Endometrial carcinoma (EC) is the fourth most common cancer in women in the developed world. Classification of ECs by histomorphologic criteria has limited reproducibility and better tools are needed to distinguish these tumors and enable a subtype-specific approach to research and clinical care. Based on the Cancer Genome Atlas, two research teams have developed pragmatic molecular classifiers that identify four prognostically distinct molecular subgroups. These methods can be applied to diagnostic specimens  with the potential to completely change the current risk stratification systems and enable earlier informed decision making. The evolution of genomic classification in ECs is shared herein, as well as potential applications and discussion of the essential research still needed in order to optimally integrate molecular classification in to current standard of care. In the field of cancer, this has meant a progression from broad categorization of tumors by anatomic site, to distinguishing subgroups by histomorphology, and more recently defining tumors by molecular features. This evolution has not happened over night and pace of change has varied by tumor site. Paradoxically, despite endometrial cancer being the most common gynecologic malignancy in women in Canada and the United States , 2 and tglobally , researcglobally –6. Howevglobally –10.Renewed research focus on this disease site has been prompted by a dramatic increase in incidence observed in the developed world , 11–13. There has been a call for the incorporation of molecular features in to both classification and risk determination of ECs in order to better assess the biological behavior of an individual’s disease and ultimately to improve treatment decisions and outcomes , 22. TheThirty years ago, Bokhman hypothesized there were two pathogenetic types of endometrial carcinomas driven by very different metabolic and endocrine signals . Type 1 Tumor grade and histologic subtype assessment are subjectively assigned according to appearance under the microscope and predefined pathologic criteria. Nuclear features and the proportion of solid tumor vs. identifiable glands defines grade 1–3. Histologic subtype is assigned by morphologic criteria and often aided by immunostains. Pathologic accuracy is hampered by poor diagnostic reproducibility, especially in the case of high-grade subtypes . Studies describe inter-observer disagreement or lack of consensus on histologic subtype diagnosis in one-third or higher of ECs –27. The Endometrial carcinoma has been a surgically staged disease since 1988. Surgery traditionally involves hysterectomy with bilateral salpingo-oophorectomy +/− lymph node dissection or sampling and omentectomy with several safe options in surgical approach , 33–35. after surgical staging (stage is a component of risk assignment). There is great need to obtain earlier and more biologically informative data from EC tumors that could assist in planning the optimal course of treatment for the individual. In addition, diagnostic tools that could objectively and consistently categorize ECs into distinct subgroups would enable stratification of clinical trials and study of treatment efficacy within biologically ‘like’ subgroups. Stemming from clinical need and a recognized inadequate/unsustainable system a call was made for the integration of molecular features.The adequacy of risk stratification systems in EC have recently been compared and challenged , 49. TheSeveral research teams have defined immunohistochemical and/or mutation profiles to aid in distinguishing EC subtypes –58. In oPOLE ultramutated, MSI hypermutated, copy-number (CN) low, and CN high - that correlate with progression-free survival.The most comprehensive molecular study of ECs to date has been The Cancer Genome Atlas (TCGA) project, which included a combination of whole genome sequencing, exome sequencing, microsatellite instability (MSI) assays, and copy number analysis . MoleculPOLE subgroup was a novel finding from the TCGA, and generated interest due to its very favorable outcomes even within high-grade tumors. In TCGA, ultramutated cases were characterized by POLE exonuclease domain mutations (EDM), a high percent of C > A transversions, a low percent of C > G transversions, as well as more than 500 SNVs. POLE encodes the major catalytic and proofreading subunits of the Polε (Polymerase Epsilon) DNA polymerase enzyme complex responsible for leading strand DNA replication. The exonuclease proofreading function and the high fidelity incorporation of bases by POLE ensures a low mutation rate in the daughter strand. In ECs, POLE EDMs are mostly found in hotspot regions with V411L and P286R being the most common mutations. Substitutions in DNA polymerases were shown to inactivate or suppress proofreading abilities, thus causing increased replicative error rates and resulting in the ultra-mutated phenotype. In the TCGA, whole genome or exome sequencing was used to assess POLE status. Other series have subsequently assessed POLE status using more focused methods including Sanger sequencing [POLE aberrant ECs.The ultramutated quencing , 69, genquencing , digitalquencing –74 or fuquencing  and confTCGA also described a molecular subgroup that exhibited microsatellite instability (MSI). MSI arises from defects in post-replicative DNA mismatch repair system. In the TCGA, MSI was determined by a panel of four mononucleotide repeat loci  and three dinucleotide repeat loci  in addition to the recommended markers from the National Cancer Institute , tumor DPOLE ultramutated group, the MSI group, or the CN high group, were termed CN low. The appeal of objective molecular categorization of new EC cases in to one of four prognostic subgroups was immediately apparent. However, methodologies used for the TCGA study were costly, complex and unsuitable for wider clinical application.Finally TCGA distinguished a distinct molecular subgroup by copy number analysis. Copy number was determined using Affymetrix SNP 6.0 microarrays using DNA originating from frozen tissue. Hierarchical clustering identified significantly reoccurring amplifications or deletions regions and a ‘copy number (CN) high’ subgroup. All remaining samples that did not belong to the TP53 mutational testing and p53 IHC to determine p53 status obtained from sequencing as a surrogate for CN high TCGA subgroup. The promega MSI analysis system was used to determine MSI status. For tumors exhibiting low levels of instability or from which extracted DNA quality was poor, immunohistochemistry for mismatch repair (MMR) proteins  was performed. POLE EDM hotspot mutations were identified by Sanger sequencing. This team also tested for hotspot mutations (159) across 13 genes . Testing ultimately yielded four molecular subgroups: group 1 - p53 (mutation identified), group 2- MSI, group 3 –POLE (POLE EDM identified), and finally group 4 –NSMP, a group with ‘no specific molecular profile’ , with known high risk features. Recurrence-free survival and time to distance metastasis were assessed within the four molecular subgroups. They observed that patients belonging to the POLE and the MSI subgroups showed similar and much better survival outcomes in comparison to the p53 mutant group and the NSMP group which exhibited worse recurrence and distance metastasis outcomes even within the endometrioid histology cases. Differences in survival patterns relative to the TCGA results were attributed to a greater proportion of high-risk features in the PORTEC 3 cohort.Two research teams, including our own, have subsequently developed more pragmatic methodologies to evaluate molecular features of ECs, working in standard formalin-fixed paraffin-embedded tissue. These methods do not identify molecular subgroups that are identical to TCGA but do recapitulate the four survival curves observed in TCGA , 73, 77 le’ Fig. . Tumors The Leiden/TransPORTEC group has since applied the same series of molecular tests to a larger, more diverse cohort . Howevern = 141) [n = 319) [n = ~500) of ECs from collaborators at the University of Tübingen (Germany). Molecular decision tree analysis for ProMisE is outlined in Fig. POLE exonuclease domain mutations (‘POLE EDM’). Finally, cases are assessed using IHC for p53 . We have demonstrated that women within each molecular subgroup have clinicopathological characteristic that have consistently been shown to be typical of that group. For example, the p53 abn subgroup usually encompasses the highest proportion of high grade, advanced stage, non-endometrioid histotypes and arises in older, thinner women. Similarly, the emerging phenotype of women whose EC harbor POLE EDMs is of particular interest since it generally includes younger, thinner and with surprisingly aggressive pathologic features  yet consistently exhibit favorable outcomes. The MMR-D subgroup have very similar ‘uterine factors’ (clinicopathologic features in the uterus itself) [POLE subgroup, i.e. a comparable proportion of high grade tumors and deep myometrial invasion and LVSI, yet they have worst observed outcomes of any group next to p53abn [Our research team has also developed a molecular classification system that uses practical methodologies to assign ECs to one of four molecular subgroups with distinct survival outcomes. We have followed the Institute of Medicine (IOM) guidelines for the development of ‘omics based tests , initialn = 141) , next lon = 319) , 79 to p itself)  to the Po p53abn , 79. On Both ProMisE , and the Leiden classifier (within the intermediate-risk group examined) demonstrate comparable risk discriminatory ability to the ESMO risk stratification system. Furthermore when clinical and pathological features were integrated with molecular features they resulted in improved risk stratification. Through evaluation of the collective cohort  we plan to evaluate which key clinicopathological parameters can add value to molecular classification giving high priority to those features available at time of diagnosis .Our goal has consistently been to develop a molecular classification tool that could be applied to diagnostic specimens  and therefore inform treatment at the earliest time point. Biologically relevant information about an individual’s tumor could guide surgical urgency and aggressiveness, fertility or hormonal function sparing management options, adjuvant therapy, and/or surveillance schedules. We have demonstrated high concordance between ProMisE molecular classification in diagnostic vs. final hysterectomy samples, far superseding concordance of grade, or histotype as assigned on original pathology reports or within or between reviews by expert gynecologic cancer pathologists . The LeiUltimately, integration of molecular classification by either method into current practice, as performed on diagnostic specimens or final hysterectomy, will need to be studied in the context of a prospective clinical trial; comparing survival outcomes, quality of life and health economic implications to conventional/historical standard of care.POLE exonuclease domain mutations and aberrant p53. Similarities and differences are shown in Fig. The Leiden/TransPORTEC and Vancouver/ProMisE pragmatic molecular classification systems incorporate the same integral components: identification of ECs with mismatch repair deficiency/microsatellite instability, There are different techniques for the identification of mismatch repair deficiency , 83–87. Although all MMR deficiencies are often grouped together, for inherited mutations (Lynch syndrome), the lifetime risk and age of penetration of Lynch-associated cancers can vary substantially according which gene is aberrant . This maMLH1 methylation, and MMR protein expression as part of a combined NRG Oncology/Gynecologic Oncology Group Study (GOG210) [p = 0.1) but this association  was not maintained after adjusting for other factors, including the highly relevant parameter of age in this cohort. In addition, the authors observed a trend to improved PFS in tumors with MMR mutations and a suggestion that these patients received greater benefit from adjuvant chemotherapy compared to women with normal mismatch repair. Similar results for probable germline/Lynch syndrome mismatch repair deficient tumors were observed in a smaller series of 221 ECs, with no prognostic nor predictive associations noted in the tumors with methylation events [Prognostic and predictive implications of mismatch repair may also vary according to specific MMR gene mutation or protein loss identified. It has been hypothesized that epigenetic/methylation events in mismatch repair likely have different implications on tumor characteristics and clinical outcomes than germline defects e.g. an age-related somatic event would not be expected to promote the development of tumor that is equivalent to one arising in a young individual harboring a germline mutation. Immune environment, intrinsic biologic behavior, toleration of adjuvant therapy/response to cell injury may vary significantly in these individuals. This may partially explain the relatively wide range of response to immunotherapy within MMR-D cases. At present, all mismatch repair deficiencies are lumped together but further interrogation of these differences (e.g. subgroups of subgroups) is warranted. Recently, over 1000 women with EC had their tumors evaluated for microsatellite instability, (GOG210) . Categorn events .POLE mutated tumors by histomorphology and immune environment [POLE mutated tumors would suggest that costly targeted therapy might better be reserved for the very rare cases of recurrent or advanced disease [POLE somatic mutations are found in less than 10% of endometrial carcinomas and recurrence is seldom observed; thus, it has been difficult for a single study to be adequately powered to determine optimal management of women whose tumors harbour this molecular feature. Adjuvant treatment is commonly administered due to the frequency of ‘high-risk’ features in ECs with POLE EDMs  but whether this is over treatment of women who would do well based on their POLE genotype alone or whether treatment is needed and favorable outcomes are secondary to exquisite sensitivity to DNA damaging agents in these tumors is as yet unclear.Several research teams have characterized ironment –101. Obv disease , 103. POPOLE EDMS .The paradox of observed aggressive histopathologic features but excellent survival outcomes may in part be explained by the high neoantigen load and immune rich microenvironment in tumors with POLE mutation testing are more costly than IHC and utilize methods that require a skilled team to perform and interpret. We, and others, continue to search for surrogates for POLE sequencing. Although the clinical and pathological phenotype of women with POLE mutated tumors is beginning to be characterized; on average younger, lower BMI, high proportion of grade 3, LVSI+, predominantly endometrioid, and low stage, these parameters overlap with other molecular subgroups. At present there is no single pathognomonic surrogate for this feature.Both sequencing and functional assays currently employed for TP53 mutations within ECs was recently described in Schultheis et al. [TP53 mutations in the ‘CN high’ TCGA category but also seen in 35% of the POLE genomic subgroup. No clinical correlative data was provided with their paper but our series and others confirm the highly favorable outcome of POLE mutation carriers even with the identification of other mutations traditionally associated with high risk disease. The order of our categorization: identification and removal of POLE subgroup prior to p53 stratification thus seems to be of great importance (see tumors with >1 molecular feature below). Also described in this series was the presence of frameshift or nonsense TP53 mutations (22% of TP53 mutant subset) of which they acknowledge would yield different IHC results (loss or IHC score 0) than missense variants (IHC score 2). Identification of both aberrant states is essential. Our team, in collaboration with others is in the process of further characterizing both TP53 mutational and IHC status in ECs in order to better guide interpretation in this disease site.The mutational spectrum of s et al. , both inPOLE proofreading defect and a DNA mismatch repair defects varies in the literature, but in series where co-occurrence is higher, this has been attributed to somatic MMR mutations which may be secondary to the ultra-mutated POLE phenotype. [POLE and MMR-D subgroups of ECs with high mutational loads, tumors may also harbour TP53 mutations  [POLE status prior to p53 testing will categorize a given EC as POLE EDM. Favorable outcomes are therefore anticipated for that individual, and indeed for cases reported thus far with dual features, that has been observed [POLE status  which is not currently integrated into treatment algorithms. Ultimately, distinguishing between what are likely passenger mutations or late events without functional consequence as compared to mutations that define biologic behavior is essential. Molecular classification tools that utilize large gene panels may detect a plethora of coexisting mutations in POLE EDM ECs and need to be interpreted with caution e.g., discovery of BRCA1 or BRCA2 mutation in a POLE mutated EC may not indicate homologous recombination deficiency / PARPi efficacy [Both Talhouk et al. , 79 and enotype. , 70, 98. on IHC) , 79, 104observed . We beliefficacy .Clinical outcomes may be harder to discern between ECs demonstrating both MMR deficiency and p53 mutations and the ‘best’ categorization of these tumors remains to be determined. At present, molecular classification will first identify the MMR deficiency at least enabling patients to be referred for hereditary counselling and providing opportunities in genotype specific clinical trials.Genotype-phenotype interactions have been appreciated and characterized in recent years. Although not the focus of this review, we will take this opportunity to describe one highly relevant example.It is now appreciated that PTEN loss has different prognostic implications in lean vs obese individuals. Mutations in the central relay pathways of insulin signals (phosphatidylinositol 3-kinase (PI3K) pathway including mutations specifically in PIK3CA, PIK3R1 and PTEN) are extremely common in ECs yet prior studies on the prognostic significance of PTEN mutations had markedly discordant results. Westin et al. stratified cases by body mass index (BMI) revealed improved progression free survival in obese (BMI >30) women with endometrioid endometrial carcinoma suggesting an interaction between metabolic state and genetics . SubsequThe role of molecular classification in rare histotypes of endometrial carcinoma has not been determined. The TCGA was restricted to cases of endometrioid and serous histology, however, the TransPORTEC cohorts and our own series included other histologies; 15% clear cell, and a combination of 6% clear cell, carcinosarcoma, undifferentiated, and mixed, in the cohorts respectively , 77, 79.Intratumoral heterogeneity in EC has been described , 114, anWe have harboured too long in a system of irreproducible categorization of endometrial carcinomas, inconsistent management within and across cancer centers, and inappropriate research investigations that grouped diverse tumors for study, making advances in research and clinical management slow or impossible in this disease site. It is essential that biologically relevant molecular features are assessed and considered for categorization of tumors, and in deciding surgical management and adjuvant therapy. This does not require abandonment of clinicopathologic parameters, many of which have been demonstrated to maintain prognostic relevance even in the post-TCGA era, but rather not to rely on them as the only or most important feature to guide management.POLE  EDM. All components of the molecular classifier together can be achieved at a cost* comparable to other commonly utilized clinical assays in cancer care. At minimum, this system provides objective reproducible categorization of EC’s. Familiarity with MMR and p53 IHC testing and interpretation lends to rapid adoption in any pathology department. The reproducibility of ProMisE across Canadian cancer centers is currently being evaluated.We have shown that in the hands of two independent research teams molecular classification of endometrial carcinomas is feasible, and identifies four prognostically distinct subgroups. Historical segregation of Type I  and Type II ECs  is inadequate and do not account for the approximately 30% of cases that are MMR-D or POLE mutation status within an individual’s EC will impact her clinical management, as favorable outcomes observed in these individuals may be either independent or secondary to increased sensitivity to DNA damaging agents , and withholding treatment cannot yet be advised. Plausibly, women with p53 abn tumors with higher association of metastatic disease and aggressive clinical course would be recommended to undergo more comprehensive surgical staging and closer surveillance.  Conversely, biologically indolent tumors may be cured by simplified surgery alone and perhaps spared toxic treatment and managed by community gynecologists.Additional benefits of molecular classification include early identification of women who may have an inherited genetic syndrome (Lynch) who would benefit from additional screening or interventions for other Lynch-associated cancers or in whom specific therapies for their endometrial carcinomas may be more effective. For young women with EC considering delay of hysterectomy for fertility reasons , molecular classification of her diagnostic endometrial specimen could help guide management as either MMR-D  or p53 abn categorization would discourage a conservative approach. It is still unclear how knowing the POLE? Are favorable outcomes in POLE patients independent of treatment  Although there may be many questions to address we anticipate that molecular classification will facilitate rapid progress in research and clinical care as has been achieved through a subtype specific approach in other tumor sites.We are at an exciting juncture, but aware of the many questions still remaining Table . ThroughIn summary, whilst the combination of histomorphology and clinical factors has proven to be insufficiently reproducible, prognostic and predictive, two molecular classifiers based on the TCGA study show great potential as pragmatic and effective tools to stratify patient risk and subsequent care decisions. Given the high and increasing incidence of endometrial cancer and the societal cost of over- and under-treatment there is urgent need for prospective clinical studies to determine how best to utilize these tools.*For ProMisE; the materials, assay and interpretation costs total < $300 USD


Bovine tuberculosis (bTB) is a chronic, zoonotic, multi-species disease of cattle caused by Mycobacterium bovis. In developed countries, effective surveillance and enforcement of regulations on bTB control resulted in significant reduction of infections in cattle and hence, humans. However, in developing countries, weak surveillance systems affect accurate and timely reporting of bTB in humans and cattle. In Ghana, transhumance movement of cattle increases the risk of bTB importation and spread, however, the extent to which surveillance detects bTB is unknown. We therefore evaluated the bTB surveillance system in the Greater-Accra Region to determine its performance and assessed its attributes.We interviewed stakeholders, and reviewed bTB surveillance data for all ten districts in the region from 2006-2011 using the CDC Guidelines for Evaluation of public health surveillance systems.From 2006-2011, bTB was suspected in 284/244,576 (0.12%) cattle slaughtered, of which 7/284 (2.5%) were submitted for laboratory confirmation and all tested positive. Predictive value positive was 100%. There is no standard case definition which guides bTB detection. Fifty percent of carcasses slip through inspection, and confirmed cases are not traced back. There were 99/284 (34.9%) condemnations from suspected carcasses and 57/97 (58.8%) from positive reactors from screening. Ninety percent (9/10) of districts submitted reports late to the region whereas representativeness was 30%. Regional and district data were manually stored with no electronic backups. The region's cattle population is unknown.Although the bTB surveillance system is sensitive, it is under performing, and the possibility of bTB transmission from cattle to humans is high. Bovine tuberculosis (bTB) is a debilitating, chronic disease of cattle caused by Mycobacterium bovis from the Mycobacterium tuberculosis complex of bacteria. The bacterium is ubiquitous and has worldwide distribution, however, developing countries are most affected –3. BovinStudy area, design and sources of data: the study area was the Greater-Accra Region of Ghana which is located in the south-east of the country along the Gulf of Guinea, between latitude 50 330 North, 00 130 West. It occupies a land surface area of 3,245 square kilometres, with an estimated population of 4,010,054  and compCase definitions: there was no official written case definition for suspected and probable cases of bTB. However, its suspicion and detection during meat inspection was based on the identification of granulomatous lesions in the lungs, intestines and other affected organs. All stakeholders interviewed were familiar with bTB infected carcasses and described bTB lesions as whitish granular nodules, which “sounded like a knife cutting through sand upon incision”. Similarly, animals in contact with suspected or confirmed cases were referred to as probable cases, whereas confirmed cases were referred to as samples testing positive to Ziel-Neelson's acid fast stain test for tuberculosis.Cattle population: the number of cattle herds and cattle in each district, and the region for that matter, is unknown.Morbidity and mortality: there was an upward trend in bTB suspicion during slaughter from 2006-2008, but this took a dip in 2009, rose slightly in 2010 and dipped again in 2011 as shown in Data flow: the flow of data in the bTB surveillance system in the Region is shown in Resources for bTB surveillance: The cost of bTB surveillance is covered under the budget allocation for scheduled diseases surveillance. On average $1,667.00, $500.00 and $444.00 respectively is allocated to the national, regional and district levels every quarter for disease surveillance and control activities including bTB. However, in 6/10 (60%) of the districts evaluated, these funds were sometimes not made available for that purpose by the District Directors of Agriculture. They were rather used for other administrative activities [tivities .Types of surveillance: two main types of bTB surveillance were observed, namely, active and passive. Active surveillance comprised detection during meat inspection, targeted screening of cattle in an area based on detection of more than five bTB cases from that particular area within a month, and laboratory testing and confirmation using Ziehl-Neelsen staining technique. There was no systematic routine screening in place, but screening upon request in farms that were mainly public occurred. Passive surveillance was based on voluntary reporting by peri-urban dairy farms and cattle herdsmen to district veterinary staff. However, bTB detection by this method was rare. Out of nine such reports, only one (1/9) farmer reported suspicion of bTB in a cow slaughtered for domestic consumption. Otherwise all the remaining 88.88% (8/9) voluntary reports came from public farms.Documentation and data quality: the Accra Veterinary Laboratory records all animal and carcass sample test results for bTB, however, documentation was not uniform. Results were recorded as AFB, positive, Acid Fast Bacilli, TB positive, MTB, some were scored, whereas others were not. Notwithstanding this, there was clear client and specimen identification, and detailed information regarding receipt and processing of samples at the laboratory. Results were entered into a log book, and correspondence on bTB stored in a file.Attributes of the bTB surveillance system. Sensitivity, predictive value positive and usefulness: the surveillance system was found to be sensitive as it was able to detect cases of bTB. The number of cattle slaughtered from 2006-2011 was 44,460, of which 284 (0.12%) were suspected to be bTB infected. Of these, 7/284 (2.46%) were sent to the laboratory for confirmation and all tested positive . The number of cattle which tested positive out of 3,367 screened was 97 (2.9%). The usefulness of the surveillance system was found to be low; out of the 284 carcasses suspected to be bTB infected, there were 99 (32.04 %) total and partial condemnations; 8/99 (8.08%) of these were totally condemned. Similarly, out of the of 97 animals detected to be bTB infected from screening, 57 (58.76%) were culled, as shown in Simplicity: the bTB surveillance system was found to be complicated in structure as detection requires special training, particularly in the screening of animals and laboratory confirmation of the disease. Also, only the Accra Veterinary Laboratory, which is at the regional capital, has the facilities to test for bTB in the region, as such, all suspected samples should be transported for confirmation. This situation was further complicated by the lack of transportation to send samples.Acceptability: twelve percent (3/25) of stakeholders interviewed admitted willingness to comply with bTB reporting. Butchers and cattle merchants explained that a disincentive for voluntary reporting of suspected bTB cases was the lack of compensation for cattle farmers. This is because animals which are condemned on suspicion of bTB imply financial loss to the farmer.Stability: the system was found to be unstable for the following reasons: at regional and district levels, surveillance data were stored manually or on personal computers of staff, and some monthly reports could not be traced. Additionally, most districts had inadequate staff who were poorly resourced to keep surveillance on scheduled diseases including bTB. This was exacerbated by irregular release of surveillance funds.Flexibility: At the district level, bTB suspected carcasses at slaughter were reported on Veterinary Form 9 which is also used to capture other diseases at the end of the month. Hence the system was flexible. However, there were no daily records of slaughter findings.Representativeness and completeness: three districts out of ten (30%), reported on bTB in their monthly reports, and one district did this consistently. Moreover, not all cattle were slaughtered under veterinary supervision; hence some incidence of bTB may have been missed in official reports. In all districts, Veterinary Form 9 was missing for some of the months, making reporting incomplete.Timeliness: one district out of ten (10%) regularly submitted monthly reports on time. Delays ranged from 1-26 days. In contrast, reports from laboratory investigations of samples submitted for bTB testing were released to clients in a timely manner, on the same day.Data analysis: data was neither analysed at the district nor regional levels. They were merely compiled and forwarded to the Epidemiology Unit, which performed the analysis. There was feedback from laboratory testing to the districts; however, feedback from the regional veterinary office to the districts was absent.Statement of Principal Findings: the bTB surveillance system of the Greater-Accra Region is able to detect infection both in live animals and carcasses; however, there was no officially written case definition for bTB. Diagnosis, confirmation and screening were all based on the presence of lesions or reactions which are characteristic for bTB infected carcasses or animals; therefore all the criteria used were acceptable. However, this is in contrast to human tuberculosis which has well defined case definitions as found in the Integrated Disease Surveillance and Response System of Ghana [of Ghana . The preof Ghana , 29, butof Ghana , 31. Theof Ghana –34. Therof Ghana . Additioof Ghana . Additioof Ghana –38. Furtof Ghana , 40. ThiThe usefulness of the surveillance system was found to be very low; only 32% of infected carcasses were either totally or partially condemned, whereas 58.7% of positive reactors from screening were culled. It is unclear what was done to the remaining infected carcasses. There was also no evidence to indicate that epidemiological trace back was done to identify the herds from which these animals originated. This could have been due to lack of a proper cattle identification system in place in the country. In a study in England on post slaughter detection of bTB in carcasses, improper identification of animals was found to be a major factor contributing to the inability to trace back infected carcasses to their herds . This meStrengths and weaknesses of the study: this study has clearly demonstrated the areas where there are lapses in the bTB surveillance system, however, it was unable to establish by how much bTB detection is being missed. A more detailed study would be required to investigate the actual prevalence of bTB at slaughter, national herd prevalence and the bTB status of cattle moved for breeding purposes. The latter, in turn would depend on an effective cattle identification program, which currently, is lacking in Ghana.Although the bTB surveillance system is sensitive and flexible, reporting is neither complete nor representative of the actual situation on the ground, timeliness is poor and there is generally no feedback from the region to the districts. Usefulness of the methods applied in bTB surveillance were found to be low, as was acceptability. The system was found to be unstable and data quality was poor, and the availability of resources for bTB surveillance untimely and woefully inadequate. Additionally, denominator data is lacking; actual cattle populations in the ten districts remain largely unknown. Therefore the BTB surveillance system is achieving some of its objectives; however, there is room for improvement. The possibility of bTB transmission from cattle to humans is high. The Ministry of Food and Agriculture should invest more resources to support bTB surveillance and the implementation of its control as this will go a long way to reduce transmission of the disease to humans. This should include compensation of farmers whose animals are detected to have bTB as this will improve acceptability. The VSD should increase bTB awareness creation among farmers and other key stakeholders. It should also train all veterinary officers on the importance of bTB surveillance in order to improve on representativeness, timeliness and completeness of reporting. All district officers should compile a database of cattle farms in their district for easier surveillance. Animals which are moved for breeding purposes should be certified to originate from bTB free herds. The head of the Epidemiology Unit has since drafted case definitions not only for bTB but also for all other scheduled diseases. Districts which were formerly not reporting on bTB surveillance have started doing so in their monthly reports following training on the importance of bTB reporting. Discussions have been held with the Director, VSD and plans are under way to organize bTB awareness creation in collaboration with the Ghana Health Service, Butchers Association and other key stakeholders.


We narrow this gap and investigate how employees reciprocate after receiving immaterial gifts and material gifts over time. We purchase (1) ice cream from fast food restaurants, and (2) durum doner, a common lunch snack, from independent vendors. Prior to the food’s preparation, we either compliment or tip the salesperson. We find that salespersons reciprocate compliments with higher product weight than in a control treatment. Importantly, this reciprocal behavior following immaterial gifts grows over repeated transactions. Tips, in contrast, have a stronger level effect which does not change over time.Reciprocation of monetary gifts is well-understood in economics. In contrast, there is little research on reciprocal behavior following  It is indisputable that employees appreciate monetary rewards. They expend additional effort when they receive wages exceeding a theoretical minimum wage. Akerlof , for insSurprisingly, the role of purely immaterial gifts—e.g., private compliments and individual expressions of appreciation and respect—in economic contexts is less clear. This is remarkable, as the desire for approval and being esteemed is deeply rooted in human behavior, likely because esteem is associated with material and reproductive benefits  and extra effort/kindness from the salesperson is valued greatly.In this paper we narrow these research gaps by reporting results from two natural field experiments  and material gifts (tips)—given in advance—induce positive reciprocity, i.e., salespersons provide more product weight. While monetary gifts trigger a larger level effect, only the effect of immaterial gifts increases significantly over repeated interactions. Finally, we show that the increase in product weight does not necessarily suffice to compensate the customer for the increased cost of the tip, which means that immaterial gifts are more effective when accounting for transaction costs  in our settings.With our approach we extend the literature along four dimensions. First, we explore reactions to material gifts and particularly to immaterial gifts in the same settings (ice cream and doner), making them comparable. Importantly, our approach of defining immaterial gifts differs from those of Kosfeld and Neckermann , BradlerBoth settings of this study (ICECREAM and DONER) were carefully selected to fulfill the following four requirements, ensuring a high degree of experimental control. First, the entire consumer–salesperson interaction, including accepting the order, preparing the product, and accepting the payment is attended to by a single salesperson. Many comparable settings are characterized by a division of labor on the salesperson side. For instance, a waiter in a restaurant has to rely on the barkeeper and on the kitchen staff when aiming to provide high quality drinks and food in minimum time. Second, the amount of additional food provided is measurable. One might get better treatment with tipping or complimenting in advance in many service settings—e.g., getting a massage, or a haircut—, yet this better treatment may not be easily measurable. Third, the salesperson has the discretion to choose a higher than normal amount of food. Many services are standardized with no possibility for the salesperson to intervene by adding any additional benefit. For example, beverages are frequently filled to the brim, with no chance to add any extra. Finally, the consumer–salesperson interaction reflects an everyday life situation. Going to snack bars or restaurants to order ice cream or durum doner is a common occurrence, even when repeated on five consecutive days.In our first experimental setting, ICECREAM, the experimenters ordered ice cream in McDonald’s restaurants. The restaurant clerk takes the client’s order in a face-to-face interaction, accepts payment, fills the ice cream into a cone and hands it over to the customer. Since the salesperson only has to press and release a button to start and end the filling process, it is easy for her to vary the amount of ice cream per cone. See Fig. In treatment NORMAL the experimenter ordered using the standardized wording (translated from German): “One vanilla cone without topping, to take away please.” This standardization ensured that the products were comparable and extra benefit provided by the salesperson could be clearly measured and quantified. The only way in which the salesperson could provide such extra benefit in the interaction  was to increase the amount of ice cream. We consciously refrained from ordering toppings, because they might have added noise. The experimenters also did not start conversations with the salespeople. In case they were asked questions, they answered naturally but succinctly.Treatment COMPLIMENT was identical to NORMAL, but the experimenter made a compliment about the product, prior to the product’s preparation. The standardized wording was: “One vanilla cone without topping, to take away please. You have the best ice cream in town.”Treatment TIP was identical to treatment NORMAL except that the experimenter gave a tip to the salesperson. As a percentage of price, tips varied between 10.0 and 14.3%, with a mean of 13.1%. This was due to the requirement of total payment amounts being multiples of €0.10 in order to remain inconspicuous. The experimenter took great care to ensure that the tip was recognized by the salesperson at the time the order was placed. The experimenter put the product price plus the tip on the counter and simultaneously augmented the order by adding “The rest is for you” to the standardized wording. The experimenter also chose the coins handed over to the salesperson such that the latter could see that the tipping was intentional and not caused by, e.g., rounding to the nearest integer amount.All observations were collected by three male experimenters aged 23–25 years. We randomized experimenter roles and arrival orders to control for experimenter fixed effects. Specifically, each experimenter ordered 12 times each using the procedures for treatments NORMAL, COMPLIMENT and TIP. We also randomized the order in which experimenters entered restaurants, such that each experimenter was the first to enter a restaurant 12 times, was second 12 times and was last 12 times. Finally, we randomized the order in which the experimenter playing a specific role entered a restaurant, such that the experimenter in treatment NORMAL was the first to enter a restaurant 12 times, was second 12 times and was last 12 times (and the same for treatments COMPLIMENT and TIP).After conclusion of each transaction, the experimenter stepped outside the restaurant to a place where he was not visible from inside and immediately weighed the ice cream cone on small letter scales which he carried in a backpack. He noted the weight and usually gave the ice cream to a passerby. The experimenter then filled in a form recording details about the transaction. These were the restaurant ID, product price, and tip amount (if any), as well as salesperson characteristics like gender, estimated age and ethnicity.In the top panel of Table In addition, Fig. t test of raw weights, COMPLIMENT vs. NORMAL: t(35) = 4.9802, p = 0.0000) and a tip in advance by 20.6 g (TIP vs. NORMAL: t(35) = 6.3761, p = 0.0000; TIP vs. COMPLIMENT: t(35) = 3.0996, p = 0.0038). In our regression model in Table ,On aggregate we find that a compliment increases mean raw cone weight by 11.2 g  how reciprocity in response to both immaterial and material gifts develops over time, and (2) whether the observed effects are robust to using a setting where providing extra food requires greater effort by the salesperson.In setting DONER, the experimenters ordered durum doner in restaurants and snack bars. A durum doner, pictured in Fig. Experimenters bought durum doner from the same salesperson on five consecutive days. An individual experimenter’s role  was fixed for each salesperson and we again randomized the experiment (see below for details). We conducted the experiment in Graz (GRZ) and Innsbruck (IBK), Austria, and in Munich (MUC), Germany. In each of the three cities, three experimenters visited the same 18 restaurants, yielding a total of 54 restaurants and 801 individual observations.Table five consecutive days. An individual experimenter’s role  was fixed for each salesperson. We furthermore strove to ensure that all experimenters always interacted with one and the same salesperson in a given restaurant. We thus designed the experiment to obtain 15 observations per salesperson . To control for experimenter fixed effects we randomized the experiment. We applied each of the six possible assignments of treatments to experimenters  three times to cover the 18 restaurants in each town. Thus, each experimenter played each role six times (for five visits each) in each town. Again, the experimenters entered each restaurant independently from each other and were never present in the restaurant at the same time.All selected restaurants are organized in such a way that the salesperson takes the client’s order personally, collects the money, prepares the doner in front of the client and hands over the finished product. Thus, the entire service process is executed by one and the same salesperson. We employed eight male experimenters of similar age (22–26 years), but only three of them collected data in a particular town. One experimenter was active in two towns. Each experimenter ordered one durum doner from a given salesperson on each of After exiting the restaurant, the experimenter weighted the doner as is, i.e., including the tin foil the doner was wrapped in . The experimenter noted the weight and put the doner into his backpack for later hand-over to a charitable agency.In treatment NORMAL the experimenter used the standardized wording (translated from German): “One durum doner without sauce, to take away please.” The only way in which the salesperson could provide extra benefit in the interaction  was to increase the amount of meat or other ingredients, since the durum wraps are standardized. We deliberately refrained from ordering sauce, because it has high relative density and might add noise. One could argue that getting more doner weight may not be considered beneficial by every customer. However, getting something extra is a typical act of kindness in the service industry .“One durum doner without sauce, to take away please. You have the best durum doner in town.”;“[...] It tastes best at your place.”;“[...] By the way, your durum doner tastes great.”;“[...] I never had a better durum doner than at your place.”;“[...] There is no place where the durum doner tastes better.”Treatment TIP was conducted analogously to the corresponding treatment in ICECREAM. As a percentage of price, tips varied between 8.1 and 10.3%, with a mean of 9.2%.The five standardized wordings in treatment COMPLIMENT, which were used in randomized order, were:,Table Table F = 7.44, p = 0.0080). With regard to developments over time, we find that the time trend of treatment COMPLIMENT is significantly positive, with an average weight increase of 4.9 g per visit. The other treatments’ time trends are insignificant by themselves. Furthermore, while the difference in time trends between treatments NORMAL (with a positive and insignificant coefficient) and TIP (with a negative and insignificant coefficient) is not significant  = 1.82, p = 0.1815), the trend difference between COMPLIMENT and TIP is highly significant  = 7.46, p = 0.0079).Focusing on raw doner weight, we find a significantly positive overall effect of both interventions compared to treatment NORMAL. The difference between treatments COMPLIMENT and TIP in model 3 is highly significant as well  who pays the cost of the increased goods and material employed. At the same time, the principal may profit most from a satisfied customer, because the latter will be more likely to return and to spread the word among friends, thereby increasing the restaurant’s future sales. In the case where the owner herself serves as salesperson, she presumably derives the same direct utility as an employee salesperson from being tipped or complimented. Thus, the effect sizes in COMPLIMENT and TIP could differ for employee salespeople and owner salespeople, even though the direction of this difference is difficult to predict Akerlof .17Fortunately, doner are frequently prepared not by employee salespersons, but by the restaurant owners themselves (the same is not true for in the ICECREAM setting). This allows us to study differential effects between employee and owner salespersons. As noted before, the final experimenter to interact with any particular salesperson inquired whether the salesperson was the owner of the restaurant. Since some salespersons changed during the experiment, we were able to obtain this information for all but 84 salespersons.In Table The findings from both settings allow us to answer all research questions with results using the same numbering scheme as we used for the questions themselves.Result 1 Immaterial gifts in the form of compliments prior to the product’s preparation induce positive reciprocity by the salesperson.Result 2 A monetary gift, given prior to the product’s preparation, induces positive reciprocity as well. On the aggregate, the effect is more pronounced than following an immaterial gift before accounting for the cost of the gift. After accounting for the cost of the gift, results reverse, indicating that immaterial gifts are the strongest intervention when controlling for costs.Result 3 Immaterial gifts by the consumer lead to increasingly reciprocal behavior over time. Thus, compliments to the salesperson yield positive time effects compared to (1) reciprocity induced monetarily by tipping first, and compared to (2) normal orders.Reciprocation of monetary gifts is well-understood in economics. In contrast, there is little research on reciprocal behavior following immaterial gifts like compliments and how these effects develop over time. It is for this reason that we investigate how employees reciprocate after receiving immaterial or monetary gifts. Moreover, we investigate the stability of immaterial and monetary gifts over time with repeated gift exchange transactions.We find that (1) immaterial gifts in the form of compliments significantly increase salespersons’ reciprocal behavior. Salespersons prepare ice cream and durum doner weighing more than ones obtained without intervention. We also report that (2) monetary gifts (tips) induce positive reciprocity which is on average stronger than that from immaterial gifts before costs. However, the increase in product weight does not necessarily suffice to compensate the customer for the increased cost of the tip, indicating that immaterial gifts are more effective when accounting for transaction costs. Finally, we show that (3) only reciprocation upon immaterial gifts grows significantly over repeated interactions, increasing by around 6 percent over the course of five visits. Reciprocal behavior conditional on monetary gifts, in contrast, does not vary significantly over time.It is important to note that other factors than those discussed could have contributed to the reported effects. First, altruism among sellers could drive part of the results. The compliment and the tip could serve to update seller’s belief about how much the buyer values the product and, in case of an altruistic seller, could result in increased food weight. However, the effects of conditional altruism are difficult to quantify, particularly as conditional altruism and reciprocity are considered to be related concepts , making them comparable. We particularly consider the immaterial gift in the form of a compliment to constitute an important contribution to the literature. Compared to other studies, a private compliment like in our study is entirely immaterial, cannot be “stored”, cannot be used as physical evidence for impressing others, and thereby expresses respect directly  to the recipient.Second, we investigate both approaches in natural consumer–salesperson interactions in everyday life situations. We consider this an important aspect of our study. By studying immaterial and monetary gift exchange in customer–salesperson interactions, we test the robustness of the existing evidence from employer–employee interactions. We believe that customer–salesperson interactions are of high importance, because they occur very frequently  and monetary and immaterial incentives  play a major role.Third, we use a repeated setting to analyze how monetary and particularly immaterial gifts work over time. The increasingly reciprocal behavior upon repeated provision of immaterial gifts is a novel finding and adds to the literature which heretofore has focused only on a single workday, and on situations with a single (or double) exchange of monetary gifts (Gneezy and List Fourth, we investigate the robustness of our findings by analyzing whether our results replicate from the first to the second setting, i.e., from the ice cream to the doner setting.Finally, we would like to emphasize that our results hold in a situation where the behavior exhibited in our treatments—while not entirely out of the ordinary—is not a general norm among consumers. We would expect our treatment effects to diminish with increasing frequency of tipping and complimenting, respectively, in the general consumer population. In the extreme case that tipping or complimenting were to become a social norm, we would expect negative reciprocity for consumers not providing an immaterial or monetary gift. In other words, we conjecture that consumers might be punished by the salesperson if no tip or compliment were given in advance when it is the norm to tip or compliment.


A small proportion of patients with Parkinson’s disease (PD), chronically under dopamine replacement therapy, may undergo an addiction-like behavioral disturbance, named dopamine dysregulation syndrome (DDS). This behavioral disorder is characterized by the increase of doses beyond those required for motor control, and its management remains difficult; thus, early recognition and careful monitoring of at-risk individuals are crucial. We report the cases of two PD patients with a previous unsatisfactory switching from an immediate release (IR) to an extended release (ER) pramipexole formulation who developed DDS. PD patients unsatisfactorily switched from an IR to an ER formulation of dopamine agonists should be considered as at-risk individuals for DDS development.


Unlike other drugs of abuse such as alcohol, nicotine, opiates/opioids, the FDA has not approved any agent to treat psychostimulant dependence. Certainly, it is widely acceptable that dopaminergic signaling is a key factor in both the initiation and continued motivation to abuse this class of stimulant substances. It is also well accepted that psychostimulants such as cocaine affect not only the release of neuronal dopamine at the nucleus accumbens (NAc), but also has powerful inhibitory actions on the dopamine transporter system. Understandably, certain individuals are at high risk and very vulnerable to abuse this class of substances. Trace-amine-associated receptor 1 (TAAR1) is a G -protein coupled receptor activated by trace amines. The encoded protein responds little or not at all to dopamine, serotonin, epinephrine, or histamine, but responds well to beta-phenylethylamine, p-tyramine, octopamine, and tryptamine. This gene is thought to be intronless. TAAR1 agonists reduce the neurochemical effects of cocaine and amphetamines as well as attenuate addiction and abuse associated with these two psychostimulants. The mechanism involves blocking the firing rate of dopamine in the limbic system thereby decreasing a hyperdopaminergic trait/state, whereby the opposite is true for TAAR1 antagonists. Based on many studies, it is accepted that in Reward Deficiency Syndrome (RDS), there is weakened tonic and improved phasic dopamine discharge leading to a hypodopaminergic/glutamatergic trait. The dopamine pro-complex mixture KB220, following many clinical trials including neuroimaging studies, has been shown to enhance resting state functional connectivity in humans (abstinent heroin addicts), naïve rodent models, and regulates extensive theta action in the cingulate gyrus of abstinent psychostimulant abusers. In this article, we are hypothesizing that KB220 may induce its action on resting state functional connectivity, for example, by actually balancing (optimizing) the effects of TAAR1 on the glutamatergic system allowing for optimization of this system. This will lead to a normalized and homeostatic release of NAc dopamine. This proposed optimization, and not enhanced activation of TAAR1, should lead to well-being of the individual. Hyper-activation instead of optimizing the TAAR1 system unfortunately will lead to a prolonged hypodopaminergic state and as such, will cause enhanced craving for not only psychoactive substances, but also other drug-related and even non-drug related RDS behaviors. This hypothesis will require extensive research, which seems warranted based on the global epidemic of drug and behavioral addictions. Borowsky et al.  identifiIn this article, we are proposing a role for TAAR1 in “Reward Deficiency Syndrome” (RDS) involving both a hypodopaminergic and hypoglutamatergic trait/state. Certainly, the informative paper by Revel et al.  providedTheir elegant review points out that TAAR1 knockout mice show increased sensitivity to dopaminergic activation while TAAR1 agonists reduce the neurochemical effects of cocaine and amphetamines as well as attenuating addiction and abuse associated behavioral effects of these two psychostimulants. TAAR1 agonists R05166017) block the firing rate of dopamine in the limbic system, thereby blocking a hyperdopaminergic trait/state [166017 blIt is well characterized that a hypoglutamatergic trait/state causes a reduced release of dopamine at the nucleus accumbens (NAc) due to an enhanced activation effect on GABAergic transmission in the NAc . In theiThe NAc comprises medium spiny neurons (MSNs) that constitute >90% of all neurons in both the shell and core . TherefoIt is now believed that dopamine alone does not produce great changes in MSN current, but potentiates or impedes the volume of glutamate to depolarize neurons. Several of these intracellular methods are well characterized and include a variation of calcium (D1) and potassium (D2) channels. Gating of glutamatergic activity can also be a lengthy process because of dopamine’s ability to control long-term excitatory synaptic plasticity. Physiologically, dopamine is at least partly colocalized with glutamate, and hence, these afferents are capable of inducing fast changes in membrane conductance . SecondlWhile we point out the importance of other neurotransmitter (other than just dopamine) systems in addiction behavior, there is an emerging and substantial literature on the role of glutamate signaling  in long-term addiction behavior. The NAc receives glutamate input from the VTA and PFC; both of these inputs have been well characterized in addiction behavior . GLT-1 and xCT expression decreases following long-term drug use; GLT-1 down-regulation specifically is known to contribute to the glutamate overflow that occurs during relapse. Whether or not these changes occur as a direct result of altered dopamine signaling has not been, to our knowledge, determined. Also, it is important to note that the dopamine overflow that occurs in response to relapse behavior diminishes over time , whereasThis hypothesis points out that short-term hyperactivation of TAAR1 will block dopamine firing, and potentially long-term inhibition of TAAR1 will enhance dopamine-firing rate in the VTA. If we were to consider this a target for psychostimulant abuse, then it will follow the proposal of Blum et al.  suggestiRhodiola rosea. They carried out a number of clinical trials using various forms of this amino-acid based formulae finding anti-craving benefit for psychostimulant abuse [Blum et al.  proposednt abuse . The Bront abuse  paper rent abuse  showed tnt abuse , reporteThis early work is supported by more recent evidence by Blum et al.  involvin+/K+− ATPase [RDS, insensitivity, and inefficiency in the reward system has been the subject debated by many investigators and remains controversial in terms of “liking” and wanting” –34. Howe− ATPase . Dopamin− ATPase . Therefo− ATPase .Glutamate and DA represent potential targets for novel treatments that modulate not only cocaine seeking behavior, but also other RDS behaviors and functional connectivity. Both substrates are affected by chronic psychostimulant administration , 40. In Additionally, it was recently shown that as cocaine intake escalates, phasic DA signaling in the ventromedial striatum is reduced . The DA Rhodiola rosea that inhibits COMT activity in the synapse and MAO-A activity in the mitochondria. KB220 variant has been tested in abstinent psychostimulant abusers and found to normalize quantitative electroencephalographic (qEEG) abnormalities as well as similar effects in alcoholics and heroin addicts [Key ingredients in this complex act synergistically to replenish the pool of L-DOPA and facilitate its conversion to DA. The formulation is directed at re-establishing baseline connectivity through the DA biosynthetic pathway amongst other ingredients  , 54. Unl addicts , 55. MorTo reiterate, Willuhn et al.  observedIt is well-known that the central nervous system (CNS) rewarding properties of ethanol, cocaine, and heroin may activate a common catechoaminegic (primary Dopamine) reward system in the mesolimbic circuitry of the brain. TAAR1, a member of the TAAR receptor family , is a G At the moment, there remains no Food and Drug Administration (FDA) approved drug treatment for cocaine addiction. Though several neurotransmitter systems include the pharmacological effects of cocaine, dopamine plays one of the most vital roles that resolve the addiction-related behavioral effects of cocaine. Numerous pharmacological methods have been suggested to regulate the dopaminergic system to offset the abuse-related effects of cocaine. For example, antagonists that focus on diverse dopamine receptor subtypes  have been observed to block specific effects linked to cocaine abuse. Nonetheless, important clinical triumph has not been established, mainly due to the modest effectiveness and severe side effects, especially in the long–term treatment, including down regulation of dopaminergic receptors and potential mood changes (suicide ideation). Traditional “agonist replacement therapy”  has been explored both in animals and in human subjects, and only on an acute basis, exhibits some encouraging clinical results. Conversely, anxieties about the abuse accountability of the substitute drugs per se make this method less attractive. Through this perspective, discovering substitutes that indirectly regulates the dopaminergic system could be a productive plan. Current findings strongly advise that trace amine associated receptor (TAAR) 1 could be a favorable new treatment target to fight cocaine addiction as well as other RDS behaviors.While we have had evidence of trace amine presence in the mammalian brain for decades, their autonomous physiological responsibilities have been debatable up until the detection of TAARs. Specifically, TAAR 1 has been duplicated from both rodent and primate brains and symbolizes the most extensively studied TAAR as of yet. Along with the help of genetically modified mice, we now understand that TAAR 1 contributes to the regulation of dopaminergic function. TAAR 1 knockout mice express a behavioral phenotype of supersensitivity to dopaminergic stimulation, with augmented behavioral reaction to amphetamines. In contrast, certain brain overexpression of TAAR 1 produces a behavioral phenotype that is hyposensitive to amphetamines. These outcomes indicate a functional variation of dopaminergic system by TAAR 1 , which iImportantly, it was discovered that TAAR 1 agonists exhibit extremely encouraging antipsychotic-like outcomes in preclinical studies and show excellent therapeutic profiles than current antipsychotics because they subdue feeding and reduce body weight in animals; hence, lacking the main unfavorable effect of weight gain as seen when taking some antipsychotics. These data align with the view that TAAR 1 agonists could functionally regulate the dopaminergic system and therefore, it is an expected hypothesis that these compounds may also modify stimulant abuse. However, we do not agree that this approach over the long-term would be beneficial and in fact, it may be harmful .TAAR 1 agonists can stop addiction-linked outcomes of cocaine use in rats , 59. In The Thorn et al.  study emFinally, we are cognizant of other important systems now being recognized such as the Neurokinin-1 (NK1) receptor system. While additional work is required in this fruitful area of research and understanding that that SP/NK1 receptor system is involved in processing of positive incentive anticipation and plays a role in accentuating positive valence in association with the primary dopaminergic pathways in the reward circuit, any definitive conclusions of our hypothesis and even other endogenous systems linked to appropriate psychostimulant therapy must await additional scientific evidence .While these reviewed data are noteworthy because they examined the concept that pharmacologically regulating TAAR 1 can decrease cocaine addiction utilizing animal representations with suitable translational and predictive scores, we are proposing that TAAR1 agonists may be beneficial only in the short–term, but should not be utilized in long-term treatment in humans due to interference with natural dopaminergic regulation.Moreover, KB200z, is a formulation containing both a D1 agonist and NAC. We hypothesize that this compound should lead to a normalized and homeostatic release of NAc dopamine. NAC has been well studied in the treatment of cocaine relapse and has shown promising therapeutic results. However, others have demonstrated its effects to be due to normalization of xCT and basal glutamate levels. Thus, KB200z may be effective in the treatment of addiction not through regulation of the dopamine but rather the glutamate system. We must await these anticipated studies to help clarify our hypothesis.Along these lines, we are further hypothesizing that the dopamine pro-complex mixture known as KB220z (a putative indirect dopamine agonist containing NAC) may induce its action on resting state functional connectivity. For example , by actuWhile others have suggested that it is important to balance both glutaminergic and dopaminergic systems to treat addiction  unlike u


Synthetic molecular machines are promising building blocks for future nanoscopic devices. However, the details of their mechanical behaviour are in many cases still largely unknown. A deeper understanding of mechanics at the molecular level is essential for the design and construction of complex nanodevices. Here, we show that transient two-dimensional infrared (T2DIR) spectroscopy makes it possible to monitor the conformational changes of a translational molecular machine during its operation. Translation of a macrocyclic ring from one station to another on a molecular thread is initiated by a UV pulse. The arrival of the shuttling macrocycle at the final station is visible from a newly appearing cross peak between these two moieties. To eliminate spectral congestion in the T2DIR spectra, we use a subtraction method applicable to many other complex molecular systems. The T2DIR spectra indicate that the macrocycle adopts a boat-like conformation at the final station, which contrasts with the chair-like conformation at the initial station. A deeper understanding of the mechanics of molecular machines is limited by the fast motions which are in the nanosecond or picosecond timescale. Here the authors present a real-time observation of structural changes in a rotaxane-based molecular shuttle by transient two-dimensional infrared spectroscopy. In some respects, biological and synthetic molecular machines resemble macroscopic machines. However, many physical concepts governing macroscopic motion are not applicable to microscopic motion5. Therefore, the physical principles underlying the operation of nanomachines are actively investigated, both experimentally7 and theoretically8. Synthetic molecular machines are ideal systems to investigate such nanomechanics, due to their simplicity and superior robustness compared to biological molecular machines9.The development of supramolecular chemistry has made it possible to synthesise externally addressable molecular devices. These synthetic molecular machines are often inspired by the functionality of biological molecular machines6. Transient one-dimensional infrared (henceforth T1DIR) spectroscopy is very suitable for this purpose11, in particular in the special case that the molecular vibrations are localised on specific chemical bonds and are sensitive to conformation. However, T1DIR spectroscopy only probes changes in the frequencies and absorption intensities of molecular vibrations, and these properties are related to the conformation only in an indirect manner. Transient two-dimensional infrared (T2DIR) spectroscopy is a much more direct probe of structural changes at the molecular level17. The two-dimensional infrared (2DIR) spectrum of a molecule reveals the couplings between its vibrational modes19. These couplings are directly related to the relative orientation and distance of the coupled vibrational modes19. A 2DIR spectrum therefore provides direct conformational information, similar to 2D-NMR spectroscopy, but with a time resolution of typically 1 ps or less . In time-resolved 2DIR spectroscopy, one triggers a conformational change (e.g. by a UV-induced photoisomerisation or a temperature jump), and subsequently records 2DIR spectra at specific time delays with respect to the trigger. By recording a series of 2DIR spectra at increasing time delays, a molecular movie of the conformational changes can be obtained20. T2DIR spectroscopy is a relatively new method, but has already been applied successfully to investigate photo-13 and temperature-induced21 folding of peptides, tautomerisation of organic compounds16 and structural rearrangements in catalytic complexes14. Here, we use T2DIR spectroscopy to investigate the structural changes in a molecular machine during its operation cycle.To obtain a deeper understanding of the mechanics of molecular machines, one ideally would like to probe the conformational changes during their operation in real time. Since molecular motions typically occur on the nanosecond or even picosecond time scale, this requires a conformational probe with ultrafast temporal resolution22, which are known to couple strongly and in a distance- and orientation-dependent manner24. Thus, the relative distance and orientation of the components of the molecular shuttle can be probed by measuring the interactions between their respective CO-stretch and amide I vibrations;25 and by triggering the shuttling motion and recording a series of 2DIR snapshots, we can track the conformational changes as the shuttle progresses through its operation cycle.The molecular machine investigated here is a rotaxane-based molecular shuttle. The shuttling macrocycle and its two docking stations each contain amide groups, and the corresponding vibrational modes provide excellent localised vibrational chromophoresWe find that the cross peaks in the T2DIR spectra of the molecular shuttle recorded at different stages of its operation cycle directly reveal the proximity of the shuttling macrocycle to the different stations on the thread. Interpreting the T2DIR spectra using quantum-chemical calculations, we find evidence that the macrocycle adopts two distinct conformations depending on the station to which it is hydrogen bonded. To isolate the macrocycle/thread cross peaks from the other contributions to the T2DIR spectrum, we use a method for decomposing the congested equilibrium- and transient-2DIR spectra that exploits the presence of a localised electronic chromophore in the investigated molecule. We believe this method may be useful in other cases as well, especially because spectral congestion will occur more frequently as larger and more complex systems are investigated with 2DIR spectroscopy.11. Prior to the absorption of UV light, the macrocyclic ring , the macrocycle shuttles back to the succ station11.The chemical structure of the rotaxane-based shuttle is shown in Fig. A). T1DIR spectra comprise positive and negative contributions which arise from the vibrational modes of the UV-induced transient species and depleted electronic ground state, respectively. (3) Steady-state 2DIR spectra, which reveal the vibrational couplings in the electronic ground state. (4) T2DIR spectra, that show the UV-induced time-dependent changes in the 2DIR spectrum. The T2DIR spectra comprise contributions from the UV-induced transient species and contributions due to the electronic ground-state depletion. Both in the T1DIR and T2DIR spectra, the signals associated with the depleted electronic ground-state species have the opposite sign of those of the UV-generated transient species. Specifically, the T2DIR spectrum is obtained by subtraction of the steady-state 2DIR signal ΔA2DIR from the UV-pumped signal ΔAUV,2DIR:tUV is the waiting time between the triggering UV pulse and the probing 2DIR pulse pair. Coherent interactions caused by temporal overlap of the IR pulse-pair are avoided by using a fixed 1-ps delay between the IR-pump and IR-probe pulses. Figure 11. Coordinates  of spectral features in the 2DIR and T2DIR spectra refer to the negative feature for both diagonal and cross peaks.In the following, we will present four types of spectra: (1) steady-state infrared (1DIR) absorption spectra, which provide information on the molecular shuttle in thermal equilibrium. (2) UV-pump 1DIR-probe difference-absorption spectra (T1DIR), that show the UV-induced time-dependent changes in the vibrational absorptions (ΔtUV = 0 ns). Supplementary Table HB⋯succ, 1663 cm−1) and the hydrogen-bonded succ station  are the most relevant probes for determining the conformation of the macrocycle when bound to the initial station.In thermal equilibrium, the macrocyclic ring is hydrogen bonded to the succ station Fig. . The imiA in IR absorption induced by a spectrally narrow (16 cm−1 FWHM) tunable IR pump pulse, as a function of the probe frequency and of the centre frequency of the pump pulse (double-resonance 2DIR spectroscopy). When the pump frequency is resonant with one of the vibrational modes, the v = 1 state of this mode is populated, resulting in a signal on the diagonal peak of the 2DIR spectrum. Each diagonal peak comprises a negative (blue) and positive (red) ΔA part: the former is caused by vibrational ground-state depopulation and v = 1 → 0 stimulated emission, the latter by the v = 1 → 2 transition (which occurs at a lower frequency due to anharmonicity). Two diagonal features at  =  and  are observed in the 2DIR spectrum, which correspond to the mcHB⋯succ and succHB resonances, respectively. Off-diagonal peaks resembling the diagonal features appear in the spectrum at  =  and  when two modes i and j are coupled. The cross peak observed between the mcHB⋯succ and succHB bands at  is an indication of the coupling (and thus proximity) of the succ station and macrocycle CO groups. However, the mcHB⋯succ and succHB modes have significant spectral overlap with one of the naphthalimide aromatic-stretch (niAr) modes and with the antisymmetric naphthalimide CO-stretch (nias) mode. The latter two ni modes are strongly coupled (because they are located in the same molecular moiety), giving give rise to a strong niAr/nias cross peak. Due to the spectral overlap, we cannot obtain the separate contributions of the niAr/nias and succHB/mcHB⋯succ cross peaks from the congested 2DIR spectrum. Below, we will demonstrate how T2DIR can be used to solve this 2D-spectral congestion problem.Figure *) is in its triplet state due to rapid intersystem crossing. Figure tUV = 50 ns) of the operating cycle of the molecular shuttle. In this transient spectrum, both ni* absorption and ni ground-state bleaching peaks are observed. Comparing the tUV = 50 ns T2DIR spectra of the rotaxane : the disappearance of the antisymmetric and symmetric CO-stretch modes  symmetric CO-stretch vibration •− species at 1613 cm−1 . Figure tUV = 100 ns. The most notable diagonal feature is due to the •− station is still spatially separated from the other functional units  of the molecular shuttle. The fact that all signals in the T2DIR spectrum arise from the ni unit indicates that at this waiting time the macrocyclic ring and the succ station still have the same conformation as before the UV trigger (so that ΔΔA = 0 for these components).With increasing waiting time , systematic errors due to, e.g., beam drift can to some extent be cancelled in the depletion-T2DIR spectrum. (2) Excitation by the linearly polarised UV pulse generates an anisotropic distribution of excited molecules. For cancellation of the depleted 2DIR spectrum to occur, the waiting time tUV should be sufficient for the UV-excited molecules to scramble this anisotropy by rotational diffusion, so that the cancelling contributions in Eq. 28, but this is not possible for the perpendicular IR–IR polarisations. In the next section, we will demonstrate how we can separate the 2DIR contribution of the residual (non-excited) parts of the molecular device from the full 2DIR spectrum.Highlighting specific contributions in a congested 2DIR spectrum through selective depletion is a generally applicable method: the 2DIR spectrum of an excited moiety of a molecular system can be measured separately from that of the other parts of the system provided that the external stimulus  generates a sufficient change in the 2DIR signal of the moiety. The depletion will manifest as an inverted 2DIR signal in the T2DIR response. The method is also applicable if the UV/Vis excitation leads to non-reversible changes, such as photobleaching of particular moieties of interest: the signals belonging to the remainder of the molecule are unchanged and therefore cancel in the T2DIR spectrum, and one only observes the depleted contributions of the photobleached moiety. Practical aspects to be considered for the depletion method to work are 1) the signal-to-noise ratio and (2) the polarisation dependence of the signals. (1) If the 2DIR spectrum of depleted part of the molecule has a much larger amplitude than that of the remainder of the molecule, the difference-2DIR spectrum will be difficult to measure. Even if the 2DIR spectra have similar amplitudes, one has to average longer to obtain the same signal-to-noise ratio in the difference measurement as in the raw 2DIR spectra the signs absorption. Subsequently, the spectra are subtracted from one another to generate the 1DIR spectrum  and macrocycle mcHB⋯succ (amide I mode of the macrocycle hydrogen bonded to the succ station). We can use an analogous method to obtain the ni-free 2DIR signal. We add the appropriately scaled tUV = 50 ns T2DIR spectrum . Two diagonal features at  and  are observed in the resulting 2DIR spectrum  and   Fig.  arise soRij of a cross peak between oscillators i and j is directly related to the angle θij between their transition-dipole moments:19A⊥ and ΔA|| are the cross-peak intensities for perpendicular and parallel polarisations of the IR pulse pair, respectively. We use Eq. -optimised structure (at the B3LYP/6-31G(d) level, see Supplementary Fig. HB and mcHB⋯succ transition-dipole moments. These DFT calculations reproduce the transient vibrational spectra very well . This angle corresponds to the chair-like conformation . The stronger hydrogen bonding to the ni•− station as compared to the succ station causes the mc amide I mode frequency to red shift upon shuttling, from 1663 to 1654 cm−1.In the T1DIR spectra at  ns Fig. , we obsetUV = 1000 ns is significantly more complex than at the earlier tUV delays. The shuttling causes the amide I and CO-stretch frequencies of all three components of the shuttle  to change. As a consequence, the ground-state depletion signal at tUV = 1000 ns  which is due to the •− station), and  which is due to the tUV = 1000 ns, a small fraction of the macrocycles in the sample still reside at the succ station, observable as a low-intensity ΔA at 1613 cm−1  indicates the proximity of the ni•− station and the macrocycle. This cross-peak feature is a direct demonstration that the molecular device has successfully switched. We expect to observe a complementary cross peak at  = . In the calculated structure, the θij and the CO group geometry, see Supplementary Note To complement the T2DIR experiments, we perform DFT calculations on a model compound comprising the macrocyclic ring, a short thread and the niure Fig. , the macR6 for through-space dipolar coupling), the IR chromophores in the moving parts must be in close contact to give rise to observable cross peaks.In the above, we have shown that the conformational changes of a molecular device can be followed during its operation with the combined temporal and structural resolution of T2DIR. T2DIR spectroscopy can be used to investigate other molecular machines in a similar manner, with certain practical boundary conditions: (1) the motion must be externally triggered and (2) the number of IR-active modes in the investigated spectral region should be sufficiently small to enable unambiguous interpretation of the spectra. This somewhat limits the applicability for peptide-based molecular devices, although isotope-substitution can be used to limit the spectral congestion. (3) Since the cross-peak intensities decay rapidly with increasing distance  final state of the molecular shuttle. In addition, we demonstrate that we can obtain the electronic ground-state 2DIR spectrum of a UV/Vis chromophore from its optically excited ground-state depletion spectrum, which can be a useful method to simplify complex 2DIR spectra. In fact, using this subtraction method was essential to determine the macrocycle conformation in the equilibrium state, since the conventional 2DIR spectrum would have been too congested to do so. A similar procedure can be used in any molecular system that contains a moiety whose electronic state can be modified , for instance by photo- or electrochemical methods.32. The spectroscopic experiments are performed on a solution of rotaxane (5 × 10−4 M) and 1, 4-diazabicyclo[2.2.2]octane  in CD3CN . The solvent and DABCO have no absorption bands in the investigated IR-spectral region. To remove dissolved oxygen, argon is bubbled through the solution for >15 min prior to the experiments. The sample is kept at 70 °C in a thermostated IR cell which consists of two CaF2 windows separated by a 5-mm spacer. To avoid photochemical degradation and heating due to accumulated UV-pump pulses, we move the sample cell in a Lissajous figure using two computer-controlled translation stages.We synthesised the rotaxane as described previously−1, 800 fs FWHM) using a Fabry-Perot interferometer25. The absorption changes were monitored using frequency-dispersed detection of the broad-band probe pulses using a 2 × 32 pixel HgCdTe array detector. The 2DIR spectra were obtained with a repetition rate of 450 Hz in a similar fashion without the UV pump. Conventional (steady-state) Fourier-transform infrared (1DIR) spectra are measured with a Bruker Vertex 70 spectrometer (resolution 2 cm−1).The T2DIR spectrometer is described in detail in the Supplementary Methods, see Supplementary Fig. 11, we have used the B3LYP functional with the 6-31Gd basis set. The effect of the solvent acetonitrile was included by using the Polarizable Continuum Model. In the present work, we have explored the effects of using a larger basis set and different functionals on the structure and spectra for a representative conformation of the naphthalimide anion−macrocycle complex. In the model systems  but also a gauche-pentyl chain on the imide nitrogen, in order to test the effect of further symmetry lowering. We conclude from the calculated data (summarised in Supplementary Table −1).Quantum-chemical calculations were used to obtain structural models of the rotaxane, and to support the interpretation of the T2DIR spectra based on empirical assignments. In the previous workems Fig. , all fea•− was ~100 cm−1 (directly obtained from the difference between the symmetric and antisymmetric CO-stretch modes), which is much larger than the linewidth of the ni•− CO-stretch IR peaks; hence in this case no localisation on single CO bonds occurs, and the ni•− CO-stretch peak at ~1590 cm−1 originates the combined in-phase motion of the two ni•− CO groups. Exactly the opposite is found for the amide I mode of the macrocycle: the calculations (regardless of the model used) show that the frequencies of the four possible combinations fall within 10 cm−1, which is much less than the experimentally observed linewidth, so that in this case the modes are localised on individual CO bonds.The coupling between the two CO-stretch modes in niThe datasets generated and analysed during the current study are available from the corresponding authors on reasonable request.Supplementary Information