Adjuvants are critical components of vaccines that enhance the host immune response to the vaccine antigen, however, only a small number of adjuvants are used in vaccines approved for human use. This is in part due to the slow process of novel adjuvants advancing from preclinical models to human studies, and modest mechanistic insights obtained using standard immunological methods to justify selection of a particular adjuvant for clinical evaluation. Here, we discuss several aspects of current adjuvant research and strategies to better assess the complex pathways triggered by adjuvant candidates that can increase adjuvanticity and vaccine efficacy while minimizing reactogenicity. We propose a more systematic use of broad immunoprofiling, coupled with data integration using computational and mathematical modeling. This comprehensive evaluation of the host immune response will facilitate the selection of the most appropriate adjuvant for a vaccine, ultimately leading to the expeditious evaluation of novel adjuvants for vaccines against emerging infectious diseases, which will prove especially valuable during a pandemic where speed is of the essence when developing vaccines. Countless novel adjuvants are in preclinical development, and many novel adjuvants are evaluated in early-stage clinical trials, yet adjuvant selection for a particular vaccine continues to be mostly an empirical process. “Rational adjuvant selection,” the targeted pairing of an adjuvant with a vaccine antigen, is an attractive, but still elusive concept. Selection of the most appropriate adjuvant can determine the efficacy of a vaccine candidate, based on both strength and profile of the immune response being triggered. Even though data on the preclinical and clinical performance of different adjuvants with various antigens are noted in the literature, for example dmLT,Here, we discuss current limitations of key aspects and stages of adjuvant research: adjuvant discovery, preclinical evaluation, reactogenicity, and species-specific differences in the immune response. We offer suggestions on how to incorporate immunoprofiling into these aspects; and highlight how the establishment of comprehensive immune profiles induced by different adjuvants could expedite and standardize vaccine adjuvant.in vitro screening of compounds, with one (or few) marker(s) as the readout to determine whether a new compound has the potential to be used as an adjuvant.3–5 This approach assumes that expression of a specific signaling molecule  or cytokine  correlates with adjuvanticity in vivo. However, since a single in vitro parameter is unable to reliably predict in vivo adjuvanticity, the approach can result in a significant number of false positive hits, and potentially many false negatives. The high failure rate is due to a lack of defined in vitro correlates of in vivo adjuvanticity. Multiplex assay platforms that expand the number of cytokines being assessed partially address the issue,6 but this approach still relies on a limited number of parameters that may not necessarily correlate with in vivo adjuvanticity and can be prohibitively expensive when scaling up an adjuvant screening campaign.Modern adjuvant discovery involves systematic in vivo immunoprofiling studies with different adjuvants that have well-documented in vivo adjuvanticity, paired with “standard” antigens, to identify in vivo markers (not limited to cytokines and chemokines) associated with adjuvanticity. The inclusion of antigens in the profiling study assures that contributions of the antigen to the overall immune profile are recognized. To facilitate and refine the process of identifying in vitro immune markers that directly correlate with in vivo adjuvanticity, derivatives of strong adjuvants could be used, such as those that are generated in the process of structure-activity-relationship studies of novel adjuvants.8 The derivatives may have different levels of adjuvanticity and potentially mediate different immune profiles from which common biomarkers that directly correlate with adjuvanticity may be deduced. Computational modeling can help identify the minimally required biomarker signature suitable for predicting in vivo adjuvanticity  and be employed as an in vitro tool for identifying novel adjuvants. Considering the complexity of innate immune receptors, signals, and downstream effector molecules, it is likely that different (but overlapping) patterns will emerge from such analyses depending on the pattern recognition receptor (PRR) being targeted. For example, immune signatures shared between agonists of different PRRs would identify a pan-PRR adjuvanticity profile.We propose that adjuvant researchers employ a multi-step adjuvant discovery process , that coThe application of immunoprofiling extends beyond providing insights into an adjuvant’s mechanism of immune enhancement and understanding differences between different classes of immunostimulators. The ultimate goal in vaccine research is the rational rather than empirical pairing of antigen and adjuvant by selecting adjuvants and formulations capable of driving the immune profile associated with protective efficacy . While i9–11 for influenza, hepatitis B, and malaria respectively), but currently, it also represents the most technically feasible readout. For some vaccines, a functional activity of antibodies has been described as a correlate of protection, e.g., such as opsonophagocytic activity in case of malaria vaccines.12 Relying on antibody titers as the main parameter for choosing a vaccine adjuvant is limiting because it does not consider other immune parameters that may contribute to vaccine efficacy,13 nor does it help distinguish adjuvants from each other. More useful parameters include a broader assessment of the antibody repertoire, such as antibody avidity and breadth of epitope specificity, and the analysis of vaccine-induced T cells, particularly CD8 T cells. The mere assessment of antibody titers may also be misleading since antibody responses even against immunodominant epitopes may not correlate with efficacy, such as in the decoy antigens/epitopes of the HCV envelope glycoprotein.14 In addition, while studies to identify in vivo markers of adjuvanticity have been conducted in humans, for example with adjuvants from the GSK Adjuvant System (AS-series),15 such studies are complicated and expensive, which highlights the need for preclinical models.Antibody titers currently serve as the primary marker and predictor of a promising vaccine-induced response in preclinical models of adjuvant development. The choice of this readout is not only driven by the fact that antibody titers are the primary accepted correlate of protection for most licensed vaccines , profiling data from preclinical adjuvant comparison studies can still help in down-selecting a small number of adjuvant-antigen combinations to be tested in humans. Human organoid systems are still early in development and not commonly used but could be a valuable non-animal tool for down-selecting adjuvants in the future.16 Reactogenicity can be mitigated through 1) adjuvant design and modifications that skew the response toward signals that contribute to adjuvanticity  improved formulation, such as the targeting of immunostimulators to secondary lymphatic organs, which prevents systemic and injection site availability ,20 or a combination of CRP and IL6, respectively.21 However, this approach does not address the potential differences in the response to an adjuvanted vaccine by different species (reviewed in ref.22) or the translatability of findings to humans. Thus, methods are still needed that predict reactogenicity of novel adjuvants and adjuvant/antigen combinations in vitro. Ideally, identifying problematic novel adjuvant candidates at an earlier stage would be based on in vitro cell culture systems. Even when systems immunology approaches are applied to identifying in vivo profiles of reactogenicity,23 few attempts are made so far to correlate in vivo and in vitro reactogenicity profiles. While it is preferable to use a defined cell line ,24 it remains to be shown whether a single cell type can reliably predict reactogenicity considering the differential expression of innate immune receptors on different cell types in vivo.Reactogenicity is defined as the inflammatory response induced by a vaccine. Symptoms of reactogenicity can vary, ranging from localized injection-site pain, redness, and swelling, to systemic symptoms, such as headache, fever, or myalgia. The primary concern that slows advancement of novel adjuvant candidates into clinical trials is the risk of reactogenicity and adverse events, mostly as a result of the induction of innate immune cells, triggering inflammatory pathways. Traditionally, strong inflammation is associated with good immunogenicity, although there is little evidence to support this association.in vitro markers  to establish innate immune profiles that may predict reactogenicity. Since the antigen component of a vaccine can significantly contribute to a vaccine’s safety profile,25 it will be necessary to test adjuvants systematically, i.e., by themselves, with different antigens, and also the formulation that will be used in vivo. Licensed vaccines with different in vivo reactogenicity characteristics can be used to establish a “reactogenicity score” that would be linked to the vaccine’s in vitro reactogenicity profile. The reactogenicity scores from adjuvants used in licensed vaccines would be helpful in down-selecting novel adjuvants and adjuvant-antigen combinations. However, the reactogenicity score would still have several shortcomings, such as being limited to predicting reactogenicity after a primary immunization considering that adaptive recall responses can enhance innate immune stimulation and, thus, increase reactogenicity after a boost. The in vitro model would also not be able to address vaccine dose-related reactogenicity. As mentioned above, in the future, human organoid systems could also be a valuable tool for addressing adjuvant-induced reactogenicity that is enhanced due to pre-immune status.We are advocating for the establishment of a “reactogenicity score” that is based on a broader panel of 26 2) PRR expression patterns differ across species (reviewed in ref.27) 3) responses to antigen and adjuvant are dose dependent, thus results obtained with an “optimized” vaccine dose in a small rodent model may not translate to a large animal model or humans; and 4) downstream signals induced by PRR agonists can be species-specific.28 Not surprisingly, the profiling of different adjuvants used for a SARS-CoV−2 antigen yielded significantly different immune profiles in mice and non-human primates,29 and using immune profiles obtained in immunized animals to predict immune profiles induced by the same vaccine in humans can be challenging. This observation complicates the characterization of vaccine-induced immune profiles when different host species are used and may call into question the value of down-selecting adjuvants for vaccine candidates in animal models. One approach to address species-specific responses to defined immunostimulators has been to create humanized mouse models, where the mouse PRR is replaced by its human counterpart, ideally not only using the PRR-gene but also flanking genomic sequences to include regulatory elements and obtain “human-like” gene expression .30 Nevertheless, humanizing every component of the immune response to an adjuvanted vaccine is constrained by the complexity of the response and the number of regulatory pathways and circuits involved that cannot all be replaced.Animal studies are essential for determining the utility of vaccine formulations, but suffer from several shortcomings, most notably: 1) fine-specificities of PRRs differ between species;32 Mathematical and computational approaches that take into account species-specific differences in translational analyses would allow the identification of previously unappreciated networks and mechanisms that are critical for adjuvanticity and vaccine efficacy. Rather than aligning parallel pathways (such as the release of specific cytokines), this approach takes a holistic view of the overall immune profile the same vaccine induces in different species. This type of analysis can provide unprecedented insights into cellular interactions, tissue responses, pathways, adjuvant mechanisms of action (MoA), and also account for various modes of immunization to provide a better understanding of vaccine efficacy. In addition, it can potentially enable the dissecting of adjuvant properties in adjuvant comparison studies, and help “translate” findings in an animal model into corresponding human immune profiles in silico.To reconcile data obtained in different species, we are endorsing the concept of “humanizing computational models.”33 have limited value when generated for just individual adjuvants. They are most valuable when they can be compared to the profiles of other adjuvants, ideally obtained by using the same experimental protocols, and particularly when compared to “benchmark” adjuvants. Several studies have included alum as a comparator or benchmark adjuvant. However, no single adjuvant can serve as a universal benchmark due to the complexity of pathways and signals induced by different immunostimulators. Even the utility of alum as a benchmark adjuvant is limited by the fact that a variety of aluminum salt-based formulations are commercially available that do not have the same physiochemical characteristics and induce somewhat different immune profiles.34 Thus, in profiling studies, it may be advisable to use two distinct reference adjuvants.34 A reference adjuvant should meet at least two requirements: used in a licensed vaccine, and easy access to quality-controlled material. Including adjuvants used in licensed vaccines establishes “relevance” of the response measured and relying on a commercial source of the reference material assures that the same material is used across laboratories. Currently, these requirements limit the choice of reference adjuvants to aluminum salt-based adjuvants and Addavax, a commercially-available mimic of MF59.Immunological profiles, or “fingerprints,” of adjuvantsThe National Institute of Allergy & Infectious Diseases (NIAID) of the National Institutes of Health (NIH) continues to support research focused on the characterization of the immune response, the development of vaccines, and the discovery and development of novel adjuvants. To keep pace with the advances in vaccine design, to address the gap in knowledge regarding rational selection of adjuvants, and to leverage the power of computational modeling, NIAID launched two adjuvant comparison programs,


Human-pet attachment can impact the life of both parties, and the identification of underlying characteristics related to attachment style can improve human-pet relationships. We employed structural equation modeling (SEM) to explore associations between human, dog, and cat personalities, owner mental well-being, unwanted pet behavior, and attachment styles in a sample of 2,724 Finnish pet owners (92% women) and their 2,545 dogs and 788 cats. Our findings reveal that owner neuroticism and poor mental well-being are linked to anxious pet attachment in both dog and cat owners. Pet characteristics, such as unwanted behavior and lower human sociability are associated with avoidant attachment style. Overall, this study highlights the significance of individual traits in both pets and owners contributing to insecure attachment styles and underscores the potential to enhance the well-being of both pets and their owners through a deeper understanding of these traits.  •Anxiously attached owners scored lower in mental wellbeing and higher in neuroticism•Cats' activity and dogs' fear-behavior were linked with higher attachment anxiety•Attachment avoidance was associated with aggression and ADHD-like behavior in dogs•Human sociability was associated with lower attachment avoidance in both pet species Anthrozoology; Canine behavior; Feline behavior; Psychology These owners had filled at least one survey module of themselves and one of their animals. Of all the pet owners, 2,508 of the pet owners identified as women, 79 as men, 28 as other or did not want to tell, and for 109 gender was unknown, and the modal age group was 25–29 years. Descriptive statistics are presented in Overall, the first model reached best model fit for cat owners and dog owners . In thisIn the final model of dog owners, absolute fit indices indicated good model fit: RMSEA . Moreover, unlike many previous studies examining the relationship between the human and the pet, we used a validated method to assess the quality of the relationship. However, due to the gender bias and the primarily Finnish sample, caution is needed when applying these findings to pet owners of different genders or other cultural contexts.,,,This study follows previous findings of Reevy and Delgado,,,Other personality traits of the owner were also associated with attachment insecurity. Based on previous studies, we expected that extraversionHuman sociability of dogs and cats was associated with lower avoidant attachment in this study. In other words, the pet’s own tendency to seek closeness and interaction in the relationship was associated with a similar tendency in the owner. Human sociability in dogs has been previously linked to more positive owner-perceived attributions of the dog.Activity/playfulness in cats was associated with higher attachment anxiety in pet owners. To our knowledge, the association of these personality traits with attachment insecurity has not been studied. High activity level of a cat could elicit feelings of inadequacy in owners, if these cats do not seem content despite extensive environmental enrichment.,Unwanted behavior of dogs was associated with attachment insecurity. Based on previous studies, we expected that aggression of dogsIn our study, we did not explicitly delve into the direction of causation or the extent of interdependence between the personality traits of humans and their pets, which would require longitudinal research. Personality traits are influenced by both genetic factors and environmental experiencesIn conclusion, traits of pet owners, more specifically poor well-being and neurotic personality, are linked to the anxious pet attachment style. In turn, the pet’s personality and behavior, such as lower human sociability and unwanted behavior, seem to be related to the avoidant attachment style. Human sociability appeared to be a favorable trait of both pet species regarding attachment avoidance. These results may have practical implications for enhancing human-pet relationships. For example, recognizing the link between owner neuroticism and poor mental well-being with anxious pet attachment enables pet owners to become more conscious of their emotional responses and potentially seek ways to offer greater comfort and stability to their pets. Additionally, when pet owners seek assistance for their pet’s behavioral issues, it may be possible to enhance interventions by also considering the owner’s attachment style. Furthermore, a deeper understanding of these connections can assist prospective pet owners in making more informed decisions when acquiring a pet. It is crucial to acknowledge that obtaining a pet while experiencing poor mental well-being may not necessarily meet expectations of improving one’s psychological health. Instead, acknowledging the potential risk of an insecure attachment is crucial, as it could potentially harm the well-being of both the pet and the owner.,,,Future research holds exciting prospects for investigating the causal mechanisms behind the associations we have observed. Potential differences in the training methods of avoidantly and anxiously attached pet owners and associated factors to secure attachment style (when low levels are observed in both dimensions) would be exciting topics for future research. In addition, complementarity between pet owners and their pets has been previously linked to higher relationship satisfaction.,,,,,This study has limitations. Directionality or causal relationships between personality traits and attachment cannot be derived due to the cross-sectional design of this study. However, these results provide an initial understanding of how cross-species personality may be related to attachment, and future studies should aim to study the causal mechanisms underlying the associations. The second limitation is the limited diversity of the sample, as participants predominantly identified as female and were of Finnish origin. Previous studies have indicated gender differences in how an individual is attached to one’s pet,hannes.lohi@helsinki.fi).Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Hannes Lohi .To collect behavioral, personality and background information from dogs, we used the validated dog personality and unwanted behavior questionnaire.To collect behavioral, personality and background information from cats, we used the validated questionnaire of feline behavior and personality.lavaan,,,,We used structural equation modeling (SEM) with the package


Seeing the palpable impact of food poisoning associated with catering operations, we surveyed caterers with and without past hygiene violations to examine staffing, food safety practices and correlations with microbial counts in food and the processing environment. Past infringements did not negatively impact the current execution of food safety measures nor the microbial quality of food. In preference to added stringencies for errant operators, we discuss alternative efforts to augment food safety, as well as the policy implications thereof. Commercial catering premises are frequently identified as settings of foodborne disease outbreaks . FoodborTo the best of our knowledge, data on the causative agents, contamination events or hygiene non-conformities leading to food poisoning at catering sites are limited, and epidemiological investigations following such incidences may be challenged by the lack of contemporaneous information to establish links between contamination sources and affected cases. However, studies have reported food safety concerns in catering services to include cross-contamination due to bulk food preparation, food hygiene non-conformities by caterers taking orders beyond capacity and time-temperature abuse due to prolonged storage of ready-to-eat (RTE) dishes at improper temperatures ,5,6.Noting the reported risk factors and with a general guiding principle to identify practices within a company’s activities that are critical to food safety, caterers’ manpower resources and hygiene control practices during bulk food preparation were surveyed through interviews and on-site observations. The study aimed to elucidate whether hygiene non-conformities and poor microbiological counts could be associated with the allocation of manpower resources ; or with the practice or omittance of certain hygiene measures. The rationale for stratifying caterers according to outcomes of past hygiene inspections was based on the objective to explore whether there could be notable differences in the way caterers with and without prior hygiene non-conformities implemented food safety procedures.Gathering such food safety insights on catering businesses is important for awareness of how food safety measures including good personal and food hygiene practices are implemented among catering establishments. Such awareness could help authorities and industries to inform food safety policies and strengthen food safety practices towards risk mitigation.Via stratified random sampling, we selected participants for this descriptive analysis—10 caterers with at least 1 hygiene-related non-conformity found during routine hygiene inspections, and 10 caterers without.To select potential study participants, a list of inspection records conducted on caterers within a 2-year period (2017–2018) was generated; each caterer in this list was then assigned with a unique identifier. The Microsoft Excel randomization function was applied and subsequently used to randomly select 10 unique identifiers with and without history of hygiene non-conformity. The caterers corresponding to these unique identifiers were contacted and verbal permission was sought, prior to the study team’s visit to the catering premise for collection of data and samples.To gain insight on business practices that may impact food safety, data on the caterers’ typical scale of operations, corresponding staff strength and on the management of increased orders during peak seasons were collected through a researcher-administered questionnaire. One food safety manager, senior-level staff or the business owner themself was interviewed at each premise.Time-temperature and contamination controls as well as food handler hygiene practices were assessed using an observational checklist developed with reference to the Singapore Environmental Public Health (Food Hygiene) Regulations  and on gA total of 86 samples of food and 120 swabs of direct food contact surfaces  and surfaces in close proximity to food  were collected for the microbiological analysis. At each catering site, ~250 g of each sample from up to 6 types of dishes designated for delivery on the same day were aseptically collected and kept in sterile bags. Sterile wooden cotton-tipped applicators were used to swab food contact surfaces; these were suspended in 9 mL of Butterfield’s phosphate buffer. Both food and swab samples were then stored in cooler bags and transported to a commercial testing laboratory and refrigerated immediately (~4 °C) until the time of testing.As a proxy measure for growth of viable bacteria, the laboratory determined aerobic plate counts of the samples . This was done to determine potential sources of cross-contamination during mass production of food, and to establish correlations between microbial findings and the implementation of food hygiene controls.p-value of <0.05 was considered significant.Statistical analyses were performed using IBM SPSS Statistics version 26. Kruskal–Wallis test was used to compare the microbiological profiles across different food samples (dishes) and swabs (food contact surfaces). A p > 0.05). While it could be expected that a lower volume of orders would allow for more careful attention to food safety, our results instead showed that caterers preparing daily orders at/below median had higher Aerobic Plate Counts  in food samples compared to caterers with daily orders exceeding the median. As the association between caterers’ past record of hygiene violation and scale of operation could not be established, it appears that other factors such as staff strength and the company food safety culture could play a role in the occurrence of hygiene infringements and the execution of food safety measures when preparing food in bulk.Caterers’ scale of operation and manpower resources are summarized in During local festive periods when orders would surge, caterers reported preparing up to five times the number of meals normally made. During these peak seasons, however, 40% of respondents informed us that no adjustments are made despite the increase in workload, while some caterers coped by increasing temporary hires, adopting automation, outsourcing certain food preparations, purchasing RTE food or by limiting orders . WithoutThe above scenarios highlight the importance of having a food safety-oriented company culture, where value is placed on the formation and ownership of positive food safety habits by all members of the organization, at each stage of the catering operations. This begins with business owners and managers who recognize the significance and are committed to ensuring the safety and traceability of food produced in their facilities . A food Food hygiene practices observed across catering sites are given in In terms of cross-contamination controls—the majority of caterers (≥80%) practiced the segregation of raw and RTE food throughout its operations, but soiled premises  and food contact surfaces  were observed when there was no on-going food preparation activities both at premises with and without prior hygiene non-conformities , see p-value > 0.05) in microbiological profiles were observed between RTE food samples collected from premises with and without prior hygiene non-conformities. Similarly, no significant associations could be established between microbiological profile and the implementation (or lack) of hygiene controls in premises where they were prepared. Rather, poor microbiological profiles were observed to be correlated to certain food types, whereby dishes consumed raw or containing raw ingredients ; and dishes subjected to heavy post-cooking manipulation such as desserts accounted for the ~10% of food with high APCs. Noting the differences in the natural microflora in food, dishes that contain ingredients with inherently higher APCs (such as raw vegetables) should therefore be handled with extra precaution, to avoid further contamination of the ingredient, or cross-contamination of other food items. p-value > 0.05). Chopping boards tended to have significantly higher bacterial counts  as compared to other Zone 1 food contact surfaces such as knives/slicers, food trays, and ladles as well as Zone 2 surfaces such as preparation tables, doorknobs and entrance/exit buttons. This finding is not surprising; a substantial bacterial load can remain on boards with hard-to-clean cracks or crevices from prolonged use and can persist even with frequent rinsing before and after every use [About 10% (9/86) of RTE food collected from catering establishments had poor microbiological profile (as approximated by APC exceeding 5 log CFU/g). The median APC of all food samples collected was ~2 log colony forming units per gram (CFU/g), and no significant differences  to closer monitoring of errant operators, it may thus be more fruitful to focus efforts on ensuring the safe preparation of higher risk food items, as well as on staff training. In Singapore, as in other nations ,28,29, fTo further contextualize food safety training for catering operations, module content may also mention the adoption of practices such as those noted in this study—the use of automation to cope with larger orders, the use of blast chillers to rapidly cool cooked food and the use of thermometers to ensure thorough cooking—as well as the clear articulation of how each food handler’s habits can impact the entirety of an order and the wellbeing of multiple consumers. Organizations can further facilitate a food safety culture by providing ancillary training for staff to upskill in terms of numeracy ; in risk assessment ; as well as in book-keeping . With ancillary training, management may also consider providing subsequent support and reward structures to drive the application of concepts learned; a study on the attitudes of industrial caterers towards training programs indicated that follow-up and support from management fosters a positive attitude and motivation among employees to apply learnings in daily operations . Such prNotably, the majority of catered foods sampled for this study had total bacteria loads below 5 log CFU/g, affirming sound efforts by the industry and by local authorities in safeguarding catered food. However, given the heterogeneity and volume of food prepared at catering establishments, a careful analysis of the product’s characteristics and the process conditions required to minimize associated hazards would be worthwhile, whereby the preparation of food types more prone to spoilage, undercooking and higher microbial loads  are afforded as much attention allowable, without compromising the microbiological safety of lower-risk food types . A similar risk-based approach may also be adopted during inspections such that the preparation of higher-risk foods or food operations catering to vulnerable communities be more closely observed and immediate guidance be provided, should food safety measures be found lacking, or should reluctance to practice safe measures be perceived among food handlers. It has been shown that a food safety regimen guided by food type, process controls and intended population could mitigate or reduce food-safety risks ,33.Lastly, in highly industrialized places such as Singapore where space is limited and manpower is costly, the commitment of food business operators to instill food safety culture, the competence of workers to hygienically handle food, and the ability of food service managers to effectively coordinate resources and leverage on automation are vital to ensuring the safe bulk preparation of food. Likewise, implementing effective controls at points critical to catered food (such as maintaining food-holding temperatures from kitchen to consumer) and having easy-to-follow education modules that emphasize safe mass production of food and encourage a wider adoption of best practices  could further strengthen the catering industry’s acumen against potential food safety risks. Although our study could have benefitted from a larger sample size, it shows that when large amounts of food need to be prepared in a short amount of time, prioritizing vigilance on high-risk food types and on key processing areas are imperative to minimize downstream incidences of foodborne illness and to provide consumers with safe and wholesome food. Nevertheless, our findings put forth important considerations for the formulation, review and further strengthening of food safety policies.


Among the numerous contributions of Geoffrey Bodenhausen to NMR spectroscopy, his developments in the field of spin-relaxation methodology and theory will definitely have a long lasting impact. Starting with his seminal contributions to the excitation of multiple-quantum coherences, he and his group thoroughly investigated the intricate relaxation properties of these “forbidden fruits” and developed experimental techniques to reveal the relevance of previously largely ignored cross-correlated relaxation (CCR) effects, as “the essential is invisible to the eyes”. Here we consider CCR within the challenging context of intrinsically disordered proteins (IDPs) and emphasize its potential and relevance for the studies of structural dynamics of IDPs in the future years to come. Conventionally, dynamics of globularly folded proteins are modeled and understood as deviations from otherwise rigid structures tumbling in solution. However, with increasing protein flexibility, as observed for IDPs, this apparent dichotomy between structure and dynamics becomes blurred. Although complex dynamics and ensemble averaging might impair the extraction of mechanistic details even further, spin relaxation uniquely encodes a protein's structural memory. Due to significant methodological developments, such as high-dimensional non-uniform sampling techniques, spin relaxation in IDPs can now be monitored in unprecedented resolution. Not embedded within a rigid globular fold, conventional  By considering his experiments within the challenging context of intrinsically disordered proteins (IDPs), we want to emphasize their potential and relevance in the future years to come. Arguably, this rediscovery might require some collective effort, as current trends appear to point in the opposite direction. As Paul Schanda put it recently: “The popularity of detailed spin-relaxation measurements in liquids, clining; … with lHowever, with increasing protein flexibility this apparent dichotomy becomes blurred as structure and dynamics can no longer be considered independent of each other. While complex dynamics and ensemble averaging obfuscate mechanistic details even further, the structural information content of relaxation parameters becomes increasingly apparent. In comparison to simple population-averaged quantities, such as chemical shifts or scalar couplings, spin relaxation uniquely encodes a system's structural memory, i.e., the temporal persistence of concerted motions and structural arrangements. Somewhat counterintuitively, spin-relaxation experiments are among the prime sources of structural information available for disordered systems. However, due to a general lack of analytical descriptions for IDP dynamics This aspect alone suggests a systematic reassessment and evaluation of less commonly employed experiments. Far more pressing, in our opinion, is the inherently local nature of spin relaxation in IDPs. In contrast to folded proteins, spins in IDPs are not embedded within a fixed molecular tumbling frame. Thus, a single More immediate in its structural implications would be the presence of diffusion anisotropy, which has been hypothesized to be of substantial size even in highly disordered proteins such as Arguably, this somewhat ambiguous body of evidence illustrates the inevitable difficulties that come with extending concepts of folded proteins to IDPs. In fact, many of the above observations might very well be case-dependent. In the present study, we want to approach this question in a more agnostic manner. Are there experimental ways to better detect the signature of anisotropic dynamics in IDPs? At what level of evidence could we evoke the mental image of extended chains and For IDPs in particular, 2Our aim is to define an experimental measure for anisotropic segmental dynamics in IDPs. While the measure itself should be general, the considered source of anisotropy will be rather specific. To assess the sensitivity of the proposed protocol, we resort to the simplified image of extended chain and For most processes, the TCF can be described as a sum or distribution of exponential decays The spectral density of Eq.  corresponds to a simplification of the MF-like extension proposed by 3We will assume the canonical peptide plane geometry of  is the reduced Planck constant, As demonstrated by To complement these rates, we consider their counterparts for the While the measurement of transverse CCR is well established, longitudinal CCR has been studied considerably less. In part this is due to subtleties of the involved relaxation pathways which involve multi-exponential auto- and cross-correlated relaxation effects. Another reason lies in technical limitations as longitudinal relaxation rates are generally smaller due to their Larmor frequency dependence. Notably, this effect is far less pronounced for the smaller correlation times present in IDPs (cf. Fig. Aside from the symmetrical reconversion principle of Bodenhausen and coworkers To obtain sufficient spectral resolution, the CCR rates are measured directly from the intensity difference in a To suppress With the general feasibility of the measurements demonstrated, we can now define a ratio To assess the sensitivity of 4Experimental considerations necessarily result in compromises. The fully anisotropic Figure Besides the obvious influence of We conclude that, if the concept of anisotropic tumbling of segmental Thus, while introduced and assessed in terms of diffusion anisotropy, we expect the combination of transversal and longitudinal 5On the occasion of Geoffrey Bodenhausen's 70th anniversary, we built on his extensive body of work to conceptualize experimental means for the investigation of anisotropic segmental dynamics in IDPs. Spectral density mapping protocols based on transversal and longitudinal CCR of l'art pour l'art pulse sequence design is also a telling testimony for the unforeseeable impact of non-utilitarian basic research driven by and inspired by scholarly thinking.While detecting and quantifying the presence of anisotropy in IDP dynamics might seem like a humble academic endeavor, we believe it to be an important step not only towards a better understanding of this important protein family but also towards immediate applications in biological and biomedical research as well as drug design. We thus take particular delight from the fact that Geoffrey Bodenhausen's 10.5194/mr-2-557-2021-supplementThe supplement related to this article is available online at: https://doi.org/10.5194/mr-2-557-2021-supplement.


Editorial on the Research TopicHelping meet oral health needs in underserved communitiesThe turn of the century was a seminal moment for oral health in the United States (U.S.). In the year 2000, Surgeon General Dr. David Satcher released the first ever Surgeon General's Report on oral health titled, “Oral Health in America: A Report of the Surgeon General” . In addiHelping Meet Oral Health Needs in Underserved Communities.” The primary objectives of this topic were to disseminate current research that is elucidating the increasingly complex drivers of oral health inequities and highlight innovative interventions aimed at improving the oral health of underserved populations.Over the last 20 years, the oral health community and its stakeholders have taken heed to the call to action to address oral health inequities through several local and national initiatives. While these efforts are to be commended, inequities in oral health status still exist for many underserved groups, as highlighted by the more recent “Oral Health in America: Advances and Challenges,” a 2021 follow-up report to the original Surgeon General's report on oral health . RecogniLee et al. contributed findings from their community health worker (CHW) intervention. As part of overall oral health promotion, CHWs attempted to address social determinants of health (SDOH) and psychosocial factors that often plague caregivers in underserved communities and are associated with poor oral health behaviors. Although the intervention did not result in positively changing psychosocial factors as hypothesized, the results are beneficial in informing future oral health interventions. The results demonstrated the importance of targeting SDOH in community-based interventions and highlighted the need for validated instruments that better capture psychosocial factors prevalent in underserved communities.Frameworks that highlight multi-level influences on oral health and inequities have been developed to guide interventions. For example, a model by Fisher-Owens demonstrates that a child's oral health is influenced not only by their individual factors, but also those of their family and community . ConsideSmith reinforced the ethical obligation oral health stakeholders have in addressing health inequities. He proposed a systems-oriented ethical decision-making framework that stakeholders can use to consider the social and structural determinants of health that disadvantaged populations face when making care delivery decisions. If implemented, use of this ethical framework could result in improved quality of oral health care delivery to underserved communities, ultimately improving oral health outcomes and reducing inequities.Other multi-level frameworks have been developed to conceptualize influences on oral health, such as one proposed by Lee and Divaris . ImportaSmith et al. discussed a model of dental education that could be used to increase students' awareness of the impact of SDOH on oral health inequities so they are better prepared to deliver holistic, patient-centered care to diverse communities as practitioners. Findings from reflection assignments demonstrated that foreign-trained dentists who participated in community-based dental education (CBDE) expressed having a better understanding of: 1) how social and structural determinants of health impact access to oral healthcare for underserved communities, and 2) their professional responsibilities in addressing these barriers. Therefore, utilizing CBDE during dental training can be a promising workforce strategy used to increase future dental practitioners' awareness of drivers of inequities, so they are better prepared to address them when they enter practice.Atchison et al. presented findings on emergency department (ED) use among developmentally disabled (DD) adolescents with oral health complications. Using data from the National Survey of Children's Health, they found that DD adolescents with oral health problems were more likely than their counterparts to use the ED. They also found that not having a medical home was associated with increased ED use. Their results highlight the important role that integrated medical-dental health systems can play in reducing oral health inequities, particularly among those with DD and other medically-vulnerable groups.Individuals with developmental disabilities are an underserved group that is often overlooked. Previous research has shown that these individuals commonly have unmet dental needs, and face barriers in accessing preventive dental care –10. AtchBurns et al. analyzed state Medicaid administrative claims data to assess receipt of root canal therapy (RCT) and subsequent treatment outcomes in Medicaid insured children and adolescents by demographic factors. They found that African American and Hispanic children were less likely to receive multiple RCTs, less likely to have a permanent restoration placed after RCT, and more likely to have failed RCT treatment, compared to their White counterparts. While the results observed cannot be directly attributed to racism, the unequal treatment provision and outcomes based on race/ethnicity, highlight the need for more research, policies, and interventions aimed at mitigating structural racism and implicit bias, as a way to achieve equitable health outcomes. Addressing structural racism is a dental public health priority (More research is needed to better understand disparities in the provision of oral health services and treatment outcomes. priority .While there is still much work to be done to meet the oral health needs of vulnerable and underserved communities in the U.S. and globally, we believe that the diverse research we assembled provides important findings that can help inform future research, practice, and policies to achieve the vision of oral health equity for all.


The hippocampus has a well-established role in spatial and episodic memory but a broader function has been proposed including aspects of perception and relational processing. Neural bases of sound analysis have been described in the pathway to auditory cortex, but wider networks supporting auditory cognition are still being established. We review what is known about the role of the hippocampus in processing auditory information, and how the hippocampus itself is shaped by sound. In examining imaging, recording, and lesion studies in species from rodents to humans, we uncover a hierarchy of hippocampal responses to sound including during passive exposure, active listening, and the learning of associations between sounds and other stimuli. We describe how the hippocampus' connectivity and computational architecture allow it to track and manipulate auditory information – whether in the form of speech, music, or environmental, emotional, or phantom sounds. Functional and structural correlates of auditory experience are also identified. The extent of auditory-hippocampal interactions is consistent with the view that the hippocampus makes broad contributions to perception and cognition, beyond spatial and episodic memory. More deeply understanding these interactions may unlock applications including entraining hippocampal rhythms to support cognition, and intervening in links between hearing loss and dementia. •Hippocampus responds to sound during passive exposure, active listening and learning.•Auditory experience shapes the structure and function of hippocampus.•Sound can entrain hippocampal oscillations, with potential clinical implications.•Recording, imaging and lesion studies are reviewed in species from mouse to human. One ideThere are also practical and clinical motivations for understanding interactions between sound and the hippocampus. Auditory stimulation can entrain hippocampal rhythms, with implications for enhancing memory and mitigating cognitive decline . AuditorRecognizing the relative preservation of hippocampal anatomy and physiology across species, and the advances in understanding function this affords , we inclDominant accounts of hippocampal function, as well as key physiological properties, are briefly introduced as required, but readers are referred to detailed reviews  cortices, which connect to ERC from anterior and posterior directions respectively as shown in In rodents, subcortical pathways also carry auditory information to the hippocampal formation, including one from cochlear nucleus via pontine nuclei and medial septum , and anoEstablishing the extent to which both these standard and non-canonical pathways in rodents and non-human primates are mirrored in humans is difficult. In the absence of axonal tract tracing or post-mortem studies, indirect measures of structural connectivity such as diffusion tensor imaging (DTI) can be informative. A neurosurgical atlas based on data from the human connectome project highlighted the absence of direct connections between medial and lateral temporal structures, although indirect pathways were functionally established that correspond to some of those outlined in non-human primates, such as from auditory association areas on the superior temporal gyrus to posterior parahippocampal fields then entorhinal cortex . One ultassociation areas in humans, with the possible exception of olfaction, compared to the primary sites that dominate in rodents  activity measured with functional magnetic resonance imaging (fMRI) has revealed distinct connectivity between different parts of the hippocampus and neocortical regions. Whereas activity in posterior hippocampus and parahippocampal cortex correlates with activity in lateral parietal cortex and midline sites, activity in anterior hippocampus and perirhinal/entorhinal cortex correlates with that in lateral temporal regions including superior temporal gyrus extending to temporal pole . Cluster rodents .Electrophysiological and fMRI studies coupled with electrical or optogenetic stimulation also reveal pathways relevant to auditory processing. The hippocampus orchestrates activity across cortex, propagating theta oscillations (4–7 Hz in rodents) for temporal control of information processing, and sharp-wave ripples for memory consolidation . For exaThe influence of the hippocampus on auditory processing elsewhere is also revealed by lesion studies in animals, and in patients. For example, the neonatal ventral hippocampal lesion rat, a model for neurodevelopmental aspects of schizophrenia, shows altered responses to sound in inferior colliculus and auditory cortex compared to controls, such as reduced power of the 40-Hz auditory steady state response (ASSR) . ASSRs aIn sum, multiple pathways are available for auditory information to reach the hippocampus, and for the hippocampus in turn to influence activity at canonical auditory structures. Although the most direct have so far only been anatomically verified in rodents, some electrophysiological studies hint at their presence in humans.3Sounds that hold no meaning for a passively listening animal elicit a number of forms of hippocampal response see  as well Different classes of hippocampal cells can be distinguished based on anatomy and physiology, including the relationship of their firing to ongoing hippocampal theta oscillations . InhibitHippocampal responses to sound can be brief, persist throughout or beyond the stimulus, or be phasically modulated by its temporal structure ; Fig. 3CMany of the hippocampal responses described above, whether at the single cell or evoked potential level, are not specific to particular sounds but rather scale with intensity. Because the hippocampus is involved in a range of cognitive and spatial processing, such unselective responses are hard to attribute to auditory processing per se rather than to general arousal or orienting. However, there are exceptions to this lack of selectivity during passive exposure: A key factor influencing the magnitude of hippocampal responses to sound in the absence of behavior is stimulus history. Although habituation to a repeated auditory stimulus is a widespread neural phenomenon , it is pSelective habituation to identical stimuli gives rise to a neural indicator of the presence of novelty. In many species, any perceptible change in stimulus after repetition can be sufficient to restore a large hippocampal response, with a greater effect for larger differences and more rarely occurring sounds . Althoug4. Such differential responses can arise between tones of different frequencies, or between more complex sounds such as artificial vowels with different formant structure  with a subsequent reward or punishment (unconditioned stimulus) elicits changes in hippocampal activity over the course of learning see  D. Diffetructure . Condititructure . This cotructure  and can tructure .The temporal pattern of firing that develops during conditioning may reveal something of what is learned. During eyeblink conditioning, animals learn to associate a tone with a subsequent air-puff . As the Although most hippocampal recordings during conditioning with auditory stimuli have been made in rats and rabbits, some of the principal findings have been replicated in cats . Humans Despite these extensive learning-related changes in neural activity, animals with hippocampal damage are not impaired in many conditioning settings  H. Multi5Once sounds have acquired behavioral relevance, hippocampal activity can be examined as animals perform tasks relating to them see  E. For eIn human subjects, behavioral relevance can be instilled in the absence of explicit reward through task instructions, such as to count particular target sounds or to make certain judgments. By having subjects report target counts at the end of a trial or block, explicit motor confounds are removed. In contrast to the passive listening case in humans described earlier, active detection of rare frequency  or intenEven if a subject is not aware of what distinguishes a particular sound from others, previous exposure can result in the formation of distinct hippocampal representations that are revealed during subsequent active listening. For example, 6As mentioned earlier, whereas animals with hippocampal lesions are able to learn the association between a conditioned stimulus and unconditioned stimulus if these are presented in an overlapping or abutting fashion (“delay conditioning”), the insertion of a silent interval between the two (“trace conditioning”) renders these animals impaired. In rodents, persistent firing of individual hippocampal neurons over the trace interval is rare . HoweverThis bridging of a silent interval by assemblies of hippocampal cells, each with its own temporal firing field, is not limited to conditioning paradigms in which a sound is to be associated with a subsequent appetitive or aversive stimulus. Hippocampal “time cells”  are alsocritical role for hippocampus in maintaining sound features over a temporal interval, outside of a conditioning setting? Lesioning the fimbria-fornix input to rat hippocampus impairs their ability to remember the presentation rate of click trains, as well as their duration  could be linked based on the phase in a theta cycle at which corresponding neural assemblies fire. Direct experimental support for this kind of auditory phase precession is currently lacking. We know that in their firing  to rats  or image to rats . Additio to rats , mirrori to rats . Neuroim to rats .In addition to these univariate markers of increased activity during sequence learning, representations of specific sound sequences emerge in hippocampus over the course of exposure. In one fMRI experiment, multivariate patterns of activity in left hippocampus came to encode the identity of ordered sequences of spoken letters that repeated over the experiment, even though the individual elements were shared across all sequences . AnotherSome have argued that sequences of sensory content  become associated with pre-existing hippocampal cell assemblies that fire in a particular order, while the sensory elements themselves are represented in neocortex . Relatedreduced in tone sequences in which simple or hierarchical rules concerning pitch and duration were violated compared to when they were met , comes from findings that BOLD activity there scales with the vividness of a retrieved episodic memory, including its auditory content . FurtherWhich memories get consolidated in humans during sleep can be biased by presentation of relevant sounds. In one study, subjects learned object-location pairs while presented with an object-specific sound, then slept during a scanning session . Greater14Speech  F, J andBeyond online speech perception there is substantial neuropsychological evidence for left hippocampus in particular playing a key role in the learning and recall of verbal material . Neuroimencoding of memory for words, deficits in consolidation have been identified in groups with presumed hippocampal damage. Patients with transient epileptic amnesia  for explicit recollection of an episode, compared to general familiarity likely to be supported more by parahippocampal/perirhinal circuits . However16Also hard to dissociate from sound familiarity are sound preference and other emotional drivers of hippocampal activity, both due to “mere exposure” effects of repetition  and the Medial temporal sites - and the focus is more parahippocampal gyrus than hippocampus proper - are involved in processing emotion in music, beyond the familiarity effects described above. Lesion  and neurNot only simple musical emotional contrasts  but also subtler differences involving arousal or valence alone modulate (para)hippocampal BOLD activity and its relationship with that in auditory and reward networks , and indMusic is not the only means of expressing emotion through sound. (Para)hippocampal areas are also sensitive to different types of laughter , as well17Aversive auditory experiences do not require an external stimulus. Tinnitus see  L usuallThese negative links between hippocampal structure and tinnitus stand in distinction to functional studies that often  is an independent risk factor for dementia, estimated to account for 9% of cases . The hip20We have reviewed imaging, recording, lesion and neuropsychological work across species, charting diverse interactions between sound and the hippocampus. These include a hierarchy of responses, from reacting non-selectively during passive listening, through tracking associations between particular sounds and rewards, to mapping out auditory dimensions during behavior. We identified hippocampal involvement in linking sounds with each other, with stimuli in other sensory modalities, and with spatiotemporal context to form episodic memories. We also detailed roles of the hippocampus in processing music, speech, emotional sound, and aberrant auditory percepts, as well as how hippocampal structure and function can be shaped by auditory experience. We described how the machinery that supports spatial navigation and memory may be harnessed in more general sequential processing, including that relating to sound. Questions remain as to the extent to which the hippocampus helps build representations of auditory objects and scenes, which are structured in time, frequency and space. However, the synthesis of auditory-hippocampal interaction that emerges does not support an adequate account of hippocampal function based on spatial navigation and episodic memory. The auditory work requires an explanation based on broader aspects of perception and cognition.What does it mean to say the hippocampus is “involved in” a function? It may not be critical for a given task, but the stimulus and behavioral information it tracks becomes available either for an optimal solution to the current problem, or as part of a more complex representation than is available upstream and/or that may be drawn on subsequently. Although single-neuron tuning to specific auditory features is most pronounced when they are behaviorally relevant  we have What characteristics of hippocampal circuits might lend themselves to auditory cognition? A non-exhaustive list could include: theta phase precession to track past, present and future in sound sequences; a monosynaptic pathway that can learn probabilistic contingencies between sounds and other stimuli; the means to separate patterns of signals arising from distinct auditory objects or in different contexts through sparseness and inhibition in dentate gyrus; the means to compare stored representations in CA3 with, or to complete, or predict from, current auditory input; connectivity with subcortical and cortical sites that provide auditory information processed with respect to distinct features or to different extents.A number of steps can be taken to further test the idea that circuits in hippocampus and entorhinal cortex that support memory and navigation in physical space can be harnessed for auditory processing. Neuroimaging work that has indirectly found evidence for grid-cell like structures supporting non-spatial navigation (e.g. of semantic spaces) could be extended to cover auditory dimensions. Human intracranial recordings may provide more direct evidence for tuning to auditory features if coupled with the right task. Methodological advances in magnetic sensory technology, headcasts and source localization may facilitate temporally resolved magnetoencephalographic hippocampal recordings during listening .An important outstanding question is the extent to which spatial coding and processing in the hippocampus dominates over other dimensions of experience. Relatedly, is the hippocampus only engaged in forming auditory scenes or objects when there is an element of spatial variation rather than when spectral and/or temporal information alone are present? Experiments are needed in which subjects have to perceive, remember or mentally construct combinations of sounds composed of different feature conjunctions . Comparable experiments with visual objects by Maguire and colleagues  have beeMore straightforward analogs of experiments in other sensory modalities can answer pressing questions that have already been addressed for olfaction (in rodents) and vision (in humans and non-human primates), and that might be even more relevant for audition for which stimulation always unfolds over time. For example, does the phase at which rat hippocampal neurons fire for sequential sounds precess as the sequence unfolds c.f. ? Can calWe have seen how rhythmically structured sound at a range of timescales provides a non-invasive means of driving or entraining neural oscillations, which hippocampus plays a role in orchestrating across cortex. There are promising signs that such auditory interventions can improve memory and even disrupt pathology in animal models of disease. Compared to electrical, magnetic and invasive stimulation, an acoustic approach is accessible, inexpensive and generally non-intrusive/intimidating. To harness its full potential it will be important to understand how the auditory pathway and hippocampus work together with other regions including prefrontal cortex in bringing about these changes. More broadly, although we have focused on the hippocampus throughout this piece, considering such a richly connected structure in isolation will have only given part of the picture - whether with respect to conditioning, sound sequences, spatial and temporal aspects of sound, auditory memory, musical emotion, or phantom percepts.If the findings from animal models concerning links between hippocampus, hearing loss, tinnitus and dementia are to translate into to clinical advances in humans it is important that we understand the degree of overlap between species in terms of anatomy, connectivity, physiology, and function. Recent results are reassuring in this regard. For example the identification of human hippocampal place cells, time cells, and phase precession - originally discovered in rodents - indicate that at least some of the coding and computational principles overlap. At the same time, post-mortem anatomical data on human auditory-hippocampal pathways is almost non-existent. We also acknowledge that different species evolved in the context of unique environmental constraints, with a range of sensory and higher-level capabilities . DespiteIn conclusion, the hippocampus receives all manner of auditory information regardless of its behavioral relevance at the time. Any tuning to acoustic features or criticality of involvement is strongest when there is a requirement to associate sounds with locations, rewards or punishments separated in time, other sounds, or stimuli in other sensory modalities - either for perception or memory. The structural, synaptic and biochemical components that facilitate such processing are themselves sensitive to the organism's auditory experience - both in the short and long-term.