and increased DANCR expression was positively correlated with higher histological grade and advanced TNM stage. Knockdown of DANCR could inhibit malignant phenotypes and EMT of bladder cancer cells. And they found that DANCR was distributed mostly in the cytoplasm and functioned as a miRNA sponge to positively regulate the expression of musashi RNA binding protein 2 (MSI2) through sponging miR-149 and subsequently promoted malignant phenotypes of bladder cancer cells in vitro and metastasis of tumor xenografts in nude mice. They found that TIMP2/3, which are critical metastasis inhibitor of prostate cancer, were down-regulated by DANCR synergistically with EZH2 through epigenetically silencing their promoter. DANCR expression was repressed by androgen-AR signaling pathway and DANCR knockdown facilitated the upregulation of TIMP2/3 and the suppression of invasion and migration by androgen-AR, while DANCR knockdown decreased the promotion of invasion and migration in prostate cancer cells by enzalutamide treatment In 2015, Jia et al. found the expression of DANCR was increased in prostate cancer tissues and cells compared to normal prostate cancer tissues and cells. Moreover, DANCR promoted prostate cancer cell invasion and migration Wen et al. reported that DANCR was upregulated in nasopharyngeal carcinoma, especially in those with lymph lode metastasis, and its upregulation could predict poor survival. And DANCR was responsible for nasopharyngeal carcinoma metastasis and hypoxia phenotype. DANCR could stabilize HIF-1α mRNA through interacting with NF90/NF45 complex, leading to nasopharyngeal carcinoma metastasis and invasion In recent days, some studies also reported that DANCR played important role in other cancers such as papillary thyroid cancer, esophageal squamous cell carcinoma, ovarian cancer and retinoblastoma.Zhang et al. demonstrated that the expression of DANCR was notably decreased in papillary thyroid cancer tissues in comparison with adjacent normal tissues. DANCR expression level was correlated to T grade and TNM stage. Their results suggested that DANCR was an independent factor for TNM stage of papillary thyroid cancer Figs. 4).DANCR abnormal expression in various human cancers has been shown to play active roles through regulating cancer-related phenotypes, as promoting cell proliferation, migration, invasion, inhibiting apoptosis. Furthermore, the regulatory mechanisms of DANCR are complex and involve multiple factors . Wang and Tao et al. demonstrated that DANCR could directly interact with miR-216a-5p to regulate the expression of KLF12, Nanog, OCT4 and SOX2 CeRNAs is a novel regulatory mechanism whereby lncRNAs function as ceRNAs to sponge microRNAs and simultaneously target both noncoding RNAs and genes Fig. 2).EZH2 is one component of Polycomb Repressive Complex (PRC2), which catalyzes the trimethylation of Lys-27 of histone H3 (H3K27me3) to suppress the transcription of target genes. EZH2, as a key epigenetic regulator, participates in a variety of cancer metastasis Fig. 3).Previous reports indicated that the activation of β-catenin pathway promoted tumor cell proliferation and metastasis Fig. 3).The PI3K pathway plays a key role in the regulation of multiple cellular events, including cell growth, proliferation, cell cycle progression and survival Fig. 3).TGF-β signaling pathway interacts with multiple signaling pathways to participate in the development and progression of different types of human malignancies Fig. 4).Xu et al. found that DANCR overexpression markedly upregulated mRNA and protein levels of MDR-related genes, such as MDR1 and MRP1 to contribute to the development of MDR With the repaid development of next-generation sequencing technology, lncRNAs have been identified dysregulated as oncogenes or tumor suppressor genes, which play important regulatory roles in tumor formation and development. Since its discovery, DANCR has been widely investigated in various human malignancies. DANCR is overexpressed and plays an oncogenic role in most types of tumors, including breast cancer and non-small-cell lung cancer. But Li et al. found that ANCR was downregulated in breast cancer and in particular, they demonstrated that a variable ANCR expression pattern and biological function in different cancer types and different cell lines The molecular functions and cellular mechanisms by which DANCR mediates its actions are complicated and associated with multiple factors. Several experiments have been identified that lncRNAs play a role in epigenetic, transcriptional, and post-transcriptional levels to contribute carcinogenesis and cancer development. Therefore, the molecular mechanism of DANCR in each cancer type should be clarified. Recently, lncRNAs have been demonstrated not only in tumor tissues but also in body fluids including plasma, serum, urine, and so on. The expression of DANCR in body fluids including plasma should be detected. And multiple effects between DANCR and molecular target markers should be explored in most available diagnostic samples like blood to apply it in clinical use. Further studies on the clinical value of DANCR in the diagnosis and treatment of cancers require more attention.


Though social sciences are expectedly instrumental in combating antimicrobial resistance (AMR), their research on AMR has been historically lacking.This study aims to understand the current academic literature on AMR within the social science field by investigating international contributions, emerging topics, influential articles, and prominent outlets, to identify research gaps and future directions.Bibliometric data of 787 peer-reviewed journal articles published in the period of 2010 to 2019 were extracted from the Social Science Citation Index in the Web of Science database. Bibliographic networks of the extracted articles were examined.Social science research on AMR has grown rapidly in the past 5 years. While western developed countries contributed the most to the field in the past decade, research within developing regions such as Asia and Africa have increased in the last 2 years. Social sciences have been contributing to AMR research in several different domains from surveillance and risk assessment of AMR, to promotions of appropriate use of antimicrobials in primary care and clinical settings. Though the idea of one health has been incorporated into research on AMR within the medical and microbial science fields, it has not been well recognized by social sciences.Social science research on AMR is a new, while rapidly developing, research area that requires continued and intense global efforts from an interdisciplinary and one health approach. Research on social issues surrounding AMR transmissions between human, animal, and environments should be emphasized in the future. Antimicrobial Resistance (AMR) occurs when microorganisms develop resistance to antimicrobial medications such as antivirals and antibiotics. AMR is increasingly becoming a global threat, both economically as well as in terms of health. AMR can lead to the treatments becoming ineffective, making it more difficult to treat infections, and can then cause an increase in the risk of the disease spreading . ParticuSeveral factors have been found to cause AMR. Human use of antimicrobial drugs, particularly antibiotics, is recognized as one of the primary drivers of AMR. Antibiotics are among the most familiar of medicines by the public, and the global consumption of antibiotics has been estimated at more than 35 billion daily doses at 2015 . SeveralDifferent approaches have been proposed to tackle AMR. One effective way is to provide interventions through antimicrobial stewardship programmes, which aims to promote the appropriate use of antimicrobials , 11. On Social sciences, encompassing a wide range of academic disciplines, such as sociology, psychology, communication, and economics, are expected to play a significant role in antimicrobial stewardship and the one health approach. For example, antimicrobial stewardship often requires theories, frameworks, and methods from behavioural and psychological sciences at a micro level. The one health approach also inspires social sciences to address AMR from the societal, historical, and economic perspectives. However, within the social science field, academic literature on AMR has historically been lacking . It is oAs the global efforts to reduce AMR rapidly increases, and social science is becoming more prominent in the field, existing gaps and future directions need to be identified. Therefore, this study investigates the social science literature surrounding this topic and how the landscape has changed and developed over the last decade. We aim to analyse the bibliometric data of the literature in the field via network analyses. Frid-Nielson et al.  have utiWe followed the PRISMA guidelines  for condA topic search in SSCI for “antimicrobial resistan*” and “antibiotic resistan*” published from 2010 to 2019 was conducted on Jan 14, 2020. Inclusion criteria included publications in English and limits were set to full-text peer-review journals to ensure the accuracy of follow-up co-citation analyses. The search produced 787 unique articles published in 294 journals with no duplicates. Bibliographic data including full reference lists of each article were extracted. Data of all articles were included in the analysis.VOSviewer is a software tool that was used to analyze and visualize the bibliographic networks. VOSviewer can provide distance-based bibliometric maps in which the strength of the relation between two items can be visualized as the distance between the two items, allowing easy understanding and visualization of clustering . Co-authFinally, the citation and co-citation network of journals were examined. The citation network, where a connection is demonstrated when a journal was cited by another journal, is used to reveal the knowledge flow in AMR social science research. The co-citation network, where a connection is demonstrated when a journal was co-cited with another journal by a research document, often indicates the topic similarity between journals. In this study, we use the co-citation network to reveal the knowledge foundation of the research field, as it can present and cluster all journals that were cited by research documents in the current dataset.As demonstrated in Fig. Table A total of 112 countries collaborated on the 787 AMR articles published in SSCI. Particularly, forty-three of the countries co-authored more than 5 articles and 31 co-authored more than 10 articles. Approximately one-third of the articles (N = 245) were co-authored by different countries, with only 3% of articles co-authored by more than five countries. The above findings reflect AMR as a global issue because a large number of countries have been involved in the academic efforts. However, the findings also indicate that there were not as many international collaborations as expected to be in AMR social science research.Figure Surprisingly, countries from the same WHO regions  were notIn addition, the colour distribution of the nodes shows that articles within countries from Asia  and Africa  have an average publication year of 2017–2018. This indicates that research coming from Asia and Southern Africa in the social science field has increased in the past two years. Notably, the research from Southern America seems to be relatively small and stagnant, with the majority of articles being published around 2015.Co-occurrence of article keywords was analysed and visualised to showcase the topical clustering and their development trajectories Fig. . In Fig.As demonstrated in Fig. Interestingly, keywords indicating environmental research and one health  were predominantly clustered within AMR epidemiology and surveillance research (Red cluster), indicating that they were more connected with AMR clinical surveillance than other research topics. They were also located peripherally at the connection area between social science research (Green cluster) and the clinical surveillance research. This suggests that the environmental and one health research has the potential to integrate the two research areas. However, keywords of one health research in the current dataset were relatively isolated as they demonstrated less dense connections with other keywords or clusters. This implies that the one health approach is at the periphery of the AMR social science discourse.Figure To further understand the literature surrounding topics emerging from the keyword co-occurrence analysis, citation and co-citation analyses of references were conducted to identify influential articles in each topic. Tables The knowledge and evidence gained from AMR surveillance and risk analytic research highlights the urgent need for social science research, which was found in half of the highly cited and co-cited articles. A global report by WHO in 2014 found very high rates of antimicrobial resistance for bacteria that cause common infections in healthcare and community settings around the world.  SpecificResearch has blamed increased and inappropriate use of antimicrobials for the resistance , 52, 56.Despite limited national and regional efforts in containing AMR in the early 2000s, , 51, 53 Notably, recent calls of AMR management and stewardship have recognized the importance of R&D and appropriate use beyond humans. Particularly, in 2017, the WHO defined antimicrobial stewardship as an overarching term that includes promotion of appropriate use in human, animal, and plant health, from individual to global levels . The scoOne of the keys to promoting appropriate antimicrobials use in human is to understand the determinants of antimicrobial prescription, dispensing, and community use. Research has revealed an antimicrobial prescribing culture in which immediate benefits  , 49, 55,There is a relative lack of recent influential studies regarding antimicrobial dispensing and community use. However, some studies have found that lack of government regulation, economic incentives, and pressure to meet customers’ needs were major determinants of dispensing antimicrobials , 58. RecLancet Infectious Disease, calling for global solutions to antibiotic resistance. The article summarized that resistance has spread worldwide and has highly burdened the world society with increasing health and economic costs. In addition, antibiotic use beyond humans is increasing and “a holistic, ecological, one health approach is needed (p. 9)”. Similarly, two years later, the WHO endorsed a global action plan on antimicrobial resistance, aiming at increasing awareness, strengthening knowledge, reducing infection, optimizing use of antimicrobials, and developing sustainable investment in new medicines through a one health approach [In 2013, Laxminarayan and the colleagues  publisheHowever, social scientists have paid little attention to antimicrobial use beyond humans in the past decade, regardless of the global calls for the one health approach. Of the listed influential articles, only three articles are related to food animal and environmental science: two articles are targeted towards antimicrobial use in animal food products , 54 and As we have identified four major topics in the literature of AMR social science research, we aim to further understand what outlets have been involved in this discussion. Table Lancet Infectious Disease, a medical journal that aims to influence health policies, and PLOS One, a non-specialized interdisciplinary journal. Medical journals were clustered at the east of the network. The west and the east clusters were connected by three journals located at the centre of the network, including Social Science and Medicine, Antimicrobial Resistance and Infection Control, and BMJ Open. Specifically, Journal of Antimicrobial Chemotherapy was clustered within the medical journals, offering knowledge and insights predominantly to medical journals in the last decades.The citation network between 44 journals that received more than 50 citation indicates the knowledge flow among journal outlets in the current dataset  in the AMR social science research. This reflects the current trend of the one health approach in tackling infectious disease and AMR issues. Nevertheless, these journals are predominantly connected only to medical and microbial science (Yellow cluster and Blue cluster). Social science outlets (Red cluster), such as eas Fig. .Fig. 6ThAMR is a global threat to the sustainability of human health and development. Social sciences can contribute to tackling this public health issue by providing domain knowledge that other sectors may not be able to provide to the field. By analysing bibliometric data of SSCI literature on AMR, this study demonstrated the landscape of social science research on AMR in the last decade and identified research gaps and future directions of AMR social science research.Though social science research within the area of AMR has rapidly increased in the past few years, the field remains relatively new and requires continued and intense international collaborations. Findings from our authorship analysis revealed that only 31.2% of social science studies on AMR involves non-high-income countries that house 84.3% of the global population. This phenomenon may be due to several important reasons besides the obvious economic factors. Countries at lower income levels may be less aware of the AMR issues than those at higher income levels. For example, AMR is largely a neglected problem in South-East Asia Region, as only 6 of the 11 regional countries carried out national AMR surveillance up to 2014 . Also, lAnother important finding that warrants attention is that regional collaboration networks of AMR social science research remains undeveloped. While WHO has been promoting political coordination in data sharing and dissemination of AMR research , our co-Importantly, our analyses of keywords, articles, and journals showed cohesive evidence that one health was marginally linked to social science AMR research, though it has been largely connected with medical and microbial sciences. Given that the one health approach has only been recognized as a fundamental approach to combat AMR in the past five years, this disconnection is understandable. Nevertheless, as agriculture and food sectors are expected to account for two-thirds of the future antibiotic use in the coming decade , it is iIn fact, under the umbrella of one health, all social science disciplines can play important roles in reducing AMR. For example, economic models can likely offer optimal solutions to balance profits and risks regarding antibiotic use, while sociology can utilize qualitative methods to understand and resolve tensions between different AMR stakeholders from the institutional perspective. The disciplines of communication and psychology can focus on a more micro-level to study attitudinal and behavioural mechanisms underlying debates of antibiotic use in food and animal sectors.Consistent with Frid-Nielson et al.’s  findingsWe only analysed bibliometric data from the SSCI WoS database, which may not reflect the complete set of research in the field. Nevertheless, as the SSCI database only includes journals that meet high research standards, the dataset ensures that our findings are based on articles that passed high standard peer-review processes and observe minimised bias.AMR is a major health threat that will become more prominent in the coming decades. The battle with AMR requires global efforts from all academic fields. Though social science research on AMR was rapidly growing in the past five years, the field is still young and requires intense international academic contributions and collaborations. The one health approach should be addressed as it provides an overarching framework for contributions from different scientific disciplines. This study demonstrates the current landscape of AMR social science research and calls for international academic efforts from the one health perspective.


The dopamine transporter (DAT) transports extracellular dopamine into the intracellular space contributing to the regulation of dopamine neurotransmission. A reduction of DAT density is implicated in Parkinson’s disease (PD) by neuroimaging; dopamine turnover is dopamine turnover is elevated in early symptomatic PD and in presymptomatic individuals with monogenic mutations causal for parkinsonism. As an integral plasma membrane protein, DAT surface expression is dynamically regulated through endocytic trafficking, enabling flexible control of dopamine signaling in time and space, which in turn critically modulates movement, motivation and learning behavior. Yet the cellular machinery and functional implications of DAT trafficking remain enigmatic. In this review we summarize mechanisms governing DAT trafficking under normal physiological conditions and discuss how PD-linked mutations may disturb DAT homeostasis. We highlight the complexity of DAT trafficking and reveal DAT dysregulation as a common theme in genetic models of parkinsonism. However, a relative preservation of dopamine synthesis and an increase in turnover is frequently observed in patients early in their symptom onset4. This supports the hypothesis that dysfunction in presynaptic dopamine neurotransmission may contribute to disease pathogenesis.Parkinson’s disease (PD) is the second most common neurodegenerative disease and is best known as a movement disorder. Motor symptoms correlate with the progressive loss of dopaminergic neurons in the substantia nigra is estimated to influence ~75,000 striatal neurons via as many as 370,000 axonal boutons, and in humans the organization may be four times as complex6. Dopamine release can originate from varicosities without termination of the axons, from en passant synapses that are often perisynaptic and extrasynaptic. Hence dopamine signaling is not restricted to a single synaptic location but has a large sphere of influence, an architecture that leads to volume transmission8. Extracellular dopamine critically modulates movement, motivation and learning by acting on its receptor. Different dopamine receptor subtypes display heterogeneity in their regional expression, substrate affinity and G-protein coupling dynamics9. Depending on the type of receptor activated, dopamine can have completely opposing effects on target neurons. Volume transmission and dopamine receptor heterogeneity both highlight the need to dynamically regulate extracellular dopamine in time and space - a function diligently executed by the dopamine transporter .Midbrain dopaminergic neurons have a very distinct morphology and mechanism of action. In the rat a single dopaminergic neuron in the 12. As a member of SLC6 gene family of Na+/Cl- dependent neurotransmitter transporters, DAT couples the transport of Na+ ions down the electrochemical gradient with the reuptake of extracellular dopamine into the intracellular space. Lowering the concentration of extracellular dopamine limits its duration of action and regulates dopamine transmission. Deletion of the DAT gene in mice greatly reduces tissue dopamine content, despite a great increase in the rate of dopamine synthesis13, suggesting DAT reuptake is the primary mechanism for maintaining intracellular dopamine storage. Beyond simple reuptake of extracellular dopamine, DAT is capable of generating a depolarizing conductance that regulates the excitability of midbrain dopamine neurons both in vitro and in vivo15. Under certain conditions, DAT also mediates the release of intracellular dopamine via a reverse transport mechanism known as DAT-mediated dopamine efflux16. Recent studies have also identified DAT as a regulator of short-term plasticity in striatal dopamine release17.DAT is a plasma membrane protein selectively expressed in dopaminergic neurons and critically regulates dopamine neurotransmission+ binding site that interfere with conformational changes during transport, resulting in dystonia-parkinsonism, and which highlight the functional significance of this transmembrane domain19. Additional rare substitutions have been implicated in attention deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD)22. DAT N- and C- terminals have the most diverse structures among SLC6 family members, and these domains account for differential interaction profiles. Notably, the N-terminus of DAT harbors several secondary modification sites, including residues subject to phosphorylation23 and ubiquitination24, that are important for the activity and trafficking of the transporter. The C-terminus contains domains for interactions with other proteins including α-synuclein25, parkin26, flotillin27 and protein kinase C (PKC)28. Recently, RAS-like GTPase, Rit2 was shown to complex with DAT and regulate PKC-stimulated DAT trafficking30. Genetic variability in the Rit2 locus has been robustly associated with PD and several other dopaminergic disorders32.DAT contains twelve transmembrane helices that harbor the primary substrate binding site and have large N- and C- terminals extended into the cytoplasm. DAT undergoes N-linked glycosylation on extracellular loop 2, which is important for surface targeting of the transporter. Pathogenic amino acid substitutions have been described near the Na33. At resting state, dDAT exists in an open and outward facing conformation. Upon substrate binding, an outward facing occluded conformation forms that is stabilized by salt bridges between key residues (the extracellular gate). Then the occluded complex transitions to become inward facing. Subsequent disruption of a separate set of salt bridges (the intracellular gate) allows substrate release into the intracellular space. Mutations that disrupt the conformational structure often lead to anomalous efflux or inefficient uptake activity22. However, it should be noted that dDAT intracellular N- and C- termini were removed to facilitate crystallization. In addition, dDAT was crystallized as a monomer34, whereas biochemical and microscopy data frequently detect dimers, oligomers and clusters of DAT molecules in DAT expressing model systems41. Oligomerization could influence DAT trafficking and surface expression. Small molecules, notably AIM100, may directly induce DAT oligomerization and subsequent endocytosis42. Readers are referred to the recent review for more detailed discussion on DAT oligomerization43.The high-resolution crystal structure of human DAT (hDAT) has not been solved as it is lipid associated and the protein aggregates during purification. Nevertheless, recent studies have resolved a partial structure of Drosophila melanogaster DAT (dDAT) in multiple conformational states. Hence, the functional dynamics of DAT and its interactions with ligands are being elucidatedPlasma membrane dopamine transporters are dynamically regulated by endocytic trafficking. Extensive studies have identified a variety of signaling pathways that lead to DAT internalization. The mechanisms of endocytic carrier formation and post-endocytic sorting of DAT largely depends on the signaling context. In this section, the mechanisms underlying DAT endocytosis are reviewed.45, which robustly drives the loss of surface DAT in heterologous systems and striatal tissues46. New data suggests PKC activation may disrupt DAT interactions with Rit2, and with cytosolic nonreceptor tyrosine kinase , that normally stabilize DAT in the plasma membrane47. AIM-100 may also inhibit TNK2 and lower surface DAT48. The effect of PKC can be arrested by blocking CME. For example, dynamin interacts with clathrin-coated pits and plays an important role in vesicle budding. Expression of dynamin p.K33E, a dominant-negative form of dynamin, or pharmacological inhibition of dynamin activity prevents phorbol 12-myristate 13-acetate (PMA)-induced loss of surface DAT in cell lines and ex-vivo striatal slices, respectively49. Down regulation of clathrin heavy chain by siRNA or acute inhibition of clathrin function by pitstop2 prevents PMA-mediated DAT internalization in DAT expressing cell lines50. However, dynamin is involved in both clathrin-dependent and clathrin-independent pathways including RhoA- and caveolar-mediated endocytosis51. Clathrin is also critically involved in a broad range of endocytic pathways including sorting of ubiquitinated proteins for lysosomal degradation53. Therefore, indirect regulation of clathrin and dynamin on PKC-mediated DAT internalization cannot be excluded. DAT complexes with Eps15, a component of clathrin-coated pits, in heterologous systems and midbrain neuronal cultures50. However, immunogold-labeling has failed to observed DAT localization in clathrin-coated pits in the axonal terminals and soma of dopaminergic neurons56. Assessing DAT’s interaction with clathrin and scaffold proteins, such as adaptor protein-2, in endogenously expressing systems may further clarify their role in DAT endocytosis.Clathrin-mediated endocytosis (CME) was the first mechanism identified for DAT regulation and is best characterized by PKC activation57. RhoA-mediated DAT endocytosis is most studied in the context of substrate exposure including amphetamine (AMPH); AMPH-induced DAT internalization was first described in HEK293 cells58, and later confirmed in mid-brain neuronal cultures59, ex-vivo striatal and mid-brain slices59. AMPH-mediated DAT endocytosis is dynamin dependent58, accompanied by the activation of RhoA and Rac1 in mouse mid-brain slices, and prevented by the inhibition of RhoA through PKA59. Similarly, dopamine treatment of rat mid-brain slices also increases RhoA activation and reduces surface DAT expression, and such effects are inhibited by pharmacological activation of the PKA-coupled D1 receptor60. AMPH and dopamine-mediated DAT internalization is blocked by cocaine which occupies the substrate binding site of DAT and prevents transport activity59. Extracellular AMPH failed to induce internalization of an uptake-impaired mutant form of DAT (Y335A-hDAT); whereas direct intracellular application of AMPH via a whole cell patch pipette restored the trafficking of Y335A-hDAT, and revealed that its endocytic regulation is triggered by intracellular AMPH61. Subsequent study has identified the G-protein coupled receptor (GPCR) TAAR1 as the intracellular effector of AMPH-mediated RhoA activation and DAT endocytosis62.RhoA belongs to the Ras GTPase family and defines a clathrin-independent endocytic pathway64 and highlights the role of actin dynamics in DAT trafficking. Actin depolymerization agents lead to reduced DAT surface expression in striatal tissue49. Piccolo, a component of the presynaptic cytoskeletal matrix, was shown to negatively regulate methamphetamine-induced DAT internalization, possibly through sequestering membrane phosphatidylinositol 4,5-bisphosphate (PIP2)65.An essential downstream effector of RhoA activation is Arp2/3, which directly regulates actin branching and polymerization to provide the necessary force for membrane remodeling in the absence of clathrin66. Although it is generally acknowledged that DAT association with lipid rafts negatively regulates the kinetics of the transporter activity68 and membrane mobility69, the role of lipid rafts on DAT endocytosis is controversial. While some studies report raft-associated DAT exhibits more resistance to constitutive and PKC mediated endocytosis66, others show DAT endocytosis arises equally from raft and non-raft associated populations49. DAT forms a stable protein complex with membrane protein flotillin-1, and that interaction appears to be required for DAT targeting to lipid rafts50. siRNA mediated flotillin-1 knock down may block PMA-mediated DAT endocytosis in heterologous systems and mid-brain dopaminergic neuronal cultures27. Paradoxically, in an independent study, the same treatment did not exhibit any effect in PMA-treated HA-DAT expressing cells50. Lipid rafts provide a scaffold for plasma membrane protein interactions with the cytoskeleton and promote DAT clustering40. It is possible that flotillin facilitates endocytosis by promoting DAT clustering prior to carrier formation, rather than being directly involved in carrier formation, as shown for other cargos71. However, the conflicting results might be due to the model system used to study raft and non-raft associated DAT trafficking. Future studies might directly examine the trafficking routes of DAT-flotillin complexes in mid-brain dopaminergic neurons expressing DAT endogenously.Lipid rafts are cholesterol- and sphingolipid-enriched microdomains on the plasma membrane. DAT is proposed to distribute equally among raft and non-raft domains72. The endosomal sorting complexes required for transport (ESCRT) family of proteins defines degradative subdomains73 and orchestrates the recruitment of ubiquitinated cargos to budding intraluminal vesicles. Conversely, tubular extensions occupy most of the membrane surface and provide subdomains enriched in cargo retrieval complexes, including the retromer and retriever complexes. These generate tubulo-vesicles for plasma membrane recycling or retrograde transport to the Golgi network76. Extensive efforts have been invested to identify the subcellular localization of internalized DAT, to define its post-endocytic fate. However, the ambiguous colocalization pattern of DAT with known post-endocytic sorting markers, and lack of methodology to reliably track endogenous DAT have led to conflicting results, as reviewed below.In general, degradative and recycling pathways are physically separated by distinct endosomal subdomains, and defined by their respective sorting machineries81. Electron microscopy studies also show that DAT localizes to the endolysosomal compartment, albeit less in axonal terminals than in soma56.Although the field cannot reach a consensus on the exact post-endocytic fate of DAT, most studies provide evidence that DAT is, at least in part, targeted for lysosomal degradation following endocytosis. Studies using either antibody feeding assays or fluorescent cocaine analogs to visualize epitope-tagged or endogenous DAT in mid-brain neuronal cultures all confirm that internalized DAT partially localizes to late endosomes and lysosomes83. Nedd4–2 has been identified to ubiquitinate DAT in a PKC dependent manner24. Either down regulation of Nedd4-2 or abolition of DAT N’-terminal lysines by mutagenesis impairs the PKC-mediated endocytosis of DAT83. However, Nedd4 regulates the trafficking and degradation of many membrane proteins so disruption of DAT trafficking could be a secondary consequence rather than a direct effect.N-terminal ubiquitination may be the tag that sorts DAT to its degradative fate. DAT undergoes ubiquitylation on conserved lysine residues in heterologous systems and mid-brain neuronal cultures, which is significantly enhanced following PMA treatment84, both of which serve to target cargos to a degradative fate by recruiting cargo to ESCRT-positive subdomains85. Supporting the theory, DAT localization to the ESCRT component ‘hepatocyte growth factor-regulated tyrosine kinase substrate (HRS)-positive’ endosomal compartment was also confirmed in PMA treated YFP-DAT expressing cells83. Conversely, modification that reduces DAT ubiquitination also reduces degradation and enhances DAT’s steady state level86. Collectively, these studies support the model where PKC-mediated ubiquitination recruits DAT to a degradative fate.Interestingly, DAT is proposed to undergo monoubiquitylation and lys63-linked polyubiquitylation81. Although PKC-induced loss of surface DAT can be robustly reproduced using a biotinylation assay of ex-vivo striatal and mid-brain slices, the total level of DAT is not significantly reduced compared to heterologous systems49. Conflicting results may be due to differences in PMA treatment times and/or use of heterologous model systems overexpressing DAT. More research is needed to validate DAT ubiquitination and subcellular localization in its native environments, including the soma and terminal regions of dopamine neurons.However, attempts to validate PKC-mediated degradation for endogenous DAT have proven challenging as multiple groups report PMA treatment fails to induce DAT internalization in mid-brain neuronal cultures56. Although DAT exhibits partial colocalization with recycling markers, such as syntaxin13, the transferrin receptor and Rab11, in cultured mid-brain neurons and in ex-vivo slices, the extent of that colocalization is quite ambiguous compared to bona fide recycling cargos such as transferrin and β2-adrenergic receptors80. Direct comparison between the trafficking of DAT and norepinephrine transporter  shows that NET is more likely to recycle via Rab11 than DAT, due to the non-conserved N’-termini of the two transporters48. However, in N2A neuroblastoma cells, expression of dominant-active Rab11 upregulates DAT surface expression and uptake activity87. Multiple groups have also demonstrated DAT plasma membrane recycling with antibody feeding or biotinylation based assays in various cell lines and mid-brain neuronal cultures89. In support of its recycling fate, DAT was shown localize to VPS35 positive vesicles in dopaminergic cell lines and dopamine axons in mouse medial forebrain bundle axons90. Either knocking down or expression of PD-linked mutant Vps35 p.D620N leads to reduced total and surface expression of DAT in heterologous systems and mouse striatal tissues91, albeit without producing a PD-like motor phenotypes in this animal model91. Internalized DAT that undergoes retromer-mediated plasma membrane recycling or retrograde trafficking may potentially account for the comparatively decreased colocalization between DAT and Rab11.Endocytic recycling pathways for DAT also remain elusive. Early electron microscopy (EM) studies in rodent brains observed DAT immunoreactivity in tubulovesicular structures in the soma, albeit less frequently in axonal varicosities, suggesting DAT may be subject to endocytic recycling93. However, the sequence responsible for DAT-retromer interaction remains to be determined. The PDZ domain is a well characterized recycling sequence recognized by retromer and its adaptor protein for plasma membrane recycling92. The same mechanism is proposed to regulate retromer-mediated recycling of DAT89. Indeed, mice harboring mutations in the putative PDZ domain of DAT exhibit reduced surface DAT expression in the striatum94. Furthermore, the DAT PDZ motif was required, at least partially, for exit out of retromer-positive endosomes89. However, unlike well-characterized retromer cargos such as beta 2 adrenergic receptor (β2AR) or glucose transporter1 (GLUT1)97 which have a type I PDZ domain, DAT has a type II PDZ domain. This is unlikely to be recognized by SNX27, the adaptor protein best characterized in retromer-mediated plasma membrane recycling99. Retromer binding is sometimes facilitated by palmitoylation of cysteine residues in the cytosolic tails of cargo proteins100, and notably, DAT undergoes C’-terminal cysteine palmitoylation101. Pharmacological inhibition or mutagenesis of those cysteine residues enhances PMA-induced downregulation of DAT in heterologous cells and rat striatal synaptosomes, suggesting palmitoylation may function to resist PKC-mediated DAT degradation101. The functional impact of palmitoylation on dopamine reuptake kinetics of surface DAT is well described102, but whether palmitoylation affects DAT endocytic sorting and trafficking is unknown.The retromer complex orchestrates sequence-dependent cargo retrieval as opposed to sequence-independent bulk recycling5. In rat a single dopaminergic neuron in the substantia nigra is estimated to influence ~75,000 striatal neurons5, via as many as 370,000 terminal axonal boutons6. DAT is found throughout the plasma membrane of the axons and varicosities of dopaminergic neurons56. It seems intuitive that with such a complex morphology, dopaminergic neurons might employ local DAT synthesis rather than long-range axonal trafficking. Surprisingly, the in-situ hybridization signal for DAT mRNA is primarily observed in the mid-brain and almost invisible in the striatum104, although that may reflect the probes used and the sensitivity of traditional in-situ methods. It remains possible that DAT mRNA may be found outside of the somatodendritic compartment. At the protein level, most DAT localizes to smooth endoplasmic reticulum in the soma and not in axonal terminals56. In mid-brain neuronal cultures from tagged HA-DAT knock-in mice, HA-DAT vesicles display rapid bi-directional movement along axons suggesting that DAT is synthesized in somal endoplasmic reticulum (ER) and subject to axonal trafficking. A recent study examined long-range DAT trafficking in medial forebrain bundle axonal tracts to reveal that most DAT is membrane localized with a more limited distribution in retromer positive vesicles90. The result raised the possibility that DAT might be trafficked by both membrane diffusion and vesicular transport90. Future studies examining DAT localization to microtubule markers and/or microtubule motor proteins such as kinesin would further clarify the mechanism of long-range DAT trafficking.Another puzzle in DAT trafficking is the mechanism of DAT axonal targeting. Midbrain dopaminergic neurons project long and extensively arborized terminals105. Supporting the role of SEC24-mediated ER export in DAT axonal targeting, siRNA knockdown against SEC24D in heterologous cell lines slows down dopamine reuptake106. In C.elegans, deletion or mutagenesis of a conserved sequence 591PYRKR595 in the DAT-1 C′-terminus leads to somatic retention107. The PYRKR sequence contains a 3 R motif that mediates the interaction between SEC24 and α2β-adrenergic receptor108, suggesting the failure of DAT-1 synaptic localization might be due to reduced interaction with SEC24-1. In HEK293 cells, mutation of the corresponding sequence in DAT does not affect its interaction with SEC24D, nor significantly reduces DAT surface expression, but does disrupt the ability of SEC24D to enhance DAT surface expression107. However, HEK293 cells are not going to be an informative model for DAT axonal trafficking as they lack the bipolar morphology of dopaminergic neurons.ER export relies heavily on coated-protein complex (COPII) coat-derived vesicles with SEC24 family proteins orchestrating cargo recognition110, and may prove useful to dissect mechanisms governing endogenous DAT trafficking in bona fide dopaminergic terminals.In conclusion, multiple molecular mechanisms are implicated in DAT trafficking and the specific trafficking route is highly dependent on signaling. Key mechanisms are summarized in Fig. Given the complex morphology of dopaminergic neurons and the onerous burden of long-term axonal trafficking, it is tempting to speculate that endocytic dysregulation of the dopaminergic transporter may lead to the selective vulnerability of dopaminergic neurons in PD. In this section, monogenic parkinsonism, and models based on those gene linkages are reviewed, albeit focused on their DAT biology. Most mice that model human gene mutations linked to PD do not exhibit overt motor symptoms but many have subtle alterations to their dopaminergic system, as illustrated in Table SNCA missense mutations and genomic multiplications111. Mice models overexpressing mutant or wild type human α-synuclein mostly exhibit reduced DAT expression and dopamine reuptake116 with some exceptions117. α-synuclein may form a stable complex with DAT in heterologous systems, rodent and human striatal tissues119. Studies in dopaminergic neuronal cultures suggest DAT/α-synuclein association negatively regulates DAT reuptake activity122 possibly by redistributing surface DAT to cholesterol-rich microdomains and altering ionic coupling of DAT mediated current121. DAT surface expression, measured by biotinylation, is also reduced with co-expression of wild type α-synuclein124. When compared to wild type α-synuclein, the PD-linked missense mutation SNCA p.A30P enhances DAT α-synuclein interaction and further reduces DAT surface expression and function; in contrast, p.A53T does not form a strong protein-protein complex with DAT, nor modulates DAT function or surface expression. Although SNCA p.A30P and p.A53T have disparate effects on DAT-binding and trafficking, they are N’-terminal located on the hydrophobic face of α-synuclein’s amphipathic helix, highlighting the importance of maintaining that protein-lipid interaction.α-synuclein (SNCA) is a presynaptic protein and a major component of Lewy pathology. Studies of familial forms of Parkinson’s disease have identified 111. All mutations lead to upregulation of LRRK2 kinase activity. Investigations of in vivo dopaminergic function with PET imaging consistently reveal increased dopamine turnover rates in asymptomatic LRRK2 heterozygotes125. Interestingly, those asymptomatic individuals showed reduced DAT levels, measured by the commonly used PET tracer 11C-d-threo-methylphenidate (11C-MP)125, suggesting early dysfunction of DAT in the absence of significant loss of dopamine synthesis and storage. In rodent models, temporal overexpression of human LRRK2 p.G2019S in dopaminergic neurons impairs DAT mediated dopamine reuptake126. Constitutive Lrrk2 p.G2019S knock-in mice also exhibit impairment in striatal dopamine reuptake at various ages128. Although there are limited number of studies that directly examine the role of LRRK2 in DAT regulation, growing evidence has established LRRK2 as an important regulator of intracellular membrane trafficking129, at a nexus between actin-dependent and tubulin-dependent cargo transport132. Mutant LRRK2 leads to dysregulated endocytic sorting133 through phosphorylation of Rab proteins135 whose coordinated action critically regulates vesicular transport and endosome maturation137. LRRK2 binding to microtubules is also associated with tau phosphorylation139, microtubule acetylation140 and impaired axonal transport132. Hence, DAT endosomal recycling and axonal trafficking may be dysregulated by chronic LRRK2 hyperactivity. Exploring the increased (or compensatory) dopamine turnover observed in asymptomatic LRRK2 heterozygous individuals may provide mechanistic insight into the physiological and pathological role of LRRK2 in DAT regulation.Leucine-rich repeat kinase 2 (LRRK2), encodes a large multidomain protein containing kinase and GTPase enzymatic activities. LRRK2 genetic variability is found throughout the gene but pathogenic mutations cluster in the Roc-COR and kinase domains, and account for this common form of familial parkinsonism76. A missense mutation, VPS35 p.D620N has been linked to a familial form of parkinsonism that is clinically indistinguishable from idiopathic late-onset PD142. VPS35 colocalizes with DAT and mediates DAT plasma membrane recycling both in vitro and in vivo89. Mice harboring Vps35 p.D620N exhibit reduced striatal DAT expression and altered striatal dopamine neurotransmission in the absence of motor deficits or neurodegeneration91. Although the exact pathological mechanism of VPS35 p.D620N remains elusive, multiple studies show the mutation disrupts the interaction between VPS35 and the WASH complex144 whose primary function is to coordinate actin branching and stabilize endosomal subdomains for VPS35-mediated cargo sorting of recycling proteins145. Failure to adequately recruit or retain WASH complex may lead to the mis-sorting of DAT, as illustrated by other retromer cargos146. These data are consistent with the hypothesis that expression of VPS35 p.D620N can impair DAT recruitment and recycling.VPS35 is a component of the retromer complex required for recycling cargo protein in the early endosome to the trans-Golgi network or plasma membrane147. VPS35 p.D620N significantly elevates LRRK2-mediated phosphorylation of Rab proteins in mouse brain and human neutrophils148. Endolysosomal dysfunction induced by mutant LRRK2 can be rescued by overexpression of VPS35149, although toxic to synapses in primary culture96. Given VPS35 and LRRK2 mouse models exhibit similar deficits in DAT mediated reuptake, it is possible that they also cooperate in the regulation of DAT trafficking. Interestingly, acute treatment with the LRRK2 kinase inhibitor, MLi2, in Vps35 p.D620N knock-in mice rescues the reduction in striatal DAT levels and the reduction in dopamine reuptake91 . However, it is currently unknown which step in DAT trafficking  is involved and whether VPS35 and LRRK2 work in concert or in competition.Increasing evidence suggests that VPS35 and LRRK2 functionally interact and converge on endocytic trafficking. VPS35 and LRRK2 directly interact, decorate endosomes, and may work in concert in synaptic vesicle recycling150. Mutations in Parkin lead to loss of function of its ubiquitin ligase activity and are linked to autosomal recessively inherited, early-onset Parkinson’s disease with a central role in mitophagy151. However, patients with mutations in Parkin often exhibit a marked reduction of DAT expression or uptake activity153. DAT ubiquitination was previously shown to be increased in Parkin transfected human dopaminergic SH-SY5Y cells26. WT Parkin, but not PD-linked mutants, significantly increase DAT surface expression and dopamine reuptake, and possibly enhance degradation of misfolded DAT26. An association between Parkin and DAT has also been shown to disrupt the interaction between DAT and α-synculein154; however, further validation using a more sensitive approach, such as Förster resonance energy transfer (FRET) or yeast 2-hybrid methods, is warranted. Parkin is reported to ubiquitinate Rab7, Eps15, and VPS35 and so contributes to the regulation of the endo-lysosomal pathway157. Parkin knock-out mice exhibit reduced striatal levels of DAT and dopamine uptake159. In patient-derived IPSCs, loss of Parkin function also leads to reduced [3H]CFT (2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane, a cocaine analog) binding to DAT and increased Ca2+-independent dopamine release, potentially via a reverse transport mechanism160. Several studies support the hypothesis in which loss of parkin function reduces DAT activity either directly, through impaired protein quality control, or indirectly through dysregulation of DAT endocytic trafficking.Parkin is a protein-ubiquitin E3 ligase which ubiquitinates and targets substrate to proteasome degradationRIT2 was identified as a PD risk gene in multiple GWAS studies and in different ethnic groups164, and Rit2 expression is significantly reduced in the substantia nigra of PD patients165. Rit2 encodes a small neuronal GTPase that associates with DAT and may act as DAT endocytic brake30. Studies in heterologous systems reveal RIT2 associates with the C-terminus of DAT, but not SERT, and that RIT2 GTPase activity is critical for PKC-mediated DAT internalization29. Although Rit2 was shown to co-immunoprecipitate with DAT in mouse striatum29 this result appears to be an artifact as the antibody used was unspecific30. Nevertheless, the effects of Rit2 in bona fide dopaminergic terminals are confirmed in mouse striatal slices46. In Pitx3IRES2-tTA shRit2 mice, where Rit2 is selectively knockdown in dopaminergic neurons, PKC-stimulated DAT internalization in acute striatal slices is significantly reduced30. Interestingly, Pitx3IRES2-tTA shRit2 mice exhibit sex-dimorphism in dopamine related phenotypes166. Conditional Rit2 knockout leads to a blunted response to acute cocaine treatment in female mice, but an increased response in males166. Accordingly, [3H] dopamine uptake in male striatal slices is enhanced without significant change in surface DAT expression166, whereas in females surface DAT is reduced in the ventral striatum30. Considering the important role of sex in DAT function and PD risk172, RIT2 poses a promising target to study the effect of sexual dimorphism in DAT regulation and in the etiology of PD.173. Direct administration of dopamine and overexpression of dopamine transporter both lead to neuronal loss175. Conversely, dopamine reuptake by DAT is the primary mechanism for the maintenance of dopamine storage, and loss of DAT leads to dopamine depletion13. The depolarizing currents generated by dopamine reuptake also directly regulate the excitability of presynaptic boutons15. Thus, DAT surface expression must be controlled to allow dopaminergic neurons to optimally function. The complexities of DAT regulation may enable multiple compensatory mechanisms to maintain dopamine signaling despite early dysfunction. Given the role of DAT in the regulation of dopamine release and the long-hypothesized connection between altered dopamine neurotransmission and PD, studies of DAT regulation in physiological and pathological conditions remain a promising avenue to identify early biomarkers and therapeutic targets for PD.A variety of signaling pathways have been identified that dynamically regulate dopamine reuptake through DAT plasma membrane trafficking, and many overlap with key molecular mechanisms compromised by PD-linked mutations as illustrated in Fig.


Functional assessment to rule out myocardial ischemia using coronary computed tomography angiography (CCTA) is extremely important and data on the Brazilian population are still limited.To assess the diagnostic performance of myocardial perfusion by CCTA in the detection of severe obstructive coronary artery disease (CAD) compared with single-photon emission computerized tomography (SPECT). To analyze the importance of anatomical knowledge to understand the presence of myocardial perfusion defects on SPECT imaging that is not identified on computed tomography (CT) scan.A total of 35 patients were evaluated by a simultaneous pharmacologic stress protocol. Fisher’s exact test was used to compare proportions. The patients were grouped according to the presence or absence of significant CAD. The area under the ROC curve was used to identify the diagnostic performance of CCTA and SPECT in perfusion assessment. P < 0.05 values were considered statistically significant.For detection of obstructive CAD, CT myocardial perfusion analysis yielded an area under the ROC curve of 0.84 . SPECT myocardial perfusion imaging, on the other hand, showed an AUC of 0.58 . In this study, false-positive results with SPECT are described.Myocardial perfusion analysis by CTA displays satisfactory results compared to SPECT in the detection of obstructive CAD. CCTA can rule out false-positive results of SPECT. Multimodal assessment and the combination of these techniques provide safe information on the anatomical and functional diagnosis of obstructive CAD, enabling better clinical and therapeutic planning.3In the last years, we have observed several coronary computed tomography angiography (CCTA) studies of patients with moderate stenosis. The patients were referred to perform complementary functional tests, such as pharmacologic stress cardiac magnetic resonance imaging and single photon emission computed tomography (SPECT) to verify the presence of perfusion defects. This approach allows for, with high sensitivity and specificity, the characterization of ischemia in patients with obstructive CAD.7Myocardial perfusion by CCTA is still little explored. Stress computed tomography (CT) myocardial perfusion imaging is a technique which has shown consistent results in the diagnosis of obstructive CAD. In its turn, myocardial perfusion scintigraphy is a well-established method for detection of CAD. The possibility of integrating anatomy and function in a single exam can enhance stratification of obstructive CAD and ensure better patient management.7 not only for grading stenosis, but also for characterizing the atherosclerotic load and the types of plaques. Recent data in the literature, on the evaluation of significant obstructive CAD (> 50%) by CCTA, have revealed good accuracy, with high sensitivity (82-99%) and specificity (94-98%), when compared to invasive cinecoronariography.8The clinical benefits of CCTA are changing the perspectives of contemporary cardiology,10Multicentric studies, published in the last years, have demonstrated the high negative predictive value of CCTA (95-100%), emphasizing its excellent performance in excluding CAD. This fact should be increasingly exploited in clinical practice, avoiding invasive exams.18 On the other hand, the use of hybrid technology, which combines the anatomical information from CCTA and rubidium-82 (Rb-82) myocardial positron emission tomography (PET) perfusion imaging, presents high accuracy in CAD detection;31 however, this approach is still expensive and difficult to implement clinically.SPECT assessment of myocardial perfusion can allow for better stratification of patients with intermediate stenosis and definition of therapeutic strategies, aiming at better prognosis.Thus, we observe that CCTA can aggregate perfusion imaging and, therefore, be increasingly used as the initial test for CAD, which remains one of the leading causes of mortality in Brazil and worldwide. Nevertheless, although several studies have demonstrated the diagnostic and prognostic value of myocardial perfusion by CCTA in patients with suspected CAD, these data are still limited in the Brazilian population. Besides, it is uncertain whether the use of CCTA analysis can replace other myocardial perfusion methods, such as SPECT, especially in places where this method may not be available. The implementation of myocardial perfusion assessment by CCTA is simple and less expensive compared to other methods.Our purposes were: to evaluate the diagnostic performance of myocardial perfusion assessment by CCTA for significant obstructive CAD detection compared with SPECT; to analyze the importance of anatomical knowledge to understand the presence of myocardial perfusion defects by SPECT that cannot be identified by CCTA; and to describe SPECT false positives.This is an observational study that assessed patients clinically indicated to undergo myocardial scintigraphy for CAD stratification. All patients accepted and signed the informed consent form to participate in this research on myocardial perfusion assessment by CCTA. The study and the Free and Informed Term of Consent were approved by the Research Ethics Committee of Análise de Projetos de Pesquisa (CAPPessq), do Hospital Universitário Antônio Pedro (HUAP)/Universidade Federal Fluminense (UFF) number número 392.966.Patient selection for this observational study included 38 patients from our institution [Antonio Pedro University Hospital - Federal Fluminense University (HUAP-UFF)], recruited in the Nuclear Medicine service .The CCTA results (anatomy and perfusion) were considered as research data and were not reported to the patient's clinical physician, except in case of identification of significant lesions in the trunk of the left coronary artery or in the LAD coronary artery detected by CCTA. The inclusion criteria were patients with medical request for stress/rest myocardial perfusion scintigraphy to assess CAD.Patients with creatinine above 1.5 mg/dl, obstructive pulmonary chronic disease, asthmatic patients, patients who were allergic to iodinated contrast material or for whom dipyridamole or metoprolol was contraindicated and any other aspect that the researcher deemed limiting to the method were excluded.99mTc) was infused at the computed tomography room.The exams were performed with the following flow: first the patitent was selected at the Nuclear Medicine Service and, after signing the free and informed term of consent, the patient was referred to the service of radiology to undergo CCTA (perfusion at rest) followed by myocardial perfusion under pharmacological stress with dipyridamole. Before the infusion of iodinated contrast material, during stress-induced hyperemia, 2-methoxy-isobutyl-isonitrile-99mTc  (0.56 mg/kg/4 minutes). We chose to infuse it by hand, after images of the ascending aorta were blurred using iodinated contrast media, because it facilitates the correct selection of the beginning of acquisition, especially in the stress phase, which must occur a little earlier than usual for other coronary studies. During dipyridamole infusion, the patients’ heart rate, blood pressure and symptoms were monitored every minute. Immediately after the conclusion of stress perfusion evaluation, 240 mg of aminophylline were administered  to reverse the vasodilatation effect of the stress agent. This CT protocol was idealized in a 64-detector tomographic angiography  and the mean dose of radiation was 12.1 ± 5.2 mSv.The first acquisition was volumetric and static, having been performed retrospectively using the following parameters: 120 KV, 240-400 mA and 512 × 512 matrix, 70 ml iodinated contrast media at a concentration of 350 mg/mL, infused at 5 ml/s. The second acquisition was performed following the same parameters and soon after 5 to 6 minutes from the beginning of dipyridamole infusion . The patient was referred to the Radiology Sector, and the injection of the radiotracer was performed at the tomography room, in the Radiology Sector. Soon after CT was finished, the patient was referred to stress imaging acquisition (first-passage perfusion) with a maximum interval of 30 minutes. After this stage and an interval between 60 and 120 minutes, the rest phase was performed with a new injection of Tc-99m sestamibi. The mean dose administered in each stage was 925 MBq. The images were acquired 30 to 90 minutes after intravenous administration of the agent. A total of 64 projection images of the chest were acquired from an arc of 180 degrees, from the 45-degree right anterior oblique view to the 45-degree left posterior oblique view. In the rest phase, the acquisition time was 30 seconds per projection; in the stress phase, the acquisition time was 30 seconds per projection as well. In both the stress and rest phases, ECG-synchronized image acquisition was performed.Myocardial perfusion scintigraphy (SPECT) was performed with intravenous infusion of Tc-To analyze the correlation between the myocardial perfusion techniques, the following criterion was used to characterize myocardial ischemia: there should be perfusion defects on stress images with no correspondent perfusion defect on rest images of both CCTA and SPECT.Myocardial perfusion and CCTA were assessed visually and semi-quantitatively by two blinded and independent observers, without any knowledge of clinical data or other exams. Disagreements were resolved by means of consensus. The degree of coronary stenosis was graded, according with visual and semi-quantitative assessment by CCTA, as non-significant  and significant .All continuous variables were expressed as mean ± standard deviation and the categorical variables as number and percentage. Fisher’s exact test was used to compare between proportions. Based on CCTA fidings, the patients were grouped according with the presence or not of significant CAD. The criterion used to define significant CAD was existence of obstruction > 50% of the lumen of coronary arteries. Sensitivity and specificity were estimated and displayed as number and percentage. The analysis of the area under the ROC curve was used to identify the efficacy of CCTA (CT perfusion) and scintigraphy (SPECT) in the diagnosis of perfusion data in this study. The research was conducted on two groups: one with stenosis > 50% on anatomical assessment by CCTA, as the "true positive" surrogate marker in this population, compared with the group with stenosis < 50% in the same method as the “true negative” . Intra- and interobserver agreement was obtained by using intraclass correlation coefficient reliability analysis . About 43% of perfusions performed using CCTA techniques (15/35) were reassessed by the same observer; the analysis was performed by a second independent observer to characterize the variability between the analyses. A total of 1,440 segments were assessed using the 16-segment model of the American College of Cardiology (ACC) and the American Heart Association (AHA), with 240 LV segments being analyzed by observer 1 at rest and, subsequently, under pharmacological stress, totaling 480 segments. Observer 1 repeated this analysis after a 3-month period, blinded to the previous analysis. Observer 2 performed the independent analysis, blind and with no previous agreement with the first observer. Both observers have more than 10 years experience in performing CCTA.® statistical software . Two-tailed p values < 0.05 were considered statistically significant.Statistical analysis was performed using MedCalcA total of 38 patients were selected; out of these, 35 were included in the study. Three patients were excluded: one patient due to long wait times to undergo the stress phase as a result of problems with schedule and other two due to technical problems in the Radiology Sector.Out of the 35 patients studied, with a mean age of 52.5 ± 9 years, 18 were women (51%). In this study, obstructive CAD (stenosis > 50%) was present in 43% (n = 15) of the patients; non-obstructive lesions were identified in 57% (n = 20) of the patients.The distribution of perfusion defects on both methods are shown in Based on the data in Twenty patients had abnormal myocardial scintigraphy, and half of them (n = 10) also presented obstructive CAD at CCTA. Based on the data in Myocardial perfusion with CT showed an AUC of 0.84 for the detection of obstructive CAD, with a confidence interval (CI) range of 0.67 - 0.94 (p < 0.001). On the other hand, SPECT myocardial perfusion had an AUC of 0.58, with a CI range of 0.40 - 0.74 (p < 0.001) .Excellent intra- and inter observer correlation was reported in the assessment of stress perfusion, with an ICC of 0.90 (0.87-0.92) and 0.94 (0.93-0.96), respectively. The intraobserver correlation of perfusion at rest was also excellent, with an ICC of 0.96 (0.95-0.97). For interobserver correlation of perfusion at rest the result was good, with an ICC of 0.71 (0.63- 0.78).99mTc-sestamibi were compared with the findings of myocardial perfusion by 64-detector row computed tomography. As a strength of this study, we highlight the simultaneous use of the same pharmacological stress agent for CT perfusion image acquisition, and the administration of the radiotracer, which enables performance of CT and subsequent scintigraphy image acquisition, because it lacks significant redistribution. Another important data was the possibility for anatomical localization and correlation with the presence of myocardial perfusion defects by SPECT. In this study, it was also possible to understand why the defect was not detected by CCTA and to describe SPECT false positives.In this study, it was possible to assess the diagnostic performance of myocardial perfusion by CCTA for the detection of significant obstructive CAD in relation to SPECT. The perfusion findings of scintigraphy with et al.32 clearly state that CCTA has better accuracy than SPECT for detecting significant obstructive CAD. Hence, it is necessary to explore this finding and understand that many patients with false-negative SPECT results are unnecessarily submitted to cardiac catheterization, due to lack of anatomical assessment.35If we assess myocardial perfusion alone, an intermediate correlation between CT and scintigraphy images will be found, especially because the sensitivity of CT perfusion sensitivity for perfusion defects detection on SPECT was 70%, with a specificity of 66%, considering that scintigraphy is the standard method used to assess perfusion. Tanami 37 In this study, we observed better ischemic catheterization by CT myocardial perfusion when compared with SPECT. It is important to highlight that catheterization was not used as the gold standard and, thus, these results may vary if other methods of reference are used, such as flow fractional reserve (FFR).39 Rochitte et al.,35 showed that combined CCTA and stress perfusion imaging accurately identifies patients with > 50% lesion in the catheterization and who presented perfusion defects at SPECT. Moreover, the rational use of these techniques and multimodality assessment are important in modern cardiology, since they are always associated with increased exposure to radiation.36An interesting finding, in line with previous studies, is the comparison between the sensitivity and specificity of the two perfusion techniques in detecting obstructive coronary lesions, considering that coronary CT is the gold standard for the diagnosis of anatomic CAD.36 greater accuracy was observed for CT perfusion imaging when compared with SPECT , but the authors used another methodology with a higher slice CT system (320 detectors), as well as a slightly different protocol, which is not a problem, according with recommendations.37 In contrast, other studies compared CCTA with SPECT and PET perfusion imaging with invasive catheterization with FFR, as a gold standard. Interestingly, perfusion PET was the exam that better correlated with the gold reference, whereas CCTA and SPECT performed similarly, showing that anatomic measures are not substitutes for functional assessment and that, even when the best method for anatomy assessment is used, functional assessment of coronary lesions is required.40In the study carried out by Arbab-Zadeh et al.,41 With regard to scintigraphy, we observed that one of the studies presented low levels of the tracer, due to tracer leakage, that was not detected during the study and, therefore, was not excluded from the analysis. We believe that further studies need to be conducted in order to better clarify these findings, because they will affect clinical decision-making.Another finding of the study that needs discussion is the presence of 10 patients (28%) with abnormal SPECT who did not present significant obstructive CAD on CCTA. Considering that CCTA is the anatomical method of reference in this study, we observed a high number of “false-positive” myocardial perfusion scintigraphy findings. We believe that a large part of these findings may be related with microcirculation disease (40%), since it was not possible to identify another cause that could explain them. The other findings (60%) were explained by anatomy assessment by CCTA. The best example is the case of a patient with myocardial bridge in which CT provided the anatomical substrate for the diagnosis of underlying myocardial ischemia detected by both SPECT and CT, already previously published by our research group.99mTc-sestamibi exhibits a roll-off phenomenon, in which there is a limitation of its regional distribution when the flow is increased above certain threshold, while the same does not occur with iodinated contrast.52There are several factors that can be potentially responsible for disagreements between the tests. Some of them are obvious, such as differences in spatial resolution between the techniques  and the distinct contrast properties used: the 44In the Brazilian context, in spite of the absence of nuclear medicine services, combined CCTA and myocardial perfusion imaging is available, thus we consider this method as a simple and enforceable strategy. Some aspects should be considered, such as the use of beta-blockers to reduce heart rate for CCTA imaging, which can have a relative influence on the ischemic area detectable by SPECT, especially in cases of microcirculation disease. Another aspect is obesity, because in these patients the quality of the images is worsened, which can cause disagreement between the techniques. Another point is that, in order to perform CT perfusion, the patient needs to be inside the equipment in the stress phase, which makes the use of pharmacological stress mandatory. If physical stress could be used, perhaps the results would have been different from what we found.36For CCTA, undoubtedly, the greatest limitation is exposure to radiation and iodinated contrast media, which are agents with potential adverse events. This protocol optimization, with new equipment, may be capable of reducing the levels of exposure; however, even so, the protocol shall only be adopted in selected patients, where information can be complemented. Studies using 320 detectors have shown that the combination of CT perfusion and CCTA can promote lower radiation exposure compared to the conventional protocol for myocardial perfusion imaging .Standardization of CT analysis is still a limitation, and the use of automatic analysis software is one of the priorities for technology development, since there are no polar maps yet, as in nuclear medicine, to display ischemic and normal patients for quantification of the level of ischemia, with validated and widely available software.Among other limitations of our study, as we detailed throughout the discussion, is the small number of individuals recruited. We believe that this is a partial limitation and should encourage further studies in different populations. We also took into account the false-positive scintigraphy results that might have influenced its performance, because we believe that the majority of cases can be explained by anatomy. Last but not least, one could imagine that the use of CCTA as an anatomical test would be limiting. In this case, numerous studies have compared CTA and catheterization with excellent results, which validates this approach.Myocardial perfusion assessment by CCTA, after dipyridamole stress, is feasible and simple, with satisfactory results, when compared with SPECT, for obstructive CAD detection. Combined assessment of anatomy and stress perfusion by CCTA shows good capacity for detecting significant obstructive CAD, while ruling out SPECT false-positive findings.


Molecular electronics promises a new generation of ultralow-energy information technologies, based around functional molecular junctions. Here, we report optical probing that exploits a gold nanoparticle in a plasmonic nanocavity geometry used as one terminal of a well-defined molecular junction, deposited as a self-assembled molecular monolayer on flat gold. A conductive transparent cantilever electrically contacts individual nanoparticles while maintaining optical access to the molecular junction. Optical readout of molecular structure in the junction reveals ultralow-energy switching of ∼50 zJ, from a nano-electromechanical torsion spring at the single molecule level. Real-time Raman measurements show these electronic device characteristics are directly affected by this molecular torsion, which can be explained using a simple circuit model based on junction capacitances, confirmed by density functional theory calculations. This nanomechanical degree of freedom is normally invisible and ignored in electrical transport measurements but is vital to the design and exploitation of molecules as quantum-coherent electronic nanodevices. The development of molecular electronics at single molecule level calls for new tools beyond electrical characterisation. Kos et al. show an optical probe of molecular junctions in a plasmonic nanocavity geometry, which supports in situ interrogation of molecular configurations. Metal-molecule-metal junctions have been extensively studied using scanning probe techniques and break junctions, typically through statistical analysis of electrical conductance in repeated transient pull-off experiments which shed little light on individual junction morphologies. Experiments which contact self-assembled molecular monolayers (SAMs) by pulling away a sharp tip7, until one final molecule bridges the tip-surface gap  are conformally coated with an insulating parylene layer, and dry-etched to expose the AuNP crown (Methods section). NPoMs are individually contacted using a conductive transparent Si3N4 tip-less cantilever, giving negligible contact resistance (Supplementary Note This scheme exploits the tight light confinement inside E > 500) optical field within the NPoM plasmonic gap enables strong surface enhanced Raman spectroscopy (SERS ∝ E4) of the molecular spacer17, giving >100 kcounts/mW/s thus allowing short integration times for tracking dynamics. The collected Raman from the λ = 633 nm focussed laser , and provide large Raman cross-sections.Each NPoM is optically accessed through the cantilever for imaging and spectroscopy in real time during electrical measurements. The enhanced  scattering spectroscopy of the electrically contacted NPoMs , the 20-fold decrease in SERS shows that the molecular Raman cross-sections reduce  to 90° (rings perpendicular to each other). The energy 23 decreasing the Raman cross-section.We explore this twisting through density functional theory (DFT) on a BPDT molecule bound to Au atoms at both thiol terminal groups. The molecule is progressively twisted by changing the dihedral angle gle Fig. , and is 17, we can also observe twist switching of individual molecules . From the charge stored vs potential drop across this bond, a capacitance θ = 90°  , the C–C twist is a broad Gaussian (100 cm−1 FWHM), suggesting each molecule is sensitive to its subtly different molecular environment within the layer, which supports the idea that Fermi energy varies slightly across the molecules in the gap. As bias is applied, a 10-cm−1 shift to smaller wavenumbers of this C–C twist is seen, in agreement with the 27% reduction in curvature of the full potential −1 reduction in vibrational energy. Adjacent SERS lines corresponding to the S–Au bond stretch show no detectable shift, while the Stokes: anti-Stokes ratio17 shows no change in temperature with bias. This nano-device measures real-time molecular twist angles, suggesting the capability for detecting binding events, for instance sensing trace-gas molecules.To further confirm this molecular twisting, we directly observe the central C–C twist of BPDT at low wavenumbers (Methods section) in both Stokes and anti-Stokes SERS Fig. . The 0 V(θ) Fig. . Comparedel Fig.  that shoWe conclude that the energies obtained from the full quantum calculation of our molecular junction can be understood as being dominated by energies from electrostatics, and modelled as a classical electrical circuit based on surface dipoles and capacitance  in ethanol to deposit a self-assembled monolayer, and are then rinsed with ethanol. Gold nanoparticles  from BBI solutions are drop-cast on the surface to obtain a number density of ~0.001 µm−2 on the Au pattern for contacting individual AuNPs through the cantilever. Sample are post-coated with a ~350-nm thick uniform layer of parylene-C (SCS Labcoter 2) at room temperature, and the parylene-C layer is then selectively etched using O2 plasma to expose the top of the AuNPs while leaving ~20–30 nm insulating layer around the sides and base  are coated with a 3-nm Cr adhesion layer and 6 nm Au by e-beam evaporation. The quality of the conductive film is verified with scanning electron microscopy and a uniform film without pinholes is obtained, with a sheet resistance 3Ω/square.Optical dark-field images are recorded in a custom-modified Olympus BX51 microscope. Samples are illuminated with a focussed white light source . The scattered light is collected through a ×100 dark-field objective  and analysed with a fibre-coupled  Ocean Optics QE65000 cooled spectrometer. We use a standard diffuser as a reference to normalise white light scattering.Sample topography is performed using an Asylum Research MFP-3D atomic force microscope in non-contact AC mode. Asylum Research AFM Software version 15 is used for instrument control and data post-processing.Raman spectroscopy is performed in the same custom-modified Olympus BX51, using a 633-nm excitation laser with 200 µW power focussed into a diffraction limited spot. The Raman emission is detected after separation through a dichroic beamsplitter and laser line filter using an Andor grating monochromator and EMCCD. Low wavenumber measurements are performed with the same system using a 785 nm excitation laser with 200 µW power.Computational spectra are calculated after geometry optimisation with B3LYP hybrid functional employing Grimme’s D3 dispersion correction with Becke-Johnson damping. The basis set of def2SVP was used including the pseudopotential definitions for gold atoms. Potential bias was modelled as a homogeneous dipole field. Raman intensities were recalculated from the polarizability derivatives according to the experimental setup, including the temperature and frequency of the excitation laser. Numerical integrals were computed on an ultrafine grid, as method implemented in Gaussian09 Rev. E , with a single-ζ basis set and the Local Density Approximation (LDA) functional with CA parameterisation. A real-space grid was defined with an equivalent energy cut-off of 250 Ry. We applied bias voltage under non-equilibrium conditions and calculated the Hamiltonian and electrostatic potential for each bias voltage. From the converged DFT calculation, the underlying mean-field Hamiltonian Gollum to calculate transmission coefficient E passing from the source to the drain. We then calculate the room-temperature current To investigate the quantum transport through BPDT molecules between gold electrodes, we first carried out geometry optimisation to the force tolerance of 10 meV/Å using the Supplementary Information


Plastic bottles are generally recycled by remolding them into numerous products. In this study, waste from plastic bottles was used to fabricate recycled polyethylene terephthalate (r-PET) nanofibers via the electrospinning technique, and high-performance conductive polyethylene terephthalate nanofibers (r-PET nanofibers) were prepared followed by copper deposition using the electroless deposition (ELD) method. Firstly, the electrospun r-PET nanofibers were chemically modified with silane molecules and polymerized with 2-(methacryloyloxy) ethyl trimethylammonium chloride (METAC) solution. Finally, the copper deposition was achieved on the surface of chemically modified r-PET nanofibers by simple chemical/ion attraction. The water contact angle of r-PET nanofibers, chemically modified r-PET nanofibers, and copper deposited nanofibers were 140°, 80°, and 138°, respectively. The r-PET nanofibers retained their fibrous morphology after copper deposition, and EDX results confirmed the presence of copper on the surface of r-PET nanofibers. XPS was performed to analyze chemical changes before and after copper deposition on r-PET nanofibers. The successful deposition of copper one r-PET nanofibers showed an excellent electrical resistance of 0.1 ohms/cm and good mechanical strength according to ASTM D-638. Flexible electrically conductive materials have gained significant demand for electronics and telecommunication systems because of their capability to convey electrical properties in this century . The incWearable electronics rely heavily on metal-coated textiles to produce textile materials with special functional properties. In the previous research studies, textile based conductive materials were widely fabricated via various deposition methods ,10. CurrThe previous textile based conductive fabrics contain a low surface area and high porous structure, which can limit the metal deposition rate. Electrospun nanofibers have gained much attraction in wide applications due to their high surface area and can create a high metal deposition rate . RecentlNanotechnology has deep roots in every field of life and in helping the research community to solve a wide a range of challenges . ElectroRecycled polyethylene terephthalate (r-PET) was obtained from water bottles at a convenience store in Japan and were utilized directly without further purification. Trifluoracetic acid, chloroform, vinyltrimethoxysilane (98%), (2-(methacryloyloxy) ethyl) tri-methyl-ammonium chloride solution (METAC), ammonium tetra-chloro-palladate (II), and copper (II) sulfate pentahydrate were purchased from Sigma Aldrich USA . Chemical reagents, potassium sodium tartrate tetrahydrate, formaldehyde, potassium persulfate, and sodium hydroxide were purchased from Sinopharm Chemical Reagent Co., Ltd. .2) and dissolved in the TFA/chloroform with a ratio of 1:3 in order to make a 15% w/w polymer composition. Electrospinning was performed using a high-voltage power supply  at room temperature. The prepared solution was poured into a syringe with a capillary tip attached having an internal diameter of 0.6 mm and keeping a 0.5 mL/h flow rate with a supplied voltage of 15 kV. The as-prepared r-PET nanofibers were collected on aluminum foil wrapped over a rotary collector system, and the capillary tip to collector distance was set as 15 cm. After electrospinning, the r-PET nanofibers were peeled off and dried overnight before further use.The r-PET nanofibers were fabricated by following the previous protocol . BrieflyGraphical abstract represents the complete fabrication process to prepare conductive r-PET nanofibers and characterized by 4 steps . Each step is explained below.The obtained r-PET nanofibers were hydrophobic, and to create hydroxyl groups before the silanization process, plasma treatment was needed. Previously, plasma treatment was used to create hydroxyl groups on textile substrates . The r-Pv/v) vinyl-tri-methoxy-silane (VTMS) for 20 min. The VTMS-treated r-PET nanofibers were rinsed with DI water and immediately vacuum dried at 50 °C. The successful VTMS treatment provided the hydrophobic surface of r-PET nanofibers due to the presence of dense vinyl groups.The plasma treated r-PET nanofibers were placed in an ethanol solution having 10% (v/v) METAC aqueous solution (100 mL) and 60 mg of potassium persulfate (KPS) for 60 min to carry out the polymerization of METAC (PMETAC) brushes while stirring at 60 °C. Finally, the polymerized r-PET nanofibers were washed with DI water and vacuum dried at 50 °C for 2 h.The silanized-treated r-PET nanofibers were immersed into a polymerization solution, which contained a mixture of 20% (4)2PdCl4) for 20 min for the ion-exchange reaction. During the ion-exchange, [PdCl4]2- catalytic ions were immobilized onto the quaternary ammonium groups of PMETAC polymer chains, and the samples were gently washed with DI water. Further, an ELD bath was prepared to contain Solutions A and B (The electroless deposition (ELD) of copper was carried out on PMETAC r-PET nanofibers by following previous protocols ,22,23,24 A and B  with a rScanning electron microscopy  with an acceleration voltage of 30 kV was used to observe the surface morphology of the neat r-PET nanofibers and copper-coated r-PET nanofibers. The EDX images and spectrum were obtained using an S-3000 N, Hitachi Ltd. X-ray spectroscopy, Japan. All samples of SEM and EDX were sputter coated with gold before analysis. The X-ray spectroscopy (XPS)  was performed to analyze the chemical composition of the neat r-PET nanofibers and copper-coated r-PET nanofibers.An OCA-40 contact angle instrument (Data physics Filderstadt Germany) was applied to determine the water contact angle of neat r-PET nanofibers and each process involved to fabricate the conductive r-PET nanofibers.σ, and E represents the stress, strain, and Young’s modulus, respectively. l shows the original length of the specimen. F is the applied force on the specimen, and A is the area of the specimen.The mechanical properties of neat r-PET nanofibers and copper-coated r-PET nanofibers after the ELD process were tested by a Titan Universal Tester 3-910, Titan Company Ltd., Hessen, Germany. The tensile strength was measured according to the ASTM D-638 standard. During the test, the speed was set at 5.0 mm/min. The values of the stress-strain curves and Young’s modulus were calculated by using the following Equations (1)–(3), respectively .(1)ε=Δl2 r-PET nanofibers with an average thickness of 120 µm. The shrinkage of r-PET nanofibers after the ELD process was calculated by using Equation (4) for lengthwise shrinkage and Equation (5) for widthwise shrinkage, respectively.The shrinkage test was performed by taking 5 × 5 cmLs is lengthwise shrinkage, L is the original length of the samples, and L∆ is the change in the original length after the ELD process. In Equation (5), Ws is widthwise shrinkage. W is the original width of the samples. w∆ is the change in the original width after the ELD process. In Equation (6), Rs is the residual shrinkage after the ELD process.In Equation (4), In this study, neat r-PET nanofiber samples were assessed by SEM (scanning electron microscopy) and analyzed to identify the surface morphology before and after the copper ELD process. The SEM images are shown in The EDX elemental mapping was applied to verify the presence of copper metal in r-PET nanofibers as shown in To investigate the specific elements and the presence of the chemical bonds, the XPS analysis was performed on neat r-PET nanofibers and copper-coated r-PET nanofibers to ensure the presence of copper elements after the ELD process, which was also confirmed by the EDX process. The water contact angle was used to examine each successful step required for the ELD process. In the electroless deposition, temperature and time had significant effects on the deposition of copper on r-PET nanofibers. After the optimization of the temperature, for the deposition of copper on the r-PET nanofibers, time optimization was also equally important, as shown in According to the experimental results, continuous copper deposition could be obtained as the deposition time was extended up to 30 min, but the optimal time was observed as 15 min for the deposition of copper on the r-PET nanofibers at a temperature of 40 °C. In the optimum condition, the deposition of copper was good, showing the lowest resistance. The mechanical properties of the pristine r-PET nanofibers and copper-coated r-PET nanofibers were investigated. As shown in The r-PET nanofibers after being exposed to the salinization and the ELD process substantially shrunk, so it was necessary to analyze the shrinkage behavior in the r-PET nanofibers after the ELD process; there was exaggeration in documenting the finding. In this research study, copper-coated r-PET nanofibers were successfully fabricated by the process of electroless deposition. The optimized temperature was observed as 40 °C, and the optimized time of 15 min was set for proper copper deposition on the r-PET nanofibers. The SEM images evidently showed the fibrous morphology of the r-PET nanofibers with an average diameter of 350 nm, and the copper-coated r-PET nanofibers retained the fibrous morphology with a slight increase in the average diameter of 700 nm. The optimized copper-coated r-PET nanofibers were composed of 71.6% copper and showed a low electrical resistance of 0.1 Ω. Moreover, the r-PET nanofibers were flexible and showed good mechanical strength. As-prepared, conductive r-PET nanofibers have good potential and could be used in many applications such as wearable electronics, flexible sensors, and energy storage.