Early and accessible testing for influenza with point-of-care testing (POCT) can be a critical factor for deciding to begin antiviral treatment. More than 10,000 pharmacies across the USA offer Clinical Laboratory Improvement Amendments-waived POCT for infectious diseases, such as influenza A/B. Knowledge of barriers and facilitators to large-scale POCT implementation may be useful in scaling POCT for influenza test-and-treat services (Flu POCT). The objective of this study was to explore the experiences of pharmacists who were early adopters of Flu POCT and treatment under collaborative practice agreement in community pharmacy settings.Qualitative research design with in-depth, semi-structured virtual video interviews of licensed US community pharmacists. Interview questions were derived from the Consolidated Framework for Implementation Research (CFIR). Interviewees were selected via a purposeful sampling of pharmacists who were enrolled in a nationwide clinical trial involving pharmacy-based influenza test-and-treat under a collaborative agreement. Interviews were recorded and transcribed. A deductive analytic approach was used via constructs from the CFIR.Six pharmacists were interviewed. Interviews ranged from 28 to 70 min, with an average length of 46 min. Four broad themes emerged from the data, and each had corresponding subthemes and supporting quotes: influence of the Flu POCT service characteristics on pharmacy implementation, influence of factors outside of the pharmacy setting in Flu POCT implementation, factors within the pharmacy setting influencing implementation, and process of implementing Flu POCT. A novel pharmacy-based Flu POCT implementation framework is presented.Implementation of community pharmacy-based Flu POCT services is feasible; but, a thorough understanding of both barriers and facilitators to their implementation is needed to increase the spread and scale of these programs. Specifically, pharmacy stakeholders should focus efforts on increasing patient and provider awareness, pharmacist acceptance, leadership support, and support of health providers external to the pharmacy to improve implementation success.The online version contains supplementary material available at 10.1186/s43058-022-00324-z. Contributions to the literatureThe community pharmacy represents an underutilized public health access point for acute infectious disease prevention and treatment as highlighted by the COVID-19 pandemic.Early and accessible testing with Flu POCT can be a critical factor for deciding to begin antiviral treatment.In many U.S. states, pharmacists may assess the patient and subsequently furnish antivirals under collaborative practice agreements using CLIA-waived POCT —expanding care access using the existing workforce.Existing literature suggests that Flu POCT is feasible and effective when implemented in several areas of the U.S.This study captures implementation perspectives from such “early adopter” pharmacies and puts forth a novel implementation framework to guide future implementation efforts.The Centers for Disease Control and Prevention (CDC) estimate during the 2019–2020 influenza season more than 39 million people developed influenza, leading to more than 18 million healthcare provider visits. Annually, between 410,000 and 740,000 Americans are hospitalized due to influenza [Guidelines on the management of seasonal influenza from the CDC and Infectious Disease Society of America (IDSA) suggest beginning antiviral treatment within 48 h of symptom onset with suspected or documented influenza infection , 3. For Early and accessible testing for influenza can be a critical factor for deciding to begin antiviral treatment. Historically, viral culture laboratory testing was the standard of practice for influenza diagnosis. However, due to cost and turnaround time for results, its use is no longer recommended for initial or primary diagnosis . The CliMore than 10,000 pharmacies across the United States offer CLIA-waived POCT for infectious diseases, such as influenza A and B, and group A streptococcus . In manyAccording to Dulaney et al., community pharmacists reported willingness to perform POCT and recommend treatment for patients with influenza . ParticiWhen implementing CLIA-waived POCT in a community pharmacy setting several logistical challenges must be addressed, including establishing collaborative practice agreements, incorporating POCT into workflow, providing training for pharmacy staff, determining methods of remuneration, and following regulatory reporting requirements . BarrierWe used a qualitative research design with in-depth, semi-structured virtual video interviews of licensed U.S. community pharmacists. A phenomenological approach was selected to best understand the subjective experience of community pharmacists implementing the novel service of influenza POCT (Flu POCT) and treatment with antiviral therapy prior to the COVID-19 pandemic . The Unihttps://cfirguide.org) interview guide tool. Subsequently, the document was shared with the expert panel and the instrument was further truncated. Interviews occurred in the Spring of 2020 by two trained researchers (KH and CC). Interview sessions were audio-recorded digitally and professionally transcribed by a third-party transcription service. Interviews were conducted over virtual videoconferencing technology, recorded, and subsequently transcribed. Field notes were also collected during interviews to note non-verbal expressions and interactions and incorporated into the data analysis process.A semi-structured interview guide was developed by experts in influenza, POCT, and community pharmacy. Questions were derived from the Consolidated Framework for Implementation Research (CFIR), and the instrument was adapted based on an expert panel comprised of clinicians , researchers, and pharmacy leadership in line with guidance from the CFIR Research Team-Center for Clinical Management Research . The intInterviewees were selected via a purposeful sampling of pharmacists who were enrolled in a nationwide clinical trial involving pharmacy-based influenza test-and-treat under a collaborative practice agreement. Flu POCT implementation champions known to the researchers were selected as part of the stratified sample of pharmacists, which included varying geographic locations, sex, organizational roles, and practice setting . Recruitment occurred over email to each of the study site organizations and continued until a point of saturation whereby no new themes emerged with subsequent focus groups .A deductive analytic approach was used via constructs from the CFIR . Two codSix participants were interviewed, one for each of the pharmacy organizations approached. Demographics for interviewees are listed in Table This subtheme centered on participants’ perceptions of the advantage of implementing Flu POCT in relation to other pharmacy services. Participants opted to implement pharmacist-delivered Flu POCT over spending time in other pharmacy services for reasons of professional satisfaction by means of providing direct patient care, ability to serve new patients within the pharmacy, and offering services not offered at other pharmacies. Participants noted unanticipated facilitator for implementation was that the service primarily attracted new patients to the pharmacy.Flu POCT complexity centered on workflow integration, sample collection, and test supply procurement. Workflow integration of Flu POCT test-and-treat was customized to each pharmacy and required small changes, mostly in preparing the team how to handle the time when the pharmacist stepped away from their computer terminal and away from drug dispensing workflow. It was noted that these changes were similar to those made when first integrating vaccinations into workflow. Participants indicated initial staff pharmacist hesitance to perform sample collection via intranasal swab, but this was overcome with training and increasing experience. A major hurdle in the implementation of Flu POCT was obtaining the testing device, something not normal to pharmacy inventory, and this delayed implementation for some participants. In these instances, the gap between procurement of the Flu POCT device and pharmacist training served as an additional barrier as there became a need to refresh pharmacists on previously taught materials while awaiting Flu POCT machines and supplies.Both patient needs and barriers to meeting those needs were discussed. Specific to patient needs, participants noted that patients prioritize convenience, speed of care, and service access, but were mostly unaware of the pharmacy’s ability to meet these needs with the Flu POCT service. Participants articulated most patients using the Flu POCT service were not patients of the pharmacy, but had come to the pharmacy based on word-of-mouth recommendations or advertising. A resounding barrier among participants was the lack of patient awareness that pharmacists can perform Flu POCT. Similarly, for participants who had performed Flu POCT for one or more previous seasons, they noted how word of mouth, advertising, referrals, and social media facilitated awareness of how this service met patient demands for convenience and speed of care. It was also noted how the SARS-CoV-2 pandemic raised awareness of the expanded role of the pharmacist and how this may facilitate further expansion of the Flu POCT.This subtheme centered on the degree to which the pharmacy’s network of prescribers, hospitals, and medical offices impacted implementation. Participants noted in some cases there was initial resistance to participants performing Flu POCT, but that this was usually limited to a single medical practice or prescriber and overcome with proactive communication and time. Related to communication, participants emphasized the importance of communicating Flu POCT care plans to the patient’s primary care physician over telephone or fax as a key policy of their service.Broadly participants discussed the challenges of community pharmacy’s current business model and the need to expand or develop new patient care services offered. Participants at independent pharmacies noted the need to offer services not seen by the national pharmacy chains to differentiate themselves. Participants at chain pharmacies noted that they too were in competition, but with both the other chain pharmacies and independent pharmacies.Both future third-party reimbursement potential and advancing scope of practice were facilitators for Flu POCT. Participants noted that continued national and state-specific conversations around third party payment models drove interest from pharmacy leadership as a supplement to cash-based payment models. Also, the provision of a prescription antiviral after receiving a positive flu result and corresponding patient assessment  was essential to program success; therefore, working within states allowing expanded scope of practice or broad collaborative practice agreement rules facilitated both implementation and scaling of services. Participants noted that even with the regulatory ability to both test and treat influenza, there sometimes existed a barrier in finding a collaborating physician to oversee the program and partner under a collaborative practice agreement.This subtheme refers to the informal and formal communication strategies used by participants within their pharmacy. Generally, independent pharmacies used a more informal communication method  to implement and sustain Flu POCT, whereas chains used existing formal communication methods . Leadership and front-line communication were noted to be critical to success. Communication across settings generally occurred over email and occasionally telephone. Face-to-face communication happened most often between front line staff members or between members of the leadership team and was due to the close physical proximity of those individuals. However, these siloed communication loops were not found to serve as a barrier for implementation.The culture of pharmacies implementing Flu POCT was described as innovative, highly efficient, and clinically focused. Participants considered their organizations as “leaders of the pack,” and expressed pride in their innovative approaches to the practice of pharmacy. Overall the pharmacies were noted to have characteristics in line with Rogers’ definition of Early Adopter and Innovator organizations . OrganizThe implementation climate of the pharmacy is the “shared perception among intended users of an innovation, of the extent to which an organization's implementation policies and practices encourage, cultivate, and reward innovation use” . Like phIncentives were sometimes provided by organizations to support implementation—but this was not seen across all pharmacies. Reasoning to not specifically incentivize their staff centered on relying on the intrinsic motivation of pharmacists to take on new clinical services for their own quality of work life and professional satisfaction, and this was found to be a sufficient incentive for those pharmacies using that incentive. This concept of professional satisfaction was articulated by both management and front-line pharmacists.Participants noted that Flu POCT was overall fully compatible with existing workflow, work tasks, and physical layout of the pharmacy; although some minor workflow adaptations were required. Some specific physical aspects required included a private patient care room located in proximity or attached to the pharmacy department itself. It was also compatible with pharmacist views of their own professional responsibilities and professional capabilities.Participants noted readiness for implementation revolved around a few core items: advertising and patient awareness, training and education, and testing equipment. Pharmacies with longer experience providing Flu POCT had achieved each of these items; similarly, those who had not yet begun testing (despite a high degree of willingness) lacked the majority of the three.Engagement from top-level management teams was a key facilitator for implementation. This was especially true given the unique circumstance whereby patients were unaware of the pharmacist-delivered service. Marketing and advertising were two areas in particular where collaboration between departments was required and was largely organized by upper management and leadership. This included social media, flyers, bag stuffers, billboards, radio advertisements, and store signage. Furthermore, top-level management and leadership also organized and facilitated training & education for their pharmacy staff and procured testing supplies and equipment.Access to both the testing equipment, supplies, and supporting training and education were also noted to be crucial elements for successful implementation. As most pharmacy suppliers do not carry POCT equipment or supplies, new supply chains or ordering procedures needed to be developed for most pharmacies—causing several pharmacies to have initial delays in implementation. Similarly, despite having foundational knowledge in therapeutics and pathophysiology , most community pharmacy practices do not involve routine use of the knowledge required for Flu POCT. Therefore, participants referred to the value of “refresher” courses in infectious diseases, laboratory diagnostics, and related administrative topics .Planning was noted to be detailed and involved the creation of new policies and procedures in line with other pharmacy-based services. Planning was mostly performed in a top-down manner, with pharmacy ownership or corporate leadership being responsible for developing and enforcing these policies and procedures.Generally, engaging participants and their support staff in Flu POCT implementation was straightforward and not resource-intensive. Given recent shifts within the pharmacy profession toward services that offer direct patient care, pharmacy staff enthusiastically participated in the implementation and scaling of the program. Though, several interviews noted a persistent minority of those who do not embrace change to their work tasks like Flu POCT, despite social norms supporting Flu POCT,Participants who had already implemented POCT in years prior noted that despite the impact of COVID-19 on the flu season, patient demand grew. For those just beginning the implementation, they noted how positive patient feedback facilitated service rollout and staff enthusiasm in implementation.Goals and measures of success for Flu POCT varied depending on the maturity of the pharmacy’s Flu POCT service. Some pharmacies set a goal of a specific number of tests, while others found setting such a goal difficult because of the variability of the flu season year-to-year. When discussing financial goals, participants articulated their goals of expanding current patient care service offerings and “breaking even” on the cost of the program.Generally, most measures of success centered around improving patient care and diversifying pharmacy service offerings.Participants noted another goal was to set up a lean experiment model centered on the voice of the consumer . Given the novelty of Flu POCT as a service and of a community pharmacy as an acute care access point, participants used the service to test patient demand and feasibility.patient demand, pharmacy workflow integration, supportive organizational climate, and supportive network of health providers external to the pharmacy. Each of these main facilitators was mediated by at least one other variable, which included (a) patient and provider awareness, (b) pharmacist acceptance, (c) leadership support, (d) protocol-driven collaborative practice agreement solely for Flu POCT test-and-treat, (e) and sharing patient load during peak season/outside normal clinic hours. Of note, these latter two mediators (mediators “d” and “e”) related to the broader concept of built trust between the pharmacist and other healthcare providers. The first of these two mediators, protocol-driven collaborative practice agreement solely for Flu POCT test-and-treat, refers to the overseeing physician’s comfortability with delegating responsibilities to a pharmacist.Figure sharing patient load during peak season/outside normal clinic hours, was a downstream mediator from the use of the collaborative practice agreement. Once the Flu POCT service had begun and patient experiences became known in the community, other clinicians within the community increased their trust in the service such that they saw an opportunity to refer low complexity cases to the pharmacist in a way that closely mirrors how clinicians refer routine vaccinations to community pharmacies presently to offload low complexity workload. Finally, a series of implementation targets are listed within the framework.The simplicity of the Flu POCT protocols used by the participants, including patient assessment, laboratory result interpretation, and prescribing guidelines, facilitated trust where prior to this collaborative practice agreement there was none. Although it is standard practice for nurse practitioners and physician assistants to work under these collaborative agreements, the inclusion of a pharmacist in such an agreement is novel and therefore may represent additional risk or liability at first to a physician who previously had not collaborated with a pharmacist. The second mediator, Emerging evidence suggests that community pharmacies may serve as a key, underutilized care access point for acute infectious disease prevention, testing, and treatment , 19–21. The study also provides deeper insights to existing published research and expert opinion which can be found in the literature. Pharmacist’s growing acceptance of POCT services can be represented by the over 5,000 pharmacists who have been credentialed by a nationally recognized POCT certificate program through 2020 . POCT seThe potential public health benefit of the community pharmacy is primarily that of patient accessibility . AccessiCommunity pharmacy-based POCT’s impact on public health has been studied to a great extent in the published literature. Several efficacy studies have shown the benefits of POCT test-and-treat in the community pharmacy setting. Results from a collaborative physician-pharmacist Flu POCT test-and-treat for patients presenting with influenza-like illness (ILI) across 55 pharmacies demonstrated the service’s feasibility and acceptability during the 2013–2014 influenza season. In total, 121 patients were screened within the program, 35% of whom did not have a primary care physician and about 40% were seeking care outside of normal clinic office hours. Of note, 37% of patients did not meet eligibility for the service according to the collaborative practice protocol and were referred to their physician or an urgent care for further evaluation. Just over 1-in-10 patients were provided oseltamivir based on physical assessment and positive Flu POCT result, and at follow-up, only 3% of those patients reported worsening symptoms and were referred for further medical care . AlthougSimilarly, another study in 2016 community pharmacy-based study resulted in 40% of patients testing positively for influenza using POCT in combination with a thorough physical assessment. Of those patients, 63% were prescribed oseltamivir under a collaborative practice agreement directly from the pharmacy and over-the-counter medications were provided to 85% of patients for symptom management . PharmacThere were limitations to this study. Given its qualitative research methodology approach, generalization of the results to the entire population of community pharmacists is not possible. A future, follow-up study which uses the present study’s results to develop and disseminate a cross-sectional questionnaire would be best suited in establishing generalizability. Qualitative research is also highly dependent upon the data analysis team and their qualifications. In the case of the present study, this limitation was mitigated in part by both data coders (KH and KM) having been trained in and previously published on qualitative research methodology, while also being licensed community pharmacists with POCT experience.Implementation of community pharmacy-based Flu POCT services, which include prescribing of antivirals under collaborative practice laws, is feasible; but a thorough understanding of both barriers and facilitators to their implementation are needed to increase the spread and scale of these programs. Specifically, pharmacies should focus efforts on increasing patient and provider awareness, pharmacist acceptance, leadership support, and support of health providers external to the pharmacy to improve implementation success.Additional file 1. Semi-Structed Interview Guide.


Glioblastoma is the most common malignant primary brain tumor. Recent studies have demonstrated that excitatory or activity-dependent signaling—both synaptic and non-synaptic—contribute to the progression of glioblastoma. Glutamatergic receptors may be stimulated via neuron–tumor synapses or release of glutamate by the tumor itself. Ion currents generated by these receptors directly alter the structure of membrane adhesion molecules and cytoskeletal proteins to promote migratory behavior. Additionally, the hyperexcitable milieu surrounding glioma increases the rate at which tumor cells proliferate and drive recurrent disease. Inhibition of excitatory signaling has shown to effectively reduce its pro-migratory and -proliferative effects. Glioblastoma is the most common malignant primary brain tumor ,2. CurreThe discovery of neuron-to- neural or -oligodendrocyte precursor cell  synapses, paired with the hypothesis that the putative cell of origin in gliomas is either an NPC or OPC, raised the possibility of neuron–tumor synapses . In suppDrosophila model [Neuron–tumor synapses are vital in the amenability of glioma cells to hyperexcitable signaling and may be identified by electron microscopy as demonstrated by Venkatesh et al. and Venkataramani et al. ,10. Workla model . The rolla model .The synaptic and intercellular networks in which glioma cells are involved create not just a hyperexcitable microenvironment but also an increased sensitivity to excitatory signaling. Consistent with the purported role of hyperexcitability in glioma progression, neuron–tumor synapses are involved in glutamatergic signaling . VenkataThe functional connections between glioma cells lend themselves to Increased sensitivity to excitatory signaling. Excitatory synapses between peritumoral neurons and glioma cells are often found on a network of tumor microtubes, and these microtubes connect glioma cells to each other via gap junctions . It has The composition of the extracellular space also contributes to hyperexcitability in glioma. In addition to EPSCs, glioma cells also exhibit slow inward currents (SICs) that are mediated by potassium channels . ProlongAdditionally, glioma cells themselves can cause peri-tumoral hyperactivity due to non-synaptic glutamate secretion such that the extracellular glutamate concentration in glioma tissue is 30 times that in peritumoral tissue ,5. This GABAergic signaling also plays a role in peritumoral hyperexcitability. Peritumoral cortical neurons exhibit downregulated GABAR expression and dysregulated chloride homeostasis, rendering them less susceptible to the increased concentrations of GABA typically found in peritumoral regions . AdditioThe ability of peritumoral neurons to depolarize glioma cells coupled with the resultant increase in peritumoral glutamate can create a feed-forward mechanism in which hyperexcitability begets more hyperexcitability . This maBoth synaptic and non-synaptic mechanisms contribute to glioblastoma cell migration. Less than 30% of glioblastoma cells receive synaptic input, and the majority of these connections are located in zones of tumor invasion . VenkataBy simultaneously recording activity-related calcium currents and cell movements, Venkataramani et al. demonstrated that glioma cell migratory episodes corresponded with increases in the frequency of inward calcium currents . The expFurthermore, autocrine activation of glutamatergic receptors has been demonstrated to lead to increased migratory behavior by modulating adhesion molecule expression and environmental interaction in glioma cells . Piao etBoth synaptic and non-synaptic excitatory neurotransmission are involved in glioma growth and proliferation. Persistently high intracellular calcium resulting from AMPA or NMDA receptor activation has been shown to promote cell proliferation and tumor angiogenesis . Co-cultOverexpression of AMPAR has been shown to increase tumor burden and reduce survival in mouse models of glioblastoma, and non-functional AMPAR led to reduced glioma growth in vivo, but not in vitro, which suggests that synaptic glutamatergic signaling contributes to glioma proliferation ,10. ConvOf note, Oh et al. demonstrated that calcium-permeable AMPARs were selectively upregulated on brain tumor initiating cells compared to differentiated glioblastoma cells . The rolNMDA receptors may also play a role in glioma proliferation. Ramaswamy et al. demonstrated that NMDAR stimulation significantly increased cell proliferation in vitro in two glioblastoma cell lines . Tsuji ePatients with postoperative seizures have been shown to have lower time to recurrence than patients who do not, even after adjusting for extent of resection, potentially reflecting a role of neural hyperactivity in recurrence . HoweverInhibitory neurotransmission may play a role in glioma proliferation. Glioma cells also express GABA A receptors, though they are often functionally inactive . GABA reOther neurotransmitters have also been found to have a role in glioma progression. Neuroligin-3 (NLGN3) is a protein, secreted in an activity-dependent fashion, that has been identified as a mitogen in optogenetic studies of neuron-glioma synapses ,34. NLGNNeuronal activity is important not just for growth and invasion of glioma but also for progression from low- to high-grade tumors. It has previously been demonstrated that gliomas are made of up four general types of cells: OPC-like, NPC-like, mesenchymal-like, and astrocyte-like, with higher proliferation indices seen in OPC-like and NPC-like phenotypes . OPC-likGlutamate signaling plays a role in malignant transformation. De Groot et al. demonstrated that high grade glioma cells have reduced expression of EAAT-2, a transporter responsible for clearing synaptic glutamate, compared to their low-grade counterparts, resulting in increased peritumoral glutamate and suggesting an association between a less-excitable tumor microenvironment and lower grade tumors . FurtherThere are multiple downstream mechanisms by which excitatory receptor activation leads to increased malignant behavior. EGFR is involved in several pathways that contribute to cell survival in glioblastoma, including the PI3k/Akt/mTOR and Ras/Raf/MAPK pathways, and can be induced by glutamatergic signaling . The PI3It has been demonstrated in vitro that NMDAR activation leads to decreased levels of gamma-H2AX, a marker of double-stranded DNA breakage, after radiation exposure, which suggests that receptor activation confers radioresistance . This isThe role of excitatory neurotransmission in promoting glioma progression offers several novel therapeutic targets. These may be directed at postsynaptic effectors, gap junctions, or the mechanisms of synaptogenesis. As mentioned above, given its role in maintaining the hyperexcitable microenvironment and conferring treatment resistance, the cystine-glutamate antiporter is a potential therapeutic target. Sulfasalazine, a medication historically used to treat Crohn’s disease, has been shown to inhibit this antiporter and lead to decreased seizure activity in mice, likely due to limitation of peritumoral glutamate secretion . SeveralArcella et al. demonstrated reduced growth rates in a mouse model of glioma with LY341945, a glutamate receptor antagonist . MK-801,Pharmacologic inhibition of AMPAR has yielded mixed results in phase II trials . TalampaDespite the limited success of targeting glutamatergic receptors and the association of these receptors with EGFR-dependent pathways involved in cell survival, targeting EGFR has not proven successful in glioblastoma treatment for several reasons . FirstlyPostsynaptic calcium currents are a potential therapeutic target. General AMPAR blockers are less effective at inhibiting glioma cell migration and proliferation than blockers of calcium permeable AMPAR . AdditioGiven their role in facilitating excitatory signaling, intra-tumoral gap junctions also represent a relevant target. In a mouse model of glioblastoma, DeMeulanaere et al. investigated the gap junction blocker tonabersat and found reduced rates of growth compared to both controls and mice undergoing standard chemoradiation only when tonabersat was added to standard treatment .NLGN3 may represent a therapeutic target, as knockout mice were observed to have otherwise normal neurologic function . InhibitWhile hyperexcitability is intimately linked with tumor progression, therapies aimed at limiting presynaptic hyperexcitability have thus far yielded mixed results. In a retrospective study of patients with newly diagnosed glioblastoma receiving standard chemoradiation, Happold et al. found no association between levetiracetam or valproic acid use and survival, though this study had several methodological limitations that curbed its validity and generalizability . HoweverTherapeutic strategies aimed at curbing excitatory signaling in glioma may address glutamate secretion, glutamate or growth factor receptor activation, calcium currents, gap junctions, or limiting presynaptic activity. While many authors have investigated such opportunities, few have demonstrated promise, reflective of the heterogenous ways in which glioblastoma can evade treatment.Both synaptic and non-synaptic mechanisms contribute to excitatory signaling in the invasion and progression of glioblastoma. Glioma cells functionally connect with peritumoral neurons and with themselves, creating a network susceptible to neural activity. Peritumoral hyperexcitability is augmented by antiporter-mediated glutamate secretion, which may contribute to additional excitatory neurotransmission. Activation of glutamatergic receptors on glioma cells leads to increased migratory behavior, proliferation, and malignant transformation of gliomas. Excitatory signaling increases intracellular calcium and leads to extension of tumor microtubes and cellular migration. AMPA and NMDA receptor activation and resultant increases in intracellular calcium also lead to prolonged cell survival, resistance to therapy, and enhanced rates of proliferation, while activation of inhibitory GABA and M2 receptors has been shown to have opposing effects. This creates a system that may be susceptible to becoming a feed-forward loop, where increasing peritumoral neural activity begets more glioma electrical activity and vice versa, thus continuously increasing rates of migration, proliferation, and malignant transformation. Whether such amplification takes place has yet to be established and should be investigated in future study. Genetic and pharmacologic manipulation of this signaling present therapeutic targets, though human studies have yielded mixed results, reflective of the heterogeneity of methods by which glioblastoma progresses and evades treatment.Glioblastoma is characterized by extensive molecular heterogeneity and multimodal treatment resistance. Current treatment is aimed at reducing the overall tumor burden and relief of symptoms but does not address the role of hyperexcitable milieu in the development and progression of this disease . The cle


Despite significant advances in research, the prognosis for both primary and secondary brain cancers remains poor. The blood-brain barrier (BBB) is a complex and unique semi-permeable membrane that serves as a protective structure to maintain homeostasis within the brain. However, it presents a significant challenge for the delivery of therapeutics into the brain and tumor. Some brain tumors are known to compromise BBB integrity, producing a highly heterogeneous vasculature known as the blood-tumor-barrier (BTB). Identifying strategies to bypass these obstacles to improve the penetrability of anticancer therapeutics has been the focus of research in this area. In this review, we discuss the strategies that have been investigated to evade or alter the cellular and molecular barriers of both the BBB and the BTB and detail the methods currently under preclinical or clinical investigation, including molecular, biological, and physical processes to overcome the BBB or BTB. Increased understanding of the BBB and BTB and the current methods of overcoming these barriers will enable the development of new and more effective treatment strategies for brain tumors. Each capillary is approximately 7.5 μm in diameter, allowing for blood supply within 10μm of each brain cell The blood-brain barrier (BBB) is a complex and unique semi-permeable membrane that serves as a protective structure to maintain homeostasis within the brain The expression of transport proteins and efflux transporters in the BBB blocks the efficacy of therapies used to treat brain tumors. Molecular size and lipophilicity determine each therapeutic's ability to enter the brain. Moreover, once in the brain, the drug must remain active and at pharmacologically therapeutic concentrations. It is known that 100% of large molecule therapeutics and 98% of all small molecule therapeutics are excluded from the brain by the BBB Tumors are known to compromise BBB integrity, producing a highly heterogeneous vasculature known as the blood-tumor barrier (BTB). The BTB is formed by brain tumor capillaries and is distinct from the BBB, characterized by non-uniform permeability and active efflux of molecules. The BTB encompasses both existing and newly formed blood vessels that deliver nutrients and oxygen to the tumor and facilitate cell migration to other parts of the brain and is considered in many cases to be 'leakier' than the BBB The BTB has three distinct microvessel populations; continuous capillaries, fenestrated capillaries, and capillaries containing inter-endothelial gaps Figure . AbnormaNumerous strategies have now been investigated to either evade or alter the cellular and molecular barriers of both the BBB and BTB. These methods can be sub-categorized into methods of drug modulation, methods of BBB/BTB modulation, mechanical disruption of the BBB/BTB and complete BBB/BTB bypass methods. Strategies to bypass or hijack the BBB are currently being established or optimized. In this review, we discuss all methods currently used in the clinic or under preclinical or clinical investigation, including molecular, biological, and physical methods to overcome the BBB.Peptide-drug conjugates are considered as prodrugs due to the covalent coupling of a peptide to a drug via specific linkers. They typically include a cytotoxic agent, a tumor-homing peptide, and a linker between them. Peptide-drug conjugates have been produced by linking drugs with BBB permeable peptides. In recent years peptide-drug conjugates have evolved, with the large-scale production and purification of peptides being simplified while an array of different tumor-targeting peptides have been discovered for different cancer types. This allows for a somewhat personalized therapy to be designed by selecting a tumor-homing peptide and desired physiochemical properties, such as solubility and stability, necessary for conjugation with the therapeutic load.For a peptide-drug conjugate to be effective, it requires specific features, including a receptor-specific peptide and a stable peptide-drug conjugate with a strong binding affinity for the receptor. The peptide must bind selectively to a specific receptor on the cell surface of the target tissue that is unique or overexpressed in the cancer cells and at sufficient levels to transport the drug into the tumor. The peptide-drug conjugate site and linker must not modify the binding affinity to the target receptor or the stability to ensure that it will reach the tumor site to release the drug, thus limiting off-target toxicity. Some of the most used linear and cyclic peptides are arginine-glycine-aspartic acid, gonadotropin-releasing hormone, somatostatin, epidermal growth factor and Angiopep-2. All these peptides are delivered to cells using endocytosis/adsorptive-mediated transcytosis except for Angiopep-2, which enters the cells via transcytosis via the Low-density lipoprotein receptor-related protein 10 (LRP-1) transporter. These peptides have commonly been paired with cytotoxic agents such as gemcitabine, doxorubicin (Dox), daunorubicin, paclitaxel and camptothecin.The tripeptide arginine-glycine-aspartic acid (RGD) motif is a widely applied peptide carrier and mediates cell attachment by targeting integrins. The RGD motif is contained in various proteins like fibrinogen, fibronectin, prothrombin, tenascin, and other glycoproteins. RGD is recognized by integrins, which play an essential role in cell proliferation, invasion and angiogenesis, and are overexpressed in brain endothelial cells, newly formed vessels and tumor cells RGD peptide drug conjugates have been used for targeted drug delivery in multiple high grade glioma models Somatostatin (SST) is a neuropeptide produced by neuroendocrine, inflammatory, and immune cells. SST's various physiological functions include working as an endocrine hormone, a paracrine regulator or a neurotransmitter GBMs express multiple SST receptors, with SSTR1 and SSTR2 being the most frequently expressed in GBM in vitro, leading to improved transcytosis. The nanoparticles were also seen to improve median survival in a brain metastatic mouse model when loaded with Dox Angiopep-2 was derived from low-density lipoprotein receptor-related protein 1 (LRP1) receptor involved in the uptake and processing of amyloid precursor protein in the intracellular compartment inside endosomal vesicles  et al. designed tunicamycin-loaded transcytosis-targeting-peptide-decorated-nanoparticles (TM@TTP) loaded with Dox to reversibly inhibit Mfsda2 and enhance transcytosis, showing efficacy in a breast cancer brain metastases mouse model Other strategies to modulate transcytosis for improved penetration of the BBB have been studied. Mfsd2a is an essential fatty acid transporter and is a regulator of transcytosis, limiting BBB penetration. Inhibition of Mfsd2a would therefore allow greater permeability. Ju,The non-covalent interaction of avidin-biotin has been utilized as a nanoscale drug delivery system for pharmaceuticals, including small molecules, proteins, vaccines, monoclonal antibodies, and nucleic acids. Avidin is composed of four identical glycoprotein subunits that bind biotin with high affinity and specificity. The avidin-biotin non-covalent interaction is highly specific and stable against manipulation, proteolytic enzymes, temperature, pH, harsh organic reagents, and denaturing reagents 90Y-biotin showed promising survival rates in a cohort of GBM patients. Survival was 98.4% at 6 months, 79.2% at 12 months, 51.7% at 18 months, and 30.7% at 24 months after treatment Avidin-biotin technology is an efficient way to deliver chemotherapeutic drugs to the site of action via monoclonal antibody carriers. TfR monoclonal antibodies function to transport the bound drug into the brain through binding to the BBB TfR. Avidin-biotin technologies have shown promise due to their ability to specifically target and accumulate in tumors, permitting non-BBB permeable drugs to efficiently reach the tumor site, minimizing off-target effects and adverse side-effects. The avidin-biotin delivery system has been used preclinically with paclitaxel (PTX) and with siRNA and clinically in GBM Recent breakthroughs and the advancement of nanotechnology offers the potential for patients suffering from CNS diseases a promising route for drug delivery. Nanotechnology is an engineering approach that can be used for medical applications including the development of nanoparticles as drug carriers to enhance delivery through the BBB Figure B. The beLipid-based nanoparticles have either bilayer or multilayers and are capable of transporting both hydrophilic and hydrophobic drugs. Their characteristics increase the stability and solubility of the drugs being delivered across the BBB. In addition, being coated in micelles or polyethylene glycol allows efficient targeting and transportation of the nanoparticles Liposomes are nanosized, spherical vesicles that are made up of one or more phospholipid bilayers where the polar groups of phospholipids are orientated inside, and there is an outer aqueous phase. They can also contain cholesterol can reduce the permeability of the lipid bilayer and increase liposome stability. Liposomes can be readily encapsulated with a range of drugs within the inside aqueous compartment, the lipid bilayers, and at their interfaces Figure A. Due toClinical results of lipid-based nanoparticles applications used to improve BBB penetration in brain tumor treatment are summarized in Table in vitro BBB model and that spherical and disc-like silver nanoparticles created an easier entry pathway Nanoparticles synthesized with metals such as gold, iron, silver, copper, and others have been studied and are of interest in the use of diagnostic probes in brain tumors. Coatings and functional groups on nanoparticles play a role in improving penetrability while limiting toxicity to surrounding tissues. Variation of the physiochemical properties of nanoparticles, including size, surface coating and charge, shape and chemical composition can significantly alter biocompatibility, distribution, and function, thus making them a potential nano-carrier in neuro-oncology. A study by Guo and colleagues tested varied sizes of different metal-based nanoparticles including cerumin oxide, iron oxide and zinc oxide, along with four different shapes of silver . Results showed that zinc oxide was able to readily cross the The use of specific coatings, such as starch, PEG, chitosan, dextran or polysorbate, can enhance penetration, bio compatibility, and systemic half-life by evading the clearance by the reticulo-endothelial system Gold nanoparticles are a promising agent for brain tumors and have gathered momentum in the advancement of modern nanotechnology Figure A. They c3O4, and are divided into paramagnetic nanoparticles (PMNPs) that are 100 nm in diameter and superparamagnetic iron oxide nanoparticles (SPIONs) that are less than 100 nm. SPIONs are favorable due to their small size, susceptibility to magnetism, prolonged circulation and retention in tissues, and low toxicity Iron oxide nanoparticles typically have a solid iron oxide core with a polymer coating Figure A. They aPreclinical and clinical studies using metal-based nanoparticles and their functionalizations for the treatment of brain tumors are summarized in Table ε‐caprolactone, and polyamidoamine dendrimers have been developed but are not without limitations. Drawbacks include low biodegradation rate due to hydrophobicity, toxicity, high cost, and purity Polymer-based nanoparticles are a form of organic nanotechnology produced by polymerizing various monomers and loaded with the desired agent Figure A. The chPolymeric nanoparticles for CNS delivery are still under investigation, with some showing promise in the preclinical setting. Other studies show that nanoparticles, combined with mechanical BBB disruption methods such as focused ultrasound and convection-enhanced delivery, are more beneficial A summary of preclinical studies for polymer-based nanoparticle delivery for brain tumor treatment is listed below in Table et al. discovered a regional variability in mannitol BBB opening effectiveness, where sucrose permeability post mannitol infusion was higher in cortex and midbrain compared to cerebellum and brainstem 86Rb+ (a marker for K+ transport), 14C sucrose and Evans blue albumin. Only sucrose and Rb had increased permeability, and sucrose showed regional differences - cortex and midbrain higher permeability than brainstem and cerebellum Hyperosmolar BBB disruption is the shrinkage of endothelial cells caused by hyperosmolar plasma conditions, which cause the vasodilation of capillaries and opening of tight junctions To date, mannitol has shown promising results in PCNSL. Intraarterial hyperosmolar mannitol infusion is considered safe and has been used to transiently open the BBB to increase the penetration of macromolecule chemotherapies by 100-fold and thereby improve the response to treatment +/Ca2+ channel blockers for 30 min. This is beneficial as the brains receive longer exposure to the drug and less systemic exposure. A prolonged opening may also be beneficial for drugs that are only active in specific phases of the tumor cell cycle, such as MTX. However, limitations for this approach include repeated hospitalisation, associated with a need for sedation, and toxicities include neurological deficits, strokes and seizures BBB disruption with hypertonic mannitol is reversible with minimal permanent brain changes. The maximum osmotic effect on the BBB lasts for 10 min but can be enhanced by NaDespite being developed in the 1970s, mannitol is still being explored to help improve chemotherapy penetration to brain tumors. A Phase I/II clinical trial (NCT00303849) is currently recruiting patients to undergo mannitol BBB disruption with chemotherapies carboplatin, melphalan, etoposide, and sodium thiosulfate for the treatment of previously treated brain tumors. In addition, intra-arterial infusion of cetuximab is being investigated with mannitol BBB disruption for newly diagnosed GBM (NCT02861898).There is currently a lack of completed studies using mannitol to treat other primary brain tumors other than GBM. The safety and feasibility demonstrated in PCNSL suggests that application of mannitol warrants further research.ATP-binding cassette (ABC) transporters are a superfamily of integral membrane proteins that transport solutes across cellular membranes by ATP. Pgp, MRP and breast cancer resistance protein (BCRP) are ABC transporters expressed in the BBB and play a role in limiting the entry of therapeutics into the brain and creating multi-drug resistance. This poses a significant problem in delivering therapeutics into the brain, especially in brain tumor treatment.et al. showed that ABCB1 (Pgp) and ABCG2 (BCRP) efflux transporters at the BBB and work concurrently to restrict access to tyrosine kinase inhibitors. Elacridar is a well-known ABCB1 and ABCG2 inhibitor and has been studied in CNS metastases models. In mice, elacridar successfully increased brain uptake of erlotinib, however, preclinical data in humans did not show the same success in vitro studies that it inhibited cell growth 2-fold that of dasatinib and had dose-dependent suppression of Pgp activity Tournier, et al. investigated PI3K/mTOR inhibitors for the treatment of GBM in mouse models where they modified GDC-0980, an inhibitor with high affinity for efflux transporters, and its analog GNE-317. Both inhibitors displayed decreased efflux and 3-fold higher drug penetration into the tumor core, as well as decreased histological staining for effector proteins Drugs have also been modified to have a lower affinity for efflux transporters. A study by Becker, et al. in 2019 investigated a more specific ABCG2 inhibitor, botryllamide G, and found that it could significantly increase brain concentrations of lapatinib. However, ABCB1 would also need to be blocked for a complete efflux transporter inhibition Currently, research of efflux transporter inhibition is still progressing toward finding potent dual ABCB1/ABCG2 inhibitors. A study by Strope, 99mTc-sestamibi had increased tumor accumulation by 22% with administration of tariquidar. Out of 29 enrolled subjects, one patient had a complete objective response, and 2 had partial responses. Toxicities from tariquidar were minimal, including transient hypotension, loss of taste and nausea. However, when administered in combination with chemotherapeutic agents' docetaxel and vinorelbine, systemic clearance was reduced thereby increasing drug exposure causing drug associated toxicities A phase 1 clinical trial with tariquidar (XR9576) for inhibition of Pgp, in combination with Dox, vinorelbine or docetaxel was conducted in pediatric patients with a range of solid tumors, including brain tumors (NCT00011414) Thus, the large availability of preclinical studies into Pgp inhibitors has not translated well into a clinical setting, and the search for more highly potent, selective, and efficacious Pgp inhibitors is still ongoing.Inflammatory cytokine tumor necrosis factor α (TNFα) has been shown to disrupt the BBB by decreasing levels of tight junction proteins, causing changes in endothelial cell adhesion, and compromising barrier function and integrity Focused ultrasound (FUS) can focus acoustic energy into a focal spot to deliver selective BBB disruption and enhanced permeability Microbubbles and FUS have been successfully combined with multiple drugs, including Dox, TMZ, BCNU -1-nitrosourea) bevacizumab and MTX in preclinical models Radiation therapy plays a prominent role in treating many types of cancer, with almost 50% of patients receiving ionizing radiation at part of their treatment in vitro and in vivo level that high doses of radiation have been shown to induce BBB permeability, tight junction morphology changes, reductions in cell density, and the formation of actin stress fibers in cerebral endothelial cells in healthy regions of the brain Previous studies have shown at both the Electric field modulation has been used in other fields of medicine and has recently been used as a novel treatment method in oncology. Electric field modulation has recently been re-termed tumor-treating fields and has been used to open the BBB for chemotherapeutic drug delivery in a method known as electrochemotherapy Preclinically tumor treating fields have been used successfully to inhibit cell proliferation in cancer cell lines, including human glioma, non-small-cell lung carcinoma, breast carcinoma as well as animal models of glioma and melanoma and in combination with chemotherapeutics et al. who introduced the hypothesis that laser light could be used to cause a concentrated disruption by focally applying a neodymium-doped yttrium aluminium garnet (Nd:YAG) laser pulse et al. proved that near-infrared ultrashort pulsed laser in combination with large molecules such as nanoparticles and genetically engineered viruses, could penetrate the BBB Laser induced thermotherapy  (LITT) was first described in 1990 by Kiesslin, Unfortunately, at the clinical trial level there is little to no reported information on LITT in brain cancers even though some clinical trials have run to completion  or are currently still in the recruitment phase  Intrathecal administration is the delivery of therapeutics directly into the cerebrospinal fluid (CSF) that surrounds the spinal cord, termed the intrathecal space or subarachnoid space, bypassing the BBB and achieving higher therapeutic concentrations due to the proximity to the target, thereby limiting potential systemic toxicities Intrathecal therapeutics can be administered by an external pump or implantable devices. Implantable infusion pumps, however, are costly, invasive and cause secondary issues such as formation of granulomas and infection which may lead to catheter dysfunctions, compression of the spinal cord and paralysis in vivo studies have been conducted using intrathecal delivered agents for leptomeningeal dissemination. A study using intrathecal MX2, an anthracycline inhibiting topoisomerase II, in a rat model of dissemination showed only a modest increase in survival, which could be explained by inadequate infiltration into parenchyma Various Intraventricular administration is the placement of a subcutaneous device, an Ommaya reservoir, with a catheter in the lateral ventricle, allowing therapeutics to enter directly into the ventricular CSF. The benefit of this technique is allowing for a more consistent drug distribution with higher concentrations achieved in comparison to intralumbar injections Intranasal delivery has been investigated in the context of neurological diseases such as neurodegenerative conditions, migraines, schizophrenia, etc Perillyl alcohol, a natural compound, was investigated via the intranasal route for the treatment of GBM where 37 recurrent glioma patients were enrolled and treated with the agent by inhalation and resulted in 14 patients showing either partial response or stable disease However, limitations of the intranasal technique includes small volumes of administration, limited retention in the nasal cavity for absorption, potential mucociliary clearance or enzymatic drug degradation Intracavitary chemotherapy is administered in the form of implantable biodegradable polymer wafers in the brain or in a tumor cavity during surgery, allowing for a continuous release of chemotherapeutics locally at the tumor site and limiting systemic exposure. Direct injection of chemotherapeutics was the earliest method for intracavitary delivery, by injecting directly into the resection cavity, surrounding parenchyma or the ventricle, using a needle or implantable catheter with reservoir. This method has been used for various therapeutics such as chemotherapies, radioactive compounds, and antibodies Bortezomib, a proteasome inhibitor, is ineffective for the treatment of GBM, however implantation of a mini-osmotic pump to intratumorally deliver the drug was shown to be successful and significantly increased survival in an orthotopic GBM mouse model in vivoTo overcome systemic toxicity of chemotherapies, Gliadel® wafer was developed, which is composed of polyanhydride polymer impregnated with BCNU for the treatment of malignant brain tumors. Development of a BCNU implantable polymer that releases the drug directly into the CNS has made it possible to achieve high local drug concentrations while minimizing systemic toxicity and circumventing the need for a drug to cross the BBB. BCNU has been successfully used preclinically and has since shown clinical success in patients with primary malignant gliomas. However, some adverse side effects have been reported, such as increased cerebrospinal fluid leaks and intracranial hypertension Another biodegradable polymer, n-butylidenephthalide, suppressed growth of subcutaneous rat and human brain tumors, reduced GBM tumor volumes and significantly prolonged the survival rate in a preclinical model and has since been incorporated into the 'Cerebraca wafer', which is the first human-use drug product and is a biodegradable implant comprised of (Z)-n-butylidenephthalide ((Z)-BP) and Carboxyphenoxypropane-Sebacic Acid Copolymer (NCT03234595) A summary of the preclinical investigations and clinical trials for the use of biodegradable polymer wafers are summarized in Table Intratumoral delivery can be a carried out in a range of techniques, such as local injection, surgical insertion of a catheter with a reservoir, or convection enhanced delivery (CED). Intratumoral delivery of bleomycin in a phase 1 clinical trial was done via an Ommaya reservoir, an implantable device with a delivery tube directed to the center of the tumor and used to treat patients with recurrent GBM. The maximum tolerated dose and recommended Phase II dose was determined and no dose limiting systemic toxicity was documented et al. in 2002 detailed the use of 131I-labeled antitenascin monoclonal antibody 81C6 in post-surgical resection cavities in 33 patients with malignant gliomas. Median survival was greatly increased to 86.7% compared to historical controls, and 79.4 weeks for specifically GBM patients. At 93 weeks follow up, 11 patients survived. Side effects included treatment-related hematologic toxicity that was treatable in 9 patients, neurologic toxicity in 5 patients, and radionecrosis in 1 patient Monoclonal antibodies may be used as vectors for delivery of local adjuvant radiotherapy. A phase II study by Reardon, In vivo studies were conducted in DIPG and pediatric HGG mouse models, where liposomal Dox was delivered by CED into the pons or the thalamus. However, excessive toxicity was seen with injection of Dox into the pons, indicating a potential issue with using CED in certain anatomical locations Convection enhanced delivery (CED) is a drug delivery technique developed in the early 1990s. This method relies on a continual hydraulic pressure to distribute a homogenous supply of any sized therapeutic molecule directly the site. Although this technique demonstrates a direct route for administration, this method is highly invasive as a catheter is inserted through burr holes into the interstitial spaces of the brain Chemotherapy drugs with low BBB penetrability, such as topotecan and PTX, have been investigated with CED delivery in patients with malignant gliomas, showing tumor regression in 69% of patients and 73% response rate, respectively. However, side effects were observed: dose-dependent meningitis and infusate leakage into the CSF Clinical investigation of CED for the delivery of topotecan into two patients harboring DIPG tumors, has demonstrated feasibility and safety of the technique at low infusions rates, however no prolongation of survival was observed (NCT00324844) et al. was published in 2017 that details ongoing clinical trials Several Phase I clinical trials are still underway for an investigation into CED for malignant gliomas and DIPG. A more in-depth review by Mehta, Anatomical differences still pose a major problem for implementing CED. Brainstem tumors such as DIPG involve delicate structures that are sensitive to invasive procedures. Further developments into improving catheter design as well as precise placement to prevent reflux and enhance distribution are currently being investigated. Additionally, imaging should also be used during CED infusions to monitor drug distribution and retention.The BBB is a unique and complex structure that is a significant impediment to effective drug delivery to the brain for the treatment of brain tumors. This review detailed the established and emerging techniques investigated to bypass the BBB, from modulation of drugs to modulation or mechanical disruption of the BBB. The future directions of improving drug penetrability into the brain for treating brain tumors greatly rely on technological advancements. Furthermore, clinical trials for adults and children are pivotal for the further development of these techniques. The discovery of a safe and efficacious approach to bypassing the BBB for clinical use for the treatment of brain tumors is urgently needed and has the potential to change the course of brain tumor therapy greatly.


Vaccination, as a public health measure, offers effective protection of populations against infectious diseases. Optimising vaccination efficacy, particularly for higher-risk individuals, like the elderly whose immunocompromised state can prevent the development of robust vaccine responses, is vital. It is now clear that 24-hour circadian rhythms, which govern virtually all aspects of physiology, can generate oscillations in immunological responses. Consequently, vaccine efficacy may depend critically on the time of day of administration(s), including for Covid-19, current vaccines, and any future diseases or pandemics. Published clinical vaccine trials exploring diurnal immune variations suggest this approach could represent a powerful adjunct strategy for optimising immunisation, but important questions remain to be addressed. This review explores the latest insights into diurnal immune variation and the outcomes of circadian timing of vaccination or ‘chronovaccination’. Vaccination is a powerful and cost-effective public health strategy to protect against infectious diseases, with ~2-3 million lives saved yearly through global infant immunisation programmes , 2. HowePioneering 2017 Nobel Prize in Physiology or Medicine recipient chronobiologists Hall, Robash and Young, elucidated the mechanistic basis of circadian rhythms Figure 1via the retinohypothalamic tract, a non-image forming mechanism of ‘entrainment’ which results in synchronisation of the internal clock to external time cues  of the hypothalamus, where it receives information on light exposure ime cues , 15, 16 ime cues Figure 1ime cues . Approxiime cues –19. Impoime cues .There is increasing evidence for tight, bidirectional crosstalk between the circadian and immune systems, resulting in temporal gating of the magnitude of immunological responses , 20. RhyA comprehensive analysis of circadian clock influence on the immune system is reviewed elsewhere . HoweverSalmonella typhimurium during their rest phase, for example, resulted in higher bacterial loads compared with those infected in the middle of the active phase, and this is CLOCK-dependent -specific IFN-γ production by cells in vitro was elevated at 3 months post- vaccination in the group vaccinated in the morning, but not in the evening  may play a key role in timed vaccine effects. Indeed, circadian clock genes have been shown to oscillate in immune cells . De BreeInterestingly, epigenetic differences resulting in increased chromatin accessibility in genes important for the mTOR pathway at 3-months post-BCG vaccination have been identified in morning- but not evening-vaccinated participants. Morning-vaccinated individuals also showed enrichment of transcription factors involved in mTOR signalling and associated with active histone marks . As the Future studies to validate these early findings might include high-dependency, frailer populations to investigate whether chronovaccination remains effective despite more complex health profiles . It is cEvidence for a diurnal response to vaccination is less convincing in younger adults. The literature for this demographic is largely inconclusive: some investigators suggest time-of-day dependent effects on post-vaccination antibody responses , 57, whiIn 2016, three landmark genome-wide association study (GWAS) analyses of self-reported chronotype –64 identThere are highly systematic age-dependent changes of chronotype . Older aphase, many studies document a decline in circadian amplitude with age  effects on study results and correlated outcomes. The most immediate influence of chronovaccination research may lie in its experimental consideration as a covariable in future vaccine clinical trial designs. Further study is certainly needed to resolve the important issues highlighted in this review before any potential future clinical role for chronovaccination can be safely implemented.In the meantime, it would seem easy and reasonable that ‘time of vaccination’ should be stratified into most vaccine trials for later analysis. ‘Omics’ based deep phenotyping approaches may be key to creating a phase translation map that would aid in identifying translational potential and challenges , 86. IndCO conceived the paper with assistance from JA, partly inspired by the Oxford Masters in Integrated Immunology Course (BC & JA). The first draft was written by CO. RT, DR, BC, and JA contributed to the writing and critical reading of the manuscript. All authors contributed to the article and approved the submitted version.BioRender.com.Figures were created with 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.


We review different methods for studying movement, such as with acoustic telemetry. Methods for connecting movement and physiology are then reviewed, by using non-lethal tissue biopsies to assay environmental contaminants, isotope composition, protein metabolism, and gene expression. Methods for connecting movement and genetics are reviewed as well, such as by using population genetics or quantitative genetics and genome-wide association studies. We present further considerations for collecting molecular data, the ethical foundations of non-lethal sampling, integrative approaches to research, and management decisions. Ultimately, we argue that non-lethal sampling is effective for conducting integrative, movement-oriented research in freshwater fishes. This research has the potential for addressing critical issues in freshwater systems in the future.Freshwater ecosystems and fishes are enormous resources for human uses and biodiversity worldwide. However, anthropogenic climate change and factors such as dams and environmental contaminants threaten these freshwater systems. One way that researchers can address conservation issues in freshwater fishes is  For exa methods . Increaseadiness . Elevati species . Physiol species .Salveliunus alpinus) in northern Canada. Using a combination of non-lethal sampling for genomics and telemetry, via outlier markers) on cardiac genes of char as migratory length and elevation gain increased. These genes were related to skeletal muscle differentiation and heart development, actin binding in the sarcomere Z-disc, glycogen breakdown into glucose, and the conversion of glycerol to glucose (ibid). In a related study, physiological metrics of increased thermal maxima, increased aerobic capacity, and decreased heart mass were also associated with increased population migratory difficulty  and anaerobic glycolysis while ascending the fishway indicated higher probability of mortality, while lower plasma glucose, earlier migration, and longer lake residency time upstream of the dam increased the probability that migrating fish reached their spawning sites  mucus was assayed for 11-ketotestosterone  (Oreochromis niloticus)  , and DNAloticus) . While tloticus) , highlig+,K+-ATPase mRNA levels were analysed and found to be depressed in the 17β-estradiol and 4-nonylphenol treatments during smolting and migration. They also found that 17β-estradiol and 4-nonylphenol delayed smolting and migration, and increased mortality during migration compared to control fish. Exposure to contaminants has also been found to heighten sensitivity to external stressors, which may impact the long-term sustainability of migrating salmon populations  to determine location-specific exposure to polychlorinated biphenyls (PCBs) and organochlorine pesticides. To measure these concentrations, they evaluated if adipose fins could be used in replacement of lethal muscle samples. They found that the concentrations of PCBs and organochlorine pesticides in adipose fin samples correlated to lethal muscle tissue samples, providing an appropriate minimally invasive substitute for this species. They also found contaminate concentrations in the mobile PSDs correlate to the concentrations found in the stationary PSDs in several locations with varying PCB and organochlorine pesticides concentrations. The concept that the movement ecology of a species can affect their contaminant burden is significant, particularly in the context of point source pollution. Reduced movement or increased use of habitat at a point source of pollution may contribute to increased contaminant burden. By contrast, more transient movements through such an area are less likely to influence individuals’ contaminant burdens. These connections between contaminant burden and movement may have long term impacts on fish populations that can be monitored through physiological metrics.Locations of high residency time  can also affect the contaminant burdens individuals face . For exaδ13C measured from muscle tissue was associated with downstream sites along the river system  of higher trophic levels moved more often into different zones than snook of lower trophic levels (Cynoscion nebulosus), using acoustic telemetry paired with isotopic signatures from scales, revealed that fish resident to a recently restored oyster reef were larger and had narrower isotopic niches than their smaller, less reef-residential counterparts (Megaptera novaeangliae) skin samples as well as satellite telemetry throughout the Chesapeake Bay, showed that whales utilize the area for foraging commonly in similar areas to shipping channels, demonstrating related isotopic signatures to whales feeding in the Gulf of St. Lawrence on likely similar prey items  movement across a large freshwater lake by non-lethal whole blood biopsy . Gene expression can be measured by quantifying messenger RNA (mRNA) abundance, which can be sampled from tissues non-lethally  migration survival was predicted via elevated transcript abundance of inflammatory responsive genes in the gill, via qPCR, associating gene expression responses with mortality or successful migration (Anguilla japonica), upregulation in prolactin gene expression revealed its importance during upstream migration from sea water to fresh water, and its role in relation to hyper-osmoregulation  were associated with gene expression in liver and brain tissue (Quantitative polymerase chain reaction (qPCR), microarrays, and mRNA sequencing are all techniques that have been used to study gene expression and have been paired with movement . With thigration . The comigration . In the gulation . Alternan tissue . AdditioO. tshawytscha) and provided additional evidence that disease status may be an important predictor of migration success in anadromous fish (via microarray and qPCR) and blood plasma parameters paired with telemetry  were analyzed from the gills and blood of Chinook salmon during upstream migration (ous fish . Diseaseelemetry . This stO. kisutch) revealed how gene expression may change with important life history events including implications for physiological preparations for migration , a direct approach of genetic assignment with DNA data detected asymmetric dispersal between two current stock designations, which had important implications to the management as it suggested spawning stock biomass was overestimated (Negaprion brevirostris), Population genetics has been useful for describing the extent to which evolution and movement interact by measuring multigenerational population connectivity (reviewed in stimated . DNA frostimated  and in bstimated . Furtherstimated . For exaOncorhynchus spp.) has revealed a strong genomic basis for early versus late-run fish linked with the genes greb1 and rock1 (Vermivora chrysoptera & V. cyanoptera), a selection-based approach was used to identify the gene vps13a as affecting wintering location choices (Catharus ustulatus) was used to identify genomic regions underlying migratory orientation, such as in endopeptidase inhibitor activity and circadian clock genes  or by snap-freezing the tissue with liquid nitrogen. The latter method is thought to be a better means of sample preservation but may not be possible in all field applications because of transportation or safety concerns  in Seton River , researchers integrated multiple techniques of telemetry, behavioural tests, molecular analyses, and hydraulic monitoring to characterize the effects of a dam fishway on the survival and reproductive success of returning adults. Their findings directly influenced new operational guidelines for dam flow releases (ibid), demonstrating that non-lethal sampling with movement information can be a powerful management tool.Specific examples of movement-based research being applied to management questions exist, as well. Dams and water diversion structures provide important goods and services to humans, but can alter freshwater systems and affect species movement . River rFreshwater systems are faced with some of the most pressing contemporary conservation issues and are important for numerous human and ecosystem-based uses. At the landscape scale, these freshwater ecosystems are characterized by waterbodies of varying connectivity, together creating mosaics of fresh water. Movement research on freshwater systems has the potential to explain key factors underlying organism movements within and between waterbodies in these mosaics of connectivity. Non-lethal sampling has enormous potential for addressing integrative movement-oriented questions. Tagging methods require an initial interaction with a research organism before release, a situation consistent with non-lethal practice. During these brief interactions, an opportunity exists for researchers to non-lethally sample a small amount of biological tissue useful for analysis in diverse approaches across environmental toxicology, stress physiology, gene expression, and genetics. Preserving a research organism’s life is practical for species of conservation concern, such as those in small populations that may be diminished by large lethal sample sizes, and ethical within frameworks of wellbeing (which underlies the three R’s) and intrinsic value. Using movement information in conjunction with other data requires careful domain-dependent interpretations of results, and careful but creative applications of integrative methods. Given the importance of movement in freshwater, the conservation issues facing freshwater systems, and the practical and ethical advantages of non-lethal sampling, resource managers would benefit from using non-lethal sampling and movement-centered questions to guide decisions. With the array of biological sampling and tracking methods becoming available to researchers, integrative non-lethal movement studies will provide deep insights into freshwater ecosystems in the future.


Only one study targeted weight loss in an overweight population. When guideline-recommended measures of body composition were used, the largest improvements occurred with combined diet and exercise interventions. These mostly employed high protein diets with aerobic and or resistance exercises for at least 8 weeks. Benefits were also observed with supplementary branched-chain amino acids. While body composition in cirrhosis may improve with diet and exercise prescription, suitably powered RCTs of combined interventions, targeting overweight/obese populations, and using guideline-recommended body composition measures are needed to clarify if sarcopenia/sarcopenic obesity is modifiable in patients with cirrhosis.Alterations in body composition, in particular sarcopenia and sarcopenic obesity, are complications of liver cirrhosis associated with adverse outcomes. This systematic review aimed to evaluate the effect of diet and/or exercise interventions on body composition (muscle or fat) in adults with cirrhosis. Five databases were searched from inception to November 2021. Controlled trials of diet and/or exercise reporting at least one body composition measure were included. Single-arm interventions were included if guideline-recommended measures were used . A total of 22 controlled trials and 5 single-arm interventions were included. Study quality varied (moderate to high risk of bias), mainly due to lack of blinding. Generally, sample sizes were small ( Advanced liver disease is a complex major health problem, impacting more than 1.5 billion individuals worldwide . Liver tThere are two key issues relating to body composition for people with liver cirrhosis. Firstly, sarcopenia is a condition characterised by a significant depletion of skeletal muscle in combination with low muscle strength and/or physical performance . SarcopeThe first challenge in addressing low muscle mass and/or high adiposity in patients with cirrhosis is accurately assessing body composition, which can be complicated by fluid retention with ascites and oedema. Triceps skinfold thickness (TSF) and mid-arm muscle circumference (MAMC) appear less affected by fluid overload than other anthropometric measures in this population . While tIt is well known that both diet and exercise have positive effects on health outcomes across multiple chronic health conditions. Low muscle mass and high adiposity are attractive therapeutic targets in advanced liver disease because they may be modifiable through diet and/or exercise interventions. Exercise training is known to reduce the progression, or reverse muscle wasting  and has Therefore, we aimed to systematically evaluate the evidence on the effect of diet and/or exercise interventions on body composition in adults with cirrhosis, with a particular interest in the impact of these interventions on patients with obesity and liver cirrhosis to determine whether muscle mass can be preserved concurrently with fat loss.This systematic review followed the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) statement  see Sup, and theDatabases were searched from inception to 15 November 2021 . Reference lists of relevant review articles were hand-searched to identify further articles. The strategy utilised a combination of key words and controlled vocabulary combining terms related to liver cirrhosis AND diet/exercise AND intervention/trial  in a standardised extraction table. Extraction was piloted across three different study designs  to ensure consistency. Where present, we extracted body composition change data between study treatment groups. If unavailable, we recorded the within-group change. All extraction was cross-checked by a second reviewer with disagreements discussed to reach consensus.For each included study, risk of bias was assessed independently by two of three reviewers  using the Cochrane risk of bias tool (Rob2)  for RCTsA meta-analysis was unable to be performed due to variability in study interventions, control groups, tools to assess body composition, and reporting of means and medians across studies. Narrative synthesis was conducted based on type of intervention and body composition measures. Where a study reported on multiple body composition measures the guideline-recommended measures were prioritised in the text results .The final search contained 10,099 articles, including three articles from hand searches . A totalFor most of the included studies, the change in muscle or fat mass was a secondary outcome, and factors such as muscle strength , aerobicn = 9) interventions prescribed a high protein and energy diet plus oral nutritional supplements either with [Twelve diet-only studies ,57,58,59her with ,56,57,59her with ,55,58. Oher with , one stuher with , and oneher with . One exercise-only intervention met the eligibility criteria. This RCT involved supervised aerobic and resistance exercise sessions of moderate intensity for 60 min three times weekly, versus a relaxation program for the control group of the same frequency and duration .Across all studies, ten different methodologies were used to measure body composition see . Most coPlots summarising the risk of bias are presented in From the nine combined diet and exercise RCTs, four showed significant improvements in lean mass measured by CT , MRI 4040, BIA , and quan = 17) may have limited the power to detect change in some measures. This cohort of transplant candidates also had more advanced liver disease compared to the other combined interventions.A combined diet/exercise RCT  which usFour of the remaining diet/exercise RCTs reported significant increases in muscle mass, however this was only reported within study groups. These four studies used either supplements in combination with a diet and exercise intervention,  with a 16-week program of supervised exercised with reduced caloric intake observed a significant reduction in fat mass with no significant change in lean mass [n = 6) all-female cohort. Both of the non-randomised combined diet/exercise intervention studies found no significant changes in lean or fat mass measured via BIA  or MAMC ean mass . A seconean mass . The finean mass . CompliaThree of nine diet-only RCTs found a significant increases in lean mass assessed by MAMC ,48,56, wThe five remaining RCTs of diet-only interventions mostly assessed lean mass using MAMC and found no significant changes with the intervention ,54,55,57The final diet-only RCT  evaluateThree non-randomised or single arm studies of diet-only interventions yielded mixed results ,58,59. OThe one exercise-only study  was an RThe aim of this systematic review was to assess the impact of diet and/or exercise interventions on body composition in patients with liver cirrhosis. While published reviews exist on nutrition and/or exercise interventions in cirrhosis, there are none, to our knowledge, that reviewed studies which specifically measured body composition across both diet and exercise interventions. Secondly, this review also sought to determine the effect of these interventions in patients with cirrhosis and obesity, given the increasing prevalence of obesity and the risks associated with this , versus Unfortunately, the 27 studies identified for this review were too heterogeneous in terms of design and outcome measures to allow meta-analysis. Small study size and failure to report adherence to interventions also impacted data synthesis. Nonetheless, on systematic review, the combined diet and exercise interventions appeared to show the greatest potential to increase muscle mass. To demonstrate an increase in muscle mass with exercise, these interventions needed to be of ≥8 weeks duration and comprise 30–60 min of moderate intensity supervised exercise (aerobic and/or resistance), on at least 3 days per week combined with protein intakes of 1.2–2 g/kg/day. In addition, there appeared to be a benefit to muscle mass from BCAA supplementation ,36,39,402) increases the risk of peri-transplant complications and death [Obesity is known to impact patients with cirrhosis as an important contributor to progression of liver disease. In patients undergoing liver transplant, severe obesity (BMI > 35 kg/mnd death . The prend death ,38,39,40nd death ,42,43,44nd death ,39. In tnd death .In relation to the heterogeneity of studies, one issue that impacted the ability to synthesise the findings of this review was that 10 different methods of assessing body composition were used across the 27 studies. Current guidelines recommend CT or MRI as optimal body composition assessment methods, in part because they are less impacted by the fluid overload and ascites that occur in decompensated cirrhosis than some of the other methods ,19. WhilWhen abdominal CT or MRI are not available, guidelines recommend using DXA and BIA to assess body composition, on the provision that fluid retention is not an issue ; howeverAn additional issue was that most diet and exercise studies in cirrhosis have small study populations with body composition measures generally underpowered and as mentioned, were often included as secondary outcomes of the studies. Some of the challenges to increasing participant numbers in studies in this area are the complexities of conducting lifestyle interventions in a population with advanced liver disease who may be quite unwell. Another factor which can impact drop-out rates in this population is inclusion of participants who are potential transplant candidates. In one of the RCTs included in this review , a 6-weeThis review also highlighted the sparsity of relevant intervention studies which have targeted patients with decompensated liver disease. Patients with decompensated disease are a complex and high-risk population, who are more likely to experience muscle wasting and adverse outcomes . Chen etWhile this systematic literature review focused on changes in body composition measured using methodology validated in liver cirrhosis, there are other diet and or exercise intervention studies that have added value to the management of patients with advanced liver disease. Several exercise RCTs in patients with cirrhosis have measured aspects of physical performance including strength, exercise capacity, and/or physical function and therefore did not meet the inclusion criteria for this review. Measures such as hand grip strength, anaerobic threshold by cardiopulmonary exercise testing and functional performance assessments such as the Short Physical Performance Battery and the Liver Frailty Index have demonstrated associations with patient outcomes ,66,67,68The field of diet and exercise interventions in patients with cirrhosis is obviously at an early and evolving stage. An important goal for future studies is to determine the significance of modest improvements in body composition both in terms of clinical outcomes, but also in patient-important outcomes and their quality of life. Given the potential range and combination of diet and exercise interventions, defining minimal clinically important differences for muscle and fat mass and thresholds for adverse outcomes patients should be a goal to facilitate comparisons between interventions.In summary, effective interventions to improve body composition in cirrhosis appear more likely to succeed if diet and exercise components are combined. There remains a paucity of studies in patients with cirrhosis and obesity despite the increasing prevalence of obesity in this population. At present, the evidence supporting diet and exercise approaches to improve body composition in cirrhosis is impacted by underpowered, short-term interventions. Future research should be directed at appropriately powered combined diet and exercise RCTs of at least 8 weeks duration. Ideally assessments of changes in muscle mass, particularly in patients with decompensated cirrhosis should rely on guideline-recommended methods in this population, specifically CT or MRI. These studies should ideally be large enough to allow for the potentially high rates of patient drop-out and include formal assessments of patient adherence to interventions to identify strategies that do and do not work in this cohort. An important goal for future studies should be to determine what are clinically meaningful changes in body composition in patients with cirrhosis as this will facilitate comparison between intervention strategies. These approaches will help clarify if sarcopenia and sarcopenic obesity are modifiable risk factors in cirrhosis.


Technological developments in high-content biological and data platforms have led to the generation of large-scale multi-omics datasets in radiomics, genomics, epigenomics, transcriptomics, proteomics, metabolomics, and phenomics . CollectLi et al. explored a putative role for circular RNA (circRNA) in predicting the immune landscape of lung adenocarcinoma. Their data suggests that a circRNA-related risk score model is associated with the level of immune cell infiltration in the tumor that could impact the efficacy of adjuvant treatments . 2) Chen et al. studied the impact of germline copy number variants (gCNV) on the prognosis of patients with non-small cell lung cancer (NSCLC). They developed a prognostic nomogram model and validated their findings in an external cohort. Whether the two gCNVs they have identified, CNVR395.1 and CNVR2239.1, regulate or are accidentally associated with  NSCLC phenotypes require experimental validation. 3) Ye et al. examined the prognostic utility of a signature of ferroptosis, a form of programmed oxidation-related cell death associated with iron accumulation, a decrease in antioxidant cellular capacity, and the accumulation of lipid peroxidation or reactive oxygen species (ROS). In addition to clinical and functional studies validating aspects of this signature, a nomogram that incorporated clinical features provided reasonable concordance with overall survival. 4) Lastly, Wang et al. studied the association of long non-coding RNAs (lncRNA) with prognostication in NSCLC. They reveal lncRNA that are associated several clinical and pathological variates including histology, tumor size, stage, and, relatedly, overall survival. Altogether, these studies represent varied attempts to link omic variates, biological pathways, and patient outcomes. Many more attempts that integrate the panoply of omic variates from bulk and single cells coupled with well annotated clinical outcomes are needed.The contributions within this Research Topic are in line with ongoing efforts to better characterize the associations between omic determinants and tumor phenotypes. 1) The convergence of technology and large-scale dataset development has created an unparalleled opportunity to couple omic information with clinical outcomes for many patients with cancer. Rigorous evaluations will require many additional validations and innovations that are guided by additional advances in tumor omic profiling, data interpretation and integration, and innovative clinical trial designs. The rigor with which these studies are validated and prospectively tested will determine whether and how comprehensive tumor information is incorporated into the routine care of patients. Despite the stated potential, there remain several pitfalls for the informed incorporation of omic data for clinical testing and use. A main hurdle is the risk that large-scale association data emerges without evidence-based clinical approaches for data interpretation and integration. It will be pivotal to proactively construct scientific frameworks with adequate guardrails that can prioritize tumor omic information for clinical use.