On the other hand demonstrated therapeutic effects suggest a necessity of rethinking our pharmacokinetic understanding. In this light this review discusses the value of plasma levels as pharmacokinetic surrogates in comparison to the more informative value of tissue concentrations. Furthermore the need for new methodological approaches is addressed like the application of combinatorial approaches for identifying and pharmacokinetic investigations of active multi-components. Also the physiological relevance of exemplary in vitro assays and absorption studies in cell-line based models is discussed. All these topics should be ideally considered to avoid inaccurate predictions for the efficacy of herbal components in vivo and to unlock the “black box” of herbal mixtures.Medicinal plants represent a big reservoir for discovering new drugs against all kinds of diseases including inflammation. In spite the large number of promising anti-inflammatory plant extracts and isolated components, research on medicinal plants proves to be very difficult. Based on that background this review aims to provide a summarized insight into the hitherto known pharmacologically active concentrations, bioavailability, and clinical efficacy of boswellic acids, curcumin, quercetin and resveratrol. These examples have in common that the achieved plasma concentrations were found to be often far below the determined IC Chronic inflammation is associated with many chronic diseases like arthritis, asthma, atherosclerosis, autoimmune diseases, diabetes, allergy, cancer, and conditions of aging . MoreoveInflammation is a result of activated cellular elements and the existence of various biochemical mediators like cytokines , interleukin-(IL)-1β, IL-2, IL-6, IL-8), kinases , extracellular-signal regulated kinases (ERKs), mitogen-activated protein (MAP) kinase), transcription factors  and matrix metalloproteinases (MMPs). Also the pro-inflammatory activity of lipid derived mediators e.g., the leukotrienes formed by lipoxygenases (LO) and prostaglandins by cyclooxygenases (COX) are well known. NF-κB is considered to be the chief regulator of the inflammatory response as it regulates the transcription of inflammatory cytokines like IL-1β, IL-2, IL-6, IL-8 and TNFα along with the genes encoding for COX-2, inducible nitric oxide synthase  and cell adhesion molecules. Typically NF-κB is localized to the cytoplasm by the inhibitory protein IκB. Upon stimulation IκB protein is phosphorylated by IκB kinase (IKK), ubiquitinated, and degraded. This allows the free NF-κB to accumulate in the nucleus, where it can activate transcription . In factBoswellia serrata and plant-derived compounds like curcumin, quercetin, and resveratrol, are the subject of hype in the media, even in scientific circles, for their anti-inflammatory potential  that may be taken as an example for a well-studied isolated compound from medicinal plants  rhizome and represents the main component (~77%) of curcuminoid mixture along with demethoxycurcumin (~17%) and bisdemethoxycurcumin (~3%). In general curcuminoids in turmeric rhizomes range from 3% to 5% of dry weight and may vary depending on the geographical conditions . Ba. Ba58]. 50 1–10 μM), NF-κB signaling ((IC50 4–11 μM) and diverse kinases e.g., Janus kinase (JAK)3 (IC50 2 μM) in THP-1 activated human monocytes and LPS-activated murine macrophages RAW246.7 leading to a reduction in transcription and expression of TNFα [In several in vitro studies quercetin proved inhibitory actions on a large number of targets including RaF/MEK1/ERK , COX-1 (IC50 15–20 μM), 15-LO (IC50 25 μM), mitogen-activated protein kinases  (IC50 25 μM), and NF-κB signaling ((IC50 30 μM) [Similarly several potential anti-inflammatory mechanisms of action have been proposed for resveratrol including inhibition of 5-LO (IC0 30 μM) .In general quercetin occurs in the plants in form of its glycoside with different attached types of sugars. Before they can be absorbed they are hydrolyzed by lactase-phlorizin hydrolase (LPH) enzyme to the aglycone, which is then passively absorbed into the enterocyte. Alternatively the glycosides can be actively transported by sodium-dependent glucose transporter (SGLT 1) into the enterocyte, where they are hydrolyzed by cytosolic β-glucosidase. The resulting aglycones then either passively diffuse into the hepatic portal vein or undergo phase I and II metabolism to produce glucuronidated, sulfo-substituted and methylated forms, which are transported via ABC transporters into the hepatic portal vein. The absorbed aglycones bound to serum albumin and the metabolites are transported to the liver. In the liver the remaining aglycones undergo phase I and II metabolism resulting also in the formation glucuronidated, sulfo-substituted and methylated forms, which are transported along with the intestinal metabolites into the systemic circulation for distribution to body tissues . AvailabThe administration of 151 mg quercetin-3-glucoside capsule and 154 mg quercetin-4′-glucoside capsule resulted in similar peak plasma concentrations of 5.0 μM and 4.5 μM, respectively. A comparison of the absorption of quercetin from different food matrices  yielded the highest absorption rate of 0.74 µM for fried onions and 0.3 µM for the other quercetin sources. Moreover the accumulation of quercetin conjugates in plasma could be shown following the periodic ingestion of onion. Hence the concentration of glucuronide and sulfate metabolites increased from 0.04 μM to 0.63 μM after the consumption of 96.3 mg quercetin per day from onion slices over one week. The effective dose of quercetin supplement reported to have beneficial anti-inflammatory effect was found to be 500 mg . EnhanceO-sulfate and two monoglucuronides were detected at maximal concentrations of 3.7–14 μM and 0.9–4.3 μM, respectively [O-sulfate, reached considerably higher plasma levels (18 μM) compared with resveratrol (4 μM) [After resveratrol is orally ingested 77–80% is absorbed in the intestine . Neverthectively . Besidesectively . Also inl (4 μM) . Severall (4 μM) .Since quercetin and resveratrol are mainly used for their anti-oxidative effects, clinical studies addressing the anti-inflammatory properties are rare. Nevertheless it can be concluded from the clinical data available up till now, that the anti-inflammatory effects observed with quercetin in vitro could not be supported by studies in humans and generally remains unclear ,80,85.In case of resveratrol contrasting results were obtained. Hence a significant increase in TNFα was observed in ten healthy volunteers 24 h after the intake of a single dose of 5 g resveratrol . In a diThen again another study based on the administration of 75 mg/day resveratrol for 12 weeks to non-obese postmenopausal women with normal glucose tolerance did not observe any change in inflammatory markers .A possible reason for the contrasting results could be that low doses or a single but higher dose exerts no effect, whereas a moderate continuing intake seems be more effective .the role of quercetin and resveratrol conjugates as well as the respective parent compounds in the mediation of the pharmacological effectspossible activation of metabolites by glucuronidases and/or sulfatases in the tissuesachievable tissue concentrations of the active substancesAll in all quercetin and resveratrol are further examples of well-studied substances in vitro that are poorly bioavailable because of extensive metabolism to glucuronidated or sulfated metabolites. Interestingly the plasma concentrations of the metabolites exceed that of the parent compound several folds, giving rise to the question of:50 values for the here mentioned exemplary anti-inflammatory herbal components is shown in For a better overview a rough simplified excerpt of the relations between different inflammatory mediators visualizing the respective points of attack and the corresponding IC50 values  determined for most of the putative pharmacological targets in vitro. Moreover these substances as well as their conjugates are highly bound to plasma proteins, reducing further the free unbound drug concentration [All the above examples have in common that the achieved plasma concentrations of the tentative active ingredients were found to be far below the ICntration . At the According to our traditional understanding of pharmacokinetics, it is assumed that pharmacological activities of drugs are closely related with the respective free unbound drug concentration in plasma . Excelled). This is reflected for example by Vd values of 1.8 L/kg or 3.7 L/m2 determined for resveratrol and quercetin, respectively [d of 9.1 L/kg [Boswellia serrata extract to rats with the highest concentration determined for βBA corresponding to 2.33 μM, falling thus in the range of pharmacologically active concentrations. Compared to KBA and AKBA with a brain-to-plasma ratio of 0.2 and 0.4, respectively, βBA yielded a brain-to-plasma ratio of 1, indicating facilitated blood-brain permeability due to its greater lipophilicity [In the blood stream, drugs are transported partly in solution as free (unbound) drug and partly reversibly bound to blood components . The extent of drug distribution into tissues after entering the systemic circulation depends on the degree of plasma protein and tissue binding. For all the mentioned compounds protein binding dominates leading to a small apparent volume of distribution  explains that plasma protein binding is a rapid equilibrium process allowing a constant concentration of free drug, and in the absence of energy-dependent process, this free drug concentration in plasma is the same in tissues and extracellular fluids at steady state. The other main principle of the free drug concentration theory is that only free drug can reach the site of action, and therefore the free drug concentration is what drives the pharmacological effect of a substance .Meanwhile it seems to be more and more recognized, that the drug concentration taken by the cells in the presence of extracellular binding proteins could be substantially higher than expected based solely on the free drug concentration. This additional increased uptake of highly bound drugs might be driven by the so-called albumin-facilitated or protein-facilitated uptake . The exaIn this sense binding of the poorly water-soluble resveratrol to plasma proteins like lipoproteins, hemoglobin, and HSA is essential for its body distribution and bioavailability. It could be shown that besides a passive diffusion influx, resveratrol enters the liver cells by an active process involving OATPs that bind the resveratrol-albumin complex and deliver resveratrol in a similar way to the fatty acid uptake .There are also indications from commonly used synthetic drugs, such as benzodiazepines, and steroids that demonstrate higher concentrations in the CNS, than their unbound fraction would suggest . In caseTranslating these observations to plant research, this would mean that in contrast with the previous assumption, that protein binding might reduce the free unbound pharmacologically active form, binding to albumin reveals to be essential for the delivery of the bound form at the cell surface, facilitating consequently membrane uptake to finally produce its biological effect . TherefoAnother important issue that should be taken into consideration when explaining the therapeutic effects of herbal substances with unfavorable plasma levels is the role of β-glucuronidases and sulfatases localized in tissues. They might release active herbal substances from its glucuronic acid and sulfate conjugates, promoting thus their pharmacological activity in tissues.3H-labelled resveratrol to rats via gastric gavage [This hypothesis may be underlined by the results of a distribution study following the oral administration of c gavage . Hence tc gavage .Similar observations were made with curcumin. Following the administration of curcumin to mice curcumin glucuronide was the primary metabolite detected in serum accounting for 99.7% of total curcumin. In contrast the aglycone curcumin was the major metabolite detected in bones accounting for 69.4% of total curcumin. Hence the aglycone concentration in bones was 6.4-fold higher than in the circulation. This might be attributed to the high deglucuronidation activity known for bone marrow cells .Inflammation might even increase the glucuronidase deconjugation activity. This was shown in mouse plasma after the i.v. injection of lipopolysaccharide (LPS), where the β-glucuronidase activity increased with time as did the levels of inflammation marker, TNFα, and soluble intercellular adhesion molecule-1. In agreement with this the peak of luteolin monoglucuronide decreased to about half and the ratio of luteolin to luteolin monoglucuronide increased in LPS treated rats .Quercetin glucuronides were also shown to be deconjugated in vitro in cultured macrophages and in homogenates from human liver and small intestine . FurtherAll together these examples show, that although the glucuronide and sulfate conjugates are in general less active, they may be easily converted back to the non-conjugated forms, acting thus as a depot for drugs in free form within target tissues. Hence the pharmacological activity of herbal substances is also linked to the kinetics of the conjugates, the reason why studying the pharmacokinetic behavior of the conjugates should receive more attention in the future.For other drugs the concentration in tissue may also provide better correlation to therapeutic effects than plasma concentrations. This is especially true for locally acting compounds in the skin as e.g., melatonin and secosteroids.Besides being a hormone controlling the sleep-wake cycle in human, melatonin has been identified in about 120 plant species in variable concentrations. Of particular interest are its cutaneous pleiotropic effects as a consequence of its local concentration and metabolism occurring directly in the skin at the site of action . CirculaThe same holds also for the hydroxymetabolites of the secosteroids vitamin D3 (D3) and lumisterol (L3), which like melatonin and melatonin metabolites also protect the skin from or reverse UV-B induced damage. This is achieved by downregulating pro-inflammatory responses via suppression of NFκB activity and its downstream cytokines including TNFα and INF-γ, inhibition of IL-17 production, and stimulation of keratinocyte differentiation . As in cThe determination of the tissue-to-plasma ratio therefore represents a much more conclusive surrogate for the pharmacological activity of herbal substances than the plasma levels. In fact, the importance of tissue binding cannot be neglected, as 98% of all proteins likely to contain binding sites for drugs are located extravascularly. However the difficulties inherent to studying the interactions with the proteins of fixed tissues hinders the readiness to carry out these essential investigations in contrast to the predestined plasma pharmacokinetics.Apart from tissue distribution studies carried out in vivo in animals the simplest way to simulate binding competitions between tissue components and plasma proteins is a simple in vitro technique named distribution dialysis that has been established long time ago ,122. In Over the years other techniques were utilized in vivo to gain insight into the pharmacokinetics at the site of action and estimating drug exposures in different compartments. A good overview on the individual techniques that may be applied for the different tissues, their advantages and disadvantages is given in the review of Rizk et al. . The mosP is used to model phospholipid partitioning and several equations are necessary to model neutral, acidic and basic drugs. A shortcoming of using log P calculated from the octanol:water partition coefficient (P) to represent phospholipid partitioning is the lack of orientation specific interactions with phospholipid membranes resulting in mechanistically unsound assumptions. By contrast depending on partitioning into microsomes (unsorted phospholipid vesicles) represents a great benefit, as it allows measuring interactions with all phospholipids for both charged and uncharged species in simple experimental settings leading to more accurate predictions  and the associated technical challenges quantitative measurements of time-dependent concentration profiles of bioavailable multiple components exceed the scope of commonly applied traditional research. This has led to significant limitations in understanding the efficacy of herbal medicines. In fact it is technically challenging to identify each metabolite and to assess the human response to exogenous substances in a complex network involving a large number of variables. Thus the majority of absorbed herbal components and their metabolites are often intermixed against the background of endogenous substances in biological samples. But recent advances in liquid and gas chromatography as well as capillary chromatography coupled with mass spectrometry  and nuclear magnetic resonance (NMR) made it possible to simultaneously detect various multiple components and their metabolites .P-values, and fold changes greater than two qualify potential unknown bioavailable components and their metabolites for further testing and identification. For these purposes numerous commercial and freely usable data-processing packages are available as well as multiple databases that are continuously improved [These are important prerequisites for metabolomic profiling techniques that have already clearly demonstrated their value in elucidating the interaction of the biological system’s genome with its environment and have been recently applied in the pharmacokinetic analysis of drugs, xenobiotics and several nutrients . In factimproved .Compared to the classical approaches metabolomic strategies have clear advantages, firstly in the capacity of handling a great number of variables allowing a shift from targeting analysis to profiling the complete set of exogenous substances and metabolites, and secondly, in the unbiased selection of variables that are significantly altered, which facilitates the discrimination between exogenous and endogenous substances. Moreover it is very useful in identifying significant bioavailable herbal components and their metabolites paving the way for the identification of pharmacologically active components of herbal medicines that have been not yet extensively studied. In fact several publications reported the use of metabolomics to capture both drug-derived and drug-induced metabolites . Due to Radix Astragali and Radix Glycyrrhizae) and the metabolic responses in healthy Chinese volunteers [The major objectives of applying metabolomics-based pharmacokinetic studies are to qualitatively identify and characterize the bioavailable herbal components “what is absorbed” and their metabolites “what is produced” and present their semi-quantitative time courses in the global subject metabolite pool following the administration of herbal medicines. An example for the successful application of phytochemical and metabolomic profiling coupled with multivariate statistical analysis is the simultaneous pharmacokinetic monitoring of several components of Huangqi decoction  may not always represent a natural physiological setting and thus may fail to provide exact predictions for the drug in vivo. In contrast, cells contained in human primary whole blood provide a physiologically relevant setting, as well as facilitate assessment of drug protein binding characteristics and the amount of free drug acting on the target. How big the influence of the experimental setting on the results obtained may be is demonstrated on the example of boswellic acids. Hence KBA and AKBA were found to block 5-LO with ICee assay . The mar2 compared with TEER values of 12 to 120 Ω cm2 in human small intestine tissue. This is attributed to the origin of the Caco-2 cells being derived from the large intestine and the smaller average junction pore radius of 3.7 Ǻ compared to 8–13 Ǻ for native human small intestine. In addition Caco-2 cell-based assays suffer from (a) weak expression of important intestinal metabolic enzymes such as cytochrome P450, (b) lack of the crypt-villus axis, which is important for fluid and ion transport, and (c) the absence of mucus producing cells. Another disadvantage of Caco-2 cell-based assays is that the high passages used in many laboratories induce variable expression levels of differentiation markers and transporters [Physiological relevance is also a very important issue when carrying out permeability studies. Hence Caco-2 cells obtained from colon carcinoma are considered the gold standard for drug absorption studies. Besides being used alone they are often applied in combination with other cell lines to investigate drug absorption, inflammation, nutrient uptake, and toxicity in the gut as well as in the frame of absorption, distribution, metabolism and excretion studies . Howeversporters . To compsporters .r2 = 0.91) compared with Caco-2 cells (r2 = 0.71). Moreover organotypic tissues allow to study the effect of a substance on isolated intestinal mucosa alone or together with immune cells or in combination with a liver-on-a-chip model rendering them a more holistic model for the investigation of drug absorption and metabolism in human gastrointestinal tract compared with Caco-2 cells. [A physiologically relevant in vitro intestinal model should ideally closely resemble the human intestinal epithelium structurally and phenotypically comprising the different cell types of the intestinal epithelium. In addition the model should be able to be cultured for long periods . Intesti2 cells. .Another important key element for evaluating drug availability at the site of action is the blood-brain-barrier (BBB) permeability for CNS active herbal components. Unfortunately in vitro experimental designs often fail to take into account binding to the brain tissue as a major factor influencing BBB permeability especially for lipophilic drugs . This miIn the future further attempts are expected to enhance the physiological relevance of in vitro assays. Even when screening pharmacological effects and absorption of compounds in two-dimensional (2-D) cell-based assays still represent the main pillar of today’s medicinal plant research, it has to be kept in mind that the in vivo complexity of three-dimensional (3-D) tissues cannot be fully recapitulated. It is therefore encouraging to see, that a lot of advances have been already made in the field of physiologically relevant in vitro models including the use of primary cells instead of cell-lines, 3D cell cultures, microtissues and organoids. Recent advances in nanofabrication techniques have led to the development of novel in vitro cell culture models that better mimic the in vivo cellular microenvironement by providing physiologically representative mechanical and structural cues to the cultured cells. Engineered 3-D tissue constructs have been already developed to simulate cardiac, musculoskeletal, skin, lung, kidney, liver, and adipose tissues that are capable of mimicking complex tissue physiology and functionalities . By and Boswellia serrata, curcumin, quercetin and resveratrol little is known about their target-site concentrations. This is however very important, because the therapeutic potential of herbal medicine is not only related to its pharmacological activity at the target, but also to its ability to attain an effective dose at the target site. The enormous lack of knowledge regarding biological dispositions is one of the major obstacles to understanding herbal medicines. Therefore novel approaches are needed in medicinal plant research that should focus on combinatorial approaches when evaluating herbal candidates like chemical and metabolomic profiling, ligand fishing and microfluidic techniques. Moreover the physiological relevance of the in vitro models applied should be usually kept in mind in order to avoid inaccurate predictions for the efficacy of herbal components in vivo. In future it is expected that tissue engineering in combination with big data and artificial intelligence will promote the use of combinatorial approaches. This is important as most of the plant components and metabolites likely work in a synergistic fashion or concurrently to give the plant extracts its therapeutic effects. Much effort should be also paid to profile the ingredients of herbal medicines to provide a profound base for pharmacological and bioavailability studies. But it is just as important to adapt the choice of lead substance serving as quality control of herbal extract to the insights gained from physiologically relevant pharmacological and bioavailability studies.Because of the complexity of mechanisms involved in most chronic diseases, it is highly questionable that a drug targeting a single gene or enzyme will be sufficiently effective in combating such diseases. Multiple target drugs are gaining thus significant importance. Botanical herbal therapeutics may represent an important source for new drug discoveries as they exert their therapeutic benefits via a multiple-component and multiple-target mode. However this requires much more knowledge about the pharmacological activity of these herbal therapeutics, which is often hampered by the superimposed complexeties of herbal medicines. Even for well-studied herbal plants and substances like


The Eph receptor tyrosine kinase member EphB6 is a pseudokinase, and similar to other pseudoenzymes has not attracted an equivalent amount of interest as its enzymatically-active counterparts. However, a greater appreciation for the role pseudoenzymes perform in expanding the repertoire of signals generated by signal transduction systems has fostered more interest in the field. EphB6 acts as a molecular switch that is capable of modulating the signal transduction output of Eph receptor clusters. Although the biological effects of EphB6 activity are well defined, the molecular mechanisms of EphB6 function remain enigmatic. In this review, we use a comparative approach to postulate how EphB6 acts as a scaffold to recruit adaptor proteins to an Eph receptor cluster and how this function is regulated. We suggest that the evolutionary repurposing of EphB6 into a kinase-independent molecular switch in mammals has involved repurposing the kinase activation loop into an SH3 domain-binding site. In addition, we suggest that EphB6 employs the same SAM domain linker and juxtamembrane domain allosteric regulatory mechanisms that are used in kinase-positive Eph receptors to regulate its scaffold function. As a result, although kinase-dead, EphB6 remains a strategically active component of Eph receptor signaling. Erythropoietin-producing hepatoma receptor tyrosine kinases (Eph RTKs) and their Eph-receptor interacting (ephrin) ligands serve as a communication conduit between cells, a function that is integral for the evolution of multicellular organisms . To dAll Eph RTKs have a common set of domains . The extUpon binding to their ephrin ligands, Eph receptors will form a heterotetramer composed of two receptors and two ephrin ligands, constituting the basic unit of an activated Eph receptor . This inThe formation of higher-ordered multimers promotes activation of the kinase-dependent and kinase-independent functions of Eph receptors in the cluster. The kinase-dependent functions involve phosphorylation of tyrosine residues in downstream effector proteins. Kinase-independent functions include the ability to act as a scaffold, whereby phosphorylated Tyr, Ser and Thr residues serve as recognition sites for adaptor proteins containing Src homology-2 (SH2) or phosphotyrosine binding (PTB) domains. Phosphorylation of these highly conserved sites can be accomplished by the tyrosine kinase activity of an adjacent activated Eph receptor or by the activity of other kinases recruited to the cluster. Together, these functions modulate the identity and intensity of forward signals initiated by an Eph receptor cluster.Pseudoenzymes are protein family homologs that have not retained the ability to catalyze a reaction due to the alteration of specific residues involved in cofactor and/or substrate binding. The incidence of these proteins is widespread, having been identified in approximately 20 protein families that include kinases, phosphatases and proteases . HigEphB6 is a protein that is expressed in all tissues and was shown to be important to maintain physiological homeostasis in areas such as the kidney , vascula897 [Accumulating evidence supports the ability of EphB6 to actively modulate cellular signaling pathways and be involved in the regulation of cellular responses. For example, several studies of EphB6 in human breast ,38 and l897 . EphB6 a897 ,37,44 an897 ,46 and p897 , neurobl897 ,49, colo897  and pros897  cancers,897 . In a st897 , the pro897  and colo897  tumors i897 . Togethe897 .Gallus gallus domesticus (chicken) species. Likewise, the intracellular domain of EphB6 in humans is 51.8% identical with that of human EphB1, yet is 58.2% identical with the intracellular domain of chicken EphB6. Therefore, comparison of human EphB6 with the EphB6 of other mammals and non-mammals allows for an opportunity to more precisely identify residues that have co-evolved with the loss of kinase function to confer the new kinase-independent mechanisms of mammalian EphB6.Phylogenetic analysis of EphB6 has identified that loss of kinase function is an evolutionarily “new” phenomenon by occurring solely in mammals . This fiIn this review, we postulate how mammalian EphB6 could function as a scaffold. To accomplish this, we will briefly describe what is currently known about the mechanistic action of kinase-active Eph receptors and how these functions are mediated at the amino acid/structural level and compare that to the amino acid sequence of both kinase-positive and kinase-negative EphB6 proteins from different species. With this analysis, we suggest that although mammalian EphB6 is kinase-negative, it has retained the allosteric regulatory mechanisms involving the juxtamembrane and the SAM domain linker that are used to regulate the kinase activity of kinase-active Eph receptors. Moreover, we suggest that the activation loop in EphB6 from placental mammals has evolved into an SH3 domain binding site whose conformational state is regulated by the activation state of the juxtamembrane domain. These analyses suggest that EphB6 has a switch-like mechanism of “active” and “inactive” states that regulate its ability to act as a scaffold that modulates functions of other Eph receptors in its cluster. How EphB6 modulates the activity of other receptors could involve an indirect mechanism by recruitment of adaptor proteins to the cluster that function to regulate the activity of kinase-active Eph receptors. Together, our analysis suggests that EphB6 is a modulator of Eph receptor cluster activity whose kinase-independent scaffold functions are precisely regulated.Tyrosine kinases utilize their kinase domain to catalyze the transfer of the γ phosphate of ATP to a tyrosine residue of a protein substrate. Kinases, in general, have a highly conserved architecture, being composed of two lobes designated the N-lobe and the C-lobe. The N-lobe is largely composed of β-sheet that, via several highly conserved regions, are used to bind to nucleotide and divalent cation cofactors. The C-lobe is primarily alpha-helical and houses both nucleotide and substrate binding sites. As shown in violet highlight coloring in A comparison of human EphB6 with kinase-positive human EphB receptors  identifiAlthough mammalian EphB6 houses several substitutions that have rendered it incapable of kinase activity, human EphB6 was shown to retain nucleotide-binding capability. A study by Murphy et al. (2014)  determinInterestingly, retaining the ability to bind nucleotide to induce protein conformational changes that are required to interact with other proteins is a common strategy employed by many pseudokinases that act as molecular switches ,19, inclIn addition to its ability to bind ATP, mammalian EphB6 has most likely retained the allosteric regulatory mechanisms involving the SAM domain linker and the juxtamembrane domain that are present in its kinase-active members.C-terminal to the kinase domain is the SAM domain linker; this linker region functions to allosterically repress the C-lobe of the kinase domain via an interaction that involves a set of highly conserved residues , except The molecular mechanism used to disrupt allosteric repression by the SAM domain linker is not known; however, a study by Kwon et al. (2018)  suggestsIn kinase-active Ephs, the juxtamembrane region, when non-phosphorylated, functions to prevent both substrate and ATP from gaining access to the kinase domain . It acco742 with the hydroxyl group of Ser768 located in the activation loop , yet a Tyr742 to Phe mutation did not . This suggests that the Tyr to Phe substitution in β6 of human EphB6 likely supports the maintenance of activation loop disorder in the absence of JX1 tyrosine phosphorylation and that mammalian EphB6 has likely retained the steric hindrance effect on the activation loop when the juxtamembrane domain is unphosphorylated.A study by Davis et al. (2008)  found thion loop . Therefo species . HoweverThe next question, however, is why would EphB6 in mammals have retained this mechanism to regulate ordering of the activation loop? Since the activation loop is used to prevent ATP and substrate from gaining access to the kinase site when a kinase-positive Eph receptor is inactive, why would this mechanism be maintained in a kinase-negative version? We postulate that the “activation sensor” mechanism that links the juxtamembrane domain with the activation loop in kinase-positive Eph receptors is used by kinase-negative EphB6 to regulate access to the activation loop because it was repurposed into an SH3 domain binding site that is used to mediate interactions with adaptor proteins.Kinase-active Eph RTKs (and EphA10) have an approximate 35 amino acid activation loop that contains a conserved Tyr residue that, when phosphorylated, serves as an SH2 domain binding site to keep the activation loop in a configuration that prevents it from occluding access of ATP and substrate to the kinase site  Figure . Mammali813 is the first amino acid in the symmetrical RLxxSPxxPSxxLR sequence and was previously shown in human EphB6 to be integral for nucleotide-binding since an R813D mutation abrogated nucleic acid binding capabilities [813 is not the orientation residue of the putative SH3 domain binding site, and instead, a histidine residue located nearer to the PxxP motif (His816) could serve this purpose  that modulate the phosphorylation state and thus kinase activity of individual members in the cluster. Retainment of allosteric regulation by the SAM domain linker and juxtamembrane allosteric regulation of an activation loop that has evolved to house an SH3 domain binding site suggests that the potential molecular switch function involving the SH3 domain binding site of mammalian EphB6 is a regulated process that occurs upon activation. We suggest that upon activation, phosphorylated Tyr residues in the juxtamembrane that serve as SH2 binding sites together with the SH3 domain binding site are used by mammalian EphB6 to recruit specific proteins that in turn modulate the signals generated by the Eph cluster. Repurposing the activation loop of a kinase domain into a protein interaction site is a strategy that was previously characterized for the pseudokinase STRADα   identifiadhesion . EmployiThe molecular switch mechanism of EphB6 may also include promoting the degradation of Eph clusters by recruiting ubiquitin ligases since a functional relationship between EphB6 and the ubiquitin ligase c-Cbl was previously identified . In the Our comparative analysis of mammalian kinase-negative EphB6 with its kinase-positive counterpart in non-mammals has provided insight into the potential mechanistic functions of the human pseudokinase EphB6. Instead of being an “inert” Eph receptor that solely prevents kinase-active Eph receptors from activating each other, we propose that EphB6 in mammals is a highly regulated pseudokinase whose function as a scaffold to recruit adaptor proteins to an Eph cluster is precisely controlled. To accomplish this, we suggest that mammalian EphB6 would require deactivation of the same allosteric regulatory mechanisms that are used to regulate the kinase activity of kinase-active Eph receptors involving the SAM domain linker and the juxtamembrane domain . Derepre


Dear Editor,2. Population-based studies in NLPHL have provided grounds for optimism that most patients with NLPHL have survival expectations that approximate those of the general population7.Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare and clinicopathologically distinct entity within the spectrum of Hodgkin lymphomas8. Therefore, the available statistics on NLPHL survival—which are all measured from diagnosis or the start of treatment—might be somewhat pessimistic about informing long-term NLPHL survivors about their prognosis over time. This problem can be tackled by estimating the survival of patients who survive a specified time since diagnosis . Estimating conditional survival accounts for time-dependent mortality rates, which are generally increased in the first few years after diagnosis. Conditional survival estimates become even more valuable when they are corrected for the life expectancy in the general population; that is, conditional relative survival (CRS).Although excess mortality in NLPHL is generally low, patients with older age and advanced disease do experience excess mortality within the first few years after diagnosisTo our knowledge, data on CRS in NLPHL are lacking. These data could help to inform patients and physicians about the changes in NLPHL prognosis over time and the intensity of surveillance and follow-up activities based on the patients’ risk of mortality over time. In this population-based study, we assessed 5-year relative survival (RS) at diagnosis and 5-year CRS for each following year after diagnosis up to 10 years post-diagnosis among adult patients diagnosed with NLPHL in the Netherlands.9. We identified all adult (≥18 years) patients diagnosed with NLPHL during 1993–2018—with survival follow-up through December 31, 2019—from the NCR. The International Classification of Diseases for Oncology morphology code 9659 was used to select patients with NLPHL from the NCR. That code was introduced in 1993 based on the REAL classification introduced in that same year10. Therefore, patient selection was restricted to those diagnosed as of 1993. The Privacy Review Board of the NCR approved the use of anonymous data for this study.Established in 1989, the Netherlands Cancer Registry (NCR) has national coverage of at least 95% of all newly diagnosed malignancies in the Netherlands11. The Ederer II method was used to estimate the expected survival using Dutch population life tables12.We calculated 5-year RS at diagnosis and 5-year CRS for each additional year survived up to 10 years post-diagnosis. RS is a measure that approximates disease-specific survival in the absence of information on the cause of death, which is not ascertained in the NCR. RS is calculated as the ratio of the patients’ overall survival (OS) to the expected survival of equivalent groups from the general population—matched to the patients by age, sex, and calendar year13. The hybrid approach was applied for patients diagnosed during 1993–2018 who were alive at some point during the follow-up interval 2007–2019, resulting in 15 years of post-diagnostic follow-up information. Supplementary Fig. RS was estimated using a hybrid approach—which is conceptually similar to the approach used to estimate the life expectancy at birth—to predict up-to-date survival statistics in an unbiased manner when incidence data lag behind mortality statistics14. Excess mortality is considered minimal when RS exceeds 95%15. Differences in CRS estimates between subgroups were considered statistically significant when the 95% confidence intervals (CIs) did not overlap. Analyses were performed using STATA Statistical Software version 16.1 .RS was computed for the overall cohort and by sex and age (18–44 and ≥45 years) and disease stage at diagnosis (I–II and III–IV). The age cutoff was based on the International Prognostic ScoreA total of 747 patients  were diagnosed with NLPHL in the Netherlands between 1993 and 2018 (Table Overall, 5-year RS at diagnosis was 93% Fig. . Five-yeThe information from this first study on CRS in NLPHL provides a better appreciation of the prognosis with each additional year survived from diagnosis. The overall prognosis of NLPHL can be regarded as favorable.6. This finding can be used to reassure most NLPHL survivors who were diagnosed with these adverse risk factors.We show that the well-established prognostic effect of older age and advanced stage mostly diminished with additional years survived after diagnosisAnother key finding of this study was that patients aged 18–44 years and those with stage I–II disease had 5-year CRS that virtually remained above 95% throughout the 15 years of post-diagnostic follow-up. This finding indicates that these patients have survival expectations similar to comparable groups from the general population. On the other hand—while the prognostic effect of stage largely diminished over time—5-year CRS did not exceed 95% throughout the follow-up period among patients with stage III–IV disease and remained around 90%, suggesting that these patients continued to endure small excess mortality over time. However, this finding should be interpreted in light of the wideness of the 95% CIs, which is related to the comparatively small number of patients in the group with stage III–IV disease. Nevertheless, the overall short- and long-term excess mortality encountered in NLPHL may be a consequence of treatment-related sequelae such as secondary malignancies, late recurrent disease or transformation to a high-grade lymphoma.6—but not all—studies5. This finding could, in part, be related to the use of RS, instead of OS, in this study.We demonstrated no prognostic effect of sex, which is congruent with someLimitations of the study include the lack of central pathology review and information on prognostic factors other than age and stage, detailed treatment strategies, and transformation and relapse rates. Nevertheless, our study provides patients with NLPHL and their physicians with valuable information about NLPHL prognosis during follow-up, which, in turn, can be used in concert with information on other risk factors to tailor surveillance and follow-up activities.CRS in NLPHL in NL - Supplemental Information


Post-hoc analyses revealed that the standard scores and intraindividual differences for successive processing were significantly lower than those for planning and attention. The WISC-IV scores did not significantly vary among the four measures, although the working memory index was the lowest among the four measures. The within-individual weakness in successive processing, a form of working memory function, may be a distinct characteristic of children with MMD. The CAS may be more sensitive than the WISC-IV for detecting this selective neurocognitive weakness in children with MMD.Although cognitive impairment is well-documented in children with moyamoya disease (MMD), selective decline in specific neurocognitive domains remains controversial. The purpose of this study was to characterize the neurocognitive profile of children with MMD using the Das Naglieri Cognitive Assessment System (CAS) and the Wechsler Intelligence Scale for Children, Fourth Edition (WISC-IV). We analyzed the neurocognitive data of 30 children  with MMD who were assessed with the CAS and the WISC-IV before surgery. We focused on the comparison of standard scores and intraindividual differences across domains. The CAS scores significantly varied across four measures (standard scores,  The age of onset of MMD has a bimodal peak: a major peak in the first decade of life and a moderate peak in the late 20s to 30s2. Long-term follow up studies have revealed that 10% to 20% of children with MMD have difficulty with social adaptation after adolescence because of cognitive impairment7. Such neurocognitive impairment is attributable to hypoperfusion in the vascular territories, which include the anterior and middle cerebral artery territories8 and the frontal lobe in particular15. Several multicentered registration studies, such as the Cognitive Dysfunction Survey of the Japanese Moyamoya Disease study16, are ongoing and aim to reveal neurocognitive and neuroradiological characteristics of moyamoya disease.Children with MMD commonly manifest ischemic symptoms, such as transient ischemic attacks (TIA) and stroke. However, children with MMD can also suffer from cognitive impairment. According to a recent systematic review, the prevalence of cognitive impairment is 30% (range 13% to 67%), and median intelligence quotient (IQ) is 98 (range 71 to 107)17. One possible reason is that the neuropsychological tests that are applicable to children are limited . To date, there have been no studies conducted in children with MMD that have applied various neuropsychological tests on a large, representative, and standardized sample. Another possible reason for the controversy is that the interpretations of most available neuropsychological tests are based on comparisons with the normal population, which may underestimate the variability of neurocognitive domains within an individual.Although the mechanisms underlying neurocognitive impairment in moyamoya disease suggest selective impairment of specific neurocognitive domains that correspond to the vascular territories, the evidence remains controversial18. The official Japanese version of the CAS was developed in 200719. The CAS comprises four measures: planning, attention, simultaneous processing, and successive processing. It is unique in that it assesses “intraindividual differences,” which are statistical fluctuations (strengths and weaknesses) across measures within an individual20. For this reason, we routinely use CAS as a preoperative neuropsychological test, in addition to the WISC.The CAS is a standardized neuropsychological test developed by Das and Naglieri that focuses on the assessment of cognitive function, rather than intelligence, in children aged 5 to 17 years21. The official Japanese version of the WISC fourth edition (WISC-IV) was developed in 201022. The WISC-IV comprises four indices: verbal comprehension, perceptual reasoning, processing speed, and working memory. The WISC-IV implements a clinical method known as “the discrepancy analysis,” which aims to detect significant discrepancies among indexes in each individual. Kazumata et al. reported that discrepancies were most frequently observed between the verbal comprehension index and working memory index of the WISC-IV23. However, the discrepancy analysis of the WISC-IV is not necessarily suitable as a research method for detecting cognitive variability within a specific population. This is because, although discrepancy analysis of the WISC-IV only detects differences between each index, the CAS allows analyses of whether an index deviates from the average value of the entire individual. Therefore, the method whereby intraindividual differences are detected is limited in the WISC-IV.The WISC is the most commonly used neuropsychological test worldwide that measures the intellectual ability of children aged 5 to 16 yearsTo date, there have been no studies that have examined the neurocognitive function of children with MMD using both the CAS and the WISC. Determining whether specific neurocognitive weaknesses exist in children with MMD would be valuable for clinicians and rehabilitation professionals who support children with MMD.The purpose of this study was to characterize the neurocognitive profile of children with MMD before surgery using the CAS and WISC-IV. We compared the scores of these neuropsychological tests and analyzed the intraindividual differences derived from these tests.This cross-sectional study was approved by the ethics committee of Kyoto University Graduate School, Faculty of Medicine (R1417). Study procedures were carried out in accordance with the Declaration of Helsinki. For prospective data, informed assent was obtained from all individual participants and written informed consent was obtained from their parents. For retrospective data, all subjects gave opt-out consent.1. All moyamoya children aged 5 to 16 years and their parents were asked at their first visit whether they were willing to undergo neuropsychological tests.The inclusion criteria were children aged 5 to 16 years who had been newly diagnosed with MMD and had undergone neuropsychological testing before surgery. Patients were diagnosed according to the criteria proposed by the Research Committee on Moyamoya Disease in JapanA total of 40 children aged 5 to 16 years were admitted to Kyoto University Hospital between June 2016 and June 2020 and diagnosed with MMD. Of the 40 patients, 10 were excluded from the analyses for the following reasons: the patients and family declined the neuropsychological tests (2 patients); preoperative neuropsychological tests were not completed because of unstable symptoms (2 patients), limited examination schedule (1 patient); deafness (1 patient); lack of Japanese ability (1 patient); judgment by the psychiatrist that the neuropsychological test results were biased because of the patient’s developmental background (2 patients); and special education for gifted children (1 patient). This resulted in a total of 30 children for the analysis.24, hemodynamic state assessed using single photon emission computed tomography (SPECT)25, laterality of disease involvement, presence or absence of posterior cerebral artery (PCA) involvement, and asymptomatic infarction on magnetic resonance imaging (MRI). PCA involvement was defined as stenosis greater than 50% in the P1 to P3 segment of either PCA, with decreased delineation of cortical arteries in either hemisphere6. The hemodynamic state for each hemisphere was graded according to SPECT findings into one of the following three stages: stage 0, normal baseline cerebral blood flow (CBF) with normal acetazolamide-challenged CBF; stage 1, normal baseline CBF with reduced acetazolamide-challenged CBF; and stage 2, reduced baseline CBF with reduced acetazolamide-challenged CBF25. Acetazolamide-challenged SPECT was not indicated for patients with severely reduced CBF in at least one hemisphere because of the risk of ischemic complication. Because this resulted in unclear differentiation between stages 0 and 1 in several hemispheres, we recorded hemodynamic state as either stage 2 or non-stage 2.Clinical data were collected, which included sex, initial manifestations , age at symptom onset and first admission, time interval between onset and first admission, Suzuki’s angiographical stageThe CAS and the WISC-IV were performed as preoperative neurocognitive assessments. All but one participant underwent both tests during the first admission. Occupational therapists, who are proficient in neurocognitive testing, conducted all tests in a consistent manner. Table 18. The standard scores are defined by a mean of 100 with a standard deviation of 15. Each PASS scale comprises three tasks, with the whole battery comprising 12 tasks. The intraindividual difference is calculated by subtracting the average of the four PASS standard scores from each PASS standard score20. The intraindividual difference is negative if an individual scores relatively low in one PASS scale compared with the other scales. To interpret the CAS, a significant level of intraindividual difference was defined for each measure based on a standardized sample19. According to the manual of the CAS and WISC-IV, we defined a significant intraindividual difference as − 11 points or lower.The CAS provides standard scores for each of the four scales of planning, attention, simultaneous processing, and successive processing (PASS) as well as a full scale score21. These standard scores are defined by a mean of 100 with a standard deviation of 15 and are based on a large and representative standardized sample. Unlike the CAS, the calculation of the intraindividual difference is not specified in the WISC-IV. However, we used the same method as the CAS for calculating intraindividual differences based on the WISC-IV for the purpose of performing analyses within each participant.The WISC-IV provides standard scores for the following measures: full scale IQ, verbal comprehension index (VCI), perceptual reasoning index (PRI), working memory index (WMI), and processing speed index (PSI)Wilcoxon rank-sum test was used for comparison between groups with and without asymptomatic infarction for mean full scale CAS and WISC-IV scores.Descriptive statistics of the CAS and the WISC-IV are presented as mean and standard deviation for both the standard scores and intraindividual differences. Normality of the data was evaluated using the Shapiro–Wilk test. To compare the standard scores across the four measures of the CAS and the WISC-IV within each individual, we used a one-factor repeated measures analysis of variance (ANOVA). To compare intraindividual differences across the four measures, we used a one-way ANOVA because it was calculated based on the average of the four measures and thus, a repeated analysis was not necessary. Post-hoc tests were performed using Tukey’s honestly significant difference (HSD) test.p < 0.05 was considered to indicate statistical significance.All statistical analyses were performed using JMP 14 . All tests were two-sided, and Table p = 0.904; WISC-IV, p = 0.749).Mean full scale scores of CAS and WISC-IV were not statistically different between patients with and without asymptomatic infarction  = 9.206, p < 0.001), but not in that of the WISC-IV  = 0.621, p = 0.603). The one-way ANOVA also revealed a significant difference in the intraindividual differences of the CAS  = 12.275, p < 0.001), but not in that of the WISC-IV  = 0.828, p = 0.481).There was a statistically significant variance across all four measures of the CAS for standard scores and intraindividual differences, as shown in Table p = 0.002; intraindividual difference, p < 0.001) and attention . The intraindividual difference of simultaneous processing was significantly lower than that of planning (p = 0.002).Table The mean value of the intraindividual difference in successive processing was lower in those with SPECT stage 2 than in those with non-stage 2 (− 7.6 ± 8.6 vs − 6.3 ± 8.9), although the difference was not significant.The proportions of patients who exhibited significant intraindividual differences in each measure of the CAS and WISC-IV were as follows: planning (0/30 or 0%), attention (3/30 or 10%), simultaneous processing (4/30 or 13.3%), and successive processing (11/30 or 36.7%) for the CAS; and VCI (3/30 or 10%), PRI (5/30 or 16.7%), PSI (4/30 or 13.3%), and WMI (6/30 or 20%) for the WISC-IV.26. Furthermore, we used both the CAS and WISC to assess children with MMD and showed that the CAS detected selective neurocognitive weaknesses that were not detected by the WISC-IV.Our study had three main findings. First, children with MMD exhibited selective within-individual weaknesses in successive processing as measured by the CAS. Second, intelligence measured with the WISC-IV was within the normal range with or without infarction. Third, no significant variability across the intellectual domains of the WISC-IV was observed, although the score for the working memory index was the lowest. The first finding is novel because CAS has not been applied to children with MMD, except for in a pioneering case report written in Japanese27. Furthermore, a validation study on the Japanese version of the WISC-IV also revealed a high correlation between the successive processing scale of the CAS and the WMI of the WISC-IV22. Therefore, it is reasonable to suggest that successive processing reflects a form of verbal working memory function. Further studies are required to determine which components of working memory are associated with neurocognitive weaknesses in children with MMD.Successive processing in the CAS is thought to be similar in concept to the WMI in the WISC-IV as both involve the auditory recall of a sequence. The successive processing scale of the CAS comprises three tasks: immediate recall of a series of words (e.g. cat-flower-book); repetition of sentences with no actual meaning (e.g. white is blue); and sentence questions as syntactic comprehension . The WMI in the WISC-IV comprises digit span and letter-number sequencing, whereas successive processing in the CAS involves language recall. Das et al., one of the groups that developed the CAS, has explained that successive processing involves the articulatory loop of working memory, as proposed by Baddeley17. The successive processing scale of the CAS, which requires the working memory of language, might better reflect the cognitive skills required in daily life and the difficulties experienced by patients, such as inability to follow schoolwork, trying to learn too many things simultaneously, and poor attention. Our results also suggest that such difficulties could occur even if overall intelligence, as defined by the full scale scores, is high. Alternatively, the WMI of the WISC-IV, which comprises only digit span, may simply be impractical and unsuitable for detecting neurocognitive characteristics in children with MMD.Interestingly, our result of selective weakness in successive processing corresponds to those of a previous study. Hsu et al. reported that selective impairment in immediate word recall, as measured by the Word List Test in the Wechsler Memory Scale, might be a characteristic of MMD children with younger onset and prolonged symptom duration23, which is inconsistent with our results. However, one reason for why the verbal working memory tasks of the CAS were more affected than the digit span task of the WISC in our study is that the former task is more complex and difficult because it requires not only basic working memory ability but also semantic processing ability. Therefore, our results are not inconsistent with the suggestion of Kazumata et al. In addition, logically, whether verbal function or non-verbal function is more impaired is defined by the laterality of disease involvement and may be less related to working memory function per se. Although the relatively complex working memory tasks of the CAS seem suitable for MMD children, the optimal working memory task for detecting working memory weaknesses specific to children with MMD remain to be ascertained. This issue should be addressed in future studies.Kazumata et al. have suggested that verbal function is relatively preserved in MMD children28. Numerous studies have demonstrated a close relationship between the dorsolateral prefrontal cortex and executive functions, including working memory30. Moreover, Lezak et al. described that the prefrontal lobe is involved in short-term memory31. Therefore, our finding of verbal working memory disturbance in MMD is consistent with brain dysfunctions typically reported in MMD patients. However, further studies on cerebral perfusion imaging are required to clarify the relationship between working memory and previously reported cerebral blood flow changes in children with MMD23.Selective impairment of neurocognitive domains has been attributed to hypoperfusion in specific vascular territories that are predominantly affected in MMD. Arterial stenosis in MMD involves the internal carotid artery by definition and thus causes ischemia of the frontal lobe35 These results may be interpreted as an underestimation of within-individual variability in assessments using the WISC, given that variability is not measured by this scale. To address this question, we applied the method used for calculating intraindividual differences in the CAS to the WISC-IV and found no significant variability among the four measures, although the WMI score was the lowest. These findings suggest that the WISC-IV is less able to detect characteristics of neurocognitive deficits in children with MMD.The fact that the scores of the WISC-IV fell within the normal range in our patients is consistent with previous studies that showed average to above average overall intelligence in children with MMDThe present study has several limitations. First, our study population was relatively small. Although our study is one of the given the rarity of the disease, larger studies are needed. Nevertheless, our study design of using two standardized tests that are rarely applied to children demonstrates some validity of our results on neurocognitive profiles. Second, our study had the potential to have been contaminated by selection bias because we only included preoperative patients. The exclusion of children under 5 years of age, for whom neuropsychological testing is inapplicable, or those who required acute surgery because of unstable symptoms may overestimate our results and limit their generalizability. Finally, this study did not include a control group.Our assessment of the CAS suggests that a selective within-individual weakness in successive processing, a type of working memory, may be a characteristic of children with MMD. The CAS was more sensitive in detecting this selective neurocognitive weakness than the WISC-IV in children with MMD. Further studies are required to determine whether the CAS has value for clinical use in supporting the education and daily life of children with MMD.


Purpose: Electromagnetic acoustic imaging (EMAI) is a hybrid imaging technique using radio-frequency irradiation to induce ultrasound (US), providing an US image in which spatial conductivity differences provide image contrast. The method is potentially clinically important in that the added diagnostic parameter has been shown to be useful in cancer detection and vascular space delineation.Approach: We report the development of coil configurations and imaging processing techniques designed to address the low signal-to-noise of EMAI and demonstrate achievable resolution and contrast in phantoms along with EMAI signals in excised animal tissue. Experiment results are compared with theoretical calculations.Results: EMAI signal intensities depend on the square of the ampere-turns in the coil radio frequency coil as predicted theoretically. Resolution is shown to be comparable to conventional US imaging with contrast and signal intensity depending on source conductivity. Optimizing signal-to-noise depends on coil design, orientation of the electromagnetic fields, and coherent processing.Conclusions: Two-dimensional EMAI images are shown to have the expected resolution of conventional US with image contrast dependent on conductivity. Achievable signal-to-noise is sufficient to form potentially clinically useful images. The induced electric fields then generate first and second harmonic ultrasound (US) by virtue of intrinsic electrical properties in the target.–,in vivo and in vitro.For medical imaging, EMAI offers a means of obtaining conductivity weighted US along with automatic overlay onto conventional US images. The contribution of electrical properties to image contrast adds an important diagnostic parameter as it has been shown to be a distinguishing factor in malignanciesIn-vivo diagnostics in cancer detection rely on techniques that exploit tissue parameters reflecting the microenvironment(s) characteristic of malignancy. Various imaging methods that leverage tissue acoustic properties, electrical properties, or a combination thereof, are currently in use or proposed and under development. Conventional US with elastography provides diagnostic information based on acoustic properties including density and stiffness.Both signals were demonstrated previously in various phantoms consisting of compartments with distinct values of electrical conductivity. Here we report further development of the imaging modality with demonstration of achievable signal-to-noise in excised tissue phantoms, qualitatively show resolution and image contrast in two-dimensional (2-D) imaging, and expand on modeling with implementation of methods for RF delivery and signal processing in EMAI.22.1For all the EMAI experiments, the general equipment configuration consisted of an RF coil assembly, a water tank with US transducer(s), an RF burst transmitter, oscilloscope, and compressible phantoms providing varying regions of conductivity (inclusive of immersion solutions). The system was set up to enable RF transmission with US reception or to operate in pulse-echo mode with US transmission and reception. Water tanks were assembled to house the US transducer and phantom and modified for the particular coil assembly in use.2.1.1An RF burst transmitter operating up to 30 kW delivered pulses at 1-ms repetition rates with pulse lengths of 3 to The final amplifier used to drive the coils was a tube type pulse amplifier circuit using a 3CPX1500 ceramic vacuum tube. The various coil configurations were incorporated as part of the plate output circuitry. In the case of the low inductance coils, a simple parallel resonant topology was used. For coils with higher inductance, a split-capacitor step-up configuration was used to maximize coil current. In all cases most of the RF power in the generated pulse was absorbed in the resistor used to set circuit 2.1.2All RF coils used were lab-developed and designed to maximize the magnetically induced The following is a description of the coil assemblies fabricated along with theoretical predictions of field and EMAI signal optimizations.For Helmholtz coils, the approximate expression for the EMAI pressure applied when the conductivity change occurs over a distance that is small compared with the acoustic wavelength of the generated US, as would be the case at boundaries between tissues of different electrical properties, is The intent of this coil design  was to mSince the magnitude of EMAI signals for Helmholtz coils depends on A modified 10-turn Helmholtz pair with a diameter of 47 mm and a 32 mm gap  was consTo investigate the effect of a circumferential current surrounding the phantom, a 10-turn solenoid was constructed as shown in An axial current configuration was tested with the coil as shown in A single 6-turn oval solenoid coil (25 mm length) was constructed as shown in 2.1.3US was detected using an Olympus A311S focused transducer with a resonant frequency of 10 MHz, a focal length of 5.1 cm, and a diameter of either 1.3 or 1.9 cm.2.2Signal averaging, coherent processing with amplitude envelope display, active damping of the transmit burst, or a combination thereof was performed.Coherent detection was implemented to provide the highest possible SNR by rejecting all nonphase coherent noise frequencies. A 40-MHz master crystal oscillator was used as the time base. This clock was divided by four with a circuit that produced two 10-MHz clock references with an exact 90-deg phase relationship (Single point data were displayed on the oscilloscope with time of flight (TOF) defining the region of interest generating the EMAI signal. The oscilloscope used was an Agilent DSO5034A which had the capability to signal average. Depending on the strength of the returned signal, signal averages ranging from 2 to 32,768 acquisitions could be performed. The oscilloscope was synchronized with a pulse produced by the coherent signal processor. 2-D scans were obtained with a single transducer by rastering across the phantom with a 2-D mechanical stage. Signal data were processed in Mathematica for 2-D images.To maximize transmitter efficiency, the coils were resonated at 5 MHz with as high a circuit 2.3Room temperature vulcanizing (RTV) silicone rubber phantoms in both silver and nickel formulations were used in a bath of either water or saline. Small RTV spheres were suspended by nonconductive rods. An emblem of RTC silicone was constructed as described below to demonstrate resolution and image contrast in a 2-D image. A simple phantom consisting of the interface between saline and air was used to track TOF and EMAI signal intensity as a function of salinity (and thereby conductivity). Tissue phantoms were untreated beef and chicken liver parts.33.13.2While the ferrite core as described above for coil 2 was intended to provide flux enhancement through the phantom volume, the increase in flux was largely restricted to the coil core rather than through the gap. Therefore, the boost in induced field in the phantom was minimal. Further, the nonlinearities associated with using ferrites at high flux levels created harmonic production, which distorted the coil waveform thereby complicating the 1f-2f methodology used with EMAI. In addition, with long mechanical scans, ferrite heating exceeded tolerable limits.3.3in-vivo tissue along with the expected spatial variability of conductivity encountered in the body. Excised tissues rather than inorganic phantoms were used to more closely simulate in-vivo conditions. Example results from chicken liver and beef liver are shown below to further demonstrate feasibility.Various phantoms were selected to approximate the acoustic properties of EMAI signals demonstrated in Figs. 8For reference, In The feasibility of detecting tissue architecture was tested with a heterogenous piece of beef liver that included vessel stubs. In Axial resolution is a function of the wavelength of the US and the pulse width. Pulse width is a function of the number of cycles used in the RF burst and the Lateral resolution was set largely by the focal waist of the focused transducer and axial phantom location. For the 10-MHz US frequency and 50-mm focal length transducer, lateral resolution is on the order of few wavelengths or With the use of coil 6, the off-center solenoid, avoiding the central 3.4The theoretical prediction of the EMAI signal intensity was compared to that obtained experimentally using the series-connected modified Helmholtz coil (coil 4) described in Sec. 3.5To demonstrate resolution and image contrast, a 2-D EMAI image of a phantom  In coil 5, the The signals and images in the experiments were all obtained with by scanning a single piezoelectric detector resonant with the US frequency. Improved signal to noise can be obtained and the data collection time considerably reduced if linear and 2-D detector arrays are employed instead of a single detector.