Humans have an exceptional ability to cooperate relative to many other species. We review the neural mechanisms supporting human cooperation, focusing on the prefrontal cortex. One key feature of human social life is the prevalence of cooperative norms that guide social behavior and prescribe punishment for noncompliance. Taking a comparative approach, we consider shared and unique aspects of cooperative behaviors in humans relative to nonhuman primates, as well as divergences in brain structure that might support uniquely human aspects of cooperation. We highlight a medial prefrontal network common to nonhuman primates and humans supporting a foundational process in cooperative decision-making: valuing outcomes for oneself and others. This medial prefrontal network interacts with lateral prefrontal areas that are thought to represent cooperative norms and modulate value representations to guide behavior appropriate to the local social context. Finally, we propose that more recently evolved anterior regions of prefrontal cortex play a role in arbitrating between cooperative norms across social contexts, and suggest how future research might fruitfully examine the neural basis of norm arbitration. Relative to other species, humans have an exceptional ability to cooperate—we are willing to incur personal costs to benefit others, including strangers, and people who we will never meet again –7 shared inractions .Fig. 2ShThere are several possibilities as to why humans came to exhibit this significant departure from the capacity of nonhuman primates. Firstly, it is likely to be an evolutionary adaptation to better cope with the cognitive demands from expanding social group size and complexity . The demEvidence for shared intentionality comes from studies comparing cooperation in human children and nonhuman primates. For example, when presented with a choice between working alone and collaborating together to obtain food, from age 2 to 3, children preferred to collaborate, while chimpanzees preferred to work alone . When insocial norms: “commonly known standards of behavior that are based on widely shared views about how individual group members ought to behave in a given situation” [cooperative norms, which we define as social norms that facilitate cooperative interactions: i.e., norms that motivate individuals to cooperate when it is not in their economic self-interest. [Shared intentionality enables humans to conceptualize tuation” . Researctuation” . Here, wnterest. , 86, 87One common cooperative norm is a norm of fairness, i.e., that windfall resources should be distributed equally among group members . FairnesWhen a cooperative norm is violated, humans will incur costs to enforce the norm by punishing the transgressor . People Cooperation based on kinship and reciprocity is common to nonhuman primates and humans. However, humans are substantially more advanced in their ability to regulate individual desires towards the achievement of collective goals. Moreover, sophisticated metacognitive abilities of monitoring our own cognitive processes as well as others through mentalizing enables us to establish common understanding of joint goals and intentions. The ensuing shared intentionality, in turn, lays the foundation for uniquely human abilities to represent cooperative norms, comply with those norms oneself, and enforce norm compliance in others. These sets of advanced abilities in humans, building on one another and working in concert, may enable humans to conceptualize informal, temporally, and socially extended social contracts that play an important role in guiding cooperative behavior.When we make decisions, we must represent the set of available choice options, assign subjective values to the expected outcomes resulting from these options, select the most valuable option, evaluate the outcome, and update subjective value representations through learning . For cooFor instance, imagine you are sitting in a park at lunchtime. Your stomach rumbles and so you begin unwrapping a sandwich you’ve just purchased at a local café. Before you can take a bite, a homeless person approaches and asks if you could spare some money for a meal. You have no cash on you. Do you offer them your sandwich?Decisions like this engage a variety of cognitive processes. Your overall decision will depend not just on how much you value your own and others’ outcomes for their own sake, but also the relative valuation of those outcomes and the valuation of normative principles like generosity more broadly . So you Rewarding and aversive outcomes for oneself are strong motivators of decision-making. Converging evidence in nonhuman primates and humans demonstrates that motivationally salient outcomes are encoded in a valuation circuitry encompassing multiple cortical and subcortical brain regions including ventromedial prefrontal cortex (vmPFC), orbitofrontal cortex (OFC), different subregions of anterior cingulate cortex (ACC), ventral striatum, and amygdala . Thvicarious outcomes—i.e., motivationally salient outcomes for others? Are others’ rewards and punishments encoded in similar or distinct neural circuits to one’s own rewards and punishments? A growing body of work implicates the medial prefrontal cortex, especially the ACC gyrus, in vicarious outcome processing [Cooperative decisions are guided not just by outcomes for oneself, but also outcomes for others. How does the brain represent ocessing . Initialocessing –118. Forocessing . A meta-ocessing . Vicarioocessing  and predocessing –123.Similar evidence for vicarious neural representations in the mPFC/ACC can be found in the aversive domain. Pain delivered to both oneself and others evokes activity in the mid-ACC and anterior insula (AI), regions implicated in the affective component of pain , 125. MuAlongside studies suggesting overlapping neural representations of outcomes for oneself and others  there isrelative values of one’s own outcomes with respect to others and vice-versa. In one study, pairs of participants took part in a random drawing where one received $50 and the other received nothing. Following this, each was scanned while observing monetary transfers to self and other. For the participant who received nothing, vmPFC and ventral striatum responded more strongly to rewards for self than other. High-pay participants, meanwhile, showed the reverse pattern, with stronger responses to rewards for the disadvantaged partner than rewards for self [Aside from representing the value of outcomes for oneself and others individually, medial prefrontal areas are also sensitive to the for self . Similarfor self , 140). Sfor self .joint values that arise from cooperative interactions where the whole is greater than the sum of its parts. Human fMRI studies of social exchange games like the prisoner’s dilemma show increased activity in vmPFC for cooperative relative to selfish decisions [In addition, there is evidence that medial prefrontal regions encode the ecisions –143, andecisions . Consistecisions . In the Social decision-making requires not only representing the value of decision outcomes for self and others, but also integrating those values into an “all-things-considered” subjective value, or “relative chosen value” corresponding to the value of the chosen course of action relative to the alternatives. The vmPFC and dmPFC , respectively, are suggested to positively and negatively encode relative chosen value, both for decisions that impact only oneself , 146–148Making decisions on behalf of others may be particularly difficult if the other person is a stranger or if their preferences are not well understood. To overcome this uncertainty, people may engage in mentalizing processes that activate a network encompassing superior temporal sulcus, TPJ, medial precuneus, and dmPFC –158. MenRepresenting outcomes for oneself and others is an important first step during cooperative decision-making. Converging evidence from studies of humans and nonhuman primates demonstrates a role for medial prefrontal regions, including dorsal and ventral ACC, in encoding the value of outcomes for self and others. Medial prefrontal regions encode vicarious rewards and punishments, and individual differences in these responses predict individual differences in cooperation. These neurons are sensitive to relative and joint values in social contexts, which may explain cooperative behavior in both nonhuman primates and humans. During social decision-making, subjective values are computed in vmPFC and dmPFC, and in humans, there is evidence that these domain-general valuation regions receive input from other areas that represent social information . Although both humans and primates represent outcomes for self and others in medial prefrontal regions, humans go considerably further in incorporating social cognitive information into integrative values of action through actively engaging in mentalizing. In addition, human cooperative decision-making cannot be understood simply in terms of these basic outcome representations. In the next section, we’ll examine how the subjective value of cooperative decisions depends not just on relative and joint valuation of social outcomes, but also on shared beliefs about what is normatively right or wrong.meaning of their actions and resulting outcomes in the context of local cooperative norms (see Glossary). As we suggested in Section 1, the ability to represent cooperative norms and use those norms to guide one’s own and others’ behavior may be unique to humans. In this section, we review work on the neural mechanisms of norm compliance and enforcement. As cooperative norms are rules that guide cooperative interactions, we begin by considering work in both nonhuman primates and humans implicating lateral PFC  in representing rules. Next, we turn to work on norm compliance and enforcement in humans, highlighting the role of lPFC and its interactions with the brain’s valuation circuitry [When people make cooperative decisions, they consider not just the possible outcomes for themselves and others, but also the ircuitry , 162. ThThe ability to represent and use rules to guide appropriate actions is a core aspect of goal-directed behavior –166. SinRecent evidence shows that human lPFC computes higher-order goals based on the associations between rules and expected outcomes to ultimately guide action selection in the striatum by modulating choice-related value signals , 170. MoSuch evidence indicates that both human and nonhuman primate lPFC is centrally involved in rule representation and flexible rule-guided behavior. It is possible that humans’ exceptional ability to comply with and enforce cooperative norms may be realized by some aspects of the lPFC function that diverged between humans and nonhuman primates through evolutionary elaboration. Next, we discuss how lateral prefrontal mechanisms, in parallel with its domain general functions, contribute to norm compliance and enforcement. We note that there are no published studies of the neural basis of norm compliance and enforcement in nonhuman primates; therefore, the remainder of this section will focus on research in humans.In line with its domain-general support for rule-guided behavior, lPFC is reliably engaged in neuroimaging studies where human participants decide whether to comply with cooperative norms. For example, in settings where fairness norms are highly salient, several studies reported increased right dlPFC activity when participants make decisions to fairly distribute money with interaction partners , 178. DiOther work has implicated lPFC in complying with a norm of honesty, using tasks where participants are tempted to earn more money by cheating or lying. Right dlPFC and vlPFC are more active when participants respond honestly in these tasks –188. EnhSimilarly, dlPFC is more active when participants uphold a norm of conditional cooperation, i.e., reciprocating others’ trusting or cooperative decisions –193. RelInitial theorizing on the role of lPFC in cooperative norm compliance proposed that this region implements a top-down inhibition of prepotent selfish impulses , drawingLPFC modulation of subjective value is hypothesized to operate via functional interactions with valuation circuitry including vmPFC and subcortical areas –205. SevWhat motivates people to comply with cooperative norms when no one is watching? Normative and descriptive theories have highlighted a role for moral emotions like guilt in guiding compliant behavior even in the absence of external punishments. Studies using formal models of guilt aversion reveal activation in dlPFC during guilt-averse decisions , 191. OnPrevious theorizing has suggested norm compliance and enforcement might rely on common psychological and neural mechanisms , 211–213Norm enforcement via third-party punishment shares neural substrates with second-party punishment , 219. StDuring third-party punishment, dlPFC is hypothesized to integrate multiple streams of information, including the amount of harm and the intentions of the transgressor . SupportCooperative norms play a central role in guiding the large-scale cooperative interactions that characterize human social life. Humans are willing to incur considerable personal costs to comply with cooperative norms and enforce those norms in others. Converging evidence implicates the lateral PFC, most commonly dlPFC, in representing cooperative norms and integrating those representations with other streams of information to guide normative behavior. Functional interactions between lateral and medial prefrontal regions suggest that the former modulates value representations in the latter in a goal-directed manner, consistent with other work highlighting a domain-general role for dlPFC in rule representation that is common to both humans and nonhuman primates.Certain cooperative norms, such as a prohibition against physically harming an innocent person for pure personal gain, apply widely across a vast range of social and cultural settings . Howeverexchange norm, where benefits are provided with the expectation of receiving a comparable benefit or payment in return, while the friend’s dinner is governed by a communal norm, where benefits are given without any expectation of compensation [Take, for example, a set of cooperative norms around dining in a Western cultural setting. At a restaurant, a diner is expected to pay for their meal . However, offering to pay for a home-cooked meal at a friend’s house would be seen as socially awkward or outright rude. This discrepancy can be explained by the fact that different norms operate in these different settings: the restaurant meal is governed by an (economic) ensation . Oftentinorm arbitration might draw on similar neural processes.To address this question, we build on studies of how decision-makers reflect on their own choices and arbitrate between different decision strategies. This work suggests that the anterior PFC, including anterior lPFC as well as frontopolar cortex , plays a key role in metacognition, counterfactual processing, and arbitrating between valuation systems. We suggest that Notably, FPC is unique to anthropoid primates , and is Successful cooperation requires an ability to reflect on one’s own thought processes and behaviors. Numerous studies have implicated a frontoparietal network that includes FPC and anterior lPFC  in metacognition (for reviews see –238). AsIn humans, FPC is dramatically expanded compared with nonhuman primates , which mOne important aspect of evaluating the quality of one’s decisions is keeping track of counterfactual outcomes: is the grass greener on the other side of the fence? Multiple studies have shown that FPC prospectively tracks counterfactual evidence, including the reward value of unchosen options  and counThere is also evidence that FPC prospectively tracks internal variables that bear on the future success of decisions. During perceptual decision-making, people prospectively estimate an internal probability of making a correct choice, and these estimations are tracked in mFPC and alPFC . A similTogether these studies suggest that FPC, in particular its more lateral aspects that underwent a dramatic expansion over the course of human evolution, plays a fundamental role in enabling people to evaluate the quality of their decisions both prospectively and retrospectively—an important aspect of adjusting cooperative behavior across social contexts. However, it is important to note that the neural evidence of metacognition is not limited to the FPC. In fact, multiple regions in the FPC network, encompassing ACC subregions and mPFC subareas, will likely synergistically contribute to the metacognitive process. For instance, confidence-related signals have been observed in the perigenual ACC , 253 andResearch on the neurocomputational mechanisms of value-based learning and decision-making reveals that there are multiple valuation systems in the brain that employ different algorithms for learning the expected value of actions and outcomes. In particular, an important distinction has been made between ‘goal-directed’ and ‘habitual’ systems, which are proposed to rely on model-based and model-free reinforcement learning algorithms, respectively , 255. ThRecent work has examined the neurocomputational mechanisms supporting arbitration between model-based and model-free control over decision-making. An early computational account suggested an uncertainty-based arbitration, whereby control is exerted by the system with the lowest uncertainty in its value predictions . BuildinEvidence for the mixture of experts model comes from fMRI studies of human subjects in learning environments where different learning strategies are variably successful over time. In a study of arbitration between model-based and model-free systems, reliabilities for both systems were encoded in vlPFC and FPC . EnhanciWe suggest that a process similar to the mixture of experts model is likely to guide arbitration between different cooperative norms in guiding context-appropriate social behavior. That is, norm arbitration might involve allocating control over behavior by weighting the reliabilities of value predictions generated by different cooperative norms. We propose that anterior PFC computes the reliabilities of the expected values of different behavioral policies prescribed by any cooperative norms being considered. These predictions may be generated based on the presence of contextual cues that indicate whether a particular norm is relevant for the current context. The posterior over the behavioral policies can therefore be obtained as a weighted sum over the predicted values of cues indicating different norms, producing graded levels of confidence over the chosen policy.Consider the example that opened this section: how do you decide whether to pay for your meal when dining? There are two potentially relevant cooperative norms that make opposite behavior policy recommendations: an exchange norm that dictates you should pay for your meal, and a communal norm that suggests you should not. When you are dining solo at a restaurant Fig. , the excEmpirical evidence for this hypothesis is so far scarce. One possible explanation for this missing evidence is that most studies of social decision-making in humans consider only a single social context or norm, applied over a very brief timescale. For instance, neuroeconomic paradigms typically consider how individuals implement fairness norms in one-shot interactions. Such paradigms generally do not require participants to arbitrate between behavioral policies, but instead to consider implementing a single policy (such as a norm of fairness or reciprocity).However, preliminary support for norm arbitration in FPC comes from neuroimaging studies of social decision-making that require participants to consider multiple decision contexts and strategies. One early study examined the neural basis of compliance with a fairness norm in two distinctive contexts: one where punishment was possible, and another where it was not. The contrast between the two conditions revealed increased activation in alPFC and vlPFC as well as dlPFC . AnotherFinally, the anterior PFC undergoes a protracted period of development, rapidly increasing its volume and dendritic complexity throughout late childhood and adolescence –269  to apply in a particular social context. Preliminary evidence for this hypothesis comes from neuroimaging studies of adaptive social decision-making, which show that the anterior PFC (including anterior vlPFC and lFPC) encodes variables that are necessary for maximizing rewards across diverse social contexts. We further hypothesize that there is a possible anterior-posterior gradient of norm processing such that norm arbitration is carried out by more anterior aspects of the PFC, whereas norm representation is mediated by more posterior aspects of the PFC. Future work can extend these findings by probing the engagement of anterior PFC in tasks where participants must use cooperative norms to guide decisions across different cultural or relational contexts.The prefrontal cortex, in particular its more anterior regions, has expanded dramatically over the course of human evolution. In tandem, the scale and scope of human cooperation has dramatically outpaced its counterparts in nonhuman primate species, manifesting as complex systems of moral codes that guide normative behaviors even in the absence of punishment or repeated interactions. Here, we provided a selective review of the neural basis of human cooperation, taking a comparative approach to identify the brain systems and social behaviors that are thought to be unique to humans. Humans and nonhuman primates alike cooperate on the basis of kinship and reciprocity, but humans are unique in their abilities to represent shared goals and self-regulate to comply with and enforce cooperative norms on a broad scale. We highlight three prefrontal networks that contribute to cooperative behavior in humans: a medial prefrontal network, common to humans and nonhuman primates, that values outcomes for self and others; a lateral prefrontal network that guides cooperative goal pursuit by modulating value representations in the context of local norms; and an anterior prefrontal network that we propose serves uniquely human abilities to reflect on one’s own behavior, commit to shared social contracts, and arbitrate between cooperative norms across diverse social contexts. We suggest future avenues for investigating cooperative norm arbitration and how it is implemented in prefrontal networks.


Despite the continuous, growing need for health professionals who are trained to work with older adults living with Alzheimer’s disease and related disorders (ADRD), research shows that recruitment and retention of gerontological health care professionals remains low. Ageism plays an important role in this resistance and continues to have societal impact, even proliferating in disturbing ways during the COVID-19 pandemic via stereotypes, discrimination, and framing in the media. Gerontologists in various health professional educational settings continue to address the need to infuse aging content in creative ways and increase the competency of all health professionals to combat ageism and understand the importance of specialized care for this population. Our gerontological research team has engaged students in various ways to increase interest in aging issues and ADRD. Current research projects involve the implementation of non-pharmacological, creative arts interventions, including Mason’s Music & Memory Initiative (M3I) and TimeSlips, both which have the potential to appeal to intergenerational partnerships and provide students with tools to communicate better with those living with ADRD. We examined attitudes about aging among undergraduate and graduate students (N=78) who have completed our training modules and/or participated in these two projects. The asynchronous trainings provide content on ADRD and the implementation of non-pharmacological, creative arts interventions. We examined students’ attitudes about aging and ADRD and analyzed their open-ended responses regarding their experiences with someone living with ADRD. Various levels of education, relationships with older adults, and life experience influenced their responses regarding their attitudes about aging.


Primary care and frontline healthcare providers are often the first point of contact for patients experiencing tick-borne disease (TBD) but face challenges when recognizing and diagnosing these diseases. The specific aim of this study was to gain a qualitative understanding of frontline and primary care providers’ knowledge and practices for identifying TBDs in patients.From fall 2018 to spring 2019, three focus groups were conducted with primary care providers practicing in a small-town community endemic to Lyme disease (LD) and with emerging incidence of additional TBDs. A follow up online survey was distributed to urgent and emergency care providers in the small-town community and an academic medical center within the referral network of the local clinical community in spring and summer 2019. Qualitative analysis of focus group data was performed following a grounded theory approach and survey responses were analyzed through the calculation of descriptive statistics.Fourteen clinicians from three primary care practices participated in focus groups, and 24 urgent and emergency care clinicians completed the survey questionnaire. Four overarching themes emerged from focus group data which were corroborated by survey data. Themes highlighted a moderate level of awareness on diagnosis and treatment of LD among participants and limited knowledge of diagnosis and treatment for two other regionally relevant TBDs, anaplasmosis and babesiosis. Providers described challenges and frustrations in counseling patients with strong preconceptions of LD diagnosis and treatment in the context of chronic infection. Providers desired additional point-of-care resources to facilitate patient education and correct misinformation on the diagnosis and treatment of TBDs.Through this small study, it appears that clinicians in the small-town and academic medical center settings are experiencing uncertainties related to TBD recognition, diagnosis, and patient communication. These findings can inform the development of point-of-care resources to aid in patient-provider communication regarding TBDs and inform the development of continuing medical education programs for frontline and primary care providers.The online version contains supplementary material available at 10.1186/s12879-021-06622-6. Tick-borne diseases (TBDs) are a group of infectious diseases caused by a variety of pathogens transmitted through the bite of an infected tick. The Centers for Disease Control and Prevention (CDC) have identified eight species of hard tick responsible for transmitting multiple bacterial, viral, and protozoan pathogens detrimental to human health in the United States , 2. In tPrimary care and urgent care clinicians are often the first point of contact for patients suffering from TBDs , 4. HoweThe Northeast Regional Center for Excellence in Vector-Borne Diseases (NEVBD) fosters research and education on diseases spread by mosquitoes and ticks in the Northeastern United States. NEVBD collaborated with a local health department, community hospital system, and the Cornell Master of Public Health program, to directly engage with primary care and urgent care clinicians on TBD education in an area endemic to Lyme and other TBDs. The purpose of this effort was to gain a qualitative understanding of the knowledge and experiences of frontline providers in diagnosing and treating TBDs to inform the development of training and resources for this community. Current evidence on provider knowledge and practice regarding the diagnosis and treatment of TBDs has been gained through survey research, and medical chart and billing reviews –10. WhilThe initial target study population included primary care and emergency and urgent (frontline) healthcare providers practicing in a small-town community endemic to Lyme disease with emerging incidence of additional TBDs. Invitation emails for focus group participation were sent to practice managers and medical directors for medical practices employing members of the study target population. Researchers coordinated with these key contacts within the practices to schedule in-person focus groups at a date and time convenient to the participating providers. Focus groups were scheduled in late fall 2018 and early spring 2019. Participants included both prescribing  and non-prescribing  providers.Academic project team members developed guided focus group questions covering current knowledge, perceived challenges, and needed resources on tick-borne illness, which were reviewed by physician and health department team members prior to finalization  developed a survey questionnaire using validated questions from the published literature , 12, 13 The project team contacted colleagues at an academic medical center within the referral network of the local clinical community for expanded distribution of the questionnaire to this hard-to-reach population. The survey questionnaire was moderately modified to adapt to the academic medical center clinical community by reducing rating scale complexity, informed by additional beta testing by an infectious disease physician at the academic medical center. This version of the questionnaire was distributed through the clinical network of the academic medical center via email and was open for responses from July 1 to August 31, 2019. Due to the limited sample size generated, analysis of survey responses consisted of calculation of descriptive statistics using Microsoft Excel .Protocols and procedures for this study involving human subjects were deemed exempt for review under criterion three by the Institutional Review Board of Cornell University, Protocol Numbers: 1806008097 and 1903008648.Three primary care practices scheduled focus groups with the study team. These practices represented internal medicine, family medicine, and pediatric medicine. Participants across the three focus groups included eight physicians (MD/DO), five nurse practitioners, and one licensed practical nurse . Six clinicians completed the survey questionnaire distributed within the small-town community, including three physicians, two physician assistants, and one nurse practitioner. Eighteen clinicians completed the survey questionnaire distributed to the academic medical center, including 14 physicians, two physician assistants, and two nurse practitioners. Table Four overarching themes emerged from analysis of the focus group transcripts: difficulty in diagnosis; challenges presented by patients and combating misinformation; resources and support to improve diagnosis and treatment; and continuing education and clinical proficiency. Table Clinicians described multiple challenges in the diagnostic process for TBDs. Clinicians from each of the three participating practices felt that differential diagnoses for TBDs were complicated by the nonspecific symptoms associated with these diseases. Clinicians had a self-professed lack of awareness of TBDs outside of Lyme disease, noting that they were unfamiliar with the signs, symptoms, and appropriate serologic testing needed to diagnose non-Lyme TBDs, as well as the prevalence of these TBDs in their local communities.“The most annoying one, for me, is the Lyme…part of the differential, but not high on the list, and what do I do about it? So, I’ve got a fever and a little bit of a cough, and my aches, my joints hurt, um, and I live in [endemic area]...So that’s, that actually, it’s frustrating.” “That’s where I have the hardest time deciphering...when we know in the literature they keep telling us this is a clinical disease, well, here’s a clinical symptom. But, is this Lyme or is it not?” Focus group participants reported confidence in diagnosing Lyme disease and developing treatment plans when serologic test results and/or empiric assessment of the patient was conclusive, particularly when the erythema migrans (EM) lesion, or rash, was present. However, interpreting test results for Lyme disease was more difficult in scenarios where patients had nonspecific symptoms in the absence of the characteristic EM rash. At least one participant from each focus group described patients associating nonspecific symptoms, such as fatigue, with Lyme disease specifically and expressed uncertainty around interpreting serology results for Lyme disease. This juxtaposition of nonspecific symptoms and unclear diagnostic testing results proved challenging for clinicians making decisions on how to treat and diagnose their patients for Lyme disease."I'll have people come in for a follow up or from seeing like urgent care... and they've put them on one dose of amoxicillin. And I'm like... the literature doesn't show that amoxicillin can be used preventively, you know...Especially I think it's urgent care."Another common challenge cited by focus group participants centered on interactions with clinicians from different specialties who had alternative and, at times, conflicting practice patterns for diagnosing and treating TBDs. Focus group participants from the pediatric practice consistently described challenges when patients visited their office following an emergency or urgent care visit related to their TBD due to differences in the diagnostic approaches taken between providers in these two medical specialties. For example, the pediatric providers stated that their first approach to the assessment of swollen joints with no history of trauma is to conduct non-invasive diagnostic testing for Lyme disease. Conversely, these providers felt that patients presenting to the urgent care setting with the same symptoms often received more invasive joint aspiration. Additionally, providers from this practice felt that urgent care and emergency medicine clinicians often prescribed treatments that did not follow established guidelines for tick bite prophylaxis.“I think when people have bad data, that’s particularly hard because no matter what I say, they’re going to be like ‘You’re not Lyme literate. I have other sources.’” Focus group participants highlighted challenges in the patient-provider communication dynamic that stemmed from patient orientations toward Lyme disease diagnosis and treatment, competing care plans from outside providers, and the need to address misinformation on TBDs during clinical encounters. Clinicians described the process of redressing misinformation as difficult when patients were attached to specific beliefs and did not accept ‘new’ information discussed during the clinical encounter. In several instances, participants described patients referencing materials on Lyme disease that were counter to the guideline-based resources and evidence they personally rely upon in their medical practice. The patient-provider relationship was at times negatively affected during these interactions.“The toughest one I ever had was a young kid who came in with his parents, just for a general checkup, but they mentioned his past history of Lyme disease and how he was being treated at a clinic...for chronic Lyme disease with sequential courses of multiple intravenous antibiotics over and over and over again. And I was just sitting there biting my tongue the whole time. And his complaints were fatigue.[chuckle/sigh]” Patient education was more difficult when patients had existing relationships with outside healthcare providers delivering contradictory information. Two focus group discussions centered around differences in care plans between participants’ primary care offices and what they termed the ‘Lyme literate’ community. These participants noted a strong disagreement with the care delivered by these outside providers. In these scenarios of conflict, participants in the family medicine focus group identified infectious disease specialists as a resource of authority to whom they could refer patients. However, providers in the other two focus groups expressed a resigned view of the issue as something they could no longer effectively address within the clinical encounter without damaging the patient-provider dynamic.Discussion around the subject of chronic Lyme disease occurred within all three focus groups and coincided with sentiments of frustration. Clinicians described patients who self-diagnose or otherwise identify with the diagnosis of chronic Lyme disease as particularly challenging in both the disease diagnostic process as well as treatment. Clinical encounters with these patients were described as time consuming and difficult. Participants in all three focus groups described experiences where they provided clinical information contradictory to their patients’ pre-existing ideas regarding the biology of Lyme disease infection, resulting in those patients challenging the proposed treatment plans. This commonly came in the form of patients rejecting results of serologic testing, wanting a different length of antibiotic course than that prescribed, or seeking an alternative treatment modality.Clinicians participating in the focus groups largely felt that existing resources to aid with patient education did not meet their needs. Clinicians felt their ability to disseminate information to patients was influenced by patient education level, and there was sentiment among participants that resources for the lay audience on Lyme and other TBDs are limited. Clinicians also described wanting point-of-care resources for patients, either through flyers, pamphlets, or a reference list of online resources. Ideally, this educational material would support clinicians' recommendations, help to minimize patient doubt, and support trust in the patient-provider relationship. Participants in two focus groups specifically described wanting resources that directly address misinformation and myths around chronic Lyme disease using evidence-based information.When describing reference materials to inform their clinical practice, focus group participants emphasized that the resources need to be both available at the point-of-care and up to date. Participants in two focus groups described using UpToDate.com as their primary source for both obtaining information for their clinical practice and to give to patients. Participants in each focus group felt that access to infectious disease specialists for patient consults and recommendations was helpful for their practice and often referred patients to infectious disease specialists when diagnoses were unclear.“To have someone be able to answer questions online while you’re watching the presentation because often there are a lot of questions, which don’t have the time to get answered…A little bit more case studies...because that’s what a lot of the primary care doctors are facing.” Participants in all three focus groups felt that the predominant web-based format for CME is not accessible or conducive to their education. Specific suggestions on effective training included interactive instruction and review of case studies."I ordered what I thought was sort of the standard Lyme titer. It came back negative and this kid continued to have a swollen knee...He went to the orthopedist. They couldn't figure it out. He's going up to the rheumatologist. And it's just because I ordered the wrong test...I think [I] ordered the PCR, which was like, you know, the quick and easy one. But actually that wasn't probably a good one to do." Specific content areas to include in CME programs included resources and information on treatment modalities for unique populations, including pediatrics and individuals with antibiotic allergies; local epidemiological data and information on tick vectors; and updates on TBD basics including presentation, diagnostics, and treatment. Clinicians felt resources that included case studies and images of EM rashes would improve their ability to diagnose TBDs. Data from the focus group transcripts also identified an issue regarding diagnostic testing for TBDs related specifically to appropriate test selection in the electronic medical record (EMR). Clinicians expressed a lack of knowledge on differentiating test options in the EMR and their appropriateness for Lyme disease diagnosis. Clinicians may not be familiar with the differences between available tests and, when given multiple options, make inappropriate selections. Clinicians felt additional training and guidance in this area would be beneficial.Approximately half of survey respondents self-rated as moderately knowledgeable on the treatment of anaplasmosis, babesiosis, and Lyme disease Table . While aHalf of the respondents reported feeling neutral on their ability to address misinformation on TBDs and misinformation on Lyme disease specifically, while 37.5% and 41.7% felt confident in their ability to address misinformation on TBDs and Lyme disease, respectively. Most respondents (91.7%) reported that patients rarely or never refused to take the antibiotic treatment they prescribe to treat TBDs. Moreover, 37.5% of respondents reported that, about half of the time, patients request a longer course of antibiotic treatment, and 45.8% reported patients try to negotiate on the length of antibiotic treatment to treat TBDs.Half the respondents reported using educational tools to help their patients better understand TBDs, but only 41.7% reported that current resources for patient education are sufficient and 83.3% reported wanting additional educational resources for their patients on TBDs. Respondents indicated they often use information on UpToDate.com, the CDC website, and the CDC tick-borne disease handbook  to educaThe majority of respondents 79.2%) reported they have access to the resources they need to update their personal knowledge on TBDs, and close to half of respondents (45.8%) reported regularly looking up peer-reviewed literature on the treatment and diagnosis of TBDs. All respondents reported sometimes or often using UpToDate.com to access information on TBDs. Other common resources respondents used (sometimes or often) included the CDC website (79.2%), medical journals (66.7%), and Infectious Disease Society of American (IDSA) guidelines (54.2%). Just over half of respondents (55.6%) reported never attending CME accredited seminars, and 88.9% reported never watching CME accredited webinars to learn about TBDs. Additional summaries on survey response distributions are available in Additional file % reporteFrom our small study in a Lyme disease endemic community, it appears that primary care and frontline healthcare providers experience uncertainty in the diagnosis and clinical management of TBDs. The first focus group theme we identified indicates that primary care clinicians lack confidence in their ability to diagnose and treat non-Lyme TBDs and to identify Lyme disease infection in ambiguous clinical scenarios. Participant statements reflected several points of frustration related to this uncertainty. Frustration in the decision-making process in the context of inconclusive Lyme disease serology centered not only on the clinicians’ internal misgivings about their own knowledge, but also on how this uncertainty resulted in lengthy and difficult conversations with patients. The study data also indicate that the approach to Lyme disease diagnosis and treatment is variable across medical specialties. Inconsistency in practice patterns and variable adherence to guideline-based care across healthcare providers negatively affected patient trust, further complicating patient education, counseling, and care plan development.Another prevalent focus group theme focused on the contentious issue of chronic Lyme disease and conflicting guidance from alternative care providers. Participants frequently mentioned conversations with patients on this topic, consistently reporting the need to address misinformation on Lyme disease during the patient encounter. Participants often felt frustrated about this misinformation, knowing it came from sources they did not regard as authoritative or guideline-based, including word of mouth, social media, and alternative “Lyme literate” care providers. As stated by the National Institutes of Health, “chronic Lyme disease” is not a universally recognized disease due to confusion on diagnosis and the lack of a clear clinical definition . SeveralWhile only 24 clinicians participated in our survey questionnaire, their responses reflect certain findings from the focus group data. Roughly one-third of respondents failed to identify the CDC-recommended treatment approach for patients with nonspecific symptoms and negative Lyme disease serology, reflecting a similar challenge identified through the focus group data. While survey respondents reported feeling confident in their ability to address misinformation on TBDs with their patients, close to half reported receiving push back from patients on the recommended treatment regimen. While our survey sample size is too small to draw broad conclusions or generalize to larger populations, these novel question items may be useful in future, larger surveys measuring clinician experiences regarding TBD diagnosis and treatment.We identified opportunities to improve clinician knowledge regarding TBD diagnosis and management, including development of a comprehensive algorithm to standardize care across specialties, providing updated epidemiological data for regional TBDs, and a primer on how to order the appropriate Lyme serology in the EMR. While our study particularly struggled to reach emergency and urgent care specialists, making connections with practicing clinicians overall is challenging , 16. OurOne issue we encountered in focus group recruitment for this study was a hesitancy of practices to participate due to differing orientations of medical staff on the issue of chronic Lyme disease. In cases where practices included clinical staff with strong and discordant beliefs on diagnosis and treatment of chronic Lyme disease, these focus groups were perceived as harmful to office dynamics. Medical directors at two practices declined to organize focus groups at their practices due to this contentious issue. While we did not specifically ask focus group participants to identify their opinions on this issue, several of our participants openly stated they did not believe in the chronic Lyme disease diagnosis. Thus, our focus group findings do not include the experiences of alternative care providers or providers who follow International Lyme and Associated Diseases Society (ILADS) guidelines.An additional issue encountered in this project was the low response rate from the emergency and urgent care clinical community, for both the focus group interviews and the online survey questionnaire. Our low response rates limit the generalizability of our findings beyond the small group of study participants. Frontline providers in the emergency and urgent care clinical community play an important role in diagnosing and treating TBDs, and additional efforts are needed to engage with this hard-to-reach community. Additionally, during the survey, clinician’s knowledge was tested on CDC-recommended treatments for specific TBDs. Due to the survey being online, it is possible that the high-performance scores were due to respondents looking up the correct answers.The gaps in knowledge identified through the focus group and online survey data, coupled with the consistent necessity to provide point-of-care counseling and education to patients, highlight a pressing need for resources and support for primary care and frontline providers treating patients for TBDs. Participants from both the focus groups and survey questionnaire indicated a desire for more educational resources to share with patients on TBDs. Focus group participants specifically described these resources as tools to support productive conversations with patients on TBDs in a wide range of subject matter, including explaining the diagnosis process, explaining the evidence behind treatment, and dispelling misinformation regarding treatment efficacy and persistent infection. Additional efforts are also needed to provide ongoing TBD educational opportunities that are accessible to these hard-to-reach clinical communities.Additional file 1. Focus group and survey questionnaire data collection tools. Full listing of focus group discussion script (primary questions and prompts) and frontline provider survey questionnaire.Additional file 2. Survey responses. Response distributions for all question items included in the frontline provider survey questionnaire.


Antimicrobial resistance not only increases the contagiousness of infectious diseases but also a threat for the future as it is one of the health care concern around the globe. Conventional antibiotics are unsuccessful in combating chronic infections caused by multidrug-resistant (MDR) bacteria, therefore it is important to design and develop novel strategies to tackle this problems. Among various novel strategies, Structurally Nanoengineered Antimicrobial Peptide Polymers (SNAPPs) have been introduced in recent years to overcome this global health care issue and they are found to be more efficient in their performance. Many facile methods are adapted to synthesize complex SNAPPs with required dimensions and unique functionalities. Their unique characteristics and remarkable properties have been exploited for their immense applications in various fields including biomedicine, targeting therapies, gene delivery, bioimaging, and many more. This review article deals with its background, design, synthesis, mechanism of action, and wider applications in various fields of SNAPPs. Diseases caused by infectious microbes go on to be the greatest challenge and warning to worldwide health care. Although significant progress has been attained over the past few decades with the introduction of novel and effective antimicrobials, current antimicrobial therapy is still experiencing some major setbacks, including lack of selectivity of usual drugs, unwanted side effects, uneconomical and time-consuming synthetic processes, and more importantly the acquirement of multidrug resistance  bacilli cover specifically the pathogens including The emergence of drug-resistant microbes and the raising concerns about the usage of antibiotics resulted in the development of antimicrobial peptides (AMPs) which are produced as a first line of defense by many multicellular organisms. AMPs are a class of naturally occuring small peptide molecules and have a wide range of inhibitory actions to directly prevent the growth of organisms like bacteria, fungi, parasites, viruses and even cancer cells. AMPs are classified based on their source, antimicrobial activity, structural characteristics and amino acid composition. Apart from medicine, they also have good application prospects in other fields like food, animal husbandry, agriculture and aquaculture  have been introduced as novel agents having more potency, stability and bioavailability. SNAPPs indicate a new class of synthetic AMP replicas; exist as star-shaped polypeptide nanoparticles containing hydrophilic lysine and hydrophobic valine amino acid residues , reversible addition − fragmentation chain transfer (RAFT) polymerization, atom transfer radical polymerization (ATRP) and nitroxide-mediated living radical polymerization (NMP).This method involves ROP of cyclic esters and derivatives or N-carboxy anhydride where the later one has been proved to be a more flexible and controls chain-growth polymerization technique for the synthesis of peptide polymers having antimicrobial properties. This method relies on employing amino acids as building blocks to obtain polymers having a close structural resemblance to naturally occurring peptides and proteins, hence specific functionalities can be easily incorporated into polypeptide materials. This approach enables preparing synthetically challenging high molecular weight biopolymers which are better compared to other polymers. Improvements in NCA–ROP have shown a superficial direction for the synthesis of distinct peptide polymers with complex macromolecular architectures, like star polymer nanoparticles though they cannot compete with specific peptide orders attained by solid-phase peptide synthesis  is the modified form of the ROP technique that operates by opening the cyclic construction, discharging the ring strain of the monomer, and introducing the monomer into the budding chain. The main advantages of this method include easy flexibility, faster reaction rate, negligible side reactions, and very simple end-group modification  for the reaction to occur Nakayama .From various studies, the following general observations are drawn from the characterization data of synthesized SNAAPs.Star-shaped SNAPPs with different arm numbers having medium (M) range arm length which was designed in preference to studying the effect of arm number on antimicrobial performance. They exhibited comparable results to the expected values.Star-shaped SNAPPs molecular weight is found to be directly proportional to arm number and the hydrodynamic diameter of the stars was also observed to be slightly increasing with arm number.Stars with varying arm lengths  have been studied with different degrees of polymerization values. There is an increase in molecular weight and a slight increase in hydrodynamic diameters.The surface charge values exhibited similar values obtained across the SNAPPs prepared. Further, any increase in arm number and arm length of the SNAPPs is also found to be increasing with the size of the SNAPPs  targeting, destabilization /fragmentation of outer membrane, and unregulated ion movements across the membranes in many Gram-negative pathogens, including MDR bacteria. This movement results in membrane disruption along with hyperpolarisation and depolarization of the membrane followed by oxidative stress and production of reactive oxygen species (ROS)  ions to show counter action against many Gram-negative pathogens. All of them have registered minimum bactericidal concentrations with superior antibacterial efficacies. Further investigation was performed with the synergy of SNAPPs with Doxycycline  methacrylamide hydrochloride (AEMA.HCl) cationic core has displayed better colloidal stability, neutral zeta potentials and minor cellular toxicity which provides a modified carrier for efficient siRNA delivery. SNAPPs that can respond to stimuli like pH  contrast agents, macromolecular ligands were chelated to complex Gd3+ and the results were compared by studying reflexivity properties (Li et al. 3+ MRI contrast product. Poly acrylate-based SNAPPs with bimodal imaging agents which is the combination of fluorescent and magnetic resonance features have also been developed (Adkins et al. 3+ and Eu3+. High molecular weight polymer drug carriers with branched star-like structural design have been proved to exhibit higher accumulation in tumor tissues due to better permeability and retention effect (Ulbrich et al. To determine the effect of SNAPPs on Gadolinium Magnetic Resonance Imaging (GdCurrently, SNAPPs, in the form of star-shaped polymers present an emerging novel approach for the treatment of various infections associated with antimicrobial resistance. The unique properties of SNAPPs make them more successful and widely acceptable for the various applications of the pharmaceutical field and also help in delivering the drug molecules with minimal toxicity and greater therapeutic effect. In the current review, we have covered the detailed synthesis and mechanism behavior of the SNAPPs. Also, various novel applications of SNAPPs are included by discussing the case studies.In the future, with the help of various approaches, the effective, less toxic and nanomaterial sized SNAPPs can be given priority to provide permanent solutions for microbial resistance developed infections. Though having many advantages over other polymers and formulations, the commercial utility of these precious SNAPPs is awarded less attention. More focus can be provided for their commercial value from researchers by keeping parallel coordination with those from other disciplines and industries. It is highly necessary to continue extensive research in the development of efficient star-polymers to acquire highly economical and still better reproducible outcomes. Further modulations of functionalities in SNAPPs development and their role in therapeutic value with minimal impurities and utility in many other important fields can be kept as a futuristic goal for the forthcoming research activities.


Health system constraints are increasingly recognized as an important addition tomodel-based analyses of disease control interventions, as they affect achievable impactand scale. Enabling activities implemented alongside interventions to relax constraintsand reach the intended coverage may incur additional costs, which should be considered inpriority setting decisions. We explore the use of group model building, a participatorysystem dynamics modelling technique, for eliciting information from key stakeholders onthe constraints that apply to tuberculosis infection prevention and control processeswithin primary healthcare clinics in South Africa. This information was used to designfeasible interventions, including the necessary enablers to relax existing constraints.Intervention and enabler costs were then calculated at two clinics in KwaZulu-Natal usinginput prices and quantities from the published literature and local suppliers. Among theproposed interventions, the most inexpensive was retrofitting buildings to improveventilation (US$1644 per year), followed by maximizing the use of community sites formedication collection among stable patients on antiretroviral therapy  andintroducing appointments systems to reduce crowding (US$9302). Enablers identifiedincluded enhanced staff training, supervision and patient engagement activities to supportbehaviour change and local ownership. Several of the enablers identified by thestakeholders, such as obtaining building permissions or improving information flow betweenlevels of the health systems, were not amenable to costing. Despite this limitation, anapproach to costing rooted in system dynamics modelling can be successfully applied ineconomic evaluations to more accurately estimate the ‘real world’ opportunity cost ofintervention options. Further empirical research applying this approach to differentintervention types (e.g. new preventive technologies or diagnostics) may identifyinterventions that are not cost-effective in specific contexts based on the size of therequired investment in enablers. Estimating the full costs of health system investments to successfully implementtuberculosis infection prevention and control interventions in South African clinics isessential for priority settingGroup model building, a participatory system dynamics modelling (SDM) technique, mayassist with designing feasible interventions and identifying activities that can becosted to overcome health system constraints and achieve implementation targetsInterventions requiring large upfront capital investments, such as retrofittingbuildings to improve ventilations or installing UV germicidal irradiation systems, arecheaper over time than interventions requiring behaviour change (e.g. ensuring windowsand doors are kept open) due to the higher relative share of enabling costs (e.g. ofintensified training and supervision) required to make them sustainableSDM-informed costing allows for a comprehensive view of the health system influences onintervention impact and feasibility, in a way that may be superior to other, lessparticipatory stakeholder consultation methodsMycobacterium tuberculosis(Mtb) in primary care clinics and other health care settings is apriority on tuberculosis (TB) infection prevention and control (IPC) agenda in South Africa.Transmission of drug-resistant (DR) Mtb is documented within healthfacilities (Mtb transmission in clinics in high human immunodeficiency virus(HIV) burden settings may be higher than contact data would suggest for both health careworkers and patients , and a set of enabler activities identified during the group modelbuilding as necessary to ensure interventions are feasible in South African clinics (e.g.increased training and district-level supervision). Pathways of action of the identifiedinterventions and enablers were described through a process of iterative review andrevision of the causal loop diagrams and free lists generated by stakeholders during theworkshops, integrated with the qualitative evidence gathered by the wider UmoyaOmuhle project.This information then fed into the design of interventions that would be effective atreducing nosocomial Umoyaomuhle study facilities, who were asked about input requirements, includingstaff time, for carrying out hypothetical tasks. Capital investments and other start-upcosts were annualized using a 3% discount rate for future costs. All costs are presentedin 2019 US$.Unit costs for core intervention activities and enablers were estimated using price andquantity data from the published literature and quotes from local suppliers. Unit costscaptured the incremental economic costs of all core activities, including the opportunitycost of staff time, recognizing that even activities that are not time-consuming and thatare already implemented to some degree, such as opening windows and doors to improveventilation or directing queuing patients, will need dedicated staff to increase theirfeasibility and impact compared to current levels , above service-level  or outside the remit ofthe Department of Health (e.g. improving transport links to clinics to ensure theviability of appointment systems). Other activities were acknowledged as central to theintervention but excluded from the costing exercise, as they did not represent an actualcost, i.e. they referred to barriers that could not be overcome through financialinvestments (e.g. having to obtain permission from the district to carry out clinicbuilding modifications).The final list of interventions included in the cost model is presented in Incremental annual costs of each intervention option, including upfront capital investmentand recurrent costs of all intervention activities and enablers, are reported in The most expensive input to intervention implementation was the time of clinic staff. Forthis reason, relatively simple but human resource-intensive interventions, such as ensuringthe regular opening of windows and doors or implementing a queuing system that allows forcoughing patients to be rapidly triaged and for other patients to wait in a sheltered areaoutdoors, were found to be more expensive than those interventions relying on capitalinvestments and technology, such as installing ultraviolet germicidal irradiation (UVGI)systems.Enablers identified ranged from relatively inexpensive capital investments, such aselectric heaters to ensure thermal comfort in winter when windows are kept open, to morecostly enhancements to training programmes for clinic staff and consultations with communityrepresentatives, to ensure lasting changes in work culture and local ownership of theinterventions. Overall, the proportion of total intervention costs represented by theenablers was inversely proportional to the size of capital investment required by theintervention; and directly proportional to the intervention’s reliance on changes in thebehaviour of patients and/or staff .CorrespThis analysis applied group model building, an established SDM approach, to elicitinformation on the health system constraints that operate in TB IPC in South Africa. Thisinformation was then used in a novel way to build a model of the local incremental costs ofa set of TB IPC interventions implemented at two clinics in KwaZulu-Natal. The interventionswere designed bearing in mind potential barriers to implementation and necessary investmentsto overcome these; then costed iteratively, based on feedback given by SDM participants. Theresulting unit costs thus reflect information linking the implementation process tooutcomes, and are a closer representation of the full opportunity costs of theseinterventions compared to those generated with standard costing methods, as they include thecosts of relaxing health system constraints.Despite the addition of enabling costs, the TB IPC interventions considered aresubstantially less expensive than other interventions for preventing TB transmissioncurrently included in the South African National Strategic Plan for HIV and TB as well as inthe Investment Case for TB, such as improving the timeliness and yield of facility-based TBscreening by using more sensitive algorithms and contact tracing . IntensiFurther cost savings could be realized by considering the proposed TB IPC interventions asa package, thus allowing the costs of those enablers that are shared by more than oneintervention to be spread across them. An example would be the costs of enhancing routinestaff training and supervision, which are shared by all the interventions analysed with theexception of building retrofits. Similarly, gains in efficiency could be realized fromscaling up the interventions to the regional and/or national level  or that are otherwise ‘uncostable’. This may bebecause the interventions and enablers consist of novel activities for which sources ofprice and quantity data cannot be readily identified, such as setting up new CCMDD pick uppoints; or they may consist of high-level activities, such as redesigning training formatsand materials based on data collected from routine monitoring and evaluation, the costs ofwhich are above-site and difficult to allocate to specific interventions; or they mayconsist of activities that fall outside the remit of the health sector, such as improvingpublic transport links to health facilities to support the implementation of a clinicappointment system that spaces patient visits throughout the day. While activities that donot incur a cost and those that fall outside the health sector might be excluded from aneconomic evaluation (depending on the perspective taken), additional data collection isneeded for costing novel activities and for allocating and scaling the costs of sharedabove-site enablers, e.g. from pilot/demonstration projects or feasibility studies.SDM-informed costing allows for a comprehensive view of the health system influences onintervention impact and feasibility, in a way that may be superior to other, lessparticipatory stakeholder consultation methods. By providing several occasions forinteraction between different TB IPC stakeholders at the national and decentralized level,as well as between stakeholders and researchers, the group model building exercise presentedin this analysis enabled a thorough process for validating and refining costing assumptions,including on the details of intervention and enabler design. For successful application ofthis approach in economic evaluation, further research is needed into ways of integratinginsights from SDM, that are well suited to identifying above-service level costs, with moretraditional costing methods, which usually focus more on service-level inputs. Such acombination can, e.g. smooth the process of linking costs into transmission model outputs,which are usually service-level units, as well as potentially inform the choice of afunctional form for modelling costs at scale. SDM can also be useful for identifyingintervention types that might not be cost-effective based on the share of total costsrepresented by the required enablers. Further analyses of interventions that are more orless reliant on capital investments or behaviour change, such as new preventive ordiagnostic technologies, are needed to fully assess its potential applications in economicevaluation and priority setting.czab155_SuppClick here for additional data file.


Preeclampsia is a pregnancy-specific condition which gets detected through hypertension and excessive protein excretion in urine. While preeclampsia used to be regarded as a self-limiting maternal condition which resolved with the delivery of the placenta, it is nowadays considered a complex and multifactorial disease that affects the offspring. Unfortunately, the etiology and pathophysiology of this multifaceted disorder remain elusive. Recent findings have confirmed that an altered maternal autonomic function may play a vital role in developing preeclampsia in conjunction with an imbalanced maternal immune system. Additionally, further evidence supports the crucial role of an exacerbated immune response driven by a non-infectious trigger during preeclampsia. Therefore, as a sterile inflammation, the elucidation of the neuroinflammatory mechanisms of preeclampsia warrants obtaining relevant knowledge suitable for translational clinical applications.Heart rate variability (HRV) is an affordable and non-invasive method for indirectly assessing the autonomic nervous system and the cholinergic anti-inflammatory pathway (CAP). Notably, the nonlinear analysis of HRV offers novel indexes to explore the neuroimmune interactions in diverse preclinical and clinical settings of inflammation. Given that the dynamics of HRV is nonlinear in health, we hypothesized that a neuroinflammatory condition in preeclampsia might be associated with changes in nonlinear features of maternal and fetal HRV. Thus, the present review aims to present evidence of the potential changes in maternal-fetal HRV associated with neuroinflammatory modifications in preeclamptic women. We considered that there is still a need for assessing the nonlinear features of maternal and fetal HRV as complementary biomarkers of inflammation in this population in future studies, being a potential route for translational clinical applications. Preeclampsia is one of the leading causes of critical maternal and fetal mortality and morbidity. It is diagnosed by pregnancy-onset hypertension and proteinuria after 20 weeks of gestation . Both prPreeclampsia’s prevalence is estimated to be seven times higher in low-income countries compared to high-income countries ; thus, iConventionally, preeclampsia has been regarded as a self-limiting maternal pathological condition which gets completely resolved with the placenta’s delivery. However, it is also considered a complex and multifactorial disease that may also have consequences for the offspring . It is kPreeclampsia remains a particular disorder of pregnancy, with an incomplete understanding of its etiology . Recent New disciplines such as neuroimmunology have emerged in order to provide a compelling integrative link between the immune system and nervous system. It recognizes that immune responses are present in the central and peripheral nervous systems during disease, considering the existence of a neuroimmune axis . Some auAccording to the literature, linear approaches of HRV have been extensively studied and are not adequate to fully describe a complex system . ParticuThis review article aims to present evidence of the potential changes in HRV associated with neuroinflammatory modifications in preeclamptic women; we first describe the immune and neuroimmune profiles in preeclampsia, followed by linear and nonlinear HRV changes during preeclampsia. Finally, we discuss the nonlinear analysis of HRV as a novel tool for exploring inflammatory mechanisms in the maternal-fetal dyad during preeclampsia.According to some studies, the immune profile of women with preeclampsia shifts from a steady state of mild inflammation; from an equilibrium of both pro-inflammatory and anti-inflammatory mechanisms to a downregulated state characterized by increased inflammatory cytokines and effector immune cells with a concomitant decrease in anti-inflammatory factors and regulatory cells . Other rPreclinical evidence has indicated that interleukin 6 (IL-6) may play a role in mediating hypertension and reducing renal hemodynamics observed in uterine perfusion reductions in pregnant rats . AccordiYang et al.  reportedNovel findings suggest that the vagal-driven activation of the α7nAChR attenuates preeclampsia-like symptoms, and this protective effect is likely result of the inhibition of inflammation via the NF-κβ pathway .McAllen et al., have provided compelling evidence in preclinical studies that indicates that splanchnic sympathetic outflow acts to suppress immune-mediated inflammation . HoweverThere is still a lack of knowledge in how preeclampsia modifies the neuroimmune pathways; for example, some studies have reported an association among maternal immune activation, the manifestation of preeclampsia, and an altered neonatal neurodevelopment . A possiNonlinear HRV analysis is based on chaos theory, in which the heart rhythm generation, besides being sensitive to initial conditions and evolving very fast over time, is considered nonlinear , 49. On Evidence has also shown negative associations between linear HRV indexes such as the standard deviation of the normal-to-normal interval with TNF-α in heart failure patients, suggesting that the immune system’s activation is related to autonomic imbalance . More spSpecifically, it is known that preeclampsia affects the cardiovascular health of both mother and offspring . StudiesNovel nonlinear measures have been introduced to describe HRV, which present the advantage of not being affected by nonstationary effects . Table 1Nonlinear HRV measures or features have shown potential as biomarkers of internalizing psychopathology, often associating lower information-based complexity with internalizing psychopathology . The relNonlinear measures of HRV have already allowed the discrimination of depressive patients from healthy subjects, consistently showing a significant decrease of HRV complexity in neuroimmune pathologies . Mainly,According to the consulted literature, no clinical studies have been conducted to simultaneously evaluate the nonlinear features of HRV and multiple inflammation markers in women diagnosed with preeclampsia. However, results indicate that preeclamptic women reveal a reduced complexity by incrementing periodicity assessed by multiscale asymmetry indexes of HRV . The autThe CAP activity can be monitored easily and non-invasively via the linear analysis of HRV data derived from either maternal or fetal electrocardiogram, Fig. The information derived from chaos theory, fractal mathematics, and the dynamic complexity of HRV has not yet been fully applied in the obstetrics field. It is a productive area for research in both healthy and pathological scenarios. Also, the complex characterization of the HRV time series during preeclampsia has rarely been studied. Theoretically, the maternal and fetal nonlinear analysis of HRV offers unique opportunities to explore the neuroimmune interactions in the mother-child binomial.Neuroinflammatory alterations in preeclamptic women may involve autonomic changes associated with a possible concomitant downregulated CAP and modifications in nonlinear features of HRV. We consider that the assessment of nonlinear measures of both maternal and fetal HRV during preeclampsia is a promising field for exploring the neuroinflammatory interactions in the maternal-fetal dyad and a route for potential translational clinical applications. The knowledge of this frontier topic promotes basic research and translational clinical applications for scientists worldwide who conduct research concerning inflammatory processes.


Hyperostosis frontalis interna (HFI) is a benign entity manifested by bony overgrowth in the frontal endocranial surface. It is most commonly reported incidentally among postmenopausal elderly women. Tracer uptake appearances of HFI can vary on planar bone scans, enabling it to be easily confounded with bone metastases. We report a case of HFI in a 69-year-old postmenopausal female with treated left breast cancer detected on bone scintigraphy, with subsequent confirmation by computed tomography. Our case highlights the importance of having awareness of HFI and its key pattern findings to avoid mistaking it for pathology, and to recognise the use of computed tomography and hybrid fusion imaging techniques as reliable diagnostic tools for HFI. Hyperostosis frontalis interna (HFI) is characterized by irregular overgrowth of bony tissue in the frontal bone inner table. The condition is often found incidentally among postmenopausal elderly women and is typically benign. However, marked calvarial thickening can cause compression of the frontal lobes. Tracer uptake appearances of HFI on bone scintigraphy studies can be variable, enabling it to be easily confounded with bone metastases. Recognition of this entity and its pattern findings is important to avoid mistaking it for pathology. In addition to the use of computed tomography (CT) as a reliable diagnostic tool for HFI, hybrid fusion imaging techniques can add value as a useful adjunct to guide identification and characterization of HFI, as well as to discriminate between benign and malignant processes.The 69-year-old female patient with a background of treated left breast cancer in 2012 presented with a history of gradual, progressive left-sided chest wall and breast pain. An initial mammography and ultrasound of both breasts were carried out which demonstrated BI-RADS 1 findings. Bone scintigraphy was performed to exclude skeletal involvement. The results of this study revealed asymmetrical abnormal increased tracer uptake involving the bilateral fronto-parietal regions of the skull . Tracer Origin: Department of Radiology, Darlington Memorial Hospital, Hollyhurst Rd, Darlington, United Kingdom.HFI is a benign entity manifested by irregular overgrowth of bony tissue in the inner table of the frontal bone, which is typically bilateral and symmetrical. Literature has shown HFI to be an age- and sex-related phenomena The condition is often found incidentally and is generally of no clinical significance On CT imaging, HFI has unique characteristics which help to distinguish it from other endocranial bony overgrowths including (1) bifrontal symmetry; (2) distinct boundaries without crossing the sagittal sinus area medially; (3) expansion over the frontal endocranial surfaces; (4) continuity with the inner table and diploe Utilizing plain radiographs, Moore Increased tracer uptake in thickened areas of the frontal bone is a frequent finding on bone scintigraphy studies Taking into consideration the increasing prevalence and severity of HFI over time In some cases, the use of hybrid fusion imaging techniques including PET/CT and single-photon emission computed tomography/computed tomography can add value by demonstrating specific findings, assisting in the identification and classification of HFI. It is important to recognise these techniques as a useful adjunct in the evaluation and discrimination between benign and malignant processes.•HFI is a benign entity manifested by irregular overgrowth of bony tissue in the frontal bone endocranial surface and is most commonly reported as an incidental finding among postmenopausal elderly women.•Tracer uptake appearances of HFI can vary on planar bone scans, enabling it to be easily confounded with bone metastases. In view of this, it is important to be aware of HFI and its key pattern findings to avoid mistaking it for pathology.•Utilising volume rendering with a CT 3-scale classification has been shown to be a reliable and valid tool for radiological diagnosis of HFI. Hybrid fusion imaging techniques can add value as a useful adjunct to guide identification and characterization of HFI, as well as to discriminate between benign and malignant processes.Written informed consent for publication was obtained from the patient.This article does not contain any studies with human participants or animals performed by any of the authors.


During development and in several diseases, endothelial cells (EC) can undergo complete endothelial-to-mesenchymal transition (EndoMT or EndMT) to generate endothelial-derived mesenchymal cells. Emerging evidence suggests that ECs can also undergo a partial EndoMT to generate cells with intermediate endothelial- and mesenchymal-character. This partial EndoMT event is transient, reversible, and supports both developmental and pathological angiogenesis. Here, we discuss possible regulatory mechanisms that may control the EndoMT program to dictate whether cells undergo complete or partial mesenchymal transition, and we further consider how these pathways might be targeted therapeutically in cancer. Endothelial cells (EC) that line the various blood vessels of the body share numerous structural characteristics and gene expression programs, yet are also remarkably heterogeneous  and plasIn addition to supporting complete mesenchymal transition, the EndoMT program can also proceed only partially resulting in the temporary and reversible appearance of intermediate cells that exhibit both endothelial and mesenchymal characteristics . Tombor Activation of the EndoMT program in EC during angiogenesis must be partial and reversible to support the formation of perfused and functional blood vessels. This implies the existence of essential regulatory mechanisms that act on the EndoMT program to determine whether it will proceed completely in some tissues, but only partially in other contexts such as angiogenesis. Here, we revisit the argument for sprouting angiogenesis as a partial EndoMT event and explore possible regulatory mechanisms that might limit complete progression through the EndoMT program during angiogenesis, thereby preventing excessive mesenchymal transition and ensuring organized and controlled new vessel growth.Sprouting angiogenesis is a complex developmental program wherein specialized endothelial “tip” cells migrate away from the parent vessel wall toward pro-angiogenic stimuli, while bringing with them endothelial “stalk” cells, thereby establishing a new blood vessel sprout . EndotheGiven the heterogeneous outcomes associated with EMT program activation, identification of an EMT event can be difficult and requires the combined observation of several cellular and molecular hallmarks, including the upregulated expression of at least one of several EMT-associated transcription factors, induced expression of mesenchymal markers, and alterations in functional properties such as cytoskeletal rearrangement, reduced cell-cell adhesions, and increased cell motility . FurtherWhen compared to this standard, the cellular and molecular changes that occur in sprouting angiogenesis are highly suggestive of partial EndoMT . During In further support of angiogenesis as a partial EndoMT event, we have found that the master transcription factors Snail and Slug are induced in ECs during sprouting angiogenesis, both developmentally  and in tSimilar to its role in EMT, TGF-β superfamily signaling is a potent activator of the EndoMT program, both in developmental and pathological settings . This prin vivo  roles in EndoMT . Slug anAside from Slug and Snail, endothelial expression of Zeb1 , Zeb2 C, and Twiin silico analysis suggest that Twist1 may operate upstream of Slug and Snail  and cell surface Notch receptor expressed on adjacent cells. Thus, Notch signaling necessarily requires that at least some cell-cell junctions be intact, suggesting an initiating role in EMT and presumably an early role also in EndoMT. Indeed, Notch drives EndoMT in development  and duriansition .instructive signal. More consistent with its multiple and differing roles in numerous developmental programs, however, would be if Notch signaling instead plays a permissive role to open a “window of opportunity” for other pathways to be active. Thus, for any process – whether positively or negatively acting pathways – both could each be dependent on Notch signaling, such that neither is able to operate without a permissive signal from Notch. In EC, Notch may thus control whether pro- or anti-mesenchymal transition signals gain the upper hand. Similar processes have been suggested for Notch during arteriovenous specification  are short single-stranded, non-coding RNA sequences that regulate post-transcriptional gene expression at the mRNA level. Several miRNAs are involved in EMT , both thBoth complete and partial EndoMT depend upon the availability of key inducers to activate core DNA-binding transcription factors that regulate the expression of downstream mesenchymal transition genes. Given this, epigenetic changes that alter access to individual genes  play a significant role at multiple levels of the EndoMT program, such as to regulate the availability of EndoMT inducers and effectors . For exaEndothelial-to-mesenchymal transition progression is also sensitive to epigenetic changes that alter access to mesenchymal genes acted upon by EndoMT master transcription factors . Under bEarly studies of EMT and EndoMT proposed that mesenchymal transition is a binary, on/off switch between two distinct cell states. However, more sophisticated studies have now refuted that idea, and instead support an alternative model wherein most cell fate changes – including both EMT and EndoMT – involve progressive transition from one cell identity to another via a fluid spectrum of intermediate cell states . In suppSimilarly, complete EndoMT also involves fluid transition through intermediate endothelial and mesenchymal substates with characteristics of both cell types. Cell subpopulations that co-express endothelial- and mesenchymal- markers – indicating cells in an intermediate state of EndoMT – have been reported in cardiac , pulmonaCell identity – even among committed cells – is dynamically established and maintained by the moment-to-moment balance of competing cell fate signals . Indeed,Several EndoMT signaling pathways likely regulate EndoMT progression by modulating the relative strength of endothelial vs. mesenchymal fate signals. Classic EndoMT activators that drive complete EndoMT to generate endothelial-derived fibroblasts  likely do so by strongly inducing pro-mesenchymal cues that overpower endothelial commitment signals, perhaps through the initiation of positive feedback loops. Others, however, may only modestly activate mesenchymal transition, either alone or in combination with the maintenance of endothelial identity signals, to limit the EndoMT program and ensure it proceeds only partially. VEGF, for example, is a classic pro-angiogenic factor that may promote  vessel growth by activating the EndoMT program to induce sprouting angiogenesis, while simultaneously preserving endothelial identity cues to prevent excessive mesenchymal transition in resulting tip and stalk cells. Underscoring its role in promoting new vessel growth, we found that VEGF signaling blocking prevents overaggressive sprouting in the context of Slug overexpression  suggestiin vitro sprouting angiogenesis assay, we found that Slug upregulation precedes that of Snail by several days, suggesting that Slug activation is independent of Snail. Moreover, Slug dominates during the early proliferative phase of angiogenesis, whereas in later stages of angiogenesis, its expression decreases as Snail expression increases  by creating a window of opportunity, as suggested above . Notch ike state . Thus, ike state  may looseled ECs . Subsequeled ECs . FurtherMature blood vessels are highly stable, and sprouting angiogenesis is a tightly regulated process that involves the temporary adoption by angiogenic ECs of pro-proliferative and migratory states (via stringent regulation of the EndoMT program). This enables the formation of new vessel sprouts, but also the return of EC to a quiescent and stable state . HoweverConsistent with this hypothesis, we recently reported that beyond its developmental role in angiogenesis, Slug is critically required to support the pathological hypersprouting of blood vessels into tumor explants. In the absence of endothelial Slug expression, tumor angiogenesis was almost completely abolished  underscoEndothelial-to-mesenchymal transition is associated with several disease settings, including atherosclerosis, where activation of the EndoMT program drives robust EC transformation into EC-derived fibroblasts that are critical for plaque formation and progression . StudiesExisting anti-angiogenic approaches in cancer treatment have largely focused on inhibiting pro-angiogenic signals. When pathological angiogenesis is reconceptualized as a problem of dysregulated EndoMT – and not merely an issue of over-active pro-angiogenic  signaling – it becomes clearer why anti-VEGF therapy [which has been a standard of care in cancer treatment for the last two decades ] largelyMost pro-EndoMT pathways appear to converge at the level of the master EndoMT transcription factors. Indeed, as mentioned above, several EndoMT transcription factors are abnormally expressed in tumor-associated ECs where they drive abnormal tumor vessel formation. Therefore, therapies that target master EndoMT transcription factors are likely to be more effective than treatments that inhibit EndoMT program activators, particularly in the context of cancer, because of the following properties of these transcription factors: (1) they serve as a common signaling nexus for most upstream pro-EndoMT activators and could therefore alleviate the issue of “escape pathway” activation; (2) they are transcription factors that directly drive expression of suites of cell identity genes, thus ensuring broad effects on target cells; and (3) their abnormal activation is often specific to the tumor environment  which suOne concern in this approach might be the challenge of targeting intracellular proteins. However, recent developments in antibody-mimicry and antibody-fusion peptides, as well as advances in viral- and nanoparticle-based delivery methods, have shown great promise for cell-type specific delivery of antibodies that target intracellular antigens  and shouAnother possible avenue for therapeutic intervention in the EndoMT program may involve targeting regulatory mechanisms that govern EC plasticity. Therapies that selectively promote EC commitment and limit sensitivity to local pro-EndoMT signals may prevent overaggressive and pathological angiogenesis in the tumor mass, particularly when applied in combination with other chemotherapeutic or anti-angiogenic treatments. Such an approach might be used to either normalize tumor vessels, or to block tumor angiogenesis altogether. If, as we propose, Dll4-Notch signaling promotes EC commitment to limit  EndoMT, therapies that activate Notch signaling specifically in tumor vasculature may help suppress pathological angiogenesis. Yet, application of Notch inhibitors have produced mixed outcomes on tumor blood vessels  likely dComplete EndoMT serves as a critical source of endothelial-derived mesenchymal cells during organogenesis and is a significant contributor to fibrosis in disease. It is also increasingly clear that partial EndoMT also plays an essential function during angiogenesis (and likely other processes), and that dysregulation of the EndoMT program may contribute to abnormal and pathological blood vessel growth. Specifically, we propose that tight regulation of a core EndoMT program dictates the extent of mesenchymal transition in a context-appropriate manner by manipulating the strength of identity and transition cues, as well as the extent of target cell plasticity, and that loss of this control contributes to blood vessel disorganization in diseases such as cancer.However, several questions remain outstanding: What are the contexts aside from angiogenesis under which partial EndoMT occurs? What specific regulatory mechanisms determine the extent of EndoMT progression, and how are they dynamically regulated during angiogenesis to maintain partial endothelial identity during sprouting? What are the regulatory mechanisms that govern cell plasticity during EndoMT, and how do they modulate progression of the EndoMT program? How stable and discrete are intermediate stages of partial EndoMT and, in addition to mesenchymal transition signals, are these cells sensitive to other cell lineage signals? Why do EndoMT transcription factors appear to be more essential in pathological angiogenesis compared to during development, and what aspects of the tumor microenvironment and other disease states lead to dysregulation of EndoMT regulatory mechanisms? And finally, how can EndoMT be targeted therapeutically in these diseases? An improved understanding of EndoMT, and specifically the regulatory mechanisms that dictate complete vs. partial mesenchymal transition in a context-dependent manner, warrant further study and are likely to reveal important insights into these and other crucial questions.JF and NH wrote the manuscript. CH provided edits and additional scientific insights. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.