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Official websites use. Share sensitive information only on official, secure websites. Address all correspondence to: Yasuhiro Yamamura, Ph. In order to assess the extent of Xylazine Xyz injection in Puerto Rico, two waves of used-syringe collections were performed. In the first, syringes were gathered, anonymously and without additional information; in the second, a short interview, also anonymous, was administered. We found Xyz in With a high prevalence of skin ulcers Surprisingly, though a higher percentage of Xyz users than non-users had college-level educations The use and consequences of speedball are well documented Colon et al. Employees of a local health-risk reduction program visiting a shooting gallery observed that several injecting drug users IDUs were disoriented and had trouble standing; many had open skin ulcers. In order to assess the extent of Xyz use in PR, two waves of syringe collections were performed and the contents of the syringes analyzed. Though there are reports on acute toxicity induced by Xyz injection or inhalation Spoerke et al. The first wave ran from October to November, ; the second, March — April, After the first wave, 22 sealed containers containing exchanged syringes without identifying information were delivered to the laboratory by NEP staff members. Ten syringes each were chosen at random from 21 containers; an additional 50 syringes were taken from Arecibo, where the evidence for Xyz abuse was first detected. In the second wave targeting eight of the 11 original towns , two syringes and a short questionnaire were received from each of the participants; the syringes and questionnaires were matched solely by coded numbers, assuring anonymity—thus exempting the study protocol from the full review requirements of the Institutional Review Board of the Ponce School of Medicine FWA Containers and questionnaires were delivered to the laboratory. All of the syringes collected were tested. In both waves, each syringe together with second-wave questionnaire was assigned a bar-code number. Data entry and analysis were conducted using the bar-codes. Data were acquired in full-scan mode; compounds were identified from the program data bank. Questions were asked to evaluate awareness of the effects and side effects of Xyz. Participants were asked whether, to their knowledge, their syringes contained Xyz. Finally, the NEP staff conducting the interviews recorded whether the participants had visible skin ulcers; if no ulcers were visible, participants were asked about ulcers in clothed areas. Categorical variables were compared using Z-test. ANOVA revealed consistent patterns of drug use from one collection wave to the next. Out of syringes containing Xyz, speedball was found in Generally, greater quantities of Xyz were found in the syringes collected from municipalities having a higher prevalence of Xyz use. Regarding age, age at first injection, and number of injections per day, there was no significant differences between the two groups. Proven Xyz abuse was somewhat more directly associated with poor health conditions than self-described use. Demographic profiles of and the presence of skin ulcer among Xyz users and non-Xyz users. Information was obtained from addicts who responded to the study questionnaire and who returned at least one syringe that contained detectable amounts of one or more drugs. The figures represent the respective ranges and the median value , respectively. Statistical significance was calculated for each category between the two groups by using Z-test ns: not significant. Xyz, a veterinary analgesic, sedative, and muscle relaxant, is used on large animals such as horses and cattle. A recent survey Hewson et al. An anecdotal case report of human Xyz use describes a patient who experienced attacks of fatigue, faintness, and blurred vision Lewis et al. Xyz users in our study were observed to suffer physical dysfunctions comparable to those seen in lower doses. Indeed, users of Ketamine another veterinary analgesic were reported to have a high incidence of skin ulcers; presumably, the pain of infections resulting from repeated scratching and cutting under the influence of the drug goes unnoticed Micallef et al. In our analysis, Xyz use was singly most closely associated with the presence of skin ulcer. Being homeless, which may compromise their hygiene, only slightly increased the risk of having skin ulcer. There are no reports detailing specific behaviors that might account for the increased incidence of skin ulcers among Xyz users. Syringes collected in these areas reflect that fact, containing greater quantities of Xyz than those from other regions. While access to Xyz at agricultural supply stores is legally controlled, apparently it is available on the illicit market. The present study explored neither how users obtain Xyz nor who prepares the Xyz-containing admixtures. Because Xyz content remained fairly consistent within a single collection site though varying greatly from region to region , it seems likely that local dealers prepare and distribute the Xyz-containing drugs. More detailed analysis of Xyz distribution patterns may help curtail the spread of Xyz abuse. Speedball was co-present in There are a number of acute toxicology reports Poklis and Mackell ; Spoerke et al. To determine the multiple impacts of these two factors, further studies are required, the results of which are likely to be quite different from those presented in existing literature. Still, one can definitively say that chronic Xyz use is associated with physical deterioration. Though we found that Xyz users are more likely to be college educated The results presented here must be interpreted with care as they may not precisely represent the overall pattern of Xyz abuse in PR: both waves of syringe collection were limited to sites where participating NEPs routinely conducted needle exchanges—sites selected for programmatic or public policy reasons, rather than for scientific sampling purposes. Consequently, large areas were not included. Nevertheless, both waves of syringe collection yielded almost identical rates of Xyz abuse the short interview given to second-wave participants did not seem to hinder the collection process , leading to the conclusion that the drug-abuse pattern determined by this study is an accurate portrayal of Xyz abuse in PR. Unfortunately; there is a dearth of information regarding the pharmacological, epidemiological, and clinical aspects of Xyz injection on the island. Though not officially acknowledged, there have been—to date—three drug-related deaths in PR possibly linked to Xyz overdose; these, coupled with our findings, argue the need for a more thorough investigation of Xyz abuse in PR. It is unclear at present if the observed abuse of Xyz with speedball represents a new trend of drug abuse pattern or how wide spread Xyz abuse is beyond this Caribbean island of PR. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. As a library, NLM provides access to scientific literature. Drug Alcohol Depend. Published in final edited form as: Drug Alcohol Depend. Find articles by Juan Panelli. Find articles by Bob Ritchie. Find articles by Yasuhiro Yamamura. Issue date Aug 1. PMC Copyright notice. The publisher's version of this article is available at Drug Alcohol Depend. Elementary School 3 7. Middle School 6 High School 21 College 9 On street 25 In a shelter 0 1 1. With relative s 5 With parent s 7 In own house 1 2. Open in a new tab. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.
Colombian, Dominican, and Puerto Rican criminal groups are the principal wholesale-level distributors of cocaine in Puerto Rico and the U.S. Virgin Islands.
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Official websites use. Share sensitive information only on official, secure websites. Sepulveda-Orengo, msepulveda psm. The use, distribution or reproduction in other forums is permitted, provided the original author s and the copyright owner s are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Clinical and preclinical research on cocaine use disorder CUD has shown that sex differences in drug seeking are influenced by hormonal fluctuations. Preclinical studies show that estradiol in females facilitates the extinction of cocaine-seeking behavior indicating a possible role in regulating extinction learning. However, it is unclear whether estradiol plays a role in male extinction learning as it does in females. Furthermore, how endogenously aromatized estradiol affects drug addiction in males is unknown. Therefore, this study investigated whether endogenous estradiol regulates cocaine seeking in male rats. We hypothesized that decreased aromatase enzyme activity, resulting in decreased estradiol synthesis in male brains, will impair extinction learning leading to increased cocaine-seeking behavior. This hypothesis was tested using cocaine-conditioned place preference CPP , and short access self-administration SA , followed by extinction and reinstatement. Before each extinction session for CPP or SA, male rats received an injection of either 1 low dose or 2. FAD groups showed dose-dependent effects on cocaine-seeking behavior compared to the vehicle group during CPP extinction. Interestingly, only the low dose FAD group had decreased active lever pressing during cue- and cocaine-primed reinstatement compared to the vehicle group. Neither dose of FAD had an effect on sucrose extinction or reinstatement of sucrose seeking. These results from CPP experiments suggest that estradiol may impact extinction learning, as a low dose of FAD may strengthen the formation of cocaine extinction memory. Additionally, in male rats undergoing cocaine SA, the same low dose of aromatase inhibitor effectively reduced reinstatement of cocaine-seeking behavior. Thus, estradiol impacts cocaine seeking and extinction in both males and females, and it may also influence the development of sex-specific treatment strategies for CUD. Keywords: cocaine, estradiol, conditioned place preference, extinction, self-administration, rats, aromatase inhibitor. Sexual dimorphism in drug addiction has been extensively studied over the last three decades Robbins et al. Clinical studies focused on the neurobiology of addiction have shown that women are at higher risk of addictive disorders than men Jackson et al. More significantly, estrogens, which have been studied from a sex-difference perspective in multiple drug-addictive disorders in which differences between males and females are observable Rubonis et al. Interestingly, estrogens are also synthesized de novo in the brain Hojo et al. Within the male and female physiological system, estrogens are required for brain development Wu et al. Up until the discovery that sex differences contribute to a wide range of diseases Schuurs and Verheul, ; Scheidt-Nave et al. As the relevance of conducting research on both males and females became recognized Wald and Wu, , new studies revealed how in many instances hormones produced sex-dependent differences, including in addictive disorders Robbins et al. Additionally, female rats exposed to cocaine self-administration SA learn to seek out and consume the drug faster than their male counterparts Lynch, Despite an increased understanding of E2 effects in females, whether E2 was important for drug seeking in males has been neglected. There are several types of estrogens, with estradiol being the most potent form. E2 is known to modulate cocaine addiction in females by increasing the sensitivity of the brain reward system Galankin et al. In a cocaine SA paradigm, female rats acquire cocaine-seeking behavior more readily than males Lynch, Importantly, Twining et al. In particular, ovariectomized female rats showed persistent CPP that did not extinguish across more than forty tests, but injecting E2 rescued extinction Twining et al. Thus, E2 plays a central role in cocaine seeking and extinction in females. In males, however, little is known about the role of E2 in cocaine seeking and extinction. The impact of E2 on the cocaine-reinforcing effects in males is still unclear. One study found that E2 administration increased cocaine SA in female rats but had no effect on gonadectomized male rats Jackson et al. However, another study found that chronic administration of E2 in male rats increased cocaine choice over food during concurrent reinforcement and under progressive ratio Bagley et al. These studies investigated the impact of E2 on the reinforcing effects of cocaine, and we extend these findings to include extinction and reinstatement. Relevant to our study, Graham and Milad have shown that inhibiting the production of E2 by the aromatase inhibitor, Fadrozole FAD , negatively affects the ability of male rats to extinguish conditioned fear. The results of this study suggest that E2 is required for the successful extinction of learned fear, prompting us to investigate the role of E2 in the extinction of drug seeking in males. Therefore, we hypothesize that E2 is a mediator of extinction of cocaine seeking in both females and males. Aromatization of androgens to estrogens along the cholesterol pathway is the last step in steroid synthesis. The aromatase enzyme, which is a Cytochrome P protein found in many body parts the brain, liver, breast, and fat; Rochira et al. FAD, a potent non-steroidal second-generation aromatase inhibitor, can efficiently halt brain synthesis of E2 Afonso et al. The aim of the present study is to examine if endogenous E2 is involved in the extinction of cocaine seeking. Two behavioral paradigms for cocaine use disorder were used to determine the role of E2 in the extinction of cocaine seeking: CPP and SA. For cocaine CPP experiments, rats were pair-housed in standard plastic cages clear and fed with pellet chow and water ad libitum , except when placed in experiment boxes. CPP experiments took place during the light phase. SA experiments took place during the dark phase. Body heat was maintained throughout the surgical procedure using heating pads. For catheter implantation, a guide cannula CG, Plastics One attached to silastic tubing 0. Catheters exiting the skin were secured by a subdermal surgical mesh Atrium and by a cannula. A catheter cap was used when the rats were not connected to infusion pumps. Before behavioral protocols, animals were provided seven days to recover from surgery. Catheters were flushed daily with 0. Cocaine hydrochloride provided by the NIDA drug supply program was dissolved in sterile saline 0. In SA, cocaine was infused at 0. Sterile saline was used for control or vehicle solution 0. The larger conditioning chambers had wire mesh with white walls in one and gold-grated flooring with a black wall in the other. The center chamber had aluminum sheeting as flooring. Removable partitions were used to isolate the rats within specific chambers during conditioning. During baseline and CPP trials, the doors were removed to allow free access to the entire apparatus. If the beam furthest from the door was broken, the rat was considered to be in the larger chamber. If only the beam closest to the center chamber was broken, then the rat was considered to be in the center chamber. During all phases of the experiments, the room was kept in semi-darkness. Rats spent equal time in the larger conditioning chambers, and less time in the center chamber as previously reported Yousuf et al. After the acclimation period, rats began cocaine place conditioning training. Two CPP experiments were conducted as follows. After conditioning, rats were assigned to different treatment groups for extinction. Thirty min before extinction training, rats were administered either saline or FAD 1. Thirty min before extinction training, rats were administered either saline or one of two FAD doses 1. This session allowed association between lever pressing and sucrose pellets. The operant chamber had two levers: an active lever which dispenses sucrose and an inactive one no action. The day before the onset of SA conditioning which started after five days of recovery from surgery , all rats were tested for patency using 0. For the conditioning phase, rats were placed inside an operant conditioning chamber and connected to an infusion line. Criteria for self-administration were ten days of at least ten infusions per session. Rats were divided into treatment groups based on active lever presses during the last three days of SA. Fadrozole 1. To evaluate the response to non-drug reward, we conducted a separate experiment. Rats were injected with FAD 1. Statistical analyses were performed using Graph Pad Prism. Significant main effects were followed by Bonferroni post hoc tests. To test whether E2 affects the expression and extinction of cocaine seeking in males, we conducted a cocaine CPP experiment using an aromatase inhibitor FAD to suppress E2 synthesis in the brain. These results suggest that suppression of E2 synthesis by inhibiting aromatase activity interferes with cocaine CPP. FAD prevents expression of cocaine seeking in extinction. A Timeline of experiment with male rats receiving an i. We examined different doses of FAD during extinction of cocaine seeking. Using the CPP paradigm, rats were conditioned with cocaine and then given extinction training Figure 2A. Rats were divided into three groups vehicle, FAD 1. Prior to each extinction session. FAD 1. A Schematic outline for the experimental timeline of cocaine CPP and i. D High dose 2. The FAD 1. The group that received FAD 2. Additionally, we examined the possibility that inhibition of aromatase activity during extinction of SA could affect extinction learning, and cue- and cocaine-induced seeking behavior in the male rats. As in the experiment described in Figure 3 , rats were subdivided into three treatments vehicle, FAD 1. In addition, within cue-induced reinstatement groups, 1. Similar to cue-primed reinstatement, only the FAD 1. In the 2. Reinstatement of drug seeking was significantly diminished by 1. A Experimental timeline of cocaine SA showing i. Insert shows number of cocaine infusions during SA. C Reinstatement to drug-paired cues was measured by active lever presses compared to average number of lever presses on the last three days of extinction for each group left. D Reinstatement to cocaine was measured by active lever presses as compared to average number of lever presses on the last three days of extinction for each group left. This experiment had the same parameters as the cocaine SA Figure 4A , except that the reward was sucrose pellets. FAD had no effect on extinction and reinstatement of sucrose SA. A Schematic illustration of sucrose SA timeline including i. B Active lever presses during SA and extinction sessions showed no significant differences between treatments across days and in number of sucrose pellets acquired in SA Insert. C Reinstatement to sucrose was measured by active lever presses as compared to average number of lever presses on the last three days of extinction for each group left. No effect of FAD was observed. The findings of this study demonstrate that FAD, an aromatase inhibitor that halts E2 synthesis, administered during extinction training dose-dependently alters extinction of cocaine CPP in male rats. A lower dose of FAD was observed to facilitate extinction, whereas a higher dose of FAD appeared to impede or prevent extinction. Results from Experiments 1 and 2 on CPP showed different outcomes on the first day of extinction. This difference may be related to the more extended training in Experiment 2, relative to Experiment 1, as it leads to a stronger CPP memory that, in turn, may not be affected by lower doses of FAD during memory recall. These findings suggest that aromatase activity, which results in E 2 synthesis, has a role in extinction learning of cocaine CPP in male rats. Consistent with our findings on high dose FAD 2. Specifically, Twining et al. Similarly, studies of fear extinction also implicate estrogens as necessary for extinction, demonstrating that lack of estrogens impairs the extinction memory formation in assigned female at birth AFAB persons and female rats Graham and Milad, In , Graham and Milad reported that male rats that received FAD had an impairment of extinction recall. Altogether, these results revealed a dose-dependent effect of the aromatase inhibitor, with higher concentrations leading to longer periods of cocaine seeking more preference for the chamber paired with cocaine , than lower concentrations. In contrast, a lower dose appeared to facilitate extinction learning or blunt CPP expression. Our findings indicate that estradiol plays a vital role in extinction memory formation. In order to determine whether FAD during extinction has an impact on cue- or cocaine-induced seeking behavior, we decided to perform extinction and reinstatement using cocaine SA. Results showed that aromatase inhibition during the extinction of cocaine SA had no effect on extinction learning. The mechanism by which a low dose of FAD alters cocaine-induced adaptations during extinction withdrawal within the reward circuitry remains to be determined. Our results show that inhibiting aromatase during extinction of cocaine SA does not affect extinction training. Nevertheless, a low dose of FAD may have an additive effect of increasing neurotransmitters during the day treatment, thus creating a stronger memory of extinction and reducing cue and drug-induced reinstatement of cocaine seeking. FAD is known to reduce sexual behavior in male rats Bonsall et al. Additionally, E2 is known to play a crucial role in synaptogenesis and memory formation by regulating transcriptional activity and protein synthesis Torres, When E2 levels decrease, this can obstruct estrogen-induced memory formation by inhibiting transcription resulting in memory impairment. Consequently, this may cause a reduction in both cue- and drug-primed SA reinstatement. Interestingly, two different doses of FAD did not affect non-drug reinstatement when using a sucrose SA paradigm. This suggests that FAD does not alter the motivational properties of reward-associated behavior or memory formation. Clearly, further research is required to test these hypotheses, but taken as a whole, these findings suggest that a decrease in aromatase activity i. Both behavioral paradigms presented in this study have distinct features, allowing for assessment of different aspects of cocaine use disorder. CPP has operant-like components that influence behavior in drug-paired contexts Green and Bardo, which can be taken as a measure of interoceptive reward without motivational aspects. On the other hand, SA involves operant conditioning through reinforcement Panlilio and Goldberg, indicating that it measures interoceptive reward modulated by motivation. This suggests that FAD impairs extinction of a reward seeking CPP associated with the environment cocaine-paired chamber , while having no effects on the motivational aspect of the operant conditioning paradigm SA during extinction. Further experiments are required to evaluate the disparity between the results from the CPP vs. SA paradigms. The effect of E2 on reinforcing effects of cocaine in males remains unclear. Preclinical studies showed that administering E2 during SA had no effect on gonadectomized male rats Jackson et al. It is important to note that these studies only examined the effect of E2 on the SA reinforcing effects of cocaine , and not during extinction learning. In our study, we observed how FAD high dose, which disrupts E2 synthesis during cocaine extinction, impairs male cocaine-seeking behavior similar to what was previously seen in females Twining et al. On the other hand, low dose FAD facilitated extinction or suppressed CPP expression in male rats, suggesting sex differences in E2mediation of cocaine seeking. Our findings indicate that E2 is important in both sexes for cocaine seeking and extinction. The dose—response effects observed during CPP extinction and SA cue- and cocaine-induced reinstatement might be due to the effects of FAD on other molecules from the estrogen synthesis pathway for example, testosterone, or last metabolites in this pathway before being aromatized to estrone and E2 by CYP19 , as a compensatory mechanism, or on some other receptor. We speculate that upon decreasing aromatized activity with FAD, testosterone levels increase due to not being converted to E2 Soma et al. These studies suggest that testosterone metabolites can contribute to changes in the brain, possibly mediating cocaine extinction learning and memory. Another recent study showed that increased testosterone levels by acute administration of a gonadotropin-releasing hormone GnRH receptor agonist enhanced extinction recall during fear extinction training in rats Maeng et al. Clearly, additional studies are needed to elucidate if and how testosterone metabolites modulate cocaine-seeking behavior. One possibility is that a decrease in E2 availability reduces interaction with its receptors, and reduced receptor activation could explain part of the duality observed when FAD was administered, causing impairment in extinction behavior. E2 scarcity, due to a higher inhibition of aromatase inhibitor, would result in no activation of estrogen receptors. On the contrary, a lower dosage of aromatase inhibitor might not fully affect E2 levels entirely. Instead, it may partially halt E2 synthesis, with a low level of E2 present that could trigger a compensatory mechanism within a cell, and generate more receptors, thus facilitating extinction and lowering reinstatement. Low concentrations of both estrogens and androgens have been shown to upregulate receptor production Smith et al. Moreover, there is new evidence showing sex differences in expression of E2 and androgen receptors within the projections from the medial preoptic area to the ventral tegmental area in rats Martz et al. This difference in hormone receptor expression between sexes may contribute to the behavioral effects of E2 on reward and motivation pathways in males and females. Further studies should examine the pharmacodynamics of FAD at both doses, and measure estrogen receptor activity in brain structures related to reward. Estrogens have an impact on various neurotransmitter systems and can upregulate and increase their activity. These systems include dopamine DA , glutamate, and serotonin 5HT , with estrogens modulating their synthesis, receptors, and transporters. These neurotransmitters are essential for learning and memory processes Becker, , ; Sumner and Fink, ; Gazzaley et al. Our data suggest that a low dose of FAD slightly increases estrogen levels, which in turn may improve the release of neurotransmitters and result in facilitated CPP extinction learning. On the other hand, a high dose of FAD could cause a more significant decrease in estrogen levels, which would have the opposite effect. Additionally, some drugs have a biphasic dose—response curve, which means that low and high doses have opposite effects. This could be due, for example, to different receptor affinities, or to differential modulation of downstream signaling pathways at different concentrations. Further research is necessary to assess these and other related hypotheses. Since the behavioral effects observed in this study are due to intraperitoneal FAD injections, the question may arise whether FAD can cross the brain—blood barrier. Wade et al. Although we did not collect blood samples or brain tissue from experimental rats, multiple preclinical studies showed that aromatase inhibitors, such as FAD, reduced levels of estrogens in the blood Soma et al. Another limitation of this study is that we focused on the effect of FAD during cocaine extinction within both paradigms but only on SA reinstatement. To learn more about this caveat and determine whether or not it has any bearing on the observed SA data, future experiments could look into the possibility of changes in CPP reinstatement and sucrose-induced CPP. The observed results are consistent with previous research that has highlighted the significance of estrogens in the developmental processes of the male brain Wu et al. Although there is no FDA-approved pharmacologic agent for cocaine use disorder, sex-difference studies have highlighted hormones, mainly estrogens, as essential elements influencing this disorder. This study adds new information on how sex steroid hormones like E2 directly or indirectly regulate drug seeking. Our findings suggest that E2 modulates cocaine extinction learning and cocaine-seeking behavior in male rats. Altogether, these results, along with all updated knowledge about estrogens as a whole, will prompt a reassessment of the significant impact estrogens have on sex differences in cocaine use disorder, and how estrogens can facilitate the recovery of those affected by this disorder. Discovering a new mechanism to diminish cocaine relapse will help many women and men overcome cocaine use disorder, ultimately improving their lifestyle. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. The study was conducted in accordance with the local legislation and institutional requirements. AS: Formal analysis, Investigation, Writing — original draft. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author s declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. This section collects any data citations, data availability statements, or supplementary materials included in this article. As a library, NLM provides access to scientific literature. Front Behav Neurosci. Find articles by John K Alvarado-Torres. Find articles by Roberto Morales-Silva. Find articles by Alexia Sanabria Ponce de Leon. Find articles by Genesis Rodriguez-Torres. Find articles by Joshua Perez-Torres. Find articles by Yobet Perez-Perez. Find articles by Devin Mueller. Find articles by Marian T Sepulveda-Orengo. Received Oct 4; Accepted Dec 26; Collection date Open in a new tab. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.
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