Cardiovascular diseases represent a major cause of mortality and morbidity in the world, and specifically, thrombotic conditions such as heart attacks and strokes are caused by unwarranted activation of platelets and subsequent formation of blood clots (thrombi) within the blood vessels during pathological circumstances. Therefore, platelets act as a primary therapeutic target to treat and prevent thrombotic conditions. Current treatments are limited due to intolerance, and they are associated with severe side effects such as bleeding complications. Hence, the development of novel therapeutic strategies for thrombotic diseases is an urgent priority. Flavonoids are naturally occurring plant-derived molecules that exert numerous beneficial effects in humans through modulating the functions of distinct cell types. However, naturally occurring flavonoids suffer from several issues such as poor solubility, lipophilicity, and bioavailability, which hinder their efficacy and potency. Despite these, flavonoids act as versatile templates for the design and synthesis of novel molecules for various therapeutic targets. Indeed, several synthetic flavonoids have recently been developed to improve their stability, bioavailability, and efficacy, including for the modulation of platelet function. Here, we provide insight into the actions of certain natural flavonoids along with the advantages of synthetic flavonoids in the modulation of platelet function, haemostasis, and thrombosis. Cardiovascular diseases (CVDs) are the biggest cause of death in humans ,2. AmongPlatelets are small circulating blood cells that are responsible for maintaining haemostasis via blood clotting upon vascular injury. Although they are anucleated cells, they are derived from megakaryocytes and possess all the organelles and proteome required for normal cellular function ,8. Along2 (TxA2). Platelets associate via fibrinogen, which binds to integrin αIIbβ3, and this binding can induce outside-in signalling via this integrin and results in stabilisation of the clot . T. T2 [50  mimetic ): apigen14.4 μM) . However 0.1 μM) . Equol ( 0.1 μM) . This in 0.1 μM) ,33,34.Justicia procumbens and Cephalotxus wilsoniana were assayed for their effects on platelet-rich plasma (PRP) stimulated with adrenaline and were demonstrated to be able to acutely supress adrenaline-induced platelet activation [Several complex yet naturally occurring flavonoids isolated from tivation . Furthertivation ,37.Moreover, thrombin is a protease generated on the surface of activated platelets that can stimulate platelets via the cleavage of extracellular portions of protease-activated receptor 1 (PAR1) and PAR4 . Some flex vivo conditions; however, a few flavonoids have been tested for their efficacy under in vivo settings. In humans, quercetin 4′-O-β-d-glucoside , which has multiple effects in platelet activation ,44. FlavIIbβ3 [IIbβ3 which enables it to bind to fibrinogen and subsequently trigger aggregation, and outside-in signalling at a later stage to promote clot retraction [IIbβ3 is affected by flavonoids such as nobiletin and tangeritin as the clot weight increased, and thus, clot retraction was diminished [Fibrinogen binding is a useful measure of platelet activation as it can be used as a marker for inside-out signalling to integrin αIIbβ3 . During traction . Clot fotraction . The outminished ,20. Chryminished ,25. Furtminished ,25.2 receptor  but instead target upstream signalling molecules, which were not established yet [2 (PGH2) by preventing its entry into the active site [2 production and determined that 26 out of 28 flavonoids tested did not have a significant effect [2 into TxA2) was achieved by 100 μM apigenin, 100 μM 7-hydroxyflavone, and 100 μM epicatechin; however, they were all significantly less potent than 1-benzylimidazole. All of the tested flavonoids in this study were able to significantly inhibit human platelet aggregation induced by U46619 [In contrast, the binding of thrombin to platelets is unaffected by the presence of flavonoids, which suggests that their effects are mediated intracellularly and not by direct receptor antagonism . Flavanoshed yet . Conversshed yet  as they ive site ,36. Furtive site . In contive site  used isot effect . Howevery U46619 . Due to y U46619 ,50.Moreover, nobiletin increased the levels of cyclic guanosine monophosphate (cGMP) from around 6 pM to 40 pM, which increases the activity of the inhibitory kinase, cGMP-dependent protein kinase (PKG). As expected, nobiletin increased the phosphorylation of vasodilator-stimulated phosphoprotein (VASP) at serine 239, which has been identified as the major site of PKG phosphorylation ,51. The 3) receptor [Direct potentiation of adenylyl cyclase is another mechanism of action for flavonoid-mediated inhibition in platelets as cyclic adenosine monophosphate (cAMP) levels were observed to be increased in the presence of EGCG. Phosphorylation of VASP on a site specific for cAMP-dependent protein kinase (PKA) was also siginificantly increased, and the inhibitory effect was partially reversible by treatment with inhibitors of adenylyl cyclase. This was suggested to be due to the removal of PKA’s inhibitory phosphorylation of the inositol trisphosphate ) that are closely associated with GPVI-mediated signalling were unaffected by nobiletin or tangeretin treatment, which suggests that the ligation and activation of GPVI proceeds as normal into the LAT signalosome; however, signalling is then restricted to this area as phosphorylation of PLCγ2 and Akt (Protein Kinase B) was inhibited in a dose-dependent manner. Furthermore, whole cell tyrosine phosphorylation was largely unaffected by this flavonoid treatment. This strongly suggests that nobiletin may inhibits the function of phosphoinositide 3-kinase (PI3K) although the binding site and mechanism of action of this inhibition has yet to be fully elucidated ,19,20. AP-Selectin exposure is a useful marker for platelet α-granule secretion , and adeDue to the wide range of naturally occurring flavonoid structures, it is possible to investigate the effect of their functional groups on specific biological pathways. In general, the molecules that lacked efficacy possessed methoxy or large glycoside groups whereas the potent molecules possessed hydroxy groups that are capable of acting as hydrogen bond donors . For exa2 (PGI2)  in huma50 concentrations. Despite the promising potential of natural flavonoids, there are currently none routininely prescribed by practitioners in medical clinics for the treatment of thrombotic diseases. This is due to the issues described in the previous sections involving compound bioavailability, stability, and multiple cellular targets. In particular, the flavonoid concentrations achieved in the blood tend to be far less than their ICSince natural flavonoids show promising but hampered potential for drug development, synthetic flavonoids have been developed by multiple laboratories to address the myriad of issues that are associated with natural flavonoids. The synthethic flavonoids have been developed primarily to overcome the issues raised in the previous sections regarding bioavailability (and interactions with plasma proteins) and stability. Many experiments have been conducted to determine the specific functional groups necessary for the beneficial antiplatelet effects that natural flavonoids exhibit.Chrysin is a naturally occurring flavonoid that is capable of inhibiting platelet activation in isolated platelets; however, its potency is significantly reduced when the experiment is repeated in PRP (in the presence of plasma proteins) ,57. In oH-pyridopyrimidin-4-one flavonoids and discovered that two of these compounds with multiple methoxy groups  were mThe development of synthetic flavonoids enables modifications to the chemical structure in order to allow investigation of the effect of different functional groups on the antiplatelet ability of the compounds. F-1 I is a syrd and 4th carbon atoms (O-propionate) and 52.7% (quercetin-3-O-butyrate) versus 43.5% inhibition for quercetin), potentially due to the change in molecular packing [Quercetin’s inhibitory potential can be increased via the addition of an acyl chain between 3on atoms M, which  packing . PMQ inh packing .ex vivo, no significant difference between the tail bleeding times of mice treated with chrysin or Ru-thiochrysin was seen at the same concentration, which also emphasised this as a safer compound for therapeutic applications [in vivo settings.3′,4′-Dihydroxyflavonol pyrimidin-4-one flavonoids decrease α-granule secretion induced by U46619 [Both chrysin and Ru-thiochrysin inhibit dense- and α-granule secretion; however, Ru-thiochrysin inhibits P-selectin exposure more than chrysin, yet there were no significant differences between their effects on dense granule secretion . Other sy U46619 . Conversy U46619 .Conjugation of ruthenium to chrysin (Ru-thiochrysin) was also significantly more effective at inhibiting calcium mobilisation, fibrinogen binding, and clot retraction in PRP whereas there was no difference between the two compounds in isolated platelets .Limited studies have investigated the ligation of synthetic flavonoids to specific molecular targets on the surface of platelets. Naturally occurring flavonoids have been suggested to have extracellular targets and 2,3-diphenyl-4H-pyridopyrimidin-4-one flavonoids, like apigenin, have been shown to antagonise TP receptor ,59.An immunoblotting analysis of Ru-thiochrysin and chrysin on the phosphorylation of focal adhesion kinase (FAK), Akt, and Src showed a decrease in the phosphorylation; however, the target protein(s) of chrysin and its synthetic derivatives were not fully elucidated .O-propionate (16.27 μg/mL) was the most soluble in water when compared to quercetin-3-O-butyrate (9.36 μg/mL), quercetin (1.98 μg/mL), and quercetin-3-O-valerate (1.07 μg/mL) [in vitro and in vivo. Longer acyl chains had a negative impact and reduced hydrosolubility and potency [Synthetic modifications of flavonoids are required to increase the therapeutic potential of flavonoids as natural flavonoids have non-ideal bioavailabilities. For example, it was calculated in rodents that to achieve an effective plasma concentration (of approximately 50 µM), 100 mg/kg of tangeretin or nobiletin would be necessary, which would equate to >7 g/day in human adults . When th7 μg/mL) . A 3 or  potency . A balanonate 16. μg/mL wain vivo. This is a key consideration as it has been shown that platelets can metabolise flavonoids on their own [Synthetic flavonoids could also be produced to resist metabolism heir own . For exaheir own . A synthheir own . Equol iheir own . InteresAdditionally, most studies examining natural flavonoids utilise isolated platelet preparations to mitigate the influence of plasma proteins ,57. Howein silico molecular docking analysis coupled with functional screening of predicted compounds.Thus far, characterisation of flavonoid efficacy has mainly been limited to adjusting the functional groups on a flavonoid backbone and testing a wide range of compounds on platelet aggregation without examining the molecular targets involved or determining why the specific functional groups are effective. However, they still provide a valuable insight into potential modifications that could be used in future drug design ,61. Thisex vivo have yet to be replicated in vivo. Indeed, flavonoids provide a good template for future drug design, but further experiments, particularly investigating the structure–activity relationships of functional groups in more detail and the specific molecular targets of flavonoids, must be conducted for the potential of flavonoids as therapeutic agents to be realised. Although studies on synthetic flavonoids are limited, they provide a robust starting point to take this research further in order to develop better antithrombotic agents using flavonoids as a basis.Platelet activation can be inhibited by flavonoids via multiple mechanisms; however, naturally occurring flavonoids raise challenges with reduced bioavailability and metabolism to form less efficacious metabolites. Synthetic flavonoids enable the tailoring of flavonoid structures into better therapeutic agents. However, the benefits observed for currently used synthetic flavonoids 2 to mitigate the size of the thrombus whilst still enabling platelets to respond to damage at the sites of injury. Flavonoids also possess the ability to influence the activity of other cell types such as endothelial cells and, therefore, may provide additional inhibitory effects not limited to direct actions on platelets.Further development of synthetic flavonoids that are more specific for effector proteins would provide a greater opportunity for achieving therapeutic agents that are as effective as current treatments but without the unwanted and dangerous side effects currently encountered. If flavonoid molecules could be biased towards the inhibition of specific sectors of platelet activity, then this could improve the tolerance of drugs. These novel compounds could be designed to only mildly inhibit collagen-induced platelet activation but to strongly inhibit platelet activation induced by secondary activators such as ADP and TxA


Barth Syndrome (BTHS) is a rare X-linked genetic disease in which the specific biochemical deficit is a reduction in the mitochondrial phospholipid cardiolipin (CL) as a result of a mutation in the CL transacylase tafazzin. We compared the phosphokinome profile in Epstein-Barr-virus-transformed lymphoblasts prepared from a BTHS patient with that of an age-matched control individual. As expected, mass spectrometry analysis revealed a significant (>90%) reduction in CL in BTHS lymphoblasts compared to controls. In addition, increased oxidized phosphatidylcholine (oxPC) and phosphatidylethanolamine (PE) levels were observed in BTHS lymphoblasts compared to control. Given the broad shifts in metabolism associated with BTHS, we hypothesized that marked differences in posttranslational modifications such as phosphorylation would be present in the lymphoblast cells of a BTHS patient. Phosphokinome analysis revealed striking differences in the phosphorylation levels of phosphoproteins in BTHS lymphoblasts compared to control cells. Some phosphorylated proteins, for example, adenosine monophosphate kinase, have been previously validated as bonafide modified phosphorylation targets observed in tafazzin deficiency or under conditions of reduced cellular CL. Thus, we report multiple novel phosphokinome targets in BTHS lymphoblasts and hypothesize that alteration in the phosphokinome profile may provide insight into the pathophysiology of BTHS and potential therapeutic targets. Subsequent to biosynthesis, CL is rapidly remodelled to yield the molecular species of CL found in the mitochondrial membrane [4-CL) [Cardiolipin (CL) is a key phospholipid involved in mitochondrial energy production ,4,5,6,7.e [4-CL) . The trae [4-CL) ,10. Altee [4-CL) .Barth Syndrome (BTHS) is a rare X-linked genetic disorder associated with skeletal muscle abnormalities, cardiomyopathy, and neutropenia and is the only genetic disease in which the specific biochemical defect is a dramatic reduction in CL ,13,14,15Here we examined the phosphokinome profile of Epstein-Barr-virus-transformed lymphoblasts prepared from a BTHS patient and compared it with those of an age-matched control individual. We observed striking differences in the phosphokinome profiles between BTHS and age-matched control lymphoblasts and hypothesize that the novel targets identified may be involved in the pathophysiology of the disease and may represent potential therapeutic targets.crd1∆ yeast mutants exhibiting reduced CL [Previous studies have indicated that BTHS lymphoblasts exhibit large reductions in CL levels ,21,22,23duced CL ,27.TM KAM-880 antibody microarray with phospho-specific antibodies and compared with that of age-matched control lymphoblasts, as described in Material and Methods. The phosphokinone profile of identified targets are presented as pathway maps in A previous study indicated that alterations in various processes involved in protein translation, amino acid metabolism, nucleotide metabolism, GTP hydrolysis, and folate metabolism were observed in TAZ knockdown mice . Given tPathway map analysis of the phosphokinome profile of proteins involved in the Lipid Metabolism pathway revealed a striking >2-fold increase in pan-specific phosphorylated adenosine monophosphate kinase subunit b (AMPKb(Ps)) compared to controls indicating activation of AMPK in BTHS lymphoblasts . AMPK isWhen we analyzed the phosphokinome profile of proteins involved in the Glucose Metabolism pathway, we determined a >3-fold increase in pan-specific phosphorylation of protein phosphatase 6C (PP6C(Ps)) and in insulin receptor/insulin-like growth factor-1 receptor (IR/IGF1R (INSR) (Y1189/Y1190)) phosphorylation . PP6C isPathway map analysis of the phosphokinome profile of proteins involved in the Inflammation pathway indicated that the pan-specific phosphorylation of interleukin-1 receptor-associated kinase 4 (IRAK4 (Ps)) was increased by >2-fold . IRAK4 aWhen we performed a pathway map analysis of the phosphokinome profile of proteins involved in the Chaperone pathway, we observed a >2.5-fold increase in pan-specific phosphorylation of a highly conserved protein in the heat shock protein 110 subfamily, APG-1 (APG1 (Ps)) . This isPathway map analysis of the phosphokinome profile of proteins involved in the Transport pathway revealed a 2-fold increase in cortactin (Y470) . CortactThe phosphokinome profile of proteins involved in the protein Translation pathway indicated a >3.7-fold increase in translation initiation factor eIF2Be (S540) , which mWe have also generated a pathway map analysis of the phosphokinome profile of proteins involved in the Apoptosis pathway. Our analysis revealed a 3.7-fold increase in pan-specific phosphorylation of death-associated, protein-related apoptotic kinase-2 (DRAK2) , which iThe Cell growth/division phosphokinome profile revealed a 3.5-fold increase in pan-specific phosphorylation of cell division cycle 25C phosphatase (Cdc25C) . The CdcPathway map analysis of the phosphokinome profile of proteins involved in the Adhesion pathway revealed a 2-fold increase in phosphorylation of lymphocyte-specific protein-tyrosine kinase (Lck) (S157) . Lck is Interestingly, the phosphokinome profiles of several proteins involved in the Neuronal pathway were modified in the BTHS lymphoblasts, for example, a 2-fold increase in phosphorylation of Disabled-1 (Dab1(Y198)) . Dab1 tyOur analysis also revealed changes in the phosphokinome of BTHS lymphoblasts in the Adrenergic Receptor pathway. For example, we report a 3-fold increase in phosphorylation of arrestin beta 1 (S412) . The arrWith respect to the phosphokinome profile of proteins involved in regulating Calcium homeostasis, a 2.6-fold increase in pan-specific phosphorylation of cAMP-dependent protein serine-kinase (PKA Ca/b) was observed in the BTHS cells compared to controls . SolubleThe phosphokinome profile of proteins involved in the mitogen-activated protein kinase (MAPK) pathway revealed a 2.3-fold increase in pan-specific phosphorylation of MEK4  and a 74Possibly related to mitochondrial dysfunction in BTHS cells, pathway map analysis of the phosphokinome profile of proteins involved in the cAMP response element binding protein (CREB) pathway revealed a 2.2-fold increase in phosphorylation of CREB1(S133) and a 51% reduction in pan-specific phosphorylation of calcium/calmodulin-dependent protein-serine kinase 1 gamma (CaMK1g) . CREB fuAnalysis of the phosphokinome profile of proteins involved in the Jun N-terminus protein-serine kinases (JUN) pathway revealed a 2.6-fold increase in phosphorylation of JNK (T183 + Y185) . JNK is Finally, pathway map analysis of the phosphokinome profile of proteins involved in the Orphan receptor pathway revealed a 2.7-fold increase in phosphorylation of crystallin αB (heat-shock 20 kDa like-protein) (S45) and a 61% reduction in pan-specific phosphorylation of Fes/Fps protein-tyrosine kinase (Fes) . αB-crysIn summary, we report for the first time that BTHS lymphoblasts, which exhibit a 90% reduction in CL and increased oxPC, express a markedly distinct phosphokinome profile compared to age-matched control cells. We hypothesize that the phosphokinome profile of BTHS cells may provide novel insight into the pathophysiology of BTHS and identify potential therapeutic targets.2 in a Thermoscientific Steri-cycle CO2 incubator HEPA Class 200 . Cell media was changed every 48 h and the cells were passaged every five days until mass spectrometry or phosphokinome analysis was performed. Mass spectrometry of phospholipids in control or BTHS lymphoblasts was performed in triplicate exactly as described [Age-matched control Epstein-Barr-virus-transformed lymphoblasts and BTHS lymphoblasts  were a generous gift from Richard Kelly . RPMI 1640 media and other cell culture components and supplements were from Life Technologies Inc. . Cells were grown in RPMI 1640 medium containing 10% FBS and 1% antibiotic/antimycotic (A/A) solution at 37 °C and 5% COTM KAM-880 antibody microarray with phospho-specific antibodies as indicated on the Kinexus website (http://www.kinexus.ca). The KAM-880 chip uses 877 distinct antibodies (~518 pan-specific and ~359 phosphosite-specific) for protein kinases and other cell signaling proteins. Data was expressed as average percent change from the control sample (%CFC) and target proteins were analyzed using the Kinections Pathway Map analysis feature to determine the category of protein family. PhosphoSitePlus® (https://www.phosphosite.org) was used to find the information regarding the protein posttranslational modifications (PTMs).Phosphokinome analysis was performed in duplicate by Kinexus Bioinformatics Corporation  using 50 μg of lysate protein from control or BTHS lymphoblasts using Kenex


The electron spin degree of freedom can provide the functionality of “nonvolatility” in electronic devices. For example, magnetoresistive random access memory (MRAM) is expected as an ideal nonvolatile working memory, with high speed response, high write endurance, and good compatibility with complementary metal-oxide-semiconductor (CMOS) technologies. However, a challenging technical issue is to reduce the operating power. With the present technology, an electrical current is required to control the direction and dynamics of the spin. This consumes high energy when compared with electric-field controlled devices, such as those that are used in the semiconductor industry. A novel approach to overcome this problem is to use the voltage-controlled magnetic anisotropy (VCMA) effect, which draws attention to the development of a new type of MRAM that is controlled by voltage (voltage-torque MRAM). This paper reviews recent progress in experimental demonstrations of the VCMA effect. First, we present an overview of the early experimental observations of the VCMA effect in all-solid state devices, and follow this with an introduction of the concept of the voltage-induced dynamic switching technique. Subsequently, we describe recent progress in understanding of physical origin of the VCMA effect. Finally, new materials research to realize a highly-efficient VCMA effect and the verification of reliable voltage-induced dynamic switching with a low write error rate are introduced, followed by a discussion of the technical challenges that will be encountered in the future development of voltage-torque MRAM. The evolving information society has triggered the rapid spread of advanced technologies, such as Artificial Intelligence (AI), Advanced Safety Vehicle (ASV), and IoT (Internet of Things), and this has led to further industrial innovation. In the society of the future, Big-Data collected from physical space will be stored and analyzed in cyber space, which creates new social values. Such a data-driven society can only be sustained by the high-speed processing of Big-Data; therefore, reducing the power consumption of nano-electronic devices is becoming increasingly crucial. One promising approach is the introduction of nonvolatile computation.It is expected that the stand-by power of future computing systems will be reduced by utilizing the nonvolatile features of spintronic devices, such as a magnetoresistive random-access memory (MRAM) while using magnetic tunnel junctions (MTJ). An MTJ consists of two ferromagnetic layers that are separated by an ultrathin insulating layer, such as magnesium oxide (MgO) ,2. Elects-d exchange interaction between the conduction electron spin and localized spin, called the spin-transfer torque (STT) effect [MRAM has great potential to be a fast, high write endurance, and CMOS-compatible nonvolatile memory, which is suitable for embedded as well as standalone memory applications. However, one of the significant remaining challenges is to reduce the energy that is needed to write information, that is, to switch the magnetization. In the long history of magnetism, magnetic fields that are produced by electric-current have been used for magnetization reversal. This indirect approach is inefficient and not scalable. Spintronics has brought us a new way of switching the magnetization through the ) effect ,5,6,7,8.) effect ,10,11,127kBT . On the other hand, the energy that is required to maintain magnetic information, i.e. the thermal stability, is about 60 kBT  has brought a drastic reduction in writing energy and expanded potential for applications; STT-MRAM ,14,15. FVarious kinds of approaches to the electric-field manipulation of spin have been proposed and experimentally demonstrated, such as using the inverse magnetostriction effect in a multilayered stack with piezoelectric materials ,17,18, tThis paper reviews recent progress in the research of the VCMA effect and the challenges that are faced in developing new types of MRAM controlled by voltage, called voltage-torque MRAM -RAM) ,37,38,39d transition ferromagnetic layer in 2007 [p orbitals near the Fermi level, which are coupled to the d states, play an important role [d orbital induced by the accumulated charges at the interface causes a change in the perpendicular magnetic anisotropy (PMA) [Weisheit et al. first reported the VCMA effect in a 3 in 2007 . They ob in 2007 . Theoret in 2007 . Nakamurant role . Tsujikapy (PMA) . Other ppy (PMA) ,45 and epy (PMA) .80Co20 layer is fixed at 0.58 nm. The bias direction is defined with respect to the top ITO electrode. A clear change in the saturation field in the out-of-plane direction can be seen, which suggests a modification in the PMA. Under the application of a positive bias, the PMA is suppressed and the in-plane anisotropy becomes more stable. On the other hand, the application of a negative voltage enhances the PMA and even the transition of the magnetic easy axis can be realized from the in-plane to the out-of-plane direction.We attempted to apply the VCMA effect in an all solid state structure, which consisted of epitaxial Au/ultrathin Fe(Co)/MgO/polyimide/ITO junctions grown on MgO(001) substrates see a to inveDue to screening by free electrons, the penetration of the electric-field into a metal is limited to the surface, unlike in the case of a semiconductor; however, if the thickness of the ferromagnetic layer is thin enough, e.g. several monoatomic layers, the modulation in the electronic structure at the interface can make a sizable impact on the magnetic properties. Details of an experimental verification for the physical origin of the VCMA effect are discussed in 80Co20(0.5 nm)/MgO(tMgO)/Fe grown on a MgO(001) substrate [KPMA, was evaluated from the resonant frequency of the free layer at each applied voltage. In addition to FMR measurements, the effect of a bias voltage on normalized TMR curves has also often been used for the quantitative evaluation of the VCMA effect, as discussed later [E, which is defined as the applied bias voltage, Vbias, divided by the MgO thickness, tMgO. The slope of the linear relationship represents the VCMA coefficient in units of J/Vm, e.g. −37 fJ/Vm for the case in One great advantage of the VCMA effect is its high applicability in a MTJ structure, which is the most important practical devices in spintronics. ubstrate . Here, wed later . GeneralThe realization of the VCMA effect in all-solid state devices, including a MTJ structure, made it possible for us to demonstrate the high speed response of this effect, such as in voltage-induced ferromagnetic resonance excitation ,52,53,54In addition to ultrathin epitaxial films with large PMA ,65,66,67The VCMA effect can induce a transition of the magnetic easy axis between the in-plane and out-of-plane directions by the application of a static voltage; however, bi-stable switching is not easily attained, because the VCMA effect does not break the time reversal symmetry. One possible way is to use the VCMA effect to assist other external fields. For example, the coercivity of the perpendicularly magnetized film can be reduced by the application of dc voltage ,96,97 orHbias. When a short pulse voltage is applied to eliminate the PMA completely, the magnetization starts to precess around the Hbias  as a function of pulse width, as shown in PSW is obtained at the timing of half turn precession; however, when the pulse width is twice this, one turn precession results in low PSW. From a practical point of view, the first half turn precession is effective in obtaining a low WER with fast switching speed. Under the condition that the PMA is completely eliminated, the amplitude of Hbias determines the precession frequency, and then the switching time, tSW for the half turn precession is expressed asα, γ, and μ0 are the magnetic damping constant, the gyromagnetic ratio, and the permeability of vacuum, respectively.The possible advantages of voltage-induced dynamic switching are as follows. (ⅰ) Fast switching ( ~1 nanosecond) can be induced with an ultralow switching power of the order of a few fJ/bit. (ⅱ) The switching transistor can be downsized, because we do not need to apply a large electric-current. (ⅲ) Unipolar switching can separate the polarity of voltages for writing and reading. In addition, the VCMA-induced enhancement in PMA has been used to propose a unique approach to reduce the read disturbance .On the other hand, the following technical challenges remain. Firstly, the realization of a large VCMA effect is the most important issue to show the scalability of the voltage-torque MRAM, as discussed in In this section, recent experimental trials conducted to understand the physical origin of the VCMA effect are introduced . The folλ is the spin-orbit interaction coefficient. L and T′ are the orbital angular momentum and part of the magnetic dipole operator, respectively. Here, z- and x- components of the spin angular momentum, respectively. The same is the case for zT term (mT), respectively, the aforementioned two mechanisms can be validated by X-ray absorption spectroscopy (XAS) and X-ray magnetic circular dichroism (XMCD) spectroscopy.Here, adrupole b. An elep core to d valence states, which is related to XMCD at the L edges of transition metals. The dichroic signal directly reflects the difference in the density of the states near the Fermi level between the up and down spin sub-bands. From the XMCD results with sum-rule analysis [mS, mL, and mT) can be determined from the measured XAS/XMCD spectra. Here, the measured orbital magnetic moments and magnetic dipole zT term have the following relationships;The XAS/XMCD experiments provide element-specific information on the electronic structure via the optical transition from the core level to unoccupied states in the valence band. Based on the use of circularly polarized X-rays, X-ray absorption techniques provide interesting features for the study of magnetic materials. analysis ,118, thezT term.It should be noted that the PMA energy from the spin-conserved virtual excitation process (first term in Equation (2)) is related to the orbital magnetic moment and the PMA energy from the spin-flip virtual excitation process (second term in Equation (2)) is related to the magnetic dipole 2 (5 nm)/Cr (2 nm)/Au (5 nm), was deposited on a MgO(001) substrate. L3 and L2 edges of Co with a magnetic field of 1.9 T (θ = 20°) to saturate the magnetization of the Fe/Co layer. The changes in the orbital magnetic moment and effective spin magnetic moment (mS − 7mT) of Co were determined while using sum-rule analysis, and they are summarized in mL of Co with an electric-field of −0.2 V/nm is larger than that corresponding to +0.2 V/nm. Moreover, the induced change in mL with θ = 20° is larger than that with θ = 70°. The experiment demonstrates that an orbital magnetic moment anisotropy change of (0.013 ± 0.008)μB between the magnetization angles of θ = 20° and 70° was generated in the presence of applied electric fields of ±0.2 V/nm. mS − 7mT of Co. As with mL, mS − 7mT is enhanced under the application of a negative electric-field. Moreover, the electric-field-induced change in the magnetic moment is anisotropic. In contrast to mT, it is known that mS is not sensitive to the magnetization direction. Hence, the anisotropic part of the induced change in the magnetic moment should be attributed to mT. A Fe/Co (1 ML)/MgO multilayer was employed to see the changes in the orbital magnetic moment in XAS/XMCD experiments . The samλCo = 5 meV, then the induced change in the PMA energy is estimated to be 0.039 ± 0.023 mJ/m2 when the applied electric-field is switched from +0.2 V/nm to −0.2 V/nm. Here, the experimentally obtained ΔmL = (0.017±0.010)μB was used. From the VCMA coefficient in the Fe/Co/MgO system (−82 fJ/Vm), the PMA energy change at ±0.2 V/nm is 0.03 mJ/m2, which is in good agreement with the PMA energy change that was obtained using the first term of Equation (2). From the discussion above, the change in the orbital magnetic moment anisotropy in Co seems to explain the VCMA effect. However, the impact of the change in the magnetic dipole zT term (mT) that is shown in E↓↑ + ΔE↑↓) is found to be negligible and that from the spin-conserved terms (ΔE↑↑ + ΔE↓↓) appeared to be dominant. Therefore, the change in orbital magnetic moment is responsible for the VCMA effect. Due to the large exchange splitting for Co, the observed changes in mT do not contribute to the VCMA effect, as described by the second term in Equation (2).As discussed in the previous section, Equation (2) can be used to analyze the VCMA effect. If we employ the spin-orbit interaction coefficient of Co, d/5d-layered transition metals are employed [L3 and L2 energy edges of Pt. A perpendicular magnetic field of ±60 mT was applied to saturate the magnetization of FePt. mS − 7mT, while there is no significant change to mL under voltage applications.It has been reported that the spin-orbit interaction energy from a spin-flip virtual excitation process makes a significant contribution to the VCMA effect when 3employed . Figure Tz term (mT) [mT, mS is not sensitive to the magnetization direction. In Ref. 116, the voltage-induced change in mS − 7mT shows large magnetization direction dependence. Thus, the observations indicate the significant induction of mT in Pt by an external voltage. A first-principles study was also conducted for the FePt/MgO system, similar to the Fe/Co/MgO study. As a result, firstly, the monoatomic Pt layer at the interface with MgO makes the dominant contribution to the VCMA effect. Moreover, while the VCMA effect from the spin-conserved terms (ΔE↑↑ + ΔE↓↓) decreases the PMA energy, the VCMA effect that is induced by the applied voltage from the spin-flip terms of interfacial Pt increases the PMA energy (ΔE↓↑ + ΔE↑↓). The total PMA energy in the FePt/MgO system increases under the condition of electron depletion at the Pt/MgO interface, as the PMA energy increase by the spin-flip terms is greater than the PMA energy decrease by the spin-conserved terms.In general, in low-symmetry systems, such as interfaces, the atomic electron orbital may possess an electric quadrupole moment. If the atom is also spin-polarized, the electric quadrupole moment induces the anisotropic part of the spin-density distribution, i.e., the magnetic dipole erm (mT) ,116,118.d-transition ferromagnetic metals, it is important to consider the orbital magnetic moment anisotropy. The validity of the Bruno model [d/5d-multilayered ferromagnetic metals, the orbital magnetic moment anisotropy in 3d-metals cannot completely explain the VCMA effect. In addition to the magnetic moments in 3d metals, those in 5d metals should be considered in treating the total PMA energy in the system. Moreover, both the orbital magnetic moments and the electric quadrupole mechanisms (second term of Equation (2) and L10-FePt, as shown in Ref. 116. As discussed in the recent review paper [To conclude, for the 3no model  /Pt (3 nm)/Co (0.9 nm)/GdOx (33 nm)/Ta (2 nm)/Au (12 nm) multilayer and found that an applied voltage changes the oxidation state and magnetization of the Co. Ref. 125 also reports real-time measurements of such an electrochemical VCMA effect. The operating speed strongly depends on the applied voltage and temperature, which strongly indicates that the electrochemical VCMA requires a thermal activation process. The reported maximum speed was in the sub-millisecond range. Therefore, such large values of the electrochemical VCMA seem attractive, but lie beyond the scope of VCMA studies for working memory applications. A similarly large VCMA effect with limited operating speed has been observed in many systems with electrochemical reactions [A much larger VCMA coefficient can be obtained when compared with that of purely electronic origin if we use a chemical reaction ,125. Foreactions ,126,127 eactions ,129.Recently, strain-induced modulation of electronic structures and its influence on the VCMA effect has attracted attention ,131. ForThe VCMA coefficient is one of the most important parameters for the scalability design of voltage-torque MRAM. When the cell size is reduced, we need to increase the PMA of the free layer to maintain the target thermal stability. As described in tfree and MS are the thickness and saturation magnetization of the free layer. iK(E) is the PMA under application of the electric-field (E), and it is given byη is the VCMA coefficient. The thermal stability Δ(Ε) of the free layer under the application of the electric-field can be expressed byHere, A and Δ0 are the area of the free layer and the thermal stability under zero electric-field, respectively.Here, η, which is required to eliminate Δ0 can be expressed as,ESW is the amplitude of the switching electric-field. Consequently, the VCMA coefficient, tfree = 1 nm and ESW = 1 V/nm for each value of Δ0. If we take cache memory applications as an example, the required KPMAtfree values range from 0.2 mJ/m2 to 0.5 mJ/m2, depending on the target Δ0 values; consequently, the required VCMA coefficient is estimated to be from 200 fJ/Vm to 500 fJ/Vm. The main memory applications need higher KPMAtfree values in the range from 0.6 mJ/m2 to 1.5 mJ/m2. As a result, the required VCMA coefficient is in the range from 600 fJ/Vm to 1500 fJ/Vm. However, in experiments that have only focused on the purely-electronic VCMA effect, the achieved VCMA coefficient that is demonstrated in practical materials, such as CoFeB, has been limited to about 100 fJ/Vm [For the curves in 00 fJ/Vm ,78,81,98tFe)/MgO (tMgO = 2.3 nm)/Fe(10 nm) on MgO(001) substrates. Here, the bottom ultrathin Fe layer is the voltage-controlled free layer with perpendicular magnetic easy axis and the top 10 nm-thick Fe is the in-plane magnetized reference layer. Vbias, and dependence of the half-MR loop measured under an in-plane magnetic field, Hex. The vertical axis is normalized using the maximum (exH = 0 Oe) and minimum (Hex = −20 kOe) resistances. The Fe thickness is fixed at tFe = 0.44 nm.We employed a fully epitaxial Cr/ultrathin Fe/MgO system as a standard system for the materials research of VCMA effect , becauseG, depends on the relative angle (θ) between the magnetizations of the free and reference layers, i.e. G(θ) = G90 + (GP−G90)cosθ. Here, G90 and GP are the conductance under the orthogonal and parallel magnetization configurations. Therefore, the ratio of the in-plane component of the magnetization of the free layer, Min-plane, to its saturation magnetization, MS, is expressed asRP is the MTJ resistance in the parallel magnetization configuration, R90 is the MTJ resistance in the orthogonal magnetization configuration, and R(θ) is the MTJ resistance when the magnetization of the ultrathin Fe layer is tilted towards the in-plane direction at angle θ under the application of an in-plane magnetic field. Using Equation (8), we can evaluate the normalized in-plane magnetization versus the applied magnetic field. The inset in M-H curve measured under Vbias = 10 mV. The PMA energy, KPMA can be calculated from Min-plane (H) with the saturation magnetization value evaluated by SQUID measurements , and the usual linear VCMA effect appears for thicker layers (red dots). Xiang et al. systematically investigated the tunneling conductance, the PMA, and the VCMA effect in a similar system to determine the origin of the nonlinear VCMA effect, but the MgO was replaced by a MgAl2O4 barrier, which has smaller lattice mismatch with Fe. Interestingly, they found strong correlation between the VCMA effect and the quantum well states of Δ1 band formed in an ultrathin Fe layer that is sandwiched between the Cr and MgO layers [The application of an in-plane magnetic field tilts the magnetization of the ultrathin Fe layer into the magnetic hard axis, while that of the reference layer remains in the film plane /Ir(tIr)/MgO (2.5 nm) with indium-tin oxide (ITO) or Fe (10 nm) top electrodes to investigate the impact of the introduction of Ir on the interfacial PMA and the VCMA effect [Z-contrast HAADF-STEM imaging enabled the identification of inter-diffused Ir atoms as bright spots that are indicated by yellow arrows. The first-principles calculation predicts strong in-plane anisotropy at the Ir/MgO interface [tFe = 1.0 nm) and an Ir-doped Fe layer formed the bilayer structure consisting of Fe (1.0 nm)/Ir (0.1 nm)). The pure Fe layer exhibits large saturation fields of about 7 kOe, which indicated an in-plane magnetic easy axis. On the other hand, the introduction of the quite thin Ir doping layer resulted in transition of the magnetic easy axis from the in-plane to the out-of-plane direction. Ki,0, on the thickness of the Ir layer. With appropriate Ir doping, Ki,0 reaches 3.7 mJ/m2, which is about 1.8 times that observed at the Fe/MgO interface (2.0 mJ/m2) [We prepared multilayer structures consisting of Cr (30 nm)/ultrathin Fe ,134. tFeIr = 0.82 nm; formed from Fe (0.77 nm)/Ir (0.05 nm)). The saturation field shifts with changes in the applied voltage, as is the case in a pure Fe/MgO structure. However, the applied electric-field dependence of KPMAtFeIr exhibits a completely different trend when compared with that observed in the Fe/MgO structure. We observed a large reduction in PMA with a VCMA coefficient of −320 fJ/Vm under positive voltages /MgO(5ML) structures to discuss the physical origin of the large VCMA effect in Ir-doped Fe. The Ir-doped bcc Fe was modeled by a supercell consisting of 4×4 unit cells as shown in A theoretical analysis using first-principles calculation was performed in Cu(5ML)/Fe↑↑ is larger than that for the minority spin states δE↓↓. On the other hand, the spin-flip terms that are by the electric-field, δE↑↓ and δE↓↑ have almost the same absolute value, but with opposite sign, so the VCMA effect that arises from the spin-flip term is small. Therefore, the large VCMA effect in Ir-doped Fe is mainly caused by the electric-field effect on the majority spin Ir-5d states and it can be interpreted by the modulation in the first term of Equation (2), i.e. the orbital magnetic moment mechanism.We also attempted to divide the MAE into contributions from the spin-flip and spin-conserved terms between the occupied and unoccupied states. d states are dominant near the Fermi level, since the minority spin 5d states near the Fermi level form bonding and anti-bonding states by hybridization with the minority spin Fe-3d states. On the other hand, the majority spin 5d states are well-localized when compared with the minority spin states near the Fermi level. d and 5d elements, therefore materials engineering using heavy metal doping has enormous possibilities for further improvement in the interfacial PMA and VCMA properties.The theoretical calculations predict the larger VCMA effect exceeding a few thousand fJ/Vm by inserting a monolayer of Ir at the Fe/MgO interface; however, such a structure can drastically degrade the TMR properties in the MTJ device, in addition to the strong in-plane anisotropy. On the other hand, if Ir doping can improve both the PMA and the VCMA effect while minimizing degradation in TMR, the MTJs should be much more manufacturable, even by sputtering processes. In fact, the enhancement of the PMA and the VCMA effect by Ir doping has also been confirmed in polycrystalline MTJs that are mainly prepared by sputtering . We stilIn this section, recent experimental trials for reliable voltage-induced dynamic switching are discussed. As shown in Although earlier experiments have characterized the basics of voltage-driven magnetization switching, it was only in 2016 that the WER in a practical MTJ was quantitatively evaluated for the first time . Figure Psw is the switching probability, tpulse is the pulse width; and, Vpulse is the voltage amplitude. When Vpulse is small, the VCMA effect cannot completely eliminate the magnetic energy barrier; therefore, the magnetization switching in this region is dominated by thermal activation. As Vpulse is increased, well-defined oscillation of Psw appears, which is a signature of precession-mediated switching induced by the VCMA effect. As discussed in Psw is obtained at tpulse that corresponds to one-half the magnetization precession cycle, and then Psw gradually moves toward 0.5 while undergoing damped oscillations. This behavior can be understood as the combined action of magnetization damping and thermal fluctuations. 80B20/W cap and experimentally demonstrated a WER of 4 × 10−3. They also demonstrated in numerical simulations that the WER could be reduced by improving the thermal stability factor, Δ and by reducing the magnetic damping, α of the free layer, as shown in −9 for 10 ns total write time by introducing the read verify processes. The introduction of read verify processes makes it possible to reduce the effective WER, however it causes an increase in the total writing time. Therefore, we need further effort to reduce the essential WER that is induced by single pulse switching. Recently, Shiota et al. showed that improvement in the PMA and VCMA properties can be achieved in the MTJ consisting of Ta/(Co30Fe70)80B20/MgO/reference layer, and demonstrated a WER of 2 × 10−5 without the read verify process [−6 to be achieved, as shown in −12.In Ref. 105, Shiota et al. employed perpendicularly magnetized MTJ (p-MTJ) that consisted of a reference layer/MgO/Fe process . FurtherM, and its time evolution can be obtained by numerically solving the Landau-Lifshitz-Gilbert equation: Ms is the saturation magnetization, t is the time, α is the damping constant, and Heff is the effective field given byE is the energy density expressed asm =  is the magnetization unit vector and xH is an in-plane bias magnetic field. As displayed in xm = −1, and one saddle point at xm = 1. By letting KPMA fall to zero, the magnetization precesses around xH associated with damping, and the appropriate duration can switch the magnetization direction. In addition to materials engineering, a physical understanding of the voltage-driven magnetization dynamics is also needed in order to facilitate reductions in the WER. Recent studies ,108 showtpulse that was observed in an MTJ consisting of a Ta/(Co30Fe70)80B20 (1.1 nm)/MgO/reference layer. The amplitude of the in-plane component of the bias magnetic field is 890 Oe. The filled circles and the line denote data were obtained from experiments and numerical simulations, respectively [tpulse, which cannot be explained just by considering the VCMA effect. A detailed analysis of the magnetization trajectory revealed that thermal agitation during the relaxation process  induces the transition of the magnetization between the precession orbits surrounding the energy minima and that the precession-orbit transition enhances the WER. The numerical simulations also revealed that the probability of the precession-orbit transition depends on tpulse (see Ref. 107 for more details). In the present case, the probability is maximized at around tpulse = 0.12 ns. This results in the appearance of a local maximum in the WER, and it narrows the operating tpulse range for which reliable magnetization switching is assured. As the appearance of the WER local maximum is related to magnetization fluctuations during the relaxation process, we need to reduce its influence by improving the PMA and VCMA properties in order to achieve a wide operating tpulse range.ectively . Good agtpulse, a recent study revealed that the WER depends in a unique manner on the rise time (trise) and fall time  [m+ towards m− (red line) and, after the pulse, the magnetization relaxes to either tpulse (green line). An important thing is that, due to the nonzero magnetization damping, the magnetization direction at the end of the voltage pulse (m′) never reaches tpulse is chosen as long as one uses square pulses , which is given byIn addition to   . Figure e pulses a. TherefHani switches according to the polarity of zm, it applies additional torque to the magnetization that tilts the magnetization to xH during trise  effect is a promising approach to realizing voltage-torque MRAMs. Bi-stable magnetization switching has been demonstrated while using precessional dynamics that are induced by the VCMA effect. The purely-electronic VCMA effect originates from electric-field induced modification of the electronic structure at the interface between an ultrathin ferromagnet and a dielectric layer, such as MgO. In a 3


Escherichia coli  and Penicilliumcyclopium . Under the central composite design (CCD) of the response surface methodology (RSM) tool, the parameters of the hybrid synthesis reaction were optimized and the result obtained was as follows: reaction pH was 11.3, reagent Zn/Al ratio was 3.27, and chitosan concentration was 1.07 g/L. After optimization, it was found that the antibacterial activity of ZnAl/CS was strengthened against E. coli as evidenced by a widening of the inhibition zone of about 41.6%. The antibacterial activity of ZnAl/CS was mainly due to the reactivation of the antibacterial activity of CS associated with the release of Zn2+ and Al3+ metal ions in addition to ZnO, Al2O3, and ZnAl2O4 compounds resulting from the method of preparation.The menace of antimicrobial resistance continues to increase and hence the need to discover new antibiotics, especially alternative and effective sources such as hybrid organic-inorganic, organic-organic materials, and other combinations. In this study, an antimicrobial hybrid supra-nano material was prepared by the bi-titration synthesis method of chitosan (CS) and ZnAl layered double hydroxide. Fourier-transform infrared spectrometer (FTIR), thermogravimetric and differential thermal gravimetric (TGA/DTG), ultraviolet-visible (UV-Vis), X-ray diffraction (XRD), and scanning electron microscopy (SEM) analyses indicated that the ZnAl/CS hybrid exhibited low crystallinity with high thermal stability. The results of ZnAl/CS characterization showed the characteristic properties of the individual components ZnAl and CS, indicating a successful preparation of the ZnAl/CS hybrid. The antibacterial tests revealed that the ZnAl/CS hybrid possessed an enhanced antimicrobial effect against both  Microbial pathogens are one of the leading causes of human morbidity in the world . Unlike Recently, the use of hybrid materials at the supra-nanometric scale is an increasingly used approach in the fight against antimicrobial resistance ,4,5. TheZnAl consists of positively charged brucite-type layers of divalent and trivalent metal hydroxides, whose excess positive charge is compensated by anions and water molecules present in the interstitial position. Among the heterostructured nanomaterials, layered double hydroxide (LDH) was the subject of sustained attention as an inorganic component applied in many fields due to its unique physicochemical and mechanical properties, which cannot be obtained from other similar compounds .Escherichia coli strains cause a variety of human diseases, which result in more than 2 million deaths each year   . The Zn2released a in the otassium b in the m fungus . The mety of ZnO . This woclopium) . The bi-E. coli.Based on previous experimental results (above) performed via “one-factor-at-a-time” experiment type, RSM was used to optimize the preparation conditions and obtain sufficient antibacterial activity from the selected materials. The selected effective variables were pH (P), ratio ZnAl (R), andCS concentration . The experimental design suggested by central composite design (CCD) was applied because it helps to optimize effective parameters and analyze interactions between parameters and also produces results within a minimum of number of experiments. The bacterium chosen to conduct this part of the study was E. coli, with different sizes of inhibition zones. A statistical analysis of variance (ANOVA) was conducted by using a software to investigate not only the suitability and relevance of the model, but also the effects of individual variables on interaction effects on the response . ANOVA showed  indicated the good precision and reliability of the experiments [The antibacterial effectiveness experiment was conducted using the CCD design, and the results are presented in A showed  that a qeriments  as well p ≤ 0.0001.When considering the independent variables individually, CCD study indicated that each parameter had a determinant role in the formation and inhibition efficiency of the antibacterial compound ZnAl/CS. Indeed, P strongly affected the formation of ZnAl/CS, first through the dissolution of CS which took place in an acidic medium, and finally through the connection between ZnAl and CS which occurred in an alkaline medium. A lack of control about this factor P, led to a modification and/or stoppage in the formation of ZnAl/CS, inducing a final compound inefficiency through a rather poor inhibition zone. Regarding R, a ratio lower or superior to 3 influenced the width of the zone of inhibition differently. With R < 3, ZnAl/CS produced a very small inhibition zone. At R > 3, the inhibition zone was enlarged, while remaining largely lower than the ZnAl/CS zone when R = 3. C represented the perfect dosage of CS at which ZnAl should be associated to produce a compound with a more effective antibacterial capacity. According to the experiment, the zone of inhibition of ZnAl was lower than that of ZnAl/CS, which showed the importance of controlling C. In terms of the significant coefficients, the independent variables P, R, and C were all highly significant with p-values< 0.05 were both significant terms for influencing the inhibition ability (inhibition zone ZI) of ZnAl/CS. It was quite remarkable that all of the significant interaction terms contained the independent variable C, implying that C played a key role in the process of optimization or improvement of ZnAl/CS inhibition ability. The quadratic terms P2, R2, and C2 were all highly significant (p < 0.0001). After analyzing the independent and dependent variables, we obtained a regression equation that could predict the response under a given range. The linear regression equation was obtained, after elimination of insignificant terms to improve the regression model for the responses (zone of inhibition). The quadratic model Equation (1) in terms of the coded factors was given as follows:In terms of interaction, PC and RC with To study the interaction between parameters in depth, the quadratic model’s regression equation graphical representation was used. The three-dimensional response surface plot in question was also used to determine the possible optimum conditions of each factor in order to maximize the ZnAl/CS inhibition capacity. The model confirmed the existence of significant particular interactions principally between C and the two other terms P and R. We further characterized the interaction in the range of the process variables. After analyzing the influencing variables and the appropriate model, the next step was to accurately determine the corresponding value of each important parameter individually or in a set that can improve the antibacterial inhibitory capacity of ZnAl/CS and to achieve a zone of inhibition broader (larger) than the previous one. This optimization was done via RSM, by applying a canonical analysis which is one of the multivariate linear statistical analyses used to locate the stationary point of response surface and to determine whether it represents a maximum, minimum, or saddle point. P: 11.27, R: 3.27, and C: 1.07 g/L were the optimal conditions for obtaining the widest zone of inhibition as predicted by the model. The theoretical inhibition zone predicted using the above conditions was 17.07 mm. In order to validate the applicability of the model, a series of experiments was carried out using the optimal parameter values suggested by the conical analysis, P: 11.27, R: 3.27, and C: 1.07 g/L. As a result, a relatively higher experimental value, but close to that predicted by the model, was obtained, see P. cyclopium and E. coli. The ZnAl/CS hybrid was more effective against P. cyclopium fungi with a zone of antimicrobial inhibition of 29 ± 0.7 mm (bi-titration) against 22.3 ± 0.5 mm (urea method of preparation) and less effective against E. coli compared to the previous study with a zone of inhibition of 17.3 ± 0.65 mm (bi-titration preparation method) against 24.2 ± 0.8 mm (urea method of preparation).One of the most striking differences between this study and our previous study  was the The urea method of preparation is a high crystallinity method with more complete bonds leading to firmly attached components . This leIn contrast to the urea method of preparation, the bi-titration preparation method produces weak crystallinity but better heat stability material , in addiE. coli growth cycle extends over a period of 24 h, we set the duration of the experiment to a growth cycle, that was why the incubation time was 24 h. This study was carried out in a liquid medium, so it was possible to deduce a kinetic model showing the amount of potassium released as a function of the microbial colony reduction rate. Also, potassium release by E. coli has already been studied and a kinetic model has already been established and described in the form of: 10 μM K+/min for 0.5 mg/mL of propyl paraben. This previous study was able to relate the variation in the size of the inhibition zone as a function of the quantity of potassium released without establishing an equational model [Since the al model .E. coli. The optical density at 600 nm (OD600) was used to measure and control bacterial growth. All the results confirmed the antibacterial efficacy of ZnA1/CS. This was further confirmed, when beyond the addition of 8 mg/mL of ZnAl/CS, the optical density no longer varied as a function of time, which indicated the total inhibition of bacterial growth  was purchased from Sinopharm Chemical Reagent Co., Ltd. . AlCl3.6H2O (>97%) and KOH were obtained from Heng Xing Chemical Preparation Co., Ltd . ZnCl2 (98%) was purchased from Fuchen Chemical Reagent Factory Co., Ltd . NaOH (≥96%) was procured from Xilong Chemical Co., Ltd . Lactic acid (Mw 90.07) was purchased Shanghai Meryer Chemical Technology Co., Ltd .Chitosan from shrimp shells (Mw 1.5 × 102 was carried out by means of a gas-washing bottle filled with a KOH solution. All solutions were prepared with deionized water (resistivity of 18.2 mX cm) which was obtained with a Millipore ultra-pure water system, previously distilled and decarbonized by boiling plus bubbling N2. The compounds studied in this study were labeled as CS, ZnAl, and ZnAl/CS.The ZnAl/CS hybrid was prepared using a co-precipitation technique proposed by Depan and Singh, 2010  with sli−1. Powder samples were molded in potassium bromide KBr pellets and analyzed at a resolution of 2 cm−1.The FT-IR spectra of CS, ZnAl, and ZnAl/CS samples were obtained with Perkin-Elmer Spectrum GX in the wave number ranging from 4000 to 400 cm−1.Thermogravimetric and differential thermal gravimetric analyses (TGA/DTG) were performed on a Perkin-Elmer TGA-7thermal analyzer in a temperature range of 50 to 900 °C. 10 mg of each composite sample was loaded in an open ceramic crucible, and heated under nitrogen atmosphere at a heating rate of 20 °C min−2 g/L) was prepared by adding a stoichiometric amount of 10−1 M perchloric acid to the chitosan solution. Given the water content and the degree of deacetylation of the chitosan, the chitosan–perchloric acid solution was then stirred at an appropriate speed to complete the dissolution before recording the measurements. Samples of ZnAl and ZnAl/CS were prepared by depositing the aqueous dispersions onto quartz glass slides and drying them in air. The concentration of ZnAl and the ratio of ZnAl to ZnAl/CS were the same for both the UV-Vis spectra.Measurements of the UV–vis spectra at room temperature for CS, ZnAl, and ZnAl/CS solids were recorded on a SHIMADZU UV-2550 spectrophotometer . In order to record the measurements, a solution of chitosan  D/max-2550PC, with Cu-Scanning electron micrographs (SEM) were obtained through a JEOL JSM-6700F instrument . The samples  were spread on a double-sided conducting adhesive tape that was pasted to a metallic stub, coated with gold (100 µg), and then observed atan accelerating voltage of 20 kV.E. coli MTCC 739 and P. cyclopium AS 3.4513 were kept at the Xiangtan University General Microbiological Culture Collection Center. The fungus P. cyclopium strain was incubated on potato dextrose (PD) agar medium at 23 °C for 3 days before dilution and inoculation, while E. coli, the Gram negative strain, was maintained in mineral salt (MS) media, pH 7.0, at 37 °C for 24 h before multiple dilution and use.E. coli bacteria and P. cyclopium fungi were used as target organisms. Müller–Hinton broth (MHB)  was chosen to prepare the microbial suspension; the turbidity of the suspension was balanced with 0.5 McFarland standard. The broth plates were inoculated with the microorganisms (bacteria and fungi) and slightly perforated with a glass capillary to create wells that were filled with 1.0 cm diameter of CS, ZnAl, and ZnAl/CS. The pills  were irradiated under a UV lamp for 30 min to eliminate microbial contamination. Each sample was added into a 10 mL Luria-Bertani LB liquid culture medium which was then inoculated with the microbial suspension. All antimicrobial experiments involving both cultures were carried out in sterile 250 mL shake flasks or 90 mm agar plates. Indeed, 100 mL cultures of P. cyclopium and E. coli strains were partially transferred onto PD and MS media in order to perform the antimicrobial tests. The plates were incubated for 24 h at 37 °C for E. Coli and 72 h at 28 °C for P. cyclopium. Finally, the inhibition zone surrounding the wells (in millimeters) was measured to evaluate antibacterial activity.The antibacterial efficiency of ZnAl/CS was evaluated through determining the antibacterial susceptibility, by the agar well diffusion method, of bacteria after exposure to the tested samples. Gram-negative The response surface methodology (RSM) is a powerful statistical tool. It relies on quantitative data from an accurate fact-finding conception to simultaneously determine and solve multivariate equations. Its advantage is the reduction of the number of experimental tests necessary to evaluate the impact of several parameters and the effect of their interactions. After deducting the best experimental conditions from the “one-factor-at-a-time” experimental method, RSM was applied to determine the effect of initial pH, ZnAl ratio, and CS concentration on the bacterial inhibition efficiency of the hybrid ZnAl/CS. The CCD tool of RSM was chosen for the experimental test with the three independent variables that were the initial pH (P), ZnAl (R) ratio, and chitosan concentration , while optimizing the zone of inhibition (ZI) as response variable (Y) of the antibacterial study. Each independent variable was coded at three levels between −1 and +1, while the variables initial pH (P), ZnAl ratio (R), and chitosan concentration  were changed in the ranges shown in E. coli was selected for the kinetic study as the index bacterium. The culture was incubated overnight at 37 °C. The bacterial suspension (106–107 CFU/mL) was treated with 1,3, 5, 7, and 10 mg/mL of CS, ZnAl, and ZnAl/CS hybrid. The samples were incubated at 37 °C in an aerobiosis and the bacterial growth rates were assessed by monitoring optical density at 600 nm (OD600) at regular time intervals using a UV–Vis spectrophotometer while using normal saline as a control.An antibacterial kinetic test was performed, and n = 3) of the standard error of mean. Response surface methodology (RSM) was employed to build the best model and optimize the antibacterial compound system using Design Expert version 8.0.5.0 (Stat Ease Inc. USA). One-way ANOVA for response surface model was carried out to assess the main effects of each parameter and its interactions, and the accuracy and quality of the fitted model.Each sample was analyzed individually in triplicate and the results were expressed as the mean value  with weak crystallinity, promoting an easy and fast dispersion of metal ions in the culture medium. Furthermore, improvement of antibacterial activity against E. coli by optimization via CCD of RSM was successful with a 41.6% widening of the inhibition zone. Finally, ZnAl/CS retained antimicrobial activity in a culture medium at pH greater than or equal to 6 with an antimicrobial activity that could be directly improved. ZnAl/CS appears to be a promising antimicrobial agent, which could be useful in many areas.Both methods used for the preparation of the ZnAl/CS hybrid in previous and current studies resulted in the synthesis of an antimicrobial compound. However, the hybrid had more or less different characteristics according to the preparation methods. In this study, ZnAl/CS exhibited a potent and prolonged antibacterial activity against


Natural gas hydrates are ice-like crystalline materials formed from natural gas and clathrate ice under high pressure and low temperature. Ice XVI, the first S-II type clathrate ice produced in the lab, was simulated by first-principles density functional theory with the CASTEP code. A 34-molecule supercell was built to mimic the hydrogen-disordered structure. The vibrational spectra were calculated as a reference for inelastic neutron scattering (INS), infrared (IR) absorption, and Raman scattering experiments. Two kinds of H-bond vibration modes corresponding to two different bond strengths were found in our previous studies. In this paper, the statistics of distribution calculated by integrating these two kinds of modes was found to match the phonon density of states (PDOS) very well. We confirmed that the two basic types of H-bonds also appeared in clathrate ice XVI. The typical normal modes were analyzed to illustrate the dynamic process of lattice vibrations. Ice XVI was later reported to have good mechanical stability and negative thermal expansion [Ice has a diverse presure temperature (P–T) diagram, with currently more than 17 crystal phases known under a variety of pressures and temperatures, including clathrate ice under negative pressure ,9,10,11.xpansion . However−9 hartree. The energy cut-off was set as 750 eV and the K-point separation was 0.07/Å. Norm-conserving pseudopotentials were used to calculate the phonon density of states (PDOS). We took the periodic structure of ice XVI from Ref. [1264. These rings were capable of enclosing large guest molecules. The geometry optimization was performed under zero pressure. Because RPBE slightly underestimates H-bond interactions, the density of the calculated crystal was 0.77 g/cm2, 5% lower than the experimental value. The H-bond lengths ranged from 1.797 Å to 1.845 Å, the length of O–H covalent bonds ranged between 0.984 and 0.990 Å, and the distance between adjacent oxygen ranged from 2.768 to 2.83 Å.Using the CASTEP code , a firstrom Ref. . The oriIn addition, we counted the numbers of four-H-bond and two-H-bond normal modes based on the calculated results using a purpose-designed, self-compiled program. To clearly characterize the complex vibration modes, which are a key property of ice hydrates, we distinguished the mode types by one molecule in the supercell possessing the largest amplitude. The standard classification of stretching modes in terms of the vibrational direction was applied. Finally, we plotted a distribution diagram to mimic the PDOS of these two types of H-bonds.Based on the harmonic approximation, the PDOS, infrared (IR) absorption, and Raman scattering spectra of ice XVI were calculated as shown in Because of the wide range of spectral intensities of Raman and IR, −1 to 320 cm−1 to mimic the PDOS spectrum of the H-bonds, as shown in The most interesting results are in the translational band. For a water molecule, there are three spatial vibration modes: stretching, wagging, and rocking. The mechanism of H-bond vibrations was the central topic in our previous studies ,27,28,29−1 shown in −1, the molecules undergo a combination of rocking and wagging along the H-bonded linkages. The vibration of each molecule only aligns with two H-bonds. Herein, the strength ratio of these two modes is approximately equal to −1 and 314 cm−1 to illustrate the dynamic process , and a slight blue-shift in the stretching region [Ice XVI, the first negative-pressure clathrate ice produced in the lab, was simulated using the first-principles DFT method. The vibrational spectra of INS, IR absorption, and Raman scattering were calculated theoretically. Four regions of the spectra are discussed individually together with some typical vibration modes. Compared with calculated results of ice Ih, its density is 0.77 kg/cmg region . In the Based on this study, we also found a rule for the number of vibration modes in the four vibration regions in the ice. Assuming that the number of molecules in one unit cell of ice is N, the total number of optical normal modes should be 3N × 3 − 3. The O–H stretching region contains 2N modes corresponding to the two kinds of molecular stretching: symmetric and asymmetric. The bending region contains N modes as there is only one such mode for each molecule. The librational band has 3N modes representing the twisting, wagging, and rocking vibrations. Finally, the number of optical normal modes in the translational band is 3N − 3. The coefficient is 3 because there are three spatial vibration modes for each molecule, while 3 acoustic modes must be subtracted. This is a general formula governing any ice phase.


It has been proposed that one of the mechanisms of taxane-site ligand-mediated tubulin activation is modulation of the structure of a switch element (the M-loop) from a disordered form in dimeric tubulin to a folded helical structure in microtubules. Here, we used covalent taxane-site ligands, including cyclostreptin, to gain further insight into this mechanism. The crystal structure of cyclostreptin-bound tubulin reveals covalent binding to βHis229, but no stabilization of the M-loop. The capacity of cyclostreptin to induce microtubule assembly compared to other covalent taxane-site agents demonstrates that the induction of tubulin assembly is not strictly dependent on M-loop stabilization. We further demonstrate that most covalent taxane-site ligands are able to partially overcome drug resistance mediated by βIII-tubulin (βIII) overexpression in HeLa cells, and compare their activities to pironetin, an interfacial covalent inhibitor of tubulin assembly that displays invariant growth inhibition in these cells. Our findings suggest a relationship between a diminished interaction of taxane-site ligands with βIII-tubulin and βIII tubulin-mediated drug resistance. This supports the idea that overexpression of βIII increases microtubule dynamicity by counteracting the enhanced microtubule stability promoted by covalent taxane-site binding ligands. Microtubule-based chemotherapy is one of the most effective treatment options for both solid tumors and hematological malignancies ,2 and coThe successful use of maytansine and auristatin loaded ADCs indicates that these potent cytotoxic agents are extremely effective anticancer drugs when used as the warheads of tumor-targeting antibodies. It could be speculated that compounds targeting the taxane-site delivered with an antibody could similarly be a promising ADC strategy with a higher therapeutic index. However, the development of ADCs necessitates compounds with an extremely high affinity or a covalent mode of action that can effectively engage the target, in spite of the lower concentrations delivered to the tumor by this method. Additionally, compounds that bind tubulin covalently are not susceptible to efflux by P-glycoprotein or other membrane efflux pumps due to the covalent nature of the interaction, thus escaping a major clinically relevant mechanism of resistance to chemotherapy ,7,8.Four classes of compounds that covalently bind to tubulin to modulate microtubule polymerization have been described . PironetRecent studies of microtubules and tubulin-bound taxane-site targeting agents ,15 have In the work described in this manuscript we have employed a multidisciplinary approach involving biochemical techniques and structural biology to understand how tubulin assembles into stable microtubules as a result of the binding of small molecules to the taxane-site. We utilize the different biochemical and structural properties of the covalent microtubule binders zampanolide, taccalonolide AJ, and cyclostreptin to reveal the structural basis for drug-induced microtubule assembly.2R-TTL complexes to perform the crystallization experiments as described in the “Methods” section. The crystal structures were solved to 1.9Å resolution by X-ray crystallography and the T2R-TTL-cyclostreptin complexes superimposed well with the one obtained in the absence of a ligand , thereby suggesting that cyclostreptin-binding does not affect the overall conformation of tubulin (To obtain the structure of cyclostreptin bound to the two previously described binding sites (the pore site (binding at βThr220) and the taxane-site (binding at βAsn228) ,19), we  tubulin A. Surpri tubulin A. We fou tubulin B,C. The  tubulin  with Leu tubulin B,C.219LTTPTYGDLNHLVSATMSGVTTCLR243. This histidine residue is contiguous with the previously characterized cyclostreptin interacting residue βAsn228 [In order to understand the discrepancy between these crystal structures and previous MS/HPLC results, we performed a higher precision MS analysis . We foun βAsn228 ,19. The  βAsn228 ,19. HighAlthough the final structure showed an overall folding essentially identical to the apo-state B, the βTTaxane-site ligands have been proposed to induce microtubule assembly by driving M-loop folding into a helical structure observed in the assembled form . The eneTo gain further insight into the detailed mechanism that controls M-loop structuring, we superimposed all available crystal structures of taxane-site ligand-tubulin complexes and compared the ligand environment in the binding site. Based on the observed structural features, these taxane-site ligands can be subdivided into two groups, those that induce helical M-loop structuring, and those that show little to no effect on M-loop conformation . All ligThe first group of compounds, which comprises zampanolide (PDB ID 4I4T), epothilone A (PDB ID 4I50) and the DDM-paclitaxel hybrid KS-1-199-32 (PDB ID 5LXS), establishes extensive interactions with the M-loop through their corresponding sidechains, resulting in the induction of helical structuring A. The seThe covalent binding of cyclostreptin to βHis229 allows establishing comparable, but less extensive hydrophobic contacts shared among both classes of compounds. Moreover, the A ring moiety of cyclostreptin  occupiesel) [2+ dependent [2+ concentrations [+2 concentrations  displayed a lag time of ~20 min [Our cyclostreptin structure indicated that taxane-site ligands may favor microtubule assembly not only by assisting  folding of the M-loop in unassembled tubulin heterodimers in solution but also by other mechanisms. However, we also considered the possibility that the differences observed between diverse ligands with regard to folding of the M-loop could be related to differences in the assembly-promoting activities of these compounds. Thus, we measured the assembly-promoting activity of all three compounds that are known to bind covalently within the taxane-site  by assaying tubulin polymerization under conditions where microtubule assembly does not occur on its own (phosphate buffer without glycerol)  Figure . Under tel) , which iel) . Since tependent , we alsotrations ,24. We ftrations A only zatrations B all thr ~20 min  with a mThese results closely correlate the M-loop stabilization states found in zampanolide and cyclostreptin structures with the assembly promoting activity of these compounds. Importantly, the DDM-paclitaxel hybrid (KS-1-199-132), which interacts with and stabilizes the M-loop (5LXS) also displays a higher assembly promotion activity than discodermolide despite a lower binding affinity .It is well established that, in addition to P-glycoprotein overexpression, βIII-tubulin isotype expression is a main clinical determinant of resistance to tubulin-targeted therapy ,27,28. TAs expected , βIII-tubin1  of the adducted protein for the microtubule end as compared to the non-adducted protein (Kel1)  there is no difference in affinity for the unassembled and the assembled forms because the binding reaction is irreversible. In this case, the assembly induction should arise from a higher affinity (Kn (Kel1) . Since 1assembly ,25, the Previous structural studies have shown that M-loop structuring is promoted by ligands with their respective side chains capable of establishing extensive interactions with the M-loop, as observed in the tubulin-complexes of zampanolide and epothilone  or the DIt should be mentioned that in chain D of the presented cyclostreptin complex the βT5-loop adopts the conformation observed for the GTP bound state despite of having GDP bound at the E-site. A closer inspection of the electron densities defining the βT5-loop conformations present in all available crystal structures of taxane-site ligand-tubulin complexes reveals a preference for the GTP state, with a few structures featuring both conformational states of the βT5-loop . One exception is dictyostatin, one of the best microtubule stabilizers having a moderate tubulin-binding affinity ,23. In tOur high-resolution structure of cyclostreptin-bound tubulin together with new MS-HPLC experiments performed at a higher resolution challenge our previous hypothesis of cyclostreptin binding to βAsn228 and βThr220  and instIn spite of the fact that cyclostreptin lacks the ability to stabilize the helical conformation of the M-loop, it weakly induces tubulin assembly, supporting its function as a microtubule stabilizing agent likely through stabilization of the βT5 loop in the GTP conformation. Although the results support the relevance of progressive M-loop structuration in the potency of tubulin assembly induction as proposed previously , this M-The two main clinically relevant mechanisms of drug resistance of tubulin-targeting agents are expression of drug efflux pumps  belonging to the ATP-binding cassette family of proteins ,33, whicIt has been proposed that the resistance of tubulin-stabilizing chemotherapeutics in βIII-tubulin expressing cells arises from defective binding to the taxane-site of βIII-tubulin ,40,41. O50 in HeLa cells of 0.045 nM. Taccalonolide AJ has an IC50 of 6.2 nM in this cell line with cyclostreptin having an IC50 of 19 nM, suggesting a direct relationship between the promotion of purified tubulin assembly and potency for inhibition cellular proliferation, as previously proposed [The use of covalent binders to investigate the mechanism of βIII-tubulin-mediated resistance has the advantage of eliminating the parameter of binding affinity. In principle, when studying a covalent binding agent, the rate and extent of drug binding will not be affected by a mutation in the binding site as long as it is able to bind with fast on rate. As an example, zampanolide has been found to be active in cells with paclitaxel/epothilone and laulimalide/peloruside resistance mutations and attempts to generate a zampanolide resistant cell line by mutagenesis have so far been unsuccessful . Consideproposed .When we assessed the antiproliferative potency of covalent microtubule stabilizers in HeLa cells overexpressing the βIII-tubulin isotype, we found that they were less susceptible to this form of resistance than paclitaxel. The finding that irreversibly binding ligands are less susceptible to βIII-tubulin-mediated resistance than reversible ligands, demonstrates that binding has a role in βIII-tubulin resistance. However, it is important to note that each of the covalent compounds still demonstrated partial (2–5-fold) resistance to the βIII-tubulin overexpressing line as compared to the parental line. The inability to completely overcome this mechanism of resistance through covalent binding supports the binding-independent roles of βIII-tubulin in drug resistance described above. Importantly, the use of the covalent microtubule destabilizer pironetin allowed us to distinguish between the proposed mechanisms of βIII-tubulin as a survival factor and as a mediator of microtubule dynamics. Pironetin completely overcomes βIII-tubulin-mediated resistance and is actually 1.7-fold more potent in the βIII-tubulin overexpressing line than in the parental cell line. Since pironetin does not bind to the taxane-site it would be expected to be completely insensitive to any change in the binding site. If the role of βIII-tubulin in the resistance is as a cell survival factor we would anticipate that both covalent stabilizers and destabilizers would be similarly impacted by βIII-tubulin overexpression, which is not the case. The fact that βIII-tubulin overexpressing cells are more sensitive to pironetin than the parental cells is instead consistent with a mechanism of resistance that involves counteracting microtubule stabilization. Therefore, as suggested previously , we propPurified calf brain tubulin, and chemicals were as described . The sta50 values were evaluated using the t-test option implemented in the Sigma Plot 13 software package .An isogenic couple of HeLa S3 and HeLa βIII  cells we2, pH 7.0) plus 0.1 mM GTP. Samples were taken at different times  and extracted three times with 1 v/v of dichloromethane. Samples without tubulin were used as a control. Ligand was quantified by HPLC-MS as described [Samples containing 20 μM tubulin and 25 μM cyclostreptin were incubated at 25 °C in PEDTA buffer  were added. The samples were warmed to 37 °C and the assembly of tubulin followed by turbidimetricaly at 350 nm in a Thermo Scientific Multiskan Sky plate reader.Samples of 25 μM tubulin in 10 mM sodium phosphate, 1 mM EDTA, 1 mM GTP, pH 7.0 were supplemented with either 3 mM or 7 mM MgCl2 pH 7.0 buffer for 4 h at 25 °C. To prepare the T2R-TTL-cyclostreptin complex, which contains two tubulin heterodimers, one TTL and one RB3 molecule, the proteins were mixed and concentrated (Amicon MWCO 10) at 4 °C to a final complex concentration of 20 mg/mL.Two approaches were performed to form the tubulin-cyclostreptin adduct for crystallization. In the first approach, the adduct was prepared by incubating 20 µM tubulin with 80 µM cyclostreptin in PEDTA 1.5 mM MgCl2, and 0.1 mM GTP pH 6.5 for 30 min, and subsequently incubated with 80 µM cyclostreptin for 3 h at 37 °C. Microtubules were then disassembled by the addition of 4.5 mM EDTA and RB3 and further incubated at 4 °C for 1 h. Finally, TTL was added and the complex was concentrated as described above.In the second approach, microtubules were first assembled from 20 µM tubulin in 3.4 M glycerol, 10 mM sodium phosphate, 1 mM EGTA, 6 mM MgCl2R-TTL-cyclostreptin samples were supplemented with 10 mM DTT, 0.1 mM GDP and 1 mM Alpha,beta-Methyleneadenosine triphosphate (AMPCPP) before setting up crystallization experiments by the sitting-drop vapor diffusion technique at 20 °C. Considering known conditions from previous structures [2, 30 mM MgCl2, and 5 mM L-tyrosine.Both the Tructures ,16,60,61For data collection, crystals were collected directly from the drop and subsequently transferred into reservoir supplemented with 16 and 20% glycerol in two steps. X-ray diffraction data were collected at the beamline X06DA of the Swiss Light Source . Images were indexed and processed using XDS . StructuTubulin samples were digested with modified porcine trypsin (Promega) at a final ratio of 1:50 (trypsin-protein). Digestion proceeded overnight at 37 °C. After digestion, samples were vacuum-dried and finally dissolved in 1% acetic acid for LC-MS analysis. The resulting peptide mixtures were subjected to nano-liquid chromatography coupled to mass spectrometry for protein identification. Peptides were injected onto a C-18 reversed phase nano-column  and analyzed in a continuous acetonitrile gradient consisting of 0–40% B in 120 min, 50–90% B in 1 min . A flow rate of 200 nL/min was used to elute peptides from the RP nano-column to an emitter nanospray needle for real time ionization and peptide fragmentation on a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). One survey full scan and the 15 most intense fragmentation spectra were analyzed along the chromatographic run. Dynamic exclusion was set at 30 s. For protein identification, tandem mass spectra were extracted and charge state deconvoluted by Proteome Discoverer 1.4.0.288 (Thermo Fisher Scientific). All MS/MS samples were analyzed using SEQUESTTM (Thermo Fisher Scientific). Sequest was searched with a fragment ion mass tolerance of 30 ppm and a parent ion tolerance of 15 ppm. Oxidation of methionine was specified in Sequest as a variable modification. Custom application in R (Vseq) was developed to calculate and generate quality control graphs to monitor the specificity in peptide identification .


Most emergency departments (ED) use patient experience surveys  that include specific physician assessment fields. Our ED group currently staffs two EDs – one at a large, tertiary-care hospital, and the other at a small, affiliated, community site. Both are staffed by the same physicians. The goals of this study were to determine whether Press Ganey ED satisfaction scores for emergency physicians working at two different sites were consistent between sites, and to identify factors contributing to any variation.We conducted a retrospective study of patients seen at either ED between September 2015 and March 2016 who returned a Press Ganey satisfaction survey. We compiled a database linking the patient visit with his or her responses on a 1–5 scale to questions that included “overall rating of emergency room care” and five physician-specific questions. Operational metrics including time to room, time to physician, overall length of stay, labs received, prescriptions received, demographic data, and the attending physician were also linked. We averaged scores for physicians staffing both EDs and compared them between sites using t-tests. Multiple logistic regression was used to determine the impact of visit-specific metrics on survey scores.A total of 1,012 ED patients met the inclusion criteria . The overall rating-of-care metric was significantly lower at the tertiary-care hospital ED compared to our lower volume ED (4.30 vs 4.65). The same trend was observed when the five doctor-specific metrics were summed (22.06 vs 23.32). Factors that correlated with higher scores included arrival-to-first-attending time (p=0.013) and arrival-to-ED-departure time (p=0.038), both of which were longer at the tertiary-care hospital ED.Press Ganey satisfaction scores for the same group of emergency physicians varied significantly between sites. This suggests that these scores are more dependent on site-specific factors, such as wait times, than a true representation of the quality of care provided by the physician. Under the Affordable Care Act, increasing emphasis has been placed on delivery of healthcare that is both patient-centered and high quality with the aim of incentivizing better value and outcomes.5Numerous studies conducted in the ED have demonstrated the many factors that influence patients’ satisfaction with their visits. While good communication, attitude and interpersonal skills demonstrated by ED staff are associated with increased patient satisfaction scores, factors such as wait time, patient demographics and acuity, as well as crowding, also influence scores.21Regarding physician-specific metrics, Bendesky et al. in 2016 showed that patient satisfaction scores differed for emergency physicians (EP) based on the setting in which they were practicing. Specifically, satisfaction scores were consistently lower in an ED setting when compared to an urgent care. This finding suggests that even metrics that attempt to narrowly assess the patient-provider relationship are subject to external factors.23In August 2015 our health system opened a second ED at a university-affiliated site that is staffed by the same emergency medicine faculty group. There are some operational differences between the sites, including consultant availability as well as the level of involvement of residents and advanced practice providers (APP) in care. However, most ancillary services offered are largely identical, including radiology studies  and lab services. This presents an ideal scenario to compare physician-specific Press Ganey ratings. Our objective was to evaluate consistency of physician-specific patient satisfaction scores between the two sites.This was a retrospective cohort study examining Press Ganey surveys at two different EDs. Site 1 is situated in a suburban area, has inpatient medicine services with limited subspecialty services available. It is approximately 12 miles from site 2 and has an annual ED volume of 11,221 (during the study period). Site 2 is an academic, tertiary-care hospital in an urban environment with an annual ED volume of 55,561 (during the study period). Both EDs are staffed by board-certified or board-eligible EPs. In addition to EP staffing, site 1  had limited APP staffing (four hours of coverage daily) during the study period, whereas site 2 (academic center) had significant resident and APP staffing with their involvement in most patients’ care.Discharged patients from both EDs received a survey (via mail or email) administered by Press Ganey Associates . We included in the analysis patients cared for by EPs who worked at both sites from September 2015–May 2016, a period chosen based on availability of data for analysis. Further requirements included a minimum of 10 evaluations per site per physician (which had the effect of limiting inclusion to full-time physicians with significant practice at both sites) and full survey responses. Returned surveys were linked to the encounter so that treating physician, demographics, date and time of visit, vital signs, and any tests performed could be obtained. We excluded from the analysis patients who were cared for by more than one EP within a visit.What do we already know about this issue?Physician-specific scores on patient satisfaction surveys are often used as a proxy for the quality of care delivered by emergency physicians.What was the research question?Do patient satisfaction scores differ for the same physicians staffing two different emergency departments?What was the major finding of the study?Patient satisfaction scores for the same physicians were lower at the higher volume/longer wait time site.How does this improve population health?Press Ganey scores, intended to measure patient satisfaction with physicians, may be more influenced by site-specific than physician-specific factors.We used patient responses to physician-specific questions. These questions included the following: overall rating of care; courtesy of the doctors who cared for you; degree to which these doctors took the time to listen to you; concern these doctors showed to keep you informed about your treatment; concern these doctors showed for your comfort while treating you; and degree to which these doctors advocated for your care. Possible ratings ranged from 1 (very poor) to 5 (very good). Additional variables were selected based on potential impact on patient experience based on prior literature; these included age, race, gender, acuity, means of arrival, time interval from arrival to rooming, time interval from arrival to leaving the ED, and whether patients received any labs or advanced imaging.202 test for categorical data. To evaluate physician-specific metrics, we evaluated the response rate for overall rating of care as well as the sum of the five physician-specific metrics. A logistic regression model was created to evaluate the impact of site and physician on scores while controlling for covariates. Given the high proportion of returned surveys with a total score of 25 , we dichotomized outputs into scores of 25 vs all other scores for the regression analysis. Additionally, we ranked all included physicians from highest to lowest in Press Ganey scores at both sites. Given our sample size, we expected to detect a difference in mean score of 0.18 from the mean Press Ganey scores at site 2 (the academic site) with a power level of 0.8 at an alpha of 0.05 based on a two-tailed test.We obtained data from the electronic health record (EHR), which exists in one continuous instance at both sites. Press Ganey data were linked to EHR data reports by departmental staff during the creation of the dataset. We analyzed data using Stata 15 . We compared demographic and Press Ganey data using t-test for continuous data and chiAfter applying exclusion criteria, we included 1012 encounters in the analysis: 457 from site 1 and 555 from site 2. The Patient demographics were similar between sites, including age, race, gender, and mode of arrival . Wait tiA total of 13 EPs  met the minimum of 10 returned surveys per site and were included in the analysis. By physician, the median number of surveys returned was 29 at site 1  and 37 at site 2 . Mean Press Ganey satisfaction scores for provider overall rating of care were higher at site 1 compared to site 2 . The samIn the regression analysis, no individual physician was associated with a significant odds ratio for achieving or not achieving high Press Ganey scores. Being seen at site 1 and shorter arrival-to-room and arrival-to-discharge times were associated with a higher incidence of high scores. Patient-specific factors such as age, race, gender, arrival mode, and acuity were not associated with differences in scores, nor were any individual physicians associated with statistically significant increases or decreases in scores. The regression model had a c-statistic of 0.68 and a non-significant Hosmer-Lemeshow goodness of fit test at 0.278 . When raThis study was conducted within one health system and trends may differ in other organizations. Additionally, while both sites are EDs with similar patient populations, one difference of note is that EPs who staff site 2 typically work with resident physicians and APPs, including physician assistants and nurse practitioners, which is less common at site 1. Differences in physician-specific-scores may be due to the fact that physicians at site 2 were rated along with their residents and APPs. While we would argue that this is one of the site-specific characteristics of site 2, with regard to this site it is important to note that the effect of residents or APPs overall was not directly measured and may be a major driver of the effect observed.The study was also limited by its retrospective design. Due to the methodology of data collection (reporting from EHR records) it is possible that physicians were incorrectly matched to patient encounters in some cases, although this is unlikely as all cases with more than one assigned physician were dropped from analysis. In our setting, as has been reported in institutions elsewhere, Press Ganey survey response rates were low. While this is a common feature of Press Ganey data in general, we cannot extrapolate our results to other scenarios in which response rates were higher, in which case physician-specific ratings may be more accurate and less dependent on external factors as observed here.This study compared physician-specific patient satisfaction scores for EPs who practice in two different EDs. We observed that Press Ganey survey scores were consistently lower for the same physicians practicing at site 2 compared to site 1. This is similar to the findings of Bendesky et al. (2016), who found that patient satisfaction scores of the same EPs differed based on the site where they were practicing.10While other investigators have found associations between satisfaction scores and factors such as patient age, race, acuity, and arrival mode, our analysis did not show any of these associations.A physician’s Press Ganey score is increasingly being used as a proxy for the quality of care they provide. While we feel that improved patient experience scores are a worthy goal for EPs given the multiple benefits that have been shown to correlate with an improved patient experience ,We found that Press Ganey scores for the same group of physicians differed between two sites. Scores were higher at the lower-volume site where wait times were shorter. These results suggest that Press Ganey scores are affected by factors outside of the physician’s control. Scores should be interpreted with caution, especially when used as a proxy for the quality of care provided by the physician.