Novel 3D organoid culture techniques have enabled long-term expansion of pancreatic tissue. This review comprehensively summarizes and evaluates the applications of primary tissue–derived pancreatic organoids in regenerative studies, disease modelling, and personalized medicine.Organoids derived from human fetal and adult pancreatic tissue have been used to study pancreas development and repair. Generated adult human pancreatic organoids harbor the capacity for clonal expansion and endocrine cell formation. In addition, organoids have been generated from human pancreatic ductal adenocarcinoma in order to study tumor behavior and assess drug responses.Pancreatic organoids constitute an important translational bridge between in vitro and in vivo models, enhancing our understanding of pancreatic cell biology. Current applications for pancreatic organoid technology include studies on tissue regeneration, disease modelling, and drug screening. The pancreas is a mixed endocrine and exocrine gland that plays a pivotal role in digestion and metabolic homeostasis. The endocrine pancreatic compartment includes the alpha and beta cells which secrete the hormones glucagon and insulin, respectively, that are required for normal glycemic control. The exocrine compartment is composed of acinar cells, which store and secrete digestive enzymes that are drained into the duodenum by a highly branched, tubular epithelial tree–like duct network within the pancreas.Pancreas biology has been intensively studied, in an effort to understand pancreas development and function, to obtain insight into pathophysiological processes, to identify disease-associated markers, and to find better treatment options for devastating pancreatic diseases associated with high mortality and health care costs, such as diabetes mellitus, pancreatic adenocarcinoma (PDAC), pancreatitis, and cystic fibrosis. Studying the normal human pancreas at the cellular level is difficult. The high risk for complications of pancreatic biopsies more or less precludes this option to generate cross-sectional and longitudinal data of normal human pancreatic tissue at the cellular level in most patients. So we are dependent on generating cross-sectional data from surgical specimens, being aware that the pathological process for which the surgery was indicated could affect the “normal” pancreatic tissue surrounding the pathological process, or from donor pancreas that is not (entirely) used for pancreas or islet transplantation. In vivo models, such as rodents, do not have this limitation but are time consuming and expensive. Moreover, results obtained from in vivo animal models are not always translatable to humans . AlthougRecent breakthroughs in three-dimensional (3D) culture methods have led to the development of organoid culture platforms, which allow researchers to perform translational research on long-term in vitro cultures not limited by barriers present in 2D culture or animal models –13. OrgaLong-term organoid cultures were originally established by the identification of conditions that recapitulate the in vivo intestinal stem cell niche in vitro. The field was spearheaded by a report that organoids could be made from isolated mouse intestinal crypts cultured in Matrigel and supplemented with growth factors that stimulated Wnt signalling, reflecting the high degree of Wnt signalling in intestinal crypts . One of In this review, we discuss the application of human fetal and adult tissue–derived pancreatic organoids in regenerative studies and their use as patient-specific tools for disease modelling.Pancreas organogenesis is a complex process requiring a dynamic spatiotemporal interplay of multiple cells and their surrounding niche. Deciphering the molecular mechanisms underlying morphogenesis and cell specification is essential for a better understanding of pancreas development and regeneration. While the use of transgenic animal models has greatly increased our knowledge of the molecular basis of pancreatic lineage decisions and cell specification, the mechanisms governing morphogenesis are not well understood. Thus, a different approach for the study of fetal development is necessary. Because there are currently no cell lines available that can act as multipotent pancreatic progenitor cells, an alternative approach to overcome this hurdle is the culture of primary fetal tissue.Due to the limited availability of human fetal tissue, rodent fetal tissue has initially been used for in vitro studies. Some of the early insights into rodent pancreatic differentiation arose from the culture of rat fetal pancreas explants, with the tissue being cultured with or without the surrounding mesenchyme. A marked increase in endocrine cells was observed in cultures without mesenchymal cells , 21..While rodent explants at least partially recapitulate endocrine differentiation in vitro, they are limited by their short-term maintenance and the contribution of multiple cell types, which makes it difficult to characterize and study the bona fide fetal pancreatic stem cells. Prior studies indicated that cells expressing SOX9 during early pancreas development can give rise to both endocrine and exocrine cells making this a suitable candidate marker for multipotent progenitors . With thA similar method was applied in an effort to investigate the morphogenesis of murine pancreatic duct development . Cells eIn an effort to obtain insight into human fetal pancreas development, Bonfanti et al. • used huAlthough large steps have been taken in the study of pancreatic development through the use of fetal organoids, limited accessibility and ethical considerations hamper the use of human fetal tissue. Nevertheless, the use of human fetal tissue in organoid culture is a valuable method to study signalling pathways and conditions necessary for normal pancreas morphogenesis and cell specification.Recent technological achievements have revealed that adult tissues may contain a more abundant source of tissue stem cells than previously anticipated, allowing organoid cultures from multiple types of tissues to study differentiation, tissue stem cell maintenance and disease. A breakthrough in adult primary tissue culture was the discovery of Lgr5(+) adult stem cells in the intestinal crypts of mice that generated organoids in vitro , 30. SinHuman pancreatic ducts have been shown to exhibit a capacity for in vitro expansion and formation of 3D hollow structures when cultured in collagen or Matrigel, hence showing potential as tools for regenerative studies . InitialMultiple groups have reported on the generation of pancreatic organoids derived from the adult human pancreas , 38••. TOrganoids derived from the adult human pancreas differ from fetal pancreatic organoids in multiple aspects, such as morphology and endocrine cell formation, most of which are likely to be explained by the apparent absence of a multipotent progenitor in the adult organ or the lack of an optimal culture medium or enrichment strategies. They are however important tools to study regeneration by identification of the cell population responsible for organoid formation and of factors or cell to cell interactions involved in this process.Unlike the small intestine, there is limited information on the presence or identity of progenitor cells in the adult pancreas  and whetLong-term organoid cultures of adult pancreatic tissue fragments largely consist of cells expressing ductal markers such as KRT19, SOX9, and MUC1, suggesting that the organoid-forming cells are derived from the pancreatic duct compartment or through transdifferentiation of other pancreatic cells. But what subpopulation of cells exactly forms complex organoids is still unclear. Multiple groups have used cell surface markers or other isolation techniques in an effort to find adult pancreatic exocrine cells capable of clonal expansion in 3D culture. Initially the pan-epithelial cell surface marker EpCAM in combination with the fluorescent chelator TSQ, which binds endocrine granules, was used on adult mouse pancreas to sort EpCAM-positive, non-endocrine epithelial cells that were capable of long-term clonal expansion in 3D culture . By usinThe cell surface marker CD133 (prominin-1), initially recognized as a surface marker for hematopoietic and neural stem cells , 59, hasThe exocrine compartment of the pancreas is for a large part composed of acinar cells. There is limited evidence from rodent studies that suggests that the adult acinar compartment can generate new exocrine and endocrine cells during pancreatic injury , 70. WolAltogether, these results indicate that enrichment for cell subpopulations with progenitor cell characteristics from the heterogeneous pancreatic exocrine compartment can yield cells with an increased capacity for clonal expansion. However, the exact location of these subpopulations and their role in organ maintenance during homeostasis or injury has not been elucidated.Since the organoid culture methods outlined here allow the long-term culture of pancreatic cells, it is now possible to use these as model to further study the mechanisms involved in adult pancreas regeneration. As only 1–2% of the pancreas is composed of endocrine cells and the remaining 98–99% of exocrine cells, insight in the processes that allow endocrine cell specification from exocrine cells could be beneficial for the creation of novel beta cells—this could be used for beta cell replacement therapy for patients with diabetes mellitus, a treatment which is currently hampered due to the shortage of donor tissue. We have demonstrated that the complex organoids derived from human adult islet-depleted tissue fragments harbor cells that have the potential for endocrine differentiation as they can develop into a limited number of hormone positive cells when transplanted in vivo under the kidney capsule of mice ••. The rIn order to improve our understanding of the signals required for progenitor cell growth and differentiation, more defined culture medium in organoid models should be used. A serum-free, conditioned medium–free 3D culture model for adult murine pancreatic progenitors was developed by using 7 defined growth factors and small molecules in culture, although extracellular matrix was formed by a mix of methylcellulose and Matrigel. These growth factors combined with ECM were shown to support self-renewal and in vitro differentiation of cells that resemble ductal, acinar, and endocrine cells on the gene expression level, indicating tri-lineage differentiation potential of these multipotent cells .Investigations into the underlying molecular mechanism that enhance endocrine differentiation could provide additional insights into the required factors for endocrine differentiation. Azzarelli et al.  used aduThe current protocols for the formation of mature and functional beta cells from adult pancreatic organoids are still far from clinical application due to limitations in efficiency, reproducibility, and scalability. Nonetheless, the in vitro platform that organoid culture offers will allow researchers in the field to further decipher the pathways and mechanisms controlling exocrine and endocrine differentiation, which could subsequently aid in the development of future clinical applications such as beta cell replacement therapy. One could envisage that patients undergoing islet autotransplantation after total pancreatectomy due to benign pancreatic disease will receive an additional infusion procedure using their own expanded and differentiated organoids as the isolated islet preparation itself is often insufficient for insulin independence. Naturally this requires more robust expansion and differentiation protocols and also sufficient evidence of genomic stability of these expanded cells before and after transplantation.Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer with 5-year survival rates less than 8% and is projected to be the second most common cause of cancer-related deaths by 2030 , 75. TheAlthough cell lines, rodent models, and patient-derived xenograft models have generated valuable insights, organoid models have emerged as a superior tool for studying PDAC behavior and assessment of the response to potential therapeutic molecules or screening for drug sensitivities in vitro –80. RomeGenomic analysis has identified molecular subtypes of pancreatic cancer and PDAC subtypes as well as their differential response to therapy , 86. PDABesides the potential to study tumor behavior and drug responses in human PDAC organoids, another advantage is the possibility to generate large amounts of human PDAC organoids from small amounts of tissue. Using endoscopic ultrasound fine needle aspiration (EUS-FNA) to collect tumor tissue, it has been demonstrated that PDAC organoids can be robustly generated with high rates of success with this technique , 88. ThiPre-stages of PDAC have also been studied using organoid models. Transplantation of human PDAC organoids into immunodeficient mice resulted in PDAC precursor lesions called pancreatic intraepithelial neoplasms (PanINs) ••. This In summary, these recent studies demonstrate that large numbers of PDAC organoids can be derived from small tissue samples and that PDAC organoids show great promise for the recapitulation of PDAC tumor characteristics in vitro. A current limitation of the PDAC organoid cultures is the limited recapitulation of tumor microenvironment in vitro, which is provided by stromal cells, blood vessels, nerves, and immune cells in vivo. This tumor microenvironment in PDAC is highly dynamic; promotes tumor progression, metastatic niche formation, and therapeutic resistance; and thus has impact on clinical outcome . Future Pancreatic organoids are also a promising tool for screening of toxic effects of drugs and other compounds, such as alcohol, on pancreatic cells. Alcohol is a major cause of acute and chronic pancreatitis in man, but little information is available on the molecular mechanisms that underly this pancreatic disease. Currently, the effects of alcohol have been largely studied on cell lines that exhibit features of acinar cells or on primary acinar cells isolated from rodents , 93. HowPancreas disease modelling in a 3D culture system can also be performed using induced pluripotent stem cells from subjects with monogenetic diseases. Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis membrane conductance regulator (CFTR), affecting anion transport and fluid secretion in the pancreas and ultimately leading to chronic pancreatitis and pancreatic insufficiency. Also, diabetes often occurs in patients with CF and pancreas exocrine insufficiency, which is the result of a complex cascade of events that has not been elucidated. Unfortunately, most of the animal models of CF do not fully recapitulate important disease aspects of human CF . Hence, Primary human intestine and rectal tissue from patients with CF have already been cultured as organoids, which could be used for quantitative assays of CFTR function and for the testing of new drugs that modulate CFTR protein function , 96. ForIn conclusion, human pancreatic organoids constitute an important translational bridge between in vitro 2D monolayers composed of a single-cell-type and in vivo animal models. While the expansion and differentiation protocols are still being improved, studies performed with human pancreatic organoids have increased our understanding of pancreas development, homeostasis, and benign and malignant pancreatic diseases. Their potential as a platform for developmental studies, disease modelling, toxicity screening, and personalized treatments for PDAC is already evident, and we envision that they will have a substantial clinical impact in the near future.


Recent investigations in neuroscience elucidate the neural basis of close other cognitive representations, which serve functions central to our health and happiness. Yet, there are persistent barriers to this research, including disparate research methods and the absence of a common theoretical background. The present review connects neuroimaging and attachment theory within a novel social, cognitive and affective framework. We apply attachment theory to understand why we would expect cognitive representations of close others to be different from other social neural representations. Developing reliable markers of attachment is a critical step in mapping close other neural representations. We then examine existing neuroimaging literature on close other representations, highlighting the recruitment of neural systems supporting reward, motivation and distress alleviation, in addition to the mirror neuron system, default network and salience network. We then review the methodologies of past studies, revealing a diverse array of self-report measures assessing `closeness’ and social cognitive tasks that, taken together, preclude meaningful synthesis of findings. Lastly, we discuss specific behavioral measures of attachment and closeness with recommendations for the field. This attachment framework integrates brain and behavioral sciences and unites theoretical principles with empirical methods to further our understanding of how the brain represents close others. I love you without knowing how, or when, or from where,I love you straightforwardly, without complexities or pride;so I love you because I know no other wayI does not exist, nor you,than this: where so close that your hand on my chest is my hand,so close that your eyes close as I fall asleep.-Pablo Neruda, One Hundred Love Sonnets: XVII, 1959;Translated by Stephen TapscottIf you are able to suspend disbelief for a moment, assume that two individuals’ bodies could so precisely synchronize as a function of mere affection. Then perhaps Neruda’s prose does accurately reflect feelings of intense love. His imagery evokes a level of intimacy and connection reserved only for certain close relationships. Humankind’s fascination with the experience of love and thinking about loved-ones stretches far back through history, long before Neruda wrote his sonnets. This curiosity flourishes today, too; the observation of biobehavioral synchrony, or the sensitization to and mirroring of another’s physiological and behavioral cues, in pair bonds ; the default network, responsible for internally-directed thought and social cognition; and the salience network in differentiation of unique close other representations. The studies we review adopted diverse approaches to operationalize and measure the construct of close other representations. In the next section of our review, we evaluate these approaches. We discuss core methodological challenges in this area, including the following: use of terminology relating to close relationships and to attachment bonds, a smaller subset of those relationships examined; assessment of relationship length; use of self-report measures of relationship closeness and quality; differing neuroimaging task paradigms; and the potential impact of demographic factors such as sex, sexual orientation and age. Of particular interest are experimental parameters reflecting how researchers measure `closeness‘ of close others in relation to neural responses. Such methodological discrepancies preclude meaningful integration of findings across concepts and fields. To conclude, we offer a series of recommendations designed to promote an interdisciplinary approach for mapping the neural representation of unique close relationships, conceptualized as attachment bonds. We review behavioral methods to assess close adult relationships, including attachment bonds, and with recommendations for the field. Developing reliable markers of attachment, grounded in social psychological theory, is a critical step in mapping close relationship representations in the human brain.et al., vs non-presence) of attachment to characterize close relationships. We do not approach attachment theory from Ainsworth et al.’s  methods. Accordingly, results show diversity in the brain regions and networks, or large-scale systems of functionally connected brain regions, implicated in creating, updating and utilizing these mental representations. Social and affective neuroscientific studies focus on motivation and reward conditioning to close others and the affect-regulatory capabilities close others impart. These investigations consistently implicate the recruitment of reward and distress-alleviation systems in the brain . Participants included individuals who, at 40-month follow-up, were together with their romantic partners from the first assessment and others who had since broken up. Results showed that partner-related activity in the tail of the caudate during the early-stage assessment was associated with remaining together 40 months later, as well as with higher self-reported commitment to the relationship. A second, somewhat counterintuitive set of activational effects also emerged, wherein lower early-stage activity in medial orbitofrontal cortex and nucleus accumbens (NAcc) was associated with greater commitment, happiness and longevity of participants’ relationships at 40-month follow-up. The directionality of Xu et al.’s (2012) second set of activational effects was brought into question by another foundational study of long-term relationship representations. Acevedo et al.  was positively correlated with activation of NAcc and caudate in response to romantic partner vs friend. There is also evidence that this pattern of findings holds across samples of non-heterosexual individuals in long-term romantic relationships  was associated with decreased activation in right dorsolateral prefrontal cortex, left caudate and NAcc, whereas decreased activation of ventral ACC and posterior cingulate cortex (PCC) was shown for both spouse- and stranger-related attenuation of threat. Using a similar paradigm, modified to tap individuals’ abstract mental representations of non-physically present partners, another experiment had female participants view partner vs stranger pictures while receiving painful heat stimulations . Incorporating elements of emotion regulation research and cognitive neuroscientific methods, one study applied a mild electric shock paradigm to look at self-focused threat, vs close friend-focused threat or stranger-focused threat , PCC, lateral prefrontal cortex, lateral temporal cortices (LTC) and lateral parietal cortices  of four characters . In 2010, Krienen, Tu and Buckner advanced this earlier work. Participants in their study made judgments about personal preferences in response to facial images of close friends vs strangers. They found, for the first time, that friends vs strangers yielded a network of brain regions including mPFC, PCC/ retrosplenial cortex, inferior parietal lobe, lateral temporal cortex and medial temporal lobe , a few studies have initiated a focus on the role of mPFC and PCC in differentiating close other from stranger or from self-representations. Some have asserted that the representation of self is `special‘, uniquely recruiting mPFC in contrast to representations of intimately known others  in a trait judgment task yielded higher mPFC and ACC activity  social representations, utilizing the same trait judgment fMRI paradigm. In particular, we were interested in how individuals neurally represent some of their closest reciprocal relationships—those with parents (young adults) and children (older adults)—and how these representations reflect perceived closeness or attachment to those figures. Patterns of neural activation for mentalizing about a parent or child significantly varied as a function of attachment; interestingly, we found that the more attached one feels to their parent/child, the lower brain activity was observed in brain regions such as ACC, left amygdala hippocampus, anterior and posterior insula, PCC, and the putative occipital face area, suggesting that bringing to mind one’s attachment figure requires less engagement of these brain regions often recruited for distress relief, memory and facial processing.Our approach across these studies has several innovations for work on known and, specifically, close others. First, our experimental stimuli included the names of real individuals who were highly relevant to each participant. Second, unlike past studies utilizing passive fMRI tasks (such as free-viewing photos of known others), our paradigm required that participants actively mentalize about each of the social targets. Third, we included numerous self-report measures in an attempt to better describe and characterize participants’ relationships with their social targets; one such measure, for example, provided us with information about who participants’ (primary) attachment figures were. We were able to provide initial evidence that the representation of adult attachment is a distinguishing feature of the neural activation differences in social cognition.If one overarching conclusion emerges from fMRI studies of close other representations, it is that we are now arriving at a novel, cross-disciplinary neuroscience of close relationships—yet from several very different perspectives. Just as our mental representations of close others reflect the complexity of rich person-knowledge, emotionally salient memories and unique regulatory capabilities, the neural regions and networks responsible for carrying close other representations are complex, as well. Bringing together past findings, these neural regions and networks appear to include: dopamine-rich regions sensitive to partner reward such as VTA, NAcc and putamen; threat-responsive regions sensitive to partner comfort such as ACC and insula; mirror neuron regions sensitive to a partner’s inner states such as inferior parietal lobules; default network regions sensitive to mentalizing about a partner such as mPFC and PCC; and salience network regions sensitive to meaningful cues associated with a partner such as anterior insula and anterior cingulate.et al., et al., This integrative model  is suppoIn the next section of this review, we employ a different approach towards integrating this literature. Here we look across recent fMRI studies of close other mental representations with respect to their methods. Of particular interest to us are experimental parameters reflecting how researchers measure `closeness‘ when studying neural responses to social cognitive stimuli. Here, we discuss the following: usage of terminology relating to close relationships; collection of relationship length data; self-report measures of closeness with relationship partners; social cognitive tasks used in the MRI scanner; and specific condition contrasts used in BOLD fMRI data analysis. We also track sample sizes, as well as gender, sexual orientation and age composition of these studies’ samples.et al., et al., et al., et al., et al., et al., et al., et al., Even a cursory glance at the terminology used by existing studies provides some rationale for their diverse findings. Authors use varied, but specific, language to operationally define their constructs of interest. They often continue to use this language in reporting and promoting research findings in their articles. Of the studies we surveyed, a majority describe their construct of interest as `romantic love‘  indicating that a full-fledged attachment bond has formed may not be completely present until 2 years or more into the relationship . Current fMRI research does not recruit or report on a wide-enough range of participant relationship lengths to capture a full understanding of how this variable relates to other neural and behavioral outcome variables.Yet, the existing literature reveals a lack of reported relationship length data. Half of the studies reviewed do not report any relationship length descriptive statistics for the target close relationships. For those who do report this variable, average (arithmetic mean) length of relationships with target relationship partner range from a few months . Of the studies that do administer other self-report closeness measures, only a few report the resulting behavioral data  or utilize participants’ responses as variables in their neuroimaging analyses , and support giving or receiving from partner vs others  et al., vs a partner hand-holding experimental paradigm .A study’s motivations are inherently connected to its results through the careful design of its experimental paradigm. As evidenced by the variety of fMRI tasks we cover in the present review, there is space for both replications and constructions of different tasks in future research. We argue that the design of neuroimaging tasks can be better-informed by understanding the affective, behavioral and cognitive processes involved in representing close others. We recommend that researchers explore different types of tasks that require active mentalization about or utilization of attachment figure representations. Passive tasks will not require participants to draw upon their attachment figure representations in replicable or consistent ways. Examples of active mentalization tasks include, but are not limited to, trait judgement, or social autobiographical memory, accompanied by target name or photograph prompts that are individualized for each participant. Other rigorous designs that would require utilization of attachment figure representations include real or simulated presence of attachment figures in threat-induction paradigms (e.g. hand-holding task).We believe this point is important enough to be a separate recommendation; it is challenging to draw conclusions about any close other neural representations if social contrasts are not intentionally included. Implications of task-based fMRI findings rely on our ability to compare patterns of activation across different conditions. We recommend that all studies of close other representations include targets such as romantic partners, family members, close friends, acquaintances, famous figures, strangers and the self as control conditions that are social in nature and possess ecological validity. Within the broad social category of known others, there may be substantial differences along the dimensions of closeness and familiarity. To parse out behavioral and neural differences between several known others, studies could require participants to think of specific exemplars of each of the following dimensions: an attached romantic partner or family member, a friend with whom the participant is close and familiar but not attached, a familiar but not close acquaintance and a known but not close or familiar famous figure. Ideally, future fMRI studies will capture patterns of brain activity for more complete spectra of familiarity and closeness.et al., et al., et al., Increased interest in neural correlates of social psychological constructs must go hand-in-hand with adherence to rigorous methodological practices needed for neuroimaging studies (for review, see Yarkoni, Importantly, we need to administer self-report measures that answer numerous questions about participants’ cognitions, behaviors and emotions within the context of their relationships. Some of the questions we would certainly want future participants to answer include: To whom are participants attached? How do participants view their social closeness with specific others? Toward whom do participants feel passionate love? Toward whom do participants feel companionate love? What attachment style do participants show in their current romantic relationships? How committed, satisfied and invested do participants feel in their current romantic relationships? How long have participants been in relationships with their romantic partners? How long have participants known specific other people?Answers to these questions could let us know, on a basic level, the core drivers of regional and network brain activation differences. If we implement a standard battery of measures addressing these and other questions across studies, we can begin to tap into a unified cognitive construct of attachment figure mental representations. Such a standard battery can provide a clear picture of the content of attachment figure mental representations, the attachment `status‘ and style, and the specific ways in which attachment figure representations are different from other social representations.et al., et al., et al., et al., et al., Our proposed standard battery can be found in Appendix A. This compilation of close relationship self-report measures includes the WHOTO (Hazan et al., Lastly, we include brief questionnaires in order to gain consistent self-report data about variables such as relationship length. The partner specific items assess commitment, exclusivity and satisfaction, whereas the general questions gauge depth of personal knowledge and emotional investment in any kind of relationship. By including each of these measures, in addition to the highly relevant IOS (Aron Various regions of the brain, including those important for reward, emotional regulation, memory and understanding of others’ actions, are recruited in the activation of mental representations of attachment figures. The neural systems involved in the formation and function of mental representations in adult attachment relationships are understandably complex. In the present work, we call attention to an emerging field of close relationship neuroscience and a gap in its literature that would benefit greatly from increasing collaborations across disciplines.We have asserted that the developing social neuroscience of attachment is based in both a rich theoretical framework and an increasingly robust collection of empirical studies. Attachment theory suggests that behavior towards certain close others differs fundamentally from behavior towards more distant others. One important characteristic of attachment bonds is the feeling of security associated with attachment figures’ proximity. As individuals undergo conditioning processes over the course of relationship development, the accessibility of attachment figure mental representations supplants the need for physical proximity. Social cognitive neuroimaging studies implicate a wide array of brain systems in supporting close other representations, including those of attachment figures. Engagement of reward systems, as well as the distress-alleviation mechanism within the limbic system, have been implicated in attachment formation and maintenance. Past work also highlights the role of the limbic system in emotional regulation provided by a close other. Memory systems support the encoding and retrieval of person-specific knowledge and social memories necessary to form rich cognitive representations. The default network has also been implicated in differentiating mental representations of oneself and of known others. Lastly, the salience network demonstrates a critical ability to distinguish primary attachment figure representations from other social representations. Past studies have, however, applied diverse approaches to operationalize and measure the constructs of close relationship representations or attachment figure representations.In light of the excitement and confusion surrounding this new area of research, we have offered a series of recommendations designed to promote an interdisciplinary approach for mapping the neural basis of attachment figure representations. We assert that administration of standardized assessments, including measures of attachment status, style and relationship quality, is necessary to develop comprehensive, reliable and replicable markers of real-world attachment representations. In addition to uniting the contributing neuroimaging fields, future research could include implementation of longitudinal designs investigating which neural structures are sensitive to the affective, behavioral, cognitive and physiological processes involved in attachment figure mental representations. In the future, it could be possible to learn how neural representations of primary attachment figures within a romantic couple or a parent/ child relationship change over the course of a lifetime spent together. As attachment theory includes such a robust developmental component, this type of longitudinal study—or cross-sectional examinations of adolescents or children—could be very fruitful in further deciphering the creation and maintenance of neural representations of attachment figures. We might also be able to examine what kinds of interpersonal experiences, specifically, recruit neural regions of interest within the context of close other mental representations. We already have evidence for how crucial attachment bonds are for our psychological and physical health throughout the life course; with cross-disciplinary communication and sharing of methodological tools, the possibilities to learn more about the brain’s function in these powerful relationships is limitless.


Unrestricted cancer growth requires permanent supply of glucose that can be obtained from cancer-mediated reprogramming of glucose metabolism in the cancer-bearing host. The pathological mechanisms by which cancer cells exert their negative influence on host glucose metabolism are largely unknown. This paper proposes a mechanism of metabolic and hormonal changes that may favor glucose delivery to tumor (not host) cells by creating a cancer-host “vicious cycle” whose prolonged action drives cancer progression and promotes host cachexia. To verify this hypothesis, a feedback model of host-cancer interactions that create the “vicious cycle” via cancer-induced reprogramming of host glucose metabolism is proposed. This model is capable of answering some crucial questions as to how anabolic cancer cells can reprogram the systemic glucose metabolism and why these pathways were not observed in pregnancy. The current paper helps to better understanding a pathogenesis of cancer progression and identify hormonal/metabolic targets for anti-cancer treatment. Metabolism consists of catabolic processes, i.e., the breakdown of molecules resulting in the release of energy, and anabolic processes, i.e., the synthesis of predecessors and components for proteins, lipids, and nucleic acids which consumes energy. Maintenance of the delicate balance between anabolism and catabolism is one of the most important requirements for organism survival, especially in critical host situations, such as embryo growth in pregnancy or unrestrained cancer proliferation. Glucose is an essential fuel for embryo and tumor cells wherein glucose uptake is independent of insulin. This contrasts with glucose uptake by insulin-dependent cells, such as skeletal muscle, fat and hepatic cells. Reduction in the ability of these cells to take glucose from the blood in response to normal circulating levels of insulin is known as insulin resistance (IR).Chronic IR is a key pathological feature of obesity, type 2 diabetes with an increased weight of the patients and cancer cachexia with a reduced weight of the cancer-bearing host . A possiThe survival of multi-cellular organisms depends on the organism ability to maintain glucose homeostasis for the time of low/high nutrient availability or high glucose requirement to support cellular proliferation. Glucose is an essential fuel for cellular growth in adaptive responses (such as pregnancy and immune protective response) or in uncontrolled proliferative cancer disease. Pregnancy is a period marked with adaptive changes in the women's hormonal status and metabolism. The ability to regulate nutrient balance during this period is critical for health of the mother and the growing fetus. It is well known that glucose is the primary source of energy and structural materials for the embryo growth during pregnancy. Embryonic consumption of glucose passes ahead of glucose availability from the pregnant mother. Because of this, the normal concentration of blood glucose in the embryo/fetus is lower by 10–20 mg/100 ml (0.6–1.1 mmol/l) than in the blood from the pregnant mother (3.3–6.6 mmol/l). This difference becomes maximal in the second-half period of human pregnancy. It is interesting to note that this period is characterized by physiological IR  that is After childbirth, the concentration of the maternal TNF-α reduces rapidly to the normal basal state, in parallel with declined IR to its original state which is typical of healthy women . Howeverin vivo. Different gluconeogenic precursors, such as lactate, alanine, and glycerol, contributing to prevention of systemic glucose deficit can be used. As an example, cancer-produced lactate is recycled to glucose by the liver through the ineffective Cori cycle. This extensive glucose recycling accompanied by increased energy expenditure in tumor-bearing hosts has been documented particularly in cancer patients , which subsequently supports tumor cell proliferation and promotes host IR while reducing circulating insulin, increasing glucose availability to the cancer cells , 41. TreHowever, cancer cells have developed another strategy that could give them, but not the host organism, an advantage in the glucose supply. This result could be achieved by a distant cancer-induced stress- and inflammatory response that gives rise to peripheral IR responsible for activation of hepatic glucose synthesis and redirection of unused glucose from muscle and adipocytes with IR to glycolytic cancer cells. The pathways integrating stress and inflammatory response with insulin action are detailed in Glucose homeostasis is also regulated systemically by insulin and glucagon acting in the opposite direction . In healThe balance points between the two antagonistic hormones (insulin and glucagon) provide a sensitive switch of gluconeogenesis activation during cancer progression. This cancer-bearing state was found to be associated with lowering insulin production  and raisNot only glucagon but also cancer-induced stress hormones such as glucocorticoids are important because they directly promote hepatic gluconeogenesis that increases the level of blood glucose , and by It is known that chronic inflammation is a key contributor to cancer progression , whereasTaken together, cancer growth and progression is related to an energy and hormonal imbalance. Metabolic re-programming of host cells supports the production of energy-rich glucose via catabolism , which is then transferred to cancer cells to promote their anabolic growth. This result is achieved by the cancer re-programming of glucose metabolism in distant host organs, thereby creating a cancer-host vicious cycle that may perpetuate its own maintenance as well as cancer growth and progression. The vicious cycle involves numerous participants, and all of them may serve as targets for specific personalized treatment of cancer patients aimed to prevent cancer progression.The cancer problem is not merely a cell problem, because it is a problem of cellular interactions not only within cancer-containing tissues, but also within normal cells in other distant tissues. Host-tumor metabolic interactions can be considered as a two-sided process in which cancer cells show the parasitic behavior, because they have no specific function other than growth and dissemination, and compete with the host cells for essential resources such as glucose, lipids and amino acids. This opinion article describes how cancer cells metabolically reprogram the host cells by redirecting the majority of glycolytic fuel from the insulin-resistant host cells to glycolytic cancer cells. In contrast to a transient retention of this metabolic asymmetry without stress and insulin deficit in pregnancy, the chronic retention of such an asymmetry with stress and insulin deficit between cancer and host cells might trigger formation of a vicious cycle , 3; its The author confirms being the sole contributor of this work and has approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Techniques employed in the field of neuroscience, such as eye tracking, electroencephalography, and functional magnetic resonance imaging, have been important in informing our understanding of the cognitive mechanisms underlying tobacco smoking. These techniques are now increasingly being used to investigate the likely impact of tobacco control policies.In this narrative review, we outline the value of these methodological approaches in answering policy-relevant tobacco control research questions, with a particular focus on their use in examining the impact of standardized cigarette packaging and health warnings. We also examine the limitations of these methodologies and provide examples of how they can be used to answer other policy-relevant questions.We argue that neuroscience techniques can provide more objective evidence of the impacts of policy measures, allow investigation where it is not possible to conduct behavioral manipulations, and facilitate a deeper understanding of the cognitive mechanisms underlying the impacts of tobacco control policies such as standardized packaging, health warnings, point-of-sale displays, and mass media campaigns.Rather than replacing more traditional methods of examining tobacco control measures, such as observational experiments, surveys, and questionnaires, neuroscience techniques can complement and extend these methods.Neuroscience techniques facilitate objective examination of the mechanisms underlying the impacts of tobacco control measures. These techniques can therefore complement and extend other methodologies typically used in this field, such as observational experiments, surveys, and questionnaires. Techniques commonly employed in the field of cognitive neuroscience (described here as neuroscience techniques) are increasingly being used to investigate the likely impact of tobacco control measures. The overarching premise of our narrative review is that these techniques can complement and extend self-report and behavioral research approaches commonly used to evaluate tobacco control measures. First, we describe how neuroscience techniques can overcome some of the limitations of these other research techniques. Second, we provide a review of studies using eye tracking, electroencephalography (EEG), and functional magnetic resonance imaging  to evaluate these policies, with a particular focus on standardized packaging and health warnings. Third, we discuss the limitations of these techniques. We conclude with a discussion of the future of neuroscience techniques for tobacco control research and provide a framework for future research.Every research methodology has its limitations (and we discuss limitations with neuroscience techniques later). However, techniques used in neuroscience can overcome some of the limitations associated with methodologies often used to examine questions related to tobacco control policies, including those relying on subjective responses.1 Of these, only 23 outcomes were objective . Despite considerable converging evidence,3 tobacco industry advocates argue that approaches relying on subjective reports do not constitute “credible evidence.”4 Tobacco industry criticisms on the grounds of a lack of credible evidence are likely to have slowed United Kingdom government decision-making on standardized packaging6 and US decision-making on health warnings.7 Indeed, as intentions are known to only play a small role in actual behavior,8 research which relies on self-reported outcomes has also been criticized by academics within the field of tobacco control who have argued that experimental designs with reliable, behavioral measures are required to provide “convincing evidence.”9 Neuroscience techniques allow us to examine processes and behaviors that are outside of the awareness of individuals and therefore are not subject to these same criticisms.Traditionally, research examining the potential effectiveness of tobacco control measures has relied on subjective response methodologies, such as surveys, focus groups, and questionnaires. A recent systematic review of 68 experimental studies comparing text-only warnings with pictorial health warnings identified a total of 278 outcome measures characterizing 61 constructs across these studies.10 while another has examined the impact of standardized packaging on actual smoking behavior.11 Similarly, in a recently completed randomized controlled trial, the effects of reducing the nicotine content in cigarettes on smoking behavior over 6 weeks were evaluated in a diverse sample of US smokers.12 The results provide more compelling evidence than would a survey study asking smokers to self-report on their opinions or imagined reactions to a reduced-nicotine cigarette. However, while studies with behavioral outcomes provide a more rigorous evaluation of potential policy measures than those relying on subjective reports, these evaluations can take years to complete and require large sample sizes and long follow-up periods in order to observe the downstream effects of these interventions.13Behavioral research methodologies can indeed provide more convincing evidence of the effects of a proposed tobacco control measure. A recent randomized controlled trial evaluated the effects of pictorial cigarette pack warnings on quit attempts,14–16 before implementing it. However, this is not always feasible, and generalizing from one country to another is an important limitation of this kind of evaluation. Neuroscience techniques can provide objective evidence of the likely effects of a tobacco control measure but in a considerably shorter timescale.In addition, behavioral outcomes are not possible when the policy manipulation is outside the control of the experimenter. For example, it is typically not feasible to randomly assign half of a country to a tobacco control measure such as standardized packaging and compare its effects on smoking outcomes to the half of the population continuing with normal practice. Instead, policymakers will wait for evidence from other countries that have introduced a tobacco control measure before implementing it themselves. For example, the United Kingdom, Ireland, and France waited for pre/postevaluations of the effectiveness of standardized packaging in Australiaimpact of policy on target endpoints, are also limited in their ability to explain why a policy intervention worked in the manner it did. Neuroscience techniques have been important in informing our understanding of the cognitive mechanisms underlying tobacco smoking, including nicotine withdrawal,18 the impact of quitting smoking,19 prediction of cessation outcomes,20–23 and cue reactivity.24–26 These techniques are increasingly being used to examine the cognitive mechanisms underlying responses to tobacco control measures. In some cases, insights into cognitive mechanism may be obvious  or unimportant. However, in other cases, a deeper understanding of how a policy manipulation exerts its influence can result in insights necessary for further refinement and even greater impact. For example, understanding why a particular health warning is effective at encouraging thoughts about quitting can inform development of future health warnings. Furthermore, while a group of participants in a study may all exhibit the same self-reported or behavioral response to one stimulus or manipulation, the underlying cognitive and neural processes by which they arrive at those outcomes may vary in meaningful ways.Studies with behavioral outcomes, while providing evidence regarding the increase purchasing behavior.27–29 Box 1 describes these techniques in more detail, and in the following sections, we provide a review of studies using these techniques to answer tobacco policy-relevant questions, with the goal of ultimately reducing purchasing of tobacco by reducing smoking initiation and encouraging cessation.By measuring processes and behaviors that are outside of the awareness of individuals, neuroscience techniques can overcome many of the limitations of methodologies relying on both subjective and behavioral responses. These techniques include eye tracking, EEG, and fMRI and are commonly used in market research to understand how neuroscience can inform consumer decision-making and Box 1. Neuroscience Techniques Explained54 While early eye trackers were invasive and relied on direct and invasive observations of the eye,80 today’s eye trackers typically measure eye movements using noninvasive optical tracking with a video-based eye tracker. At the most basic level, eye movements can be divided into fixations (when the eye is stable in a particular location) and saccades (when it is moving to another location). Researchers typically examine the location of the first fixation, the duration of fixations, or the number of fixations. These measures provide an indication of visual attention toward a stimulus . Eye trackers are either “static” or “mobile.” When using a static eye tracker, the participant is static and views 2D stimuli on screen (either still or moving images). By contrast, using a mobile eye tracker allows for analysis of eye movements when individuals are mobile in naturalistic or real-world environments.Attention to a stimulus is recognized as an important prerequisite for behavior change.Eye tracking can be used to answer questions such as “Which elements of pro/anti-tobacco messages capture attention?” “How is attention allocated in tobacco retail environments?” “How does attention to vaping cues influence later smoking behavior?”81 has been reliably shown to reflect the heightened incentive value of smoking cues among smokers.82During EEG, electrodes are placed over the skull to measure changes in the electric field being produced by the brain. Event-related potentials (ERPs), which reflect the specific electrical activity related to a specific sensory “event” , can be measured using EEG. ERPs can refer to different components, such as P300, where each component is typically quantified by its amplitude and latency related to the onset of the sensory event. Although EEG has poor spatial resolution , EEG affords excellent temporal resolution (within the millisecond range), meaning that it is useful for measuring brain activity over time. A large volume of literature has previously used EEG to explore the mechanisms underlying tobacco addiction and the P300 ERP, which is related to attentional processes, and participant arousal stateEEG can be used to answer questions such as “Do anti-tobacco advertisements successfully elicit emotional responses?” “What are the neural indices underlying smokers’ reactance to health warnings?” “What is the minimum nicotine content needed to induce change in neural activity?”The basic principle behind functional magnetic resonance imaging (fMRI) is that when a region of the brain is more active, it uses more oxygen, resulting in an increased blood flow to this region. fMRI works by detecting these changes in blood oxygenation and flow and measures blood oxygenation–level dependent (BOLD) signal in the brain. Unlike EEG, fMRI has relatively poor temporal resolution (~1 s) because of the slower speed of blood when compared with electrical impulses. However, the spatial resolution of fMRI (approximately 1–2 mm) is much greater than EEG, meaning that fMRI can be used to create maps that show the activated regions of the brain during certain tasks. fMRI is therefore important in understanding the neural mechanisms underlying behavior change.fMRI can be used to answer questions such as “How do tobacco advertisements influence neural responses to reward?” “What effect does vaping during scanning have on neural responses related to reward and craving?” “How do neural responses to anti-tobacco messages predict later choice of tobacco products above and beyond self-report?”30 to those exploring the cognitive mechanisms underlying supermarket choices.31 This methodology provides an objective measure of what visual stimuli or objects individuals are attending to, and which aspects of the environment capture attention. The value of this research technique is also recognized by the tobacco industry, who have used mobile eye tracking (see Box 1) to examine the impact of tobacco point-of-sale displays on visual attention.32 Similar research has been conducted by academics.33Eye tracking has been used for decades by consumer marketers, from studies examining visual attention to adverts in the Yellow Pages3 Research using eye-tracking technology has therefore been particularly valuable in objectively measuring visual attention to health warnings on branded and standardized packaging,34–38 observing that standardized packaging increases visual attention to warnings among adult and adolescent nonsmokers and nondaily smokers.37 Other research has found that this is observed for text-only warnings and pictorial health warnings on standardized packs.38 Eye-tracking research was cited in the legal case between British American Tobacco and the UK Department of Health regarding the legality of standardized packaging legislation39 and was described as using “objective physiological techniques.” This research adds weight to and extends findings obtained using other research methodologies, including focus groups,41 quantitative surveys,43 and naturalistic studies,45 which have reported similar effects.One of the mechanisms through which standardized packaging is expected to be effective is by increasing the salience of health warnings.36 indicating that different strategies for capturing attention are required among this population. Studies using this technique have examined the impact of varying a number of different health warning components including novelty,47 size,48 format,50 and emotional content51 on visual attention. Other research has used eye tracking to examine the impact of risk statements,52 message congruency,53 and pictorial warnings49 in cigarette advertisements on visual attention.By measuring which components of a health warning smokers are attending to and consequently which are impacting their self-reported reactions, eye-tracking technology allows greater understanding of the cognitive mechanisms underlying the influence of tobacco marketing materials on self-reported reactions and behavioral effects  and negative responses .54 By examining correlations between self-reported reactions and brain activation, EEG and specifically examination of particular event-related potentials (see Box 1) have allowed us to begin to understand the cognitive mechanisms underlying these self-reported responses.Despite considerable research showing that pictorial warnings are more effective than text-only warnings,55 used EEG to understand the cognitive mechanism underlying avoidance of health warnings among smokers, a finding previously observed using both self-report56 and eye-tracking36 research. While smokers showed no differences in early perceptual processing when compared with nonsmokers , they showed reduced later cognitive responses to the warnings  when compared with nonsmokers. This suggests that smokers’ avoidance of health warnings is not due to differences in perceptual processing of the health warnings, but rather due to reduced sensitivity to the emotional content of health warnings . Similarly, a recent study by Cochran et al. observed that disgust-based health warnings, but not health anxiety-based warnings, reduced attentional processing of smoking cues (as indexed using EEG).58 Together, these studies indicate that although smokers may avoid health warnings, graphic warnings that evoke a strong emotional reaction (such as those proposed for use in the United States) may be effective at changing relevant behaviors , thus providing new insights into the cognitive mechanisms underlying the effects of health warnings.In a similar EEG study, Wang et al.59 another which combined fMRI with eye tracking observed that when taking visual attention to health warnings into account, standardized when compared with branded packaging increased activation in the visual cortex, suggesting that standardized packaging increases the visual salience of health warnings.60 fMRI has also been used to examine neural activity when smokers are presented with cigarette package health warnings and have found that pictorial warnings activate large-scale neural networks including the hippocampus, fusiform gyrus, and supplementary motor area61 and the amygdala, medial prefrontal cortex, medial temporal lobe, and occipital cortex59  on product valuation, as well as allow for the comparison of valuation across product categories . The potential of this mixed methodological approach in the field of tobacco control has been demonstrated by Falk et al. who found that the extent to which antismoking messages activated the medial prefrontal cortex predicted the success of those same antismoking messages at the population level.65 Combining neural data with self-reported responses to the campaign accounted for the largest proportion of the variation in the population-level success of the campaign, demonstrating the added value of the neural predictor.65 Similarly, activation in the medial prefrontal cortex has been shown to be predictive of successful smoking cessation (as measured using expired carbon monoxide) over and above self-reported intentions to quit, self-efficacy, and the ability to relate to health messages.66 This technique could therefore be used to augment self-report measures which are typically used as predictors of behavior and may be particularly useful when self-reported preferences and actual behavior are likely to diverge .Future research in this field can draw on well-validated neural indices  to provide an objective assessment of the effects of product features such as health warnings on behavior . The ventromedial prefrontal cortex, for instance, has been shown to index the valuation of products across multiple different attributes of goods.Although these neuroscience research techniques can be valuable adjuncts to other experimental and qualitative research and can provide important insights into the potential cognitive mechanisms underlying the effectiveness of tobacco control measures, they also have some important limitations.27–29While these techniques afford excellent internal validity, allowing researchers to manipulate different elements of the tobacco control measure , their ecological validity is limited. However, these limitations are not insurmountable. Indeed, there is a long history of using these techniques in market research to answer questions related to consumer behavior.64 Recently, researchers have conducted the first feasibility trial demonstrating the use of an e-cigarette during fMRI scanning.67 Furthermore, with improvements in technology, neuroscience techniques are increasingly being taken into real-world environments. For example, mobile EEG68 and eye tracking69 equipment allow us the possibility of examining neural responses and visual attention in the real world, such that the impact of real, 3D cigarette packs on visual attention to health warnings can be assessed. In addition, a growing number of studies are bringing real-world, personally relevant stimuli into the lab and scanning environment for assessment of subjective and neural reactivity71; in the future, such methods could be applied to the study of real-world tobacco retail and point-of-sale environments. Furthermore, recent advances in virtual reality (VR) technology mean that some of the limitations of these neuroscience techniques can be overcome even in controlled laboratory settings. Using VR headsets, participants can be immersed in life-like scenes in combination with neuroscience techniques. This technology has been used to investigate the impact of smoking cues in VR environments on craving,72 and as a result, the impact of VR smoking cue exposure therapy on smoking cessation success.73 Moreover, at least three studies have evaluated craving and tobacco product purchasing behaviors in virtual tobacco retail outlets74–76 As far as we are aware, however, VR has yet to be combined with simultaneous eye tracking, EEG, or fMRI, or other neuroscience techniques, to answer questions relevant to tobacco control.Purchasing behavior can be modeled during fMRI by giving subjects a set amount of money that can be allocated across purchasing decisions with later, real-world delivery of those products.actual behavioral responses to tobacco control measures. For example, the extent to which visual attention to health warnings34–38 or neural responses to standardized packaging59 replicate that observed when a smoker pulls a cigarette pack from their pocket is unknown. Studies that use a combination of methodological approaches can attempt to bridge this gap. Neuroscience measures should be used in combination with questionnaires or biological measures (such as cotinine) to understand the relationship between the neuroscience measure and actual behavior. As described in the fMRI subsection, neural measures are increasingly being used to improve predictions of decision-making models, where neural activation predecision can predict later choice.64In addition to relatively poor ecological validity, the degree to which findings from neuroscience research are generalizable is also a potential limitation. Indeed, it is important to determine to what extent the behaviors observed using neuroscience techniques are predictive of 77 which also outlines other techniques that have not been mentioned here, including magnetoencephalography, positron emission tomography, near-infrared spectroscopy, transcranial magnetic stimulation, and transcranial direct current stimulation.Finally, each of the neuroscience techniques described here has several inherent limitations that researchers interested in their use should consider. These include high set-up costs and large amounts of time and skill required to run experiments and analyze the data. This technical obstacle can be overcome by forming collaborations between researchers with different research backgrounds. For a more detailed description of the techniques that have been described here, including their advantages and disadvantages, readers should refer to the review by Kable,78 and food labeling.79This review has focused on the role of neuroscience techniques in addressing research questions related to tobacco control policies, in particular, standardized packaging and health warnings. Other possibilities for future research are also discussed in Box 1. Indeed, the scope of these research techniques goes far beyond what we have described here, and our review should therefore be seen as a springboard for future research. For instance, these techniques can be used to examine other tobacco product marketing practices including point-of-sale displays, tobacco advertisements, and mass media campaigns. Similarly, neuroscience techniques can be applied to evaluate novel strategies for restricting, modifying, or banning tobacco advertisements or to study the effects of modified tobacco products and determine their likely impact. Beyond tobacco control, these techniques can answer policy-relevant research questions in other fields, such as the potential impacts of alcohol advertisementsincreasing purchasing behavior and turn these on their heads to reduce tobacco purchasing.Rather than replacing more traditional methods of examining tobacco control measures, such as observational experiments, surveys, and questionnaires, neuroscience techniques should be a useful adjunct to these methods. These techniques can provide more objective evidence of the impacts of policy measures, allow investigation where it is not possible to conduct behavioral manipulations, and facilitate a deeper understanding of the cognitive mechanisms underlying the impacts of tobacco control policies. In addition, it seems that results from neuroscience studies can be more persuasive legally than those relying on more subjective methodologies. Although our research questions should not be solely led by their potential to have “impact,” this is an important consideration when working in a field with such clear implications for policy. We hope our review demonstrates that we can use the techniques developed in the consumer neuroscience literature that focus on the goal of This work was supported in part by the Medical Research Council Integrative Epidemiology Unit at the University of Bristol, which is supported by the Medical Research Council and the University of Bristol (MC_UU_12013/6 and MC_UU_12013/7) and the Economic and Social Research Council (ES/R003424/1). JO was supported by the National Institute on Drug Abuse (K23 DA042898). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.None declared.


An unprecedented expansion of vector-borne diseases, especially those caused by viruses transmitted through mosquitoes, has posed a serious public health challenge worldwide. Chikungunya virus (CHIKV), which mainly invades tropical and subtropical regions, is one of the recently emerging pathogens associated with severe morbidity in humans. Although Pakistan is known to be endemic for arboviral infections, only Crimean-Congo hemorrhagic fever (CCHF) and dengue have been officially recognized over the last three decades, despite a recent trend of increasingly frequent chikungunya cases, first detected in December 2016.Aedes aegypti and A. albopictus) as key factors favoring efficient virus transmission in Pakistan. Likewise, even though this is the first CHIKV outbreak reported at a vast geographical scale in Pakistan, identification of concomitant cases across all four provinces indicates an increased gravity of the burden of mosquito-borne diseases in the coming years.Chikungunya infection is not new to Pakistan, as the presence of anti-CHIKV antibodies has been reported in rodent and human sera since the 1980s  assay heWithout implementation of a nationwide functional surveillance system, thorough investigations of disease determinants cannot be achieved to preempt disease trends and initiate control measures before they affect large populations. A sustainable surveillance program with potential epidemic indicators is also required to reduce the risk of virus transmission by generating early warnings, which is among the best-known interventions considering the fact that impeccable vector control is not feasible. Although Pakistan is known to be at high risk for arboviral infections, with unswerving epidemics of dengue and CCHF each year, the disease prevention and control measures are still below par. The very recent CHIKV outbreak, initially perceived as a “mysterious” pathogen , is a te


Proteasomal protein degradation exists in mycobacteria and other actinobacteria, and expands their repertoire of compartmentalizing protein degradation pathways beyond the usual bacterial types. A product of horizontal gene transfer, bacterial proteasomes have evolved to support the organism's survival under challenging environmental conditions like nutrient starvation and physical or chemical stresses. Like the eukaryotic 20S proteasome, the bacterial core particle is gated and must associate with a regulator complex to form a fully active protease capable of recruiting and internalizing substrate proteins. By association with diverse regulator complexes that employ different recruitment strategies, the bacterial 20S core particle is able to act in different cellular degradation pathways. In association with the mycobacterial proteasomal ATPase Mpa, the proteasome degrades substrates post-translationally modified with prokaryotic, ubiquitin-like protein Pup in a process called pupylation. Upon interaction with the ATP-independent bacterial proteasome activator Bpa, poorly structured substrates are recruited for proteasomal degradation. A potential third degradation route might employ a Cdc48-like protein of actinobacteria (Cpa), for which interaction with the 20S core was recently demonstrated but no degradation substrates have been identified yet. The alternative interaction partners and wide range of substrate proteins suggest that the bacterial proteasome is a modular, functionally flexible and conditionally regulated degradation machine in bacteria that encounter rapidly changing and challenging conditions. Frankia  pathway, where an AAA+ protein called Cdc48  cooperates with the proteasome [reviewed in   (Unciuleac et al., in vitro, forming ring-stacking, collinear complexes (Ziemski et al., cpa deletion strain of M. smegmatis exhibits growth defects under carbon starvation and the levels of several hundred proteins are changed more than 3-fold (Ziemski et al., in vitro degradation of model substrates by Cpa in cooperation with the proteasome has also not been demonstrated. Further studies will be needed to establish a potential in vivo connection between Cpa and the 20S proteasome and to better understand the role of Cpa in mycobacteria.It has been demonstrated that Cpa can interact with the 20S core particle Actinobacteria have acquired a proteasome by horizontal gene transfer to expand their degradation repertoire beyond the canonical compartmentalizing proteases found in bacteria, like for example ClpXP and ClpCP proteases, or FtsH. Indeed, they have not only maintained the proteasome in their genome, but have evolved multiple substrate recruitment strategies converging on the proteasome as the unifying degradation module. The interaction with multiple regulators adds to the functional versatility of proteasomal degradation, and it offers the possibility of differential regulation of access for different substrate classes to proteasomal degradation.M. smegmatis under nitrogen starvation conditions (Elharar et al., One shared theme appears to be that these degradation pathways are important during stresses encountered by the bacteria, rendering them conditionally needed. Starvation for nutrients is a common occurrence in the natural environment of actinobacteria and requires adaptation of metabolic pathways as well as adjustment of translation activities under maintenance of minimal protein synthesis. Amino acid recycling, as was proposed for From the range of conditions and cellular contexts in which proteasomal degradation appears to be involved, it is obvious that after its transfer into bacteria, the proteasome has adopted an ever-growing list of roles over the course of evolution. It can be expected that many more remain to be uncovered in the future.AM and EW-B contributed to drafting, writing and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Medical imaging is a vital part of the clinical decision-making process, especially in an oncological setting. Radiology has experienced a great wave of change, and the advent of quantitative imaging has provided a unique opportunity to analyse patient images objectively. Leveraging radiomics and deep learning, there is increased potential for synergy between physicians and computer networks—via computer-aided diagnosis (CAD), computer-aided prediction of response (CARP), and computer-aided biological profiling (CABP). The ongoing digitalization of other specialties further opens the door for even greater multidisciplinary integration. We envision the development of an integrated system composed of an aggregation of sub-systems interoperating with the aim of achieving an overarching functionality . This will require close multidisciplinary cooperation among the clinicians, biomedical scientists, and (bio)engineers as well as an administrative framework where the departments will operate not in isolation but in successful harmony.The advent of quantitative imaging provides a unique opportunity to analyse patient images objectively.• Radiomics and deep learning allow for a more detailed overview of the tumour  from many different perspectives.• As it currently stands, different medical disciplines have developed different stratification methods, primarily based on their own field—often to the exclusion of other departments.• The digitalization of other specialties further opens the door for multidisciplinary integration.• The long-term vision for precision medicine should focus on the development of integration strategies, wherein data derived from the patients themselves (via multiple disciplines) can be used to guide clinical decisions.•  Medical imaging has historically played a key role in cancer screening, diagnosis, staging, and therapeutic response monitoring. On a daily basis, treating physicians rely on input from imaging to help formulate patient management plans . This isHistorically, qualitative semantic features were used to describe tumour morphology—as observed in the patient image. These descriptions were a reflection of a scoring system based on visual assessment. Semantic features were shown in the literature to have correlations with stage, prognosis, and even response prediction . HoweverUbiquitous modern imaging modalities, such as CT, MRI, and PET in radiology , are primarily quantitative in nature. This characteristic is harnessed, using computational algorithms to extract quantitative features and generate mineable data. Rather than relying solely on subjective interpretation of images, these quantitative features can be used to objectively characterize tumour morphology.In radiomics, medical images are processed to generate quantitative features and these mineable data can then be used for clinical purposes. Radiomic features serve the purpose of describing morphological characteristics  in an objective, quantitative manner. The ambition is to find completely non-invasive radiomic features that can be used as predictive and prognostic biomarkers.The advent of radiomics has opened a brand new avenue in cancer research and presents a unique opportunity to data scientists and radiologists alike. Broadly speaking, two prominent potentials have emerged for radiomics—tumour characterization and therapeutic response prediction Fig. .Fig. 1A genetic mutations or surface expression of particular molecules) in the tumour. Normally, such biological assessment of a tumour is achieved by biopsy—a process that is highly invasive, carries potential risk for patient morbidity, and can only elucidate information for lesions in sites easily accessible to surgeons. Radiomics provides the opportunity to non-invasively assess the biological profile  of all the lesions simultaneously and instantaneously. With the increased use of computer models to diagnose conditions and predict response to therapy, this new field where biological parameters can non-invasively be assessed using quantitative features and computer models can be termed computer-aided biological profiling (CABP).The search has begun to identify imaging markers to be used to assess biological parameters  was the work of Segal et al. in human liver cancer where combinations of 28 imaging traits were shown to be capable of reconstructing 78% of the global gene expression profile (i.e. mRNA levels) of these tumours . FurtherWith the rise of deep-learning-based image analysis, computer algorithms can be used to extract radiomic features on a large scale that could then be linked to predictive and prognostic biomarkers in cancer .Unlike more traditional radiomics approaches where feature extraction and data analysis consisted of two separate steps, deep learning fuses these processes together and iteratively optimizes one with respect to the other. In other words, deep learning provides radiomics models with optimal features and optimal data analysis for a specific clinical problem. This advanced form of computer-aided biological profiling  can be termed deep learning mediated tumour profiling (DL-TP).The next application of radiomic features in cancer research was prediction of response to different forms of treatment [i.e. computer-aided response prediction (CARP)]. In non-small-cell lung cancer (NSCLC), Coroller et al. identified seven features that were predictive for pathological gross residual disease and one feature for pathological complete response . FurtherWhile the suffix “-omics” has come to denote the idea of extracting valuable information from data sets, radiomics is only the latest addition to the ever-growing list of new fields of study within the fusion of advanced technology and modern medicine. Images derived from tissue  have also been subject to quantitative analysis and new information is being generated beyond what would be observed by a pathologist, i.e. pathomics. Genomics, the branch of molecular biology concerned with the mapping of the human genome, has helped to identify many genetic mutations and pathways that have been used for prognostication or as novel targets for modern therapeutics and has heavily relied on computer models developed by skilled bioinformaticians.As it currently stands, different medical disciplines have developed different stratification methods, primarily based on their own field —quite often to the exclusion of other departments. As these traditional scoring systems were often based on subjective interpretations of analogue readouts, combining these disparate outputs is quite challenging. The rise of the quantitative aspects of various medical disciples (i.e. the “-omics”) presents a remarkably unique opportunity wherein information from different diagnostic modalities can be objectively integrated.The long-term vision for precision medicine should focus on the development of integration strategies, wherein data derived from the patients themselves can be used to guide the treating physician. Through intricate analyses that integrate clinical data, blood markers, pathomics, radiomics, and genomics, we envision that a patient can be provisionally diagnosed (via computer-aided diagnosis), stratified into a molecular subtype of their tumour , and have a recommended treatment formulated (via computer-aided response prediction). This aggregation of sub-systems cooperating with the aim of achieving an overarching functionality  is termed the integration system see Fig. . This wi


Transcranial electrical stimulation (TES) uses constant (TDCS) or alternating currents (TACS) to modulate brain activity. Most TES studies apply low-intensity currents through scalp electrodes (≤2 mA) using bipolar electrode arrangements, producing weak electrical fields in the brain (<1 V/m). Low-intensity TES has been employed in humans to induce changes in task performance during or after stimulation. In analogy to focal transcranial magnetic stimulation, TES-induced behavioral effects have often been taken as evidence for a causal involvement of the brain region underlying one of the two stimulation electrodes, often referred to as the active electrode. Here, we critically review the utility of bipolar low-intensity TES to localize human brain function. We summarize physiological substrates that constitute peripheral targets for TES and may mediate subliminal or overtly perceived peripheral stimulation during TES. We argue that peripheral co-stimulation may contribute to the behavioral effects of TES and should be controlled for by “sham” TES. We discuss biophysical properties of TES, which need to be considered, if one wishes to make realistic assumptions about which brain regions were preferentially targeted by TES. Using results from electric field calculations, we evaluate the validity of different strategies that have been used for selective spatial targeting. Finally, we comment on the challenge of adjusting the dose of TES considering dose–response relationships between the weak tissue currents and the physiological effects in targeted cortical areas. These considerations call for caution when attributing behavioral effects during or after low-intensity TES studies to a specific brain region and may facilitate the selection of best practices for future TES studies. In recent years, multi-electrode arrangements have been introduced in TES experiments to achieve more focal stimulation and supplement the prevailing use of bipolar montages  applies weak electric currents to non-invasively stimulate the human brain. TES uses either constant, oscillating, or randomly alternating currents to interact with membrane potentials. The application of constant currents is called transcranial direct current stimulation (TDCS) , the appmontages . The useThe neurobiological effects of TES depend on the magnitude of the electrical field, its direction with respect to the stimulated neural target structure, and the conductivity of the stimulated tissue. In contrast to transcranial magnetic stimulation (TMS), the electric fields induced by TES are too weak to sufficiently depolarize cortical neurons to trigger action potentials: Invasive recordings and electric field modeling have provided converging evidence that electrical fields induced by TES do not exceed 1 V/m in the brain . At thesThe mechanisms by which TDCS, TACS, and TRNS interact with intrinsic brain activity are still incompletely understood , but TESThe effects of TES on behavior have prompted scientists to make inferences about the functional localization of brain functions. In analogy to focal TMS, TES-induced behavioral effects have often been taken as evidence for a causal involvement of the brain region underlying one of the TES electrodes. This line of reasoning has been supported by a seminal paper by The notion that the cortex underlying one of the two electrodes corresponds to the main site of TES action on brain networks is too simplistic and incorrect in most instances. Simulation studies that model the electric field distributions in the brain have provided converging evidence that the properties  and location of both electrodes, along with the geometry and conductivity of the tissue compartments determine which brain areas are preferentially targeted . The comIn this review, we discuss the utility of bipolar low-intensity TES to localize human brain function. In the first part, we summarize physiological substrates that constitute peripheral targets for TES and may mediate subliminal or overtly perceived peripheral stimulation. We make the point that peripheral co-stimulation may contribute to the behavioral effects of TES and discuss how well they are controlled for by “sham” stimulation. In the second section, we discuss biophysical properties of TES, which need to be considered if one wishes to make realistic assumptions about which brain regions were actually targeted by TES. Using results from electric field calculations, we evaluate the validity of different strategies that have been used for selective spatial targeting. In the third part, we address the challenge of adjusting the dose and summarize current knowledge about the dose–response relationship of physiological effects in targeted cortical areas.The sensory side effects of TES include itching, tingling, and burning sensations under the electrode. Depending on the electrode montages, vertigo and visual phenomena such as phosphenes are also common . These nDue to the relatively quick shifts in current at the beginning of the experiment, perceptual side effects are primarily reported during the start of TDCS. An attempt of matching these side effects of non-cortical co-stimulation is made by almost every study. Minimizing the electrode-skin impedance  and usinvia peripheral input. Depending on the montage, cutaneous receptors, cranial nerves as well as other sensory organs such as the retina or the vestibular organ may be stimulated. Even when remaining below the perceptual threshold, the peripheral stimulation may modulate the input the brain receives via these routes. There is little research on the effects of concomitant nerve stimulation, but one current study found that anodal TDCS over the primary motor cortex (montage cathode/anode: Fp3/C3) modulated the trigeminal-facial reflex circuit  determines how much TES spreads into the brain; the larger the distance between the two electrodes, the larger the brain volume that is targeted by TES . Second,The statement that the distance between “active” and “return” electrode influences the spatial extent of stimulated structures sounds trivial but is often neglected. It has been argued that the use of large return electrodes “dilutes” the currents at the “return” site and thereby focalizes stimulation under the active electrode . Other avia stimulated sensory organs, and direct stimulation of remote brain regions arising from non-focal currents. Knowledge about the brain’s connectome and about the electric field distributions in the brain might help to resolve this ambiguity.The use of brain mapping studies is crucial when trying to capture how much the effects of TES spread across the brain. However, the demonstration of remote effects does not imply that TES causes non-focal brain stimulation. Remote TES effects can be caused through projections from the primary cortical target site to remote brain regions . A spreaA recent study by The interpretation of the current direction is also more complex than generally assumed. Most studies presume that cortical structures close to the anodal electrode are subjected to homogeneous anodal (inward-flowing) current while structures close to the cathode receive a homogeneous cathodal (outward-flowing) current. This assumption is based on studies in species with a smooth cortical surface . HoweverCan bipolar TES be used to link cognitive performance to specific neural structures? To address this question, we modeled the TES-induced electric fields for some recent papers to link cognitive performance to specific neural structures. Electric field modeling based on numerical methods and realistic head models is increasingly used by the community for planning and analysis of TES and TMS interventions and offers an invaluable tool for understanding current distribution in the brain during TES. Physical validation of electric field models and demonstration of their predictive power for the physiological stimulation effects is paramount, and in the recent years, a growing number of studies have compared the model-predicted electric fields of TMS and TES with physical measurements and physiological markers . They haWe have chosen the studies where we modeled the TES-induced electric fields as representative examples to illustrate how computational models can be used to aid the interpretation of behavioral or physiological TES effects. In a study from 2015, Many montages also use a bi-hemispheric montage in which anode and cathode are placed over corresponding cortical targets in each hemisphere. To illustrate such a montage, we have modeled a study that stimulated the bilateral dorsolateral prefrontal cortex (DLPFC) to show that the anodal right/cathodal left montage modulated the response to fearful faces . Our modIn a final example, we illustrate the impact of the cortical folding on the direction of the stimulation effect, using a study of A special case that further complicates the unambiguous demonstration of structure–function relationships is multi-site stimulation by TES. Especially, TACS has been used for targeting two areas to manipulate the temporal phase relationship between the local oscillations in those areas . Howeverex vivo study indicated that the skull and soft tissue surrounding the brain shunt around 60–75% of the injected currents away from the brain  further indicate that simply increasing the intensity of stimulation may not necessarily improve the efficacy of TES  from Elsevier Publishers, Amsterdam, the Netherlands, royalties as book editor from Springer Publishers, Stuttgart, and a research fund from Biogen Idec, Denmark.The remaining 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.


Driving cessation has been shown to be a potentially isolating transition in life, with important implications for mental health, social isolation, and social support. Older adults who live alone are vulnerable to social isolation in the context of driving cessation. Although some research has examined the association between driving cessation and certain kinds of social engagement activities, no research has specifically examined changes in social support, particularly among older adults most vulnerable to social isolation – those who live alone . The present study addresses this gap, using data drawn from the 2006-2014 waves of the Health and Retirement Study (HRS) to examine how social support changes in the context of driving cessation among older adults who live alone (N=412). This study specifically focuses on instrumental and emotional social support, and how different sources of the support  are influenced by loss of driving. I use a series of ordinary least squares regression (OLS) to examine four-year changes in various forms of social support among those who live alone, comparing those who lose the ability to drive relative to their continuously driving counterparts. Preliminary results indicate that driving cessation is associated with decline in perceived instrumental support of friends  for older adults who live alone. However, these effects did not extend to children or other family members. These results suggest that loss of driving may perpetuate vulnerabilities facing individuals who live alone by leading to lower levels of perceived support from non-family members.