The incidence of gastrointestinal pathologies (cancer in particular) has increased progressively, with considerable morbidity and mortality, and a high economic impact on the healthcare system. The dietary intake of natural phytochemicals with certain bioactive properties has shown therapeutic and preventive effects on these pathologies. This includes the cruciferous vegetable derivative phenylethyl isothiocyanate (PEITC), a bioactive compound present in some vegetables, such as watercress. Notably, PEITC has antioxidant, anti-inflammatory, bactericidal, and anticarcinogenic properties. This review summarized the current knowledge on the role of PEITC as a potential natural nutraceutical or an adjuvant against oxidative/inflammatory-related disorders in the gastrointestinal tract. We also discussed the safe and recommended dose of PEITC. In addition, we established a framework to guide the research and development of sustainable methodologies for obtaining and stabilizing this natural molecule for industrial use. With PEITC, there is great potential to develop a viable strategy for preventing cancer and other associated diseases of the gastrointestinal tract. However, this topic still needs more scientific studies to help develop new PEITC products for the nutraceutical, pharmaceutical, or food industries. Over time, gastrointestinal tract diseases, or disorders, have increased in incidence and prevalence, becoming a huge problem for society, with millions of people affected. Gastrointestinal disorders also have a high economic impact and lead to decreased life quality ,2,3. AmoThe WHO estimates that about 30% to 50% of cancers can be prevented by changing or avoiding the main risk factors. Thus, cancer prevention represents an ideal strategy to reduce the burden of this disease, while also offering a more economical treatment strategy in the long term for cancer control . EpidemiHere, we briefly review the current state of knowledge on the implications of PEITC for cancer prevention in the gastrointestinal tract. Simultaneously, we propose a framework to guide the research and development of sustainable nutraceutical solutions for prophylactic action on this global health system’s priority problem. Brassicaceae family), including plants such as watercress, cauliflower, broccoli, and brussels sprouts, among others [Isothiocyanates are the most abundant products of the natural enzymatic degradation of glucosinolates by the enzyme myrosinase ,11. Thesg others . To dateg others . Glucosig others ,14. Phytg others .The metabolism of glucosinolates can occur with two different types of the stated enzyme: plant myrosinase, which coexists separately with glucosinolates in plants, being activated when the plant is damaged; and bacterial myrosinase, which acts mainly in the colon (comes from gut microbiota) . This me3−), and a variable side chain (-R), which will classify the glucosinolate as aliphatic, aromatic, or indole [Glucosinolates are inert, anionic organic compounds, characterized by β-D-thioglucose, a sulfated oxime group  is a very accessible garden vegetable highly rich in glucosinolates [Watercress  in the cell. When ROS concentration is high, damage may occur at the DNA level, increasing the carcinogenesis probability . These RAs a chemo-preventive and antioxidant agent, PEITC may modulate the unregulated ROS concentration in cells, activating antioxidant defense mechanisms through the increased expression of detox enzymes, to lower ROS to basal levels ,29. LikeIn the gastrointestinal tract, cancer development is closely dependent on energy intake and nutrient availability, but it is also characterized by low-grade inflammation (a slight but chronic increase in the number of various inflammatory markers in the blood and organs) . InflammThe excessive expression of a pro-inflammatory factor results in the damage of the epithelial barrier, initiating apoptosis of epithelial cells and the secretion of chemokine. Several researchers have demonstrated the anti-inflammatory properties of PEITC through the reduced expression of this protein by inhibiting NF-κB expression ,32,33,34Brassicaceae family’s cruciferous vegetables is associated with human health benefits, such as the reduced risk of chronic diseases and several types of cancer, including gastric and colon cancers [The consumption of the  cancers ,37. Base cancers ,23,24. MCarcinogens are subjected to metabolism and elimination, mainly by phase I and phase II biotransformation enzymes. In general, carcinogenesis occurs due to the bioactivation of carcinogens by phase I enzymes. Hence, phase I metabolism products are highly reactive intermediates that can be harmful by binding to critical macromolecules, such as DNA. In contrast, phase II enzymes play an essential role in the detoxification and excretion of carcinogens from the body. PEITC is involved in the inhibition of phase I enzymes and the induction of phase II enzymes, especially CYP enzymes and transferases, which may explain the chemo-preventive activity .Moreover, it is known that cancer risk can be modified by the dietary intake of bioactive phytochemicals, such as PEITC, which can have epigenetic effects through the modulation of DNA/histone modification. However, currently, studies about the epigenetic mechanisms of these anti-tumor effects in human cells are so far very limited. Indeed, the future development of effective dietary regimes for cancer prevention will require a better understanding of the ingested phytochemicals’ effects on DNA methylation, histone modification, and/or chromatin remodeling in developing tumors . ConcernDespite the encouragement of the foregoing, what is known so far is based on purely empirical studies conducted in controlled laboratory environments. According to the vague epidemiological studies carried out so far, the anticancer properties of PEITC are still under investigation. Up to date, just inverse proportionality correlations have been established between the consumption of PEITC-containing vegetables and the risk of chronic diseases, which makes clear the need to expand the study focus to more in-depth epidemiological studies to have a more complete understanding of the effect of PEITC consumption on cancer relations .Although the antimicrobial capacity of isothiocyanates to control the proliferation of plant and foodborne pathogens has been well documented, currently, there are still few studies that assess the action of these compounds to combat human infections. Furthermore, the information that exists mainly refers to the in vitro antimicrobial activity against bacterial pathogens, and little is known about the in vivo antimicrobial effects of isothiocyanates . In thisHelicobacter pylori, blocking its carcinogenic effects in the stomach [H. pylori infection is difficult to eradicate, and it is a major cause of gastritis and peptic ulcers, a condition that may be associated with the development of gastric cancer [Clostridium difficile and Clostridium perfringens, two pathogenic agents of the genus Clostridium that can threaten human health [PEITC may act as a potential bactericidal compound against some bacterial pathogens responsible for gastrointestinal infections through the disruption of the plasma membrane, the dysregulation of the enzymatic machinery, and cell death . It is a stomach . These Pc cancer . Concernn health ,41. ThusBifidobacterium sp., one of the common bacteria belonging to the human intestinal microflora, in the hydrolysis of glucosinolates [Brassica-rich diet in healthy adults was associated with changes in the gut microbiota composition. The study concluded that a diet rich in Brassica did not significantly alter the relative proportions of intestinal lactobacilli, but was associated with a reduction in the relative abundance of SRB [Furthermore, it is of particular interest to understand its interaction with the intestinal microbiota. Isothiocyanates, as products of myrosinase−based glucosinolate hydrolysis in the human gut, are important to health, particularly their anti-cancer properties and other beneficial roles in human health mentioned above . Even thinolates ,43. Kelle of SRB . Furthere of SRB .Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Lactobacillus acidophilus, and Lactobacillus casei), and thus did not negatively affect the intestinal microbiota [According to Kim and Lee (2009), PEITC showed not to have an antimicrobial action against commensal bacteria  and considerable hydrophobicity (logP = 3.47). Its pharmacokinetic features include first-order linear absorption with a high protein binding nature [Even though PEITC has great potential as a health-promoting compound, its industrial use has been limited because of its relative instability . PEITC ig nature . Therefog nature . Till nog nature ,65. Besig nature . This op


Evaluation of the equilibrium metal association constants for the active Ca2+ binding sites of recombinant human α-PA (‘CD’ and ‘EF’ sites) from fluorimetric titration experiments and isothermal titration calorimetry data gave 4 × 109 M−1 and 4 × 109 M−1 for Ca2+, and 2 × 107 M−1 and 2 × 106 M−1 for Sr2+. Inactivation of the EF site by homologous substitution of the Ca2+-coordinating Glu in position 12 of the EF-loop by Gln decreased Ca2+/Sr2+ affinity of the protein by an order of magnitude, whereas the analogous inactivation of the CD site induced much deeper suppression of the Ca2+/Sr2+ affinity. These results suggest that Sr2+ and Ca2+ bind to CD/EF sites of α-PA and the Ca2+/Sr2+ binding are sequential processes with the CD site being occupied first. Spectrofluorimetric Sr2+ titration of the Ca2+-loaded α-PA revealed presence of secondary Sr2+ binding site(s) with an apparent equilibrium association constant of 4 × 105 M−1. Fourier-transform infrared spectroscopy data evidence that Ca2+/Sr2+-loaded forms of α-PA exhibit similar states of their COO− groups. Near-UV circular dichroism (CD) data show that Ca2+/Sr2+ binding to α-PA induce similar changes in symmetry of microenvironment of its Phe residues. Far-UV CD experiments reveal that Ca2+/Sr2+ binding are accompanied by nearly identical changes in secondary structure of α-PA. Meanwhile, scanning calorimetry measurements show markedly lower Sr2+-induced increase in stability of tertiary structure of α-PA, compared to the Ca2+-induced effect. Theoretical modeling using Density Functional Theory computations with Polarizable Continuum Model calculations confirms that Ca2+-binding sites of α-PA are well protected against exchange of Ca2+ for Sr2+ regardless of coordination number of Sr2+, solvent exposure or rigidity of sites. The latter appears to be a key determinant of the Ca2+/Sr2+ selectivity. Overall, despite lowered affinity of α-PA to Sr2+, the latter competes with Ca2+ for the same EF-hands and induces similar structural rearrangements. The presence of a secondary Sr2+ binding site(s) could be a factor contributing to Sr2+ impact on the functional activity of proteins.Strontium salts are used for treatment of osteoporosis and bone cancer, but their impact on calcium-mediated physiological processes remains obscure. To explore Sr However, strontium ranelate has serious side effects: cardiovascular disorders , venous thromboembolism, pulmonary embolism, nausea, diarrhea, headache and eczema. It is prescribed only as a second-line drug for treatment of postmenopausal osteoporosis when other drugs are unsuitable  and [Ca2+/Sr2+-aq] represent the metal cation bound to protein ligands within the binding pocket and the unbound metal, respectively. A positive free energy change for Equation (5) implies a Ca2+-selective site, whereas a negative value suggests a Sr2+-selective one.The competition between Srα atoms and the SDD basis set/effective core potential for Sr2+ was employed in the calculations. This theoretical method/basis set has been thoroughly calibrated/validated in previous publications with respect to available experimental data and has proven to be dependable, since it can properly reproduce the geometry of a series of representative metal structures [2+ Ca2+ exchange in metal-oxalate complexes (see below). The M06-2X method  in combiructures  as well ructures . Furtherel, were computed for each optimized complex. Subsequent frequency calculations were performed at the same M06-2X/6-311++G//SDD level of theory, and no imaginary frequency was found for any of the structures studied. The frequencies were scaled by an empirical factor of 0.983 [th, including zero-point energy and entropies, S. The differences  between the products and reactants in Equation (5) were used to evaluate the metal exchange Gibbs free energy in the gas phase at T = 298.15 K, ΔG1, according to the equation:Each metal-loaded binding site was optimized in the gas phase by employing the Gaussian 09 software package . Electroof 0.983  and emplThe basis set superposition error for Equation (5) was found to be negligible , and thesolvε, of each complex. The incoming Sr2+ and outgoing Ca2+ ions were considered to be in a bulk aqueous environment (ε = 78) outside the binding pocket. Accordingly, their experimentally evaluated hydration free energies of −329.8 and −359.7 kcal/mol, respectively [Metal-binding sites are located in cavities/clefts of proteins with dielectric properties that are different from those of the bulk water  but simiectively , were em2+ → Ca2+ substitution in metal complexes. Experimentally measured stability constants of Ca2+ and Sr2+ oxalate complexes [The theoretical method and calculation protocol employed here were validated with respect to experimental data on Sromplexes  were useNote that the two metals were bound bidentately to the oxalate anion and characterized by a coordination number of 7. The theoretically evaluated free energy of metal substitution of 1.4 kcal/mol was in very good agreement with the experimental estimate of 0.6 kcal/mol.E. coli strain BL21(DE3), isolated and purified to homogeneity mainly according to [2+-binding process via Phe fluorescence  was expressed in rescence A. Additi2+-binding loop in the EF domain of α-PA (EF site) by the homologous E102Q substitution in 12th position of the loop (PA-EF mutant) caused a decline in the stoichiometry of Ca2+ binding to PA down to 1:1, and facilitated Ca2+ removal via EDTA (2+-binding loop in the CD domain of α-PA (CD site) via E63Q substitution (PA-CD mutant) induced a drastic decrease in apparent Ca2+ affinity of the protein and failure to achieve the fluorescence intensity characteristic of the Ca2+-loaded state , considering a competition between the protein and EDTA for Ca2+ (scheme (2)), as shown in -EF and PA-CD mutants were approximated by the single site metal binding scheme (3)  . The ina2+ , considering the competition between the protein and EDTA for Sr2+ (Equation (2)) shows ) shows  that Sr2tio of 2 B. Approxonstants , one of 2+-induced fluorescence changes closely resembling those observed upon the Ca2+ titrations  rWT α-PA (12 μM) by Ca2+ up to 1 mM did not reveal noticeable effects  α-PA (1 μM) by Sr2+ up to a Sr2+ to Ca2+ molar ratio of 1:1 induced about a 10% increase in Phe fluorescence intensity (2+-binding site(s). An evaluation of the effective equilibrium Sr2+ association constant using single site metal binding scheme (3) gave ca. 4 × 105 M−1.Spectrofluorimetric titration of the Sr effects A, and rentensity B, which 2+/Ca2+-loaded protein at 20 °C  reveals two superimposed heat sorption peaks at temperatures above 90 °C   .2+/Sr2+-loaded CD and EF sites of PA are shown in 2+/Sr2+ in a bidentate mode, while the other carboxylic groups coordinated the metals in a monodentate manner, as observed in the X-ray structure 1PAL. The positive values of free energy changed upon substitution of Ca2+ for Sr2+  is smaller than that for the EF site . Furthermore, the theoretical results indicate that the metal competition for the CD site was slightly affected by changes in the dielectric constant  was used by Rhyner et al. K+ ions are known to bind EF-hands of PA [2+ association constants found by Rhyner et al. that were two orders of magnitude lower (2.3 × 107 M−1).Rhyner et al.  studied ds of PA  and ther2+ association with human α-PA to the amino acid substitutions inactivating its CD/EF sites  resultsdictions , revealilmodulin ) and nontalbumin .2+: 0.72 Å versus 1.18 Å. Similarly to Sr2+, affinity of PAs for Mg2+ is several orders of magnitude lower than that for Ca2+ [2+ binding and their selectivity towards Ca2+ [Magnesium, another element from group II of Periodic table but positioned above calcium, has a much lower ionic radius compared to that for Cafor Ca2+ . Thus, Erds Ca2+ .2+, which typically induces structural rearrangements resembling those observed upon Ca2+ binding but less pronounced , Sr2+ bemission A, FTIR semission , near-UVemission A, and faemission . A drastgeometry ,56, whilrdinated . Neverthtructure , Table 3te state .2+ and Sr2+ and respectively similar structural consequences of their binding to α-PA, we have revealed a secondary site selective towards Sr2+, evidently different from primary Ca2+-binding sites of the protein  that differ in stability of tertiary structure due to differences in Sr2+/Ca2+ affinities. The secondary Sr2+-selective site we discovered points to an opportunity for Sr2+ and its radioactive counterpart 90Sr2+ to affect functional activity of proteins without competition with Ca2+ for the same sites.The comparative experimental study of Sr


Blocked cellular differentiation is a central pathologic feature of the myeloid malignancies, myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Treatment regimens promoting differentiation have resulted in incredible cure rates in certain AML subtypes, such as acute promyelocytic leukemia. Over the past several years, we have seen many new therapies for MDS/AML enter clinical practice, including epigenetic therapies , isocitrate dehydrogenase (IDH) inhibitors, fms-like kinase 3 (FLT3) inhibitors, and lenalidomide for deletion 5q (del5q) MDS. Despite not being developed with the intent of manipulating differentiation, induction of differentiation is a major mechanism by which several of these novel agents function. In this review, we examine the new therapeutic landscape for these diseases, focusing on the role of hematopoietic differentiation and the impact of inflammation and aging. We review how current therapies in MDS/AML promote differentiation as a part of their therapeutic effect, and the cellular mechanisms by which this occurs. We then outline potential novel avenues to achieve differentiation in the myeloid malignancies for therapeutic purposes. This emerging body of knowledge about the importance of relieving differentiation blockade with anti-neoplastic therapies is important to understand how current novel agents function and may open avenues to developing new treatments that explicitly target cellular differentiation. Moving beyond cytotoxic agents has the potential to open new and unexpected avenues in the treatment of myeloid malignancies, hopefully providing more efficacy with reduced toxicity. Myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), the two most common myeloid malignancies, have recently seen a wave of novel therapeutics approved. While the outcomes for MDS/AML have historically been poor, this is now starting to change . InitialPreclinical drug development in myeloid malignancies, as in other malignancies, has historically focused on differential cytotoxicity as the major goal. While this approach has led to significant advances, a focus on cytotoxicity may now be delivering diminishing returns. Myeloid malignancies are unique in oncology as treatment protocols focused on inducing cellular differentiation already exist. The most striking example is the case of acute promyelocytic leukemia (APL); the development of the all-trans retinoic acid (ATRA) and arsenic trioxide (ATO) regimen (the “Lo-coco regimen”) in 2013, notable for consisting of two drugs that lack significant intrinsic cytotoxicity, has led to complete remission (CR) and overall survival (OS) rates of greater than 95% . This suIn this review, we outline the major scientific and clinical milestones in differentiation therapy, and suggest that these approaches may open avenues beyond cytotoxicity for the treatment of myeloid malignancies, ultimately producing new agents with high efficacy and reduced toxicity.CEBPA, PU.1, and RUNX1 [Hematopoiesis remains one of the best characterized differentiation pathways, and one in which differentiation blockade is known to be a prominent mechanism of malignant transformation. This has historically been modeled as a hematopoietic differentiation “cascade” with distinct, non-overlapping intermediate cell states, defined by immunophenotype, existing between stem/progenitor and terminally differentiated cells –15. Thisnd RUNX1 . These fFLT3) often being found in a single subclonal population. This clonal composition shifts in response to cytotoxic therapy, ultimately resulting in disease relapse. Indeed, a commonly observed pattern is that cytotoxic therapy will eradicate clonal populations harboring activating mutations  and achieve a morphologic remission, though persistence of low-level clones harboring epigenetic regulator mutations  can be observed [Clonal heterogeneity extends to the malignant state . AML is observed . In someobserved –26. DysrEscherichia coli) have also been shown to enforce granulopoiesis and differentiation through activation of the toll-like receptors (TLRs), as well as signaling through myeloid differentiation protein 88 (MYD88) and, downstream, granulocyte colony stimulating factor (G-CSF) [Characteristics that affect hematopoietic differentiation extend beyond those intrinsic to the HSCs. Specifically, there is emerging evidence for the role that inflammation and aging (“inflammaging”) can play in driving both normal and aberrant differentiation Fig. . There a (G-CSF) , 29. TLR (G-CSF) –33. Cyto (G-CSF) . Chronic (G-CSF) . Similar (G-CSF) . Thus, iMYD88 [IRAK1 [TRAF6 [miR-145 and miR-146a, have also been shown to be downregulated in MDS [hi) HSCs [miR-146a expression declined in older mice, and that miR-146a null young mice demonstrated premature HSC aging by depleting quiescent HSCs through IL6 and tumor necrosis factor (TNF) activation, predisposing these mice to myeloid malignancy [miR-143/145 depletes functional HSCs through the transforming growth factor β (TGFβ) pathway [Inflammation and immune activation, and the associated impacts on cellular differentiation, also have relevance in MDS. Several immune signaling related proteins have been shown to be aberrantly expressed in MDS, including TLRs  as well MYD88 , IRAK1 [8 [IRAK1  and the 1 [TRAF6 . Intrinsd in MDS . These cd in MDS , induce d in MDS , 44, and pathway . However pathway . The effAs we have outlined, the modern view of hematopoietic differentiation is more nuanced than historically appreciated. Physiologic hematopoietic differentiation is largely epigenetically regulated and characterized by a pool of precursors that become lineage-restricted in a dynamic and flexible fashion. It is also clear that the traditional immunophenotypically defined cell compartments are intrinsically heterogeneous, both on genomic and functional bases. Alterations to regulators of normal hematopoietic differentiation also seem to be a key feature in the development of myeloid malignancies. This may relate to a failure of intrinsic regulators of normal differentiation  or chronic external stimuli  that promote the development of subsequent clonal disorders. Despite the importance of abnormal differentiation to pathogenesis in MDS/AML, explicitly targeting these pathways has not been a focus in drug development for these diseases. However, as we outline below, induction of differentiation is an important part of the mechanism of action of several successful therapeutic approaches. While there have not been specific studies directly examining the impact of inflammation on therapeutic outcomes, we do know that inflammation is strongly correlated with several secondary features that result in inferior therapy outcomes . There is significant interest in integrating measures of the “immunome” into future clinical trials . CharactPML) gene with the retinoic acid receptor-alpha (RARα) gene to form the PML-RARα fusion transcript; this results in a myeloid differentiation block at the promyelocytic stage [The prototypical malignant cell differentiation therapy is the ATO and ATRA combination for APL. APL is characterized by a fulminant clinical presentation with bleeding and thrombosis and usually carries the classical translocation of chromosome 15 and 17 t , 50. Theic stage . ATRA biic stage . ATO actic stage . The dowic stage . The treic stage , 54, 55.ICAM-1 in a murine model of DS. They found that ATRA treatment increased gene expression of the CXC chemokines and ICAM-1 in the lung alveolar cells, and MIP-2 was observed on the alveolar macrophages [ICAM-1. Outside of the context of ATRA therapy, it may be that DS is primarily driven by chemokine secretion from the differentiating myeloblasts, though this has largely not been explored.It was quickly recognized with the ATO-ATRA combination that some patients would develop a syndrome characterized by the acute onset of fevers, volume overload, and pulmonary edema, which was dubbed “differentiation syndrome” (DS) . DS has rophages . OverallMN1 [NPM1 [NPM1 in AML cell lines and primary samples, leading to differentiation and apoptosis [EVI-1 may be sensitive to ATRA. Two studies demonstrated that ATRA could induce differentiation and apoptosis in EVI-1 overexpressing non-APL cell lines and primary samples [RARα agonists, such as tamibarotene (SY-1425) have been shown to induce differentiation and apoptosis in high RARα expressing AML primary cells [RARα expression and has shown encouraging results so far [There are several lessons that can be abstracted from the ATO-ATRA story in APL. The first is the striking therapeutic efficacy that this combination achieves, while provoking minimal cytotoxicity. Given that most current drug-discovery platforms utilize rapid cell death as an endpoint to assess the efficacy of compounds, neither of these compounds may have been identified in that context. However, it is important to recognize that APL is a distinct disease state with a lower degree of genetic complexity versus non-APL MDS/AML; the more pronounced clonal and genetic diversity in non-APL MDS/AML adds an additional layer of complexity to consider when examining the role for differentiation induction in therapy. In addition, ATO/ATRA directly targets the founding genetic event in APL within the leukemia stem cells, which is more difficult to achieve in non-APL leukemia stem cells. The utility of ATRA without ATO has been examined in non-APL AML. One in vitro study suggested that ATRA might be efficacious at inducing differentiation in AML with low expression of the transcriptional regulator MN1 , or in tN1 [NPM1 , 60. Twopoptosis , 60. Oth samples , 62. Des samples , 64. Othry cells . Tamibars so far , 67.Cmax of 5AZA corresponding to approximately 4 µM, suggesting this may not be the primary mechanism by which 5AZA works in patients [TET2) and IDH mutations, ascorbic acid (Vitamin C), and the epigenome, as vitamin C has been shown to stimulate TET2 catalytic activity in vitro [IDH1 mutated AML, vitamin C exposure induced a pattern of differentially methylated regions that overlapped with enhancers implicated in myeloid differentiation, specifically regions related to the hematopoietic-specific TFs: CEBPβ, HIF1α, RUNX1, and PU.1. Chromatin immunoprecipitation sequencing confirmed a loss of PU.1 and increase in RUNX1-bound elements and an increase in H3K27ac flanking sites near RUNX1-bound sequences [Another type of therapy for which differentiation is increasingly recognized as a component of its mechanism are the HMAs, 5AZA and decitabine (DEC). While hypomethylation is the most direct and obvious effect of HMAs, the downstream mechanism of action is complex and likely dose-dependent, and the connections between hypomethylation and eventual blast clearance in the marrow remain to be fully defined . It is ipatients . With repatients –73. Therpatients . This hapatients . They copatients . This supatients . Both 5Apatients . Recentlin vitro . In one equences . The autequences , though MLL fusion protein, which comprise about 10% of acute leukemias. The MLL-rearranged fusion protein binds to and dysregulates proliferation-associated genes such as the HOXA cluster, MEIS1, and PBX3. However, both native and rearranged MLL must bind with a chromatin-associated protein complex, which includes members such as disruptor of telomeric silencing 1-like (DOT1L) and Menin [MLL-Menin interaction, which have demonstrated efficacy in a patient-derived xenograft model of MLL-rearranged leukemia. This approach is being tested in early phase clinical trials [NPM1-mutations [NUP98 rearrangements [HOXA9 overexpression is thought to be a downstream transcriptional consequence of the MLL-rearrangement; it has been shown that inhibition of dihydroorotate dehydrogenase (DHODH) relieves the MLL-induced differentiation block by modulating mitochondrial metabolism [There are several other epigenetic therapies currently in clinical trials for MDS and AML Table . Specifind Menin . Two grol trials , 81. Theutations , 83 or Nngements . There hngements . HOXA9 otabolism . Inhibittabolism .Table 1SMLL-rearranged cell lines, while inhibitors for BET, DHODH, and CDK also impact non-MLL-rearranged cells. Interestingly, they identified significant differentiation induction effects for inhibitors of Menin-MLL, DOT1L, and DHODH while the BET and CDK9 inhibitors primarily acted by apoptosis induction [Another epigenetic therapy in early phase trials are the bromodomain and extraterminal (BET) inhibitors . Normally BET proteins, such as Bromodomain-containing protein 4 (BRD4), bind acetylated histone tails, bringing together the elongation complex and the promoter region , 89. Onenduction .MLL-rearranged T-acute lymphoblastic leukemia [One other class of agents that have been investigated are inhibitors of the histone demethylase LSD1 , which is a component of the MLL-protein complex. One preclinical study demonstrated that LSD1/KDM1A inhibition with a small molecule (ORY-1001) induced H3K4me2 accumulation on KDM1A target genes, resulting in blast differentiation and reduced leukemia burden in cell line and animal models of leukemia . These pIDH and FLT3 inhibitors are rapidly being incorporated into the therapeutic algorithms for myeloid malignancies. IDH inhibitors are another example of successful agents for myeloid malignancies that work partly through differentiation induction. Clinical DS has been a relatively commonly reported adverse event at 18% for ivosidenib  and 7% fSomewhat more unexpectedly, there is an evolving body of literature examining the role of cellular differentiation in the mechanism of the novel FLT3-inhibitors. FLT3-internal tandem deletions (FLT3-ITD) are classically thought of as a “driver” mutation in MDS/AML that are usually a later event in AML evolution . These mHistorically, megakaryocyte differentiation has been viewed as arising from a common myeloid-erythroid progenitor downstream of the multipotent progenitor (MPP) cell subpopulation. More recently, a new understanding has emerged that suggests most megakaryocytes derive directly from a megakaryocyte-biased HSC pool . MegakarTP53 or RUNX1 mutation, and in a rare case, loss of GATA2 function  MDS. Del(5q) is the most common cytogenetic abnormality seen in MDS, and results in haploinsufficiency for various coding and noncoding genes; this MDS subtype is uniquely sensitive to the immunomodulatory drug lenalidomide. However, at least half of the del(5q) MDS patients who respond to lenalidomide eventually become resistant. Del(5q) MDS patients who become lenalidomide resistant tend to have a either a RUNX1 or TP53 mutations? GATA2 is partially regulated through a positive-feedback loop wherein interleukin-1-beta and CXCL2 stimulate GATA2 expression [As we have outlined, aberrant cellular differentiation is a core feature of both MDS and AML. We also have seen that differentiation is a major player in the mechanism of action of many of the novel therapies being introduced in MDS and AML, and that it appears to be central to their efficacy. We have also outlined many potential benefits to exploiting differentiation induction the treatment of myeloid malignancies, such as reduced toxicity and clonal selection. One major challenge in developing therapeutics that enforce cellular differentiation is that the proteins involved are often TFs, which have not been traditionally viewed as druggable targets. However, there are creative ways to address this; in the example of lenalidomide resistance, is there a possible way to activate GATA2 in lenalidomide-resistant patients with pression . Could i


While the phototransduction-associated role of GCAP1 in the photoreceptor outer segment is widely established, its implication in synaptic transmission to downstream neurons remains to be clarified. Here, we present clinical and biochemical data on a novel isolate GCAP1 variant leading to a double amino acid substitution (p.N104K and p.G105R) and associated with cone dystrophy (COD) with an unusual phenotype. Severe alterations of the electroretinogram were observed under both scotopic and photopic conditions, with a negative pattern and abnormally attenuated b-wave component. The biochemical and biophysical analysis of the heterologously expressed N104K-G105R variant corroborated by molecular dynamics simulations highlighted a severely compromised Ca2+-sensitivity, accompanied by minor structural and stability alterations. Such differences reflected on the dysregulation of both guanylate cyclase isoforms (RetGC1 and RetGC2), resulting in the constitutive activation of both enzymes at physiological levels of Ca2+. As observed with other GCAP1-associated COD, perturbation of the homeostasis of Ca2+ and cGMP may lead to the toxic accumulation of second messengers, ultimately triggering cell death. However, the abnormal electroretinogram recorded in this patient also suggested that the dysregulation of the GCAP1–cyclase complex further propagates to the synaptic terminal, thereby altering the ON-pathway related to the b-wave generation. In conclusion, the pathological phenotype may rise from a combination of second messengers’ accumulation and dysfunctional synaptic communication with bipolar cells, whose molecular mechanisms remain to be clarified.Guanylate cyclase-activating protein 1 (GCAP1) is involved in the shutdown of the phototransduction cascade by regulating the enzymatic activity of retinal guanylate cyclase via a Ca The lhe light . The neufor Mg2+ ,5. Mg2+-channels ,7. Two ichannels  and is 2channels .GUCA1A, the gene coding for GCAP1, have been associated with various forms of autosomal dominant cone dystrophies (adCOD) [GUCA1A poses tremendous challenges to possible therapeutic options, because the compensation of the detrimental effects of the mutant allele, which in other species may arise from other GCAP isoforms, does not seem to occur in humans [To date, more than twenty point mutations identified in  (adCOD) ,22,23,24 (adCOD) . A thoro (adCOD) . The domn humans .GUCA1A result in the substitution of two adjacent amino acids in the high affinity binding site EF-hand 3 (EF3), namely, asparagine 104 with lysine and glycine 105 with arginine (N104K-G105R), both positions being highly conserved in vertebrates  examinations, each one year apart. Her visual acuity was 1.3 LogMAR (not improving with pinhole or lens correction) in both eyes and remained stable in the clinical examinations. The patient underwent color vision testing (Ishihara plates) that showed a complete loss of color discrimination, and fundus imaging  and fundus autofluorescence (FAF), OCT showed a central lesion involving the macula, characterized by thinning of the outer nuclear layer, which appeared absent in the fovea, interruption of the ellipsoid zone , and abnElectroretinography was performed on two occasions according to published techniques ,29. ERG Imaging and ERG results led us to confirm the initial clinical diagnosis of COD with negative ERG ,31. GUCA1A gene (NM_000409), the c.312_313delinsGC, p.(Asn104_Gly105delinsLysArg) . The deletion/insertion of two adjacent nucleotides resulted in the in-frame replacement of the wild-type amino acids asparagine and glycine with the amino acids lysine and arginine  coverage of targeted bases was 136 X, with 96% covered to at least 25 X. Genetic testing showed a novel heterozygous variant in the arginine . The var2+-dependent regulation of its molecular targets, namely GC1 and GC2, and comparing the activation profiles with those of wild type (WT) GCAP1. N104K-G105R displayed a small but significant reduction in GC1 activation at low Ca2+ , whereas they exhibit a shift upon Ca2+-binding proportional to their affinity [2+  proteins upon ion binding, even under denaturing conditions, can be exploited to evaluate their conformational changes  and diffaffinity . This isaffinity , which snity [2+ B, and fo2+-affinity of the double mutant with respect to the WT by an assay based on the competition of GCAP1 variants with the chromophoric Ca2+-chelator 5,5′Br2-BAPTA [2+-titrations of N104K-G105R (2-BAPTA led to the conclusion that the apparent Kd of N104K-G105R would be comparable if not higher than that of the chelator (Kdapp = 2.3 µM), which would render the double mutant unable to correctly regulate the GC in the physiological Ca2+-range (200–600 nM).We evaluated the differences in Car2-BAPTA , whose a2+ (corresponding to a molecular mass (MM) of 38.2 kDa, 2+ , pointing towards the presence of higher order oligomeric species, which appeared only as a shoulder of the main peak in the presence of Mg2+.Several lines of evidence in the last years have indicated that WT GCAP1 is a functional dimer under physiological conditions ,37; thus2+ , suggesting that the variant is considerably less compact than native GCAP1. We further investigated the oligomeric properties of the GCAP1 variant by DLS, which showed the presence of a single population with low polydispersion (PdI) both in the presence of Mg 6.85 nm , respectmax/IANS = 8.4 vs. 3.4 in the absence of ions, 4.8 vs. 2.5 in the presence of Mg2+, and 5.0 vs. 2.4 in the presence of Mg2+ and Ca2+, respectively, 2+-bound form of N104K-G105R was found to be more hydrophobic than the Mg2+-bound form  suggest that this was indeed the case . The ana2+  over 15 h C of N104for Mg2+ . Circular Dichroism (CD) spectroscopy is a valuable low-resolution tool to evaluate conformational changes in NCS proteins upon ion binding, as it provides information about the protein tertiary structure , secondary structure  and susceptibility to thermal denaturation (following the ellipticity at fixed wavelength).222/θ208) and the relative variation in ellipticity at 208 nm upon ion binding (∆θ/θ). In the absence of ions, the double mutant exhibited a smaller θ222/θ208 ratio compared to the WT (0.87 vs. 0.90), but a larger increase upon Mg2+-binding . On the contrary, Ca2+ exerted a smaller effect on the secondary structure of the double mutant, as θ222/θ208 increased only to 0.92, compared to 0.95 of the WT.The presence of two non-conservative mutations in N104K-G105R did not result in protein unfolding, as shown by the typical α-helix protein spectra in the far UV A and by 2+ addition, N104K-G105R exhibited a WT-like behavior, with almost overlapping spectra .To evaluate the thermal stability of N104K-G105R, we monitored the CD signal at 222 nm, the spectral local minimum displaying the largest variation upon ion addition. In the absence of cations, N104K-G105R was 3 °C less stable than the WT  descriptor pointed towards an enhanced plasticity of the backbone of N104K-G105R in both the Mg2+-bound and Ca2+-loaded forms compared to the WT , which are located in the C-lobe. Interestingly, the Mg2+ ion bound in the EF2 motif of N104K-G105R displayed a slightly lower RMSF compared to its counterpart in the WT , but not for EF4 (RMSF = 1.236 vs. 0.955 Å), which belongs to the same C-lobe, and even more so for EF2 (RMSF = 1.008 vs. 0.834 Å), located in the N-lobe. Overall, such observations point again to the well-known role of Ca2+-ions in stabilizing the structure of NCS proteins and are in line with the decreased thermal stability of Ca2+-loaded N104K-G105R.The double substitution N104K-G105R does not result in protein unfolding or major structural differences with respect to the WT; therefore, we ran 300 ns all-atom molecular dynamics (MD) simulations to evaluate subtle conformational and stability changes ascribable to the mutations in both Cao the WT . In deta2+ influences the release of synaptic vesicles, and therefore the communication between photoreceptors and horizontal and bipolar cells. This phenomenon has been associated with the dynamic process of disassembly and reassembly of the synaptic ribbon, which appears to be Ca2+-dependent, because lowering intracellular Ca2+ indeed reduces the synaptic ribbon size, while increases in Ca2+ concentration result in the opposite effect [The perception of complex senses, such as vision, requires precise, sustained, and extremely fast neurotransmission from receptors to downstream neurons. The presence of ribbon synapses in photoreceptors distinguishes these cells from other neurons because it provides the above features by encoding graded changes of membrane potential induced by light into the modulation of continuous vesicle exocytosis, which permits the transmission of signals to the inner retina . The pree effect ,43. While effect ,44,45. Ae effect . Beside e effect ,48. Althe effect , thus su2+ [The localization of the second guanylate cyclase GC2 within photoreceptors is not clear, but it has been established that GC2 is present in much lower amounts compared to GC1 in both bovine  and mous2+ . All theGUCA1A gene, resulting in the double substitution N104K-G105R in the translated protein GCAP1. This is the first report of a GUCA1A-related disease showing a negative ERG, indicating an abnormality of the ON-pathway related to the b-wave generation, which was not observed even in the N104K single variant [Here, we describe a clinical case of COD with negative ERG having a pathologic double missense mutation in the  variant . A negat variant ,57,58,59 variant . Alterna variant ,62. The 2+-binding motif alters the flexibility of both the N- and C-lobes (2+ (in the EF2 and EF3 motifs) and Ca2+ ions , although in a different manner. MD simulations indeed suggest that Mg2+ is slightly more tightly bound by N104K-G105R than the WT, whereas Ca2+ ions exhibit higher fluctuations . Taken together, our results point toward an impaired Ca2+-sensitivity of N104K-G105R as the molecular explanation for the pathological phenotype, similarly to D100G/E [2+-dependent conformational switch of N104K-G105R, responsible for the inhibition of the GCs, would render the enzymes constitutively active in the physiological Ca2+-concentration range  and spectral domain optical coherence tomography (OCT) were performed. 2. Responses were averaged over 10 stimulus presentations. The interstimulus interval was 2 s. Dark-adapted, mixed rod/cone ERGs were recorded in response to white 50 μs full-field stimuli with an intensity of 3 cd·s/m2. The interstimulus interval was 15 s. Five responses were averaged. Following a 20 min adaptation to Ganzfeld bowl light (30 cd/m2), Ganzfeld cone-mediated ERGs were recorded in response to white 50 μs full-field stimuli with an intensity of 3 cd·s/m2 presented on a steady white background of 30 cd/m2. Responses were averaged over 20 stimulus presentations. The interstimulus interval was 1 s. Cone-mediated ERGs were also recorded in response to 30 Hz flicker flashes of 2 cd·s/m2 presented on a steady white background of 30 cd/m2. Responses were averaged over 20 stimulus presentations. Signals were amplified (50 K), filtered (0.3–250 Hz), digitized at 2 kHz, and averaged with automatic artifact rejection. The baseline to peak rod b-wave amplitude was measured. The amplitudes of the a- and b-waves were measured. Flicker ERG amplitudes were measured peak-to-peak, and their implicit times from the stimulus onset.ERG recordings were obtained following the ISCEV standards. Following a dark-adaptation period of 30 min, Ganzfeld rod-mediated ERGs were recorded in response to white 50 μs flashes of 0.01 cd·s/m2 flashes and cone-single flash ERGs to 2 Hz, 2 cd·s/m2 flashes on a 20 cd/m2 background were recorded according to a recently published technique that was specific for cone and cone-rod dystrophy patient examination [In addition, mesopic Ganzfeld ERGs to 1 Hz, 2 cd·s/mmination .Genetic testing was performed at MAGI’S Laboratories . The proband’s DNA was extracted from saliva  and analyzed through next-generation sequencing (NGS) on a MiSeq instrument, using the PE 2× 150 bp protocol  with a custom panel comprising the following genes: ABCA4 (OMIM *601691), ADAM9 (OMIM *602713), AIPL1 (OMIM *604392), C8orf37 (OMIM *614477), CACNA1F (OMIM *300110), CACNA2D4 (OMIM *608171), CDHR1 (OMIM *609502), CEP78 (OMIM *617110), CFAP410 (OMIM *603191), CNGA3 (OMIM *600053), CRX (OMIM *602225), DRAM2 (OMIM *613360), ELOVL4 (OMIM *605512), GUCA1A (OMIM *600364), GUCY2D (OMIM *600179), IFT81 (OMIM *605489), KCNV2 (OMIM *607604), PDE6C (OMIM *600827), PITPNM3 (OMIM *608921), POC1B (OMIM *614784), PROM1 (OMIM *604365), PRPH2 (OMIM *179605), RAB28 (OMIM *612994), RAX2 (OMIM *610362), RIMS1 (OMIM *606629), RPGR (OMIM *312610), RPGR (OMIM *312610), RPGRIP1 (OMIM *605446), SEMA4A (OMIM *607292), TTLL5 (OMIM *612268), UNC119 (OMIM *604011).NGS raw sequencing data were analyzed using an in-house bioinformatics pipeline, as described elsewhere ,65. https://www.ncbi.nlm.nih.gov/snp/), ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), gnomAD (https://gnomad.broadinstitute.org/), and the Human Gene Mutation Database, professional version 2017.2 . Filtered variants were checked for their novelty and pathogenicity in databases (accessed on 24 November 2018) such as dbSNP  [All variants were also classified for pathogenicity by the American College of Medical Genetics and Genomics (ACMG) classification , with thry 2021) .Genetic testing was performed for diagnostic purposes, to confirm the clinical suspicion of cone dystrophy. The proband was invited to sign an informed consent form after appropriate pre-test genetic counseling.Saccharomyces cerevisiae N-myristoyl transferase (yNMT) [The cDNA of wild-type human GCAP1-E6S (Uniprot: P43080) was cloned in a pET-11a plasmid between NdeI and NheI restriction sites (Genscript). The E6S variant was introduced to allow post-translational myristoylation on the N-terminal Gly residue by e (yNMT) . The N10Escherichia coli BL21-DE3 cells previously co-transformed with pBB131-yNMT and purified after a sequence of size exclusion chromatography  and anionic exchange chromatography  as previously described [4HCO3 and lyophilized. Samples were finally stored at −80 °C until use. GCAP1 variants were heterologously expressed in escribed ,67, withescribed , using a2+ and either 2 mM EGTA (free Ca2+ < 19 nM) or ~30 µM Ca2+ [2, 0.3 mM ATP, 0.16 mM Zaprinast buffer and blocked with the addition of 50 mM EDTA and boiling. Samples were then centrifuged for 20 min at ~18,000× g at 4 °C; finally, the supernatant was loaded on a C18-RP column  to quantify cGMP production. Control experiments were performed in the absence of GCAP1 to measure basal cGMP synthesis for each of the independent replicas. The production of cGMP at low and high Ca2+-concentration in the presence of GCAP1 variants was subjected to two-tailed t-tests, where the null hypothesis was represented by equal averages between different conditions. All differences were statistically significant .Recombinant human guanylate cyclases 1 and 2  were expressed in permanent HEK293 cells after transfection with PEI and 10-day selection with geneticin . After c µM Ca2+ . Enzymat2+ or 1 mM Mg2+ + 5 mM Ca2+, boiled for 5 min and loaded on the gel.Cation-specific electrophoretic mobility of GCAP1 variants under denaturing conditions was evaluated on a 15% acrylamide SDS-PAGE gel. GCAP1 variants were diluted to 15 µM in 20 mM Tris-HCl pH 7.5, 150 mM KCl, 1 mM DTT buffer, incubated at 25 °C for 10 min with either 5 mM EDTA, 5 mM EGTA + 1.1 mM Mg2+-affinity of N104K-G105R was measured by a competition assay with the chromophoric chelator 5,5′Br2-BAPTA as previously elucidated [2+, <50 nM Ca2+ buffer, to a concentration of 16.2 µM . The three independent titrations were fitted to a theoretical model consisting of 3 binding sites and a chelator  using CaLigator [2+-binding constants considered unreliable due to the absence of competition. Data reported in 263, Amin and Amax are the absorbance at 263 nm, the minimum, and the maximum values, respectively.The Caucidated ,34,35. Trd assay ) in the aLigator , yieldin2+ or 500 μM Ca2+ + 1 mM Mg2+ allowed the identification of the elution volume (Ve). Apparent MM was estimated from the calibration curve of log(MM) vs. distribution coefficient (Dc) as described by Vallone et al. [c was defined as follows: v and Vt are the void (8 mL) and the total column volume (25 mL), respectively.Analytical size exclusion chromatography was performed using the same buffer  and the same calibration curve as in Vallone et al.  on a Supe et al. , in whic2+ and 1 mM Ca2+; whereas far UV CD spectra (200–250) were recorded after serial additions of 300 µM EGTA, 1 mM Mg2+ and 600 µM Ca2+. Spectra were collected after setting 1 nm data pitch, 1 nm bandwidth, 4 s integration time, 50 nm/min scanning speed and 37 °C temperature. Sample composition for thermal denaturation was the same as for far UV spectra; profiles were collected at 222 nm after setting 0.1 °C data pitch, 20–96 °C temperature range, 1 nm bandwidth, 4 s integration time, 90 °C/min scanning speed. Raw data were fitted to the following function:f and bu are the baseline of the folded and unfolded states, respectively, T is the temperature, kf and ku are the slopes of the respective states, and ∆Gt is the variation in Gibbs free energy associated with the native-to-unfolded transition. ∆Gt can be further developed in the following equation:p is the variation in heat capacity at constant pressure, and Tm is the melting temperature, whose estimation is reported in Variations in thermal stability, secondary, and tertiary structures of GCAP1 variants were evaluated using a Jasco J-710 spectropolarimeter supplied with a Peltier-type cell holder. GCAP1 samples were diluted to 42 and 10 µM  in 20 mM Tris-HCl pH 7.5, 150 mM KCl, 1 mM DTT buffer, and protein concentration was verified by a Bradford assay . Near UV2+ or 1 mM Mg2+ + 500 µM Ca2+, filtered with an Anotop 10 , equilibrated for 3 min at 25 °C, and monitored for 15–17 h . Hydrodynamic diameters and PdI for N104K-G105R variant reported in Hydrodynamic diameter and aggregation propensity were measured on a Zetasizer Nano-S  using disposable polystyrene low volume cuvettes and the instrumental setup previously developed by some of the authors . Protein2+ and 1 mM Ca2+. Fluorescence excitation was set to 380 nm, bandwidths to 5 nm, number of accumulations to 3; the spectrum of the buffer added with 0.1% DMSO was considered as reference and subtracted.The conformational changes upon ion binding of N104K-G105R were evaluated in terms of variations in hydrophobicity, using an assay based on the differential fluorescence emission exhibited by the hydrophobic probe 8-Anilinonaphthalene-1-sulfonic acid (ANS) upon binding to hydrophobic patches. Fluorescence emission spectra (440–600 nm) of 30 µM ANS in 20 mM Tris-HCl pH 7.5, 150 mM KCl, 1 mM DTT buffer were recorded with a Jasco FP-750 spectrofluorometer in the absence and presence of 2 µM N104K-G105R and 500 µM EGTA and after serial additions of 1 mM Mg2+-loaded myristoylated GCAP1 was obtained by homology modeling using the Ca2+-bound myristoylated GCAP1 from Gallus gallus [2+-bound human GCAP1 states were modeled after deletion of a Ca2+ ion in EF4 and substitution of the Ca2+ ion with a Mg2+ ion in EF2 and EF3 as previously detailed [The three-dimensional structure of human Cas gallus  as templs gallus . The mutdetailed . All-atodetailed  and CHARdetailed  force fidetailed , while tdetailed ,40, from


In this model, the high vascular density in the superior sinoatrial node places myocytes with metabolically demanding, high-frequency action potentials near vessels. The lower vascularization and electrical activity of inferior sinoatrial node myocytes could limit these cells to function to support sinoatrial node periodicity with sporadic voltage fluctuations via a stochastic resonance mechanism.The cardiac cycle starts when an action potential is produced by pacemaking cells in the sinoatrial node. This cycle is repeated approximately 100 000 times in humans and 1 million times in mice per day, imposing a monumental metabolic demand on the heart, requiring efficient blood supply via the coronary vasculature to maintain cardiac function. Although the ventricular coronary circulation has been extensively studied, the relationship between vascularization and cellular pacemaking modalities in the sinoatrial node is poorly understood. Here, we tested the hypothesis that the organization of the sinoatrial node microvasculature varies regionally, reflecting local myocyte firing properties. We show that vessel densities are higher in the superior versus inferior sinoatrial node. Accordingly, sinoatrial node myocytes are closer to vessels in the superior versus inferior regions. Superior and inferior sinoatrial node myocytes produce stochastic subthreshold voltage fluctuations and action potentials. However, the intrinsic action potential firing rate of sinoatrial node myocytes is higher in the superior versus inferior node. Our data support a model in which the microvascular densities vary regionally within the sinoatrial node to match the electrical and Ca  These APs are generated by clusters of pacemaker cells firing in unison3–9 Initiation of the AP occurs during diastole and is driven by activation of hyperpolarization-activated cyclic nucleotide-gated currents.10–13 HCN4 is the predominant HCN channel in the node, but HCN1-2 are also expressed. 11 Concurrently, spontaneous, stochastic local sarcoplasmic reticulum (SR) Ca2+ release events via ryanodine receptors 14 activate inward diastolic Na+/Ca2+ exchanger currents.15 These HCN and Na+/Ca2+ exchanger currents depolarize SAN myocytes over the threshold of activation of voltage-gated CaV3.1,16 CaV1.3,18 and, eventually, CaV1.2 channels,19 thereby initiating the AP and triggering a transient global increase in intracellular Ca2+ concentration ([Ca2+]i). Membrane hyperpolarization begins by the inactivation of Ca2+ currents and activation of voltage-gated K+ currents. As SAN myocytes approach the maximum diastolic potential, HCN channels are activated, initiating the whole process again. APs propagate within the SAN via gap junctions,20 eventually reaching surrounding atrial myocytes.The mechanisms underlying pacemaking activity in SAN myocytes have been the subject of intense investigation.22 Instead, it dynamically shifts within the SAN in response to physiological stimuli, including activation of the autonomic nervous system.2 A recent study on ex vivo rat and human SAN tissue supports the concept that 2 distinct pacemaker sites coinhabit the SAN, one near the superior section of the node and another further below in the inferior section.23 Depending on the degree of sympathetic and parasympathetic nerve activity, the leading pacemaker site in the SAN shifts between superior and inferior regions. With high sympathetic drive, the superior SAN functions as the dominant pacemaking center. The pacemaker center shifts toward the inferior SAN during strong parasympathetic activation. Accordingly, a shift in pacemaker origin has been proposed to trigger changes in heart rate.23The dominant pacemaking site in the SAN is not static.+ and K+ gradients across the sarcolemma by the Na+/K+ pump, critical for Na+/Ca2+ exchanger function. The SR Ca2+ pump, responsible for refilling the internal Ca2+ store during each beat, also requires a significant amount of energy to operate in muscle.24 Although SAN myocytes do not have as many sarcomeres as atrial and ventricular myocytes, they still consume ATP during crossbridge cycling. Furthermore, the constant generation of cytosolic adenosine monophosphate (cAMP), fulfilled by the plasma membrane-associated enzyme adenylate cyclase, is an energetically demanding process that consumes significant amounts of ATP.25 cAMP is required for protein kinase A–dependent phosphorylation of multiple proteins, including HCN and Ca2+ channels as well as SR Ca2+ pump.26–29 Even under basal conditions , SAN myocytes consume more O2 compared with a paced (3 Hz) ventricular myocyte,25 demonstrating the relatively high metabolic demands of SAN myocytes. ATP consumption is elevated during increased sympathetic input to the SAN, when cAMP levels are further amplified, and AP frequency increased.13Irrespective of pacemaker location, SAN myocytes require energy in the form of adenosine triphosphate (ATP) for normal function. For example, significant amounts of ATP are consumed for the maintenance of Na30 Deoxygenated blood exits the SAN tissue via venules and specialized Thebesian veins where blood directly enters the right atrial lumen.31 In humans, the SAN artery has multiple anatomical variations32 and damage to this artery can be proarrhythmogenic.33 Despite the high metabolic demand of the SAN, a detailed understanding of the vascular network that supports the node for aerobic respiration is limited.To sustain the energetic requirements of SAN myocytes, the SAN artery, a branch of the right coronary artery, delivers oxygenated blood to the node. During diastole, when the compressive forces of the myocardium are at their lowest, blood moves through SAN artery and capillary network to deliver oxygen to pacemaker cells.2+ imaging of isolated SAN myocytes from densely  and sparsely  vascularized sections of the SAN, we discovered that superior-derived SAN myocytes are intrinsically capable of firing higher frequency APs versus inferior-derived SAN myocytes. Based on these data, we posit that blood supply could be a determinant in sculpting the firing properties of SAN myocytes, effectively determining the dominant pacemaking site within the SAN.In this study, we used a combination of imaging approaches to generate high-resolution three-dimensional (3D) maps of SAN vascular anatomy and its relationship to SAN myocytes across the entire node. Analysis of these images shows that vascular density is high and myocyte-to-vessel distances are relatively short in the superior region of the node. However, vascular density decreases, sharply increasing myocyte-to-vessel distances toward the inferior region of the node. Using whole-cell patch-clamp techniques and CaMale wild-type C57BL/6J mice (The Jackson Laboratory) aged between 6 and 14 weeks were used in this study. Mice were euthanized with a single, intraperitoneally administered lethal dose of sodium pentobarbital (250 mg/kg). All mice were maintained, and experiments conducted, in accordance with the University of California, Davis Institutional Animal Care and Use Committee guidelines.m): 140 NaCl, 5.4 KCl, 1.0 MgCl2, 1.8 CaCl2, 5.0 HEPES, 5.5 d-glucose (pH = 7.4 with NaOH). The SAN region was dissected out of the heart and pinned flat. The SAN was briefly washed in phosphate-buffered saline then fixed in 4% paraformaldehyde in phosphate-buffered saline (0.01 M) for 30 min with gentle agitation. After fixation, the tissue was washed in phosphate-buffered saline (3 × 5 min) and then transferred to a 15 mL tube filled with phosphate-buffered saline and washed (12 h) on a low-speed tube rotator. To prepare fixed SAN tissues for labeling, tissues were dehydrated, cleared using ethanol in dimethyl sulfoxide and bleached to reduce autofluorescence as described previously.35 Briefly, SAN tissue was dehydrated through a graded ethanol series , cleared for 2 h with dimethyl sulfoxide (20%) in ethanol and bleached for 12 h with hydrogen peroxide (6%) in ethanol. Tissue was then rehydrated through a graded ethanol series, washed in phosphate-buffered saline (3 × 5 min) and permeabilized with 0.5% Triton X-100 in phosphate-buffered saline (3 × 10 min). Nonspecific antibody binding was blocked by incubating SAN tissue for 2 h with 5% normal donkey serum in Triton X-100 phosphate-buffered saline. SAN tissue was washed in phosphate-buffered saline (3 × 10 min) and incubated for 48 h in a mix of primary antibodies in phosphate-buffered saline at 4°C . After primary labeling, tissues were washed in phosphate-buffered saline (3 × 10 min) and labeled with secondary antibodies for 4 h in the dark at room temperature . SAN tissue was then washed in phosphate-buffered saline (3 × 10 min) and incubated for 2 h in a dimethyl sulfoxide and phosphate-buffered saline mixture (1:4). Tissues were then mounted in Aqua-Mount mounting medium (Thermo Scientific) and cover slipped.After confirmation of deep anesthesia, beating hearts were immediately removed and transferred to warmed (37°C) Tyrode III solution containing , ImageJ/Fiji36, and Python (3.7). Both vascular  and myocyte  image volume color channels were thresholded to generate 3D binary masks. When necessary, CD31+ positive endocardium was manually removed from the image volumes using Imaris. Vascular and myocyte fractional volumes were calculated from 3D binary masks with custom-written Python scripts, with fractional volume = (number of pixels in the 3D mask)/. The 3D binary masks were used to calculate the 3D distance from each HCN4+ pixel to the nearest CD31+ pixel . Three-dimensional masking of HCN4+ and CD31+ volumes was repeated using either Imaris or Python and obtained similar results. For 3D masking in Python, The Allen Institute for Cell Science—Segmentation package (https://github.com/AllenInstitute/aics-segmentation) was used. For 3D vascular segmentation, the total vessel segments and length per field of view were calculated using Imaris. The diameters of 1°–4° vessels were manually traced in ImageJ/Fiji using CD31+ image volumes.Imaging of fixed SAN tissues was performed using an inverted confocal laser scanning microscope  with a 40× oil-immersion lens  and acquired using commercial software (Olympus FluoView). Images were acquired at 0.5 µm/pixel with a field of view of 320 µm. Image volumes consisted of between 80 and 120 m) 140 NaCl, 5.4 KCl, 1 MgCl2, 5 HEPES, 1.8 CaCl2, 5.5 glucose (pH 7.4 with NaOH). The SAN was pinned flat and superior and inferior regions identified. The SAN myocyte isolation described in this protocol is based on a modified version of the SAN isolation detailed by Fenske et al.37 Briefly, intact tissue pieces derived from the superior and inferior SAN regions (approx. 2–3 mm2 each) were bathed for 5 min at 36°C in Tyrode low Ca2+ solution containing (in mm): 140 NaCl, 5.4 KCl, 0.5 MgCl2, 0.2 CaCl2, 5.0 HEPES, 5.5 d-glucose, 1.2 KH2PO4, 50 Taurine (pH = 6.9 with NaOH). Tissue regions were then subjected to enzymatic digestion for 30 min at 36°C in Tyrode low Ca2+ solution (pH 6.9) containing: 9.43 U elastase, 0.89 U protease, 0.27 U collagenase B and bovine serum albumin (1 mg/mL). During enzymatic digestion, tissue segments were gently agitated every 5–7 min. Once the digestion was complete, tissue segments were rinsed twice with Tyrode low Ca2+ solution (pH 6.9) and twice with an ice-cold Kraft-Brühe solution -N,N,N′,N′-tetraacetic acid, 10 mm glucose, pH 7.4 with KOH) and allowed to rest at 4°C in approximately 500 μL of the Kraft-Brühe solution. After 2–3 h at 4°C, the tissue was then warmed to 37°C for 5–10 min and mechanically dissociated using a fire polished glass pipette.Following euthanasia, SAN tissue was immediately dissected from the beating heart and placed in warmed Tyrode III solution, containing . After completion of Ca2+ reintroduction, cells were constantly perfused with Tyrode solution. The internal solution used for current clamp recordings contained (in mm): 125 K-Aspartate, 10 NaCl, 15 KCl, 1 CaCl2, 10 HEPES (pH to 7.2 with KOH). Before commencement of patch clamp recordings, 100 μm amphotericin B was added to the internal pipette solution and the internal solution was kept on ice. Fire-polished glass pipettes were first dipped in an amphotericin B-free internal solution and then back filled with the amphotericin B–containing internal solution.A drop of cell suspension was placed on a temperature-controlled recording chamber (35–36°C) and cells were allowed to settle and adhere to the chamber for approximately 10 min. Next, the external CaElectrophysiological signals were recorded using an amplifier  controlled with pCLAMP 10 software (Molecular Devices). Cells were initially patched in voltage-clamp and upon the formation of a giga-ohm seal, a short period of time was required (1–5 min) until amphotericin B permeabilized the membrane, allowing electrical access to the cell. Accessed cells were transitioned to current-clamp mode to record membrane voltage. All recordings were acquired at 10 kHz.38 The take-off potential was defined as the membrane potential when the first derivative of voltage with respect to time (dV/dt) reached 10% of its maximum value. The take-off potential was used as the time of an AP and to calculate both AP frequency (Hz) and inter–AP interval (ms). The maximum depolarizing potential was defined as the most negative membrane potential between APs. The diastolic duration was defined as the interval between the maximum depolarizing potential and take off potential. The early diastolic depolarization rate was estimated as the slope of a linear fit between 10% and 50% of the diastolic duration and the early diastolic duration was the corresponding time interval.All current-clamp recordings were analyzed using custom Python scripts for the detection and analysis of SAN myocytes AP parameters as previously described.To detect subthreshold membrane fluctuations, we used the “threshold search” function of Clampfit (Molecular Devices). A subthreshold voltage fluctuation was defined as an event that had an amplitude larger than the mean + 3 standard deviations of baseline voltage and a duration of at least 1 ms, but failed to exceed the experimentally determined AP take-off potential  of –29 mV.2+]i. Fluo-4 (5 µm) was mixed with Tyrode solution and introduced to plated cells during Ca2+ reintroduction (described above). Cells were loaded for 10 min, followed by a de-esterification period of 10 min. Temporal fluorescence fluctuations caused by [Ca2+] transients were acquired using the line-scan mode on an inverted confocal laser scanning microscope (Olympus FluoView 3000) with a 60× oil-immersion lens . Fluo-4 was excited with a 473 nm solid-state laser . Line-scan images were analyzed using ImageJ. Background subtracted fluorescence signals were normalized by dividing fluorescence at each point (F) with the baseline fluorescence (F0). Ca2+ sparks were identified automatically using the SparkMaster plugin of ImageJ.39After plating single SAN myocytes on the recording chamber, cells were loaded with the membrane-permeable acetoxymethyl-ester form of Fluo-4  for measurement of i transients at a frequency higher than 2 Hz.Analysis of whole-cell [Caectively . The dis2+]i transients was similar in superior  and inferior  SAN myocytes  was longer than that of inferior SAN myocytes .To determine the rate of clearance of intracellular Caransient . We foun2+ sparks before diastolic depolarization in both superior and inferior SAN myocytes. 2+]i in 2 regions of interest. The amplitude histograms of these diastolic Ca2+ sparks  and inferior  SAN myocytes (P = 0.57). Notably, the higher amplitude Ca2+ sparks are significantly larger in inferior (1.2 ± 0.02) than in superior (0.9 ± 0.01) SAN myocytes (P < 0.0001). This analysis is consistent with the view that there are 2 types of Ca2+ sparks in superior and inferior SAN myocytes.Next, we examined diastolic Ca+ sparks  and C co2+ spark rate ranged from 0.6 to 10.9 events/100 µm*s and 0.3 to 40.7 events/100 µm*s in superior and inferior SAN myocytes, respectively  in superior and 2.1 events/100 µm*s  in inferior SAN myocytes (P = 0.19). These Ca2+ spark amplitude data indicate that SR Ca2+ release sites vary in activity and amplitude between regions of the node, likely reflecting differences in ryanodine receptor cluster sizes and/or SR Ca2+ load .Caectively . The medP = 0.67). Together, this analysis suggests that the geometries as well as clearance mechanisms of Ca2+ spark are similar in superior and inferior SAN myocytes.The spatial spread .To address this issue, we simultaneously recorded membrane potential and [Caecording . Visuall+ sparks . To test2+ spark produce little to no membrane potential changes between APs, is the pattern of Ca2+ sparks leading to an AP different between the superior and inferior regions? To examine this, we asked if the number of Ca2+ sparks or the slope of Ca2+ sparks approaching an AP was different between superior and inferior SAN cells . Likewise, we found a significantly steeper slope in Ca2+ spark accumulation before each AP in superior versus inferior SAN myocytes .If individual CaAN cells . Interes2+ sparks to membrane potential depolarization is weak throughout the SAN, there is a regional difference in the number and rate of accumulation of Ca2+ sparks before an AP. With the superior region receiving a stronger driving force of Ca2+ sparks leading to AP generation. This could be a mechanism contributing to the higher spontaneous AP frequency of superior versus inferior myocytes.This analysis indicates that while the coupling of individual CaWe generated high-resolution 3D maps of the vasculature and SAN myocytes across the entirety of the mouse SAN. Our data show that vascular and myocyte densities vary regionally along the SAN. The superior SAN is highly vascularized, creating a redundant network whereby all pacemaking myocytes are near at least 1 vessel. This affords a high surface area for exchange and minimizes the diffusional distances in a region of the node where myocytes have highest intrinsic firing rates. In contrast, vascular and myocyte densities are lowest in the inferior SAN. The inferior SAN is also where myocytes with the lowest inherent AP firing rate are located. Thus, the SAN seems to be designed so that vascular density varies regionally to match the electrical signaling modalities of the myocytes that populate this heterogenous tissue. An important implication of these findings is that regional variations in blood supply could be a key factor determining the site of AP origin in the SAN as well as its firing frequency.Based on our detailed vascular and myocyte anatomical and functional data, we propose a new, objective identification of the superior and inferior mouse SAN. Our data indicate that SAN myocyte-to-vessel distance is relatively short and remains relatively similar from the initiation point of the SAN artery until about 3.25 mm toward the inferior section of the node. We call this region the superior SAN. We propose that the starting point of the inferior SAN is set at the lowest point of the superior node, where there is a relatively sharp, nearly 1.5-fold increase in myocyte-to-vessel distance. Using this framework, the superior and inferior SAN account for about 60% and 40% of the mouse node, respectively.An interesting observation in our study was that while the diameter of the SAN artery decreased nearly 20% from the superior to inferior SAN, the diameter of 2°–4° vessels did not. Thus, differences in vascular density are not due to fundamental differences in the structure of microvessels, rather their number.23 involving rat hearts. They found that, although the superior and inferior sections of the node have the capacity to fire action potentials, the superior node has the fastest firing rate and hence functions as the dominant pacemaking center in of the SAN. The rat inferior SAN was capable of becoming the dominant pacemaking center, but only when the superior node was electrically silenced. Although the concept of a functional heterogeneous SAN has been proposed before, our study, in combination with Brennan et al.,23 supports a model where heterogeneity encompasses all elements of nodal function, ie, vascular, neuronal, and myocyte, but in 2 distinct regions of the node, at least in rodent hearts.Our work is consistent with a recent study from the Efimov lab45 At low firing rates , ATP levels in neurons remain relatively stable, suggesting a balance between ATP synthesis and hydrolysis. However, increasing AP frequency (>10 Hz) leads to rapid ATP depletion within seconds of the onset of high-frequency stimulation as ATP consumption exceeds production.46What is the physiological significance of the reciprocal association of high excitability with high vascular density in the SAN? Our study, in combination with others, provide a potential answer to this difficult question. A reasonable hypothesis is that vessel density and hence blood supply scales up with tissue metabolic needs. It is estimated that a neuron consumes about 7–9 million ATP molecules during a single AP.Our electrophysiological data suggest that the intrinsic AP frequency is about 3.9-fold higher in superior versus inferior SAN myocytes. Although similar ATP consumption studies like those in neurons have not been performed in SAN myocytes, assuming that energetic cost of firing an AP is likely similar in these cells, our data suggest that in terms of APs alone, ATP consumption could be 3.9-fold higher in superior compared with inferior SAN myocytes. Note, however, that a relatively large fraction the inferior SAN myocytes do not produce APs. Thus, the energetic differential between superior and inferior SAN myocytes could be even larger than 3.9-fold in these cells.24 Additional ATP is consumed by myosin-actin crossbridge cycling and the constant generation of cytosolic cAMP by adenylate cyclase.25 The latter is significant as our data show that even the fastest firing superior SAN myocytes produce APs at a lower rate than the heart rate of a mouse, which is about 12–13 Hz.48 This disparity between in vivo and isolated single cell firing rates reflects strong input from the sympathetic arm of the autonomic nervous system, which increases firing rate through the activation of cAMP/protein kinase A signaling. Indeed, recent studies by Brennan et al.23 and Hanna et al.49 suggested that sympathetic innervation and drive is larger in the superior compared with the inferior SAN. Thus, a combination of higher AP firing, Ca2+ cycling, and contraction rates as well as cAMP production may conspire to produce a much larger energetic load in superior than inferior SAN myocytes. In combination with our vascular mapping, this implies that—as originally proposed Krogh50 for skeletal muscle—microvessel density scales up with the energetic needs of SAN myocytes.Although neurons and SAN myocytes have the capacity to spontaneously fire APs, their energetic needs are likely quite different. As in neurons, these include ion transport across the sarcolemma and intracellular organelles such as the endoplasmic reticulum.2+ sparks and membrane potential is relatively weak, but similar in superior and inferior SAN myocytes. However, the number of Ca2+ sparks immediately before an AP was larger in superior than inferior SAN myocytes. Thus, increased SR Ca2+ release could contribute to faster diastolic depolarization rates leading to AP threshold in superior myocytes and hence higher firing rates. In principle, these regional variations in Ca2+ clock function could be the result of differences in the activity of Ca2+ channels, SR Ca2+ pump, ryanodine receptors, and/or Na+/Ca2+ exchanger. Future experiments should determine whether regional differences in the function and expression of these proteins as well as HCN51 and Ca2+ currents contribute to differential electrical firing patterns across the SAN.Although performing a detailed analysis of the electrophysiological properties of superior and inferior SAN myocytes was beyond the scope of this study, our data provide insights into this interesting issue. We found that the coupling strength between Ca2+ pump inhibition with thapsigargin eliminated AP firing, but not subthreshold voltage fluctuations in superior and inferior SAN myocytes. In combination with our finding that Ca2+ sparks are weakly coupled to changes in membrane potential, this suggests that SR Ca2+ release may be necessary, but not sufficient to trigger an AP in superior and inferior SAN myocytes. Furthermore, our data suggest that subthreshold voltage fluctuations are unlikely produced by Ca2+ spark activation of Na+/Ca2+ exchanger currents. In a follow-up study, we will investigate the molecular identity of the proteins underlying subthreshold voltage fluctuations.An interesting finding in this study is that SR Ca52 and Bychkov et al.15 The model is illustrated in the graphical abstract of this paper. In this model, all SAN myocytes function like a bistable system, alternating from the maximum diastolic potential to a full AP. SAN myocyte in the sparsely vascularized inferior section of the node fires stochastic subthreshold voltage fluctuations and sporadic APs. These events do not lead to periodic pacemaking on their own. However, when coupled to a more periodic voltage oscillators such as superior SAN myocytes, subthreshold voltage fluctuations and rare APs integrate  and increase the probability that superior SAN myocytes reach AP threshold. Thus, inferior SAN myocytes likely increase the strength and periodicity of superior SAN activation and hence pacemaking activity. Stochastic resonance models have also been implicated in the periodic activation of APs in neurons.54 A key new insight provided by our data is that the capacity of specific regions of the node to operate as periodic oscillators or stochastic signal generators could be critically dependent on vessel density and hence blood flow. Thus, blood supply could effectively determine the dominant pacemaking site within the SAN.Finally, we formulate a new model for pacemaking activity that integrates our functional and anatomical mapping with the stochastic resonance model of SAN firing proposed by Clancy and Santana


The primary goal of this study is to be able to discern specific types of consumers in terms of their psychosocial characteristics who may need different ways of receiving dietary advice. Knowing these types will enable a better fit of advice to consumers’ psychosocial characteristics, hereby stimulating healthy eating as the probability of compliance to the advice can potentially increase. The study draws upon several psychological theories to distinguish unique underlying factors that can subsequently be used to personalize nutrition information for consumers. A number of general psychological scales  are filled out by 988 respondents, including their preferences for receiving personalized forms of nutrition advice. The set of joint items from various psychological constructs is analyzed using a Principal Component Analysis to find underlying psychological characteristics. The PCA produces four components (explaining 51% of variation), that could be interpreted as ‘intrinsic interest and capabilities for healthy eating,’ ‘perceived difficulty to eat healthily,’ ‘self-worth insecurity,’ and ‘seeking positive challenges,’ respectively. By means of a Logistic Regression these components are able to predict preferences for different forms of receiving nutrition advice. This first component shows that a mind set for maintaining a healthy diet goes together with an interest in receiving an advice on what do to and on how that will affect one’s health. The second component predicts a preference for a fixed moment to receive information/advice. This may be a strategy of those that perceive difficulties to eat healthily, to help them control their healthy food intake. The insecurity that the third component models seems to lead to a wish for receiving specific advice about their health situation at fixed moments in time. The fourth component is a small component, therefore its prediction of a wish for an advice focussing on prevention of negative consequences is probably not a strong result. The study does point out that there appear different psychosocial types of consumers, that may benefit by being addressed according to their preference for receiving nutrition advice on specific moments, of a specific level of detail or pointing at the type of consequences the advice has. A better fit of the advice to the psychosocial characteristics of the recipient, captured in the identified components in the current study, may lead to an increase in compliance, although that will have to be further investigated in subsequent work. Many people in the Western world currently have an unhealthy lifestyle, which in part is the result of consumers’ diets which overall are relatively unhealthy. For instance because they consume too much meat, have a high (saturated) fat intake, and consume too little fruits and vegetables . Such unNutrition information can be personalized in different ways. A distinction can be made between a biological/medical basis on the one hand and a behavioral/psychological basis on the other hand . The majBehavioral or psychological approaches to personalize nutrition information have been more scarce. Many behavior change techniques used in this field originate from psychological theories . PsycholWhile each theory will have its own merits, there is overlap between the psychological theories in the sense that they draw on either individual cognitions, capabilities and motivation, or on contextual and/or social variables . This ovA meta-analysis of We stress that our approach is of an exploratory nature, and not hypothesis-driven. We include a set of scales based on them potentially capturing relevant psychological traits, not based on theoretical considerations about what traits may cause a specific type of consumer to prefer specific types of food and health related information.self-regulation, self-efficacy (the extent to which one feels capable of performing a certain behavior) and social comparison in our study. Within the Health Action Process Approach model  in the current study.Personalized advice can increase the effect of a message by achieving a fit between a person and a message. According to the Elaboration Likelihood Model ELM; , informaneed for cognition), whereas an affective message is more persuasive among affect-oriented individuals . Similar effects were demonstrated by need for cognition and need for affect can be effective in stimulating people to make more healthy food choices. Therefore, we included the constructs need for affect and need for cognition in our study.promotion or prevention focus  and a mean age of 46.2 years ranging from 18 to 75 years. These, and some more, demographics can be found in An online survey was conducted in August 2018 under 1,013 respondents in the Netherlands. The survey was administered by a professional market research company (MSI-ACI Europe BV). The respondents were approached by email. As the data was gathered anonymously and only average scores were used in the analyses, no formal ethical approval needed to be officially obtained. At the start of the study participants were informed about the global goal of the study namely, to map individuals’ choices and opinions about personalized nutrition advice. The participants were explained that they could refrain from filling out the survey at any moment and could quit without providing any reason for their withdrawal. To ensure a nationally representative sample, respondents were quota-sampled based on gender, age, highest level of completed education and income. Due to a lack of variation in their responses, 25 respondents were excludedThe questionnaire started with a measurement of the psychological characteristics. Respondents then continued with several multiple choice questions which measured their stated preferences for receiving certain forms of personalized nutrition and health advice or information. Finally, the demographics were assessed.Validated scales were used to measure the various constructs. When necessary, the items were translated to Dutch using back-translation and reversed items were framed positively, because reversed items have shown to be able to lead to measurement problems like unduly complex factor structures cf. .Self-regulation with regard to healthy eating was measured using a 5-item scale developed and validated by Action self-efficacy with regard to healthy eating was measured with 7 items , based on Social comparison was measured with a 10-item questionnaire from Intrinsic motivation to eat healthily was measured with 6 items that assessed self-reports of interest and enjoyment of healthy eating  were deleted from the analyses see also . The 2ndA Principal Component Analysis (PCA) was conducted on the 68 items in the survey (using SPSS v.23), which resulted in a four dimensional solution on which a Varimax rotation was performed. The scores for this four dimensional model are used to predict reported preferences for three different forms in which dietary health information can be provided to people. These predictions have been carried out separately by means of three logistic regression analyses (using SPSS v.23). The reason to carry out three separate logistic regressions, rather than combining these into one model in order to include interactions between dependent variables is rather practical. One of the aims of this study was to explore the possibility to use the psychological profiles obtained to provide tailored dietary advice in a specialized computer program (ultimately in a smartphone-app). The three dependent variables are linked to matters that could be separately implemented in such an app. Furthermore, in hindsight, the correlations between the three variables turned out to be very low .post hoc rotation, this would have yielded a 12 component model with the component 5 through 12 together containing 12% variance). Visual inspection of the scree graph (see The amount of variance in the four component (correlation) PCA model is 51% and the choice for this four component model is not based on a formal criterion like the eigenvalue > 1 criterion  shows items that concern an intrinsic interest in healthy eating, combined with the will (and capability) to maintain a healthy diet. The first component (18% VAF), coined ‘perceived difficulty to eat healthily.”The second component (13% VAF) contains all items regarding ‘coping self-efficacy’ . These iself-worth insecurity.’ It contains the ten items from the ‘social comparison’ scale, three ‘emotion avoidance’ items from the ‘need for affect’ scale and one ‘prevention focus’-item from the ‘regulatory focus’ scale. Most items loading on this component (loadings over 0.47) point to a social comparison orientation and an avoidance to experience emotions.The third component (12% VAF) shows a general attitude which we interpret as a form of ‘seeking positive challenges.” Three other ‘prevention focus’-items also loaded on this component, however, we decided not to take this into consideration because they either had low loadings (below 0.38) or a similar (low) loading on more than one component.The fourth component contains a mere 8% of variance and contains the three items from the ‘need for cognition’ scale, the three ‘approach emotions’-items from the ‘need for affect’ scale, five ‘promotion focus’-items and one ‘prevention focus’-item from the ‘regulatory focus’ scale. These items have loadings over 0.43 on this component. Thus, the items in this component appear related to seek cognitive challenges, seek positive results and seek the experience of emotions. We interpret and label this component as “In summary, we have thus reified the four components as follows:(1)Intrinsic interest and capabilities for healthy eating,(2)Perceived difficulty to eat healthily,(3)Self-worth insecurity,(4)Seeking positive challenges.p < 0.05) results are indicated by italicizing the corresponding lines in the table.The three separate logistic regressions show that some of the four principal components predict some of the three dependent variables. The results of the three logistic regressions are given in Focus of advice (a preference to receiving advice pointing at ways to prevent negative consequences) shows a significant effect (p = 0.02) only for the 4th component , with an odd ratio of 0.80. An increase of the score on the 4th component results in a lowering of the probability of preferring to receive advice when it shows how to prevent negative consequences.The logistic regression predicting Information activity (a preference to receiving information on fixed moments) shows to be predicted by both the 2nd component  and the 3rd component . A higher score on the 2nd component results in a heightened (odd ratio 1.18) probability for a preference for receiving information on a fixed moment, analogously does a higher score on component 3 with an odd ratio of 1.35.amount of information  can be predicted by the 1st (p = 0.01) and 3rd (p = 0.00) component . A higher score on component 1 results in a 1.22 times higher probability of preferring detailed information, a higher score on component 3 in a 1.38 times higher probability of preferring detailed information.The third logistic regression shows that Personalizing nutrition information can be an effective manner to increase compliance and lead to consumers making more healthy food choices e.g., . Howeverintrinsic interest and capabilities for healthy eating, perceived difficulty to eat healthily, self-worth insecurity and seeking positive challenges.In the current study, we conducted a PCA which delivered four components: The first component  combines all the items from the action self-efficacy, self-regulation, intrinsic motivation and information processing scales. That the four scales jointly load on the first component in this study, hence correlate, can mean that they measure the same underlying construct in the context of food and healthy eating. Scores on this component increase as respondents have a higher intrinsic motivation and a higher involvement in eating healthily . These items all stem from the same self-efficacy scale . That thdifficulty to perform healthy dietary behaviors, whereas the items in the first component focus on positive motivations and capabilities with regard to eating healthily. And as mentioned before, reversed items often result in a separate component  point to psychosocial characteristics of a type of individual that compares his/her behavior to others in order to judge his/her own behavior, and at an avoidance to experience emotions. One reason why people might structurally compare themselves to others is that they are insecure about themselves  and haveThe fourth component  combines items from the ‘need for cognition’ scale, the ‘approach emotions’-items from the ‘need for affect’ scale, and ‘promotion focus’-items and ‘prevention focus’-item from the ‘regulatory focus’ scale. The latter two sets of items are not easy to reconcile as this component seems to combine a promotion focus with a prevention focus, which are traits believed to be not in line with each other. We must again stress that the fourth component represents a rather low amount of variance in the data, so it is does not represent a very strong force to explain this result. Nevertheless it appeared interpretable as ‘seeking positive challenges’, and we have included it in the prediction models.We observe that the first two components are food related components. Component 3 and 4 are related to general psychosocial characteristics, which are not specifically related to food. This suggests that, in these results, answers to the food related items are unrelated to answers to general items, as the four components are uncorrelated .This first component  shows up as predictor of the ‘information amount’ in the logistic regression. A mind set for maintaining a healthy diet seems to go together with an interest in receiving an advice/information on what do to, together with information on how the advice will affect one’s health, compared to only receiving an advice. So individuals who have intrinsic interest and capabilities to eat healthily prefer extra information on why an advice is good for their health in addition to receiving dietary advice.The second component , tallies with it predicting a preference for a fixed moment to receive information/advice. This may be a strategy of those that perceive a difficulty in themselves, to help them control their healthy food intake. They may see themselves unable to cope with a freedom to find information on their own account, hence making it more likely that a form of advice is chosen that is received on a fixed moment.Component 3 (‘self-worth insecurity’), together with the first component, predicts a preference to receiving information/advice together with instructions about what to do and how it relates to one’s health. Component 3, together with the second component, also predicts a preference for a fixed moment to receive information/advice. The insecurity of the individuals scoring high on this component seems to lead to a wish for specific information/advice about their health situation at fixed moments in time. The social comparison part of the third component, seems to be in line with wanting to receive specific information and instruction with the advice, as the advice comes from others, which enables a social comparison.The fourth component negatively predicts the preference for advice with a focus on preventing negative consequences. As this component models ‘seeking positive challenges’, this seems to make sense, with the proviso that the merger of both a prevention and a promotion focus appears strange. Note that this component contains a mere 8% variance in the data, so this component’s size is not very large, which may lead to the possibility that it combines small effects, despite their being theoretically not compatible.That four scales, adapted to focus on food and health, together make up the first component suggests that these four underlying constructs merge when seen in the context of food and health. Intrinsic motivation, self-efficacy, information processing and self-regulation appear to correlate highly when seen in the context of food and health. This points at the possibility to reduce the set of items in future surveys designed to probe relevant psychological parameters of a sample of respondents, in a food and health context.One potential limitation of the study is that for the ‘preference for feedback’ constructs single-item measures were used. However, in many cases single-item measures perform just as well as multiple-item measures . Since tFurthermore, though we aimed to keep the survey concise, potentially part of the respondents could have experienced a sense of respondent fatigue. Typically, an average maximum length of 20 min is recommended for a survey before respondent fatigue becomes an issue . This waFor the current study, an online survey was administered relying on self-reports from participants. This can have certain disadvantages as is discussed by This study shows that it is possible to use psychological characteristics to predict the way consumers would like to receive advice/information about their health status (and diet). It also points out that there are different personalities of consumers, that may benefit from being addressed according to their preference for receiving advice/information on specific moments, of a specific level of detail and pointing at the type of consequences the advice has. When developing a computer program  it is advisable to tailor the way health advice is provided to the type of consumer. The latter can be assessed by, e.g., asking the user of the app some questions upon installation of the app. The long survey in this study is not suited for this, but the PCA points at a way to reduce the length to a low number of questions.Compliance to an advice was not addressed in this study, obviously that is the ultimate ‘proof of the pudding’ that needs to be further investigated, possibly with the survey presented here as an instrument for tailoring advice. Based on the four component PCA model a reduction of items can be achieved, to in a more concise manner personalize nutrition advice. Of course this basically means the construction of a new psychological scale, specifically focused at ‘food and health’-related psychological characteristics. Such a scale will need to be studied and analyzed in detail before it can validly be applied in subsequent studies in this field.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.