In this review, we evaluate the updated data of the immunological effects of kinase inhibitors on the innate immune system and provide an in-depth analysis of the underlying mechanisms. We also discuss how this immunological effect can be harnessed to improve cancer treatment and highlight recent successes, such as the combination with anti-tumor immunotherapy. Last, we explore novel kinase targets and the incorporation of them with targeted drug delivery techniques as promising research areas.Innate immune cells constitute a plastic and heterogeneous cell population of the tumor microenvironment. Because of their high tumor infiltration and close interaction with resident tumor cells, they are compelling targets for anti-cancer therapy through either ablation or functionally reprogramming. Kinase inhibitors (KIs) that target aberrant signaling pathways in tumor proliferation and angiogenesis have been shown to have additional immunological effects on myeloid cells that may contribute to a protective antitumor immune response. However, in patients with malignancies, these effects are poorly described, warranting meticulous research to identify KIs’ optimal immunomodulatory effect to support developing targeted and more effective immunotherapy. As many of these KIs are currently in clinical trials awaiting approval for the treatment of several types of solid cancer, we evaluate here the information on this drug class’s immunological effects and how such mechanisms can be harnessed to improve combined treatment regimens in cancer. The abnormal activation of signaling pathways is one of the hallmarks of various types of cancer. Kinases function as a crucial node within the signaling network by phosphorylating surface receptors and cytoplasmatic enzymes, activating cellular functions that could potentially trigger carcinogenesis. Therefore, protein kinases serve as a promising target for anti-cancer therapy. Following the success of the first small molecule kinase inhibitor (KI), Imatinib, for the treatment of chronic myelogenous leukemia 20 years ago, along with a better understanding of the molecular mechanism of oncogenesis, an increasing number of KIs have gone through extensive investigation, which has ultimately led to the broad clinical use of KIs in cancer patients with specific mutations.Initially, the therapeutic efficacy of KIs has been postulated to be derived from their actions on malignant cells only. However, clinical evidence suggests that patients show durable responses after the discontinuation of KI therapy . SupportImmune evasion and suppression remain the major hurdles in anti-cancer therapy. Immunotherapy has revolutionized the treatment of cancer, giving hope to patients who were previously deemed incurable. However, only a minority of patients experience long-lasting benefits from these therapies, and the incidence of severe side effects remains high . To unloThe immunological effects of KIs provide a strong rationale for the combination with immune checkpoint inhibition, aiming at synergistic effects. In this review, we evaluate the most recent data on the immunological effects of KI, with a specific focus on the innate immune system. We will explore how these effects can be incorporated in the multimodal approaches against malignant tumors and to improve the patient outcome.In addition to providing protection against infections, the innate immune system serves as the front line of the non-specific defense against oncogenesis and cancer progression, but it also initiates and directs the adaptive immune response . Cells oTumor-associated macrophages (TAMs) are the largest fraction of the myeloid infiltration within TME, which is generally associated with adverse clinical outcomes, as shown in various solid tumors ,11,12,13During the past few decades, molecular pathways regulated by driver oncogenes have been extensively studied. We now know that genetic alterations of protein kinases are involved in the oncogenesis of various types of cancer. Therefore, oncogenic kinases have become highly attractive pharmaceutical targets over the past two decades . The tarThe advantage of targeted therapies, such as KIs, compared with conventional chemotherapy, is that they are highly specific, which may limit treatment-related side effects. However, as the targets of KIs are widely distributed, the receptor cross-reactivity between the targeted cancer cells and healthy cells in patients could cause off-target toxicities, such as cytopenia and gastrointestinal side effects .Based on target selectivity profiles, the development of KIs has followed two related yet independent paths ,26,27, nKIs were initially developed as tumor-intrinsic therapies; however, recent work uncovered that they are capable of modulating the composition and functionality of the tumor immune microenvironment . PreclinThe immunological effect of KIs could derive from the direct impact of drugs on malignant cells. As a notable example, KIs may invoke immunogenic cell death (ICD), an immunologically unique type of apoptosis that could elicit a potent adaptive immune response against dead cell-related antigens. Unlike the homeostatic removal of dying cancer cells , which facilitates an anti-inflammatory and tumor-promoting process, ICD induced by certain anticancer agents may elicit the immunostimulatory clearance of dying cells by APCs, where the dying cancer cells function as a vaccine that drives tumor antigen-directed immunity . The capAnother mechanism of the immunogenic effects of KIs is through the induction of pyroptosis, a hyperinflammatory type of programmed cell death that was initially observed in macrophages upon infection . The immKIs could also interfere with the paracrine signaling between tumor and immune cells as cytokines released by cancer cells can have diverse effects on myeloid cell function. For instance, in a prostate cancer murine model, tumor cells were able to recruit MDSCs and drive the immunosuppression-related gene expression in these cells through the secretion of multiple cytokines. This effect was abrogated by Cabozantinib pre-treatment by downregulating the immunosuppressive cytokine levels, including IL-1 receptor antagonist (IL-1Ra), CCL 5, CCL12, CD40, and hepatocyte growth factor (HGF) [Altogether, these observations support the idea that the anti-tumor immune response elicited by tumor-released factors/signals upon KI treatment might have profound clinical implications.VEGFR is the major therapeutic target for most MKIs. The multitarget VEGFR KIs can exert an immunological effect either through the alteration of angiogenesis in the TME or through other non-angiogenic effects on the TME.A structurally abnormal and leaky vasculature is a hallmark of most tumor tissues. VEGF, released by tumor cells and surrounding stoma, is the major driver of tumor angiogenesis. The VEGFR family contains three tyrosine kinases, of which VEFGR2 is the dominant signaling receptor for VEGFs that transmits signals to the nucleus and ultimately promotes vascular endothelial cell mitogenesis, survival, and vascular permeability during angiogenesis . VEGFRs While many of the immunological effects of VEGFR–MKIs can be explained by their VEGFR-inhibitory effects, various other cellular and environmental factors within TME may be involved as well. It has been well established that KIs have a direct impact on both malignant and other healthy cells. The tumor infiltrating myeloid cells (TIMCs) are of particular interest because some of the targeted kinases are also expressed on TIMCs . Indeed,Supporting the therapeutic relevance of these mechanisms, accumulating evidence suggests that MKIs promote an immune permissive environment by the direct immune-stimulation on myeloid cells. For instance, Sorafenib activated NK cell-mediated direct killing against a tumor by inducing pyroptosis in the macrophages and reducing the MHC-I expression on malignant cells simultaneously . Along sBesides triggering the non-specific cytolysis function of NK cells, KIs mediate an immune-stimulatory effect by priming effector T cells. For instance, in a preclinical murine model, low-dose Regorafenib was found to increase cytotoxic T cell function and antitumor immunity through the programming of macrophages toward the anti-tumor phenotype, possibly via the p38 mitogen-activated protein kinase (p38MAPK) pathway . DCs plaNeutrophils are the first line responders in the initiation of an innate immune response, and can acquire either pro-inflammatory, antitumor, or pro-tumorigenic properties through mobilization and infiltration. The differentiation of the aforementioned phenotypes requires the close interaction with the chemokine context of TME, which could potentially be modulated by MKIs . For insMDSCs are described as a heterogeneous population of pathologically activated myeloid cells at various stages of maturation. MDSCs affect multiple aspects in TME, including immune suppression, promotion of angiogenesis, and tumor invasion; thus, the functional alterations caused by KIs might assist or impair their efficacy . SupportIn summary, the majority of the FDA-approved MKIs have been shown to mediate the robust immune-stimulatory effects that might amplify the therapeutic anti-tumor efficacy. However, inconsistencies in the literature should be noted, possibly because of the difference in the selectivity and targeted kinome among the MKIs and the relative shortage of clinical studies. Hence, the therapeutic relevance remains a matter of debate.Besides the VEGFR pathway, many other growth factors and RTKs have been shown to work in a complementary and coordinated manner to regulate tumor growth and angiogenesis. Although practically all kinase inhibitors have been reported to exert antitumor activity somehow via an immune-mediated mechanism , certainThe TYRO3, AXL, and MERTK (TAM) family is a group of RTKs that uniquely co-express on immune cells. Besides its role in tumorigenesis and metastasis, the TYRO3/AXL/MERTK family plays a major role in maintaining tissue homeostasis and wound healing via various mechanisms, including efferocytosis, reprogramming macrophages towards anti-inflammatory phenotypes, and terminating TLR signaling in APCs. The TYRO3/AXL/MERTK family can be triggered by apoptotic cell materials, which are abundant within the TME, hence promoting a immunosuppressive landscape . TherefoFunctioning as an RTK binding with HGF, c-MET is aberrantly expressed in cancers, regulating cell proliferation, motility, migration, and invasion . InteresActivated RTKs are able to recruit and regulate a wide range of intracellular protein kinases to regulate cell proliferation and differentiation, including RAS/MAPK and PI-3K/AKT/mTOR signaling. Those cytoplasmic kinases are crucial mediators of extracellular signals perceived by RTKs, and oncogenic mutations in these kinases are also common in various solid tumors . The MAPTaken together, these observations complemented and extended the findings of the immunomodulation of KIs, which could serve as possible therapeutic targets or be used as combinatorial regimes with other immunotherapies.Immunotherapies, especially ICI that targets PD-1/PD-L1 and CTLA-4 pathways to re-activate T effector cells, play an emerging role in cancer treatment . HoweverPreclinical studies have shown the synergistic antitumor efficacy of such combinations rely on the innate immune system ,40,89. FSeveral trials using combined ICI and KI therapies are ongoing or have been completed , a member of the TYRO3/AXL/MERTK RTKs family, one of the latest identified oncogenes with concomitant immunoregulatory functions in the innate immune system, due to its broad expression in various TIMCs responsible for wound healing and maintaining homeostasis under physiological conditions ,99,100. Secondly, another important area of investigation for the clinical oncologists is to try and understand the heterogeneity of the therapeutic response to KI treatment. We should investigate the factors influencing this heterogeneity (why do some patients respond better than others?) and how we can identify these patients (what are the biomarkers that can tell us beforehand that a patient responds effectively to KI therapy?).Thirdly, despite these successes, the field still requires a better understanding of how to fully exploit KIs for a therapeutic benefit. On the one hand, the poor accumulation at tumor sites and severe off-target effects that cause intolerable systemic side effects in patients have greatly offset their therapeutic efficiency and limited further clinical application. Nanoparticles that improve the selective delivery of drug payloads to the cancer cells or tumor-promoting immune cells would offer a great opportunity to overcome the drug toxicity profile and amplify antitumor activity, as recently shown in experimental models . On the In conclusion, in the present review, we highlighted the myeloid cells’ indispensable role in anti-tumor immune responses, and the effect KIs exhibit to modulate these important immunological mechanisms. We described KIs’ potential to improve combination therapies for solid cancers, and we anticipate that ongoing clinical trials will ultimately guide the implementation of optimal combinatorial approaches to maximize the immune system’s anti-tumor activity.


Macro and micronutrient deficiencies pose serious health challenges globally, with the largest impact in developing regions such as subSaharan Africa (SSA), Latin America and South Asia. Maize is a good source of calories but contains low concentrations of essential nutrients. Major limiting nutrients in maize-based diets are essential amino acids such as lysine and tryptophan, and micronutrients such as vitamin A, zinc (Zn) and iron (Fe). Responding to these challenges, separate maize biofortification programs have been designed worldwide, resulting in several cultivars with high levels of provitamin A, lysine, tryptophan, Zn and Fe being commercialized. This strategy of developing single-nutrient biofortified cultivars does not address the nutrient deficiency challenges in SSA in an integrated manner. Hence, development of maize with multinutritional attributes can be a sustainable and cost-effective strategy for addressing the problem of nutrient deficiencies in SSA. This review provides a synopsis of the health challenges associated with Zn, provitamin A and tryptophan deficiencies and link these to vulnerable societies; a synthesis of past and present intervention measures for addressing nutrient deficiencies in SSA; and a discussion on the possibility of developing maize with multinutritional quality attributes, but also with adaptation to stress conditions in SSA. Deficiencies of essential macro and micronutrients in human diets pose serious health challenges worldwide ,2, althoBecause of over-reliance on maize in SSA, diseases induced by nutrient deficiencies such as kwashiorkor and pellagra that are caused by lack of proteins and tryptophan, respectively ; night bThe term biofortification, in general, refers to the biological enrichment of food crops with either macro or micronutrients by means of agronomic practices, conventional plant breeding or genetic engineering . ThroughBecause of these reasons, it is important to look at possibilities of stacking nutritional quality traits in a single cultivar. During the breeding process, breeders should also pay attention to the production environments to which biofortified cultivars are adapted. For instance, maize production in SSA is constrained by abiotic stresses such as heat, drought and low soil nitrogen ,22, as wFood inequalities still exist even in the 21st century, where some people in the world subsist on monotonous, cereal-based diets. For instance, in SSA maize alone provides more than 30% of total calories in more than 20 countries ,24. BecaDifferent maize food products are consumed across the world, and in African countries maize meal is commonly prepared from dried grain, and sometimes consumed as boiled or roasted green maize . In thisStatistics show that the global burden of both macro and micronutrient deficiency rose by more than 50% in the period between 1990 and 2010 . Figure In as much as Zn and vitamin A are important, the improvement of protein quality has long been a priority, dating back to the 1950s when the main focus was to alleviate protein-energy malnutrition prevalent in developing countries . However−1 per day respectively [Dietary Reference Intakes (DRIs) is a generic term used for nutrient reference values, including Recommended Dietary Allowance (RDA) and Estimated Average Requirement (EAR). It defines the lowest continuing intake level of a nutrient that is required to maintain a target level of nutrition in an individual . DRI of ectively . Nutrienectively . DRIs arVitamin A, Zn and essential amino acids such as lysine and tryptophan are important nutrients for growth and development in humans. Lysine and tryptophan are building blocks of proteins, whereas vitamin A and Zn are required in minute quantities but play crucial roles in metabolism . For insProtein, Zn and vitamin A malnutrition negatively affect cognitive development, reduces the ability to do physical work, and increase susceptibility to acute respiratory infections, diarrhea and a weakened immune system ,31,34. LSeveral interventions have been used to increase dietary intake of nutrients. These include industrial fortification , clinicaEnriching food with essential nutrients such as minerals and vitamins either comes as a commercial choice by food processors or a stipulated government policy to curb certain nutritional deficiencies . In SSA,Despite its wide coverage in curbing nutrient deficiencies, food fortification has a number of limitations. It can cause health hazards due to toxicity, especially when food processors exceed the stipulated dose  due to l4), Zn acetate, Zn gluconate and Zn oxide (ZnO) and Zn amino acid chelates [Dietary supplements could be a useful intervention to mitigate the effects of various forms of nutrient deficiency in targeted communities . Dietarychelates ,62. The chelates . Vitaminchelates . Proteinchelates . BecauseSuccess stories for the use of dietary supplements have been reported across the world, including SSA. For instance, the administration of dietary Zn supplements has been reported to increase the linear growth patterns and weight gain in children, and corrected hormonal imbalances in adolescents, in low-income countries ,64. Zn sDespite all the health benefits of dietary supplements, this intervention has been associated with several limitations. Toxicity from overdose of dietary supplements may arise. Zn and vitamin A toxicity were reported to cause severe abdominal pains, nausea and vomiting in some patients . In addiDietary diversification is an intervention to change household diets to increase the variety and quantity of micronutrient-rich foods and animal-based food sources . People Whilst consumption of diversified foods is unaffordable for a large fraction of the population, reduced nutrient bioavailability in some foods prohibits maximum absorption . For insAgronomic practices to improve Zn content in maize kernels has been widely reported through application of Zn fertilizers such as ZnO, Zn-EDTA and ZnSO4 ,81. ThisPlant breeding holds great promise for contributing to improvement of the nutritional status of maize and other staple cereal crops across the world . Both naDespite its potential widespread coverage and sustainability, this intervention is facing serious challenges that may need prompt attention. Whilst breeders have succeeded in accumulating high levels of Zn, provitamin A and essential amino acids in maize, all these single nutrient varieties are of less benefit to small-landholder farmers in SSA, whose limited arable land limit crop diversification of these biofortified varieties. Hence, the development of multinutrient maize cultivars, with at least two nutrients among Zn, provitamin A and protein, is an attractive approach to effectively reduce malnutrition challenges in SSA and other maize-based developing countries. It is imperative for breeders to understand the genetic mechanism of QPM, provitamin A and Zn-enhanced maize to facilitate successful integration of these nutritional traits in a single variety.opaque-2 mutation in the maize genome was the advent of QPM breeding [opaque-2 locus from homozygous dominant or heterozygous to homozygous recessive alleles that confer higher tryptophan and lysine content than in normal maize [opaque-2 mutation came with undesirable phenotypic characteristics of the maize grain [opaque-2 mutation coupled with genetic manipulation of the opaque-2 enhancers or modifiers genes [Since the 1960s, scientists have shown great interest in looking for gene mutants that could provide better protein quality in maize grain ,95. The breeding . After trs genes . The modrs genes .opaque-2 locus; (ii) enhancers or modifiers that result in the opaque-2 gene to confer high lysine and tryptophan and (iii) modifier genes that change the opaque-2 induced soft endosperm to the desired hard endosperm [To retain the QPM genetic background, the development of QPM-based multinutrient maize therefore involves manipulating three distinct genetic systems: (i) the homozygous recessive ndosperm . In addindosperm . QPM, inndosperm .Provitamin A carotenoids are derived from a large class of isoprenoids. There are two main classes of carotenoids, the xanthophylls and carotenes . XanthopMultinutrient maize inclusive of high levels of provitamin A should, therefore, have substantial amounts of dietary carotenoids, in particular β-carotene, α-carotene and β-cryptoxanthin, compared to conventional yellow or white maize. Among cereals, provitamin A maize has been reported to have the highest carotenoid concentration . The mosLarge genetic variation exists for these carotenoids in maize germplasm, making it possible to develop multinutrient cultivars with an added vitamin A nutritional advantage. Provitamin A content in yellow and orange varieties ranges from less than 2 to 25 µg/g . The tarKnowledge of the genetic basis of any trait is important in crop improvement. The genetic basis of high grain Zn content in maize was reported to be controlled by many genes, each contributing a small effect to the overall phenotypic expression of this trait . Such poSeveral studies have suggested the involvement of many QTLs in the accumulation of Zn in maize ,106,107.The development of multinutrient maize employs a wide range of both conventional and nonconventional breeding strategies. Breeding methods such as introductions, hybridization and mutation breeding, and modern techniques such as marker-assisted and genomic selection, could be used interdependently to increases the rate of genetic gain in breeding for multinutrient maize . Other hThe development of multinutrient maize requires the existence of adequate genetic variability for targeted nutrient concentration . This enSeveral studies have been conducted to evaluate genetic variability of Zn ,108,111 Apart from these findings, several studies have reported higher Zn content in QPM germplasm than maize from other nutritional profiles ,103,115.Considering the available genetic variability for Zn and provitamin A content in maize, and the EAR of Zn and provitamin A, HarvestPlus, in collaboration with plant breeders, scientists and food processors, has set a target of 33 and 15 µg/g of Zn and provitamin A content in maize kernels, respectively. The baseline content for Zn is 20 µg/g and considering the wide variability of Zn content in maize an increase of 13 µg/g is feasible .Only a few countries have sufficient plant genetic resources to fulfil their food requirements . ImproveMultinutrient biofortified maize cultivars can be developed through hybridization. Although different population improvement procedures exist, the development of multinutrient cultivars through hybridization is highly influenced by several factors, including availability of resources, genetic variability, breeders’ expertise and high-throughput phenotyping tools. Using this breeding method, breeders can develop either multinutrient hybrids or improved open pollinated varieties (OPVs) . NutritiPrevalence of additive gene action for traits such as Zn, provitamin A and quality protein, facilitates the development of improved multinutrient OPVs through recurrent selection. This can be achieved by crossing an improved OPV with a trait donor, followed by backcrossing with the recurrent parent to restore desirable traits in the original cultivar. Therefore, intrapopulation recurrent selection and pedigree selection are useful breeding methods that can be used for developing multinutrient maize OPV. The main disadvantage of recurrent selection is that some of the desirable traits come as favorable alleles in haplotype blocks and, therefore, some of these alleles can be lost during the integration of targeted traits . MaintenHybrid development is another breeding approach for developing multinutrient maize cultivars. Whilst other breeding methods are useful, the genetic improvement of maize across the world remains centered on hybrid development . MultinuThe success of multinutrient hybrid maize breeding is highly dependent on the level of heritability of targeted nutritional traits, mode of gene action and general and specific combining ability (GCA and SCA) of the parental inbred lines. For kernel Zn content, high narrow-sense heritability of more than 72% was reported in QTL mapping studies ,40. MediLcyE) [CrtRB1) [PSY1), facilitates development of multinutrient maize on a provitamin A background, by tracking the presence of these favorable alleles that confer increased β-carotene content. Identification of these favorable alleles using PCR-based markers, coupled with high-throughput phenotyping tools, such as high-performance liquid chromatography (HPLC), is crucial in accelerating genetic gains in developing multinutrient maize in a provitamin A background.Marker assisted selection (MAS) or breeding could bring a high level of success in developing multinutrient maize. MAS is an indirect selection process where selection of the desired trait is done based on a specific marker, which can be morphological, biochemical or DNA/RNA markers (known as molecular markers). This modern breeding tool has been used in developing provitamin A cultivars, where tropical and temperate germplasm with high carotenoid content was selected based on the presence of reduced-function alleles of the lycopene epsilon-cyclase (LcyE)  and β-ca[CrtRB1) ,126. Theopaque-2 locus in QPM donors to be used for QPM-based multinutrient maize. Marker-assisted backcrossing (MAB) could be useful in ensuring successful introgression of the opaque-2 loci and other genetic systems controlling amino acid content [CrtRB1 from a provitamin A donor (HP321-1) to two QPM inbred lines (CML161 and CML171) was reported to improve both provitamin A and protein quality [CrtRB1, LcyE and opaque-2 loci in the QPM genetic background, resulting in cultivars with high levels of provitamin A, lysine and tryptophan content [MAS can also be used to confirm the presence of the  content . Success quality . High ba content .opaque-2 locus have been identified as phi057, phi112 and umc1066. Among these, the phi057 and phi112 were reported to be highly polymorphic and, therefore, can be used for introgression of the opaque-2 gene in other biofortified germplasm with a non-QPM background [The use of DNA-based markers such as intersimple sequence repeat (ISSR), simple sequence repeat (SSR), random amplified polymorphic DNA and single nucleotide polymorphism (SNPs), can also be used for estimating genetic distance between inbred lines, studying population structure and classifying multinutrient germplasm into heterotic groups . The useckground . The preMutation breeding could be useful in developing multinutrient maize genotypes. This strategy has been used for many years to create genetic variability in both quantitative and qualitative traits. Different mutagens, including X-rays, gamma-rays and chemical mutagens, such as ethyl methyl sulfonate (EMS), have been widely used in different crops to induce random changes in DNA. Exposing maize seed to different levels of mutagen doses can help to identify optimal doses that can cause significant point mutations without causing much of the undesirable characteristics. M1 generation kernels are advanced and desirable phenotypic effects can be identified, depending on the dominant or recessive nature of alleles. Although there is little evidence on the development of multinutrient maize genotypes using this breeding method, few studies have reported significant changes of yellow to orange maize when EMS was used on M1 yellow maize segregating populations ,42. MutaMapping studies to identify loci with favorable alleles in mutation breeding can be done using Targeting Induced Local Lesions in Genomes (TILLING). This high-throughput technique can identify the extent of genetic variability for a particular trait in locally available germplasm. TILLING is a reverse genetics tool based on conformational electrophoresis for identifying point mutations in plant populations . MultinuAgrobacterium tumefaciens-mediated transformation, microparticle bombardment, and whiskers-mediated transformation [Genetic engineering of maize to develop nutritionally superior maize genotypes could be an efficient biotechnological breeding approach that could reduce some of the hurdles faced by breeders in developing multinutrient maize. Transgenic techniques have been advocated as breeding tools for some traits, due to limited success of conventional breeding methods to incorporate desired traits . Introgrormation . Transgesb401 encoding a lysine-rich protein into the maize genome was observed to increase lysine and total protein content in the transgenic QPM [crtB and crtL, resulting in a 34-fold increase of total carotenoids in the maize endosperm [Multinutrient maize in a provitamin A genetic background could be developed through manipulation of the carotenoid biosynthetic pathway . Severalenic QPM . The devenic QPM . A similndosperm . This isGenome editing is a powerful biotechnological tool that can be used to stack nutrients in maize. Unlike in GM crops, where transgenes are involved, genome editing techniques insert edited genes of interest into specific genomic regions and the procedure mimics the natural hybridization process . TechniqGene editing has been used to improve traits for several crops. For instance, yield and stress tolerance in rice , β-carotThe development of multinutrient maize with increased concentrations of both macro and micronutrients creates great opportunity to alleviate malnutrition in SSA. However, factors that may contribute to relatively low adoption of multinutrient cultivars should be considered. Reflecting on challenges previously experienced in adoption of these single nutrient varieties, provides a guideline to researchers in pursuit of stacking these nutrients in maize cultivars.opaque-2 locus that comes with the QPM genetic background is homozygous and recessive, extra care has to be taken in seed production to reduce xenia effects from non-QPM cultivars [Multinutrient maize cultivars developed in a QPM background may face similar adoption challenges to those faced by the conventional QPM varieties . Firstlyultivars ,113. Xenultivars . HoweverKnowledge gaps, cultural beliefs and behavioral patterns are still creating barriers to production and consumption of any orange-colored maize in developing countries ,143. HowIt is encouraging that the orange-fleshed sweet potato (OFSP) was accepted in Mozambique and all its neighboring countries due to proper consumer education. Lessons learnt from the acceptance of orange-sweet potato raises optimism for the acceptance of multinutrient maize rich in provitamin A . CurrentPricing of the multinutrient orange colored maize should be done in a strategic manner to ensure both affordability by poor consumers and acceptance by rich consumers who might associate lower prices with low quality foods . Since pThe development of maize cultivars with increased micromineral content is currently being advocated by HarvestPlus. Previously, research and development has been focusing on development of provitamin A and QPM cultivars. Hence, Zn-enhanced maize is quite new to the majority of people in SSA. This implies that research organizations should invest in promoting Zn biofortified maize. Although the rate of acceptance of newly biofortified maize may differ across different countries and regions, gradual changes in consumer behavioral patterns may be expected through consumer education .Access to seed by farmers also influences adoption of Zn biofortified maize . In factTurcicum leaf blight, fall armyworm, drought, heat and low nitrogen, can also help to increase yields.Unfortunately, all biofortified maize are perceived as low yielding compared to normal maize. Some studies report that the accumulation of nutrients in maize kernels through biofortification carries a yield penalty . HoweverMultinutrient maize requires efficient monitoring systems to ensure that the original nutritional quality is maintained. One of the strategies to ensure production of high quality multinutrient maize is the use of isolation distances to protect the crop from contamination. Isolation by time can also be used by farmers, where planting is done on different times to ensure that flowering periods of the varieties do not coincide. Quality analysis should also be performed at all stages during cultivar development, production and storage to ensure that nutritional quality is guaranteed ,96. QualNational policy regulations have an impact on the adoption of multinutrient maize. Without support from governments, the development and production of multinutrient maize can be a challenge. Enabling policy environment in this context includes supporting the whole value chain to develop and deploy multinutrient cultivars. Strengthening of seed systems to ensure easy access of subsidized biofortified seed and inputs is also important. However, cultivars developed using genetic engineering are prohibited in some countries in SSA. Such restrictive government policies limit the World Health Organization to reach its sustainable development goals for 2030 to end hunger in all its forms . TherefoThe development of multinutrient maize with Zn, provitamin A and QPM can reduce malnutrition in developing countries. Multinutrient maize cultivars can be developed using the available genetic variation for these traits, and integration of both conventional and modern high-throughput breeding methods. Such breeding methods include conventional pedigree selection, marker-assisted breeding, genetic engineering and genomic selection. Breeders can make use of the gene bank collections preserved at CIMMYT to acquire maize nutrient donors that can be accessed by both national and international breeding programs. In addition, screening of the existing germplasm for carotenoids, Zn and tryptophan content is important and can identify nutritious maize genotypes that are highly adapted to local growing conditions. Promotion of multinutrient maize cultivars should be done to facilitate quick adoption by farmers and consumers. Availability of affordable multinutrient maize in shops, and provision of seed subsidies, can facilitate its quick adoption. Apart from the nutritional attributes, multinutrient maize cultivars should have good agronomic traits such as tolerance to biotic and abiotic stresses. Quality assurance at all breeding and seed production stages should be monitored to ensure the nutritive value of multinutrient maize.


Tumor cells shed DNA into the plasma. “Liquid biopsy” analysis of mutations or other genomic alterations in circulating cell-free DNA (cfDNA) may provide us with a tool to detect minimal residual cancer, comprehensively profile the genomic tumor landscape in search of druggable targets, and monitor cancers non-invasively over time for treatment failure or emerging treatment-resistant tumor subclones. While liquid biopsies have not yet entered routine clinical management in patients with gastric and gastroesophageal junction cancers, this group of diseases may benefit from such advanced diagnostic tools due to their pronounced genetic spatiotemporal heterogeneity and limitations in imaging sensitivity. Moreover, as the armamentarium of targeted treatment approaches and immunotherapies expands, cfDNA analyses may reveal their utility not only as a biomarker of response but also for precision monitoring. In this review, we discuss the different applications of cfDNA analyses in patients with gastric and gastroesophageal junction cancer and the technical challenges that such liquid biopsies have yet to overcome. Despite overall declining incidences, gastric and gastroesophageal junction cancers—which are mostly adenocarcinomas—remain common malignancies. This group of diseases is the third to fourth most common cause of cancer death worldwide .In the non-metastatic setting, the prognosis has improved through perioperative chemotherapy with fluoropyrimidines, platinum, and taxanes . In non-Despite these advances, a number of challenges remain. These include the reliable detection of actionable lesions for targeted therapies and the sensitive monitoring of disease and resistance to open up avenues of precision targeting to more patients.In this review, we discuss our current understanding of gastric cancer genetics and the role of cfDNA genomic analyses as an emerging diagnostic tool in precision medicine of this spectrum of tumors. Since cfDNA is extracted from blood plasma, this so-called liquid biopsy is a non-invasive technique and as such opens up the possibility for tight monitoring intervals of otherwise hardly accessible solid tumors. In metastatic settings or cases with a heterogeneous tumor architecture, such as in gastric and gastroesophageal junction cancer, liquid biopsy has the advantage of more broadly reflecting all tumor subclones than conventional biopsies. This increases the chances that relevant mutations and resistance mechanisms are detected. The work summarized in this review shows how liquid biopsies may promote our diagnostic abilities to improve precision diagnostics, tumor and resistance monitoring, andearly relapse detection, thereby refining our care for patients with gastric and gastroesophageal junction cancers.Genomic analyses have shown that gastric and gastroesophageal junction adenocarcinomas have highly complex genomes characterized by both mutations and somatic copy number alterations in key genes ,16,17,18One of the therapeutically most important genes is ERBB2. The ERBB2 gene is a proto-oncogene whose protein product HER2 is a membrane-bound tyrosine kinase receptor that generates proliferative and anti-apoptotic signals when activated and is an important driver in tumor development and progression . In gastAnother gene that is amplified in up to one-third of patients and that has lately receiving increased attention is the fibroblast growth factor receptor (FGFR). This receptor is implicated in tumorigenesis and chemoresistance in many tumors . Gen ampIn routine clinical practice, HER2 and FGFR2 overexpression is determined by immunohistochemistry staining (IHC) or fluorescence in situ hybridization (FISH) on tumor slides. In both cases, tumor material obtained from a resection or biopsy is needed. The detection of copy number alterations (CNAs) from liquid biopsy would circumvent the need for surgical procedures in cases where not enough primary material is acquired or in the case of re-testings during the course of treatment. CNAs can reliably be detected from cfDNA  and wereIn the context of The Cancer Genome Atlas research project (TCGA) , gastricWhile, so far, the TCGA subtypes remain without clear predictive utility, they should be taken into consideration in future clinical trials with targeted agents and combination approaches. It is noteworthy that specific mutations are overrepresented in the four subtypes, such as CDH1 and RHOA mutations in genomically stable tumors. A high PIK3CA mutation rate was observed in the EBV and MSI groups, which may be of future therapeutic relevance.The correlation of these subtypes with distinguishable genetic patterns opens up the possibility to classify gastric and gastroesophageal junction adenocarcinomas via sequencing of cfDNA, which is suitable to detect mutations, copy number variations, and aberrant DNA methylation, and may ultimately aid in prognostic assessment and treatment decision making.Many solid tumors show spatiotemporal heterogeneity. In gastric and gastroesophageal cancers, this heterogeneity is pronounced ,33,34. VcfDNA is released from somatic tissue into the blood stream after cell death. Cell-free DNA fragments deriving from apoptotic cells are usually only ~180 bp long , which hThere are many challenges in the analysis of liquid biopsies in cancer patients as recently reviewed in . These iDue to the stage-specific amounts of tumor-derived cfDNA, caution must be exercised when selecting a technology platform with adequate sensitivity for the respective research question. A general problem of all mutation-based quantification strategies consists in inadequate representation of the whole tumor mass by single mutations in cancers with pronounced subclonal genetic heterogeneity. For prognostication and monitoring, a number of studies have, therefore, used quantification of plain cfDNA concentrations per plasma volume based on the observation that cfDNA concentrations correlate with tumor burden ,54,55,56MRD detection by cfDNA analysis has been prospectively investigated in a number of studies of early stage gastric cancer being resected with curative intent ,61,62,63Together, these studies show that post-surgical detection of mutant cfDNA is prognostically adverse and most patients with persistent MRD eventually relapse at some point. Time to relapse appears to be generally shorter in patients with post-surgical detection of mutant cfDNA and liquid biopsy positivity typically precedes clinical relapse by several months.The general problem of MRD detection in this group of diseases is the profound inter-patient heterogeneity of mutations. The use of mutation-agnostic MRD monitoring approaches, therefore, requires large gene panels. This is in conflict with the high sensitivity needed for MRD detection. CAPP-seq approaches and tumor-informed approaches may achieve much higher sensitivity. The latter require prior sequencing of the tumor followed by the design of patient-specific probes that can be used for highly sensitive over-time monitoring . Some of the limitations of tumor-informed monitoring the relatively high costs, the turn-around times for the establishment of the patient-individual assays, and the potential of losing the monitored subclone as a result of shifts in the genomic tumor landscape, e.g., on selective treatment pressure.In gastric and gastroesophageal junction cancer, there are currently two biomarkers guiding treatment decisions: ERBB2/HER2 amplification/expression for trastuzumab treatment and PD-L1 tissue expression for treatment with immune checkpoint inhibitors.For determination of the HER2 amplification status, a genomic test is necessary, which is usually performed on DNA from formalin-fixed paraffin-embedded tissue. The amplification of ERBB2 may, however, also be done on cfDNA by a ddPCR method, which showed comparable effectiveness to immunohistochemistry and fluorescence in situ hybridization . In addiThe U.S. Food and Drug Administration (FDA) granted Breakthrough Therapy Designation for the monoclonal antibody bemarituzumab as first-line treatment for patients with FGFR2b-overexpressing metastatic and locally advanced gastric and gastroesophageal adenocarcinoma in combination with chemotherapy. The licensing is expected to be based on an FDA-approved companion diagnostic assay showing overexpression of this target in at least 10% of tumor cells ,25. FGFRTogether, this data suggests that the integration of cfDNA profiling at diagnosis or repeated profiling at relapse may help to offer a targeted treatment option to a larger number of patients with gastric and gastroesophageal junction cancer.Finally, serial analysis of cfDNA may provide insights into the level of tumor control and the development of resistance traits in gastric and gastroesophageal junction cancers over time.Moreover, these above-mentioned trials showed that the resistance mechanisms to systemic targeted therapy are genetically heterogeneous with different routes to resistance that can even co-exist in individual patients. In HER2 targeting, the emergence of other genomic aberrations in alternative pathways, such as MYC, EGFR, FGFR2, and MET amplifications, and PIK3CA/R1/C3, ERBB2/4, and NF1 mutations may occur refer to . In a laA large body of evidence from different trials confirms that in gastric and gastroesophageal junction cancer, quantification of cfDNA can be used to assess the risk for disease recurrence or progression. A number of challenges still remain. These pertain to the sensitivity levels of detection methods, standardization of protocols, and the clinical implementation of complex tumor-informed monitoring approaches. Moreover, when predicting a lack of treatment efficacy, caution must be exercised to exclude false-positive liquid biopsy results that may result from the presence of clonal hematopoiesis or rare germline single-nucleotide polymorphisms in the patient. If these challenges can be tackled, pre- and post-surgical liquid biopsies may become a transformative technical tool to guide tailored adjuvant systemic treatment. New studies should be designed to evaluate whether adjuvant treatment intensification or drug rotation in patients with persisting liquid biopsy positivity may lead to higher cure rates.In the metastatic setting, early drug rotation based on cfDNA dynamics may also help to reduce the percentage of patients that die early due to insufficient tumor control by their first-line treatment regimen. Admittedly, however, the numbers of active drug regimens in this spectrum of tumors are still rather limited, hampering the design of such trials.Treatment personalization is geared toward boosting patient care in gastric and gastroesophageal junction cancer. Liquid biopsy testing will become increasingly relevant in this respect. In addition to their value in disease monitoring, liquid biopsies may more faithfully capture tumor heterogeneity. As a technical assay independent of tissue analysis, they may help to identify more patients for targeted treatment approaches.Before liquid biopsies can become a standard of care in the guidance of personalized medicine in gastric and gastroesophageal junction cancer, large prospective validation trials are required.


Providing care to an aging society in the new normal requires increased attention to the informal caregivers who support the health and well-being of older adults with chronic conditions. Hispanic caregivers carry a high caregiver-associated burden. Health disparities experienced by Hispanics, coupled with the emotional, social and physical demands of caregiving, may set an unprecedented risk for lower health-related quality of life (HRQoL). In a quantitative analysis, we investigated the relationship between spirituality, loneliness and HRQoL in Hispanic cancer caregivers using baseline data from the Support for Latinas with Breast Cancer study (N= 234 Hispanic caregivers). Findings suggested an indirect effect of spirituality on HRQoL through reduced loneliness among more spiritual caregivers, effects that were independent of age. The second study was conducted using qualitative semi-structured interviews (N= 10) with Hispanic caregivers. Interviews evaluated spirituality and HRQoL in Hispanic cancer caregivers who reported variable levels of loneliness. Five themes emerged: caregiver experience, coping strategies, loneliness, religion to gain strength or support, and spirituality to gain strength or support. Results supported the role of spirituality in promoting higher HRQoL in Hispanic cancer caregivers and elucidated pathways to intervene on HRQoL through spirituality. With Hispanics often underutilizing formal services, having an improved understanding of caregiving experiences, particularly related to spirituality, will support the development of culturally-relevant strategies and programming to promote HRQoL for Hispanic caregivers.


The working definition of health is often the simple absence of diagnosed disease. This common standard is limiting given that changes in functional health status represent early warning signs of impending health declines. Longitudinal assessment of functional health status may foster prevention of disease occurrence and modify disease progression. The LIFEHOUSE  longitudinal research project explores the impact of personalized lifestyle medicine approaches on functional health determinants. Utilizing an adaptive tent–umbrella–bucket design, the LIFEHOUSE study follows the functional health outcomes of adult participants recruited from a self-insured employee population. Participants were each allocated to the tent of an all-inclusive N-of-one case series. After assessing medical history, nutritional physical exam, baseline functional status , serum biomarkers, and genomic and microbiome markers, participants were assigned to applicable umbrellas and buckets. Personalized health programs were developed and implemented using systems biology formalism and functional medicine clinical approaches. The comprehensive database  will yield novel interdisciplinary big-health data and facilitate topological analyses focusing on the interactome among each participant’s genomics, microbiome, diet, lifestyle and environment. Many chronic diseases are related to lifestyle and aging. In the United States, approximately 50 percent of the population suffers from a chronic disease. While contributing to 86 percent of healthcare costs , the humAdvances in science and technology have led to a significant revision and modification of the medical models in practice today. Hood  has suggThere is a significant unmet need to identify the value of personalized lifestyle interventions for improving functional health outcomes. Whereas disease is well understood from traditional pathology-based indices, health is less easily defined. Historically, healthy has been the default term applied to an individual who is not recognized as having a disease. This definition of health as the absence of disease has resulted in the delivery of health and wellness as often lying outside the purview of medicine.An evolving definition of health recognizes that health is directly related to the functional capacity of the individual. Changes in functional health status over time can represent early warning signs of later disease. Functional capacity may well be categorized in four assessment areas: metabolic, physical, cognitive and emotional Assessment of these four areas reflect the organ reserve sustaining the physiologies reflected in the Functional Medicine Matrix, specifically assimilation, defense and repair, energy, structural integrity, biotransformation and elimination, communication and transport. Our capacities to work, think, communicate and to feel are quantifiable by both qualitative and quantitative metrics exploring the four areas ,4.Achievement of health through a P4 model  assumes Creating health through evaluation of function and an understanding of disordered pathophysiology requires practitioners to identify core variables while utilizing a diagnostic approach different than the standard development of a differential diagnosis list or even a more targeted split into most important diagnoses and most likely diagnoses. Indeed, evaluating a large dataset, inclusive of genomic and microbiome interactions beyond the typical biomarkers, presents challenges even in generating these standard approaches. In LIFEHOUSE, we employ the model of P4 medicine, personalized lifestyle medicine and functional medicine to address this need. In a 2013 article, Hood and colleagues wrote the following:“Ten years ago, the proposition that healthcare is evolving from reactive disease care to P4 was regarded as highly speculative. Today, the core elements of this vision are widely accepted and have been articulated in a series of recent reports by the US Institute of Medicine…. It will provide the basis for concrete action by consumers to improve their health as they observe the impact of their lifestyle decisions ”.In another article published that same year, Hood describes P4 medicine in terms of individual engagement, behavioral changes to improve health outcomes and personalization of diet and lifestyle . As firsNew and emergent tools and technologies can be utilized to measure and monitor functional health status in an ongoing manner, leading to opportunities for early intervention and improved patient outcomes prior to the onset of disease. The LIFEHOUSE  longitudinal research project is designed to evaluate the impact of a personalized lifestyle intervention program on functional health determinants. Utilizing an adaptive tent–umbrella–bucket (TUB) design, the LIFEHOUSE study follows the functional health outcomes of a group of adult volunteers.LIFEHOUSE is a series of ongoing clinical surveys; and as such, the tent and the individual umbrellas with their respective buckets have been reviewed and approved by the Aspire Independent Review Board (IRB) , now a member of the Western-Copernicus Group IRB. Our survey has been registered with ClinicalTrials.gov as NCT04005456.Within this structure, we have developed a comprehensive multi-component data set of information from all participants in a relational structure that allows integration of genomic, microbiome, biometric, wearable device, phenomic, lifestyle, environmental (exposome), psychosocial and nutritional information. Our data set provides the opportunity to assess the effectiveness of current and new analytic techniques that enable practitioners to manage and evaluate this flow of information so it can be utilized to facilitate creation of health and maintenance of lifestyle change by the patient.We desire to identify the distinction between health as the simple absence of disease and as the creation of a journey to wellness by developing tools to evaluate an individual’s functional capacities. To enable fruitful interventions and behavioral change, practitioners need to suggest effective personalized lifestyle medicine interventions. Effective measures should be those that both increase functional capacity and are meaningful to the patient. Measurements  and utilization of a broad range of questionnaires facilitate a functional baseline assessment, a metric by which therapeutic interventions can be judged for their efficacy. Additionally, we will explore the data set to develop an understanding of determinants of functional fitness and resiliency characteristics and their relationship to composite polygenic risk scores.The annual physical examination is a core component of the medical model. Information obtained during this once-yearly interaction between clinician and patient ideally would provide insight into an individual’s health status. Mehrotra and Prochazka address the failure of the routine health physical in terms of its ability to reduce both illness and premature mortality from non-communicable chronic diseases (NCD) . KrogsbiOur clinicians faced the challenge of dealing with large data sets during their sequential 30–45 min visits; initially focusing on obtaining a detailed 45-component nutritional exam  and progA primary interest of ours was to demonstrate the efficacy of personalized lifestyle medicine. LIFEHOUSE as an adaptive tent–umbrella–bucket N-of-one design is uniquely suited to explore this important clinical question, by having longitudinal measurements of participants receiving intervention, with the potential to comparing to their baseline, as a control. In particular, our goal was to determine the impact of a personalized lifestyle medicine intervention program on wellness by exploring the five categories of function: metabolic, physical, cognitive, emotional and behavioral.The target population was generally healthy adults. Approximately 400 participants, male and female, ages 18–80, from a single company’s insured employee population were recruited in the N-of-one IRB-approved study; this population represents the tent. Exclusion criteria were serious unstable illnesses inclusive of cardiac, hepatic, renal, gastrointestinal, respiratory, endocrinologic, neurologic, immunologic/rheumatological, infectious, hematologic and psychiatric diseases, such as unstable angina, recent myocardial infarction, viral hepatitis, cirrhosis, end stage renal disease, gastro--intestinal bleeding advanced chronic obstructive pulmonary disease, tuberculosis, thyrotoxicosis, Parkinson’s disease, acquired immunodeficiency syndrome, major depression, schizophrenia and ongoing addiction to controlled substances. At regular intervals, participants were reassessed using validated functional assessment tools and a variety of biometric and laboratory evaluations.Our preliminary data consists of 369 participants who had met the minimal requirements for data analysis which was deemed to consist of initial clinical visit and baseline anthropometrics and serum biomarkers.Many of the participants participated in a previous Aspire IRB approved protocol . Upon completion of this 6-month observational period, participants were offered the opportunity to participate in LIFEHOUSE. At their first LIFEHOUSE visit, participants were enrolled in the overall N-of-one tent and were assigned to an umbrella and/or bucket if requirements were met. Assignment was determined by the clinician during visits. The structure of the TUB design is illustrated in Participants only received one specific umbrella/bucket assignment at a time, and once completed, they re-entered the observation phase. If participants qualified for more than one umbrella/bucket assignment, they were later offered an opportunity to enter additional umbrella/bucket assignments. Inclusion criteria for umbrellas/buckets are described below.The wellness umbrella includes participants with minimal physical complaints and broadly normal, yet perhaps not optimal biomarkers. Within this umbrella is the elevated homocysteine bucket, for individuals with elevated homocysteine levels (≥10.4 µmol/L). A level of ≥10.4 µmol/L was selected given our reference laboratory’s Cleveland Heart Lab  identification of this level as the upper limit of normal for women. It has been proposed and accepted in integrative medicine circles that a significantly lower level may be representative of optimal function. Serial evaluation of healthy individuals may clarify whether homocysteine is best thought of as a biomarker or a target for intervention in the creation of wellness .The dental health umbrella includes participants with established dental disease, such as gingivitis, periodontitis, caries, painful teeth or fractured teeth, requiring active care.The neurological health umbrella includes participants experiencing a broad range of neurological degenerative diseases, neurocognitive issues, and psychological/emotional complaints.The metabolic health umbrella included individuals classified as desirable weight with markers of dysglycemia/dyslipidemia and classified as overweight/obese with and without markers of dysglycemia/dyslipidemia. The consequences of metabolic function/dysfunction bucket−crossover design compared three dietary and nutritional supplement programs . Final assignments into applicable umbrellas or tubs reflected a joint decision between participant and clinician.The gastrointestinal health umbrella was designed for individuals with conditions associated with issues of gastrointestinal health and of environmental toxicity or dysfunction involving metabolic transformation. Participants with irritable bowel syndrome (IBS) were enrolled in the IBS bucket. Generally healthy participants were eligible to enroll in the wellness detoxification bucket. Participants demonstrating significant health challenges were eligible to enroll in the detoxification bucket.The immune health umbrella was designed for individuals with autoimmune/inflammatory conditions (excluding metabolic disorders/atherosclerosis). Assignment to the elevated anti-nuclear antibody bucket required elevated antinuclear antibodies (ANA) levels and preclinical symptomatology only. The autoimmune conditions bucket included participants with established autoimmune conditions . The symptomatic fatigue and myalgias bucket enrolled participants with symptoms of persistent fatigue and myalgias.The reproductive health umbrella was designed for men and women with conditions associated with reproductive and hormonal health . The perimenopausal and menopausal transitions bucket enrolled for women with symptomatic perimenopausal and menopausal transitions. The premenstrual syndrome bucket was created for women with premenstrual syndrome and alterations in estrogen/progesterone balance. The PCOS bucket is a unique bucket with enrollment criteria capturing the mixed metabolic dysfunction and reproductive hormonal disturbances typical of women diagnosed with PCOS and is thus shared between the metabolic health umbrella and the reproductive health umbrella.Baseline data collection was extensive and included detailed medical history, questionnaires, anthropometrics, nutrition physical exam, functional evaluations, microbiome analysis and single-nucleotide polymorphism assessments . SpecialRand MOS SF-36 Health Symptom QuestionnairePROMIS-43 Questionnaire Depression, Anxiety and Stress Scale Beck Depression Inventory Beck Anxiety Inventory PROMIS Anxiety SF PROMIS Depression SF PROMIS Sleep Disturbance SF PROMIS Cognitive SF VIA Strength Finder University of Rhode Island Change Assessment (URICA) An 18-page history form was completed by all participants. Additional questionnaires included:Assessment conducted during clinical visits included the following measures: anthropometrics, vitals, body composition and nutrition physical exam.comprehensive metabolic panelchemistries, including gamma-glutamyltransferase (GGT), uric acid and osteocalcinmarkers of dysglycemia including insulin and HbA1ccomplete blood countnutritional markers, including 25-OH vitamin D3, homocysteine, ferritin, folate, magnesium, omega-3/omega-6 fatty acid profileadvanced lipid panel, myeloperoxidase (MPO), oxidized low-density lipoprotein (Ox-LDL) and lipoprotein(a) (Lp(a))inflammatory markers, including high-sensitivity C-reactive protein (hs-CRP), antinuclear antibodies (ANA), anti-cyclic citrullinated peptide (Anti-CCP) and rheumatoid factor (RF)endocrine markers, including cortisol and comprehensive thyroid panelmale and female sex hormones including pregnenolone, progesterone (women), dehydroepiandrosterone sulfate (DHEA-S), testosterone, dihydrotestosterone (DHT) (men), estradiol, follicle-stimulating hormone (FSH) (women), luteinizing hormone (LH) and sex hormone binding globulin (SHBG)immunoglobulin G (IgG) food allergy testingprostate-specific antigen (PSA) (men over 50 years old)Phlebotomy was performed for both plasma and serum to measure:The biomarkers were routinely measured by Cleveland Heart Lab and Quest Diagnostics.For microbiome assessment, stool was collected and analysis conducted by Genova Diagnostics, Asheville, NC. Saliva was collected for the assessment of genomic data by 23&Me, Sunnyvale, CA . SelecteFunctional status of all participants was tested at baseline and included (beyond the questionnaires mentioned above) forced expiratory volume , grip stSpecialized testing could be offered to participants based upon protocol rules and geographic location. This testing could include urinary toxic elements , peripheral artery tonometry using the Endo-PAT device , adrenal stress profile with awakening cortisol response . Participants demonstrating compliance with protocol requirements received an Apple Watch, series 3  for biomonitoring. Selected participants were asked to do continuous glucose monitoring and heart rate variability testing.Participants were seen by a study clinician at baseline and every six months during periods of active participation. Additional in-person visits were dictated by applicable tent, umbrella and bucket requirements. Virtual visits were conducted at 3-month intervals between the 6-month in-person visits. Additional virtual visits were scheduled to assess progress and acute adverse events. The principal investigator had latitude to order off-protocol laboratory testing to initiate evaluation of recognized laboratory and physical exam findings to facilitate further medical evaluation. Per protocol, history and questionnaires, anthropometrics, vitals, body composition, nutrition physical exam, laboratory biomarkers, microbiome analysis and specialized testing were repeated at regular intervals of 6 and 12 months.n = 83), the immune health umbrella (n = 72) or detoxification buckets (n = 18). Intervention programs were personalized within the scope of adaptively designed TUB design. These participants were followed for an average of 9 months before moving once again into an inactive observation phase  designed for the study, directly entered in Microsoft  Excel 2007 databases or received electronically as de-identified data directly or using secure file transfer protocol sites. Source documents included all recordings of observations or notations of clinical activities and all reports and records necessary for the evaluation and reconstruction of the clinical survey including use of an electronic medical record (EMR) . Data for CRFs was collected during participant visits, phone calls between participants and health care providers and from participant submissions (questionnaires and data logs).Methods for the analysis of N-of-one case series have been evolving quickly. A detailed discussion of these methods is available in a monograph on the conduct of N-of-one case series by the Agency for Health Quality and Research . Senn isIn LIFEHOUSE, the analyses are being performed on per-protocol data sets, including an intent-to-treat data set and a data set consisting of all participants who finish the survey. Individual analyses will be similarly conducted for each umbrella and bucket. Missing data will not be imputed. Subgroup analysis based upon gender, age, body mass index (BMI), presence/absence of defined biomarkers at baseline, readiness to change (based upon URICA questionnaire score), manufacturing versus corporate work settings, and compliance are being conducted.As applicable, non-parametric tests, including the sign test and Wilcoxon signed-rank test, will be conducted. As our survey design features possible consecutive and sequential participation in the TUB and a rolling enrollment, applicable techniques to account for continuous outcomes, effects dependent on time, autocorrelation of measurements  and carryover effects will be conducted. Standard estimation techniques, both paired and unpaired t-tests, were conducted as appropriate. Finally, principal component analyses were conducted. Primary data analysis was conducted by Bennett Data Sciences, San Diego, CA, USA.2. The corporate/sales group has a slightly lower BMI of 26.5 ± 5.1 kg/m2, compared with the manufacturing group, which had a BMI of 30.5 ± 7.4 kg/m2 (p < 0.001). BMI ranges in kg/m2 are 18.5–24.9 desirable; 25–29.9 overweight; ≥30.0 obese [.0 obese  Almost 6To address a broad series of current therapeutic challenges, LIFEHOUSE is an adaptive, function-focused survey incorporating features of randomized, placebo-controlled trials to answer targeted questions while maintaining an N-of-one approach to focus on our broad themes of defining health by function, exploring efficacious models of behavioral change and support for a personalized lifestyle medicine approach in addressing the chronic diseases of lifestyle and aging.An adaptive clinical trial is an innovative trial design that conserves both resources and time and allows for multiple interventions to be tested simultaneously with the goal of identifying “best practices” for individuals residing within specific clinical definitions or subgroups . AdaptivAs illustrated by Kravitz et al. , advanceAlthough the use of the basket-and-umbrella trial design has been limited primarily to the evaluation of drugs used in oncology, the concept has tremendous potential for the study of personalized lifestyle medicine interventions. A variation of this type of study design has already been used successfully by researchers investigating the impact of lifestyle and behavior on frailty. The Doetinchem Cohort Study evaluated physical, cognitive, and emotional function in adults aged 40–81 years who were living with different degrees of frailty . To undeWhen applied to a personalized lifestyle intervention trial, the N-of-one design would allow for the creation of a program tailored to each participant based on their individual metabolic, physical, cognitive, emotional and behavioral needs and determined through genomic analysis, medical history and data sets of metabolic, microbiomic, physical, cognitive, emotional, lifestyle and behavioral status. The aggregation and segmentation of data sets across the participant population can identify groups with similar clinical presentations yet segmented by genomic, microbiomic and functional uniqueness. N-of-one designs allow for this uniqueness to address, with personalized lifestyle medicine recommendations and clusters, these individuals according to shared similarities into several different subgroups  of the group at large (tent) to be analyzed. The design described here encourages the development of clinical guidelines based on these individual patient experiences analyzed through this lens of a TUB stratification.Additionally, this approach also allows for the development of a nested family of case reports, which could potentially identify areas of health care in which personalization may positively impact clinical outcomes .In 2004, the American College of Lifestyle Medicine (ACLM) was founded with the following stated purpose: “To educate, equip, and empower individuals to provide the information and resources that a patient needs to protect their health and fight chronic disease .” WidespDisease-risk reduction is closely linked to public health initiatives related to prevention. However, though based on inherent good intent, such efforts have limited success in terms of long-term behavior change. In addition, prevention is a metric that is difficult to measure at the individual level. How does an individual know, without ambiguity, that actions they took resulted in the prevention of a disease? While prevention cannot be easily quantified, improvement in function can be assessed, documented and tracked. For this reason, health status is likely better defined by function than by disease-risk reduction and prevention. Function results from the ongoing interaction of key personal (and highly unique) variables, including the following: genetics, age, gender, ethnicity, health history , lifestyle, diet, stress patterns, activity, sleep, medication and dietary supplement use and environmental exposures. These variables represent opportunities to apply a systems biology approach to personalized lifestyle medicine.In 2017, Bland, Minich and Eck co-authored an article describing an approach to improving individual health and wellness using systems biology and unique functional assessments to improve the effectiveness of lifestyle medicine . TechnolAs noted above, function in an individual can be segmented into five subgroups for assessment: metabolic, physical, cognitive, emotional and behavioral function. Validated tests and/or questionnaires are available for all these subgroups. Tests to measure functional reserves may differ from those used to determine the presence of disease, but this does not diminish their value to both clinicians and patients. An example of highly useful functional assessment tools is continuous blood glucose monitoring, which permits the evaluation of lifestyle’s impact on glucose metabolism . SimilarAs has been noted by Price et al. , a signiAggregation of such functional assessments has several advantages over a traditional disease-risk evaluation. By mapping the functional capacity of an individual, the patient gains access to quantitative tracking tools to evaluate how lifestyle changes influence personal function. At the same time, clinicians now have the robust information needed to personalize and manage therapeutic recommendations. One goal of the approach described here would be the high level of patient engagement that may come with the ability to self-track intensely personal metrics. The definition of health is very personal, and this approach allows the individual to determine which functional indicators they are both most interested in and willing to modify. Overall, this concept moves lifestyle medicine intervention from a population-based, risk-factor-reduction model to one that is focused on individual function and performance.In a clinical observational research study using an N-of-one tent–umbrella–bucket design, it is logical to use P4 medicine, personalized lifestyle medicine and functional medicine methodology as core clinical tools for intervention. As functional medicine has been successfully utilized in the implementation of personalized lifestyle medicine programs , it is uThus far, we have described the many strengths of the LIFEHOUSE design. However, our initial work within the scope of the survey has also demonstrated some of the weaknesses. Our study clinicians and study staff have faced several challenges. The primary challenge was the struggle to manage the wealth of data collected. Due, early on, to the lack of integrated data management systems, data were not always immediately accessible to the clinicians as they made care decisions within the bounds of the protocol rules. Additionally, study clinicians struggled with the tension between collecting data and using the data to assist participants in making personalized lifestyle medicine therapeutic choices in a meaningful way. We additionally have found that our participants and study clinicians/staff in many cases have not shared similar goals; presented with complex interventions detailed by study protocols, participants not infrequently have struggled with maintaining compliance, if not interest. The wealth of choices within the latitude of the tent and the individual umbrellas also created challenges for the staff to coordinate the delivery of care.In looking at our next steps and in recognizing our design’s strengths and limitations, we have been engaged actively in process improvements in our data collection tools and creating new tools to foster behavioral change which will be introduced in future intervention phases. We are currently writing the protocol for LIFEHOUSE 2.0, a data registry survey evaluating the personalized lifestyle medicine experience in the two Personalized Lifestyle Medicine Centers.LIFEHOUSE is a living, evolving study that will continue to produce a wealth of exploratory opportunities related to our key research goals. This work currently has resulted in several planned papers and will be continuing. New opportunities will be created as we modify the survey design and increase both the diversity and amount of data available for analysis. We welcome discussion with interested potential research partners.Every individual has unique and ever-changing patterns in five functional capacities: metabolic, physical, cognitive, emotional and behavioral. New personalized and adaptive research approaches are needed for investigators to understand the impact of gene-environment interactions on health as measured by function and to demonstrate the value of interventions suggested by these interactions.LIFEHOUSE represents an innovative approach to studying functional health outcomes in a personalized lifestyle medicine program using a tent–umbrella–bucket protocol. New data management and analysis methods provide tools to determine areas of similarity among different individuals that will allow for valid conclusions to be drawn from the data. It is important to note that LIFEHOUSE addresses multiple important clinical questions, including “What is Health?” and “How can we promote Health effectively and equitably?” Our design aims to provide data demonstrating the effectiveness of a P4 medicine model . Combining a functional medicine intervention model in the P4 medicine context represents a unique approach to examining the impact of a systems biology formalism and the delivery of personalized lifestyle medicine on health outcomes. LIFEHOUSE, as a thriving, broad series of research initiatives, is expected to reveal much about factors that may be key drivers of effective behavioral change and positive functional health outcomes, about the clinical application of personalized lifestyle medicine and about the successful execution of a large-scale adaptive N-of-one protocol.


Most cardiac studies focus on evaluating left ventricular (LV) systolic function. However, the assessment of diastolic cardiac function is becoming more appreciated, especially with the increasing prevalence of pathologies associated with diastolic dysfunction like heart failure with preserved ejection fraction (HFpEF). Diastolic dysfunction is an indication of abnormal mechanical properties of the myocardium, characterized by slow or delayed myocardial relaxation, abnormal LV distensibility, and/or impaired LV filling. Diastolic dysfunction has been shown to be associated with age and other cardiovascular risk factors such as hypertension and diabetes mellitus. In this context, cardiac magnetic resonance imaging (MRI) has the capability for differentiating between normal and abnormal myocardial relaxation patterns, and therefore offers the prospect of early detection of diastolic dysfunction. Although diastolic cardiac function can be assessed from the ratio between early and atrial filling peaks (E/A ratio), measuring different parameters of heart contractility during diastole allows for evaluating spatial and temporal patterns of cardiac function with the potential for illustrating subtle changes related to age, gender, or other differences among different patient populations. In this article, we review different MRI techniques for evaluating diastolic function along with clinical applications and findings in different heart diseases. The left ventricle (LV) of the heart fills via two separate mechanisms. In early-diastole, the LV fills passively through active relaxation. In late-diastole, the remaining blood contributing to the total end-diastolic volume enters the ventricle via active contraction of the left atrium (LA). During isovolumetric relaxation, LV volume increases due to alterations in principal strains and untwisting of the ventricles . CompletDiastolic dysfunction is an indication of abnormal mechanical properties of the myocardium, characterized by slow or delayed myocardial relaxation, abnormal LV distensibility, and/or impaired LV filling . DiastolHeart catheterization is the current gold standard for demonstrating the characteristics of diastolic heart failure, but due to the risks and costs involved with invasive hemodynamic evaluation, it is not practical for the diagnosis of diastolic dysfunction. Echocardiography, with significantly less risk compared to heart catheterization, is currently the method of choice for diagnosing diastolic dysfunction. Nevertheless, echocardiography has limitations related to poor acoustic windows, geometric assumptions, suboptimal spatial resolution, and high dependency on the operator’s skills. Some of these limitations restrict the technique’s ability to accurately measure annular velocities and assess diastolic function if regional dysfunction is present . Cardiac31P magnetic resonance spectroscopy (MRS) is another technique for non-invasively quantifying the energy required for active relaxation of the myocardial tissue through calculating the ratio of myocardial phosphocreatine to adenosine triphosphate (PCr/ATP).Different MRI techniques are currently available for evaluation of diastolic cardiac function, such as cine imaging for global function analysis, phase contrast (PC) imaging for flow analysis, and myocardial tagging for regional function analysis. Simple measurements, such as longitudinal fractional shortening, can be measured quickly and easily by MRI, which has reliably identified echocardiography-evidenced diastolic dysfunction in patients with preserved LV EF . AssessmGlobal diastolic function is assessed using time-volume curves generated by tracing the epicardial and endocardial borders of the myocardium in cine MRI images throughout the cardiac cycle or from 4D flow images across atrioventricular valves . StudiesThe heart function can also be studied using MRI tagging . MRI tagThe introduction of complementary spatial modulation of magnetization (CSPAMM) by Fischer et al.  helped rMRI feature-tracking (MRI-FT) is yet another technique for evaluating regional cardiac function directly from the cine images . Ng et aPhase-contrast (PC) MRI offers an alternative method to echocardiography for assessing vascular blood flow and myocardial tissue velocity. PC MRI has the potential to measure hemodynamic parameters that are important for assessing diastolic function in clinical routine  Figure , with inFour-dimensional flow (4D-Flow) MRI is superior to Doppler echocardiography for evaluating intracardiac velocity as it is not limited by flow direction or inconsistency in transducer alignment. Previous studies using 4D-Flow MRI demonstrated significant flow reduction and end-diastolic volume and kinetic energy increase in patients with moderate-to-severe dilated cardiomyopathy . The res, resulting in underestimation of the absolute volume change of respective indices . Ch. Ch97]. 31P-MRS predicted hypertrophic changes before they happen, possibly due to the lower myocardial phosphocreatine levels leading to calcium overload and impairment of cardiac cells [Li et al.  showed tac cells ; furtherac cells . Russel ac cells  showed tac cells  demonstrThe risk for diastolic dysfunction is known to increase with age, obesity, and hypertension in the general population , but theConstrictive pericarditis is another cause of diastolic dysfunction and is reflected by the abrupt displacement of the interventricular septum in the LV during diastole. Alternatively, aberrant septal systolic motion into the RV is a common finding following cardiac surgery in constrictive pericarditis . In theiCancer treatment is known to have adverse cardiac effects on both systolic and diastolic functions and tissue composition . DeleterThe precise mechanism leading to the development of cardiac allograft vasculopathy is still unclear. Some suggestions include stiffness of the transplanted heart due to hypertrophy of cardiac myocytes and endocardial fibrosis, immunologic mechanisms combined with other risk factors for vascular injury, and the combination of systemic hypertension and microvascular stenosis . Given tMRI is gaining ground as a routine clinical tool for the evaluation of diastolic HF. Multiple cardiac MRI techniques have demonstrated superiority over methods that are currently considered the gold standard or are most commonly used. Furthermore, new methods for applying these techniques are being developed to eliminate time-consuming post processing and burdensome image acquisition. Armed with these advances, MRI is becoming a feasible and convenient tool for the accurate prediction, diagnosis, and long-term monitoring of diastolic dysfunction. It should be noted that although cardiac MRI provides several parameters that allow for evaluating heart morphology and function, myocardial contractility and tissue characterization, and hemodynamic parameters, its clinical utility could be affected by its relatively high cost, long scan time, and the need for experienced operators. Nevertheless, recent advances in MRI hardware, pulse sequences, and artificial intelligence (AI) capabilities allow for mitigating these limitations. For example, phased-array coils allow for parallel imaging capabilities that reduce scan time . Further