Cocos nucifera L.) is a perennial palm with a wide range of distribution across tropical islands and coastlines. Multitude use of coconut by nature is important in the socio-economic fabric framework among rural smallholders in producing countries. It is a major source of income for 30 million farmers, while 60 million households rely on the coconut industry directly as farm workers and indirectly through the distribution, marketing, and processing of coconut and coconut-based products. Stagnant production, inadequate planting materials, the effects of climate change, as well as pests and diseases are among the key issues that need to be urgently addressed in the global coconut industry. Biotechnology has revolutionized conventional breeding approaches in creating genetic variation for trait improvement in a shorter period of time. In this review, we highlighted the challenges of current breeding strategies and the potential of biotechnological approaches, such as genomic-assisted breeding, next-generation sequencing (NGS)-based genotyping and genome editing tools in improving the coconut. Also, combining these technologies with high-throughput phenotyping approaches and speed breeding could speed up the rate of genetic gain in coconut breeding to solve problems that have been plaguing the industry for decades.Coconut ( Cocos nucifera L.) is a perennial palm with 32 (2n = 16) chromosomes that grows on tropical islands and coastlines [Coconut , an association comprised of 39 coconut-growing countries worldwide. Since its establishment in 1992, COGENT has gathered 1760 accessions which are maintained as ex situ collections in 23 COGENT member-countries, for a total of 25 field genebanks . All theIn order to promote a more efficient and effective system of germplasm conservation, research, and sharing activities, a multi-site International Coconut Genebank (ICG) has been established by the COGENT in five regional genebanks; Brazil, Côte d’Ivoire, India, Indonesia, and Papua New Guinea ,12. In 2Most of the earlier studies on germplasm characterization and evaluation have focused on morphological and agronomical traits ,15,16. TThe genotypically characterized germplasm can be further studied for the signatures of local adaptation, especially to abiotic factors using current genomic analytical tools coupled with environmental variables, such as genome-wide selection scans (GWSS)  and genoTo uncover the genes that underlie the important traits from the screening of ex situ and in situ coconut germplasm collections, various modern breeding approaches could be utilized such as high-throughput genotyping based on next-generation sequencing, precision phenotyping, and available genomic resources. Genomic-assisted breeding, marker-assisted breeding, and genome editing all can be employed to transfer useful genetic variation from naturally adapted genotypes identified from the germplasm. About 8000 years ago, the selection and domestication of coconuts for uncontaminated water sources  changed Inter-varietal crossing, such as dwarf and tall, has resulted in hybrids with a higher quantity of nuts but a lower yield of copra.  has repoDespite all these achievements, it is still not practical to produce high-performing hybrid seeds on a large scale. This is because production of hybrid seed is time consuming and requires wide seedbeds with sufficient pollen for assisted pollination, which leads to a hike in total cost of producing one hybrid seed . TherefoMore studies using multi-omics and utilization of recent advancements in biotechnological tools should enhance and simplify the selection of varieties with desirable traits including resistance towards biotic and abiotic challenges. This will ensure the coconut industry rise in the era of molecular breeding.Weather unpredictability has a significant impact on crop growth, development, and yield. Heat stress will affect the reproductive development and cause a decline in the productivity of plants . AbioticCoconuts are also often exposed to these extreme weather conditions throughout their entire productive lifespan . SeveralTherefore, tackling the effects of global climate change on coconut must be given similar weight. Even though the essential chemical mechanisms behind these features are important, there is a paucity of research devoted to them, particularly in the case of coconut.Major adaptive improvements are rarely overlooked by natural selection. The existing in situ coconut germplasm collections may contain genotypes that have naturally evolved to cope with heat and drought conditions since they have inhabited local environment long enough to be influenced by natural pressures . As propThus, similar approaches could be employed to effectively utilize the current coconut germplasm in identifying novel sources of genetic variation associated with their adaptation to changing climate. However, uncovering cryptic adaptive variation may be hampered by the fact that the abiotic stress tolerance is typically governed by multiple genes, each with minor effects . Marker-Severe damage caused by pests and diseases has been a major source of concern in various crop industries. Despite advances in chemical and biological management in agriculture, biotic infestation has had a substantial detrimental impact on crop productivity. Similarly, many biotic factors hinder coconut performance in various locations and has occasionally resulted in significant economic loss. Aceria guerreronis), a small insect that lives beneath the perianth of nuts is a devastating pest that attacks growing nuts. It is reported that chemical control of these mites is less effective and unsustainable due to the high expense of recurrent applications [Coconut mites  uth Asia . In Malauth Asia . Since tuth Asia . This suOryctes rhinoceros L. is another serious pest of coconut. In 1909, it was inadvertently introduced from its native Southeast Asia to the island state of Samoa in the Pacific Ocean [Coconut rhinoceros beetle (CRB) ic Ocean . Since tic Ocean , most reic Ocean . During ic Ocean ,53,54,55ic Ocean . With thLethal Yellowing Disease (LYD) is one of the major coconut diseases in the Caribbean and Latin America. More than thirty different varieties of palm trees, including coconut, are afflicted by this deadly disease . Even thProutista moesta) and lace wing bugs (Stephanitis typica) and conforms to any other phytoplasma. This disease is difficult to identify as it cannot be cultured in vitro [Root Wilt Disease (RWD) is another devastating phytoplasma disease which is transmitted by plant hoppers , plant biotechnology has emerged as a key component of comprehensive research studies in various crops. MAB benefits plant breeding programs by reducing the time it takes to develop quality attributes, which could otherwise take more than ten years . There hDNA fingerprinting which defines the unique molecular pattern of a genotype can be applied using various molecular markers such as codominant markers, microsatellite (SSR), Single Nucleotide Polymorphism (SNP), Randomly Amplified Polymorphic DNA (RAPD), Cleaved Amplified Polymorphic Sequence (CAPS), and Sequence Characterized Amplified Region (SCAR). In coconut, molecular marker was utilized to determine the palm’s height using RAPD . Ref. 6 employedCocos nucifera genome (2.42 Gb). This study revealed that 72.5% of the coconut genome is composed of transposable elements, majority of which were long-terminal repeat retrotransposons (LTRs) (92.23%) [Over the past several years, the cost of sequencing has continuously dropped, enabling more extensive studies of whole genome of important crops including coconut. A high-quality reference genome will facilitate the discovery of novel genes responsible for economically important traits such as pest and disease resistance, drought tolerance and high yield, among others, which consequently could drive genetic improvement of coconut. Also, the availability of full genome sequence can render a better understanding of the evolutionary history of coconut. In 2017, the first draft coconut genome was released from the cultivar Hainan Tall. A total of 419.67 gigabases (Gb) of clean reads were obtained by the Illumina HiSeq 2000 platform, representing 90.91% of the predicted (92.23%) .A couple years later, the first whole genome sequence of a dwarf coconut, cv. Catigan Green Dwarf (CATD) was generated using PacBio Single Molecule, Real Time (SMRT) sequencing at 15× coverage of the 2.15 Gb expected genome size. After improvement using 50× Illumina paired end MiSeq and Chicago sequencing, the final assembly with 97.6% coverage of the estimated genome size was obtained. The data analysis resulted in a total of 34,958 protein-coding genes that are involved in several important traits, including such as resistance to pests and diseases, drought tolerance, and coconut oil biosynthesis .More recently, Nanopore single-molecule sequencing and Hi-C technology were used to generate de novo assembly of two coconut individuals representing tall and dwarf varieties. A total of 29,897 and 28,111 gene models was annotated in the tall and dwarf genome, respectively. Notably, this study revealed the key genes involved in the divergence of tall and dwarf heights traits in coconut . One of the main next-generation sequencing (NGS)-based approaches for SNP marker discovery is genotyping-by-sequencing (GBS) due to its adaptability, cost-effectiveness, and efficiency . The ideThe reported studies on the sequencing of the entire coconut genome demonstrated that many novel genes and genetic markers have been discovered as well as some insights into the evolutionary history of the coconut. In the coming years, it is anticipated that the number of studies on the whole genome of coconuts will increase significantly, providing a large amount of genomic data that will facilitate future functional genomics and molecular breeding in this crop species. Transcriptomics has been intensively explored in many organisms and provides substantial insights into gene structure, expression, and regulation ,76,77. AThe transcriptome profiling of coconut was obtained using Illumina RNA-Seq technology together with de novo assembly from a mixed tissue sample, which has substantially aided in providing basic information for molecular breeding and additional molecular biological study . It is rApart from that, Illumina paired-end sequencing was utilized to obtain gene expression pattern during somatic embryogenesis in coconut. The importance of somatic embryogenesis in coconut for bulk production of high-quality palms cannot be understated. Yet, the recalcitrant nature of coconut creates a bottleneck in somatic embryogenesis . UnderstTranscriptomics serves an immense potential towards disease management strategies since it provides a lot of information about a sequence and a basic biological process . The advWith its enormous seed and maintaining endosperm even at maturity, the coconut offers a unique opportunity to study epigenetic mechanisms involving both small RNAs and RNA-directed DNA methylation in developmental context . To findA study to understand coconut’s response to salt-induced stress provides interesting facts about the current population size of coconut and signaling pathways involved in salt stress response. The abrupt decline in population size is due to changes in ocean levels brought on by glaciations, which may have triggered the recent invasion of transposable elements into the genome of coconuts . A plantIn model plants i.e., rice and Arabidopsis, an Abscisic Acid (ABA) dependent pathway expressing protein phosphate 2C (PP2C) gene on chromosome 14 is upregulated when exposed to salinity stress , which iCurrently, over half of the world’s cultivated coconut palms are senile and need to be replanted immediately to ensure adequate production to meet the ever-increasing demand for coconut products. In addition to ageing palms, biotic and abiotic stresses also affect coconut productivity. To address this issue, mass replanting using conventional means from the seed is a time-consuming and inefficient procedure to generate high quality and true-to-type planting materials. Alternatively, the rapid large-scale production of the new high-quality and disease-free coconut palms can potentially be achieved using in vitro culture. Several somatic tissues including young leaves, young stems and rachillae of young inflorescences were used as starting materials to form embryogenic calli in the early attempts of coconut somatic embryogenesis (SE) ,97,98. LIt is well known that coconut is among the most recalcitrant species to in vitro regeneration, and it remains a major bottleneck in coconut research. Among the reasons include the heterogeneous response of various coconut explants, the genotype-dependent regeneration efficiency, and the slow growth of regenerated plants under in vitro as well as ex vitro conditions . For SE,With the recent development of novel molecular techniques, further improvement of SE process in coconut can be made through a better understanding in the underlying mechanism and identification of key genes related to the embryogenic competence. In recent studies, several genes and micro RNAs (miRNAs) involved during different SE stages in coconut have been identified using genomic and transcriptomic sequencing approaches ,111. ThiThe advent of genome editing tools has revolutionized and accelerated the plant research over the last decade. Meganucleases, zinc finger nucleases (ZFNs), and transcriptional activator-like effector nucleases (TALENs) are dubbed as first-generation of genome engineering nucleases, while clustered regularly interspaced short palindromic repeat/CRISPR-associated system has gained more attention as the second generation due to its simplicity, efficiency and cost-effective. These genome editing tools share the same underlying principle by generating DNA double-strand breaks (DSBs) at a targeted genomic location which then being repaired either via non-homologous end joining (NHEJ) or homology-directed repair (HDR) by plant endogenous repair mechanism . The genDespite of its wide application in other crops, the genetic improvement of coconut via this approach remains challenging. To date, no genome editing tools have been successfully applied and reported for coconut. This little progress is due to the lack of established tissue culture and genetic transformation methodologies. At present, there is only a single report on the transient genetic transformation of embryogenic callus of coconut . IdeallySpeed breeding is a promising innovative technology for accelerating genetic gain by manipulating the growth environment of plants . This teImplementing a speed breeding algorithm in coconut breeding would be beneficial due to the long generation cycle of coconut. Coconuts undergo a lengthy juvenile period before entering their reproductive stage . As repo−2s−1 indicates a typical C3 plant nature of coconut [−1.A saturated photosynthetic photon flux density of about 1400 µmoles photons m coconut . Slow gr coconut . The higOn the contrary, protocols on growing conditions such as soil media composition, lighting, and temperature are not available to accelerate the growth of palm. Although parameters for bread wheat and durum wheat (C3 plants) could be adapted for coconut, more research on the spacing and coordination of sprouting coconut is necessary to assess whether the benefits of speed breeding outweigh the costs. Apart from this, according to , prolongPhenotype information is the link between an organism’s genetic make-up (genotype) and environmental factors. A genotype-phenotype map (G-P map) of coconut would enable breeders to understand the dynamics of the entirety of the palm’s lifespan. The G-P map is the result of extremely complex dynamics that include environmental factors, and understanding these dynamics is synonymous with bridging the genotype-phenotype gap .The morphological study of coconut palms, particularly the tall variety, is difficult. On the other hand, phenotyping the dwarf variety, which will eventually grow taller, would pose a similar problem. Studying yield-contributing traits like number of bunches/palm/years, number of fruits/bunches, stem height, petiole length, and overall frond length is not only time-consuming, but it can also be destructive. Nevertheless, the documentation of morphological traits is crucial as it will benefit farmers and breeders in future crop selection and improvement. Therefore, fast, accurate, and high-throughput methods need to be developed for collecting the phenotyping data.The current state of the global coconut industry necessitates an increase in yield to meet rising demand and ensure food security. Even though other biotic and abiotic stresses are equally important, the focus of this paper will be on a method for morphological characterization of traits that contribute to yield. The novel approach described here could serve as a springboard for the development of phenotyping for other traits. First, a clear understanding of morphological traits that contribute directly to yield and inheritability must be identified for a successful application of next generation phenotyping (NGP). Other studies have found that coconut leaves make a significant contribution to the yield. In comparison to inflorescence and fruit traits, leaf characters are more stable . A detaiThe Standardized Research Manual in Coconut Breeding (STANTECH) lists the length of the petiole, petiole color, length of the whole frond, and measurements of the width and length of four middle leaflets from the 14th frond as the minimum measurements needed for leaf characterization of coconut. However, sampling the wide range of germplasm that is available in producer countries requires a lot of labor and can be destructive as the whole 14th frond must be removed at the point where the petiole connects with the stem. The above-mentioned measurements are necessary for leaf characterization in palms such as coconut and palm oil, to estimate the total area of leaves/canopy of individual palms. Thus, measurements of the width and thickness of the petiole are employed to calculate cross sectional area, and indirect estimations of the dry weight of the entire leaf are obtained . A non-destructive method for estimating the leaf area of oil palms was developed , in whicBy integrating the PMS with the UAV, the number of bunches and fruits as well as observed crown appearances will be easier to measure, particularly in tall varieties . As a rehttps://biodiversityinformatics.amnh.org/open_source/dotdotgoose/) (accessed on 14 March 2020) is another open-source program for manually counting objects from an image. This program can be employed to manually count the number of palms in a population using aerial images captured by a UAV. With increased sensitivity, the counting of individual fruits and bunches will be feasible and useful for tall varieties.DotDotGoose   provide A phenotypic and physiological parameter for selecting drought tolerant traits among coconut is demonstrated via simulation modelling from calibrated and validated data from previous studies on various perennial crops in India . SimilarMost of the previous studies relied on manual observation to obtain quantitative data such as plant height, leaf color, chlorophyll content, disease sensitivity, yield, biomass, and drought tolerance, which is not only time consuming but also labor-intensive. The biological nature of the coconut including high stature, long generation time and low multiplication rates make the large-scale screening difficult and destructive. As a result, coconut phenotyping has lagged behind genotyping, contributing to the slow progress of fundamental research. As reported in European Plant Phenotyping Network , the chaThus, advanced phenotyping protocols are required to understand the relationship between environmental factors, genotypes, and phenotypes in order to enhance coconut performance . To addrThe combination of fundamental biology, data sciences, and engineering, among others to obtain multidimensional phenotypic data of an organism as a whole is known as phenomics . This hiThere are various methods that can be utilized to obtain phenotypic data from coconut. Infrared (IR) and hyperspectral imaging are two of the easiest and most cost-effective ways to identify drought and high temperature tolerant cultivars. These methods are based on recent experiment by NASA using satellite platforms equipped with multi and hyperspectral sensors, Ecosystem Spaceborne Thermal Radiometer Experiment on Space station (ECOSTRESS), Soil Moisture Active Passive (SMAP) and Hyperspectral InfraRed Imager (HyspIRI), which collect high-resolution spectral and environmental data providing applications for water use efficiency ,153,154.Stomatal conductance and canopy temperature are surrogate measurement tools whereby a cool canopy indicates a high transpiration rate of the palm while a high temperature denotes a lower transpiration rate . This alAgriculture is a one-of-a-kind industry with economic, biotic, and abiotic influences. Plants and soil have long been the only inquisitors as well as sensors available for quantitative and qualitative characterization. These manual methods are widely utilized to guide operational decisions, irrigation management, pest and disease prediction, monitoring, and ideotype selection. However, manual interventions in the hope of improving the yield and quality of a crop are less reliable, laborious, and prone to error. In the coconut domain, previously used quantitative and qualitative characterization methods and tools were ineffective for current and future challenges. The coconut’s heterozygosity, monotypic, perennial nature, and long generation cycle are significant deterrents. On the other hand, the experimentation process necessitates a large amount of land. Furthermore, because there are no known wild or domesticated related species of coconut, the genetic pool sources for extracting useful relationships and patterns in collection, selection, and breeding process are absent.The transformation of traditional quantitative and qualitative tasks is required as coconut cultivation intensifies. Data mining, which includes machine and human-generated data then uses the Internet of Things (IoT) as a booster, is a channel for combining findings from previous biotechnological studies, phenotype and genotype data from coconut cultivation improvement efforts, and phenotype and genotype data from farm output expansion and diversion .The fields of genetics and genomics have seen progress with the application of machine learning (ML), such as for ecological niche modelling  and funcML-based phenotyping is feasible to accelerate the rate of genetic gain in crops and the throughput of crop screening, which facilitates the early detection of diseases for a reduction in crop yield loses . Thus, MIntegrating artificial intelligence in data mining for coconut cultivation and breeding regimes could result in considerable progress in applying smart farming to rapidly revive the coconut industry. However, proper management of data and sharing protocols must be developed so that farmers and researchers can have a quick access to the data and gain profound effects. Farmers’ accessibility in performing smart farming must be studied.The application of individual tool or technology in isolation would hamper crop improvement. Thus, to attain a profound effect in farm output, integration of novel biotechnological and precision agriculture tools with selected conventional methods in breeding pipeline is crucial. To accelerate coconut genetic improvement, genomic-assisted breeding, transcriptomics, next-generation sequencing (NGS)-based genotyping, genome editing, precision phenotyping and speed breeding, among others need to be integrated which could facilitate breeding and selection decisions in breeding program. The high-throughput and cost-effective marker systems derived from NGS-based platforms will enable rapid genotyping process. As the sequencing cost is expected to continuously drop, more coconut genotypes can be re-sequenced to construct coconut “pan genome” which would provide novel insights into the genetic basis of complex traits such as yield and drought tolerance. At the same time, considerable efforts should be made to train more experts on handling the “big data” generated from the NGS platform as well as enhancing the computing capability. The genetic dissection of resistance mechanism towards important pests and diseases in coconut can be further elucidated with the increased knowledge at genomic and transcriptomic levels. With a growing number of crops being successfully optimized for speed breeding, similar achievement in coconut will become a game changer in coconut breeding pipeline. Field data collection especially related to yield trait might be seen as a major constraint due to the biological nature of coconut. The proposed method using image capture app coupled with UAV in this paper could potentially address this issue and serve as a catalyst for further development of high-throughput approaches. Prioritizing the utilization of different tools in parallel with the objectives of the breeding program is vital to resurrect the coconut industry with current increasing demand as well as a preparation for future challenges. Molecular markers, precision phenotyping tools such as UAVs, and spectral satellite imaging are readily available to be used for identification of high-performing palms in the germplasm collections. As genomic resources are expected to further expand in the coming years, biotechnological tools such as NGS-based genotyping, whole genome sequencing, transcriptome sequencing, and pangenome analysis can be applied in the medium-term to advance coconut breeding. The application of clonal propagation and genome editing may take a while as further research work is required to optimize the tissue culture protocol of coconut. Although it is still early days, success in adopting speed breeding technology to shorten the generation time of coconut will be a breakthrough in coconut research.The potential of natural variation as valuable sources of resistance to biotic and abiotic stresses has been discussed in this review through the study of local adaptation in germplasm collections assisted by GWAS, GWSS, and GEA using available phenotypic, genotypic, and climate data. To speed up coconut breeding cycles, marker-assisted selection and marker-assisted backcrossing provide an alternative to traditional breeding for the introgression of target genetic variants from exotic germplasm into elite cultivars. However, these approaches have proven to be useful for traits which are governed by a few genes such as resistance to biotic stresses but may be inefficient for tracing quantitative traits which are often regulated by many genes with small effects such as tolerance to abiotic stresses. In the near future, genomic-assisted backcrossing (GABC) may become more common to facilitate the introgression of polygenic traits into the cultivated coconut genepools. Another exciting tool that can be employed for this purpose is genomic prediction (GP), which promises to expedite selection and pre-breeding for complex polygenic adaptive traits from natural sources. We envision that this predictive breeding method will also help future genebank characterization to mine the molecular signature of selection and adaptation of coconut for climate adaptation.


Smoking cessation after a cancer diagnosis can improve health outcomes, but the Coronavirus disease 2019 (COVID-19) pandemic significantly altered healthcare patterns and strained resources, including for smoking cessation support for cancer patients. A Network that included all 13 provinces and territories (jurisdictions) in Canada received funding and coordinated support from a national organization to implement access to smoking cessation support in cancer care between 2016 and 2021, including throughout the COVID-19 pandemic. Descriptive analyses of meetings between the organization and jurisdictions between March of 2020 and August of 2021 demonstrated that all jurisdictions reported disruptions of existing smoking cessation approaches. Common challenges include staff redeployment, inability to deliver support in person, disruptions in travel, and loss of connections with other clinical resources. Common adaptations included budget and workflow adjustments, transition to virtual approaches, partnering with other community resources, and coupling awareness of the harms of smoking and COVID-19. All jurisdictions reported adaptations that maintained or improved access to smoking cessation services. Collectively, data suggest coordinated national efforts to address smoking cessation in cancer care could be crucial to maintaining access during an international healthcare crisis. The Coronavirus disease 2019 (COVID-19) pandemic has severely disrupted medical care patterns worldwide. Patients attended fewer medical visits due to COVID-19 concerns and healthcare systems modified medical treatment protocols to accommodate COVID-19 related risks . Many heSmoking is a proven risk factor for the development of many cancers and continued smoking after a cancer diagnosis is associated with increased overall mortality, cancer-related mortality, and added costs from cancer treatment ,13,14,15Starting in 2015, the Partnership began to support the development and implementation of evidence-based smoking cessation in cancer care in Canada, which included providing initial funding for nine provinces and territories (“jurisdictions”). In 2019, two- to three-year funding was provided to all 13 jurisdictions to implement smoking cessation approaches tailored to the resources and needs of each province or territory. Each jurisdiction was encouraged to develop or enhance approaches that would promote access and support to all cancer patients, including use of the 3A  or 5A  model for smoking cessation ,20. In CThe Pan-Canadian Tobacco Cessation and Cancer Care Network (Network), first convened by the Partnership in 2017, consists of officials working in tobacco control and cancer programs representing each jurisdiction, and from the federal government. The Partnership provides Network members centralized support, including access to expertise in smoking cessation and logistical support to assist with program development and evaluation. Network meetings are convened by the Partnership twice annually. The Network further serves as an engaged collaborative where members share information, results, problems, solutions, and expertise.Network activities were designed to support the implementation of smoking cessation activities across jurisdictions and were not designed as a research project. Network meetings and individual jurisdictional calls were recorded or documented with non-standardized notes, rather than through standardized questionnaires. Each individual jurisdictional call was focused on the specific accomplishments and needs of that jurisdiction. As an example, a jurisdiction that had not yet effectively implemented screening for smoking could be focused on improving those activities. A jurisdiction that had already achieved referrals to treatment programs could be focused on providing medications. Jurisdictional calls focused on tracking progress, assisting in problem solving, and sharing knowledge, including connecting jurisdictions to each other for experiential support.The COVID-19 pandemic required shifting the Network meetings from in-person to online. The online meeting format was used in July 2020, January 2021, and June 2021. At the 2020 meeting, each jurisdiction was asked to provide specific examples of challenges and opportunities they experienced because of the COVID-19 pandemic, including their ability to implement or maintain core functions of counseling and pharmacotherapy for cancer patients. In addition, virtual calls were conducted approximately every two months and as needed between the Partnership and the project team for each jurisdiction. Prioritization was given to sharing the status of funded projects and implementation challenges posed by the COVID-19 pandemic and discussing strategies to help projects maintain or restore smoking cessation activities in cancer care within their jurisdiction. Meetings and jurisdictional calls were documented through unstructured, free-text notes by Partnership staff.Data reported describe findings from a review of free-text notes collected during Network meetings, individual calls with jurisdictions, and jurisdictions’ self-assessed performance during the COVID-19 pandemic between March 2020 and August 2021. Descriptive data were extracted from meeting notes to examine challenges faced by jurisdictions and describe real-world adaptations over time. In addition, in March 2021, each jurisdiction provided the Partnership with self-reported information on performance metrics using the scale shown in Structured screening and smoking cessation processesIncorporation of smoking cessation as a standard medical directive for cancer careSupport and/or buy-in from administrative leadership, physicians, and other staffAbility to collect and analyze dataReporting of results to staff and leadershipPartnerships with community resourcesTraining and educational materials for clinicians and patientsDevelopment of feedback loops between smoking cessation services and cancer careCross-jurisdictional collaborations to share information about addressing smoking across cancer careEfforts to engage at-risk populations and incorporate culturally competent support for Indigenous patients with cancerThough implementation of smoking cessation in cancer care settings progressed at different rates across jurisdictions between 2017 and 2020, several common program elements had been achieved by many of the jurisdictions by the outset of the COVID-19 pandemic in 2020, including:At the first online Network meeting in July 2020, jurisdictions began reporting challenges and opportunities associated with the COVID-19 pandemic. In response to challenges posed by the pandemic, jurisdictions reported unique opportunities to adapt so that they could continue to offer patients smoking cessation support . SeveralThroughout the COVID-19 pandemic, jurisdictions were supported with financial and implementation support through the Partnership. Challenges and recovery adaptations were recorded at the bi-monthly meetings. By August 2021, most jurisdictions reported that changes in staffing, leadership or support had stabilized or improved. Many programs had transitioned to refining delivery of smoking cessation, such as changing smoking assessments, using electronic medical records, improving data collection and analysis, and expanding education to clinicians and patients. However, concerns were raised about upcoming clinician retirements and physician shortages, restructuring of health services such as quitlines, and anticipated resurgence of COVID-19 waves. While some jurisdictions reported concerns about strained healthcare resources, such as views that clinicians were ‘just surviving’, there were parallel views that providing smoking cessation support had ‘strong physician engagement’ and helped reduce burden for clinicians. Among programs that were in a pre-implementation or early adoption phase, all were able to develop smoking cessation strategies within changing healthcare systems. Through August of 2021, all jurisdictions were able to adapt to challenges and maintain or improve self-reported assessments for counseling or pharmacotherapy.Data suggest that national efforts to address smoking by cancer patients were maintained during an international pandemic with the support of centralized coordination focused on assisting local resources to adapt to significant shifts in health care. Widespread disruptions experienced across most cancer care settings included staff redeployments and changes in non-smoking cessation-related care patterns, requiring shifting from in-person to virtual visits. Common opportunities and adaptations included increased use of virtual care, education about risks of smoking and COVID-19, and increased collaborations with other healthcare providers and community resources. While not explicitly stated, data suggest that jurisdictions adapted to loss of in-person counseling by advancing alternative cessation-related activities, such as developing innovative approaches to increase access to counseling or medications. Importantly, jurisdictions reported the ability to maintain or increase access to behavioral counseling and pharmacotherapy between 2020 and 2021.There are limited data on how smoking cessation programs adapt to changes caused by COVID-19. Previously, a single institution supported as a part of C3I reported on the reach and effectiveness of implementing smoking cessation in cancer care prior to and during the COVID-19 pandemic [A common element among jurisdictions in Canada was the need to adjust smoking cessation in cancer care program budgets and workplans during the pandemic. As with any medical practice, business plans and financial support are critical to sustaining an intervention. Cancellation of hospital procedures resulted in rapid and significant changes in health system revenues ,21,22,23While these data are the first to report on how a national coordinated effort could assist with maintaining smoking cessation in cancer care during a pandemic, there are several limitations. Results report on real-world coordination through Network meetings and individual jurisdictional calls focused on jurisdictional needs and priorities. It is likely that data collected represent the most timely and concerning issues discussed at jurisdictional calls, and underreport the full spectrum of issues that would be captured through a standardized research format. However, it is unlikely that any structured national research instrument could be developed and administered within the rapidly developing changes in health care during the pandemic. Another limitation is that scoring of jurisdictions’ ability to deliver counseling and pharmacotherapy was self-reported and could not be verified by alternative means. These self-reported assessments may overestimate true performance among jurisdictions and no alternative verification method, such as patient-reported outcomes, were available across all jurisdictions for comparison. Best-practice alternatives, such as patient-level data or independent verification of smoking cessation program access, were not performed or available for comparison. Data cannot support conclusions about whether individual smoking cessation rates changed during the pandemic. However, data suggest that national coordinated efforts such as the Partnership and Network may help maintain access to smoking cessation support during a pandemic.The worldwide COVID-19 pandemic could be viewed as a unique opportunity to rapidly accelerate changes in health care that better adapt to patient needs. Some jurisdictions reported that shifting to telephone-based smoking cessation counseling increased access for rural and remote populations and provided opportunities for improved long-term follow-up. Reviews of telehealth initiatives during the pandemic suggest that patients and providers view telehealth favorably , and tel


Exercise-induced pulmonary hypertension EIPH has been defined as an increase in mean pulmonary arterial pressure (mPAP) during exercise in otherwise normal values at rest. EIPH reflects heart and/or lung dysfunction and may precede the development of manifest pulmonary hypertension (PH) in a proportion of patients. It is also associated with decreased life expectancy in patients with heart failure with reduced ejection fraction (HFrEF) or left ventricle (LV) valvular diseases. Diastolic dysfunction exacerbated during exercise relates to increased LV filling pressure and left atrial pressure (LAP). In this context backward, transmitted pressure alone or accompanied with backward blood flow promotes EIPH. The gold standard of EIPH assessment remains the right heart catheterization during exercise, which is an accurate but invasive method. Alternatively, non-invasive diagnostic modalities include exercise stress echocardiography (ESE) and cardiopulmonary exercise testing (CPET). Both diagnostic tests are performed under gradually increasing physical stress using treadmill and ergo-cycling protocols. Escalating workload during the exercise is analogous to the physiological response to real exercise. The results of the latter techniques show good correlation with invasive measurements, but they suffer from lack of validation and cut-off value determination. Although it is not officially recommended, there are accumulated data supporting the importance of EIPH diagnosis in the assessment of other mild/subclinical or probably fatal diseases in patients with latent PH or heart failure or LV valvular disease, respectively. Nevertheless, larger, prospective studies are required to ensure its role in clinical practice. Exercise-induced pulmonary hypertension (EIPH) has been defined as an exaggerated increase in mean pulmonary artery pressure (mPAP) >30 mmHg and total pulmonary resistance (TPR) > 3 Wood units during exercise, while an mPAP <25 mmHg is present at rest . PatientIt is increasingly recognized that EIPH may represent an early, mild stage of pulmonary arterial hypertension, and preliminary data suggest its association with an overall higher morbidity and mortality . Past evThe term “exercise induced pulmonary hypertension” was suspended at the 4th World Symposium of Pulmonary Hypertension in Dana Point, in 2008, largely due to failures at reaching a consensus. Since then, a plethora of studies have been published about EIPH, its pathophysiology and methods of assessment. A body of data increasingly supports EIPH as an important and promising entity for the research and clinical community. Despite this, its unambiguous association with exertional dyspnea and its clinical usage in the prevention, early diagnosis and management of cardiopulmonary pathologies remains controversial. For all these reasons, we have conducted this literature narrative review to shed more light on the underlying mechanisms, the diagnostic methods and the potential clinical applications of EIPH.For the needs of this narrative review paper, we searched MEDLINE and EMBASE, Web of Science, Cochrane and Google Scholar databases for English language publications from 1990 to May 2022. We also checked the reference lists of the identified articles to find any additional relevant articles. Our search included the titles, abstracts and medical subject headings (MeSH), and we used the following search terms or abbreviations: pulmonary hypertension, exercise-induced pulmonary hypertension (EIPH), pulmonary artery systolic pressure, pulmonary artery pressure, pathophysiology, right heart catheterization (RHC), exercise stress echocardiography (ESE), cardiopulmonary exercise testing (CPET), HFpEF, HFrEF, aortic stenosis, mitral stenosis and mitral regurgitation. Two investigators (N.V. and E.K.) performed the literature search independently.In our search, we included clinical studies  as well as experimental studies. To draw firm conclusions, we excluded the studies fulfilling the following criteria: unavailable full texts, publication language other than English and conference abstracts.Using the abovementioned terms, we initially found 1457 hits. After the abstracts’ screening, we removed 126 duplicated studies and 1137 irrelevant studies; 194 full-text studies were screened for eligibility. After removing the studies with wrong design, irrelevant outcomes and unavailable full text, we ended up with a total of 78 studies, 3 systematic reviews and meta-analyses of clinical data and 30 additional clinical studies which were not included in those meta-analyses.Significant progress has been made in understanding the pathophysiology of the hemodynamic abnormalities of cardiopulmonary circulation during exercise. Although EIPH pathogenesis is still not completely understood, it can be attributed to alterations in pulmonary artery flow affected by cardiac output (CO), pulmonary vascular resistance (PVR) and left atrial pressures (LAP) during exercise, as indicated by the Poiseuille–Hagen equation: mPAP = PVR × CO + LAP . EIPH ap(A) Increased pulmonary flow: Unambiguously, cardiac output (CO) and, consequently, pulmonary blood flow significantly increase during exercise in response to the increased oxygen uptake. In parallel, there is a significant pulmonary vascular dilatation as a compensatory mechanism. The magnitude of the inevitable elevation of PAP and, to a lesser extent of wedged PAP (wPAP), is a product of increased blood flow balanced by pulmonary vascular resistance (PVR). In healthy individuals, PVR as a result of pulmonary vascular distensibility is widely adapted to high pulmonary blood flow in order to maintain PAP within a normal range . PreviouAnother factor determining pulmonary blood flow and PAP elevation during exercise is the type of exercise. During aerobic exercise, pulmonary blood flow has shown a linear progressive increase and thus a linear adjustment in the mPAP–O correlation may occur, whereas resistance exercise is associated with exaggerated pulmonary vasoconstriction and higher PVR ,6. The f(B) Increased LAP: Evidence supports the relationship between EIPH and increased LAP as a result of left cardiac disorders, such as advanced diastolic dysfunction or severe mitral valvular diseases . Notably(C) Increased pulmonary vascular resistance (PVR): Another determinant of mPAP is PVR. Normally, an increase in CO and LAP, during exercise, distends the pulmonary circulation, leading to decreased vascular resistance ,9. When Overall, EIPH includes precapillary PH with abnormal pulmonary circulation and post-capillary PH with early rise in wPAP . The occCurrently there is not a consensus regarding the ideal way of detecting EIPH. Diagnostic modalities include ESE, CPET and RHC during exercise. Up until now, the latter has been considered the gold standard method for EIPH. However, there is no widely accepted strategy for EIPH diagnosis and thereby established presence.ESE seems to be a useful tool for the diagnosis of EIPH. The test is performed under gradually increasing physical stress, and different protocols have been proposed . The BruUsing treadmill patterns of exercise, all echocardiography-related measurements are conducted just before the start for the treadmill protocol and immediately after the cessation of walking on the treadmill. That time, the patients should be transferred from the standing to the supine position on a coach, always next to the treadmill device, in order to obtain the transthoracic echocardiographic measurements as soon as possible within 30 s). This is very challenging because pulmonary vascular pressures and flows return rapidly to baseline resting state after the completion of exercise [0 s. ThisAnother important limitation of ESE regarding the diagnosis of EIPH is the need for trained and experienced operators to get accurate and reproducible results. Respiratory motions of the chest during exercise may prevent the acquisition of optimum echocardiographic images or the loss of the tricuspid regurgitant (TR) blood flow jet. The latter is a prerequisite for TRVmax and PASP calculations. A study compared mPAP measurements derived from ESE with measurements using exercise cardiac MRI and concomitant invasive pressure registration. This showed a strong correlation between non-invasive and invasive measurements, although non-invasive ESE tended to overestimate mPAP . Some stFinally, ESE provides the PASP values and the ratio of early diastolic transmitral flow velocity to early diastolic mitral annular velocity (E/e’). Both of them can be easily calculated compared to the CPET parameters  and have2  ratio is greater than 37 and PETCO2  is below 30 mmHg at an anaerobic threshold, there is a great probability of EIPH [2 below 20 mmHg are highly suggestive of EIPH and uncommon in other diseases [CPET is a diagnostic modality that is useful for the evaluation of unexplained dyspnea, when medical history, physical examination and common tests, such as electrocardiography, spirometry and cardiac or lung imaging, fail to identify a cause. A wide variety of diseases and pathological situations can be identified using CPET . The exe of EIPH ,30. Notadiseases . CPET exdiseases . A greatdiseases . NeverthAdditionally, cardiac magnetic resonance (CMR) imaging during exercise is a recent diagnostic modality that is used only for research purposes so far and combines a bicycle featured in a CMR set-up . AlternaThe first invasive pulmonary hemodynamic measurements during exercise in normal subjects were reported in the late 1940s. According to Fowler et al. (1969), the mPAP–flow relationship could be described using a linear approximation until the upper physiologically possible flows . HoweverRegarding the hemodynamic differences between upright and supine positions during exercise, the mPAP and cardiac output may be slightly decreased, and the heart rate increased in the former position . FurtherRHC during exercise tests is considered the gold standard for the evaluation of individuals with unexplained dyspnea . A recenDespite the aforementioned promising results, more studies need to be undertaken in order to evaluate the sensitivity and specificity of non-invasive modalities, such as ESE and CPET, as surrogate or adjunctive methods for EIPH diagnosis. A widely accepted strategy for EIPH diagnosis cannot be drawn. Based on previous evidence, it would be wise to start the investigation of EIPH with a non-invasive technique, either ESE or CPET, depending on the underlying clinical condition , the local facilities and the experience of the center. In case the results of those tests are inconclusive, and the suspicion of EIPH remains high, then clinicians should proceed to the invasive RHC during exercise in order to confirm or reject EIPH.EIPH accurate diagnosis has been proposed as an essential component of the following clinical entities:The term PH diagnosis, included in the past EIPH, is as the elevation of mPAP above 30 mmHg in RHC during stress. However, in the 4th World Symposium on Pulmonary Hypertension (WSPH), this definition was abandoned due to a lack of evidence . The latEIPH is considered by several researchers as an early, precursor form of PAH, which may have a prognostic role in connective tissue disorders, such as systemic scleroderma ,46,47,48Comparing ESE with RHC at rest, a recent study demonstrated a good diagnostic accuracy of exercise-induced PASP >50 mmHg, as it could unmask PH in selected patients with systemic sclerosis and baseline echocardiographic-based measurements of pulmonary pressure within the grey zone . RegardiMitral stenosis (MS): EIPH reflects exercise-triggered hemodynamic changes and can be an indicator of MS severity. De Castro et al. (2020)  showed aMitral regurgitation (MR): The European Society of Cardiology has reported that ESE is an important diagnostic tool to evaluate hemodynamic changes in patients with MR. In the recently updated guidelines, it was commented that ESE may be helpful for diagnosis and prognosis stratification when there is a mismatch between the symptoms and MR severity . In asymAortic stenosis (AS): As with other valvulopathies, the optimal timing of aortic valve replacement in asymptomatic patients with AS remains controversial . AlthougHFrEF: PH at rest or during exercise is an important marker of a poor prognosis in patients with heart failure . In patiHypertrophic cardiomyopathy (HCM): A recently published study examined the prevalence and prognostic significance of EIPH in patients with HCM . Those pUnexplained dyspnea is defined as a chronic, persistent dyspnea, where after basic laboratory and imaging investigations, the underlying etiology remains unknown . DyspneaAlternatively, invasive tests can be used when HFpEF is suspected. When normal measurements of LV filling pressures and mean pulmonary capillary wedge pressure (mPCWP) are invasively obtained at rest, but HFpEF suspicion persists, then RHC during exercise is recommended . A PCPW EIPH refers to mPAP greater than 30 mmHg during exercise, and it is often an unidentified cause of exertional dyspnea in patients without obvious resting cardiac or respiratory pathology. Moreover, it is related to increased morbidity and mortality in patients with HFrEF or HFpEF, valvulopathies or respiratory diseases. The pathogenesis of EIPH is still not completely understood. Alterations in cardiac output, pulmonary vascular resistance and pressures in the left heart upon exertion may affect pulmonary pressures; hence, they are involved in EIPH. Although right heart catheterization remains the gold standard for EIPH diagnosis, because of its invasive nature, proposals have been made for the alternative use of ESE or CPET. The former is an easily conducted, cheap and repeatable technique. CPET offers a great variety of information, providing the capability of the differential diagnosis of unexplained dyspnea. Both techniques have significant advantages and disadvantages; however, more studies are deemed necessary to validate their results.Regarding the clinical application of EIPH, it has recently raised by many scientists as a precursor stage of PH, which may trigger early initiation of the appropriate treatment. Similarly, the presence of EIPH, when there is a mismatch between symptoms and LV valvular disease severity, may objectively contribute to decision-making and earlier interventional therapy. Finally, EIPH may contribute to the diagnostic algorithm of HFpEF and HF patients’ risk stratification. The identification of normal pulmonary pressures at rest in patients with left-side heart diseases does not exclude cardiopulmonary dysfunction upon exertion. EIPH may be a valuable index of the severity, chronicity and prognosis in a wide spectrum of cardiopulmonary diseases.


Exploring the relationship between various neurotransmitters and breast cancer cell growth has revealed their likely centrality to improving breast cancer treatment. Neurotransmitters play a key role in breast cancer biology through their effects on the cell cycle, epithelial mesenchymal transition, angiogenesis, inflammation, the tumor microenvironment and other pathways. Neurotransmitters and their receptors are vital to the initiation, progression and drug resistance of cancer and progress in our biological understanding may point the way to lower-cost and lower-risk antitumor therapeutic strategies. This review discusses multiple neurotransmitters in the context of breast cancer. It also discusses risk factors, repurposing of pharmaceuticals impacting neurotransmitter pathways, and the opportunity for better integrated models that encompass exercise, the intestinal microbiome, and other non-pharmacologic considerations. Neurotransmitters’ role in breast cancer should no longer be ignored; it may appear to complicate the molecular picture but the ubiquity of neurotransmitters and their wide-ranging impacts provide an organizing framework upon which further understanding and progress against breast cancer can be based. Breast cancer is the most common cancer among women. Despite research progress, it remains the leading cause of cancer death among women  and muchIt has been established in recent decades that neurotransmitters are involved in signaling pathways which influence the course of various malignancies, including breast cancer. Emerging data suggest that cancer cells take advantage of neurotransmitter-initiated signaling pathways, including VEGF, p53, AKT, and MAPK, to activate uncontrolled proliferation and metastasis Fig. . In addiAn appreciation of the role played by neurotransmitters is critical given both the numerous ways that neurotransmitters influence cancer and the number of factors that greatly influence neurotransmitter signaling, ranging from common medications (such as beta blockers and antidepressants) to stress (adrenergic activation)  Table 22. BreastAn exhaustive detailing of the entire landscape of neurotransmitter interactions with cancer is beyond the scope of a single paper. This review emphasizes the following critical topics, selected as likely closest to affecting standards of breast cancer care and representative of the myriad range of neurotransmitter impacts on breast cancer: epinephrine, norepinephrine, and adrenergic activation; acetylcholine; serotonin; dopamine; histamines; gamma-aminobutyric acid (GABA); and neurotensin; and drugs, such as beta blockers and monoamine oxidase inhibitors, impacting pathways mediated by these neurotransmitters Table . Key queThe wide-ranging impacts of neurotransmitters also provide consistent support for seemingly disparate areas of cancer research, ranging from the microbiome to fasting to exercise.Epinephrine  and norepinephrine  are endogenous catecholamines or monoamine neurotransmitters. Release of these hormones mediates the fight-or-flight response as they bind to adrenergic receptors. Breast cancer tissue has been found to overexpress β-adrenergic receptors .2AR signaling may also stimulate tumor growth by promoting DNA damage and p53-associated apoptosis suppression [Stress induced neuroendocrine activation or pharmacologic activation of adrenergic receptors has been found in mouse models to result in substantial increases to proliferation and distant metastasis . This phpression .Chances of recurrence appear to be increased by stress. Perego et al. proposed, and supported with murine evidence for lung and ovarian cancer, a mechanism for the awakening of dormant cancer cells . They prIn metastatic breast cancer, the skeleton is a frequent site of spread. Sympathetic nerves densely network across bones. It has been shown that activation of β2-adrenergic receptors, as in sympathetic nerve activation, in osteoblasts leads to increased bone vascular density and a more favorable bone microenvironment for breast cancer cells –14.If adrenergic activation stimulates breast cancer progression, then a natural question is whether inhibiting this activation can provide clinical anticancer benefits. This question has been aggressively pursued, particularly given the ready availability of beta blocker drugs –18.Beta blockers are primarily used for cardiac purposes—to treat abnormal heart rhythms and as a second- or later-line treatment for hypertension. Beta blockers are competitive antagonists that block receptor sites for epinephrine and norepinephrine on adrenergic beta receptors. Several of the preclinical studies examining the impact of adrenergic activation on breast cancer growth and migration also tested the impact of beta blockers and found them to have an inhibitory effect .On the clinical side, results have charted a more winding path. A 2017 meta-analysis of six studies, with a cumulative breast cancer patient population over 18,000, found no benefit of beta blockers on overall deaths, cancer-specific deaths, or recurrence . HoweverOther studies have reported more promising conclusions, particularly in regards to metastasis and recurrence; this evidence is stronger, though not definitive, for certain cancer subtypes . An examFurthermore, a meta-analysis of studies published between 2010 and 2013 found that women with breast cancer who took beta blockers had a significantly decreased risk of breast cancer death than women with breast cancer who did not take beta blockers .Propranolol is a beta blocker that was approved for use in the US in 1964. It is still in widespread use and it nonselectively blocks β-adrenergic receptors. In a triple-blind placebo-controlled trial reported in 2020, 30 patients with early-stage breast cancer were given propranolol for 7 days prior to resection and 30 were given placebo . Those gMontoya et al.’s study based on a single stage III breast cancer patient found that neoadjuvant propranolol reduces pro-proliferative Ki-67 and pro-survival Bcl-2 markers, and significantly increases p53 expression and induces apoptosis . MontoyaIn a randomized placebo-controlled biomarker trial, perioperative inhibition of COX-2 and β-adrenergic signaling was found to inhibit multiple cellular and molecular pathways related to metastasis and disease recurrence in early-stage breast cancer . CombinaThe combination of propranolol with non-chemotherapy medications is also showing promise. For example, Murugan et al. investigated the combination of propranolol with naltrexone  and found it to substantially inhibit tumor growth and improve survival in rat xenografts. The effects were attributed to decreasing tumor cell proliferation, inducing cellular apoptosis, and preventing the epithelial–mesenchymal transition in the tumor . In follCombating drug resistance is also an area of promise. β2-andrenergic receptors form a positive feedback loop with Her2 in breast cancer cells . Liu et While the interplay with β-adrenergic receptors has received more attention, other adrenergic receptors also appear to be relevant. Szpunar et al. showed that, in mice, treatment with a highly selective α2-adrenergic receptor agonist, dexmedetomidine, increased tumor growth and metastasis . Dexmede1 receptor partial agonist and 5-HT2 receptor antagonist.On the other hand, the opioid analgesic tramadol could inhibit proliferation, migration, and invasion of breast cancers by inactivating the α2-adrenoceptor signaling pathway . Based oAcetylcholine (ACh) functions in the nervous system as a neurotransmitter at the autonomic ganglia, the parasympathetic innervated organs, and the neuromuscular junction between motor nerves and skeletal muscle. Acetylcholine receptors (AChRs) fall into one of two categories; the relatively slow activating G protein-coupled metabotropic muscarinic receptors (mAChRs) and the faster activating ionotropic nicotinic receptors (nAChRs). ACh helps regulate cellular proliferation, differentiation, and apoptosis.Providing a partial explanation for the role of smoking in breast cancer, Nishioka et al. showed a decade ago with MCF10A (benign) and MDA-MB-231  breast cells that when nicotine is ligated with nAChR, it promotes EGFR and Src signals . Huang eLike nAChRs, the mAChRs also appear to influence breast cancer, specifically in that they are upregulated in breast tumors and absent in normal breast cells and tissues. Sales et al. has reported that mAChR agonists can act against breast tumors in a dose-dependent manner and be effective even at low doses .Serotonin  is synthesized from the essential amino acid tryptophan and mediates motility in the gastrointestinal tract and is a vasoactive agent in the blood. As a monoamine neurotransmitter, it also acts in the central nervous system. It regulates epithelial homeostasis in the breast. Serotonin is believed to impact immune signaling and stimulate growth of breast cancer cells –61. Olfa2A receptor. Proliferation and invasion is also facilitated by the 5-HT7 receptor in MDA-MB-231 cells. In these cells, 5-HT is essential to enhance the expression of TPH1 (tryptophan hydroxylase 1) and VEGF, supporting the mitogenic and oncogenic impact of 5-HT on breast cancer.Serotonin also initiates angiogenesis by the proliferation, invasion, and migration of endothelial cells . Sonier Tramadol, mentioned earlier in the context of α2-adrenoceptor, also interacts with serotonin receptors. Kim et al. reported that patients who received tramadol after breast cancer surgery had a decreased risk of postoperative recurrence and mortality, with the anti-tumor effect of tramadol appearing to involve inhibition of proliferation, induction of apoptosis, and effects on the serotonin 2B receptor and transient receptor potential vanilloid-1 expression . More geIf lowering serotonin activity reduces breast cancer recurrence, then a logical question is the impact of medications that raise serotonin levels. Evidence has been conflicting on this long-standing question. Two decades ago, researchers reported that use of tricyclic medications was associated with significantly increased breast cancer risk , 67 and However these studies were far from definitive and other analyses have reached the opposite conclusion. For example, a 2012 meta-analysis by Eom et al. on the relationship between SSRI use and breast cancer risk observed only marginal association attenuated over time, with no clinical significance . SimilarWhile the effectiveness of SSRIs for their primary indication may be in debate, there is little argument that they increase serotonin levels . FurtherBreast cancer overexpresses the hormone prolactin, and prolactin is implicated in breast cancer growth, metastasis, and chemoresistance –81. The Dopamine or its receptor agonists seem to exhibit inhibitory effect on tumor growth in breast and several other cancer types. However, dopamine fails to diminish the proliferation and invasion of breast and colon cancer cells, indicating that factors such as tumor type, receptors expressed, and doses used play a role . An impoA small study of giving patients with metastatic breast cancer cabergoline, an agonist of D2 dopamine receptors that has an inhibitory effect on pituitary prolactin secretion, had an inconclusive outcome . Later, Best known for its role in allergies, histamine is an important monoamine neurotransmitter. Four G-protein-coupled histamine receptor subtypes mediate neuronal histamine’s effects . The hisSpeisky et al. reported that the H4 receptor may be a useful biomarker for predicting triple negative breast cancer prognosis . Based oA receptor alpha3, which is normally exclusively expressed in adult brain, is expressed in breast cancer, with higher tumor expression being associated with poorer survival [A receptors increase brain metastases in breast cancer patients. The receptor could therefore represent a promising therapeutic target [The primary inhibitory neurotransmitter in the human brain is GABA. GABA may promote cancer cell proliferation and migration and is amplified in multiple cancers including breast. Gumireddy et al. observed that the GABAsurvival . As showc target , 95.A receptor and causes actin reformation and migration of breast cancer cells by collagen matrices. Observational epidemiological studies also show that benzodiazepine use increased the risk of breast cancer and many other cancers in a dose-dependent manner. Mimics of GABA, an inhibitory neurotransmitter, are frequently used to reduce peripheral nerve pain caused by chemotherapy. However, there is concern that GABA treatment could increase breast cancer metastasis [Propofol is a drug with agonist activity for the GABAtastasis . ImproveFrom 1419 primary breast tumors in a French institute, neurotensin receptor-1 overexpression was found in about one-third of breast tumors from patients undergoing primary surgery . NeuroteNeuropeptide Y is a 36 amino-acid neuropeptide that plays a role in various physiological and homeostatic processes in the nervous systems including the osteogenic response. Its major receptors are overexpressed in multiple tumors including breast cancer metastasis . NeuropeImmune checkpoint blockade therapy has become a key tool in the treatment of several types of cancer, including triple negative breast cancer, in recent years. There is an active line of research towards improving and broadening the efficacy of such immunotherapies by modifying the tumor microenvironment. Neurotransmitters appear to play an important role here. Targeting tumor-associated macrophages to reduce their inhibition of antitumor T-cell reactivity may improve an immunotherapy’s activity against breast cancer .Monoamine oxidase A is an enzyme bound to the outer mitochondrial membrane. In the brain, it is involved in degrading serotonin, dopamine, epinephrine, and norepinephrine . Given iAn excellent demonstration of this direction was provided by Wang et al. . They rePiecing together the puzzle of neurotransmitters’ influence on breast cancer is vital to better understand breast cancer biology and discover novel treatment approaches. It is also urgent to clarify the effects—positive, negative, or neutral —on breast cancer of commonly used medications, including beta blockers, SSRIs, MAOIs, and many more. Even the anesthesia used for breast cancer surgery may need thoughtful examination .While all this potentially complicates the picture of breast cancer prevention and treatment, one reason that neurotransmitters provide a compelling framework for new approaches to breast cancer is the manner in which they link seemingly disparate observations and research directions. This is a source of intricacy, even outside the domain of pharmaceuticals. For example, exercise is advised as preventive against breast cancer, and cancers in general but is also a stressor that releases adrenaline and, as discussed, β2-adrenergic receptor signaling appears to be detrimental in regards to breast cancer. Jensen et al. propose combining exercise and beta blockers for breast cancer patients, such that the positive adrenergic signaling advantages of exercise can be obtained while avoiding chronic adrenergic signaling in the tumor microenvironment . WackerhPsychological stress has been observed to be a negative prognostic factor for survival among breast cancer patients  and betaNeurotransmitters may also provide an underpinning for observations related to the influence of the intestinal microbiome on breast cancer. The gut microbiota has been found to influence treatment side effects and prognosis in breast cancer patients . This isAs indicated in prior sections and Table At the other end of the spectrum, insights can also be expected from advances in sophisticated molecular experimental techniques. McCallum et al.’s recording of neural activity in a triple-negative mammary cancer mouse model while the tumor grew and metastasized is a recent example . AnotherLastly, it is worth considering why neurotransmitters would have a substantial impact on breast cancer and whether it is reasonable for this to be the case. Indeed, a reason why this relationship was not meaningfully considered for decades is that it was not self-evident that there should be one. However, findings over recent years reveal the ubiquity of neurotransmitters in the body’s various signaling pathways. Evidence even suggests that certain neurotransmitter precursors evolutionarily predate the appearance of neurons and animals . NeurotrNeurotransmitters appear to complicate the clinical and mechanistic picture of breast cancer, but denying this complexity is unlikely to pay dividends. Taking a signaling oriented view of breast cancer, particularly incorporating neurotransmitters, unifies various observations and provides clear direction for obtaining clinically significant short-term and long-term results in improving therapeutic strategies.


Arteriosclerosis is an important age-dependent disease that encompasses atherosclerosis, in-stent restenosis (ISR), pulmonary hypertension, autologous bypass grafting and transplant arteriosclerosis. Endothelial dysfunction and the proliferation of vascular smooth muscle cell (vSMC)-like cells is a critical event in the pathology of arteriosclerotic disease leading to intimal-medial thickening (IMT), lipid retention and vessel remodelling. An important aspect in guiding clinical decision-making is the detection of biomarkers of subclinical arteriosclerosis and early cardiovascular risk. Crucially, relevant biomarkers need to be good indicators of injury which change in their circulating concentrations or structure, signalling functional disturbances. Extracellular vesicles (EVs) are nanosized membraneous vesicles secreted by cells that contain numerous bioactive molecules and act as a means of intercellular communication between different cell populations to maintain tissue homeostasis, gene regulation in recipient cells and the adaptive response to stress. This review will focus on the emerging field of EV research in cardiovascular disease (CVD) and discuss how key EV signatures in liquid biopsies may act as early pathological indicators of adaptive lesion formation and arteriosclerotic disease progression. EV profiling has the potential to provide important clinical information to complement current cardiovascular diagnostic platforms that indicate or predict myocardial injury. Finally, the development of fitting devices to enable rapid and/or high-throughput exosomal analysis that require adapted processing procedures will be evaluated. Human arteriosclerotic disease is a complex systemic inflammatory disorder characterized by key interactions between several different biological modifiers. These include various lipids, enzymes, endothelial cells (ECs), vascular smooth muscle cells (vSMCs), cytokines and both circulating and resident vascular stem cells and mononuclear cells . Initialin vivo using wildtype mice following flow restriction due to carotid artery ligation , transplant arteriosclerosis, pulmonary hypertension and in-stent restenosis (ISR)] . This vaSonographic measurement of carotid intima-media thickness (cIMT) is widely used as an indicator of not only local, but generalised atherosclerosis, even in asymptomatic individuals . Indeed,Pre 2015 studies reveal little standardisation relating to the methods used for cIMT measurement, which gave rise to a disparity in the ability of cIMT to predict risk classification . From 20Chronic coronary artery disease typical of AMI is usually associated with a rupture of atherosclerotic plaque, thrombus formation and obstruction of blood flow leading to necrosis of myocardium. However, in some patients with AMI, there are no significant lesions present in coronary arteries interrogated by angiography, thus confounding early identification of atherosclerosis. ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation myocardial infarction (NSTEMI), unstable angina and stable coronary artery disease (CAD) all carry significant morbidity and mortality . As a re1) partial somatic cell reprogramming following de-differentiation  of mature differentiated vSMCs or endothelial-mesenchymal transition (EndoMT) of ECs to vSMC-like cells (2) partial differentiation of immature multipotent “mesenchymal-like” vascular stem cells (MVSCs) that reside within the vessel wall or migrate from the circulation  partculation . Severalculation . Howeverculation  SRY-relaculation , in addiculation , in contculation .Myocardial infarction with non-obstructive coronary arteries (MINOCA) also involves a heterogenous group of atherosclerotic and non-atherosclerotic patients resulting in myocardial damage that is not due to obstructive coronary artery disease . While tInitially thought to be merely a disposal mechanism for cellular waste, extracellular vesicles (EVs) have recently become a focal point of research due to their multifunctional role in many biological processes, including cell-cell communication , proteinEVs are released in various shapes and forms by mammalian and non-mammalian cells. Due to their small size, differentiation between EVs of different origin proves challenging; however, they can be characterised to some extent by their size, content, and sub-cellular origin . There aThe second method of EV generation involves the shedding of membrane-bound sacs, known as microvesicles, from various cell types. During this process, small cytoplasmic vesicles protrude from the host cell membrane, which then detaches through a blebbing process. The mechanism that drives this process is not fully understood; however, the rate of the shedding process is known to be dramatically increased by a rise in cytosolic concentrations of calcium ions. The increase of calcium ions activates scramblase and calpain, resulting in a loss of membrane phospholipid symmetry and degradation of various proteins, thus facilitating outward budding of microvesicles from the cellular membrane . These Evia exosomal secretion of transferrin receptors in the bloodstream . Following this, invagination of late endosomal membranes results in the formation of intraluminal vesicles (ILVs) within large MVB’s  fusion to the plasma membrane and 2) fusion with lysosomes and the degradation and recycling of their content   . In summExosomes can also be formed in an ESCRT-independent manner. There are several ESCRT-independent mechanisms which demonstrate the successful generation of ILVs in MVBs following complete disruption of ESCRT function . Althoug2, ADP ribosylation factor 6, and syntenin. The formation of ILVs is dictated by the association of syntenin with ALIX and relies on the bioavailability of heparan sulfate, syndecans, ALIX, and ESCRTs  that control the cellular response and antigen presentation during cellular stress, in addition to assisting protein folding and trafficking . MultiveExosomes are released during physiological and pathological conditions by many cell types. There are two main pathways by which exosomes are released and secreted from their host MVB: the constitutive release pathway, associated with general physiological secretion of exosomes ; and/or drosophila S2 cells attenuated exosome secretion  . The mosecretion . Furtherecretion  while Raecretion . By contecretion , glycan ecretion  and actiecretion .N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), tethering factors, Rabs, and other Ras GTPases facilitate MVBs fusion with the plasma membrane. To date, the exact molecular requirements for MVB membrane fusion are not well established; however, many studies have reported the crucial role of SNARE proteins and synaptotagmin family members  and three Q-SNAREs forming four coiled-coil helices . Increased levels of intracellular calcium following monensin or calcium ionophore A23187 increases exosome secretion in human erythroleukemia K562 cells  endocytosis or 2) direct fusion with the target cell , C-reactive protein, and plasminogen activator-1  and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways contributing to the development of heart disease and potentially other chronic diseases , which are unique extracellular membrane-bound microparticles that serve as initial sites for mineral formation . ExosomeOther important mechanisms driving exosomal control of VC include regulation on cell autophagy  and oxidIn summary, a large body of research provides substantial evidence in favour of the active involvement of endothelium-derived exosomes in the development and progression of associated vascular diseases. Moreover, as endothelial exosomes efficiently deliver their cargo into recipient vascular cells to change function, they represent an important potential biomarker for disease detection and a therapeutic agent to effectively treat disease.As exosomes are remarkably stable in biofluids, such as plasma and urine, and can be isolated for clinical evaluation even in the early stages of the disease, exosome-based biomarkers have quickly become adopted in the clinical arena with the first exosome RNA-based prostate cancer test introduced for early prostate cancer detection . CurrentIn the past decade, several studies have shown an association between elevated numbers of EVs, including exosomes, and prediction and development of CVD. Circulating EVs have been described during the development of atherosclerotic plaques , peripheLiquid biopsy-based exosomal profiling offers a promising platform to assist clinical diagnosis and prediction more accurately. In particular, exosomal miRNAs possess promising protective functions in CVD while circulating exosomes have shown great potential for diagnosis and risk assessment in CVD. In particular, substantial progress has been made in identifying the therapeutic role of circulating EC-derived EV miRNAs, particularly miR-92a-3p, in regulating the phenotype of ECs and SMCs under atherosclerotic conditions . FurtherApart from miRNAs, exosomal proteins may also play a significant role in CVD diagnostics. P-selectin-expressing microparticles, CD3/CD45, SMA-α circulating exosome levels, and exosomal cystatin C, Serpin F2, CD14 levels are all significantly correlated with a high risk for incident CVD and mortality . In all,+/Annexin V+) to mononuclear progenitor cells is elated to adverse clinical outcome in patients with acutely decompensated chronic HF , polymer-based precipitation, immunoaffinity, chromatography and filtration and are often chemically lysed after isolation . MoreoveHospitals and clinics rely heavily on central laboratories to analyse patient samples and deliver accurate and timely results. This common operational model places the vast majority of blood/sample analysis responsibility on the central laboratory, irrespective of the relative exigency of the test. However, the layout of this model, in which sample transport and handling contribute to delays, is unfavourable for streamlining the triage process and ensuring critical needs are fulfilled. By contrast, point-of-care-testing (POCT) requires conducting a diagnostic test in a patient’s proximity and incorporates the use of a compact device to facilitate rapid biomarker measurement. Unlike central laboratories in which a variety of sophisticated instruments and measurement equipment are routinely utilised, a point-of-care (POC) platform is generally robust, with the capacity to perform measurements in a range of test environments and without the need for highly specialised training.1) “laboratory testing is performed in the direct proximity of the patient,” 2) “results available quickly” and 3) “results lead to a rapid diagnosis or consequences for treatment.” Evidently, they all relate to improving patient management and the efficiency of the process. As a result, this format of diagnostic testing is ideally placed to positively impact cardiovascular assessments due to the critical need to triage patients without delay.This diagnostic approach is ideally suited to cardiovascular assessments in which immediate action is a priority. For instance, minimising the system delay—which is the time period between emergency department presentation and insertion of a catheter—correlates with reduced infarct size and improved left ventricular function following myocardial injury . A promiThe development of compact and user-friendly platforms that can accommodate exosome analysis, whilst adhering to quality assurance and compliance with accreditation standards, in addition to the aforementioned recommendations, requires a combination of meticulous design and careful consideration of both the isolation and measurement processes . Moreove1) Ultracentrifugation, 2) Size-Based Isolation Methods 3) Immuno-Affinity Purification 4) Polymer-Based Precipitation and 5) Microfluidics-Based Isolation Techniques , for instance, is a technique that enables controlled manipulation of particles in a fluid and can separate particles based on their dielectric properties, shape, size and the properties of the surrounding fluid using a non-uniform electrical field to induce a dipole moment in the targeted particles . Moreove2 to facilitate EV isolation. Fe3O4@TiO2 core-shell nanoparticles were synthesised, in which the outer TiO2 shell supported binding of serum exosomes, whereas the Fe3O4 core enabled particle manipulation by magnetic separation from the sample and hence, exhibits some favourable characteristics for POCT  on the surface of magnetic beads to bind to phosphatidylserine  is a key metric that can be assessed to validate the proficiency of an enrichment technique and thus support the direct comparison of different approaches. However, the recovery rate was not determined in several studies, thus restricting direct comparison with other approaches. The apparent lack of cohesion between reported performance characteristics of these exosome isolation techniques increases uncertainty regarding the most promising methods. Moreover, determining the purity of the enriched exosomes is quite important as it can assist in confirming that a biomarker has originated from EVs and not just been co-isolated with the exosomes . The co-seeA crucial aspect of adapting analytical techniques for POCT is the ability to refine and compartmentalise standard laboratory procedures to simplify the measurement process. A POC assay for instance, should measure biomarkers directly from samples without manual pre-treatment, thus eliminating the need for ancillary instrumentation and preventing inter-operator variability. Bodily fluid samples can be quite complex, however, often requiring pre-treatment to support accurate measurements. Whole blood, for example, comprises a multitude of biomolecules, cells and cellular debris which can be challenging to process. Therefore, plasma or serum are often the media of choice as red and white blood cells are removed, the main distinction between plasma and serum is that serum lacks the blood clotting agent fibrinogen. Crucially, before processing whole blood for serum extraction, the collected blood must be left to clot for approximately 30 min, whereas plasma separation can begin immediately, and can be complete within a few minutes. Various analytical approaches incorporate automated on-board plasma extraction prior to exosome isolation see. These pCentrifugal microfluidic lab-on-a-disc (LoaD) platforms are particularly suited to plasma extraction from whole blood with one group incorporating on-disc filtration to extract EVs from separated plasma . Whole bAn alternative automated microfluidic-based plasma separation approach that involves an inertial separation structure has been reported . FundameSeveral of the platforms excluded pre-treatment methods, with one group developing an acousto-fluidic device to isolate exosomes directly from saliva by generating standing surface acoustic waves (sSAW) . SurfaceOther groups report the recovery of sample EVs using tailored microfluidic-based devices, such as the nPLEX  and iMERIt must be emphasised that embedding multiple stepwise operations within a microfluidic device to isolate exosomes and measure their contents requires extended periods of time to accommodate the analytical process. Combining some of the more rapid exosome enrichment microfluidic-based techniques with emerging accelerated PCR assays may prove to support shorter TATs and permit integration within rapid sample-to-answer POC platforms . AlthougIt is well established that endothelial dysfunction is a key atherosclerotic mechanism that without the balanced presence of corresponding athero-protective factors, leads to intimal thickening and progression of arteriosclerotic disease. Currently, cIMT is regarded as one of the primary surrogate indices of atherosclerosis. However, in some AMI patients, interrogation by angiography reveals no significant lesions within surveyed coronary beds. Fundamental to individual risk profiling and the diagnosis of ACS is the measurement of biomarkers, typically from collected whole blood specimens. Endothelial exosomes found in liquid biopsies may present as ideal cardiac biomarkers because they are involved in the progression of atherosclerosis and enhanced secretion has been demonstrated during periods of cellular stress.Performing liquid biopsies is a potentially minimally-invasive route to elucidating underlying functional disturbances; however, the prospect of exploiting exosomes as diagnostic biomarkers of CVD is intrinsically dependent on the efficiency at which they can be acquired for analysis. Currently, isolating exosomes requires laborious processes that are conducted in laboratory settings and exhibit differing capabilities in attaining consistent yields and extract purity. Many of these approaches offer suboptimal performances and are inherently unsuited to POCT or efficient high-throughput measurements. Hence, tailored microfluidic-based devices favour adaption of exosomal analysis for diagnostic platforms and provide greatly reduced processing times. These devices—some of which incorporate automated pre-treatment—can efficiently enrich exosomes from whole blood, perform lysis and supply a nucleic acid, protein and lipid rich extract for subsequent analysis. The significance of exosomes and the cargo they harbour remains in its infancy, yet it is anticipated that greater detail will be revealed regarding their role in CVD and how best to interpret their fluctuating presence during disease progression. Ultimately, their clinical value for predicting CVD will be defined by a combination of diagnostic insight which they afford, therapeutic strategies which they permit and the ease at which they can be reliably and consistently isolated from samples.