DNA methylation is an epigenetic mechanism that is essential for regulating gene transcription. However, aberrant DNA methylation, which is a nearly universal finding in cancer, can result in disturbed gene expression. DNA methylation is modified by environmental factors such as diet that may modify cancer risk and tumor behavior. Abnormal DNA methylation has been observed in several cancers such as colon, stomach, cervical, prostate, and breast cancers. These alterations in DNA methylation may play a critical role in cancer development and progression. Dietary nutrient intake and bioactive food components are essential environmental factors that may influence DNA methylation either by directly inhibiting enzymes that catalyze DNA methylation or by changing the availability of substrates required for those enzymatic reactions such as the availability and utilization of methyl groups. In this review, we focused on nutrients that act as methyl donors or methylation co-factors and presented intriguing evidence for the role of these bioactive food components in altering DNA methylation patterns in cancer. Such a role is likely to have a mechanistic impact on the process of carcinogenesis and offer possible therapeutic potentials. Cancer is an outcome of aberrant genetic and epigenetic events. Epigenetic mechanisms are responsible for regulating gene expression without changing the DNA sequence. These mechanisms mainly include chromatin remodeling, histone modification, and DNA methylation, the latter being the most investigated mechanism and the focus of the current review . The proIt is widely accepted that DNA methylation profiles are dynamic and prone to modifications in response to normal development and aging, environmental factors, and pathological conditions ,5. For eIn the present review, we summarized preclinical and clinical studies that provide intriguing evidence on the interrelation between nutritional methyl donors and carbon one metabolism co-factors and cancer through regulating DNA methylation patterns. Additionally, this review characterizes the differential effects of micronutrient methyl donors on global versus gene-specific DNA methylation and the effect of variables such as health status, source of biological samples that were analyzed, mode  and quantity of nutrient exposure, and other confounders in an effort to unravel potential sources of discrepancies in the literatureDNA methylation is an epigenetic mechanism that is essential for regulating gene transcription. This mechanism is mediated via the addition of a methyl group to the cytosine residues in a cytosine-guanine (CG) pair generating 5-methylcytosine. In the intergenic regions and repetitive sequences of the human genome, CpG sites are sparse and mostly methylated. Hypomethylation of CpG sites in these regions may lead to genomic instability and loss of gene imprinting, which eventually result in the development of neoplastic cells . On the The process of DNA methylation is catalyzed by a group of enzymes called DNA methyltransferases (DNMTs), namely, DNMT1, DNMT2, DNMT3a, DNMT3b, and DNMTL. DNMT1 maintains DNA methylation during cell replication while the rest of the DNMT family, mainly DNMT3a and DNMT3b, are responsible for de novo DNA methylation .de novo DNA methylation are highly expresses in developing embryos than adult tissues; yet, there is growing evidence that they play a role in maintaining DNA methylation patterns. For instance, combined genetic deletion or silencing of DNAMT1 and DNMT3b reduced DNA methylation to a greater extent than deleting or silencing either genes alone, supporting the critical role of de novo DNMTs in maintaining DNA methylation [DNMTs that are responsible for hylation ,11,12. Ohylation  showed thylation ,15,16. TWhen it takes place at gene promoters, DNA methylation results in transcriptional repression either via interfering with the binding of transcription activating factors or by recruiting transcriptional repressors such as methyl-binding proteins, histone deacetylases (HDACs), and histone methyltransferases that reduces chromatin accessibility . Absent INK4a, p15INK4b), apoptosis , or antioxidation  [The two main salient features of aberrant DNA methylation in cancer are 1) global DNA hypomethylation that takes place in the intergenic regions and repetitive DNA sequences and 2) local DNA hypermethylation in CpG islands located in specific gene promoters . The latase P-1) . The extase P-1) .There is also growing evidence indicating that global hypomethylation may lead to genome instability and DNA breakage . FurtherUnder normal conditions, cells possess standard methylation profiles by maintaining a balance between DNA methylation and demethylation processes. This balance is disturbed under pathological conditions such as inflammation, oxidative stress and cancer resulting in diverse phenotypes . Due to There is a cumulative body of evidence indicating DNA methylation adaptability to environmental factors including diet and nutritional elements ,44. NutrOne of the proposed mechanisms by which nutrients may modify DNA methylation is by participating in a cellular process known as “one-carbon metabolism” that provides methyl groups for biological methylation of DNA, protein, or phospholipids . One carThe negative feedback mechanism in this cycle is catalyzed by two enzymes, glycine N-methyltransferase that converts s-adenosylmethionine to s-adenosylhomocysteine and s-adenosylhomocysteine hydrolase that converts s-adenosylhomocysteine to homocysteine . This reThe “one-carbon metabolism” cycle is an example of how levels of nutrient exposure impact DNA methylation in the human body . In thisFolate naturally occurs in a wide variety of foods. Rich sources include leafy green vegetables, beans, peas, lentils, fruits like lemons, bananas, and melons, and fortified and enriched products, such as breads and cereals . Folate Conflicting results regarding the effect of folate status on DNA methylation have been reported. For example, global DNA hypomethylation was reported in three different murine studies in response to either a folate-deficient or a folate-rich diet provided during gestation, lactation, or after weaning ,117,118.Similarly, data from human studies that assessed the contribution of folate deficiency or folate supplementation on cancer risk or progression is inconsistent and varies significantly based on factors such as folate dose, mode of intake (dietary versus supplementary), stage of development during exposure , and the pathological status  of tissues ,123,124.Global DNA hypomethylation and gene-specific hypermethylation are significant characteristics of some cancers such as colorectal and prostate cancer and reversing these epigenetic changes has been of great interest for cancer researchers ,128. FolHowever, conflicting evidence has emerged, indicating that the mechanisms associated with folate effect on DNA methylation are more complex than previously thought and confounded by other dietary, genetic, or tissue-related factors. For example, a prospective nested case-control study of 331 cases and 662 matched controls in the population-based Northern Sweden Health and Disease Study demonstrated an association between low plasma levels of folate and reduced risk of colon cancer . This stThis inconsistency extended to clinical trials. For example, in a randomized clinical trial, folate supplements (600 μg/day) for two years significantly increased tissue folate and global DNA methylation in twenty post-polypectomy Patients . SimilarThus far, studies that measured the association between folate intake and global DNA methylation in cancer have yielded inconsistent results as shown above. This inconsistency could be related to the use of varying doses over varying periods; however, a critical confounding factor that should be considered is the type of methylation assays that were used and what they actually measure . Some ofCompared with global DNA methylation, gene-specific methylation analyses in response to folate status have demonstrated more consistent findings. A study by Wallace et al.  assessedTogether, these data suggest that folate might be protective against some cancers via inducing promoter methylation of proto-oncogenes; however, different genes and CpG sites are not equally susceptible to DNA methylation changes in response to folate intake. Accordingly, studies that utilize a candidate gene-specific analysis to investigate the effects of folate on DNA methylation are expected to produce more accurate results than global methylation studies. Furthermore, studies that examine thousands of CpG loci simultaneously will help elucidate the interaction between DNA methylation and folate intake. These studies should take into consideration modifying factors such as age, gender, genetic background, time of exposure (pre-cancer versus post-cancer development), mode of exposure , and other dietary habits and lifestyles.B vitamins are water-soluble vitamins That are found in a variety of foods such as meat, wholegrains, eggs, dairy products, legumes, nuts, dark leafy vegetables, and fruits  . There aIn examining the relationship between vitamin B12 levels and homocysteine levels in the blood, several human studies have reported an inverse association ,150. FurCholine is an essential nutrient that acts as an indirect methyl donor and is involved in several physiological processes such as methylation reactions, cellular membrane integrity, neurotransmitter synthesis, and metabolism . The humSeveral studies reported inverse correlations between dietary choline or betaine intake or their plasma levels and homocysteine bioavailability in humans. Melse-Boonstra et al. , reporteIn addition to these observational studies, supplementation with either betaine or choline has been shown to reduce homocysteine concentrations in clinical trials. For example, a study by Brouwer et al. , reporteSince choline and betaine can act as methyl donors and modify the bioavailability of homocysteine, several studies, mostly animal-based, investigated the association between choline and betaine status and global and candidate gene methylation . AnalyseSecondary to their role in modifying DNA methylation, choline and betaine status is associated with carcinogenesis . Low choIn human studies, four case-control studies demonstrated an inverse relationship between choline and betaine intake and the risk of developing breast cancer , colon cMethionine is an essential sulfur-containing amino acid that plays a significant role in regulating metabolism and synthesizing other amino acids, such as cysteine and taurine, and proteins, such as carnitine and melatonin ,176. MetThese contradictory effects might explain the lack of robust correlations between methionine intake and DNA hypermethylation. For example, rats fed a methionine-rich diet showed no changes in p53  or cystaChronic alcohol consumption was a strong predictor of the association between methyl donor nutrient deficiency and the disturbed one-carbon metabolism in several human studies ,159,161.There is growing evidence that maternal nutrition during pregnancy and early postnatal period affects epigenetic profiles of their offspring . SeveralHuman studies also supported this assumption and a role of maternal diet in modifying fetal DNA methylation of growth and metabolic genes has been shown in individuals exposed prenatally to famine, and conceivably extreme folate deficiency, during the Dutch Hunger Winter at the end of World War II ,208. ThiPositive associations were observed in the Maternal Nutrition and Offspring’s Epigenome (MANOE) study for maternal intake of betaine and folate and gene-specific DNA methylation in infants, mainly RXRA  gene . FurtherIn summary, these studies showed the possibility that methyl donor nutrients in the maternal diet could induce DNA methylation levels in the offspring. However, null, even inverse, associations between maternal methyl donor nutrient intake and fetal global or gene-specific DNA methylation have been reported in other clinical studies ,211,212.Nutrients can regulate several biological processes in our body via modifying epigenetic mechanisms that are critical for gene expression such as DNA methylation. These epigenetic modifications may contribute to the status of our health or the health of our offspring. This dynamic interaction between nutrients we consume throughout our lifetime and our epigenetic signature has now been identified as Nutri-epigenomics. In the current review, we summarized the current literature that reported findings and concepts related to the influence of direct and indirect methyl donor nutrients on DNA methylation and cancer risk. Despite the promising insight Nutri-epigenomics could provide for how to target health from a nutritional standpoint, our knowledge in this topic is still limited and mostly animal-based. Data related to human consumption of methyl donor nutrients were mostly collected from observational studies that are inherently prone to inaccuracy and recall bias. DNA methylation status was assessed by either global methylation approach that lacks a robust clinical relevance or candidate gene-driven approach with limited genome coverage and sensitivity. Further studies in human subjects utilizing sensitive, high-throughput quantitative technologies with a broader range of coverage to systemically analyze the role of methyl donors in modifying DNA methylation and subsequently cancer risk or progression are required. Future work is needed to understand better the interactions among methyl donors involved in one-carbon metabolism and strengthen our understanding of their biological role in health and disease states. The fact that epigenetic marks are potentially reversible and sensitive for nutritional supplementation or pharmaceutical therapies makes the subject of Nutri-epigenomics very attractive, promising, and worthy of study. Yet, we must acknowledge the complexity of the interaction between nutrients and DNA methylation, which is not indicative of a single nutrient. Instead, DNA methylation is a highly regulated process that is gene-sensitive and tissue-dependent and reflects the outcome of several pathobiological processes and environmental exposures. Accordingly, future research should be designed in a way that dissects the effect of single versus combined intake of methyl donors, dietary versus supplementary mode of intake, and dose response relationship. Furthermore, researchers should consider the differential response to methyl donors between healthy tissues and cancerous lesions where aberrant methylation profiles might exist and modify the outcome. Finally, large scale clinical trials in both healthy people and cancer patients are needed in order to provide specific recommendations for methyl donor intake that maintains normal methylation profiles in each group.


Rumination, the act of dwelling on negative, unwanted thoughts, can stoke depression and disrupt sleep, both of which may threaten older adults’ well-being. In line with a support buffering hypothesis, a previous study of younger and middle-aged adults found that social support mitigated the positive association between rumination and negative mood. To extend this research, we distinguished between spousal and family/friend support as moderators of rumination’s links both to depressive symptoms and sleep quality among older adults. Data came from a sample of 128 adults who were, on average, 77 years old at study onset. Rumination was measured via the Rumination-Reflection Questionnaire. Perceived support was measured by items utilized in multiple nationally representative studies of older adults. Depressive symptoms were measured via the NIH PROMIS measure, and sleep quality was measured via items from the Pittsburgh Sleep Quality Index. Results indicated that support from family/friends (but not spouses) buffered the positive association between rumination and depressive symptoms, even after controlling for depressive symptoms six months prior. Conversely, when sleep quality served as the outcome, support from spouses (but not family/friends) buffered the negative association between rumination and sleep quality, even after controlling for sleep quality six months prior. Findings highlight the potential for specific sources of social support to buffer different consequences of rumination on older adults’ health and well-being.


To improve food security, plant biology research aims to improve crop yield and tolerance to biotic and abiotic stress, as well as increasing the nutrient contents of food. Conventional breeding systems have allowed breeders to produce improved varieties of many crops; for example, hybrid grain crops show dramatic improvements in yield. However, many challenges remain and emerging technologies have the potential to address many of these challenges. For example, site-specific nucleases such as TALENs and CRISPR/Cas systems, which enable high-efficiency genome engineering across eukaryotic species, have revolutionized biological research and its applications in crop plants. These nucleases have been used in diverse plant species to generate a wide variety of site-specific genome modifications through strategies that include targeted mutagenesis and editing for various agricultural biotechnology applications. Moreover, CRISPR/Cas genome-wide screens make it possible to discover novel traits, expand the range of traits, and accelerate trait development in target crops that are key for food security. Here, we discuss the development and use of various site-specific nuclease systems for different plant genome-engineering applications. We highlight the existing opportunities to harness these technologies for targeted improvement of traits to enhance crop productivity and resilience to climate change. These cutting-edge genome-editing technologies are thus poised to reshape the future of agriculture and food security. To sustain life, food must provide an adequate supply of calories and nutrients. Food insecurity, the lack of access to an adequate food supply, threatens millions of people worldwide with malnutrition. Moreover, the problem is getting worse; the global population is growing rapidly and is expected to reach 8.3 billion by 2030 . As a reTo improve crop yield, current approaches aim to increase the amount of food produced per unit of area cultivated, and to prevent crop failures. To increase yield per area in grain crops such as rice, breeders have targeted traits that increase the number of grains produced per plant, the number of plants that can be cultivated per unit area, and the size of each grain. Many of these traits involve manipulation of plant architecture through balancing meristem activity and hormone signaling. To prevent crop failures and thus improve yield stability, breeders have targeted traits that help crops tolerate stresses. For abiotic stress, researchers have targeted tolerance to heat, cold, high light, high salt, heavy metals, and other stresses. For biotic stresses, which have become an increasing problem as globalization and weather accelerate the spread of pathogens, researchers have identified loci conferring resistance to various viral, bacterial, and fungal pathogens, as well as loci affecting interactions with animal and plant pathogens, including nematodes and parasitic plants such as Striga . The chaTo increase the nutrition of crops, current approaches aim to provide diverse and balanced diets with adequate levels of vitamins and minerals that enhance human health. Recent developments in crop biotechnology make it possible to manipulate the key enzymes in certain metabolic pathways, thereby enhancing the contents of key nutrients such as vitamins and iron, and reducing the contents of unfavorable compounds such as phytic acids and acrylamide-forming amino acids. Several biofortified crops such as rice, maize, and wheat have been produced to solve the problem of nutrition deficiencies . A well-Nature has been altering genomes for thousands of years, with natural selection enabling plants with certain genomic variants to survive. Moreover, humans have been using artificial selection to domesticate crops for more than 10,000 years. This process produced modern corn from its wild ancestor teosinte, among many other examples. Indeed, all crops grown today have undergone extensive genetic changes. Genetic changes or variations are key to crop improvement, but our ancestors had to make do with naturally occurring mutations. In the twentieth century, once it was recognized that DNA and genes shape all life, it became clear that altering DNA sequences induces phenotypic variations. Therefore, researchers developed and tested reagents, including radiation and chemical mutagens, to induce DNA mutations and have examined the resulting phenotypic variations . This muAgrobacterium tumefaciens (Agrobacterium), the bacterium that causes crown gall disease, is a natural genetic engineer that introduces a piece of its own DNA into the genome of a plant it infects, potentially carrying along a DNA sequence provided by a researcher  for genome editing . A laterGenerating DSBs allows many possible mechanisms of genome editing to be accomplished by harnessing the cell’s repair machinery. The big question was how to generate a site-specific DSB. Proteins that can be engineered and reprogrammed to bind and cleave DNA do not exist in nature. However, it is possible to program a DNA-binding domain to bind to any user-defined site-specific sequence. This domain can be fused with another domain that can cleave the DNA specifically where it binds. These bimodular fusion proteins are the key to precise genome engineering because they can be programmed to bind to any user-selected sequence and generate a DSB. Such programmable site-specific binding proteins can carry other functional domains capable of effecting other genetic and genomic changes, including transcriptional regulation, epigenetic regulation, and even base editing without generation of DSBs . The genXanthomonas oryzae (Xanthomonas) produces TAL effectors (TALEs), which enter the plant cell nucleus and reprogram the transcription machinery to benefit the pathogen , possesses Ralstonia TALE-like proteins (RTLs) with similar structure but completely different repeats, along with enriched numbers of the RVDs that determine repeat specificity , which facilitates and stabilizes the contact, and amino acid 13, which confers binding specificity . The DNAcificity .Deciphering the code of RTL binding to DNA revealed that these RVDs provided a rich resource for TALEN-based engineering . For exaIn nature, bacterial and archaeal species fend off invading phages and foreign genetic elements through the use of clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) adaptive immune systems. About 40% of bacteria and most archaea have with several CRISPR/Cas systems capable of targeting DNA, RNA, or both for degradation, thereby defending themselves against foreign genetic elements . When a CRISPR systems are classified into two main groups, classes I and II , 2015. IFor high-efficiency genome engineering in any eukaryotic cell, it is necessary to ensure that delivery of the genome-engineering reagents to the appropriate species be feasible and that editing of the target genome is both highly specific and efficient . TherefoNicotiana benthamiana or Arabidopsis thaliana and the subsequent delivery of sgRNAs via Tobacco rattle mosaic virus (TRV). After establishment of the TRV infection in the Cas9 overexpression line, we assayed for modification of the genomic target sequence. To further improve the efficiency of this approach and increase the recovery of seed progeny carrying the modification, we recently tested delivery with Pea early browning virus (PEBV), which is capable of infecting the germline  tissue-culture-free genome editing, in which the CRISPR/Cas9 machinery is active in the germline, and (2) tissue-culture-dependent genome engineering. Some RNA viruses are capable of infecting germline cells, albeit at low frequency, and this would enable the recovery of progeny carrying the intended genomic modification, as discussed in more detail below . We can Among prokaryotic vectors, Agrobacterium is a natural genetic engineer because of its ability to transfer a piece of its genome, the transfer DNA (T-DNA), into the plant genome . This inCurrent plant genome-engineering efforts are primarily conducted through classical transformation and tissue culture, as with transgenesis approaches. This limits the application of CRISPR/Cas technologies in crop species, especially those that are recalcitrant to Agrobacterium transformation or to regeneration. There is thus a pressing need to develop technologies that do not rely on classical transformation and regeneration of transformed cells. The ideal target cell types for this approach are the germline cells, where delivery of CRISPR/Cas9 machinery in DNA or protein form can permanently change the genotype. RNP-mediated engineering of germline cells would be ideal given the regulatory hurdles associated with DNA-based editing and the need to produce plants that are free of foreign DNA.As mentioned above, some viral systems can deliver sgRNAs to germ cells. Several other approaches can be used, including direct delivery of the reagents via Agrobacterium and isolation of the germline cells for polyethylene glycol (PEG)-mediated transfection . Other aBecause the CRISPR/Cas machinery is easy to engineer and has robust activity in plant cells, making individual cells with engineered genomes is quite efficient. However, producing whole plants from these cells remains challenging. For example, regeneration is often genotype dependent, and in most cases the cultivars used in laboratory experiments are not the elite germplasm used in agriculture . MoreoveEfficient single-cell regeneration will be a major achievement in plant biotechnology, and research in this area is ongoing on multiple fronts. Recent efforts have been made to deliver CRISPR/Cas9 in RNP form into the protoplasts of lettuce and tobacco, with subsequent editing and regeneration from single protoplast cells . RegenerRecently, morphogenic factors have been used to enhance regeneration frequency . Other pCRISPR/Cas systems offer the ability to produce a variety of genetic and epigenetic modifications that could be instrumental to testing gene functions and regulation in the genomic context. It is feasible to develop CRISPR genome-wide screens as a gene discovery platform whereby sgRNAs are used to generate mutations or epigenetic changes in single or multiple genes . In the Once plants with the desired phenotype are identified, the causal genes can be easily identified by cloning and sequencing the sgRNA. The results can then be confirmed by expressing the wild-type allele of the candidate causal gene. In the past, this required time-consuming and laborious mapping efforts, which are quite challenging in many crops that are key for food security. Not only is CRISPR much more efficient at generating mutations than older  mutagens, but the nature of the mutations is different, meaning that adding CRISPR to the plant breeders’ toolbox will enrich plant populations and enhance the gene and trait discovery process.These screens can be applied to either loss-of-function or gain-of-function platforms depending on which CRISPR/Cas system is used. Of particular utility would be the application of the CRISPR/Cas9 and CRISPR/Cas12a platforms, since they produce permanent changes in the genome and do not require the presence of the CRISPR/Cas9 machinery . An inteOne major drawback to CRISPR/Cas9 systems is that they are prone to off-target activities , owing tin planta, corroborating the general assumption that these off-target activities occur at very low levels in plants unlike a mammalian system where off-target activities is a serious problem  in the rice cultivar Zhonghua 11 by CRISPR/Cas9. Three of the resulting knockout mutations, gn1a, dep1, and gs3, showed enhanced yield parameters in the T2 generation, resulting in improved grain number, dense, erect panicles, and larger grain size, respectively  using a CRISPR/Cas9-mediated multiplex genome-editing system. The resulting mutants showed a significant increase in thousand-grain weight. GASR7, a negative regulator of kernel width and weight in bread wheat, by CRISPR/Cas9 and obtained an increase in the thousand-kernel weight. Similarly, using CRISPR/Cas9 to target a tomato cis-regulatory element in the CLAVATA-WUSCHEL stem cell circuit (CLV-WUS) that controls meristem size produced an edited tomato with an increased number of locules (seed compartments) and thus larger fruit size  and other loci has improved resistance to fungal pathogens in several species. CRISPR/Cas9 and TALEN were successfully used to generate resistance to powdery mildew by simultaneously targeting the three homologs of the MILDEW-RESISTANCE LOCUS (MLO), TaMLO-A, TaMLO-B, and TaMLO-D, in wheat  gene OsERF922, a negative regulator of rice blast resistance, enhanced resistance to the blast fungal pathogen (Targeting homologs of in wheat . In anotSPR/Cas9 . Recentlpathogen .SWEET14). These mutations affect the survival and virulence of the bacterial leaf blight pathogen Xanthomonas oryzae pv. oryzae (Xoo), resulting in resistant rice lines. CRISPR/Cas9 was also successfully implemented to create mutations in four rice SWEET type S genes , Beet curly top virus (BCTV), Merremia mosaic virus (MeMV), Bean yellow dwarf virus (BeYDV), and Beet severe curly top virus (BSCTV)  . Interes (BSCTV) .Turnip mosaic virus (TuMV), in plants, and thus demonstrated that Cas13a can mediate plant immunity to RNA viruses  and ethylene responsive factor 3 (TaERF3), further confirmed that CRISPR/Cas9 can be used to manipulate abiotic stress genes for future crop improvement.Owing to the complex nature of abiotic stress, fewer genome-editing studies have so far been done in this area than in the field of pathogen resistance . In one SPR/Cas9 . They ust stress . Recent esponses . A recenWeeds compete with crop plants for resources such as water, nutrients, light, and space, causing considerable reductions in yield. Numerous techniques have been used for weed management, especially chemical herbicides and genetic engineering approaches. Herbicides usually target a vital step in a plant metabolic pathway, and therefore completely kill weeds and may cause considerable damage to crop plants as well. The herbicides bring economic benefits by increasing the food supply worldwide, but they can endanger human and animal health and have negative impacts on the environment. The advent of biotechnology has revolutionized farming practices by making it possible to transfer a specific herbicide-resistance gene to multiple crops  Table 1Table 1, EPSPS and ALS, to produce herbicide-tolerant plants , resulting in genotypes with elevated glyphosate tolerance  gene in potato. The edited potato produces only amylopectin and lacks amylose-containing starch (Wx1) through CRISPR/Cas9  stated in 2012 that plants edited with ZFNs and meganucleases using the NHEJ pathway should not be considered as, or regulated as, GMOs . The USDIn practical terms, genome-editing technologies offer a great chance for improving crops and ensuring global food security. We should grasp this opportunity to increase crop productivity and potentially save the lives of millions of people around the world, particularly in developing nations. Treating genome-edited crops like those produced naturally or by older artificial mutagenesis will have a number of positive impacts on global food security, including (1) reducing the time and cost of regulatory scrutiny, which will encourage more small biotechnology companies to adopt genome editing; (2) increasing the number of researchers using these tools and encouraging them to improve the system’s efficiency and develop more robust techniques; and (3) allowing the technology to be applied to more crops, including food and horticultural species. As a result, revolutionary changes in crop improvement can be expected in the near future to help meet the increasing demand for food and ensure global food security.CRISPR/Cas systems have revolutionized plant genome engineering and democratized their application through their high efficiency, facile engineering, and robustness. The current state of this technology enables many applications suitable for improving plant productivity, disease resistance, and resilience to climate change. Various technological improvements are still needed, especially precise editing and delivery of genome-engineering reagents to germline cells to bypass the need for tissue culture. In addition, regulatory and ethical considerations may limit the wide applications of these technologies. We must learn from past experience and improve the technology to avoid regulatory hurdles and ensure that its fruits are within reach for the poor and for subsistence farmers. Genome-editing technologies are poised to reshape the future of plant agriculture and food security to feed the world’s burgeoning population.KS, AM, and MM wrote the manuscript. KS, AM, and MM prepared the figures and table. KS and MM edited and finalized the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Aphids vector many plant viruses in a non-persistent manner i.e., virus particles bind loosely to the insect mouthparts (stylet). This means that acquisition of virus particles from infected plants, and inoculation of uninfected plants by viruliferous aphids, are rapid processes that require only brief probes of the plant’s epidermal cells. Virus infection alters plant biochemistry, which causes changes in emission of volatile organic compounds and altered accumulation of nutrients and defence compounds in host tissues. These virus-induced biochemical changes can influence the migration, settling and feeding behaviours of aphids. Working mainly with cucumber mosaic virus and several potyviruses, a number of research groups have noted that in some plants, virus infection engenders resistance to aphid settling . However, in certain other hosts, virus infection renders plants more susceptible to aphid colonisation. It has been suggested that induction of resistance to aphid settling encourages transmission of non-persistently transmitted viruses, while induction of susceptibility to settling retards transmission. However, recent mathematical modelling indicates that both virus-induced effects contribute to epidemic development at different scales. We have also investigated at the molecular level the processes leading to induction, by cucumber mosaic virus, of feeding deterrence versus susceptibility to aphid infestation. Both processes involve complex interactions between specific viral proteins and host factors, resulting in manipulation or suppression of the plant’s immune networks. The vecBecause virions of non-persistently transmitted viruses bind very loosely to the acrostyle receptors, these viruses are acquired rapidly during initial probes of an infected plant’s epidermal cells and also are lost rapidly when an aphid ejects saliva through its stylet , 2018. WThe remainder of this article focuses on non-persistent virus transmission. Our objective is to integrate current understanding of viral manipulation of plant vectors in non-persistent virus transmission with the roles of specific viral gene products in manipulating the host and vector, and to consider the epidemiological consequences of viral manipulation of host-vector interactions.2There is compelling evidence that certain genes of plant viruses exert extended phenotypes i.e., these parasite genes influence the expression of host genes in ways that ultimately benefit the virus . Among tMyzus persicae and Aphis gossypii) with CMV-infected cucurbit hosts, showed that CMV manipulated its own transmission in two ways. Firstly, CMV engenders the emission by the plant of increased levels of aphid-attracting volatile organic compounds (VOCs), and secondly the virus induces synthesis of anti-feedant compounds in plant tissue (antixenosis). These virus-mediated changes in plant phenotype cause aphids to be at first attracted by the VOCs to feed upon CMV-infected plants and acquire viral inoculum, only to be subsequently repelled by the taste of the antixenotic compounds , especially within the phloem tissue, and electrical penetration graph analysis showed that ingestion of phloem sap by M. persicae is consequently discouraged , are likely to encourage the spread of viral inoculum by aphids from infected plants to neighbouring uninfected hosts Myzus percouraged . The vired hosts  A. thaliana plants infected with turnip mosaic virus (TuMV) (Nicotiana tabacum) plants infected with CMV (A. gossypii) survival and reproduction is enhanced on PRSV-infected squash plants but there is no benefit for the whitefly species, Bemisia tabaci, which does not vector PRSV  , 2015, awith CMV . The bentor PRSV . In the 3Several viral genes have been identified that cause changes in the host, resulting in extended phenotypes affecting host-vector interactions and likely to enhance virus transmission. These include genes of geminiviruses (DNA viruses transmitted in the persistent manner by whitefly vectors) and their satellite DNA molecules, several of which encode factors that suppress signalling by, or responses to, the phytohormone jasmonic acid (JA) . JA regu2b genes in transgenic A. thaliana or N. benthamiana suppressed the induction of gene expression following spraying with methyl-JA  is the nuclear inclusion-a protein, which appears to operate via interference with ethene (ethylene)-mediated plant signalling, rather than through interference with JA-regulated signalling  showed no indication of bias towards settling on either CMV-infected or mock-inoculated tobacco plants  on CMV-infected squash plants  are more vulnerable to attack by parasitoid wasps (Aphidius colemani) (Manipulation of vector behaviour by pathogens can promote epidemic development in diverse host-vector-pathogen systems . Buildinolemani) , which polemani) A.Fig. 2AThe second, and perhaps surprising, conclusion drawn from the model is that induction of a ‘retain’ phenotype can contribute to epidemic development by fostering longer-distance dissemination of viral inoculum . Those aAn additional effect flowing from the virally-induced ‘retain’ phenotype may be that vectors also benefit. It has been suggested that the changes in whitefly-host interactions induced by persistently transmitted geminiviruses provides an example of mutualism where the virus is ‘paying back’ its insect vector by providing host plants on which vector growth and fecundity are improved . Mutuali5Recent years have witnessed significant advances in our understanding of how semiochemicals and other plant metabolites assist aphid vectors to locate plants and subsequently choose or reject them as hosts, and how viruses manipulate synthesis of these host cues to promote their own transmission. Additionally, we are beginning to understand at the molecular level how specific viral gene products, often working in concert, reprogram host genetic and biosynthetic networks to modify host-vector interactions. At larger scales, we are modeling the consequences of viral manipulation of host phenotypes on the development of viral epidemics.These insights are timely. Current approaches to control of insect-vectored plant viruses in agriculture depend largely on the application of pesticides . HoweverNone.


Autophagy is a tightly controlled process comprising lysosomal degradation and recycling of cellular proteins and organelles. In cancer, its paradoxical dual role of cytoprotection and cytotoxicity is context-dependent and controversial. Autophagy primarily acts as a mechanism of tumour suppression, by maintenance of genomic integrity and prevention of proliferation and inflammation. This, combined with immune-surveillance capabilities and autophagy’s implicated role in cell death, acts to prevent tumour initiation. However, established tumours exploit autophagy to survive cellular stresses in the hostile tumour microenvironment. This can lead to therapy resistance, one of the biggest challenges facing current anti-cancer approaches. Autophagy modulation is an exciting area of clinical development, attempting to harness this fundamental process as an anti-cancer strategy. Autophagy induction could potentially prevent tumour formation and enhance anti-cancer immune responses. In addition, drug-induced autophagy could be used to kill cancer cells, particularly those in which the apoptotic machinery is defective. Conversely, autophagy inhibition may help to sensitise resistant cancer cells to conventional chemotherapies and specifically target autophagy-addicted tumours. Currently, hydroxychloroquine is in phase I and II clinical trials in combination with several standard chemotherapies, whereas direct, deliberate autophagy induction remains to be tested clinically. More comprehensive understanding of the roles of autophagy throughout different stages of carcinogenesis has potential to guide development of novel therapeutic strategies to eradicate cancer cells. Autophagy is a highly conserved process of catabolism, which recycles and degrades intracellular components, primarily as a mechanism of cellular survival . It was Atg genes) orchestrate the formation of the autolysosome, through a series of sequential steps: initiation, nucleation, elongation and maturation [Autophagy is a tightly controlled process, influenced by multiple signalling pathways. Autophagy-related genes  . Commonlma lines , 12. Addma lines . Reducedma lines –16. UnsuAtg 5 and Atg7 causes spontaneous formation of benign liver tumours [Deletion of  tumours , further tumours , 18. Fur tumours , 20. Alt tumours .The mammalian proteins p62 and NBR1 selectively undergo autophagic degradation and can act as cargo receptors, or adaptors for autophagy of ubiquitinated substrate targets . A key rAs alluded to, autophagy also has a tumour suppressive role in established cancer cells. Autophagy is critical for optimal immune function, indeed many studies have shown autophagy to be essential for the recruitment of immune cells to the tumour bed, including dendritic cells and cytotoxic T lymphocytes, which are key in the stimulation of anti-cancer immune responses , 27. IndThus, it may be argued that activating autophagy might be a relevant clinical objective to enhance anti-tumour immune surveillance, and potentially increase the efficacy of current conventional therapies.Autophagy also acts in a tumour suppressive manner via its capacity to induce cell death. Autophagic cell death is referred to as type II programmed cell death and is characterised by the presence of cytoplasmic vacuolisation, with increased autophagic flux. Unlike apoptosis, autophagic cell death occurs independently of caspase activity , 26; it A variety of clinical anti-cancer therapies have been reported to induce cytotoxic autophagy including resveratrol , arsenicAlthough there have currently been no attempts to directly induce autophagy in cancer models, modulation of this process could comprise a new anti-cancer strategy in the future . The rolTo summarise, it is of clinical interest to investigate further whether autophagy induction can be exploited to prevent tumour initiation, reduce tumourigenicity in established cancers, enhance efficacy, or mediate cytotoxicity of chemotherapy. Observations have also indicated that induction of autophagic cell death may provide an attractive therapeutic strategy, particularly in cancer cells able to evade apoptosis, which is a common hallmark of many cancers .A deeper understanding of the process of lethal autophagy, and how molecular mechanisms interact with other cell death pathways, will be crucial in the development of effective strategies to accomplish cancer cell toxicity.Although autophagy has been widely implicated as a form of cell death, controversy remains concerning the extent of its actions. It is possible that autophagy is merely associated with dying cells and not a direct cause of the toxic fate. Indeed, autophagic tumour cell death has only been reported in very few circumstances and instead has been shown to activate alternative mechanisms executing cell death , 46. It Although autophagy has been shown to act in a tumour suppressive manner, its role is context-dependent; once tumours are established, they can exploit this process to enable survival. This paradoxical change from tumour suppressor to tumour promoter has been termed the ‘autophagy switch’ . Cancer Autophagy promotes tumourigenesis by providing an escape mechanism to cellular stresses. Within the tumour microenvironment, cancer cells experience harsh conditions including hypoxia and acidity. Cytoprotective autophagy provides a mechanism to promote cancer cell survival, removing otherwise toxic proteins and providing cellular substrates to sustain growth . HypoxicAutophagy is robustly activated in tumour cells by cancer-associated stressors such as growth factor deprivation and hypoxia. In the majority of cases, autophagy induction promotes survival in response to stress (as will be exemplified); survival by autophagy becomes dangerous if apoptosis is disabled—resulting in quiescence/dormancy of tumour cells. Hypoxia and glucose deprivation (conditions rife within the tumour microenvironment) upregulate autophagy—enabling survival. Autophagosomes are most evident in tumour cells of hypoxic regions, and deletion of autophagy-regulating genes has been shown to result in tumour cell death especially in hypoxic regions, strongly indicating that cancer cells exploit autophagy to survive and promote tumourigenesis . A role Atg5 and Atg7 limited tumour growth [Certain tumour types have shown elevated levels of autophagy, thus ‘autophagy addiction’ has been proposed as a form of tumour maintenance or promotion . Tumoursr growth , 51, 52.r growth . It has r growth . Autophar growth . TogethePancreatic tumours typically harbour Ras mutations and frequently show constitutive levels of autophagy . GeneticIn addition to its role in tumour promotion and survival in hostile environments, autophagy has also been implicated in resistance to conventional anti-cancer therapies, another form of stress imposed on cancer cells . ReturniAll autophagy modulating drugs in clinical trials are inhibitors of the process, primarily seeking to increase tumour sensitivity to conventional therapies . It is wChloroquine and its derivatives, previously used as anti-malarial agents, are leading the field and are currently the only FDA-approved modulators of autophagy . Pre-cliAtg16L1. Together these data provide evidence that Lys05 targets autophagy, and validate the therapeutic potential of this agent [Although clinical trials for hydroxychloroquine appear promising in both solid and haematological malignancies, the requirement for more specific and potent inhibitors remains. Lys05, a bisaminoquinoline derivative of chloroquine (Fig. is agent , 67. Veris agent . This hiTo summarise, increasing our understanding of the processes of autophagy and its roles in different stages of cancer will underpin developments in this field. Evidence primarily supports autophagy as a tumour suppressive mechanism, with autophagy-related genes commonly deleted or defective in different cancer types. Autophagy suppresses tumour initiation by preventing inflammation and oncogenic proliferation. Maintenance of genomic stability is also a key property of autophagy tumour suppression and is most likely achieved by the prevention of damaged mitochondrial accumulation and subsequent generation of oxidative stress. Autophagy and immune-surveillance are intimately linked to prevent tumourigenesis. As an alternative form of programmed cell death, autophagy has been implicated as a mechanism of cytotoxicity in current anti-cancer treatments.Thus, it is of therapeutic interest to induce autophagy to enhance tumour prevention or elimination. Such a strategy could be beneficial in patients with increased risk of developing cancer, particularly in cancer types with known and defined pre-cancerous evolution. Identifying biomarkers, such as p62, could help to identify autophagy-deficient patients who would best respond to this form of treatment.Inducing autophagy in apoptosis-resistant cells could provide an alternative route of controlled cancer cell death, which could be crucial in surmounting therapy resistance. Furthering our understanding of the interactions between apoptosis, autophagy and other forms of cell death will be the key in developing alternative mechanisms towards targeting cancer cell toxicity.The elusive autophagic switch from tumour suppressor to tumour promoter is evident once tumours are established. Cancer cells exploit autophagy to sustain increased metabolic demands and survive under various stresses including hypoxia, acidity and anti-cancer therapies. This poses one of the key challenges in the clinic: therapy resistance. With this in mind, autophagy inhibition poses an attractive therapeutic strategy, with clinical advances already taking place using a combinatorial approach with hydroxychloroquine in phase I and II trials. The important role of autophagy in the anti-cancer immune response must also be considered; autophagy inhibition could be combined with immune-stimulatory drugs, such as checkpoint inhibitors, to prevent immune down-regulation . It is aTo conclude, the role of autophagy in cancer is complex, context-dependent and far from fully elucidated.Under normal physiology, autophagy can be a suppressor of tumourigenesis—a mean of ridding the body of damaged cells and ‘recycling’ nutrients. There is a role for autophagy in immune-surveillance and inhibition of malignant transformation; however, paradoxically, autophagy may support progression of established tumours, as the process can be subverted to ‘feed’ tumour cells. Autophagy may contribute to chemotherapy resistance, therefore its inhibition may sensitise cancer cells to therapy; yet the importance of chemotherapy-induced cytotoxic autophagy is evident, specifically in apoptosis-resistant tumour cells. Evidence supporting each of these statements has been discussed in this review and must be considered if, or when autophagy is to be exploited in a therapeutic setting. Fully understanding the autophagic switch will help to expose novel targets and mechanisms to target cancer cells.In the future, it is likely that autophagy modulation will be specific and tailored to different cancer phenotypes, depending on tumour evolution and autophagy-dependency.Today, autophagy exemplifies a controversial issue and an intense area of research; nevertheless, its modulation potentially represents an exciting novel approach in cancer treatment.


Motivated behaviors are often initiated in response to perturbations of homeostasis. Indeed, animals and humans have fundamental drives to procure (appetitive behaviors) and eventually ingest (consummatory behaviors) substances based on deficits in body fluid  and energy balance . Consumption, in turn, reinforces motivated behavior and is therefore considered rewarding. Over the years, the constructs of homeostatic  and reward (within the purview of mesolimbic circuitry) have been used to describe need-based vs. need-free consumption. However, many experiments have demonstrated that mesolimbic circuits and “higher-order” brain regions are also profoundly influenced by changes to physiological state, which in turn generate behaviors that are poised to maintain homeostasis. Mesolimbic pathways, particularly dopamine neurons of the ventral tegmental area (VTA) and their projections to nucleus accumbens (NAc), can be robustly modulated by a variety of energy balance signals, including post-ingestive feedback relaying nutrient content and hormonal signals reflecting hunger and satiety. Moreover, physiological states can also impact VTA-NAc responses to non-nutritive rewards, such as drugs of abuse. Coupled with recent evidence showing hypothalamic structures are modulated in anticipation of replenished need, classic boundaries between circuits that convey perturbations in homeostasis and those that drive motivated behavior are being questioned. In the current review, we examine data that have revealed the importance of mesolimbic dopamine neurons and their downstream pathways as a dynamic neurobiological mechanism that provides an interface between physiological state, perturbations to homeostasis, and reward-seeking behaviors. Motivated behaviors are fundamentally rooted in homeostasis. To survive, animals, including humans, have adopted behavioral strategies to efficiently procure and ingest substances based on homeostatic perturbations, particularly deficits in body fluid  and energy balance . Historically, researchers have dichotomized physiological underpinnings of ingestive behaviors into “homeostatic” and “non-homeostatic” [occurring in the absence of need and based on positive feedback ] neural processes—that is, as separate mechanisms. Indeed, as this field progressed, there have been attempts to conceptualize these two constructs as a means through which to view maladaptive motivated behaviors, particularly behaviors underlying obesity development and drug addiction. As such, reward-related neural substrates are thought to override processes that maintain homeostatic balance –6. HowevMidbrain dopamine neurons in VTA and SNc (substantia nigra pars compacta) exhibit distinctive firing patterns involving a combination of regular, pacemaker-like firing, irregular single spikes, and occasional high-frequency trains of action potentials, known as bursts –20. BursPhasic dopamine signaling has frequently been studied in the context of motivated behaviors that result from positive reinforcement. For example, dopamine is thought to reinforce learned associations between predictive stimuli and primary reward , 37. As However, the complex roles of phasic dopamine signaling extend beyond positive reinforcement. Indeed, as goal-directed behaviors for positive reinforcement are highly adaptive, forming associations between aversive stimuli and negative reinforcement is also essential for survival. Consideration of these complexities will be critical in understanding how mesolimbic dopamine signaling can regulate behaviors in response to homeostatic perturbation as well as how changes in physiological state can profoundly change neural computations within mesolimbic circuitry. In humans, expectation of pain relief  results in transient increases in NAc blood oxygen level-dependent (BOLD) activity as well as increased functional connectivity between the NAc and key mesolimbic nodes  , 52. SimMesolimbic dopamine responses to aversive stimuli comprise a substantial amount of complexity and heterogeneity , for exain vivo fiber photometry) along with precise control of behavioral and physiological outcomes  will be critical in understanding the interactions between behavior, physiological state, and mesolimbic phasic dopamine signaling.Given that feeding and drinking may be produced through negative reinforcement processes –67, it iHomeostasis is tightly regulated by a multitude of peripheral physiological processes as well as actions within the brain. These peripheral processes include feedback from various organs , which use both neural  and hormonal routes to relay information regarding homeostatic balance to central nodes that subsequently generate the appropriate behaviors poised to maintain and reinstate homeostatic balance . Here, were provide a brief overview of the central neural processes that are traditionally thought of as “homeostatic” and how the mesolimbic system has gained prominence as a neural substrate that is sensitive to homeostatic perturbation.Energy balance is generally well-maintained by a variety of peripheral signals relating to hunger and satiety. However, feeding behaviors come with their own complexities that often deviate from traditional notions of homeostatic balance. Recent work has focused intensely on investigating digressions in homeostatic energy balance in the context of the obesity epidemic and these studies have been reviewed in a number of recent manuscripts , 68–73. In states of hunger and satiety, hormonal mechanisms and post-ingestive effects on peripheral organs that are relayed to the central nervous system are often critical for initiating and halting feeding behaviors. Indeed, many feeding-related hormones readily enter the brain to control food intake and feeding behaviors. Hypothalamic and hindbrain nuclei have been focused on as primary targets for these hormonal and neural feeding signals. These two brain regions are traditionally associated with maintaining homeostatic energy balance, and their anatomical proximity to ventricular areas with a permeable blood brain barrier allows for heightened sensitivity to circulating hormones. The pancreas-derived hormone, insulin, a critical hormone for blood glucose regulation, enters the brain to promote satiety and reduce feeding behaviors [reviewed in ]. The adPerhaps most interesting, however, is the ability of these feeding hormones to engage motivated behaviors via signaling to hypothalamic and hindbrain substrates. For example, insulin administration into the arcuate nucleus of the hypothalamus reduces sucrose self-administration  and ventTo uncover the underlying neural mechanisms that regulate these parallel homeostatic and reward-related phenomena, agouti-related peptide expressing (AgRP) neurons in the arcuate nucleus of the hypothalamus have received a considerable amount of attention, and tremendous effort has been dedicated to examine these substrates as key regulators of energy balance. Besides the classic notion that AgRP neurons convey hunger signals , the abiAbsorption of nutrients and eventual post-ingestive consequences primarily act to engage satiety mechanisms, with hormonal signals described as one of several downstream outcomes. Alternatively, mechanisms that sense nutrients can profoundly influence motivated behaviors independently of hormonal modulation. For example, flavor-nutrient experiments have been enlightening in understanding the concept of appetition, where the post-ingestive consequences of calorie intake can induce goal-directed behaviors to further consume food. Studies from Sclafani and colleagues have demonstrated that pairing intragastric infusions of carbohydrates  or fat with particular flavors can lead to preferential intake of those flavors –95. TherTaken together, these data suggest that the presumed “homeostatic” processes of hunger and satiety are complex and capable of engaging behaviors that are often considered “reward-related.” Moreover, the collective evidence is consistent with the theme that there are parallel and overlapping circuitries that integrate changes in physiological state with reward-seeking behaviors.Body fluid homeostasis is tightly regulated as sodium deficit and dehydration pose highly threatening challenges to survival. Moreover, the rewarding value of fluids like water and hypertonic saline is heavily dependent on physiological state. A key example of this is the phenomenon of sodium appetite, where motivation to obtain and consume sodium changes drastically depending on body fluid balance. Indeed, sodium deprivation produces a robust, selective, and innate appetite for sodium that is in turn reflected in goal-directed behaviors to seek out and ingest sodium . Furthe libitum . Finallyil below . These rad libitum food access. During test sessions in which the levers were available but no reward was administered , food restricted rats exhibited enhanced heroin-seeking behaviors, as reflected by increased lever responses, relative to food sated rats. Furthermore, these effects can be modulated by both duration of food restriction or re-feeding. As such, reduced duration of food restriction and re-feeding attenuates this enhanced heroin-seeking behavior , but also enhance behavioral sensitivity toward other rewarding substances, such as drugs of abuse. Moreover, these processes are a clear example of how mechanisms that are designed to respond to homeostatic perturbations can be molded into behaviors that are maladaptive and viewed as “reward-related.” In an experiment using a model of drug relapse, rats were trained to self-administer heroin via lever presses, which was followed by a 14-day abstinence period where the rats were removed from operant chambers and underwent either mild food restriction or were allowed behavior . Importabehavior . These obehavior . Furtheruced CPP . Thus, iWhile much work has focused on the mesolimbic system in the context of food reward [, 29, 133Several studies have provided a glimpse into the role of dopamine signaling in controlling body fluid homeostasis and drinking behaviors –137. HowTo carefully parse the temporal, behavioral, and mesolimbic components involved in body fluid homeostasis, recent studies from our laboratory combined real-time recording of NAc dopamine release via fast-scan cyclic voltammetry with intraoral delivery of hypertonic saline during varying states of body fluid homeostasis . First, Based on the work described above, changes in physiological state and homeostatic perturbation have a key role in modulating mesolimbic pathways and their relevant behavioral outputs. What remains unclear are the gating mechanisms that (1) provide information regarding physiological state to the mesolimbic system and (2) how mesolimbic pathways integrate and relay this information. Fortunately, there are many investigations that provide insight into the mechanisms linking peripheral signals  and mesolimbic circuitry  as well as the transmission of homeostatic signals through central relays to mesolimbic pathways .ex vivo brain slices  but not the VTA directly , not only has receptors expressed in the VTA and NAc , but alsdirectly —supportidirectly . Thus, bBesides the role of hormonal signaling within mesolimbic dopamine pathways, other post-ingestive consequences of nutrient consumption can impact central neural substrates and subsequently guide goal-directed behaviors. In regards to the neural correlates that mediate caloric sensing and cues associated with calories, mesolimbic dopamine neurons again arise as potential nodes that integrate and relay post-ingestive information.trpm5-/- mice, which lack sweet taste transduction, these behavioral outcomes persist only with sucrose, albeit with slower temporal occurrence. These results further suggest that motivated behaviors can occur independently of hedonic value, although hedonic value certainly acts synergistically with nutritional value  to generate cue-reward associations and that these processes are in part modulated by mesolimbic phasic dopamine signaling , 167–171al value . Furtheral value . This isal value , 172. OvInterestingly, mesolimbic dopamine can be highly sensitive to the precise caloric content of nutrients. Fat, similar to sucrose and glucose, also elicits post-ingestive feedback mechanisms that influence goal-directed behaviors , 173–175The mechanisms regulating caloric sensing and hormonal regulation of energy balance, while intertwined, can also exhibit dissociable processes. In experiments involving intragastric infusions of glucose, disruption of glucose metabolism with intravenous 2-DG was shown to reduce striatal dopamine levels. Interestingly, this reduction was rescued with subsequent intravenous glucose administration . In a seThe data described above have covered dopamine signaling in response to calories in both the dorsal and ventral striatum—brain regions that have been previously attributed to dissociable functions in regards to motivated behaviors . Indeed,While physiological state information can be relayed to the mesolimbic system directly via hormones, the VTA and NAc also receive extensive neuronal projections from a multitude of neural substrates that are involved in processing homeostatic information. This provides an alternative, yet complementary, mechanism through which physiological state information is integrated prior to being transmitted to the mesolimbic dopamine system.Hindbrain neural processes are capable of modulating goal-oriented behaviors and reward , 84, 144Brainstem subregions that regulate body fluid homeostasis also have projections to the mesolimbic pathway. Within the NTS, a subset of neurons expressing 11-β-hydroxysteroid dehydrogenase type 2 (HSD2) are particularly sensitive to sodium depletion and signals that relay sodium deficiency  and in tBesides hindbrain projections, the VTA also receives extensive input from midbrain substrates that regulate homeostatic processes. In particular, the lateral dorsal tegmental area (LDTg) projections to the VTA , 186 havOther midbrain inputs to the VTA, including the pedunculopontine tegmental nucleus (PPTg) can modulate goal-directed behaviors and putative homeostatic functions. For example, the PPTg sends cholinergic and glutamatergic input to the VTA –196, modHypothalamic nuclei in the forebrain, as we have briefly discussed, consist of classic homeostatic neural regulators. In parallel with hindbrain and midbrain pathways, hypothalamic brain regions, in particular the lateral hypothalamic area (LHA), send direct and reciprocal projections to the VTA and NAc , 201–207in vitro that leptin administration can reduce excitatory synaptic strength between LHA orexin neurons and the VTA and that these effects can be attenuated by fasting or high-fat diet induced obesity  as opposed to consummatory behaviors , 50. TheFinally, the interaction between sex differences, homeostasis, and phasic dopamine signaling requires extensive examination. Several recent studies have demonstrated that the effects of central homeostatic signaling are sex dimorphic. For example, female rats have higher levels of LHA ghrelin receptor expression than males, and acute blockade of LHA ghrelin receptors in females, but not males, reduces food intake, body weight, and food seeking behaviors . CentralWhile questions remain, a putative mechanism arises whereby neurons in the VTA are readily able to burst fire in response to homeostatic perturbation and the presence of state-relevant stimuli ; this in turn modulates the degree to which phasic dopamine increases occur in striatal targets—in particular the NAc. The result of the phasic increase could be to alter ongoing NAc activity as well as plasticity in the service of guiding motivated behaviors. Future research conducted with a special emphasis on the impact of physiological state on mesolimbic dopamine signaling will be critical in furthering our understanding of maladaptive behaviors with the eventual goal of effectively treating prominent health issues such as obesity and drug addiction.TH, JM, and MR conceived the scope of the manuscript. TH generated drafts. JM and MR provided critical discussion, edits and comments. TH, JM, and MR approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the past decade, direct-acting oral anticoagulants (DOAC) have been introduced to medical practice for several indications, with a wide range of dosing regimens. As both over- and under-dosing might lead to life-threatening events, development of methods promoting safe and effective utilization of these agents is imperative. The Hadassah Clinical Pharmacy team initiated a hospital-wide program, for monitoring and promoting safe and effective prescription of DOAC during hospitalization. This study describes the types of drug related problems addressed and the program’s performance in terms of consultation rates and physician acceptance.Electronic medical records throughout the hospital were screened for DOAC orders. All DOAC orders were assessed by a clinical pharmacist for potentially-inappropriate prescribing. When potentially-inappropriate prescribing or a drug-related problem was identified, the clinical pharmacist provided consultation on management options. In specific cases, additional guidance was provided by coagulation and pharmacology specialists. Data on patient characteristics, clinical pharmacist consultations, and physician response was retrospectively retrieved for the first six months of 2017. Characteristics of patients with and without consultations were compared, consultations were categorized by the recommended management of the drug related problem, and physician acceptance rates were evaluated by category.During the evaluated period, 585 patients with DOAC orders were identified. Patients were evenly distributed by gender, and age averaged 78 years. Most patients received apixaban (75%) followed by rivaroxaban (14%) and dabigatran (11%), and most (63%) received “reduced dose” regimens. Clinical pharmacists provided 258 consultations for 210 patients, regarding anticoagulation management, such that more than one in three patients on DOAC had potentially inappropriate prescribing or drug related problems. Consultations included alerts regarding potentially inappropriate DOAC doses and recommendations to increase (29%) or decrease (5%) the dose, potentially inappropriate concomitant antiplatelet agents (20%), need for DOAC level monitoring (23%), and alerts regarding other drug related problems (23%). More than 70% of recommendations were accepted by the attending physician.Due to the complexity of DOAC management, potentially-inappropriate prescribing and drug related problems are common. Multidisciplinary collaborative projects including review and consultation by clinical pharmacists are an effective method of improving management of patients on DOAC.NCT03527615.Retrospectively registered at clinicaltrials.gov,  The introduction of direct acting oral anticoagulants (DOAC) into clinical practice over the past decade has dramatically changed the field of stroke and thrombosis prevention and treatment. These medications include the direct thrombin-inhibitor dabigatran, and the direct factor Xa inhibitors rivaroxaban, apixaban, edoxaban, and betrixaban. The DOAC have been found to be similarly effective in preventing thromboembolic events when compared to traditional anticoagulant treatment in a variety of health conditions, and tended to have a lower risk of major bleeding, particularly intracranial hemorrhage , 2. The Perhaps the main consideration driving the rapid adoption of these medications into clinical practice is their relative simplicity of use compared to traditional older anticoagulant agents. The most commonly used older anticoagulants are vitamin-K antagonists (VKA), such a warfarin, and low-molecular-weight heparins (LMWH), such as enoxaparin. VKA have an unpredictable response, requiring individualized dosing, continuous and frequent laboratory monitoring, and special care and awareness regarding drug-drug and food-drug interactions. LMWH have a more predictable dose-response profile, however they require once to twice daily injections, and their use has not been extensively evaluated in indications requiring long-term use. In contrast, DOAC have been evaluated in studies utilizing nearly uniform doses, and do not require laboratory monitoring nor multiple injections. DOAC have thus far been formally evaluated and approved for: (1) treatment and prevention of stroke and thromboembolic events in patients with atrial fibrillation, (2) treatment and prevention of deep vein thrombosis (DVT) or pulmonary embolism (PE), and (3) prevention of DVT/PE following total knee replacement (TKR) or total hip replacement (THR).7,8 and are of considerable significance in light of the continuously-expanding utilization of DOAC in clinical practice.Despite their relative safety profile and seeming simplicity of use, the adoption of DOAC in practice has introduced several significant challenges. Each of the DOAC has been tested using a range of indication-dependent doses, leading to a wide range of doses approved for each DOAC, varying based on indication, treatment duration, renal function, patient weight and concomitant medications. The introduction of several DOAC within a short time period further contributed to the complexity related to DOAC utilization. Consequently, errors, misunderstandings, confusion and inappropriate DOAC prescribing and use are common,The optimal utilization of DOAC is a clinical imperative, as anticoagulant therapy is a double-edged sword, with significant associated risk, requiring a careful watchful balance of the expected benefit in reduction of thromboemoblic events with the increased risk of bleeding. Recent studies show that anticoagulation-associated hemorrhage is among the most common causes of drug-related emergency department visits , while iThe effectiveness of medication management by clinical pharmacists has been documented in a wide variety of areas, including the treatment of diabetes, blood pressure, and dyslipidemia, and management of polypharmacy, medication safety, and adherence . StudiesIn this study, we describe and evaluate an initiative to improve DOAC utilization via programmatic review by clinical pharmacists in collaboration with coagulation specialists, including two hematologists and a clinical pharmacologist, in the Hadassah University Hospitals in Jerusalem. The study describes the types of drug related problems addressed and assesses its performance in terms of consultation rates and physician acceptance.This study is a retrospective evaluation of an initiative to improve DOAC utilization in hospitalized patients. The study was performed at the Hadassah Hebrew University Medical Center, which consists of two hospitals: an 800-bed tertiary care hospital at Hadassah Ein-Kerem, and a 350-bed community hospital at Hadassah Mt. Scopus. Both hospitals operate in Jerusalem, Israel, and share institutional resources, including an electronic health record system.The initiative included all patients prescribed a DOAC during hospitalization, regardless of indication, as the aim was to reduce all potentially inappropriate prescribing of these high-risk medications. As identifying DOAC prescriptions required a computerized query of electronic health records in all departments. The only patients excluded were patients hospitalized in departments not using the institution electronic health records – i.e. the emergency rooms and intensive care units.The retrospective evaluation and retrieval of data from patient records reviewed in this program was approved by the institutional Helsinki committee (365–15-HMO).The initiative to improve DOAC utilization via programmatic review by clinical pharmacists was developed in collaboration with clinical pharmacology and hematology specialists. The clinical pharmacists were all licensed pharmacists with advanced degrees in clinical pharmacy, whose clinical training included education and experience in medication therapy management in general, and with anticoagulant therapy specifically.The initiative was designed following an extensive literature review, including the detailed evaluation of phase III DOAC studies  , 15, revAll institutional electronic medical records were screened for physician DOAC orders, using an automated query, at least twice-weekly. All DOAC orders were thoroughly reviewed by a certified clinical pharmacist. The clinical pharmacist review included assessment of appropriateness of DOAC indication, selection, dose and regimen, taking into account patient age, weight, estimated renal function, potential drug-drug and drug-disease interactions, and other factors with potential impact on clinical management. A national medical database linking ambulatory and hospital data was accessed for obtaining additional clinical information, as needed.When potentially inappropriate prescribing or drug-related problems were identified, the clinical pharmacist alerted the attending physician and provided consultation on possible management options. Potentially inappropriate prescribing was defined as prescribing of DOAC which was not in accordance to the guidance of regulatory and medical authorities, including the Ministry of Health approved prescribing information for apixaban (Eliquis), dabigatran (Pradaxa), and rivaroxaban (Xarelto), –18 as weThe institution’s coagulation specialists, including two hematologists and a clinical pharmacologist, provided guidance for general management strategies, as well as consultation and support in specific cases, as required. Consultations and alerts were recorded in the electronic medical record. Performance measures for program activity, including follow-up of acceptance and implementation of consults, were collected and summarized for the first six months of 2017.Data summarized included characteristics of patients with DOAC, and characteristics of interventions by clinical pharmacists. Patient characteristics included age, gender, and DOAC dose. To facilitate retrospective evaluation of clinical pharmacist consultations, they were broadly categorized by the type of recommendation given into the following categories: monitor DOAC levels, increase dose, decrease dose, stop concomitant antiplatelet, and others. In addition, the acceptance of the recommendation by the physician was recorded.A larger range of characteristics was retrieved for a subset of 289 patients from the internal medicine department to compare characteristics of patients with and without clinical pharmacist consultation. Characteristics compared included age, gender, weight, serum creatinine, DOAC dose, concomitant antiplatelets, and concomitant medications which inhibit CYP3A4 and Pgp (P-glycoprotein) and are therefore expected to interact with DOAC .Continuous variables were expressed as means with standard deviation (SD). Categorical variables were expressed as frequencies and percentages. Characteristics of patients with and without consultation were compared using univariate tests. Significance between groups was assessed by the Chi square test or Fisher’s exact test for categorical variables, and the t-test or Mann-Whitney U test for continuous variables. Normality of continuous variables was evaluated by visual inspection of density plots, Q-Q plots and Shapiro-Wilk test for normality. Analyses were conducted using R version 3.4.3 .During the period evaluated, 585 patients with orders for DOAC were identified. Patients’ age averaged 78 years SD 10), with gender approximately evenly distributed . Most DOAC orders were for apixaban (75%) followed by rivaroxaban (14%) and dabigatran (11%). The majority of orders (63%) were for a reduced DOAC dose . These included recommendations to monitor DOAC and anti-Xa levels , increase (29%) or decrease (5%) DOAC dose, discontinue concomitant antiplatelet drug (20%), and other recommendations pertaining to DOAC therapy management (23%), such as need for acid-suppressing medications and anticoagulant duplication.Of the 258 consults provided, 189 were accepted and implemented in clinical management, for an overall physician acceptance rate of 73%. Physicians’ acceptance rate was highest for recommendations to stop concomitant antiplatelet agents (87%) and lowest for recommendations for drug level monitoring (47%)  contrast dramatically with the very low rates reported with computerized methods to reduce medication error and drug-related problems. Studies of computerized alerts have reported much lower acceptance rates, with some studies reporting acceptance rate as low as 4–5% , 25. SimWhile the bleeding risk associated with the use of anticoagulation is intuitive, and readily apparent, over-cautious use of DOAC might also pose significant health hazards. Patients with an indication for DOAC generally face a significantly increased risk of thromboembolic events, and under-treatment, or use of lower doses than required, might result in increased morbidity and mortality. The danger of under-dosing of DOAC has been suggested in recent studies reporting higher risk of stroke in patients receiving lower doses , 29, 30,The use of antiplatelet agents in patients using anticoagulants is associated with a significant increase in the risk of major bleeding –35. TherOf the consultations provided, advice encouraging DOAC concentration monitoring was accepted least often (46%). DOAC were studied, approved, and marketed for use without a need of continuous and frequent laboratory monitoring, and there is limited data and evidence to guide the clinical interpretation of DOAC concentration monitoring. It is therefore reasonable for physicians to be reluctant to conduct such testing. However, there is increasing recognition that monitoring may be helpful in certain clinical settings –38. ThesIn our institution, laboratory monitoring is often also considered in unique patients likely not represented in phase-III trials, when they have characteristics likely to influence DOAC’s pharmacokinetics and pharmacodynamics. These include patients with very advanced age (patient ages in this study ranged up to 103 years), morbidly-obese patients, those with severe renal failure, and those with potentially-interacting medications. Studies have suggested a potential for differences in response among morbidly-obese and post-bariatric patients patients –41. LikeOur study presents the feasibility and effectiveness of a collaborative multidisciplinary initiative, involving clinical pharmacists, hematologists, and clinical pharmacologists, to improve prescribing of DOAC. Inappropriately high or low doses of DOAC, concomitant use of antiplatelet medication, and DOAC use in high risk patients , have been clearly associated with increased risks for bleeding and thromboembolic events. In our study, clinical pharmacists provided continuous monitoring of anticoagulation prescribing, and provided alerts and consultation on a wide range of medication related problems. This activity was met by a high level of acceptance by the physicians, thereby reducing potentially inappropriate prescribing of DOAC and improving patient safety.This initiative and its evaluation have several strengths and limitations. The study provides data on DOAC prescribing in “real world” hospitalized patients in Israel, across two hospitals, in a wide variety of departments, and highlights some important challenges in prescribing these high-risk medications. Additionally, the study provides an overview of a project aimed at reducing potentially inappropriate prescribing and drug-related problems and evaluates its effectiveness in terms of acceptance by treating physicians. However, the study has a number of limitations. The study did not include an evaluation of the impact of this project on clinical outcomes (such as the rate of bleeding or thromboembolic complications). The project was based on computerized query of physician orders for DOAC and therefore did not include a few departments not using this system (the intensive care units and emergency departments). Additionally, the intervention included both patients who initiated therapy with DOACs in the community as well as those initiating therapy in the hospital, and we could not retrospectively evaluate possible differences between these groups. Lastly, the evaluation of patient characteristics linked to clinical pharmacist consultation was not performed on the full cohort but only on a subset of patients from the internal medicine department.Our study has significant implications for public health policy. The high prevalence of inappropriate prescribing and drug-related problems with DOAC identified in this study highlight a pressing need to promote strategies to reduce risk with these medications. The effectiveness of medication management by clinical pharmacists has been previously documented in a wide variety of areas and settings –11. ThisAs anticoagulants are one of the leading culprits of drug related adverse events , additioAdvanced clinical pharmacy training has been provided by the Hebrew University of Jerusalem for the past two decades on a very small scale. There are currently an estimated 150 clinical pharmacists practicing in Israel, mostly trained in this program. Recently the Ben-Gurion University has opened an advanced degree in clinical pharmacy as well. These programs would need to grow significantly to train enough clinical pharmacists to provide professional monitoring and consultation of medication related issues throughout the Israeli health-system. To achieve this goal, universities should consider transitioning pharmacist training in Israel to the U.S. model, where pharmacy training and licensing exists only as an advance degree incorporating significant clinical training. However, such a transition must be accompanied by concrete steps by the Ministry of Health to regulate and define licensing and staffing standards, as well as requires that the Ministry of Finance provide the necessary funding to facilitate the wide-spread incorporation of clinical pharmacy services in the Israeli health system.In addition, automated computerized methods should be developed to increase safe prescribing of medications in general, and DOACs specifically. While studies of clinical decision support systems have reported high rates of “alert fatigue” poor uptake by clinicians , 25, it Many of the drug related problems identified in this study existed prior to hospitalization, and chronic medication management is primarily the responsibility of primary care physicians. It is therefore clear that improving the safety of medications also necessitates the development and provision of programmatic, focused, and continued education to primary physicians in this area. There is a shortage of physicians and nurses in the Israel health system, and there are only a select few with advanced training in clinical pharmacology. Increasing the number of clinical pharmacists in Israel, could therefore also provide the professional personnel needed to develop and provide guidance and education on medication management and safety in the primary care setting.Due to the complexity of DOAC management, potentially-inappropriate prescribing and drug related problems are common. The high prevalence of inappropriate prescribing and drug-related problems with DOAC identified in this study highlight a pressing need to promote strategies to reduce risk with these medications. Multidisciplinary collaborative projects including review and consultation by clinical pharmacists with the involvement of hematologists and clinical pharmacologists are an effective method of improving management of patients on DOAC. Implementation of similar projects on a country-level scale would require significant changes in the training of pharmacists in Israel, and in the scope of hospital-pharmacy activity in the Israeli health system. Additional studies are needed to measure the impact of such projects on clinical outcomes.