People who used illicit drugs (PWUDs) represent an important population group for acquisition and viral dispersion. In Brazil, high rates of the human T lymphotropic virus 1 (HTLV-1) and 2 (HTLV-2) have been reported in epidemiological studies. However, the epidemiological scenario of HTLV-1/2 infections in PWUDs is still poorly understood. Thus, this cross-sectional study determined the prevalence, frequency of subtypes and factors associated with HTLV-1/2 infections among PWUDs in the Brazilian state of Pará, an area considered endemic for this virus and with poor health services. Among 826 PWUDs, 53 (6.4%) presented anti-HTLV-1/2 antibodies by EIA and 44 (5.3%) presented proviral DNA by PCR. HTLV-1 and HTLV-2 were detected in 25 (3.0%) and 19 (2.3%) PWUDs, respectively. Subtypes 1a (25/44), transcontinental (23/44) and Japanese subgroups (2/44), 2b (6/44) and 2c (13/44) were identified. Involvement in illicit/criminal activity, daily use of illicit drugs, illicit drug use over 12 years, unprotected sex with other PWUDs, changes in genitalia (including ulcers and wounds), and more than 12 sexual partners were associated with HTLV-1/2 in PWUDs. This high prevalence and intense circulation of subtypes and subgroups of HTLV-1/2 is very worrying, and indicate the need for urgent actions for its control, prevention and treatment. Based on the nucleotide diversity of its long terminal repeat (LTR) region, HTLV-1 has been classified in seven genetic subtypes (1a-1g), and HTLV-2 in four subtypes (2a-2d)4. HTLV-1 and HTLV-2 infections are widely distributed worldwide, with high or moderate endemicity in some regions such as southwestern Japan, sub-Saharan Africa, and specific areas of Iran and Melanesia4. In the Americas, high rates were recorded in Brazil and in Caribbean countries3. It is estimated that there are 2.5 million Brazilians infected with HTLV, and the highest rates were detected in the Brazilian states of Bahia, Maranhão and Pará5. In Brazil, HTLV-1a or Cosmopolitan subtype Transcontinental subgroup, HTLV-2a and HTLV-2c have shown high frequencies in different populations1.The human T-lymphotropic virus (HTLV) belongs to the family Retroviridae, subfamily Oncovirinae, and genus Deltaretrovirus. There are four types , but only HTLV-1 and HTLV-2 are associated with chronic infections in humans, and more than 95% of infected individuals remain as asymptomatic carriers for the duration of their lives7. People who use illicit drugs (PWUDs) represent an important population group for acquisition and viral dispersion, such as HTLV-1 and HTLV-28. Infections with HTLV-1/2 have been recorded in PWUDs in different countries, mainly in people who have used injection drugs11. In Brazil, the number of PWUDs has increased significantly in recent decades. Crack cocaine is the main illicit drug consumed by Brazilian users, and has become a major public health problem12. To date, there is still limited information on HTLV infections in PWUDs in Brazil. In the state of Rio Grande do Sul (Southern Brazil), injecting cocaine users were five times more likely to be infected with HTLV-1/−2 than non-injecting users of cocaine11.HTLV-1/2 can be transmitted through the transfusion of contaminated blood or blood products, unprotected sexual contact, sharing of contaminated syringes and other instruments, or via transmission from mother to child14. The intense flow of people and illicit products associated with the lack of infrastructure and equipment for collective use has produced several problems in northern Brazil, such as abuse and sexual exploitation of children and adolescents, prostitution, consumption and trafficking of illicit drugs16. Recently, studies have reported a high prevalence of adolescents and young adults who used illicit drugs17. In northern Brazil, asymptomatic infections and diseases associated with HTLV-1/2 have already been reported in different groups: blood donors, pregnant women, Japanese immigrants, riverside people, and indigenous from more than 25 Amazonian tribes25. However, the epidemiological scenario of HTLV infections in PWUDs is still unknown in this immense Brazilian region, as well as in other vulnerable groups. Thus, this study determined the prevalence of HTLV-1/2 infections, the frequency of viral subtypes, and the factors associated with these infections among PWDUs in the Brazilian state of Pará, an area considered endemic for HTLV-1/2 and with poor health services.The Brazilian epidemiological scenario presents numerous social, economic and geographical differences that may facilitate the spread of viruses, such as HTLV-1/2. Among the regions, northern Brazil is notable for containing a significant portion of the Amazon rainforest. Historically, this area is an important route for trafficking in illicit drugs due to the geographical characteristics that make it difficult to control and facilitate the transportation and commercialization of drugs produced in South American countriesIn this study, 907 PWUDs were accessed, but 81 PWUDs were excluded (49 had <18 years old and 32 used illicit drugs <3 months). In total, the sample number of this study was 826 PWUDs. Samples and information from PWUDs were collected in all regions in the Brazilian state of Pará Fig. .Figure 1In this study sample (n = 826 PWUDs), most were men, young, singles, had up to 10 years of education, had a reduced monthly income, and declared themselves to be heterosexual  PWUDs presented anti-HTLV-1/2 antibodies using EIA and 44 (5.3%) PWUDs presented proviral DNA using different PCR protocols. There was no disagreement between the results provided by the two HTLV proviral DNA detection protocols. All samples with anti-HTLV-1/2 antibodies by EIA, submitted to DNA isolation, presented positive results for the presence of fragment of the albumin gene, indicating the success in obtaining genetic material. Proviral DNA was not detected in nine seropositive samples, possibly indicative of non-specific seropositivity. Among PWUDs with proviral DNA, most of them (59.1%) were living in small municipalities in the state of Pará . HTLV-1 and HTLV-2 were detected in 25 (3.0%) and 19 (2.3%) PWUDs, respectively  and 2c (29.5%)  in the last 12 months, and more than 12 sexual partners in the last 12 months Table . These s26. Moreover, the differences between NIDUs and IDUs were also identified. Initially, the route of administration of illicit drugs was the main difference but other distinct features relevant to the HTLV spread were also established.This study is the first epidemiological report on the HTLV-1/2 in PWUDs from the Amazon region. In a large sample of hard-to-reach people from a variety of sites and good geographical coverage in a remote area of Brazil, several key characteristics were identified and associated with acquisition and spread of HTLV-1/2. In general, the characteristics of the PWUDs are consistent with the findings of other studies conducted in Brazil30. The seroprevalences detected in this study are relatively similar to those found in studies conducted with IDUs in different countries such as Argentina (19.1%), the Dominican Republic (6.0%), Sweden (3.2%) and Spain (3.5% to 6.2%)34. This is indicative of the influence of the drug use profile on the potential exposure to HTLV-1/2 of the present study.The seroprevalence of HTLV-1/2 found in PWUDs and its subgroups NIDUs and IDUs are higher than those reported in other studies conducted in HTLV endemic areas in Brazil 4. HTLV-1 has a worldwide distribution, but it is considered endemic in Brazil with low prevalence in the general population35. A geographic gradient has been reported in blood donors with lower prevalence in the south and rising to the north35. In the state of Bahia, an endemic area in northeastern Brazil, high rates were observed in women and in people over 50 years27, as well as in men who have sex with men and illicit drug users36. In this study, the predominance of HTLV-1 observed in PWUDs and NIDUs can be considered an example of the endemicity of this viral type reported in numerous Brazilian studies, and especially in the state of Pará – an endemic area in northern Brazil28. On the other hand, the HTLV-2 is endemic among IDUs in the United States, Europe, and Asia39, and particularly among indigenous tribes of different linguistic families inhabiting the Brazilian Amazon, where subtype 2c was described for the first time24. The predominance of HTLV-2 among IDUs in this study corroborates this information.Different epidemiological characteristics of HTLV-1/2 have been reported in geographic areas and specific population groups28. Two PWUDs with the Japanese subgroup were descendants of Japanese immigrants from the municipality of Tomé-Açú, one of the colonies of Japanese migrants from the region of Kyushu in southeastern Japan. The presence of this HTLV-1 subgroup was also registered in another epidemiological investigation with 168 Japanese immigrants living in the municipality of Tomé-Açú20. Another interesting fact observed in this study was the high frequency of the HTLV-2 subtypes. In South America, the HTLV-2 predominates among indigenous groups, with subtype 2b clearly prevailing in Amerindian populations, except in Brazil where the subtype 2c predominates25. The latter is endemically distributed in the Amazon region and maintained under continuous transmission  between members of different indigenous communities, being also detected on a smaller scale in urban populations24. In endemic populations, the HTLV-2b replication pattern exhibits high proviral loads resulting from extensive proliferation of infected cells40. The predominance of this subtype in NIDUs and IDUs has already been reported in Brazil1. Brazil has received both intense internal and external migration from Africa, Europe and Asia over the past five centuries. As a consequence, different HTLV subtypes and subgroups were introduced and distributed in the Brazilian population over the years24.The findings of this study also indicate an intense circulation of the HTLV-1/2 subtypes and subgroups among PWUDs. The subtype 1a (Cosmopolitan) was predominant among PWUDs being also detected the predominance of subgroup A . This HTLV-1 subtype and this HTLV-1 subgroup of are very common in northern BrazilThe findings of this study clearly demonstrate the contribution of the migratory process to the dispersion of HTLV-1/2 among the PWUDs in the state of Pará and possibly among the general population in the Amazon region. The latter hypotheses is strongly supported by the association between HTLV-1/2 clusters in phylogenetic trees with the profile of illicit drug use and risky sexual behavior. Accurate phylodynamic analysis using other genomic regions and more epidemiological details of PWUDs will be done in the future in order to evaluate the epidemic potential and effective targeting of preventive actions.7. HTLV-infected lymphocytes are considered the primary vectors of sexual transmission41. The presence of inflammation or sores on the genitals and sexually transmitted infection may facilitate the HTLV transmission because these conditions expose target cells through genitomucous lesions41. Unprotected sex associated with lesions on the genitalia of PWUDs was an important contributor to HTLV-1/2 spread and possibly other pathogens , similar to that reported in the Brazilian state of Bahia - high number of HTLV-1 infections associated with T. pallidum infection through the sexual route27.Overall, this study shows the HTLV-1/2 acquisition and spread among PWUDs occur mainly through sexual contact. This is most evident in NIDUs due to the HTLV-1 predominance among them. Daily and long-term use of crack is associated prostitution and unprotected sex with multiple partners (within and outside of the PWUD groups), all of which facilitate the acquisition and spread of HTLV-1. The same goes for the HTLV-2, although on a smaller scale. This scenario corroborates the assertion that many factors are associated with HTLV shedding and HTLV proviral load in genital  secretions42. Involvement with illicit/criminal activities facilitates greater contact with drug traffickers in the Amazon region and therefore enables access to a greater quantity and diversity of drugs, thus contributing significantly to chemical dependence and multifactorial exposure to the HTLV-1/2 and other pathogens.The pattern of illicit drug use enhances the risk of exposure to the HTLV-1/2. This is most evident in IDUs due to the predominance of HTLV-2. In IDUs, however, the HTLV-1/2 spread occurs through both sexual and parenteral route. Long-term cocaine use (including injectable use) associated with unprotected sex and multiple sexual partners have enabled the spread of HTLV-1/2, especially the HTLV-2. In Brazil, injecting drug use, sharing of drug use paraphernalia and unprotected sex with IDUs have already been reported as factors associated with HTLV-1/2 infection. The use of injecting drugs has also been indicated as an important factor associated with the transmission of HTLV-2 in Brazil, Argentina and Spain43. Mental comorbidities are disproportionately common in people who regularly used illicit drugs44. A program for drug treatment, including the availability of resources to minimize the harm associated with the use of illicit drugs, should be implemented. The availability and access to diagnosis, prevention and treatment must be improved urgently in the state of Pará.The present study points to several key implications for public health intervention to prevent the HTLV transmission in the Amazon region. First, none of the PWDUs were aware of HTLV-1/2 infection status and therefore were not under medical supervision. This points to a major deficiency in the state of Pará regarding the availability of laboratory HTLV-1/2 tests. This situation needs to be urgently addressed through improved required resources, services and technical skills because the diagnosis is an essential tool for surveillance, prevention and clinical monitoring, especially in an endemic area. Furthermore, it points to the need for improved targeted prevention concerning HTLV-related risks. Given that primary risk factors for HTLV identified risky sexual behavior, there is evidently an urgent need for improved prevention of unprotected sex through targeted education and distribution of prevention resources or materials (e.g. condoms). It will furthermore be important to understand to which extent the common sexual risk behaviors in the study population are directly associated with behavioral or economic motivation of drug use or procurement, as this will determine the need for other or more specific prevention targets/measures (e.g. drug dependence treatment) towards reducing HTLV risk behavior. Overall, considering the duration and intensity of illicit drug use reported by PWUDs in this study, most participants probably require treatment for chemical dependence/disorders45. Confidential information on criminal or illicit activities such as trafficking and drug use or risky sexual behavior could have omitted by PWUDs while providing information. Furthermore, HTLV-1/2 screening used EIA, so recent infections with small concentration of anti-HTLV-1/2 antibodies may have not been detected, thus resulting in false negative test result, as previously reported in the Amazon region46. Finally, the ability to establish causality is limited in cross-sectional study.This study has limitations and should be considered. The study restriction to 28 of the 147 municipalities of the state of Pará was the first limiting factor, indicative that the sample may not represent the population of PWUDs. Second, PWUDs who initiated illicit drug use during adolescence were excluded by the age limit of 18 years or older. In addition, there are other alternatives for sampling hidden populations as respondent-driven samplingThis study is unique in bringing together phylogenetic, epidemiological and behavioral risk factors for the HTLV-1/2 transmission in the Amazon region. High HTLV-1/2 prevalence was found among PWUDs in the Brazilian state of Pará, with a distinct predominance in NIDUs and IDUs. Sexual risk behaviors contributed significantly to the virus acquisition and spread within this vulnerable population, and also to the general population. The inefficiency of the health care system makes the epidemiological scenario even more worrying since there is an urgent need to implement actions for the control, prevention and treatment of the HTLV-infected people.47.This cross-sectional study was based on biological and self-reported socio-behavioral data from a convenience sample of 826 PWUDs from 28 municipalities located in the state of Pará, northern Brazil Fig. . All samAlbumin gene fragment amplification  using real-time polymerase chain reaction (PCR)48. The presence of HTLV-1/2 DNA was evaluated using two protocols: (1) Amplification and detection of non-homologous fragments of the pol gene using real-time PCR48; and (2) Amplification of pX region fragments using Nested-PCR followed by enzymatic digestion with TaqI49. HTLV-1/2 infection was defined by the presence of proviral DNA by either of two methods.All PWUDs samples were tested for the presence of anti-HTLV-1/2 antibodies  by enzyme-linked immunosorbent assay (EIA). EIA-positive samples were subjected to nucleic acid isolation using the Wizard Genomic DNA Purification Kit . The presence of DNA was evaluated by 49. These fragments were subjected to 1.5% agarose gel electrophoresis and subsequently purified using a commercial kit . The nucleotide sequencing was performed using the BigDyeTerminator 3.1 kit  by capillary electrophoresis in the system . The nucleotide sequences obtained were edited and aligned with the program CLUSTAL W, implemented in BioEdit software50. Based on Akaike’s (AIC) and Bayesian (BIC) information criteria, the most suitable phylogenetic cluster model for the HTLV-1 and HTLV-2 data was indicated by JModelTest2 software51. BIC was used in Bayesian inference using MrBayes 3.2.1 software52. AIC was used in the Maximum Likelihood (ML) and Maximum Parsimony (MP) analyzes using PAUP* 4.0b10 software53. The Bayesian analysis consisted of two independent runs with chain length of 2,000,000 MCMC runs each and 10% of burn-in, sampled every 1000 interactions. The robustness of the groups was evaluated using 1,000 bootstrap replicates. Topologies of the phylogenetic trees were visualized using TreeView software54. Reference sequences of the HTLV-1/2 subtypes, available from GenBank, were added in the alignment  < 0.05 significance value was considered for all analyses.SPSS 20.0 for Windows was used to perform all statistical procedures. Chi-square test was used to identify significant differences between subgroups of PWUDs. Odds ratios (OR) and 95% confidence intervals (CI) were used as measures of the strength of association between HTLV-1/2 infections (outcome) and independent variables by logistic regression models (bivariate and multivariate)The present study was approved by the Ethics Committee on Research Involving Human Subjects of the Federal University of Pará, Brazil (CAAE: 37536314.4.0000.5172). All procedures performed in this study were in accordance with the relevant guidelines and regulations. All PWUDs were included after providing informed and written consent, and participants with positive results for HTLV-1/2 infections received counseling and were directed to care in the public health network.SUPPLEMENTARY MATERIALS


HTLV-1 infection is endemic in Brazil. About 1 to 2% of the Brazilian population is estimated to be infected, but most infected HTLV-1 individuals do not know about their own infection, which favors the continuity of sexual and vertical virus transmission. In addition, HTLV-1 associated central nervous system diseases and their pathophysiologic mechanisms are not fully understood. This study aimed to evaluate the correlation of spinal cord metabolism, viral and inflammatory profiles with features of neurological presentation in HTLV-1 infected individuals.AG (N = 21), symptomatic-SG (N = 11) and HAM/TSP-HG (N = 16) and a nested case-control study with HTLV-1 infected individuals-HIG (N = 48) and HTLV-1 non infected controls-CG (N = 30) that had their spinal cord analysed by Positron Emission Tomography with 18F-Fluordeoxyglucose (18F-FDG PET/CT). HTLV-1 infected individuals had 18F-FDG PET/CT results analyzed with clinical and demographic data, proviral load, cytokines and chemokines in the blood and cerebrospinal fluid (CSF).This is a cross-sectional study of a cohort including 48 HTLV-1 infected individuals clinically classified as asymptomatic-11), IL-6, IL-12p70 in the plasma; and ITAC, IL-4, IL-5, IL-8 (CXCL8) and TNF-alpha in the CSF. Using regression, thoracic spinal cord SUV  and CSF ITAC level were identified as the HAM/TSP predictors in the multivariate model.18F-FDG PET/CT showed hypometabolism in the thoracic spinal cord in HTLV-1 infected individuals. The method had an accuracy of 94.4% to identify HAM/TSP. A greater involvement of the thoracic spinal cord was observed, although hypometabolism was also observed in the cervical spinal cord segment in HTLV-1 infected individuals. Individuals with HAM/TSP showed a pro-inflammatory profile in comparison to asymptomatic and symptomatic groups, with a higher level of Interferon-inducible T-cell alpha chemoattractant  structures despite of the lack of clinical symptoms. To explain the found hypometabolism, the role of microcirculatory and metabolic factors in the pathogenesis of neurological diseases associated with HTLV-1 infection must be further investigated. It is paramount to evaluate the central nervous function and to compare the performance among HTLV-1 infected individuals considered asymptomatic to the uninfected controls. For the past 30 years, human T-cell lymphotropic virus type-1 (HTLV-1) has been isolated and associated with neoplastic, inflammatory, and infectious diseases. It is known that the neurological disorder associated with HTLV-1 comprises HTLV-1-associated myelopathy (HAM/TSP) or any other isolated signals and symptoms. Despite all the knowledge accumulated so far, the association of neurological diseases to HTLV-1 infection remains difficult and neglected. We designed this study in order to assess the degree of neurological impairment associated with HTLV-1 infection through a metabolic evaluation with 18F-FDG PET/CT. Our results evidenced a more pronounced hypometabolism in the spinal cord of individuals with neurological impairment, but also evidenced hypometabolism in asymptomatic HTLV-1 infected individuals. We believe that areas of the CNS with lower circulatory and perfusional balance are more vulnerable to HTLV-1 infection. Mechanisms of cellular entry of the virus may be associated with loss of microcirculatory homeostasis and predisposition to a breakdown of the blood-brain barrier in these areas. Further studies are still necessary to shed light on the mechanisms associated with brain and spinal hypometabolism. The Human T-cell lymphotropic virus HTLV-1) was associated with tropical spastic paraparesis (TSP) and HTLV-1 associated myelopathy (HAM) in the eighties  was asso. Both diThe combination of specific virus characteristics and the varied elicited individual immune response by the host leads to HAM/TSP in only 3% of infected individuals . Female The most common and non-specific finding observed in thoracic spinal cord magnetic resonance imaging (MRI) is atrophy of the thoracic spinal cord, which appear several years after disease onset . In a stHTLV-1 infection is chronic over decades, and may evolve with milder neurological impairment of defined HAM/TSP. The differential diagnosis of HTLV-1 milder neurological impairment is often neglected or confused. In order to evaluate central nervous system involvement, we designed this study to analyze blood and cerebrospinal fluid immunomarkers and thoracic spinal cord 18F-FDG PET/CT. Neuronal activity and function are measured indirectly through metabolism of glucose by 18F-FDG PET/CT . PET hasSubjects of this study were grouped according to their clinical neurological conditions. Significant statistical difference was not observed between groups with respect to age and follow-up time at GIPH. There was a significant difference regarding gender due to the higher proportion of female in the symptomatic and HAM/TSP group. The degree of neurological impairment was measured using the Expanded Disability Status Scale (EDSS) , which sNeurological symptoms and signs were statistically different between HTLV-1 infected groups. Prevalent concomitant clinical diseases, smoking and alcoholism were not statistically different between HTLV-1 infected groups, except sicca syndrome, with 19% prevalence in the HAM/TSP group. Only two HAM/TSP patients were in use of low-dose corticosteroids. The prevalence of depression was investigated in HTLV-1 infected individuals using the Geriatric Depression Scale– 15 items (GDS -15)  and may s = —.526, R2 = 0.28, p < .001.A reduction in metabolism in the spinal cord was observed in the 18-F FDG PET/CT images in HTLV-1 infected groups compared to uninfected controls and the degree of hypometabolism correlated with the degree of neurological impairment. Thoracic spinal cord SUV and EDSS score had a negative correlation rSignificant statistical difference was not observed between gender with respect to cervical (p = 0.514) and thoracic (p = 0.602) spinal cord SUV. Thoracic and cervical SUV were significant difference between the four groups, and between the HTLV-1 infected and the control group .In the multiple comparison between groups, post ROC analysis, there was significant difference between the control group compared to asymptomatic, p = 0.041  and HAM/TSP, p = 0.006  in the cervical spinal cord segment and between the control group and all HTLV-1 infected groups: asymptomatic group, p = 0.043 (CI 95% 0.005–0.415); symptomatic, p < 0.001 (95% CI 0.232–0.740); and HAM/TSP, p < 0.001 (CI 95% 0.273–0.719) in the thoracic spinal cord segment. The asymptomatic and HAM/TSP groups also showed a significant difference in the thoracic spinal cord SUV, p = 0.012 (CI 95% 0.047–0.525). Thoracic spinal cord SUV of the symptomatic group was similar to that observed in HAM/TSP group, p = 1.00. Thoracic spinal cord SUV presented accuracy of 94.4%, 85.7% and 81.6%, in the correct classification of groups: HAM/TSP and control , HAM/TSP and asymptomatic  and HTLV-1 infected and control (p < 0.001 CI 95% 0.72–0.91), respectively. In the ROC curve, thoracic spinal cord SUV ≤ 1.15 discriminated individuals of HAM/TSP group with 75% sensitivity and 100% specificity in relation to the control group and with 76.5% sensitivity and 85.7% specificity in relation to the asymptomatic group and cervical spinal cord SUV ≤ 1.32 discriminated HAM individuals with 68.8% sensitivity and 80% specificity in relation a control group. Cervical spinal cord SUV ≤ 1.42 discriminated between HTLV-1 infected and control groups, with accuracy of 72.2%, 64,6% sensitivity, 70% specificity, p = 0.001; CI 95% 0.60–0.84. Cervical spinal cord SUV was not able to discriminate between the HTLV-1 infected groups. Another point observed in the cervical and thoracic SUV was a progressive reduction in the standard deviation as the neuronal impairment increased, higher in control group and lower in HAM/TSP group.e 90; N 20) asymptomatic; 29  symptomatic; 336  HAM/TSP in the blood and 515  asymptomatic; 50  symptomatic; 1228  HAM/TSP in the CSF, p < 0.001, copies/103 cells  copies/103 cells in the symptomatic. On the other hand, CSF proviral load was higher than blood proviral load in all individuals with detectable CSF proviral load, except in an individual of the HAM/TSP group in which CSF proviral load was 33% lower than blood. In the other subjects with proviral load detectable in the CSF (N = 22), the ratio between CSF and blood proviral load showed an average increase of 9 ± 19.6 (1.38 to 95.64) times, median of 3.5 times. There was no statistically significant difference between the groups with regard to CSF and blood proviral load ratio (p = 0.670): N = 8, 4.6 ± 3.1 (1.4–9.4) times, median of 3.1 times in the asymptomatic group; N = 3, 5.3 ± 5.2 (1.4–11.2) times, median of 3.2 times in the symptomatic; and N = 11, 13.2 ± 27.5 (2.1–95.6) times, median of 4.2 times in the HAM/TSP group. Blood proviral load ≥ 193.5 and CSF proviral load ≥ 399.5 discriminated individuals between HAM/TSP and asymptomatic groups, with accuracy of 74,1%, 71,4% sensitivity, 75% specificity, p = 0.018; CI 95% 0.573–0.909 and accuracy of 81.5%, 91.7% sensitivity, 72.2% specificity, p = 0.004; CI 95% 0.660–0.969, respectively.Blood (p = 0.566) and CSF (p = 0.721) proviral load distribution did not show a gender difference. There was a statistically significant difference in the mean proviral load of blood and CSF among the three HTLV-1 infected groups: 142  in relation to gender in asymptomatic group and plasmatic IL-17A, IL-21 and CSF Fracktal in the symptomatic group. The cytokines that presented significant statistical difference between HTLV-1 infected groups were: ITAC (r = 0.43), IL-12p/70 (r = 0.26), IL- 17 A (r = 0.28) in the plasma, and ITAC (r = 0.41), IFN-gamma (r = 0.25), IL-5 (r = 0.33), IL-8 (r = 0.34) and TNF-alpha (r = 0.32) in the CSF .s = 0.56, p < 0.001). Plasmatic (N 47) and CSF (N 46) ITAC correlated with disease time , ambulation index , respectively, but not correlated with thoracic SUV.The cytokines and chemokines that presented significant statistical difference between asymptomatic and HAM/TSP groups were: plasmatic ITAC (p = 0.003), CSF ITAC (p = 0.001) and IL-8 (p = 0.010). Symptomatic and HAM/TSP groups showed statistical difference only CSF ITAC (p = 0.003). Asymptomatic and symptomatic groups did not present significant statistical difference in relation to the cytokines and chemokines analyzed. Only plasma ITAC correlated with ITAC in CSF , IL-5  and CSF ITAC  presented significant statistical difference in relation to high or low blood proviral load, respectively. However, when only the asymptomatic group was analyzed, there was no statistically significant difference of the cytokines in relation to high or low blood proviral load.Plasmatic ITAC ; ambulation index ; thoracic SUV ; CSF ITAC  and CSF IL-8  ; plasma ITAC  CSF IL-5 ; CSF IFN-gamma  and plasma IL- 17 A .EDSS correlated strongly with: disease time  . A moderIn this analysis, only the asymptomatic and HAM/TSP groups were considered, since the subjects of the symptomatic group were considered clinically undetermined. Univariate analysis indicated that female gender, thoracic spinal cord SUV, blood proviral load, plasmatic IL-6, CSF ITAC, IFN-gamma and IL-8 were predictors to HAM/TSP, when taken as independent risk factors. Only thoracic spinal cord SUV and CSF ITAC fit in the multivariate model, with a correct classification between asymptomatic and HAM/TSP individuals of 81.3%, as shown in In this study we sought to better understand the HTLV-1 clinical manifestations and its correlations with imaging and immune features. We enrolled and classified infected individuals according to their neurological status in asymptomatic, symptomatic and HAM/TSP groups. These groups were statistically different regarding neurological symptoms and signs, EDSS score and gender, but showed no difference regarding age and distribution of systemic diseases. Female gender was more frequent in the symptomatic (4.5:1) and HAM/TSP groups (4.3:1). This higher proportion of female gender in the symptomatic and HAM/TSP groups is explained by the higher incidence and prevalence of HAM/TSP in females, as observed in other epidemiological studies . For thiFirst, we discussed two questions. Are there HTLV-1 infected individuals without CNS impairment? Is HTLV-1 related CNS impairment restricted to the thoracic spinal cord? Analyzing this same population , we divided the sample according to the EDSS score  and processed each PET brain image using the statistical parametric mapping toolbox. Gray and white brain matter density image were generated for each subject. Analysis was able to classify with the accuracy of 85.7% (p = 0.001), 75% (p = 0.024) and 85% (p = 0.003) groups with EDSS score 0 (asymptomatic), 1–2.5 (intermediate) and > 2.5 (HAM/TSP), respectively, in relation to the control group. Diffuse cerebral hypometabolism was observed in HTLV-1 infected groups, including those with zero EDSS score compared to controls . Other sThe 18F-FDG PET/CT study shown hypometabolism in the cervical and thoracic segments of the spinal cord in all the three groups of HTLV-1 infected individuals including the group considered asymptomatic compared to controls. The 18-F FDG PET/CT was able to show greater involvement of the thoracic spinal cord, with excellent accuracy in the discrimination between HTLV-1 infected individuals (81.6%) and HAM/TSP patients (94.4%) from control group, as well as between HTLV-1 infected asymptomatic and HAM/TSP groups (85.7%).Currently, the major pathophysiological mechanism considered to explain CNS involvement in HAM/TSP is an immuno-mediated chronic inflammatory process in response to HTLV-1 infection, which damages nearby CNS components . We expeThe 18F-FDG behaves like glucose in the vascular and interstitial compartments and is transported to the intracellular compartment by passive transport, using glucose transporter proteins (GLUT), especially GLUT-1, present in most tissues and in the CNS. In the cell, 18-F FDG is phosphorylated and trapped in the intracellular compartment allowing the measurement of tissue metabolism . PET/CT High proviral load is a known risk factor for HAM/TSP development –32. The About the immune profile, there is no specific immunological marker for diseases associated with HTLV-1 infection , 34. TheITAC is induced by INF-gamma and it plays an important role in leucocyte trafficking, principally acting on activated CD4 + Th1 cells, CD8 + T cells and NK cells . IL-8 haIn the regression analysis, several independent predictors did not adhere to the multivariate model. It is possible that this was due to the small sample number and the multiple correlations observed between cytokines, chemokines and proviral load. Thoracic spinal cord SUV and CSF ITAC that adhered to the multivariate model were jointly good predictors of HAM/TSP, correctly classifying 81.3% individuals into asymptomatic and HAM/TSP groups. The results of multivariate logistic regression predicted that for a decrease of each unit of thoracic SUV, there was an increase of 99 times at risk of HAM/TSP and for each unit of increase of CSF ITAC, the risk of HAM/TSP increases 0.33 times.We are aware of limitations of our study, such as the small sample size, and the not HTLV-1 serological testing in PET/CT control group. Regarding this last point, we considered a very low risk of HTLV-1 infection in the control group given that the estimated prevalence of this infection in the general Brazilian population is around 1% . HoweverIn conclusion, HTLV-1 infection involves the CNS of most infected individuals even in absence of clinical symptoms. The thoracic spinal cord is definitely affected by HTLV-1, but other CNS segments are also compromised. 18F-FDG PET/CT seems a promising diagnostic tool to evaluate HTLV-1 CNS commitment, especially when associated to high CSF-ITAC levels might explain clinical outcome in different individuals. Our findings suggest that HTLV-1 infection may promote CNS vascular-neuronal dysfunction, propitiating breakdown of CNS protection barriers, lymphocytic infiltration with pro-inflammatory response that contributes concomitantly to neuronal damage. CNS segments with lower blood flow are more susceptible to vascular-neuronal dysfunction, which may be related to HTLV-1 cell-entry mechanisms. Genetic or acquired pathologies related to microcirculatory angiogenesis may be a possible factor to explain the development of HAM/TSP in specific and familial population groups. Further investigation is necessary to assess possible microcirculatory impairment and immune mediated mechanism in HAM/TSP pathophysiology. Our findings showed new venues for studies about potential metabolic and microvascular involvement in the neurological impairment of HTLV-1 infected individuals.All the procedures performed in the study, involving human participants, were approved by the Research Ethics Committee of the two participating institutions: Hemominas Foundation and Federal University of Minas Gerais, according to the 1964 Helsinki Declaration and its subsequent amendments.Informed written consent was obtained from all participants included in the study.This is a cross-sectional study of a cohort of HTLV-1 infected individuals and a nested case-control study between HTLV-1 infected and not infected controls in relation to spinal cord analysis by 18-F FDG PET/CT.The sample was non-probabilistic and composed of 50 individuals participating in the GIPH cohort, Belo Horizonte, Minas Gerais, Brazil. The GIPH cohort has a record of 1,200 participants among former blood donors diagnosed with HTLV-1 and 2 infection, their sexual partners, relatives, patients referred, and controls. It is an open cohort started in 1997. The sample for this study was a group of 300 HTLV-1 infected cohort participants who underwent neurological evaluation performed between 2007 and 2014. Participants were admitted in the period from January 2013 to October 2015 and data was collected and analyzed until December 2016.Two of the 50 subjects invited for this study were excluded. One due to HIV co-infection and the second although showed positive ELISA test for HTLV-1, confirmatory WB was inconclusive and qualitative PCR in blood was negative in two tests. The 48 HTLV-1 infected individuals who remained in the study were subdivided in three groups, according to neurological impairment, observed in the first appointment. Complementary tests were performed up to 60 days after this appointment. The group referred to as asymptomatic (AG) was formed by participants who did not present any symptom, or just few or common symptoms observed in the general population, insufficient to characterize neurological impairment. All individuals classified asymptomatic had a normal neurological physical examination . The symAG and HG) evaluated in this study is representative when considering the magnitude of the effect between the symptoms and signs observed in asymptomatic (<10%) and HAM/TSP group (> 50%), for α = 0.05  and β = 0.20. Sixteen individuals per group were predicted. The same cannot be attributed to the symptomatic group.The sample  or real-time PCR; four or more years of formal education, minimum of 18 and maximum of 70 years old. Co-infection evidenced by positive serological tests for human immunodeficiency virus, hepatitis C and B virus, Chagas disease, syphilis, neurological impairment or disease due to another etiology were exclusion criteria.Clinical—Individuals of the GIPH cohort, eligible for the project, were called for an individual appointment. In this first contact they were informed about project objectives and tests. Those interested in participating signed the Term of Consent and then underwent a neurological appointment, neuropsychological assessment and blood collection to perform serology of possible co-infections. The clinical and neurological data collected in the appointment were systematized and recorded into a database built in the Epidata 3.1 software. Complementary tests–Blood and CSF proviral load, cytokines, chemokines and 18F-FDG PET/CT of the whole body were performed at the Molecular Imaging Center (CTMM/UFMG). The participants were instructed to fast for at least 6 hours. First, capillary glycemia was measured, venous access was obtained and used to collect blood for proviral load, cytokines and chemokines assays. Venous access was maintained with 0.9% saline until the end of the procedure. Subsequently, participants received a 18F-FDG radiotracer bolus, 5.18 MBq/Kg, by intravenous administration. Participants were kept resting for 50 minutes in a room with dimmed light and low stimulation, prior to acquisition of PET/CT images in a time interval of approximately 10 minutes, and reconstructed in a 192 x 192 x 47 matrix using the OSEM (Ordered Subsets Expectation Maximization) algorithm, with 2 iterations and 20 subsets. Attenuation correction was performed using computed tomography (CT) images. A specific protocol was performed for targeted analysis of the central nervous system, especially the spinal cord. Standardized uptake value (SUV) of the thoracic and cervical spinal cord was measured. In the thoracic spinal cord, SUV was measured on T6, T8 and T10 levels, and at the cervical spinal cord on C5, C6 and C7 levels. The spinal cord levels were located using the CT image, having the sagittal plane to define the levels and axial plane to locate the medulla in the central canal of the spinal column. The SUV was measured in an oval area with 12 mm and 6 mm diameter, lateral-lateral and anterior-posterior, respectively  was used to quantify the expression levels of 19 cytokines in plasma and CSF. The HSTCMAG-28SK kit was designed to detect the following analytes: ITAC, GM-CSF, Fractalkine, IFN-gamma, TNF-α, MIP-3α, IL-1b, IL-12, IL-13, IL-17A, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-21 and IL-23. Plates were analyzed using a Luminex100/200 system. Median fluorescence intensity (MFI) was converted to concentration (in pg/ml) using an equation derived from the standard range of each analyte using Milliplex Analyst Software. Each sample was measured in duplicate. Cytokines in the blood and CSF results were not obtained in some HAM/TSP (blood 1 and CSF 2) due to technical difficulties in collecting the material or in the processing of the test. Proviral load in blood and cerebrospinal fluid material were immediately centrifuged at 3,000x g for 5 minutes to obtain the buffy coat layer and the cell pellet, respectively, used to obtain DNA. The DNA was extracted using the QIAamp DNA Blood Kit , following manufacturer’s instructions. The quantification of proviral load was performed by real-time PCR using SYBR Green. Real-time PCR was performed on the ABI Prism 7300 Sequence Detector System (Applied Biosystems). The proviral charge value was calculated as  x 10,000 and expressed as the number of proviral copies/10,000 cells. The proviral load in blood and CSF were not obtained in some participants due to technical difficulties during collection of the material or the processing of the test: asymptomatic (blood 1 and CSF 3) and HAM/TSP (blood 2 and CSF 4).2 test or Fisher test for categorical variables, t test or ANOVA for quantitative variables with normal distribution, Kruskal-Wallis and Mann-Whitney test for non-parametric data. Pearson or Spearman correlation coefficients were used according to the parametric or non-parametric distribution of the data, respectively. The ROC curve was used to calculate the accuracy, sensitivity, specificity and cutoff of some variables. In order to define which variables would form the best predictor model for HAM/TSP, univariate and multivariate regression were performed. Statistical significance was considered when p ≤ 0.05, except for multivariate entries where p = 0.10 and Bonferroni correction where p = 0.0167 were considered. The graphics were edited using the software: GraphPad Prism 7.0d and Microsoft Exel 2008 for Mac, version 12.3.1.Demographic, clinical and laboratory data were collected and analyzed using the SPSS 22.0. The descriptive analysis of the groups included the distribution of frequency and percentage of categorical variables, measures of central tendency according to the best suitability for quantitative variables. The comparative analysis between groups included XS1 Checklist(DOCX)Click here for additional data file.


The nucleus accumbens (NAc) is clearly implicated in reward processing and drug addiction, as well as in numerous neurological and psychiatric disorders; nevertheless, the circuit mechanisms underlying the diverse functions of the NAc remain poorly understood. Here, we characterized the whole-brain and monosynaptic inputs to two main projection cell types – D1 dopamine receptor expressing medium spiny neurons (D1R-MSNs) and D2 dopamine receptor expressing medium spiny neurons (D2R-MSNs) – within the NAc core and NAc shell by rabies-mediated trans-synaptic tracing. We discovered that D1R-MSNs and D2R-MSNs in both NAc subregions receive similar inputs from diverse sources. Inputs to the NAc core are broadly scattered, whereas inputs to the NAc shell are relatively concentrated. Furthermore, we identified numerous brain areas providing important contrasting inputs to different NAc subregions. The anterior cortex preferentially innervates the NAc core for both D1R-MSNs and D2R-MSNs, whereas the lateral hypothalamic area (LH) preferentially targets D1R-MSNs in the NAc shell. Characterizing the cell-type-specific connectivity of different NAc subregions lays a foundation for studying how diverse functions of the NAc are mediated by specific pathways. The nucleus accumbens (NAc), a brain region located in the ventral aspect of the basal ganglia, is widely recognized to act as a limbic-motor interface . The NAcThe NAc, however, is an inhomogeneous tissue that is often divided into two primary segments: the shell and the core . The funOverall, to dissect the diverse functions of NAc, it is crucial to determine the direct synaptic inputs to different anatomical subregions in a cell-type-specific manner. Previous studies using traditional tracers have provided valuable information about the connections of the NAc . HoweverIn the present study, we utilized such a virus-based labeling technique to identify monosynaptic inputs to D1R- and D2R-MSNs within different NAc subregions. We then compared the input distribution patterns among four groups defined by cell type and location . Although the input distributions of different cell types were highly similar, different NAc subregions had significantly different biased inputs from numerous brain areas. These findings provide an anatomical foundation for future studies of the neural circuits that underlie the diverse functions of the NAc.ad libitum. For all experiments, adult male mice (2–3 months old) were used.All the experimental procedures were approved by the Hubei Provincial Animal Care and Use Committee and the experimental guidelines of the Animal Experimentation Ethics Committee of Huazhong University of Science and Technology. BAC-transgenic D1R-Cre [MMRRC Tg (Drd1acre) EY262Gsat], and D2R-Cre [MMRRC Tg (Drd2-cre) ER44Gsat] mouse lines were acquired from the Mutant Mouse Regional Resource Center . These mice were backcrossed with C57BL/6J mice. C57BL/6J mice were purchased from Beijing Vital River (Beijing). All animals were housed with their littermates in a dedicated housing room under a 12/12 h light/dark cycle, and food and water were available 8 infecting units per milliliter. For adeno-associated viruses (AAVs), AAV9-EF1a-FLEX-EGFP-2a-TVA and AAV9-EF1a-FLEX-RG were packaged into 2/9 serotypes with final titers at 1–5 × 1012 genome copies per milliliter.All viral tools used in this study were packaged and provided by BrainVTA . The detailed production and concentration procedures for modified RV were previously described . The finBefore virus injection, experimental mice were anesthetized with 2% chloral hydrate, 10% ethylurethane, and 1.7 mg/mL xylazine in a 0.9% NaCl mixture  and mounted in a stereotaxic holder . After the skulls of the experimental mice were adjusted to be parallel to the reference panel, a craniotomy (∼0.5 mm diameter) was made above the targeted areas with a dental drill. For cell-type-specific tracing, 25–30 nl of helper AAV  was stereotactically injected into the NAc core  or the NAc shell  using a glass micropipette connected to a syringe pump . The micropipette was held in place for 10 min after the injection before being slowly retracted from the brain. Two weeks after helper AAV injection, 150–200 nl of RV-EnvA-ΔG-dsRed was injected into the same location under biosafety level 2 conditions through the same procedure mentioned above.One week after injection of RV, mice were perfused intracardially with 0.01 M phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in 0.01 M PBS. Mouse brains were carefully removed and placed in 4% PFA solution overnight for postfixation. The procedures for agarose embedding were generally performed as reported previously . BrieflyTo characterize the starter cells, sections near the injection site were selected for immunofluorescent staining. These sections were blocked with 3% bovine serum albumin (BSA) in PBS-0.3% Triton X-100 for 1 h at room temperature, incubated with the primary rabbit anti-DARPP-32 monoclonal antibody  overnight at 4°C, washed with PBS (three times for 10 min each at room temperature), incubated with Alexa-Fluor 647 goat anti-rabbit  for 2 h at room temperature, incubated with DAPI (1 ng/ml) for 10 min, and finally washed with PBS (five times for 7 min each at room temperature) prior to mounting the slides. We imaged the sections using an LSM 710 inverted confocal microscope (Zeiss).t-tests, the Mann–Whitney U test or one-way ANOVA followed by the Bonferroni correction were performed using SPSS (version 13.0). To quantify the similarity in input patterns, we calculated Pearson’s correlation coefficients. Error bars indicate the SEM.The locations of labeled neurons were manually registered, and the number of cells in each region from both hemispheres was quantified using Fiji (NIH) according to a standard mouse brain atlas . The inpFigures 1A,B). This modified RV can infect only those cells expressing TVA and requires RG to spread retrogradely to presynaptic cells. Mice were perfused 1 week later; the whole brain was sectioned at 50 μm, and every second section was selected for further analysis.Our aim is to trace, determine and compare the direct inputs to D1R- and D2R- MSNs in the NAc core and shell . We genetically targeted different projection neuron subpopulations in the NAc by using D1R- and D2R-Cre mouse lines, which have been shown to label D1R- and D2R-expressing neuron populations with high specificity . To demoFigures 1C,D). Combined with DARPP-32 immunostaining, a reliable marker of both populations of MSNs . For each group, the location of the vast majority of starter cells was restricted to the injected subregion of the NAc , although we found a small number of eGFP- and dsRed-double-positive neurons in neighboring nuclei: the lateral septal nucleus (LS), bed nucleus of the stria terminalis (BST), anterior olfactory nucleus (AON), and caudate putamen (CPu). However, these neurons made up a small fraction of total starter neurons in most animals . We found 7336–78192 dsRed-labeled presynaptic neurons in each brain (n = 24) . Variation of the cell numbers across animals was partly due to different injection volumes. Nevertheless, the numbers of transsynaptically labeled neurons had a linear relationship with the numbers of starter neurons .The starter cells were identified based on the coexpression of TVA-eGFP and dsRed. We observed a large number of double-labeled neurons near the injection sites , indicating the dependence of the RV infection on AAV-induced expression of TVA. Next, we repeated the retrograde transsynaptic tracing strategy in wild-type animals. Consistent with previous studies using similar methods , most likely due to the leaky expression of a low level of TVA. Note that all these non-specifically labeled neurons were found near the injection sites, within the NAc. Thus, to rule out the impact of this nonspecifically labeling on the mapping of long-range inputs, the following analysis excluded data originating from the NAc. Furthermore, to verify that the transsynaptic spread is under the tight control of RG expression, we injected only AAV9-EF1a-DIO-eGFP-TVA and SAD-ΔG-dsRed (EnvA) into the NAc core of D1R-Cre mice. A large number of labeled neurons were found near the injection site, and almost all dsRed-positive neurons coexpressed eGFP . In contrast, we did not detect any labeled neurons outside the NAc . Together, these results demonstrated that our tracing approach was suitable for mapping the long-range monosynaptic inputs to the NAc and that labeled neurons outside the injection sites represent monosynaptic inputs to projection neurons in the NAc.A number of control experiments were conducted to define the specificity of our tracing approach. We first injected SAD-ΔG-dsRed (EnvA) into the NAc core of wild-type animals  without prior AAV injection. This treatment resulted in no dsRed-labeled neurons . We found that NAc (including core and shell) neurons integrate inputs from diverse brain regions, from the anterior neocortex to specific midbrain areas . The dsRed-labeled neurons were predominantly found ipsilateral to the injection site, although sparser labeling was also observed in the contralateral hemisphere (data not shown). To quantify the distributions of long-range monosynaptic inputs, we counted the number of dsRed-labeled neurons within individual regions of each brain. Since the total number of labeled neurons varied across brain samples, to directly compare between experiments, we normalized the data in each region by the total number of labeled neurons in each brain (Supplementary Table S1).Representative coronal images from the transsynaptic tracing of inputs to each group show the whole-brain distribution of dsRed-labeled neurons , the hippocampus contributed most of the long-range inputs to the NAc, followed by the cortex, thalamus, olfactory areas, amygdala, pallidum, hypothalamus, septum/diagonal band of Broca, striatum (excluding the NAc), midbrain, and hindbrain . Even at this gross anatomical level, the pattern of inputs targeting the NAc core versus NAc shell shows a notable difference, particularly in the cortex and hippocampus : the cortex preferentially projects to the NAc core, whereas the hippocampus preferentially projects to the NAc shell.When the brain was divided into 11 major brain areas  and D2R-MSNs  were similar regardless of whether they were located in the NAc core or NAc shell. All regions projecting to D1R-MSNs also provided direct inputs to D2R-MSNs and vice versa. For both D1R-MSNs and D2R-MSNs, most of the inputs to the NAc shell were concentrated in a few brain regions , and conversely, inputs to the NAc core were broadly scattered . For example, the subiculum (S), paraventricular thalamic nucleus (PV), hippocampal CA1 region and ventral pallidum (VP) were the four major presynaptic regions that projected to the NAc shell, and inputs from these four regions accounted for ∼50% of total inputs to the NAc shell . These results suggested that both types of MSNs in the NAc might be regulated by many common upstream regions, whereas the NAc core might integrate information from a broader brain area than the NAc shell.To investigate input tracing in more detail, we further divided the brain into 84 regions. As mentioned above, the overall input distribution patterns of D1R-MSNs and D2R-MSNs in both NAc subregions show a high degree of similarity; therefore, we will consider these two types of neurons in each subregion together in the next analysis.Figures 5A,B, 6A,B; also see Figure 4). This difference was especially pronounced in the anterior cortex. For example, we found dense labeling in the medial and ventral orbital cortex (MO/VO), agranular insular cortex (AI) and prelimbic cortex (PrL) when tracing inputs to the NAc core, with significantly fewer labeled neurons when tracing inputs to the NAc shell. In addition, other anterior cortical areas, including the lateral and dorsolateral orbital cortex (LO/DLO), cingulate cortex (Cg) and secondary motor cortex (M2), also preferentially project to the NAc core .When the virus injection was targeted to the NAc core, the labeled neurons were widely distributed across cortical areas. In contrast, only sparse labeling was found in cortical areas when we targeted the injection to the NAc shell . Despite being near the NAc (the injection site), the dorsal striatum, which prominently includes the CPu, receives only a small fraction of the total inputs in both NAc core- and shell-targeted cases. In fact, labeled neurons in this structure may be “spill-over” from the NAc . That is, labeled neurons were often present at the border between the CPu and NAc, whereas the center of the CPu showed very sparse or no labeling. Conventional tracing studies have identified a strong reciprocal connection between the NAc and VP .Among the rostral forebrain areas, NAc core injection led to dense labeling in the AON and piriform cortex (Pir), whereas NAc shell injection caused the densest labeling in the tenia tecta (DTT/VTT) . While the BL makes up a similar proportion of the total inputs to the NAc core and shell, the BM makes up a significantly larger proportion of inputs to the NAc core . We found a sparser distribution of labeled neurons in the central nucleus (Ce) and medial nucleus (Me) for both NAc core- and shell-targeted cases . Interestingly, the lateral amygdaloid nucleus (La) shows a notable preference to project to the NAc core .In the amygdala, two subdivisions account for nearly all amygdala projections to the NAc: the basolateral amygdaloid nucleus  and basomedial amygdaloid nucleus  . The connectivity between the hippocampus and the NAc has attracted great interest as a circuit involved in spatial navigation and recognition of novelty . The NAc shell also received dense inputs from the CA1 region of the hippocampus, whereas the NAc core received relatively few inputs from this region .Consistent with conventional retrograde tracing studies , we foun novelty . Here, wFigures 4, 5C, 6C). These results correspond well to previous experiments using traditional retrograde tracers to label inputs to different subregions of NAc . Additionally, we detected a large number of labeled neurons in the parafascicular nucleus (PF) for both NAc core- and shell-targeted cases .In the thalamus, the projections to the NAc core and shell were roughly spatially segregated. For example, more dorsal and medial structures, such as the PV, particularly the anterior dorsal part, project preferentially to the NAc shell. In contrast, more ventral and lateral structures, such as the paratenial thalamic nucleus (PT) and mediodorsal thalamic nucleus (MD), show a notable trend toward projecting to the NAc core . We found that there was a slight preference for hypothalamic nuclei, such as the lateral preoptic area (LPO), medial preoptic area (MPA), and substantia innominata (SI), to project to the NAc core. In these areas, the lateral hypothalamic area (LH) is unique in that its inputs to the NAc are strongly biased toward the shell. Interestingly, our cell-type-specific tracing revealed that the LH preferentially targets D1R-MSNs over D2R-MSNs in the NAc shell (see section “Discussion”).Hypothalamic nuclei play a critical role in the regulation of feeding and circadian rhythms . Many hyFigure 4). The ventral tegmental area (VTA) and substantia nigra pars compacta (SNc), known to contain a high density of dopaminergic neurons, provided the major midbrain inputs to both NAc subregions. Some labeling was also sparsely distributed in the dorsal raphe nucleus (DR), median raphe nucleus (MnR), rostral linear nucleus of the raphe (RLi), periaqueductal gray (PAG), pedunculopontine tegmental nucleus (PPTg), retrorubral field (RRF), locus coeruleus (LC), and parabrachial nucleus (PB), suggesting rather weak inputs from these structures to the NAc.There were very few inputs from the midbrain and hindbrain to either the NAc core or the NAc shell , and the diagonal represents the same input proportion for each pair. Comparing the input patterns of D1R- and D2R-MSNs, we found that the vast majority of the brain regions, represented by the open circles centered around the diagonals, showed that they provided similar numbers of inputs to D1R-MSNs and D2R-MSNs in both NAc subregions . In the NAc core- and shell-targeted cases, we found that only 2 and 24 of 84 brain regions, respectively, provided significantly different inputs to D1R-MSNs and D2R-MSNs. The correlation coefficients between inputs to D1R-MSNs and D2R-MSNs in both the NAc core and the NAc shell were extremely high , confirming their high degree of similarity as observed earlier. On the other hand, when we computed the correlation coefficients between inputs to the same cell type in different NAc subregions, we found relatively low correlation coefficients . The open circles representing brain regions were relatively scattered from the diagonal. The D1R-MSNs in the NAc core and shell received significantly different inputs from 57 of 84 brain regions . There were 52 of 84 brain regions that provided significantly different inputs to D2-MSNs in different NAc subregions . The results are presented in Table 1.In our analysis of long-range tracing, we found that although the input patterns to D1R-MSNs and D2R-MSNs are highly similar, the input patterns to different subregions of the NAc differ significantly. To validate our previous findings, we quantified the similarities and differences in the distribution patterns of inputs to the four groups . ll pairs . Each opFigure 7E). Both D1R- and D2R-MSNs in the NAc core are more likely than those in the shell to receive monosynaptic inputs from the anterior cortex, while D1R-MSNs in the NAc shell receive disproportionally higher input from the LH. Through quantitative comparisons, we identified a list of brain nuclei that provided significantly different inputs to each comparison pair (Table 1).Using rabies-mediated transsynaptic tracing, we mapped the whole-brain monosynaptic inputs to the two main projection cell types (D1R-MSNs and D2R-MSNs) in different NAc subregions. Our experiments confirmed many previously demonstrated connections, but with cell-type specificity and quantitative analyses. Our data showed an extremely high degree of overall similarity between inputs to D1R-MSNs and D2R-MSNs in both NAc subregions. However, the input patterns to different NAc subregions differ significantly (Accumulated evidence has indicated that D1R-MSNs and D2R-MSNs in the NAc (core + shell) play differential and often opposing roles in reinforcement learning . A recenThe transsynaptic tracing strategy allowed us to quantitatively compare inputs to different cell types of different NAc subregions. Although inputs to D1R-MSNs and D2R-MSNs in the NAc show a high degree of similarity, we identified multiple nuclei that provide significant biased inputs to different NAc subregions. The difference observed in the anatomical connections of the NAc core and shell likely underlies their distinct behavioral functions. For example, we found that many anterior cortical areas  were preferentially labeled when starter cells were located in the NAc core but not the shell. Interestingly, most of these structures have long been implicated in value-based decision making , 2011. TThe NAc shell-to-LH pathway is well documented to play an important role in feeding control . For exaIt is worth mentioning that we detected only a small proportion of total inputs from the VTA and SNc, which contain the major dopaminergic populations, as well as from the DR and MnR, the sites of the major serotonergic populations. These results appear to differ from some traditional tracing studies indicating that the striatum (the CPu and NAc) receives substantial dopaminergic  and seroFigure 1C). It is possible that other regions that were not targeted in our experiments could show different input patterns.Rabies-mediated transsynaptic tracing has been widely used for elucidating neuronal connectivity. As previously published studies have done before us, we must emphasize again the limitations of this technique , includiAnother potential caveat arises when we consider that some MSNs coexpress D1 and D2 receptors (D1/D2-MSNs) , posing In summary, we mapped the organization of inputs to D1R-MSNs and D2R-MSNs in different NAc subregions (core and shell) using transsynaptic retrograde tracing with a modified RV. Although the input distributions of different cell types were highly similar, different NAc subregions had significantly different biased inputs from numerous brain areas. The similarities and differences observed in our study may provide new insight into the diverse functions of the NAc.All data generated or analyzed in this study are included in the manuscript.ZL and TX conceived and designed the project. ZL performed the tracing experiments. ZL, GF, AL, and JY performed the whole-brain data acquisition and analyzed the data. ZL and ZC generated the figures. ZL and TX wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


There has been an increasing interest in urban agriculture (UA) practice and research in recent years. Scholars have already reported numerous beneficial and potential adverse impacts of UA on health-related outcomes. This scoping review aims to explore these impacts and identify knowledge gaps for future UA studies.A systematic search was conducted in seven electronic bibliographic databases to identify relevant peer-reviewed studies. Articles were screened and assessed for eligibility. From eligible studies, data were extracted to summarize, collate, appraise the quality and make a narrative account of the findings.A total of 101 articles  were included in our final analysis. Among these articles, 38 and 37% reported findings from North America and Sub-Saharan Africa respectively. Quantitative studies revealed evidence of positive impacts of UA on food security, nutrition outcomes, physical and mental health outcomes, and social capital. The qualitative studies reported a wide range of perceived benefits and motivations of UA. The most frequently reported benefits include contributions to social capital, food security, health and/or wellbeing. However, the evidence must be interpreted with caution since the quality of most of the studies was assessed as weak to moderate. While no definitive conclusions can be drawn about the adverse impacts of UA on health, paying particular attention to contamination of UA soil is recommended.More peer-reviewed studies are needed in areas where UA is practiced such as Latin America and Caribbean. The inconsistency and the lack of strong quality in the methodology of the included studies are proof that more rigorous studies are also needed in future research. Nevertheless, the substantial existing evidence from this review corroborate that UA can influence different determinants of health such as food security, social capital, health and well-being in a variety of contexts.The online version of this article (10.1186/s12889-019-6885-z) contains supplementary material, which is available to authorized users. Until recently, food systems were given little attention in the agenda of urban planners . Urban aNumerous beneficial and potential adverse impacts of UA have been reported in urban planning and public health fields , 4. StudA study conducted in two large community garden networks in Salt Lake City, Utah has demonstrated that UA is a good physical activity that can prevent obesity. This study revealed that the community gardener participants had significantly lower body mass index (BMI) compared with their neighbors who did not participate in community gardening activities . The posDespite these potential positive effects on a variety of health determinants, researchers are demanding for further clarity on the benefits of UA . AdverseThe diversity of evidence in the literature could be explained by different methodological approaches, a focus on a specific aspect of UA, or the socioeconomic context where UA is implemented. This scattered knowledge makes it difficult to help urban planning stakeholders and could possibly misguide decision making; and would benefit from a synthesis of scientific knowledge on this matter.To our knowledge, there is only a limited number of systematic reviews on this topic –21. WhilFurthermore, beyond how UA was defined by authors, reviews showed a lack of diversity in the socioeconomic context and geographic scope in included primary studies. While Poulsen et al.  and WarrBased on these observations, there is still a need for systematic reviews that explore the impacts of UA in a broad socioeconomic context and geographic scope. By synthesizing vast amounts of literature, a systematic review can provide insights into understanding the general or common characteristics of individuals and communities involved in UA and how this activity affects their health.For this paper, the determinants of health are personal, socioeconomic, environmental and cultural factors that influence a person’s or community’s health. They include lifestyle, food, social and community networks, sanitation, environment etc. .The aim of this study was to explore the impacts of UA on the determinants of health and identify knowledge gaps for future UA studies by conducting a scoping review of peer-reviewed literature. The following research questions were investigated: i) what are the impacts of UA on the determinants of health? and ii) how do these impacts differ according to countries’ income level  and sociodemographic characteristics of participants? The responses to these questions will allow us to present the geographical location of UA studies, the type of impacts (positive or adverse) studied, and the methods utilized by scholars to assess the impacts of UA on the determinants of health.A systematic literature review on the impacts of UA on health determinants was performed. The wide range of health determinants, methods and results used in UA research suggests the use of a scoping review as described by Arksey and O’Malley  and LevaOriginal peer-reviewed articles published in English language journals from January 1980 to December 2017 were obtained from systematic searches of seven electronic bibliographic databases that include: PubMed, Embase, MEDLINE (Embase), CINAHL Plus with full text, Academic search premier (EBSCO host), CAB Abstract (ovid), and Web of science in January 2018. The final search strategy for PubMed can be found in an additional file  wereBy applying the eligibility criteria, two reviewers (PPA with background in agriculture and MAF with background in nutrition) have screened the articles for selection. The first selection was from title and abstract screening and the second one was from a full-text screening. All conflicts generated through the screening stages between the two reviewers were discussed until consensus was reached. When needed, a third opinion from two other authors (AL and GC) was consulted to reach consensus.Once the articles were selected, the following data were recorded in a spreadsheet: author(s), year, city, region, country’s income level, level of influence , characteristic of participants , type of UA , study purpose, study design , measurement methods, outcomes measured, and key findings. One author extracted the data, and another validated them to ensure accuracy prior the quality appraisal phase.For the quality appraisal of the included articles a checklist  that led to a total of 6683 titles and abstracts that were screened after the removal of duplicates. We retrieved a total of 418 full-text articles from our different libraries. Six records were unable to be obtained in full-text format. The full-text screening’s stage led to 118 potential articles relevant to our scoping review. Additional articles were excluded after full-text assessment for the reasons mentioned in the flowchart  countries. In addition, there is a diversity of countries n = 51 used quantitative methods, n = 29 used qualitative methods, and n = 21 have explored mixed methods .Seven studies discussed challenges related to UA Table . Among tThe evidence from mixed methods studies presents a set of UA impacts similar to those described in the previous sections for the quantitative and qualitative studies. However, the findings were dominated by qualitative evidence. Only six of the studies –73, 125 The remaining studies described a wide range of motivations, perceived benefits, and challenges of UA. Among the challenges documented is the fear due to potential food contamination and exposure of UA practitioners and their families to contaminants . GallaheThe included studies were categorized into three different influence levels  to measure or demonstrate the influence of UA on the determinants of health. Most of the studies from high-income countries demonstrate or measure the impacts at individual or community levels. On the other hand, studies from middle- and low- income countries explored the impacts mostly at household and individual levels Fig. .Fig. 3NuThis scoping review used standard systematic review methods to identify, select, and synthesize findings from 101 studies that reported impacts of UA on the determinants of health. We documented the state of UA peer-reviewed literature by analyzing the geographic scope, country-level income, type of UA activities, and key findings on the main reported determinants of health. Below, we provide important information on the implications of the findings and the gaps that emerged from the results of this review that can be relevant for UA practitioners, researchers, and policy makers.The results from the included quantitative and mixed method studies revealed some substantial evidence on the positive impacts of UA on food security and nutrition outcomes with increasing F&V consumption, improving food security status of urban farmers or nutritional status of children, food diversity, and/or dietary intake. However, this evidence has to be interpreted with caution. The outcomes reported are mainly based on cross-sectional surveys that rely on the participants’ self-reported responses. Most studies did not use validated tools for food security and nutrition outcomes’ measurement. In addition, in most cases, the authors do not always provide rigorous statistical evidence to sustain their findings. Other studies , 70, 123Although social capital is a determinant of health with limited reliable and valid measurement tools , it is lSeveral studies reported the adverse impacts of UA on health by assessing the risks related to consumption of food grown in contaminated urban soil. However, the findings do not allow to draw definitive conclusions on this topic. Most of the findings are based on authors’ assumptions of the amount of produce consumed or soil accidentally ingested by the population. This method is limited since it does not always reflect reality. In addition, in regard to ethics, it may be difficult to find the right way to assess health risks. This is because it is unethical for researchers to intentionally ask participants to consume contaminated produce in order to take the correct measurements. In order to improve the reliability of this type of data, it is probably better to record the real amount of produce consumed by the studied population.The findings from qualitative studies highlight a wide range of perceived benefits and motivations of UA. The benefits reported by UA practitioners were similar to their motivations. Supplying food in adequate quantity or quality, building social capital, improving physical and mental health, and saving on food expenses were the most common reasons and benefits perceived by UA practitioners. Other less common but important reasons include income, heathy lifestyle, and education and environmental consciousness , 90, 101In this case, most of the determinants of health’ themes emerged were explored in high-, middle-, and low- income countries. Lifestyle and cultural connection were the only two themes that appeared in high-income countries but did not in middle- or low- income countries. We expected some outcomes such as food security and nutrition to be associated more with middle- and low- income countries. However, they were also importantly assessed in various studies from high- income countries. This highlights a fact that other authors have already pointed out that food is also an important function of UA in the context of high-income countries .We also found that scholars from high- income countries are more likely to study the impacts of UA at individuals and/or community levels while studies from middle- and low- income countries are more likely to explore the contributions of UA on determinants of health at household and individual levels without considering the community aspect. This trend can be explained by the fact that community gardening is a type of UA with more presence in high-income countries  comparedAnother important aspect that was observed from our review is the lack of transnational or multi-city studies. Only one included study, Frayne et al. , which pAside from the US and Sub-Saharan Africa, there is limited peer-reviewed research in other world regions where UA is highly recognized and practised. For example, we did not find eligible studies in the Latin American and Caribbean’s region. However, cities such as Belo Horizonte in Brazil, Havana in Cuba, Rosario in Argentina and Quito in Equator from this region have been widely recognized as successful UA cases for their urban and peri-urban food practice and policy . Among tAll types of studies  were predominantly qualified as weak or moderate. The inconsistent or incomplete reporting of results from some included studies were due to lack of details on study settings, sample size justification, data collection, ethical issues, statistical evidence for quantitative studies, and theoretical approaches for qualitative studies. These arguments strongly support a lack of methodological rigor in the evidence of the impacts of UA peer-reviewed literature and add on the evidence already mentioned by several authors , 19, 21.This review applied a systematic and rigorous search strategy that retrieves several articles to answer our research questions and objectives. As our topic was focused on UA and health, several well-known electronic bibliographic databases related to health, nutrition, and agriculture were used as primary sources. Each element from the PICOS framework was searched with multiple keywords in order to target all relevant studies . HoweverNo study on air pollution and UA was included in our final analysis. This can be explained by the fact that we have unintentionally omitted air pollution as a key word in our search strategy. Additionally, we only considered peer-reviewed articles without assessing the existing evidence in the grey literature. The non-consideration of the grey literature restricts our findings to what was reported by scientific journals and possibly prevent the analysis of relevant cases that were rejected for publication by scientific editors.Our study reveals a need for more rigorous studies to demonstrate the impacts of UA on health-related outcomes and the possibility of exploring more transnational and multi-city research approaches to enrich the understanding on different contexts. This will help document best practices that can be implemented across different settings and contexts. As we stated earlier, UA remains a topic studied in specific or local contexts and that partly limits the capacity to generalize its potential impacts on specific determinants of health.By combining positive and adverse impacts of UA on the determinants of health, this review takes a holistic approach to invite practitioner, and policy makers to address UA challenges while promoting it. The insights gained from this study will encourage practitioners to test the urban soils prior to growing UA produce.This study illustrates a global picture of the current academic literature on the impacts of UA on the determinants of health. The study also designs the paths for future research in public health and urban planning domains. The inconsistency and the lack of strong quality in the methodology of the included studies are proof that more rigorous studies are needed to demonstrate the positive and adverse impacts of UA on different determinants of health. Nevertheless, the substantial existing evidence from this review corroborate that UA can influence different determinants of health such as food security, social capital, health and well-being in a variety of contexts . In addition, UA practitioners can be motivated by social benefits such as supplying quality food and building social capital. There are also many physical and mental health benefits to different population groups. In a holistic sense, the evidence suggests benefits of UA on multiple dimensions of health with few adverse effects; thus, UA can be recommended as an intervention that positively influence the determinants of health. Concerns regarding urban soil contamination have to be addressed by analyzing physical and chemical proprieties of the soil and applying decontamination techniques when needed to ensure that there are no health risks to UA users.Finally, we advocate for greater impact assessments by including transnational and multi--city approaches to compare the findings in different countries’ income level and geographic contexts. We also need a unified language to deal with heterogeneity in different types of UA identified.Additional file 1:Full electronic search strategy for PubMed. (PDF 30 kb)Additional file 2:Quality Appraisal for Quantitative studies and mixed methods studies. (XLSX 21 kb)Additional file 3:Quality appraisal for qualitative and mixed methods studies. (XLSX 19 kb)Additional file 4:Description of included studies. (XLSX 53 kb)


To prepare Ni(OH)2-PtCu octahedra, CO gas was directly introduced in a reaction process as selective capping agents on the PtCu(111) facet. Ni(OH)2 was naturally deposited on the Pt-Cu octahedra during the synthesis. Carbon supported Ni(OH)2-PtCu (Ni(OH)2-PtCu/C) as an EOR catalyst showed enhanced CO tolerance due to the existence of oxophilic Ni(OH)2 on the surface of Pt-Cu, facilitating water dissolution to produce OH adsorption and to promote complete CO oxidation to CO2. In addition, Pt-Cu alloy composition also showed improvement of CO tolerance because of modified d-band structure of the Pt atoms, thereby weakening the binding strength of CO on the catalysts. Therefore, the Ni(OH)2-PtCu/C showed enhanced EOR activity and durability compared to the Pt-Cu octahedra and commercial Pt/C counterparts.Here we report the synthesis of 9 nm Ni(OH) Moreove) Koper, . Additio) Koper, . These r2-Pt-Cu electrocatalyst for EOR. In this context, we here successfully synthesized 9 nm Ni(OH)2 decorated Pt-Cu octahedra in one-pot synthesis. We designed Pt-Cu octahedra as PtCu(111) alloy substrates and Ni(OH)2 decorated on the surface of Pt-Cu octahedra as the site for OHads, improving water dissociation. The as-obtained Ni(OH)2 decorated Pt-Cu octahedra showed enhanced catalytic EOR activity and durability compared to binary Pt-Cu octahedra and commercial Pt/C.Recently, facet-dependent electrocatalytic EOR has been intensively studied with the nanocatalysts of unique morphologies  acetylacetonate  were obtained from Alfa Aesar. Nickel(II) acetylacetonate , oleic acid , oleylamine , and benzyl ether  were obtained from Sigma-Aldrich. All chemicals were used as received without further treatment.Platinum(II) acetylacetonate [Pt(acac)2 (20.0 mg), Cu(acac)2 (13.3 mg), Ni(acac)2 (10.0 mg), OAm (4.0 mL), OA (4.0 mL), and BE (2.0 mL) was heated to 190°C within 20 min under Ar atmosphere and magnetic stirring. When the temperature reached 190°C, CO gas was bubbled into the mixture with a flow rate of 1 mL min−1, and Ar purging was stopped at the same time 2-PtCu octahedra were precipitated out by sequential addition of toluene (5 mL) and ethanol (10 mL). The supernatant was discarded by centrifugation at 3,000 rpm for 5 min. The resulting Ni(OH)2-PtCu octahedra were dispersed in toluene for further treatment.In a standard synthesis, a mixture of Pt(acac)2-PtCu octahedra was added into a toluene solution containing 31.5 mg of Vulcan XC-72 R carbon and kept under ultrasonic wave agitation for 2 h. The resulting Ni(OH)2-PtCu/C catalyst was centrifuged 3 times with toluene at 3,000 rpm for 5 min, and then dried under Ar protection at room temperature.A suspension of Ni(OH)2-PtCu/C catalyst (10 mg) was dispersed in HOAc  and then heated at 60°C for 2 h under magnetic stirring. After heating, the catalyst was washed three times with ethanol and dried under Ar protection at room temperature.Ni(OH)Transmission electron microscopy (TEM) images were obtained using a HT 7100 microscope  operated at an acceleration voltage of 120 kV. High-resolution TEM and energy dispersive X-ray spectroscopy (EDS) studies were carried out in JEM-2100F  and Titan G2 ChemiSTEM Cs Probe  operated at an acceleration voltage of 200 kV. The metal contents in catalysts were determined using inductively coupled plasma-optical emission spectroscopy . X-ray diffraction (XRD) patterns were obtained with a D2 phaser X-ray diffractometer . X-ray photoelectron spectroscopy (XPS) was carried out using a spectrometer  with Al Kα X-ray (1486.6 eV) as the light source. All spectra were aligned using the C-1s peak at 284.5 eV as reference.2) was used as a counter electrode. A glassy carbon disk  was used as a working electrode. The GC surface was previously polished using 0.05 μm Al2O3 suspension. Catalyst ink was prepared by mixing a specific amount of catalyst powder, deionized water (1.0 mL), isopropyl alcohol (0.25 mL), and 5 wt% nafion (5 μL) using ultrasonication for 10 min. The ink (10 μL) was dropped on a GC electrode and dried to form a thin film. The final Pt loading of electrocatalyst on a GC electrode was 10.2 μg cm−2. The each electrocatalyst loaded on electrode was pre-cycled in an Ar-saturated 0.1 M HClO4 solution between 0.08 and 1.20 V at a scan rate of 100 mV s−1, and then cyclic voltammograms (CVs) were recorded between 0.08 and 1.20 V at a scan rate of 50 mV s−1 in an Ar-saturated 0.1 M HClO4 solution at room temperature. In a typical CO stripping experiment, the electrode potential was held at 0.05 V for 5 min in a CO-saturated 0.1 M HClO4 solution for a fully adsorption of CO on the catalyst surface followed by flowing Ar for another 10 min to remove the CO in the solution, and then the CO stripping curve was recorded by cycling between 0.08 and 1.20 V at 50 mV s−1. For the EOR measurements, CV was conducted in an Ar-saturated solution containing 0.1 M HClO4 and 1 M ethanol between 0.08 and 1.29 V at a scan rate of 50 mV s−1. Chrono-amperometry curve of the catalysts was recorded for 3000 s in an Ar-saturated 0.1 M HClO4 and 1 M ethanol solution at 0.67 V. Long-term durability of the catalysts for EOR was performed in an Ar-saturated 0.1 M HClO4 and 1 M ethanol solution by applying cyclic potential sweep between 0.65 and 1.05 V at a scan rate of 100 mV s−1 for 2,000 cycles. Then, CV for EOR was recorded again in a fresh Ar-saturated 0.1 M HClO4 and 1 M ethanol solution for comparison with the initial CV curve of EOR.All electrochemical measurements were carried out using a standard three-electrode cell connected to potentiostats (CHI 600E from CH Instruments and VSP from Bio-Logic). A Ag/AgCl electrode was used as the reference electrode, and potential values were calibrated with respect to reversible hydrogen electrode (RHE). Pt mesh 2, Pt(acac)2, Cu(acac)2, OAc, OAm, and BE was heated to 190°C under Ar protection with magnetic stirring and then CO gas was bubbled into heated mixture with a flow rate of 1 mL min−1 while Ar purging was stopped. The mixture was heated again to 210°C, and CO bubbling was stopped when the temperature reached to 210°C. After the reaction was kept at 210°C for 40 min, a dark suspension including Ni(OH)2-PtCu was obtained. In previous studies, CO gas released from metal carbonyls was used as a selective capping agent for {111} facets of Pt-Ni and Pt-Cu alloy nanocrystals under an OAm/OA co-surfactant system 2-PtCu octahedra shows uniform size and shape  of face-centered cubic (fcc) bulk (2-PtCu octahedron (2-PtCu octahedra loaded on carbon supports (Ni(OH)2-PtCu/C) were shown in 2-PtCu/C was then treated with HOAc at 60°C for 2 h. 2-PtCu/C after HOAc treatment (PtCu/C-HOAc), respectively. After the acid treatment, the average edge length of PtCu/C-HOAc was 9.2 ± 1.1 nm and d-spacing for Pt lattice fringes was 0.222 nm, similar to the pristine Ni(OH)2-PtCu octahedra. STEM with EDS mapping image of a single PtCu/C-HOAc shows an octahedral nanocrystal consisting of Pt and Cu but absence of Ni nd shape . High-recc) bulk  (Choi etcc) bulk . Scannintahedron . The Ni2-PtCu/C were also leached out and thus the surface composition of PtCu/C-HOAc is assumed to be Pt-rich.To confirm the elemental composition of the as-prepared catalysts, ICP-AES was carried out and the resulting data are listed in 3/2 spectrum of Ni(OH)2-PtCu/C shows a peak assigned to Ni(OH)2 (855.9 eV) and Ni(OH)2 satellite (861.2 eV), indicating the formation of Ni(OH)2 on the surface of Ni(OH)2-PtCu/C 2-PtCu/C and PtCu/C-HOAc showed a peak assigned to Cu(0) metal (932.3 eV), Cu(OH)2 (934.6 eV), and Cu(OH)2 satellite (942.7 and 944.7 eV) 2 leaching similar to Ni(OH)2. The Pt 4f spectra of both catalysts show a peak fitted with doublets corresponding to Pt(0) metal (71.2 and 74.5 eV) and Pt2+ (72.0 and 75.3 eV)  and Cu2+ ions (0.340 V) were primarily reduced to Pt and Cu metals forming Pt-Cu alloy nanocrystals but Ni2+ (−0.257 V) was dissolved in the mixture. Under the CO gas, Pt-Cu octahedra enclosed by {111} facets were mainly produced, and then rest of Ni2+ was deposited on the surface of Pt-Cu as Ni(OH)2 form   (Moulder44.7 eV) . The Cu(75.3 eV)  (Xia et 75.3 eV) . Figure t and Cu , indicat2-PtCu/C and PtCu/C-HOAc catalysts were evaluated as anodic oxidation electrocatalysts for EOR. For comparison, we benchmarked their electrochemical properties against the commercial Pt/C catalyst. Electrochemical measurements were conducted by using a potentiostats with three electrode system. The three different catalysts loaded working electrodes were pre-cleaned in Ar-saturated 0.1 M HClO4 solutions between 0.08 and 1.20 V (vs. RHE) with a scan rate of 100 mV s−1 at room temperature. 2-PtCu/C after cleaning, showing similar morphology and composition to its status before cleaning. CVs were then recorded with the same potential range at a scan rate of 50 mV s−1 as shown in 2 g−1 for Ni(OH)2-PtCu/C, PtCu/C-HOAc, and the commercial Pt/C, respectively (2-PtCu/C relative to that for the Pt/C is due to the bigger size and the presence of surface Ni(OH)2 2 from the catalyst surface after HOAc treatment 2-PtCu/C, PtCu/C-HOAc, and the commercial Pt/C, respectively  and Pt/C (0.80 V) catalysts. This result imply weaker CO adsorption strength on the Ni(OH)2-PtCu/C and PtCu/C-HOAc catalysts than that on the Pt/C. 2 on Pt-Cu surfaces reduced the onset potential compared to the PtCu/C-HOAc owing to the presence of more OH adsorption sites to promote the CO oxidation, namely bifunctional effect  at the potential showing highest current density in a forward scan exceeded those of the PtCu/C-HOAc (7.76 mA cm−2) and the Pt/C (0.65 mA cm−2), respectively. The EOR mass activity of Ni(OH)2-PtCu/C is recorded the highest value of 1.97 A mg−1 compared to PtCu/C-HOAc (1.89 A mg−1) and the Pt/C (0.39 A mg−1) 2, which can sufficiently promote the water dissociation and thus formation of OHads species to oxidize the toxic CO on the neighboring Pt surfaces 2 on the surface was prone to dissolve in acidic media, the Ni(OH)2-PtCu/C showed improved catalytic EOR performance and CO oxidation ability compared to the PtCu/C-HOAc, implying the influence of surface Ni(OH)2. Consequently, the cooperation effect of Ni(OH)2 and Cu leads to a significant increase in EOR activity and kinetic.The resulting Ni(OH)ectively . The low A mg−1) . The enh2-PtCu/C, PtCu/C-HOAc, and the commercial Pt/C were recorded at 0.67 V for 3,000 s to evaluate the CO tolerance on each catalyst 2-PtCu/C has a much higher CO tolerance than the PtCu/C-HOAc and the Pt/C. Long-term durability tests of Ni(OH)2-PtCu/C, PtCu/C-HOAc, and the Pt/C for EOR were performed by applying cyclic potential sweeps between 0.65 and 1.05 V at 100 mV s−1 in an Ar-saturated solution containing 0.1 M HClO4 and 1.0 M ethanol at room temperature. Then, CVs were recorded in a fresh Ar-saturated 0.1 M HClO4 and 1.0 M ethanol solution between 0.08 and 1.29 V at 50 mV s−1 2-PtCu/C, mass activity was slightly increased even after 1,000 cycles. Since the Ni(OH)2 can be dissolved out from the Ni(OH)2-PtCu/C during EOR cycles under acidic electrolyte, more exposed surface of Pt-Cu and optimized amount of Ni(OH)2 can be assumed to show slightly enhanced EOR activity after 1,000 cycles 2-PtCu/C has improved catalytic activity and durability toward the ethanol electrooxidation.Chrono-amperometry (CA) curves of Ni(OH)catalyst   facet. Ni(OH)2 was naturally deposited on the Pt-Cu alloy octahedra due to the difference of standard reduction potentials of Pt, Cu, and Ni. The presence of Ni(OH)2 on the surface of catalysts and Pt-Cu alloy octahedra were confirmed by TEM, EDS, XPS, and XRD analyses. Electrochemical CO stripping experiment showed that enhanced CO tolerance of the Ni(OH)2-PtCu/C compared to that of the PtCu/C-HOAc and the commercial Pt/C due to the existence of Ni(OH)2 on the surface of Pt-Cu and incorporation of Pt with Cu. Pt-Cu alloy composition showed improved CO tolerance because of the down-shifted Pt d-band structure, thereby weakening the CO binding strength on the Pt. In this context, Ni(OH)2-PtCu/C and PtCu/C-HOAc showed enhanced catalytic activity and durability toward ethanol electrooxidation in acidic electrolyte compared to the commercial Pt/C.We have successfully synthesized the Ni(OH)All datasets generated for this study are included in the manuscript/YH, HK, and S-IC designed experiments, finished the synthesis and characterization analysis of materials, and carried out electrochemical experiment. HL and JK offered valuable suggestions to analysis of materials. YH, HK, and S-IC wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.