Mycobacterium bovis infection serve as a recurrent source of infection for domestic livestock. Alternative control measures are urgently required and vaccination is a promising option. Although the M. bovis bacille Calmette-Guérin (BCG) vaccine has been used in humans for nearly a century, its use in animals has been limited, principally as protection against TB has been incomplete and vaccination may result in animals reacting in the tuberculin skin test. Valuable insights have been gained over the past 25 years to optimise protection induced by BCG vaccine in animals and in the development of tests to differentiate infected from vaccinated animals (DIVA). This review examines factors affecting the efficacy of BCG vaccine in cattle, recent field trials, use of DIVA tests and the effectiveness of BCG vaccine in other domestic livestock as well as in wildlife. Oral delivery of BCG vaccine to wildlife reservoirs of infection such as European badgers, brushtail possums, wild boar, and deer has been shown to induce protection against TB and could prove to be a practical means to vaccinate these species at scale. Testing of BCG vaccine in a wide range of animal species has indicated that it is safe and vaccination has the potential to be a valuable tool to assist in the control of TB in both domestic livestock and wildlife.Bovine tuberculosis (TB) continues to be an intractable problem in many countries, particularly where “test and slaughter” policies cannot be implemented or where wildlife reservoirs of  Mycobacterium bovis, Mycobacterium caprae, and other members of the Mycobacterium tuberculosis complex, including M. tuberculosis whose role in animal TB is being increasingly recognised, particularly in studies from Africa and Asia (M. bovis infection.Tuberculosis (TB) in domestic livestock and wildlife is caused by and Asia . Livestoand Asia . The disand Asia . FurtherM. bovis include the Australian brushtail possum (Trichosurus vulpecula) in New Zealand, the European badger (Meles meles) in United Kingdom (UK) and Ireland, white-tailed deer (Odocoileus virginianus) in Michigan, USA . The usM. bovis , 106. Pr animals , 107. FuM. bovis and there was no difference in the levels of protection induced by Pasteur and Danish sub-strains of BCG  ~48 h after the higher dose of BCG was administered and by one of the nine vaccinated animal (80 CFU/g) ~24 h after receiving the lower dose of BCG. No evidence was found for the transmission of BCG to non-vaccinated, sentinel, badgers housed with the vaccinated animals despite the occasional excretion of BCG in faeces. The target dose of BCG for the oral vaccination of badgers is yet to be defined.A practical limitation to the extensive use of BadgerBCG is the need to trap badgers before the vaccine can be injected and the use of an oral bait delivery system would be advantageous. BCG has been incorporated in a wide variety of baits, including encapsulation in the same lipid matrix used to deliver BCG orally to possums. Administration of BCG orally to captive badgers, either directly to the back of the throat, or indirectly via ingested bait has been shown to protect badgers against experimental challenge with s of BCG , 108. To CFU BCG . No adveM. bovis culture confirmed lesions in the fully (100%) vaccinated zone (9%), compared with the non-vaccinated (0%) zone (26%).A field trial was recently completed in Ireland that provided the first estimate of oral BCG efficacy under field conditions . Lipid-eM. tuberculosis complex (MTC) in the Mediterranean regions of the Iberian Peninsula, Spain and TB prevalence in wild boar has been associated with TB occurrence on cattle farms  vaccine has produced significant protection (70–80% lesion score reduction) in laboratory challenge trials  in Montes de Toledo, Spain  can become infected with M. bovis via feeding on tuberculous carcasses, particularly possums and potentially can become a source of infection for other wildlife or cattle  and the buffaloes were challenged with virulent M. bovis via the intratonsilar route. The study did not reveal significant differences in the number of lesioned animals between the vaccinated and control groups   . Similarper se and/or increased productivity post-BCG vaccination could potentially have benefits in developing countries. Strategic use of BCG vaccine for livestock could also be implemented in regions where wildlife serve as reservoirs of infection, particularly where it is not feasible to contain the spread of infection from wildlife. In these situations, DIVA tests, particularly skin tests utilising specific M. bovis antigens, could be used in livestock in association with vaccination to allow vaccination to be integrated with “test and slaughter” control measures.Experimental challenge studies in domestic livestock including cattle, goats, sheep and farmed deer have demonstrated that BCG vaccination can moderate the severity of the disease, while field trials in cattle and goats have indicated that vaccination can also reduce infection. No single vaccine has been shown to be better than BCG in cattle, although combinations of BCG with various subunit TB vaccines have produced encouraging results and could have application in the future . V5-106 CFU parenterally or 108 CFU orally and no differences have been detected in protection induced by two of the most commonly used BCG strains, Pasteur and Danish. Protection has been shown to wane between 1 and 2 years post-vaccination and revaccination is recommended every 1–2 years to maintain levels of immunity. BCG vaccine has been shown to be safe in cattle and vaccination of cattle pre-infected with M. bovis is not likely to exacerbate or cure infection. Vaccination is likely to produce false reactions in traditional TB diagnostic tests in the first 12 months post-vaccination and as protection induced by BCG is not complete, DIVA tests should be used if “test and slaughter” control policies are in place. It would be preferable to use BCG vaccination in association with other TB control measures such as minimising the chance of early exposure to M. bovis by feeding young calves with colostrum or milk from non-reactor cattle or with heated milk, segregating reactor and non-reactor cattle into separate herds and keeping vaccinated calves with the non-reactor animals.A number of recommendations can be made from the experimental challenge and field experiments in cattle. Calves should be vaccinated with BCG as young as possible, optimally by 2–4 weeks of age, at doses of 10The field testing of BCG vaccine in possums and badgers administered via oral or parenteral routes have resulted in the induction of significant reductions in infection of these animals and a parenteral BCG vaccine has now been licensed for use in badgers in the UK. In wild boar, feral deer and ferrets, BCG vaccine has been shown to induce significant levels of protection against experimental challenge with TB. Practical systems for delivery of oral bait TB vaccines to wildlife have now been established, but further research is necessary to improve oral bait formulations with appropriate attractants, systems for optimising bait distribution and avoiding bait uptake by non-target species. BCG vaccine has been shown to be safe in all animal species tested, although BCG has been isolated from lymph nodes draining vaccination sites and from faeces of animals for a short period following oral vaccination. There was evidence that vaccinated white-tailed deer could transmit BCG to non-vaccinated pen-mates, but not to cattle exposed to the room previously occupied by the vaccinated deer.M. bovis, the protection should be sufficient to markedly reduce onward transmission to others animals. This feature could ensure that BCG vaccine could be particularly valuable for reducing infection in wildlife populations and in domestic animals where infection is currently very high and where “test and slaughter” control strategies are not able to be undertaken. There are numerous technical hurdles still to be overcome before an economically viable oral vaccine for use in badgers in the UK might be available. In the meantime it is beholden on stakeholders to make the best use of the existing tools available, this includes the intramuscular BadgerBCG vaccine. Cattle BCG vaccination in countries using “test and slaughter” control strategies also face significant hurdles. For example, a BCG vaccination-compatible DIVA test needs to be validated to allow vaccination to continue alongside traditional “test and slaughter” control programmes; currently BCG vaccination is prohibited under EU and some other countries' legislation and this would need to change; finally, cost-benefit analyses would decide whether deployment would proceed.In summary, there have been major advances in the past 10–20 years in our understanding of the factors influencing BCG vaccine efficacy for domestic livestock and wild animals. To optimise the use of BCG vaccine, it will be important to continue to field test BCG vaccine in the various animal species in different environments, husbandry systems and in the presence of varying levels of disease prevalence as well as evaluating the practical application of DIVA tests. Although BCG vaccine may not provide complete protection against exposure to BB, HV, MC, and L-MdK-L wrote sections of the manuscript. All authors contributed to the manuscript revision, read and approved the submitted version.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.


Congenital heart disease (CHD) is one of the most common birth defects, and recent studies indicate cilia-related mutations play a central role in the genetic etiology of CHD. As cilia are also known to have important roles in kidney development and disease, it is not surprising that renal anomalies were found to be enriched among CHD mutant mice recovered in a large-scale mouse forward genetic screen. Indeed 42% of mutations identified to cause both CHD and renal anomalies were cilia-related. Many of these cilia mutations comprise cilia transition zone or inversin compartment components, consistent with the known role of these cilia proteins in a wide variety of ciliopathies. The high prevalence of CHD with congenital anomalies of the kidney and urinary tract (CAKUT) observed in mice was also corroborated with clinical studies that showed 20–30% of CHD patients have renal anomalies. Together these findings suggest CHD patients may benefit from early screening for renal anomalies to allow early diagnosis and intervention to improve outcome for this vulnerable patient population. Congenital heart disease (CHD) is the most common birth defect, occurring in up to 1% of live births –4. In faCAKUT represent a broad range of kidney and urinary tract defects. This can include abnormalities in the shape, size, or structure of the kidney including kidney agenesis, kidney hypoplasia or dysplasia, horseshoe/fused kidney, cystic kidneys, or duplex/multiplex kidney, and multiple collecting ducts or ureters . These dTBX1, a transcription factor within the T-box family, has been shown to contribute to the cardiac phenotypes associated with this disorder  of which were cilia-related . Ci. Ci32). N = 34) were cilia-related, including 11 affecting transition zone or inversin compartment components  also had some form of renal defect such as renal cysts, kidney agenesis, cystic dysplastic kidneys, and horseshoe kidney Figure , Table 2Studies in both CHD mutant mice and CHD patients showed CAKUT is highly associated with CHD. This is supported by the finding of CAKUT and CHD in various genetic syndromes, and the finding that many mutations identified to cause CHD can also cause CAKUT phenotypes. Among genes identified to cause both CHD and CAKUT there is a significant enrichment of cilia genes, indicating a central role for cilia in the pathogenesis of CHD and CAKUT phenotypes. However, as half of the mutations identified to cause CHD and renal anomalies are not known to be cilia-related, the association of CHD with renal anomalies likely extend beyond the role of cilia in heart and kidney development. The clinical relevance of these findings in mice were shown with the observation that 23–30% of CHD patients also exhibited renal anomalies. This would suggest CHD patients may benefit from routine evaluation for renal anomalies. This might improve the long-term outcome of this high-risk patient population by reducing potential renal complications with early diagnosis and therapeutic intervention.All authors listed contributed intellectually to this work and approved it for publication.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.


Humans often learn new things via imitation. Here we draw on studies of imitation in children to characterise the brain system(s) involved in the imitation of different sequence types using functional magnetic resonance imaging. On each trial, healthy adult participants learned one of two rule types governing the sequencing of three pictures: a motor-spatial rule  or an object-based rule (in the cognitive task). Sequences were learned via one of three demonstration types: a video of a hand selecting items in the sequence using a joystick (Hand condition), a computer display highlighting each item in order (Ghost condition), or a text-based demonstration of the sequence (Text condition). Participants then used a joystick to execute the learned sequence. Patterns of activation during demonstration observation suggest specialisation for object-based imitation in inferior frontal gyrus, specialisation for spatial sequences in anterior intraparietal sulcus (IPS), and a general preference for imitation in middle IPS. Adult behavioural performance contrasted with that of children in previous studies—indicating that they experienced more difficulty with the cognitive task—while neuroimaging results support the engagement of different neural regions when solving these tasks. Further study is needed on whether children’s differential performance is related to delayed IPS maturation. Imitation, the ability to copy others’ responses, has intrigued scientists at least since Darwin linked imitation with human cognitive evolution . Work inWhile imitation of single, familiar actions has been widely studied, imitation of action sequences has received less attention. Studies of children’s behavioural performance in sequence imitation tasks suggest different developmental patterns for imitation of sequences defined by objects and imitation of sequences defined by actions to locations ,5. The aOur present project draws heavily on a detailed series of studies of how children –7 and noFor both tasks, the correct sequence can be learned in several different ways. If no instructions are available, participants can learn individually by trial and error. A demonstration from a knowledgeable person (presented live or via video) gives the opportunity to learn by imitation. A partially incorrect demonstration from another person can allow participants to infer the correct sequence. A verbal or written instruction cue could allow participants to perform correctly, but without any self-other matching. Finally, participants could learn by seeing the correct sequence of items highlighted on the computer without seeing another person, and matching the observed goals and results but not body or hand actions ; in thisspecific difficulty with imitating in the spatial task, which is independent of their sequencing performance in other conditions with both the spatial task and the cognitive task.By using these sequencing tasks and various learning conditions, researchers have demonstrated that the imitation capacity does not develop in a concerted fashion but rather comes online unevenly in the preschool years ,5,7. ThaBoth children and non-human primates (macaque monkeys) improve their performance on the cognitive task when given the opportunity to learn from a conspecific via imitation ,9, compaThe research presented here aims to explore the neural systems which underlie imitation performance in these tasks, potentially helping to shed light on the differential imitation behaviour observed in children. In particular, we aim to test whether imitation (as opposed to emulation or other forms of vicarious learning) in the cognitive and spatial tasks relies on different neural substrate(s). In this study, we adapt the cognitive and spatial sequencing tasks previously used with children and non-human primates –10 to beOur overall design for the present study was a 2 × 3 within-subject factorial design, with the factors task  and demonstration type (Hand/Ghost/Text), as illustrated in To make predictions for which brain areas are likely to be engaged in our sequence learning tasks, we can draw on the wide range of past studies of action and imitation. Individual studies and meta-analyses indicate that parts of the ‘mirror neuron system’ are robustly activated during imitation tasks –18, althNineteen right-handed adults  were recruited via local publicity. Four additional adults took part in the study but were excluded from analysis due to completion of less than two full scanning runs or excessive errors . Written informed consent was obtained from all participants before testing. All procedures were approved by the University of Nottingham Medical School ethics committee.Two touchscreen-based tasks used previously with typically developing children and children with autism ,5,7,9,14Cognitive Task  > [Ghost-cognitive–Ghost-spatial]; [Hand-cognitive − Hand-spatial] > [Text-cognitive − Text-spatial]; and [Ghost-cognitive–Ghost-spatial] > [Text-cognitive–Text-spatial]. Each interaction was calculated with a A behavioural analysis of response times  and error rates was performed, and results were compared via 2 × 3 (task type × demonstration type) ANOVA in SPSS . Data from trials in which errors were made were not included in the behavioural analyses.F = 64.77, p < 0.001] and demonstration type , as well as a significant interaction between task type and demonstration type . A one-way repeated-measures ANOVA to test for simple effects showed that response times after different demonstrations for the cognitive task were not significantly different from each other . However, response times after different demonstrations for the spatial task differed significantly . Response times after Hand demonstrations of the spatial task  were slower than those after Ghost demonstrations , and those after Ghost demonstrations were slower than those after Text demonstrations , F = 134.032, p < 0.001, partial η2 = 0.882.Response times showed task-based as well as demonstration-based differences . A repeaF = 25.02, p < 0.001] but not of demonstration type, and a significant interaction between task type and demonstration type . A one-way repeated-measures ANOVA to test for simple effects showed that error rates after different demonstrations of the cognitive task were not significantly different ; error rates also did not differ after the different demonstrations of the spatial task . However, one-way ANOVAs found that significantly more errors were made for the cognitive task than the spatial task following both Hand  and Ghost demonstrations . There was no difference between errors made in the cognitive and the spatial tasks following Text demonstrations . Error trials were removed from fMRI analysis so differences in error rates cannot impact on the fMRI results.For error rates , a repeaGreater brain activation when observing demonstrations of the cognitive task compared to the spatial task was found in left fusiform gyrus across all three demonstration types in a conjunction analysis . Both HaGreater brain activation when observing demonstrations of the spatial task compared to the cognitive task was found in the left intraparietal sulcus across all three demonstration types in a conjunction analysis . In the Observing Hand demonstrations compared to observing Text demonstrations resulted in a substantial activation across lateral occipitotemporal cortex in both the cognitive and spatial tasks . In the Observing Hand demonstrations in both the cognitive and spatial tasks, compared to Ghost demonstrations, led to activations in occipitotemporal cortex . Left anRegions in the parietal cortex were engaged in several of the contrasts described above. To understand these patterns of overlap, we plotted different combinations of contrasts on the same brain image, and examined the parameter estimates  in regions where contrasts overlapped. We found that a region of the left anterior intraparietal sulcus (MNI coordinate −35 −40 52) was more active for the Hand-spatial task than the Hand-cognitive task, and was also more active for the Hand-cognitive task than for the Text-cognitive task . A regioThree interaction contrasts were tested, each in both positive and negative directions, and full results of all interactions are given in While many neuroimaging studies have explored the imitation of single, familiar actions such as the lifting of a finger or the grasping of objects , few stuPrevious studies pointed to different developmental trajectories for imitation in the cognitive and spatial tasks, and one of our aims in this study was to determine if there are different neural systems underlying learning in these tasks. Stronger activation for the cognitive task compared to the spatial task was found in both left fusiform gyrus and left orbital gyrus for all three demonstration types . This acTo link these areas to specific cognitive components would require reverse inference, which is outside the scope of this paper. However, we note that previous work shows that object recognition and memory tasks, especially those that use pictorial material, engage fusiform cortex . In addiAnother of our aims in the present paper was to examine neural regions involved in sequence imitation; it is important to know if these regions respond only to imitation, or are also engaged by other types of learning. Classic neurocognitive models of imitation imply a dedicated imitation system instantiated in the human mirror neuron system . Such a Searching beyond the traditional mirror system areas, we find that lateral occipital regions are strongly activated for viewing Hand demonstrations compared to Text or Ghost demonstrations  and are Overall, we found two areas that were more active for Hand than Text demonstrations: middle IPS and lateral occipital cortex. However, these differences could be due to specialisation for tasks other than imitation, and we consider these more in the section below.To understand the specificity of imitation learning, it is important to compare how people learn by imitation to how they engage in other forms of learning. Ghost control conditions are commonly used in developmental studies, to contrast the imitation of actions against the emulation of a goal or the end state of an action . Here weDespite previous findings of differences at the behavioural level, few differences between Hand and Ghost conditions were apparent in the neuroimaging data in this study. Observing the Hand demonstration compared to the Ghost demonstration engaged occipitotemporal regions bilaterally for both the cognitive and spatial tasks. This result is in line with outcomes of previous studies, which show that these areas are engaged in higher-order visual processing. For instance, middle and inferior occipital cortex are engaged when viewing hands and body parts even without any motor responses . There wDevelopmental data showing that children can perform cognitive imitation tasks before spatial imitation tasks might be taken to imply that there are specialised systems in the brain to enable cognitive imitation ,49,50. STo place this in the context of previous findings, many studies have linked IFG to imitation . IFG is Overall, our data suggest three regions within the classic mirror system which are engaged in our imitation tasks: the IFG, middle IPS, and anterior IPS. We suggest that IFG and middle IPS work together in the Hand-cognitive task, while anterior IPS and middle IPS work together in the Hand-spatial task. The finding that there is no single brain network involved in imitation tasks is consistent with prior fMRI studies showing that the entire MNS network is not active for every imitation task ,20,24.Novel imitation  may be distinct from familiar imitation ; in this study, participants engaged in novel imitation, as sequences were unique on each trial. While the observation and execution of familiar actions engages canonical MNS regions ,53, the The tasks we use in the present study are closely modelled on our developmental research tasks, but differ from other tasks commonly used in fMRI studies of immediate imitation. Thus, some might argue that our tasks do not tap ‘imitation’ as typically studied in cognitive neuroscience. Following the conventions of the behavioural sciences, we have operationalised imitation as the copying of a novel sequence ,56–58; rIt is important to consider that participants might have used other strategies. For example, it is possible for participants to solve the Hand-cognitive task by ignoring the central video clip of the hand operating the joystick, attending only to the identities of the objects in each sequence, and converting them to a subvocal list. The present study controlled for this possibility using the Text instruction condition. If subjects used a subvocal list strategy in each condition, then there should be few or no significant differences between Hand and Text conditions. Yet there were robust activation differences between the Hand and Text conditions for both tasks . Thus, wAn aim of the present study was to use studies of sequence imitation in children to inform our characterisation of the neural systems of imitation in the adult brain. This means it is important to consider how behaviour on this task compares in children and adults. Adult participants in this study were slower to select sequences in the cognitive task than the spatial task, and they experienced greater error rates when doing so. Slower and more error-prone performance indicates that the cognitive task may have been somewhat more difficult for adults than the spatial task. These behavioural results are in contrast to preschool children’s performance, which is less accurate when imitating in the spatial task than the cognitive task. Additionally, children’s performance is similar (if slightly less accurate) when given ghost demonstrations of the cognitive task compared to social demonstrations (imitation) . In the Consistent with behavioural differences observed when children imitated in the cognitive versus the spatial task, in the present study with adults, differences emerged in brain involvement between the tasks. Specifically, several brain areas  showed consistent activation differences when observing demonstrations of the two tasks Figs  and 5. FThe present study focuses only on the demonstration phase of the task, rather than the subsequent performance of imitation. This is because the richness of the present data leaves little space to discuss further results. Recent research indicates that observing with the intention of imitating an action  and passively observing an action with no intention of imitating activate different brain regions, including inferior parietal lobule and parts of premotor cortex . FurtherIt is possible that for adults performing the cognitive task, the spatial information from the observation phase interferes with the correct selection of items in the execution phase, resulting in less accurate performance than in the spatial task. However, if this is the case for adults, it is not the case for young children, as 3-year-olds’ imitation performance is more accurate in the cognitive task.Finally, the differences between conditions were subtle. Though participants could have adopted similar sequencing strategies for both the cognitive and spatial tasks and for the different demonstration conditions , the finding of robust activation differences between tasks and demonstrations shows that they did not. It remains possible that larger differences in brain activation could be found if our tasks and demonstrations differed more, but in such a study it would also be harder to interpret the results, in which case larger differences in brain activation could simply be due to the corresponding differences between the tasks. Our closely matched stimuli provide precise control of the task and the level of social information in each trial, and thus provide a strong test of the brain systems for sequenced imitation behaviour.Much remains to be explored regarding how people learn new things from others and how different brain areas work together to achieve this. After controlling for language and working memory (Text condition), we find two distinct brain regions used in imitation learning. Specifically, observing to imitate novel object-based rules (cognitive imitation) from a Hand demonstration selectively activated IFG, while observing to imitate novel location-based rules  from a Hand demonstration activated left anterior and middle IPS. These results indicate that non-overlapping brain systems are involved in the imitation of different types of sequences, and may help to explain differential imitation performance in these tasks in children. Future work can trace how these regions change in typical and atypical development, and how the neural systems underlying these two exemplars of imitation relate to other forms of imitation behaviour.S1 Table(XLSX)Click here for additional data file.


Working memory is the ability to hold information “online” over a time delay in order to perform a task. This kind of memory is encoded in the brain by persistent neural activity that outlasts the presentation of a stimulus. Patients with schizophrenia perform poorly in working memory tasks that require the brief memory of a target location in space. This deficit indicates that persistent neural activity related to spatial locations may be impaired in the disease. At the circuit level, many studies have shown that NMDA receptors and the dopamine system are involved in both schizophrenia pathology and working memory-related persistent activity. In this Hypothesis and Theory article, we examine the possible connection between NMDA receptors, the dopamine system, and schizophrenia-linked working memory deficits. In particular, we focus on the dopamine breakdown product homocysteine (HCY), which is consistently elevated in schizophrenia patients. Our previous studies have shown that HCY strongly reduces the desensitization of NMDA currents. Here, we show that HCY likely affects NMDA receptors in brain regions that support working memory; this is because these areas favor dopamine breakdown over transport to clear dopamine from synapses. Finally, within the context of two NMDA-based computational models of working memory, we suggest a mechanism by which HCY could give rise to the working memory deficits observed in schizophrenia patients. The purpose of this Hypothesis and Theory article is to lay groundwork for linking two findings that are typically observed in subjects with schizophrenia: spatial working memory deficits and elevated homocysteine (HCY) levels. We briefly explain these two findings, simultaneously illustrating their possible relationship within a framework of dopaminergic physiology. We next present a theory within the context of computational models of persistent neural activity through which HCY may affect neural circuits that mediate spatial working memory. Finally, we suggest future experiments based on our theory.Schizophrenia patients frequently show deficits in spatial working memory. When a subject with schizophrenia is asked to remember a spatial location over a short delay of 5 or 30 s, they perform significantly worse than both healthy controls and cohorts with bipolar disorder Figure . The errThe perseverative feature of answers during working memory tasks appears to reflect a general tendency of schizophrenia patients to perseverate in other tasks. For example, during the Wisconsin Card Sorting Test, schizophrenia patients have repeatedly shown to hold on to previously correct sorting rules even when their currently implemented sorting scheme is incorrect .A seemingly unrelated characteristic of schizophrenia patients is the presence of high levels of the amino acid HCY in the blood ; however, the mechanism by which HCY is related to cognitive deficits and schizophrenia is unknown. Granted, high HCY levels may be an epiphenomenon of schizophrenia: heightened HCY may not induce symptoms but could instead be a byproduct of an unknown dysregulated process that occurs more often in patients. If this were the case, however, it would be surprising that nutritional strategies used to reduce levels of homocysteine have proven to reduce schizophrenia symptoms and improve performance of schizophrenia patients on the Wisconsin Card Sorting Test  occurs widely in the brain. Indeed, most dopamine cell groups, including the midbrain ventral tegmental area (VTA) and substantia nigra (SN), express DAT and recycle dopamine for repeated use in their axons’ target zones , also known as the optic tectum. The SC expresses a striking mosaic pattern of dopamine receptors, with D1 receptors localized to its superficial visual layers and D2 receptors enriched in its multimodal motor layers  appears to be a hybrid of the SC and BG in terms of DAT and COMT expression. COMT activity and dopamine levels are similar between frontal cortex and the SC ), we focus here on HCY due to its association with schizophrenia.astrocytes, as revealed by elegant culture experiments in Huang et al.  and metabolize (MTHFR TT/CC) HCY. The Val COMT polymorphism produces an enzyme that is ~40% more active in metabolizing dopamine than the Met polymorphism (Chen et al., Interestingly, COMT and MTHFR polymorphisms that enhance HCY levels combine to produce working memory deficits in schizophrenia patients (Roffman et al., in vivo patch clamping (Ahrens et al., Second, a systems neuroscience approach should be taken to examine the role of HCY in working memory maintenance. Working memory paradigms should be utilized that resemble the tasks schizophrenia patients fail using model systems that are more tractable than humans or monkeys (e.g., Felsen and Mainen, It is already suspected that dopamine release is critical to WM maintenance due to DA neuron activity patterns and the effects of dopamine antagonists and agonists on working memory performance. However, fluorescent probes or electrophysiological methods indicating the presence of DA and HCY in persistently active populations haven’t been employed in the context of working memory (i.e., amperometry, two-photon imaging; Sarkar et al., MC-P supervised all graduate work by AB that lead to the development of the presented theory. AB conceived the theory and drafted the manuscript in consultation with MC-P. AB and MC-P edited and revised the manuscript and approved the final version.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.


Smoking cessation is a key step towards improving cancer care and outcomes. However, smoking cessation interventions are underprovided in oncology settings. Within Jordan’s only comprehensive oncology center, we sought to evaluate receipt of care at a smoking cessation clinic and the effect of assisted abstinence through the smoking cessation clinic on short-term (two-year) survival after a cancer diagnosis.We employed a retrospective cohort study design. Cancer registry and smoking cessation clinic data for adult Jordanian cancer patients diagnosed between 2009 and 2016, who also were cigarette smokers, and who received full treatment at King Hussein Cancer Center, were analyzed. Specifically, descriptive statistics of patients who visited the smoking cessation clinic were generated, and short-term (two-year) hazard of death of patients based on whether or not smoking cessation clinic-assisted abstinence occurred, were evaluated.There were 3403 patients who met our inclusion criteria. Approximately 21% of cancer patients were seen at the smoking cessation clinic, and significant demographic and clinical disparities in who was being seen [at the smoking cessation clinic] existed. In 2387 patients with available survival data, smokers who never went to the smoking cessation clinic  had a hazard of death 2.8 times higher than smokers who had visited the smoking cessation clinic and who also confirmed they had not smoked on atleast two of their 3-, 6- or 12-month follow-up visits . Non-abstainers at the smoking cessation clinic exhibited a similar disadvantage .Although evidence-based smoking cessation interventions increase the likelihood of abstinence and can lower the short-term hazard of death during cancer treatment, there is a deprioritization of smoking cessation interventions during cancer care, as indicated by low proportions of patients seen at the smoking cessation clinic. Our findings emphasize the importance of promoting interventions to avail smoking cessation interventions in oncology settings within the cancer treatment phase. Given the rising global burden of cancer, ensuring that cancer care is optimized and availed equitably has become an increasingly challenging priority for most countries, and resource constraints have rendered many therapeutic interventions difficult if not impossible to access . In respSmoking cessation management for cancer patients offers one such example of a cost-effective intervention to improve patient outcomes, and has been called for by many –10. ConvWe were interested in demonstrating the benefits of smoking cessation during cancer treatment by examining the two-year survival benefits associated with smoking cessation clinic-assisted abstinence, among a diverse group of cancer patients. We also were interested in identifying which patient subgroups were less likely to receive smoking cessation care, thereby potentially foregoing the benefits of smoking cessation during cancer treatment. We hypothesized that patients receiving care at the smoking cessation clinic and reporting abstinence would be at survival advantage to cancer patients who did not receive such care. We also hypothesized that being older, having an advanced stage of cancer, and not having a tobacco-related cancer in the conventional sites known to be associated with tobacco use  were associated with a decreased likelihood of receiving care at the smoking cessation clinic.King Hussein Cancer Center (KHCC) in Amman, Jordan is a Joint commission, disease-specific accredited comprehensive cancer center which offers cancer care to a substantial proportion of the Jordanian population, and also serves as a regional cancer treatment hub for the Middle East . ApproxiA retrospective cohort study design was used to analyze hospital-based cancer registry records and clinical records of cancer patients receiving care at KHCC’s smoking cessation clinic.The analysis was based on records from KHCC, and included adult Jordanian cancer patients who were current smokers and receiving care at the Center. Records spanning patients diagnosed between the years 2009 and 2016 were included. Inclusion criteria were being 18 years or older, being a Jordanian, having a documented status as a current [cigarette] smoker in KHCC’s registry records, and receiving complete care at KHCC.n = 2387, those diagnosed after 2011).Two-year hazard of death after diagnosis was measured to evaluate whether or not visiting the smoking cessation clinic within a year of diagnosis (when most interventions to treat patients are undergone) imparted survival benefits . Reliable survival data were available from years 2012 onwards at the Center. Survival analyses were therefore conducted on a subgroup of patients  indicating positive point-prevalence abstinence (1)Patients with only one follow-up point (from a maximum of three follow-up points) indicating positive point-prevalence abstinence (2)Patients with more than one follow-up point (from a maximum of three follow-up points) all indicating negative point-prevalence abstinence (3)Patients visiting the smoking cessation clinic more than once [within a year of diagnosis] but not having accrued sufficient follow-up information (4)Patients who visited the clinic only once and did not return to the clinic (5)Patients who came to the clinic after a year of diagnosis (6)Patients who visited the smoking cessation clinic: Generally, patients who visit the cessation clinic are followed to record 3-, 6- and 12-month repeated seven-day point-prevalence [self-reported] abstinence status, a measure that is used in the smoking cessation literature , 27. SpePatients who never visited the smoking cessation clinic (7)With regards to predictors associated with two-year survival, we were interested in ascertaining whether or not a survival advantage may have been observed in patients visiting the smoking cessation clinic (as a result of their abstinence due to the receipt of smoking cessation care). The following section describes the possible profiles for our sample of cancer patients [who smoked]:Group A : Patients who were seen [within a year of diagnosis] more than once at the clinic and whose records indicated abstinence at atleast two follow-up points from a maximum of three follow-up points Group B : Patients who were seen [within a year of diagnosis] more than once at the clinic and whose records indicated abstinence at only one follow-up point from a maximum of three follow-up points;Group C : Patients who were seen [within a year of diagnosis] more than once at the clinic and whose records indicated no abstinence at any follow-up point. Patients who did not accrue sufficient follow-up records to measure abstinence also were included in this group (i.e. assumed to be non-abstinent);Group D : Patients who were never seen at the clinic or seen only once, or were seen after a year from diagnosis.According to the above profiles, four main groupings of patients were created to describe abstinence trajectories:various sociodemographic and clinic variables were included as control variables given their potential for being associated with our outcome variables of interest. These specifically included patient demographics ; diagnostic clinical variables ; treatment prescribed ; and comorbidities .Descriptive and multivariable analyses were first run to compare the characteristics of patients visiting the smoking cessation clinic with those who never visited the clinic. Specifically, multivariable logistic regression was performed to determine which demographic of clinical factors were significant predictors of whether or not a cancer patient received smoking cessation care.In order to ascertain survival benefits possibly associated with becoming abstinent through the smoking cessation clinic, survival curves were compared across the four groups of patients described earlier (Groups A through D). A multivariable Cox regression was performed to determine whether or not the various patient profiles were significantly associated with survival benefits, controlling for demographic, geographic and clinical factors.From 14,813 registry records, we were able to identify 3403 unique Jordanian cancer patients documented as current smokers. A descriptive table of the patient population, categorized by whether or not they received smoking cessation care at the clinic, is provided in Table When a multivariable logistic regression was conducted to determine significant predictors of being seen at the clinic Table , femalesWithin the subsample of 2387 patients included in the survival analysis, Kaplan-Meier survival curves across the different patient groups are displayed in Fig. Our study sought to demonstrate the value of clinic-assisted abstinence in a diverse cancer patient population in Jordan, and to provide insight into which patient subgroups were at higher risk of not being seen at the smoking cessation clinic.When survival of cancer patients who received smoking cessation care was compared to that of patients who did not receive any care, our results indicated that patients who did not receive care at the smoking cessation clinic and those who did not report any abstinence despite visiting the clinic fared significantly more poorly  than those who visited the clinic and reported being abstinent at more than follow-up point. The advantage in the lattermost group persisted after running a multivariable Cox proportional hazards regression analysis adjusting for sociodemographic and clinical factors that may also have impacted hazard of death. While our data did not measure the specific pathways by which survival benefits were accrued during receipt of smoking cessation services, it is plausible that the short-run survival benefit may be explained by the averted many risks of continued smoking among cancer patients  , 28–33. While most studies have focused on the positive impact of smoking cessation on long-term survival in cancer patients, our findings reveal significant differences in hazard of death within the two-year period after diagnosis, in a cancer patient sample that included various sites and stages; and imply that integrating specialized smoking cessation interventions to induce smoking abstinence during the crucial period of cancer treatment can yield considerable survival benefits. Given the on-going debate in the scientific community about the value of costly cancer medications , and theall cancer sites being treated, regardless of clinical as well as demographic characteristics. We have previously reported that a substantial proportion of cancer care providers do not refer cancer patients to the smoking cessation clinic, and that knowledge gaps among these providers exist with regard to the detrimental outcomes associated with continued tobacco use after a cancer diagnosis [Despite our evidence of lower hazard of mortality in the sub-group of patients who visited the clinic to receive care and were able to abstain, our findings also revealed that a substantial proportion of cancer patients are not seen at the smoking cessation clinic. This is in-line with what has been reported in other cancer centers . Specifiiagnosis . Our datOur analysis was an observational retrospective one using cancer registry and clinic records, and data limitations existed. Despite controlling for several clinical and demographic characteristics that may impact survival, unobservable or residual bias cannot be completely ruled out. For example, socioeconomic status indicators were not available in the Center’s registry data and thus could not be accounted for in our survival analysis. However, in a sub-group of patients with available income and educational status information (namely those visiting the smoking cessation clinic), neither income nor educational status significantly impacted mortality in the short-run (results not presented). We also could not include more detailed information that could have enriched our analysis, such as information on specific chemotherapy regimens being used; and clinical outcomes other than survival . Furthermore, while cigarette smoking status is documented in patient records, we did not have information on use of other forms of tobacco such as the waterpipe, nor did we have information about the smoking status over time of patients who did not visit the smoking cessation clinic, or their receipt of other smoking cessation interventions that were not based at the Center. However, KHCC operates the only fully equipped, evidence-based smoking cessation clinic in the country, offering both pharmacotherapy and intensive counseling. Although the Jordanian Ministry of Health runs three clinics in the country, these are under-equipped in both staff and medications, and comprehensive treatment is not availed through them.  Receipt In summary, our results demonstrate the short-term survival value of clinic-assisted abstinence in cancer patients who were smokers on diagnosis, thus underscoring the importance of availing smoking cessation services to assist smokers in achieving abstinence. Our findings also support the need to heavily promote and increase the receipt of smoking cessation care in cancer patients. Because we address a diverse ‘real-world’ cancer patient population, our results have the potential to resonate with oncology practitioners.Our study on Jordanian cancer patients at KHCC who smoked demonstrates that smoking cessation achieved through specialized smoking cessation care (pharmacotherapy and counseling) can yield survival benefits for patients within the short-run (during cancer treatment), and emphasizes the need for availing and promoting evidence-based smoking cessation interventions to cancer patients as a means of integrating in oncology practice a cost-effective tool for improving patient outcomes.


Chronic widespread pain (CWP) is one of the most difficult pain conditions to treat due to an unknown etiology and a lack of innovative treatment design and effectiveness. Based upon preliminary findings within the fields of motivational psychology, integrative neuroscience, diaphragmatic breathing, and vagal nerve stimulation, we propose a new treatment intervention, motivational non-directive (ND) resonance breathing, as a means of reducing pain and suffering in patients with CWP. Motivational ND resonance breathing provides patients with a noninvasive means of potentially modulating five psychophysiological mechanisms imperative for endogenously treating pain and increasing overall quality of life. Chronic widespread pain (CWP), including fibromyalgia syndrome (FMS), is a particular type of chronic primary pain where biological causalities may or may not be present and can not be identified as either musculoskeletal or neuropathic pain . CWP is Many of the symptoms associated with CWP overlap with those of functional somatic syndromes (FSS)  and mediInterdisciplinary theories which include the neurovisceral integration model , 2009, tmotivational nondirective resonance model attempts to bridge the aforementioned theories and describe a mechanism-based interventional approach, motivational non-directive (ND) resonance breathing, for treating CWP. To our knowledge, we are the first to propose such a unified theory and describe how it can be applied and tested as an innovative treatment for CWP. Interventions which have been previously developed in order to apply these theoretical findings to the development and delivery of new pain treatments have failed to sufficiently treat CWP.Our unified theoretical approach herein referred to as the None of the most commonly used pharmacological, psychological, medical, or surgical treatments are, by themselves, sufficiently able to remove pain or to significantly enhance physical and emotional functioning for patients suffering from CWP . HoweverNon-pharmacological treatments for FSS and MUS that involve the active participation of patients, such as exercise and psychotherapy, have been shown to be more effective than treatments which only involve passive physical measures  . TherefoIn particular, research on mindfulness-based meditation interventions show contradictory findings , differeSubsequent data shows a strong bidirectional relationship between pain and respiration . Pain caDue to the strong bidirectional relationship between pain and respiration, a recent systematic review  called fIn this paper, we propose that the autonomic and cardiovascular mediators that link respiration and CWP are baroreceptor sensitivity (BRS) and heart rate variability (HRV); the physiological mechanism needed to reduce pain in those with CWP is diaphragmatic breathing (DB) ; the central mechanism responsible for producing respiratory hypoalgesia is vagus nerve stimulation; and the cognitive and affective psychological mechanisms needed to reduce pain in those with CWP is ND attention and motivation.We believe that DB practiced at one’s RF has the potential to increase HRV and BRS by stimulating the vagal nerve in those suffering from CWP. Indeed, stimulation of the vagus nerve targets several pathophysiological factors associated with CWP. If diaphragmatic RF breathing is practiced with a ND quality of attention, we believe that this may be the ideal means of modulating specific brain activity  and thus remodel the relationship between CWP and emotion. Furthermore, motivating CWP patients to practice ND diaphragmatic RF breathing everyday while increasing their positive treatment expectations may aid in targeting the immune-mediated parameter of CWP. We hypothesize that motivational ND resonance breathing (MNRB) is a potential psychophysiological intervention for endogenously treating pain intensity and disability in those who suffer from CWP.Baroreceptor sensitivity and heart rate variability are among the most important factors for evaluating the health and functionality of the cardiovascular and autonomic systems in those suffering from CP . BRS is Among all of the time- and frequency-domain HRV parameters, the standard deviation of NN intervals (SDNN), the percentage of successive RR intervals that differ by more than 50 ms (pNN50), the high- frequency power (hf), and the root mean square of successive RR interval differences (RMSSD) are considered to reflect cardiac vagal tone . HoweverThe largest and broadest population study to date  has showA large study  has alsoPioneering research within the field of respiratory hypoalgesia has shown that DB performed with a high respiratory volume and low frequency could activate the anti-nociceptive effects of BRS  and concDiaphragmatic Breathing (DB) is a pattern of expiration and inspiration in which most of the ventilatory work is executed by the diaphragm . The diaThe normal respiratory frequency for an adult at rest is around 12 to 20 breaths per minute whereas the respiratory frequency for performing DB can range from 5 to 8 breaths per minute . Many stBreathing at a rate of 6 breaths per minute causes spontaneous oscillations in blood pressure (BP) to synchronize with BP oscillations caused by DB . In turnHarmonic coupling between HRV, respiration, peripheral BP, and skin blood flow in a 0.15 Hz rhythm band (range: 0.12–0.18 Hz) has been demonstrated in healthy long- term practitioners of autogenic training (AT), a practice which uses visual imagery, body awareness, and DB exercises to promote a state of deep relaxation . In regaEfferent parasympathetic activity to the heart  elevates during expiration relative to inspiration due to the central respiratory gating of vagal outflow  and stimThe vagal nerve is the tenth cranial nerve composed of approximately 80% afferent fibers  and 20% efferent fibers  . Vagus nExperimental studies on animals , 1988 anVNS as a means of pain treatment has been traditionally administered through invasive procedures, known as invasive VNS (iVNS), which typically involve the surgical implantation of electrodes around the cervical vagus nerve . iVNS haEven though a number of studies using high intensity tVNS have not found any major side-effects, tVNS can still be accompanied by slight pain, burning, tingling, or itching sensations near the sight of the electrodes . tVNS deAnother important factor to consider is that the vagus nerve is not one uniform structure. It is instead composed of a diversity of molecularly distinct neuron types with different anatomical projections and functions . ArtificVarious forms of paced slow breathing have shown to influence brain electrical activity which may be mediated by VNS arising from the diaphragm , 1993. TAfferent vagal axons enter the brain bilaterally and primarily target the nucleus of the solitary tract (NTS), the first synapse in the baroreflex. This brainstem nucleus transmits sensory information to the limbic system and other deeper brain structures  and actsPsychologically, pain can be perceived cognitively   and affeMeditation encompasses a broad family of complex emotional and attentional regulatory training regimes that can be roughly categorized into three separate groups dependent upon the type of attention being practiced. Focused Attention (FA) meditation entails the voluntary focusing of attention on a chosen object or stimulus . Whenever attention wanders away from the breath, the meditator tries to quickly detect mind wandering and gently, but firmly, brings their attention back to the physical sensation of the breath . Open MoChronic widespread pain patients who report high pain intensity display cognitive deficits and show significantly impaired performance on cognitively demanding tasks when compared to CWP patients with low pain intensity and healthy controls . The diffMRI analyses show that among the five major resting-state networks, only the DMN consistently exhibits altered spatial extent and functional connectivity properties in those suffering from CP when compared to healthy controls . The DMNIt has been suggested that abnormal DMN coupling and communication with other affective brain systems in CWP may be driven by attention to pain in daily life . Those wND meditation, which permits mind wandering, involves a more extensive activation of brain areas associated with episodic memories and emotional processing, than during FA meditation, OM meditation, or regular rest . Most miStronger structural connectivity between the periaqueductal gray and the DMN is associated with the ability to mind wander away from pain and thus treat it as a non-distractor . By engaFunctional connectivity between the DMN and brain regions associated with emotion regulation  in patients diagnosed with major depressive disorder (commonly co-morbid in those with CWP) has also been shown to decrease after 1 month of tVNS compared to sham stimulation. The change in depression severity significantly correlated with functional connectivity changes between the DMN and regions that are implicated in both pain modulation and emotion, such as the anterior insula and anterior cingulate cortex . HoweverA study  comparedThe affective interoception seen in these diary statements as a result of an ND and DB- based therapy intervention reflect the neurobiological activity seen in brain regions, such as the insula, which result from VNS . The anaPreclinical and clinical evidence shows that tVNS simultaneously modulates both pain and mood, yet little is still known about possible indirect descending effects of altered mood states on pain perception for those suffering from CWP. Previous studies have shown that both positive and negative mood states can modulate the affective dimension of pain unpleasantness . CWP patPatients with CWP show significant anatomical and functional changes within reward/motivational circuitry within the brain that strengthen emotional and affective pain mechanisms . These mPain can be considered a homeostatic emotion  such as. TherefoMultilevel modeling analyses  indicateEmerging evidence suggests that systemic low-grade inflammation is associated with CWP and that the presence of inflammation could promote the spreading of pain, a hallmark sign of CWP . A multiThe psychological and emotional state of an individual can have a significant impact on pain perception . PredictA higher degree of optimism in subjects randomized to a training program consisting of meditation, breathing exercises, and cold exposure was associated with profoundly higher plasma epinephrine levels during the intravenous administration of the bacterial endotoxin followed by a more rapid and profound increase in anti-inflammatory IL-10 levels when compared to those in the non- trained group. A more positive expectation of the effects of the meditation, breathing, and cold exposure training on the psychophysiological reaction to the bacterial endotoxin was associated with lower flu-like clinical symptoms and lower subsequent pro- inflammatory TNF-α, IL-6, and IL-8 levels than the non- trained group . The resThe signaling of systemic inflammation is communicated to the brain via neural (predominantly vagus nerve) pathways, humorally via circulating cytokines, and directly via immune cells . ChronicEvaluating pain-motivated behaviors can provide a path for the assessment of new treatment efficacy for CWP with a high likelihood of translational relevance. Due to the current notion that CWP may partly be an immune-mediated condition, this evidence raises the question as to whether motivation and expectation should be targeted and implemented within CWP treatment, especially in regards to a ND meditation and DB intervention.In order to test whether MNRB is an effective psychophysiological intervention for endogenously treating pain intensity and disability in those who suffer from CWP, a randomized controlled clinical trial  lead by the lead author (C.E.P.) and co-author (H.B.J.) in the Spring of 2019 will compare and investigate the treatment efficacy of MNRB and tVNS on patients diagnosed with CWP.N = 112) who are referred to the Department of Pain Management and Research at Oslo University Hospital, Ullevål, in Oslo, Norway, will be randomized into one of four independent groups. Half of these participants (N = 56) will be randomized to either an experimental tVNS group or a sham tVNS group. The other half (N = 56) will be randomized to either an experimental MNRB group or a sham MNRB group. Both experimental and sham treatment interventions will be delivered twice per day at home, 15 min/morning and 15 min/evening, for a total duration of 2 weeks. Participants are invited to the clinic twice for pre- and post-intervention data collection. The primary outcomes are changes in photoplethysmography measured HRV and self-reported average pain intensity measured by the numeric rating scale. Secondary outcomes include changes in pain detection threshold, pain tolerance threshold, and pain pressure limit determined by computerized pressure cuff algometry as well as blood pressure and health related quality of life.Consenting CWP patients  and engineered by Dr. Marcin Czub at the University of Wrocław in Wrocław, Poland. The MNRB program and BarTekTM device work in-sync in order to deliver and guide CWP patients in both the sham and experimental versions of MNRB. The BarTekTM respiratory gating device is attached to an elastic strap which is placed around the patient’s abdomen, below the rib cage and an inch above the navel (behind which the thoracic diaphragm is located)  . ThroughP < 0.001 for both)  with feet flat on the floor, hands on thighs, and palms facing upward. Ideally, MNRB should be practiced with a head tilt no more than 30 degrees from the horizontal  due to tor both) . This reor both)  immediatTM respiratory gating device around their abdomen, open the MNRB program on the smartphone, and are guided (p < 0.001) and increases hfHRV (p < 0.05)  . Partici < 0.05) .The MNRB session begins with a transition period during which patients are taken from an average respiration frequency of 12 breadths/min to a RF of 6 breadths/min. During this transition period, patients are to attend to a respiration guide while listening to a 110 Hz frequency which has been shown to increase hfHRV . While bEmotional information derived from their MNRB practice can help CWP patients to become motivated to solve problems and achieve goals on a daily basis. In turn, this may be helpful for increasing emotional intelligence . EmotionMNRB screen begins to darken and the respiratory guide disappears while the inhale/exhale sound guide along with the 110 Hz background frequency remains. At this moment, patients are invited to close their eyes and engage in a ND state of mind. Cardiac parasympathetic nervous activity indicated by hfHRV has been found to increase more while listening to a 110 Hz sine wave when the eyes are closed as compared to when they are open  could be an innovative, effective, and noninvasive means of CWP treatment. To our knowledge, we are the first to propose such an intervention that could potentially target CWP etiological factors we believe to be imperative for successful treatment.CP is the lead and corresponding author for this work, responsible for the original conception and design of the chronic widespread pain treatment program presented and motivational ND diaphragmatic breathing. HJ interpreted and analyzed the biopsychosocial framework and psychological theory of the program in respect to chronic widespread pain treatment. CP and HJ revised the work critically for important intellectual content, approved the final version to be published, and are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.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.


Staphylococcus aureus (MRSA) offers a way of verifying adherence to the regimen. However, absence of the biomarker in some patients needs explanation, to ensure that efficient decolonisation has not been compromised by confounding circumstances, and that additional resistance to mupirocin has not unwittingly been encouraged.The recent demonstration for the first time of urinary monic acid A as a clinical urinary biomarker of exposure to intra-nasal mupirocin during medication for methicillin-resistant  In a context of expected variation in adherence to medication for intra-nasal infection, as is widespread amongst pharmaceuticals, a biomarker for compliance with the mupirocin regimen has been proposed. Demonstration of reliability in a clinical setting has been achieved in a pilot study. However, understanding inconsistencies is necessary through further experiment, not only for nasal decolonization but also to avoid encouragement of mupirocin-resistant pathogens.Decolonisation of MRSA within nares is a precautionary part of surgical premedication, for which mupirocin  is widely prescribed . The rapUnreliable adherence to regimens for prescribed pharmaceuticals is a well-recognised problem in clinical medicine, to which antibiotics are particularly prone, particularly amongst out-patients. Usually there is no means of monitoring compliance in real time or in retrospect. From clinical experience, a challenge for the first time to devise a method to monitor efficient intra-nasal application of mupirocin was addressed, because its degradation in humans to monic acid A is known and a unique reference sample was available from its original synthesis and subsequent scientific use ,3. ThereHowever, a matter of concern in the recent pilot study is the other two patients, whose urines during mupirocin medication showed no evidence of monic acid A. The significance is unclear but, although elderly and with several morbidities, they were currently residing in an environment of exceptional clinical care. Thus, what factors might limit detection of urinary monic acid A in a hospital context? Could any impair efficient nasal decolonisation of MRSA by mupirocin?Intra-nasal application of a cream, as mupirocin is formulated, can cause an uncomfortable sensation, in contrast to oral medications with tablets or liquids. There may be dis-inclination to sniff the antibiotic cream fully up and across nasal membranes. An inevitable outcome of comprehensive nasal distribution would be that traces of mupirocin are swallowed; this is harmless but potentially facilitates detection of monic acid A in urine as a biomarker of efficient medication. It is important to reiterate that mupirocin is not absorbed through skin, even that lining the nares. Therefore, cursory application of the cream within nares would not be reflected by detectable monic acid A in urine. A natural reflex to inhale via mouth instead of nose could also appear to have satisfied the need for further distribution. Do nurses who administer routine medication understand the importance of optimising trans-membranal distribution when using mupirocin cream for decolonization of MRSA fully across nasal membranes? Is application always supervised or may a patient sometimes prefer to, or indeed have to, self-medicate? Can patients’ confounding morbidities make administration less efficient? Could mupirocin be made more pleasurable to receive?Pseudomonas fluorescens producing pseudomonic acid A, using the classical dilution-plate bioassay with Staphylococcus aureus. Occasional bacterial colonies within inhibition zones, implying selection of resistant forms of S. aureus, were regularly encountered, even though that culture had never previously been exposed to S. aureus. Development of a high-level resistance to mupirocin is of concern [Escherichia coli while progressing across a thousand-fold antibiotic concentration gradient [There is also concern that variable distribution of mupirocin across nasal membranes may positively encourage selection of mupirocin resistance. Mupirocin resistance has been recognised in MRSA since increasing after a 1990 Canadian hospital epidemic from 2.7 % to 65 % through its 3 year study [ concern . Anyone gradient . NotablyConsidering a wider context, analytical extrapolation from mass spectral fragmentation characteristics of monic acid A may be valid also for pseudomonic acid B, by taking account of an oxygen atom added to pseudomonic acid A at position 8 though not conveying useful bioactivity. Biosynthetic inter-relationship between pseudomonic acids B and A has been demonstrated both by radiolabelling and molecular genetic methodologies ,12. OtheProtecting the very particular mode of antibiosis of mupirocin may be easier than discovering new antibiotics. Methodology is now in place  to verifWider discussion of recent demonstration of urinary monic acid A as a biomarker of rigorous intra-nasal medication with mupirocin emphasizes as yet unexplained variation in its expression in a hospital setting. Use of the analytical methodology for further clinical experimentation is encouraged.


To evaluate the impact of the WHO Framework Convention on Tobacco Control (FCTC) on global cigarette consumption.Two quasi-experimental impact evaluations, using interrupted time series analysis (ITS) and in-sample forecast event modelling.71 countries for which verified national estimates of cigarette consumption from 1970 to 2015 were available, representing over 95% of the world’s cigarette consumption and 85% of the world’s population.The FCTC is an international treaty adopted in 2003 that aims to reduce harmful tobacco consumption and is legally binding on the 181 countries that have ratified it. Main outcomes were annual national estimates of cigarette consumption per adult from 71 countries since 1970, allowing global, regional, and country comparisons of consumption levels and trends before and after 2003, with counterfactual control groups modelled using pre-intervention linear time trends (for ITS) and in-sample forecasts (for event modelling).No significant change was found in the rate at which global cigarette consumption had been decreasing after the FCTC’s adoption in 2003, using either ITS or event modelling. Results were robust after realigning data to the year FCTC negotiations commenced (1999), or to the year when the FCTC first became legally binding in each country. By contrast to global consumption, high income and European countries showed a decrease in annual consumption by over 1000 cigarettes per adult after 2003, whereas low and middle income and Asian countries showed an increased annual consumption by over 500 cigarettes per adult when compared with a counterfactual event model.This study finds no evidence to indicate that global progress in reducing cigarette consumption has been accelerated by the FCTC treaty mechanism. This null finding, combined with regional differences, should caution against complacency in the global tobacco control community, motivate greater implementation of proven tobacco control policies, encourage assertive responses to tobacco industry activities, and inform the design of more effective health treaties. Tobacco consumption is one of the leading causes of preventable death worldwide. Each year, tobacco is responsible for about seven million deathsThe solution is often said to be the WHO Framework Convention on Tobacco Control (FCTC). Adopted under the auspices of WHO, this international treaty aims to reduce harmful tobacco consumption; lower smoking rates among children; and counteract the tobacco industry’s lobbying, advertising, and promotion activities. Treaty negotiations were called for in 1995, commenced in 1999 after the election of Gro Harlem Brundtland as WHO’s Director-General, and completed on 21 May 2003.But has the FCTC actually reduced global cigarette consumption? Only a few attempts have been made so far to empirically evaluate the impact of this international treaty. National level studies have examined the impact of the treaty on domestic tobacco control policy and smoking prevalence in individual countries or regions.What these studies do not address is whether international law—so often advocated as the solution to health challenges—is an effective tool for changing health behaviours.International law is proposed, negotiated, adopted, ratified, and implemented in public political processes, making it impossible to conduct a randomised controlled trial or limit crossover effects between countries. Not only are there known capacity constraints in the ability of countries to implement international laws,Therefore, the best methodological approaches to measuring international laws’ effects are quasi-experimental impact evaluations.In this study, we sought to improve understanding of the global tobacco epidemic and to advance the way we understand and evaluate international laws more broadly. We took an exploratory approach to quasi-experimentally evaluate the FCTC’s impact on global cigarette consumption under different models, assumptions, and scenarios. Despite their limitations, large scale quantitative approaches allow for the incorporation of data from many more countries and time periods than would be practical with in-depth qualitative approaches.This study used a previous systematic collection and quality appraisal of national cigarette consumption data from 1970 to 2015.0) each country’s cigarette consumption data on the year that country ratified or acceded to the FCTC. These robustness checks compared the time before the FCTC was binding on each country  with the time after the FCTC was binding  and would find any legal effects of the FCTC. Finally, while our primary focus on the FCTC’s overall real world effect is best evaluated with global consumption data, we also a priori designed stratified region and income level analyses to uncover any masked group effects and to identify causal mechanisms that might explain differences in cigarette consumption trends.Theoretically, the FCTC could have achieved socialisation effects observed through the negotiating process (1999-2003), normative effects observed after the treaty’s adoption (2003), or legal effects observed through countries formally ratifying it 2005-present).-present.Given the exploratory nature of this study, considerable efforts were undertaken to identify any potential impacts using different models, assumptions, and scenarios. We used two different statistical approaches to calculate whether observed changes were statistically significant discontinuities: interrupted time series analysis and in-sample forecast event modelling. The population weighted effects of the FCTC were also calculated for different groups of countries by UN region, country income level, and membership in the Organisation for Economic Co-operation and Development (OECD), and individually for the top 10 cigarette-consuming countries, in order to maximise the chances of finding effects.Interrupted time series analysis has previously been used to evaluate the effects of different health policiesi,ty=xi,tβ+εi,t; i=1,…,N; t=1,…T . This statistical test used the annual estimates of cigarette consumption per adult directly and did not require first differencing.Event modelling is based on panel data time-series regression models with time varying coefficients. We constructed an event model based on a highly interacted series of annual country specific variables for 70 countries  to obtain a predictive model of national cigarette consumption per adult selected using k-fold cross validation. This model rests on the assumption that a series of country specific variables relating to its economy, political system, tobacco industry, and human development (table A9) would be similarly correlated with cigarette consumption both before and after the FCTC’s adoption. In the absence of change, we would expect that forecasting consumption after 2003 based on in-sample correlations prior to 2003 between consumption and country specific variables  would result in forecasted consumption estimates for the post-2003 period that are not statistically different than actual consumption. This in-sample forecasted counterfactual is represented by the following linear regression equation: Further methodological notes and details of robustness checks performed for the interrupted time series analysis and event model are available in appendix 1. Our systematic effort to detect any potential FCTC effects included two quasi-experimental approaches at the global level, for subgroups by region, country income, OECD membership, and top cigarette-consuming country. We also conducted an additional 42 statistical analyses as robustness checks. Stata software codes used to implement all analyses are available in appendix 2.Patients were not involved in this study. Findings will be actively disseminated through conference presentations, publications in academic journals, plain language policy notes, personalised briefings to leading global tobacco control organisations, and commentary in news media.We calculated annual, population weighted cigarette consumption per adult by UN region, country income level, and OECD membership in units of cigarettes consumed per adult per year . As expeWe saw no statistically significant change in either level or slope of change in global population weighted cigarette consumption per adult after 2003 . CoefficEvent modelling indicated that cigarette consumption per adult did not decrease faster than would be expected following adoption of the FCTC in 2003. According to the event model , the gapWe also segmented the overall global event model by UN region and country income level. As We then grouped countries by UN region , which rAfter numerous statistical analyses, we could not find evidence that the rate at which global cigarette consumption per adult had been decreasing over the past three decades was accelerated by the adoption of the FCTC in 2003, whether through socialisation, normative, or legal pathways. Nevertheless, this null overall finding obscures a large discrepancy in trends after 2003, between high income countries and low and middle income countries, as well as between European and Asian countries. Although the interrupted time series analysis and event model did not allow us to reject the overall null hypothesis , the stratified analyses did uncover accelerated reductions in high income countries and European countries compared with predicted consumption based on trends before 2003. These stratified analyses also indicate that low and middle income countries and Asian countries have acutely reversed their previously decreasing consumption trends before 2003, to the point of consuming at least as many cigarettes as high income countries and European countries avoided in the period after FCTC adoption.The FCTC promotes evidence based tobacco control policies in nearly every country worldwide.The first apparent contradiction arises between the definitive evidence supporting the efficacy of key tobacco control policiesBy considering both paradoxical findings together, a compelling potential explanation of these findings emerges. Varied implementation of tobacco control policies and shifting trends in cigarette affordability across countries may have generated market equilibrium effects incentivising the tobacco industry to move its lobbying, marketing, and promotion activities away from high income countries (where they faced increasingly stringent regulations) and towards low and middle income countries and Asian countries (with far less stringent measures).Our quasi-experimental evaluations quantitatively assess the FCTC’s effects using a new open access dataset of national cigarette consumption estimates for 71 countries from verified data sources, covering 95% of global cigarette consumption and 85% of the world’s population.This study was limited by the number of countries for which data were available, including limited availability of supply side data. The data did not include consumption of water tobacco, chewing tobacco, or loose leaf tobacco. Furthermore, cessation of tobacco use has a stronger protective effect on health than reduction in use, so aggregate consumption might not have fully captured the FCTC’s effects. Ten years might not have been long enough to observe the effects of the FCTC’s adoption, and low and middle income countries could increasingly benefit from measures such as legal defence against the tobacco industry’s use of international trade law to weaken tobacco control policies.The quasi-experimental methods implemented in this study have underlying assumptions. The interrupted time series analysis assumes a constant underlying time trend in the absence of an intervention, and the in-sample forecast event model assumes a constant association between cigarette consumption and the model’s explanatory variables before and after the intervention. Finally, we are unable to state with certainty that the associations observed are causal, owing to the exploratory nature of the study that purposefully included multiple testing under various models, assumptions, and scenarios. Quasi-experimental methods can be affected by omitted variable bias or confounding factors, which could lead to the masking of a true effect or finding a spurious association.Analysis of cigarette consumption trends has allowed us to discern patterns that could be useful in supporting future tobacco control efforts, including identifying countries to prioritise, the need for country specific strategies, and the importance of counteracting the tobacco industry. The divergence in cigarette consumption patterns between high income countries and low and middle income countries, and between European and Asian countries, should be studied in more detail. This emerging problem could continue to worsen owing to population growth, increasing living standards, and intensification of tobacco industry activities in more low and middle income countries and for a greater proportion of people within those countries. The degree to which governmental implementation capacity, market equilibrium effects, or any other factors have contributed to this difference should be identified and addressed in order to limit global tobacco use.This research has demonstrated that more publicly available data are needed for the study of tobacco control. We could not have conducted quasi-experimental impact evaluations of the FCTC without a recently compiled dataset, which should have been part of treaty reporting requirements. Like all population health interventions, we should think critically about international laws and subject them to rigorous evaluation to determine whether their impact matches their rhetoric. Research should be conducted into the effects of the mechanisms underlying international law, the forums in which they are negotiated, and the parties involved in their negotiation to see how these factors might influence the ability of international laws to achieve their objectives.The World Health Organization’s Framework Convention on Tobacco Control (FCTC) has received nearly universal approval in academic literature, news media, and political speechesThe FCTC aims to reduce harmful tobacco consumption; lower smoking rates among children; and counteract the tobacco industry’s lobbying, advertising, and promotion activitiesSo far no studies have used a more rigorous quasi-experimental approach to evaluate the global impact of the FCTC to account for the fact that cigarette consumption had already been falling for at least 10 years before the international treaty was adopted in 2003Using quasi-experimental approaches to evaluate the FCTC’s impact on global cigarette consumption per capita, this study showed no evidence of an acceleration in the global consumption rate (which had been decreasing over the past three decades) after adoption of the FCTC in 2003High income and European countries showed accelerated decreases in consumption, whereas low and middle income and Asian countries showed increased consumption above what would have been anticipated without adoption of the FCTCThese results should motivate accelerated implementation of proven tobacco control policies in countries with uneven implementation and more assertive responses to transnational activities of the tobacco industry


Cancer of unknown primary (CUP) designates an enigmatic cancer entity with histologic confirmation of malignancy from a metastasis but no identifiable primary tumor in spite of a thorough diagnostic work-up. In this review, we discuss the validity of CUP as a distinct cancer entity as well as diagnostic pitfalls. As arguments against a distinct entity, the diagnosis of CUP is erroneous in some cases. Diagnostic pitfalls include incomplete diagnostics, uncertainty in classifying a lesion as either primary or metastasis and mistaking a relapse of an antecedent malignancy as CUP due to histologic and immunohistologic disparities. Given the high frequency of prior malignancies in CUP patients, relapse of an antecedent cancer should always be carefully excluded. Gene expression profiling-based classifier assays aim at aligning the molecular profile of CUP patients with established primary cancer patterns for highest congruency in order to identify the putative primary and treat accordingly. However, the spectrum of predicted putative primaries by molecular techniques is somewhat at odds with the primaries identified in autopsy series. Also, a first randomized clinical trial did not show superiority of primary-tailored therapy over unspecific platinum-based chemotherapy. CUP cases share an aggressive clinical course, atypical metastasis pattern, rapid progression of metastases, a generally poor response to chemotherapy and dismal outcome as distinct clinical features. Metastatic spread appears to take place in the early stages of tumor evolution, with CUP metastases subsequently undergoing genetic evolution toward a chromosomally highly complex and instable karyotype independent from the primary tumor. In clinical practice, the diagnosis of CUP is valid when no primary tumor is detectable. Treatment should ideally offer broad spectrum coverage across numerous malignancies and be well-established in CUP as is the case for carboplatin/paclitaxel and cisplatin / gemcitabine in particular, but it should also cover the most likely putative primary. The diligent diagnosis of CUP is warranted for clinical trials, making the eligibility process particularly laborious. In conclusion, we deem CUP a distinct cancer entity and the diagnosis accurate in most patient cases. It often fosters lingering resentment against chemotherapy, which is unavoidably empiric given the failure to detect the primary. Honestly, CUP specialists do not fare better with some of their fellow oncologists (to say nothing of pathologists), who taunt them that CUP has ceased to exist as a valid diagnosis in the era of molecular diagnostics. In this review, we therefore aim to put the spotlight on the accuracy of CUP diagnosis and the validity of CUP as a distinct cancer entity.Cancer of unknown primary (CUP) is an enigmatic cancer entity. It is diagnosed in malignancies, where metastases have been histologically confirmed, but where no primary site can be identified in spite of a comprehensive diagnostic work-up –4. When All oncologists in the field, who get CUP patients referred from local hospitals, are aware that some diagnoses of CUP are premature or even outright erroneous. Keeping obvious misdiagnoses aside, where histologic confirmation of imaging findings suspicious of a primary tumor is missed, there are also cases with an incomplete diagnostic work-up. Typically, the mandatory diagnostic standard as laid down in the European Society of Medical Oncology (ESMO) guidelines  is routiAt least at our center, we repeatedly observe delicate cases where a relapse of a prior malignancy has to be considered as a differential diagnosis to a new CUP. Some patients referred as CUP in truth suffer instead from relapse of an antecedent malignancy which was disregarded due to histologic or immunohistologic disparities between the two tumors. Given a high rate of prior malignancies of around 20–25% among CUP patients , the ideAnother delicate aspect is the clinical judgement whether a malignant lesion should be classified as primary cancer or metastatic site, thus entailing the diagnosis of CUP. A typical example is a patient with isolated CK7+ adenocarcinoma metastases of the liver, where cholangiocellular carcinoma (CCC) has to be considered as a differential diagnosis to CUP with hepatic metastases. Likewise, a CK7+, TTF1- adenocarcinoma lung mass might represent a primary lung cancer or alternatively a CUP with pulmonary metastasis.In some CUP patients, the primary tumor unmasks itself over time during the disease course, or is finally detected at autopsy, ultimately leading to a revision of the CUP diagnosis. The emergence of the primary tumor during the lifetime of the patient is rare with frequencies in the 20% range reported in the literature  and evenAdvances in molecular diagnostics and microarray technology have raised expectations that molecular profiling might provide hints toward the likely primary tumors. For that purpose gene expression profiling-based classifier assays have been brought forward in CUP, which aim to align the respective profile of a particular CUP case with the best match from previously established profile databases from all sorts of cancer entities. Hereby, the assignment of molecular signatures to cancers is typically developed and validated in cancers with known primary before the molecular profile of a CUP tumor is aligned to the established primary pattern with the highest congruency . These cCurrent studies are investigating response to treatment tailored to the primary predicted by classifier assays as proof-of-principle trials. In one small study in 45 CUP patients the identification of a putative primary tumor routinely treated with carboplatin/paclitaxel indeed predicted an actual response to this regimen . In a laIn addition to gene expression profiling, Moran and coworkers have employed epigenetic profiling by DNA methylation profiles in the search of the presumed primary . In theiTP53 are by far the most abundant mutations found in CUP cancers (ALK translocations for lung cancer are scarce in CUP.Whereas the classifier assays discussed above fully focus on hints toward the primary tumor, panel sequencing strategies aim at identifying targets for molecularly driven therapies independent of the tissue of origin instead –27. Acco cancers , but thiAs discussed above at least in the German health care system many patients have received an extensive diagnostic work-up far in excess of the minimum requirements of the ESMO guidelines . GastrosAs reviewed by Pentheroudakis et al.  CUP caseNew insights into the mechanisms of metastatic seed in cancers also provide explanatory models for the enigmatic phenomenon of an undetectable primary tumor. Experimental data from mammary cancer animal models imply that metastatic spread takes place in the early stages of tumor evolution , 35. In Indeed, some findings in CUP seem to support the validity of this model, including the clinically observed high systemic relapse rate in CUP patients with localized disease treated by surgery and / or radiotherapy in curative intent . CytogenSpontaneous tumor regressions have been reported throughout a variety of malignancies, which are attributed to tumor cell elimination by the immune system in view of the frequent association with infections observed in these cases. The concept of immune-mediated cancer surveillance is further supported by an increased cancer incidence in transplant recipients on immunosuppressants and the recent success of immune checkpoint inhibitors in numerous cancer entities. Thus, it is possible that the lack of a primary tumor in CUP is an immune mediated event at least in some cases as well. Recently, it was demonstrated across several cancer entities that a high degree of CIN confers resistance to immune mediated therapies . TherefoInterestingly, evidence suggests that primary tumors in general actively modify future metastatic sites by tumor-secreted factors to make them susceptible to metastatic seed, a phenomenon called “premetastatic niche formation” , 42. It TP53 mutations are by far most abundant (TP53 mutations are found throughout all types of carcinomas this does not permit conclusions regarding a putative primary site. Likewise, other frequent mutations in CUP including RAS, CDKN2A, MYC, ARID1A, PIK3CA, or BRAF are not tissue specific.In spite of unquestionable progress, mutational profiling of CUP has its limitations. Nowadays, panel sequencing is increasingly performed on a routine basis –27, 43. abundant . Given tThe classifier assays discussed above, although designed to detect the putative primary, have not established themselves in clinical routine so far. None of these tests is either marketed or insurance-covered in Germany nor are these procedures recommended in the ESMO clinical guidelines for CUP diagnostics and treatment .Last but not least, no molecular profiling test can substitute for a clinically identified primary tumor. Even putting aside the disquieting frequency of discrepancies in the distribution of primary sites between autopsy and molecular profiling patient series , a CUP tIn most cases, the CUP diagnosis is correct, because metastatic spread has been histologically confirmed and a primary tumor has remained elusive in spite of a thorough work-up according to the ESMO guidelines , therebyFor reasons described above, in cases with a putative primary, treatment should follow the treatment algorithms for the suspected primary cancer. For example, if a patient is diagnosed with a CK20+, CDX2+, CK7– adenocarcinoma with liver and peritoneal metastases, both the immunohistologic profile and the distribution of metastatic sites is in tune with colorectal cancer, and treatment should be administered according to protocols for metastatic colorectal cancer –47. LikeData from clinical trials in CUP are scarce , 29, witThere is broad consensus that the ESMO guidelines  should bCUP cases are biologically characterized by early and aggressive metastatic spread, poor response to chemotherapy and poor prognosis, which has led to the postulation of a unifying underlying pro-metastatic signature in CUP.In the era of molecular work-up further tools beyond histology and immunohistochemistry have become available to characterize cancers. CUP classifier assays have been developed which determine the putative tissue of origin of a CUP cancer by alignment with molecular profiles established for cancers with known primary. Even if the molecular signature points toward a putative primary tumor, the diagnosis of CUP remains still valid as long as no primary tumor is detectable. However, molecular analysis, immunohistochemistry and clinical picture should weigh in to adjust treatment to the putative primary. It remains at the discretion of the treating physician to weigh clinical, immuno-histochemical, and increasingly molecular findings as well.Some patients receive a diagnosis of CUP prematurely and the diagnosis should always be questioned by an experienced oncologist. Relapse of a prior malignancy should be meticulously excluded.Being committed and dedicated to advancing research in the field of CUP, we as authors admit to be biased. Nevertheless, we feel that CUP appears as a valid cancer entity and that most, though not all, patients diagnosed with CUP indeed suffer from a “true” CUP.Both authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.Both authors work as study oncologists for the CUPISCO trial, which is sponsored by Roche, and have received reimbursement for study related travels as well as remuneration for their work as study oncologists for the benefit of their employer.