Necroptosis is a novel form of programmed cell death that is independent of caspase activity. Different stimuli can trigger necroptosis. At present, the most informative studies about necroptosis derive from the tumor necrosis factor (TNF)-triggered system. The initiation of TNF-induced necroptosis requires the kinase activity of receptor-interacting protein 1 and 3 (RIP1 and RIP3). Evidence now reveals that the ability of RIP1 and RIP3 to modulate this key cellular event is tightly controlled by post-translational modifications, including ubiquitination, phosphorylation, caspase 8-mediated cleavage and GlcNAcylation. These regulatory events coordinately determine whether a cell will survive or die by apoptosis or necroptosis. In this review, we highlight recent advances in the study of post-translational modifications during TNF-induced necroptosis and discuss how these modifications regulate the complex and delicate control of programmed necrosis. The ability of receptor-interacting protein 1 and 3 (RIP1 and RIP3) to modulate necroptosis is tightly controlled by post-translational modifications, including ubiquitination, phosphorylation, caspase 8-mediated cleavage and N-acetylglucosaminylation (GlcNAcylation).κB activation, whereas K63-polyubiquitinated RIP3 supports the formation of the RIP1–RIP3 complex and subsequent induction of necroptosis.K63-polyubiquitinated RIP1 is retained at the plasma membrane and serves as a docking site to mediate NF-RIP1 and RIP3 autophosphorylate themselves and also phosphorylate each other.Caspase 8 cleaves RIP1 on Asp324 and RIP3 on Asp328, which abolishes the activity of RIP1 and RIP3 and thus inhibits necroptosis.Which enzymes mediate the ubiquitination of RIP3?Is Ser161 phosphorylation critical for the pro-necrotic function of RIP1?Knockdown of phosphoglycerate mutase family member 5 (Pgam5) or dynamin-related protein 1 (Drp1) was able to inhibit tumor necrosis factor (TNF)-induced necroptosis in HeLa cells, whereas it had no effect on necroptosis in MEF, L929, SVEC and NIH-3T3 cells. Is Pgam5–Drp1 axis cell-type-specific?Does acetylation mediate necroptosis?2 Similar to apoptotic cell death, programmed necrosis can also be initiated by death ligands (such as TNF and Fas), interferons, Toll-like receptor (TLR) ligands and microbial infection.9 In the aspect of host defense against infection, necroptosis could be considered as a back-up mechanism for host cells to contain pathogen replication and spread when the apoptotic pathway is compromised.10 Unlike apoptosis, which is a caspase-dependent process characterized by chromatin condensation and nuclear fragmentation, necroptosis is a caspase-independent pathway characterized by morphological features resembling necrosis.1 This death pathway is involved in many pathological processes, such as chronic inflammation,12 kidney ischemia/reperfusion injury,13 neurodegenerative disease,14 retina detachment15 and atherosclerosis.16Programmed necrosis or necroptosis is an alternative form of programmed cell death that is triggered when apoptosis is inhibited.17 Subsequently, RIP3 was reported to physically and functionally interact with RIP1 to generate a super molecular complex during necroptosis.18 To distinguish this complex from other complexes that induce apoptosis or nuclear factor-κB (NF-κB) activation, the RIP1–RIP3 complex has been termed the 'necrosome'.19 Interestingly, RIP1 and RIP3 belong to the same kinase family, and each of them has an N-terminal kinase domain. Under the stimulation of TNF/zVAD, RIP1 and RIP3 form a heterodimeric scaffold through their respective RIP homotypic interaction motif domains.20 The necrosome recruits and activates downstream substrates such as mixed lineage kinase domain like protein (MLKL) to execute necroptosis.21 Therefore, a better understanding of the regulatory mechanism of necrosome will be helpful to reveal the process of necroptosis.The implication of RIP1 in necroptosis was first discovered in 2000 and was definitively confirmed by the identification of necrostatin-1 in 2008, which specifically targets RIP1 kinase and thus inhibits necroptotic cell death.Post-translational modifications have crucial roles in regulating protein function, and thereby control several fundamental aspects of eukaryotic biology, including signal transduction, protein trafficking, cell–cell interactions, cell proliferation and differentiation. Indeed, emerging evidence indicates that necrosome assembly and activation is tightly controlled by post-translational modifications. The aim of this review is to give an overview on the role of post-translational modifications in the process of necroptosis, especially focusing on protein ubiquitination, phosphorylation, caspase 8-mediated proteolytic cleavage and GlcNAcylation .22 For example, K48-linked polyubiquitination mainly targets protein for proteasomal degradation, whereas K63-linked polyubiquitination typically regulates protein function, subcellular localization and protein–protein interactions.Ubiquitylation is among the most widely used protein modifications involved in regulating cellular signaling and homeostasis. As an ubiquitin contains seven lysines , there are seven possible types of homotypic linkage as well as linear ubiquitin linkage. The ubiquitin can be inducibly and reversibly attached to a range of proteins and then regulate a multitude of cellular functions.α-induced NF-κB signaling pathway, RIP1 is a dual-functional signaling molecule that is capable of either pro-survival or pro-death, depending on the state of K63-linked polyubiquitination.23 Upon TNF stimulation, TNF receptor 1 (TNFR1) recruits multiple proteins to form a transient molecular complex, named complex I. This complex consists of TRADD (TNF receptor-associated death domain), TRAF2 (TNF receptor-associated protein 2), cIAP (cellular inhibitor of apoptosis protein) and RIP1. In complex I, RIP1 is modified by multiple forms of ubiquitination, including linear, K63 and K11-linked polyubiquitinations. K63-linked polyubiquitination of RIP1 functions as a docking site for the TAK1–TAB2–TAB3 complex and for NEMO, both of which are crucial for the activation of NF-κB and provide survival signals. Deficient complex I activity, such as deubiquitination of RIP1, inhibits NF-κB activation and leads to the formation of one of the two alternative cytosolic complexes, complex IIa or complex IIb.24 Complex IIa includes Fas-associated death domain (FADD), caspase 8 and RIP1, which activates downstream caspases and subsequent apoptosis. When the activation of caspase 8 is inhibited, RIP1 and RIP3 interact with each other to form complex IIb, which can induce necroptosis. The conversion from complex I to complex II is an important regulatory step. To date, multiple deubiquitinating enzymes (DUBs), E3 ubiquitin ligases and linear ubiquitin ligases, have been identified to modulate the deubiquitination or ubiquitination of RIP1 in the TNF signaling pathway.In the TNF25 It is worth noting that the Znf domain of A20 activates not only K48-linked but also K63-linked polyubiquitin chains. In human glioblastoma, the A20 Znf domain mediates K63-linked polyubiquitination of RIP1 and thus inhibits cell death.26 The N-terminal ovarian tumor domain of A20 acts as a DUB by removing K63-linked polyubiquitin chains from RIP1, which consequently downregulates TNFα-induced NF-κB signaling.25 As discussed above, the role of A20 is much more complicated than that of an individual DUB or E3 ligase for its two opposite-function domains. Therefore, exploring its role in cell survival and death will shed light on the precise biological function of A20.It is known to all that both the deubiquitination and ubiquitination are tightly mediated by DUBs and ubiquitination enzymes. Interestingly, A20 is an unusual protein that has dual ubiquitinating and deubiquitinating activity. The C-terminal zinc-finger (Znf) domain of A20 acts as an E3 ligase by polyubiquitinating RIP1 with K48-linked ubiquitin chains, thereby targeting RIP1 for proteasomal degradation.κB activation and promote TNFα-induced cell death by removing K63-linked polyubiquitination chains from RIP1 in the TNFR1 complex.29 Another DUB, cylindromatosis (CYLD), may also function as a switch in regulating the activation of necroptosis over NF-κB activation and cell survival in three aspects. First, CYLD inhibits NF-κB signaling by deubiquitinating NF-κB-positive regulators, such as TAK1 (TGF-β-activated Kinase 1), TRAF2 and NEMO/IKKγ.31 Second, caspase 8-mediated cleavage of CYLD generates a survival signal, whereas the mutation of caspase 8-mediated cleavage site on CYLD switches cell survival to necrotic cell death in response to TNFα.32 Last but not least, CYLD interacts with and deubiquitinates RIP1.33 However, it is still controversial whether CYLD affects the ubiquitination of RIP1 in complex I or in the necrosome.35 Given that the above DUBs can remove ubiquitin chains from RIP1, how is RIP1 ubiquitinated?The ubiquitin-specific proteases (USPs) become DUBs by cleaving ubiquitin adducts from specific protein substrates, thereby regulating protein activation or protecting the substrate from proteasome-mediated degradation. The USP family members USP2a, USP4 and USP21 are believed to inhibit RIP1-mediated NF-κB activation after TNFα stimulation.38 These researches revealed that cIAP1/2 have a critical role in protecting against TNFα-induced cell death by promoting K63- and K48-linked polyubiquitination of RIP1. cIAP1/2 also mediate the assembly of K11-linked ubiquitin chains on RIP1.39 Unlike K63 and K48 linkages, K11-linked ubiquitin chains have been less studied and are considered to serve as a degradation signal. However, Dynek et al.39 demonstrated for the first time that K11 ubiquitin linkage may take a part in TNF signaling, as the adaptor protein NEMO can be recruited to K11 ubiquitin chains of RIP1. As discussed above, cIAP1/2 act as E3 ligases to promote K63, K48, as well as K11-linked polyubiquitination of RIP1 in the TNFR1 complex, which facilitate cell survival and protect against cell death. Nevertheless, the role of another E3 ubiquitin ligase TRAF2 within the TNFR signaling complex is controversial. On the one hand, genetic evidence indicates that TRAF2 is responsible for the K63-linked ubiquitin chains of RIP1, as RIP1 polyubiquitination is not observed in response to TNFα in TRAF2 KO cells.41 The RING domain of TRAF2 with E3 activity is responsible for polyubiquitination of RIP1 and inhibition of cell death induced by TNFα.40 On the other hand, it is believed that TRAF2 is critical for recruitment of cIAP1/2,42 E3 ligases that are indeed essential for RIP1 ubiqutination within the complex. The recent finding that TRAF2 suppresses TNFα-induced necroptosis through competing with RIP3 for MLKL binding provides a crucial element in our understanding of the biological function of TRAF2.43It is worth noting that cIAP1/2 are RING domain-containing E3 ubiquitin ligases that have exceptional capacity to promote K63-, K48- and K11-linked polyubiquitination of RIP1. At first, it was found that cells deficient in cIAP1/2 are defective in RIP1 ubiquitination and NF-44 This kind of ubiquitin chain is assembled by a specific ligase complex called linear ubiquitin chain assembly complex (LUBAC), which contains two RING-type E3 ligases, heme-oxidized IRP2 ubiquitin ligase 1 (HOIL1) and HOIL1-interacting protein (HOIP), and an adaptor protein Sharpin. LUBAC catalyses the formation of a peptide bond between the N-terminal methionine of one ubiquitin molecule and the C-terminal glycine of the next, which is the so-called head-to-tail ubiquitination.46 The importance of linear ubiquitylation to necroptosis was brought to light by knockdown of HOIL1/HOIP, and mutation of Sharpin. It was worth noting that the repression of LUBAC component HOIL1 or HOIP enhanced TNF-induced necroptosis.47 Similarly, the mutation of Sharpin led to TNF-dependent inflammatory syndrome, characterized by dermatitis, liver inflammation, splenomegaly and loss of Peyer's patches.48 Although loss of a single allele of Casp8 was strikingly effective in delaying the onset of dermatitis, it did not have an impact on other aspects of phenotype. Unlike Casp8 heterozygosity, RIP3 and MLKL deletion resulted in reduced splenomegaly and completely rescued liver inflammation.49 Taken together, these results indicate that the absence of Sharpin leads to aberrant apoptosis in certain tissues and necroptosis in others. Given that a direct regulation of RIP1 by LUBAC is not yet clear, it will be necessary to delve further into the mechanism of LUBAC-mediated control of RIP1.The most recently described linear (head-to-tail) ubiquitin chain is another type of non-degradative signaling modification that functions as an important cellular regulator.50 This finding sheds new light on the importance of RIP3 ubiquitination in the process of necroptosis . However, the functional significance of these phosphorylation sites has not been well demonstrated, yet except for Ser161. The homology modeling of RIP1 on B-RAF suggested that Ser161 phosphorylation may be involved in the regulation of RIP1 kinase activity. Following this, some researchers observed that S161A mutation significantly decreased RIP1 kinase activity and attenuated 40% necroptosis.17 However, other studies found that S161A only reduced RIP1 kinase activity by ~20%, which was not sufficient to reverse programmed necrosis.55 Considering these points, it is not easy to draw a conclusion whether Ser161 phosphorylation is critical for the pro-necrotic function of RIP1. However, it is certain that Ser161 contributes to necrostatin-1 (Nec-1)-mediated inhibition of RIP1 kinase activity.17As a serine–threonine protein kinase, RIP1 can be phosphorylated either by itself (auto) or by other kinase(s). It is noteworthy that RIP1 phosphorylation was completely abrogated in RIP3-knockout cells.55 As S89A mutation resulted in hyperactive RIP1 kinase activity and increased TNFα-induced programmed necrosis, it is suggested that Ser89 phosphorylation inhibits RIP1 kinase activity and suppresses RIP1-dependent programmed necrosis. Protein kinase (PK) A, PKC or c-Jun N-terminal kinase may act as the kinase that phosphorylates RIP1 at this site. After that, another inhibitory phosphorylation site of RIP1 is reported to be mediated by the inhibitor of NF-κB kinase (IKK) complex.56 It is demonstrated that IKKα/IKKβ directly phosphorylates RIP1 in complex I, which either represses RIP1 kinase activity or interferes with RIP1's ability to bind complex IIb components, thereby protecting cells from RIP1 kinase-dependent death. The potential candidates for IKKα/IKKβ-mediated phosphorylation of RIP1 are S25, S166, S296, S331 and S416. The findings from the above research suggest that RIP1 phosphorylation has a dual role in the regulation of programmed necrosis.Recently, the inhibitory phosphorylation sites of RIP1 have been reported. At first, researchers identified a novel phosphorylation site on RIP1, Ser89, which can dampen the pro-necrotic function of RIP1.7 McQuade et al.55 revealed that the phosphorylation on Ser204 in mouse RIP3, which regulates the pro-necrotic function, is facilitated by RIP1. Furthermore, RIP3 appears to be autophosphorylated at Ser199 and Ser227 in human cells, and the Ser227 phosphorylation is required for human RIP3 to interact with human MLKL in the necrosome.57 Similarly, the Thr231 and Ser232 autophosphorylation on mouse RIP3 is essential for the recruitment and activation of mouse MLKL.58 Although there are many other phosphorylation sites on mouse RIP3 , they seem to be unnecessary for mouse RIP3 to mediate necroptosis.58 Recently, protein phosphatase 1b (Ppm1b) has been identified first as a nonspecific RIP3 phosphatase that negatively regulates necroptosis through dephosphorylating RIP3 and suppressing MLKL recruitment to the necrosome.59 According to what has been discussed above, RIP1 phosphorylation could promote or inhibit its kinase activity, which is required for necrosome formation, whereas RIP3 phosphorylation is essential for RIP3–MLKL interaction  type III effector NleB can GlcNAcylate a conserved arginine in death domains of TNFR1, TRADD, FADD and RIP1 in EPEC-infected cells, which disrupts the assembly of the TNFR1 complex and blocks host cell apoptosis and necroptosis. This kind of unexpected and pathogen-related modification revealed that under certain circumstances, other types of post-translational modification may also be involved in the process of necroptosis. Currently, it is still controversial whether acetylation mediates necroptosis. With the deepening study of necroptosis, it is reasonable for us to believe that this problem will be solved in the near future.In addition to the above-mentioned post-translational modifications, a previously unappreciated modification, GlcNAcylation, has recently attracted much attention. Li


In this study, we aim to develop a narrow-diameter and long-bore device for minimally invasive surgery that achieves the simultaneous cutting and suction of body tissue such as the diseased part of an organ. In this paper, we propose a screw made of a thin metal plate, and we developed a prototype device using this screw. For smooth operation, the suction performance must be superior to the cutting performance. Therefore, we performed experiments and evaluated the suction performance of the developed device assuming the crushed tissue pieces correspond to a highly viscous fluid. From the results, we confirmed that the suction volume is almost proportional to the rotation speed of the screw in the low speed range, and the device has an upper limit of suction volume at a certain rotation speed. Considering practical use, its proportional speed range is suitable for the device controllability of cutting and suction volume, and the size of the device tip needs to be 1 mm or more. Based on these conditions, we are planning to examine the shape of the cutting edge for realizing efficient cutting and suction and we will complete the device. Minimally invasive surgery has come into use in a wide range of fields in recent years, with the aim of improving patients’ quality of life. Surgical instrumentation has kept pace with this, with the development and use of instruments such as narrow-diameter, long-axis forceps and electric scalpels, which are widely used in neurosurgery, for example .The use of minimally invasive surgery can minimize physical and mental stress on the patient. On the other hand, the burden on the surgeon is very large because precise work is required in a limited space. For example, if removing lesions using a device such as narrow-long forceps, since resectable tissue mass in a single operation is small, it is necessary to remove tissue in many separate operations or to use a long, narrow suction tube. However, narrowing the diameter and increasing the length of negative pressure suction instruments are known to greatly reduce their suction performance. For these reasons, the increased operation time is a particular problem.2 laser and a negative pressure suction tube [In order to improve the efficiency of minimally invasive surgery, it has been desired to develop a device for realizing the cutting, transport, and discharge simultaneously. For this purpose, devices have been developed as surgical instruments that are capable of simultaneously resecting and removing body tissue, such as the CUSA system   and Sonoion tube  and a suion tube  has beenFor these reasons, in this study, we develop a narrow-diameter, long-bore device capable of efficient cutting and suction without becoming clogged. In this paper, we investigated the mechanism for realizing cutting and suction. In addition we developed a prototype device and evaluated its performance.Outside of the medical field, screw mechanisms are widely used for foods such as minced meat or pastes, and for the transport of highly viscous fluid substances such as resins and cement , 7. AtteAs the screw undergoes the physical rotation, it is possible to achieve the avoidance and elimination of clogging during suction. At the same time, it is also possible to use the rotary motion for the cutting by an edged screw tip.In this study, we similarly adopted a screw construction in the prototype device. Cutting and suction performance of the device is determined by the shape of the screw and the rotation speed. Especially in the case in which the suction performance is inferior to the cutting performance, suction causes disruption. First of all, we should be clear about the suction performance.However, analysis of the flow with the screw is very difficult , 11, RobThe maximum length of narrow screws that consist of a helical shape surrounding a rotational axis, e.g. 1 mm diameter screws, is only about a dozen millimeters because of processing limitations. Therefore, the long-axis, narrow-diameter screws required for this project are technically difficult to produce. On the other hand, the rotational suction device for ENT use , 9 shownIn this study, we addressed this issue by producing a “twist blade screw.”As shown in This device was based on a dental cutting instrument. It is equipped with an internal gear, which transmits rotation supplied by a motor drive to the screw at the tip. It is also constructed of a two-gear system that can increase the rotation speed by up to approximately five times.Most regular suction pipes are around 100 mm long, including the portion gripped by the operator, and the length of the small-diameter portion is approximately 50 mm. We used this as a reference for the length of the suction pipe in our prototype device, which was set at 55 mm to allow a degree of leeway.In order to expel the substance being suctioned and transported into the device, we provided a drainage port with an internal diameter of 4 mm that could be attached to a vacuum pump. As shown in We verified the performance of prototype device using fresh chicken meat as a target and confirmed that cutting and suction of biological tissue can be realized , Fig 4.In order to evaluate the suction performance of the device, we investigated the association between the screw rotation speed and the suction amount.Given that soft solids in paste form behave in a similar way to highly viscous fluids , 14, in A dental brushless motor  with a maximum rotation speed of 40,000 rpm and a dedicated controller were used as the drive sources of the device. The multiplying gear system fitted to the prototype device enabled the maximum rotation speed to be increased to 200,000 rpm. The controller enabled the internal control of rotation and torque, but we suppressed these functions and used only the simple ON-OFF switch and manual control of rotation speed.An electronic balance  capable of measuring to an accuracy of 0.001 g was used to measure the amount of target liquids before and after suction, and the suction volume was calculated from the accumulation using the density.A medical vacuum pump (maximum negative pressure -70 kPa) was used to expel the suction target liquids transported to the inside of the device. A silicon tube  was attached to the drainage port in the device shown in Graded screw rotation speeds of the device were set, suction was applied to the target liquid at the set rotation speed, and the weights before and after the suction time were measured and recorded. Measurements were performed ten times, and the mean changes in weight were regarded as the suction amount at the set rotation speed. During the experiments, the device was fixed and suction was applied in such a way that the tip was perpendicular to the surface of the liquid.However, if the rotation speed decreased by more than 10% from the set rotation speed due to the load during suction, this was regarded as mechanical over-load and no further measurements were performed.We also made a simplified vacuum  using thIn preparation for the experiments, the target liquids were allowed to stand for an adequate time in a room maintained at room temperature (15°C). External disturbances such as changes in the internal resistance of the suction line before and after the application were also eliminated by applying suction to the target liquid so that it filled the entire line before the start of each experiment.The first point to note with respect to all the results is that the supplementary suction volume was less than that seen with the simplified vacuum with the same tip diameter. This may have been because the rotational axis was not completely enclosed, and the pressure was consequently reduced by the internal gaps.A comparison of the results for water showed that the volume suctioned by the device was far less than that suctioned by a simplified vacuum with the same tip diameter, as can be clearly seen in The experimental results for JS2000, shown in The experimental results for JS14000, shown in The experimental results for JS52000, shown in The experimental results for JS160000, shown in The experimental results showed that for the highly viscous liquids JS2000, JS14000 and JS52000, the suction performance of all size devices exceeded that of vacuum devices with the same tip diameter. With the exception of the case where the 3 mm tip was used, similarly high levels of suction performance were also seen for JS160000.The difference in suction performance between our device with a 3 mm tip and a vacuum device was smaller than the corresponding differences in performance for other tip sizes. This may have been caused by a structural issue: the wider the diameter, the greater the rotational resistance due to friction between the screw and the inner diameter of the pipe. In our experiments, we set the rotation speed of the device to a constant level and did not control it in line with the load, which made it easy for the rotation speed to decrease. There may thus be room to improve the performance of the device by adjusting the control system.Our results showed, however, that the twist blade screw is not very effective at suctioning low-viscosity fluids similar to blood. This may be because little friction is generated between low-viscosity fluids and the screw, as well as because the amount of fluid that cannot be properly transported increased due to effects such as leakage from gaps between the screw and the inside of the pipe. Due to the supplementary suction from the drainage vacuum, however, some suction is possible, although its suction performance is inferior to that of the vacuum device itself. Blood also poses the problem of its clotting action causing narrow-diameter suction tubes to become clogged, whereas in our device the rotation of the screw physically detaches clotted areas, reducing the problem of clogging. Should blood coagulate on the screw itself, however, this may decrease the suction amount, and further testing is therefore required.We obtained the results for suction volume versus device size, target viscosity and rotation speed. On the basis of these results, we estimated the experimental equation. The result of water and 0 rpm plots were excluded from estimation because the effect of the screw is very small.uave is proportional to the screw rotation speed N and the head (energy) is proportional to N2. Therefore, the suction volume Q is given byS is the effective cross-section, A is a constant value, d is the inner diameter of the tip pipe, p is the screw pitch and t is the screw thickness.Ignoring the effect of friction and viscosity, the average flow velocity uave and N is expressed as follows,P is the pressure difference generated by the screw, ρ is the density of the target, ΔL is the flow path length and B is a constant. Considering the effect of friction, viscosity and other losses, λ is the friction coefficient and C is a constant. Therefore, from Eqs λ is assumed to be proportional to the dynamic viscosity ν, u′ ave is as follows,From Bernoulli’s principle concerning flow direction, the relationship between C)uave′22where uaFflow. The relationship between the screw angle θ and Fflow at any given radius r is expressed by the following equation.T is the screw torque, F is the force applied to the target liquid. When the twist blade screw is assumed to have a constant twist angle in the radial direction, the sum of Fflow is as follows,As shown in T serves to generate the liquid flow. Thus, from Eqs Q′ is expressed by the following equation.C1, C2 and C3 are the values to estimate.The suggested angle component C2 and C3, however, would be dependent on the pipe diameter d, and the estimation was performed individually for each size of the device. In addition, the least squares method was processed using the standard deviations of the measured suction volume as weighting factors.Using curve fitting with the least squares method, estimation was performed concerning the range of 50,000 rpm or less except for some of the plots apparently reduced. Step 1C3 will be dependent on d and ν and the others will be constant, the relationship between C3, d and ν is estimated, e.g. C3 × d/ν + C4/ν, by repeating curve fitting.Assuming Step 2C1 will be independent of d and C2 will be constant, an approximate value is determined from the estimated value of C1 for each condition.Assuming Step 3C2 will be dependent on only d, the relationship between C2 and d is estimated and an approximate value of C2 is determined.Assuming After repeating Step 1–3, each coefficient was estimated as shown in The estimation process consisted of three steps.C3 is retained as a term dependent on each devices, and it will not be considered losses like heat loss. In addition, C4 of a 3 mm tip device is also retained. Since the difference of suction volume between the device with a 3 mm tip and the vacuum is about at most about a factor of 1.4, the work of the screw is relatively reduced. From the result with water, it is clear for low viscosity liquid that the relationship between suction volume of the vacuum Qvacuum and of the screw Qscrew is Qvacuum > Qscrew and Q = Qscrew + Qvacuum can’t be established in such a case. Since the estimation had been performed by assuming that the effect of the screw is equal among the devices, it is probable that C4 of a 3 mm tip device was relatively reduced.As shown in Qupper would be depended on d and ν and expressed by these two parameters. The relationship between Qupper, d and ν was clarified by the same estimation process of On the other hand, the graph from the results with a 0.8 mm tip for JS52000 and JS160000 is close to the shape of an asymptote in form. This means that it is possible that there is an upper limit for the flow velocity by viscosity resistance. Hence, the upper limit of the suction volume Q versus the screw rotation speed N without contradiction. The suction volume is substantially proportional to rotation speed in the case in which the suction volume is sufficiently small versus Qupper and is closer to Qupper according to the increase of the rotation speed. However, the graph from the results with 0.8 mm and 1 mm tips for JS14000 is reduced after closing to Qupper. This is probably because the flow velocity around the axial center of the screw, with the force applied only in the rotating direction, and/or the flow velocity of the gap would become 0 or a minus value (reverse flow) because the volume conveyed by the screw can’t exceed to the input volume. Since, from Qupper became small according to the increase of the viscosity because the high viscosity reduces change in speed (acceleration/deceleration) of the flow and the leakage of the high viscosity liquid is small under the same condition.The relationship between Eqs 3 mm3) in size at approximately 15 minutes, if the viscosity of the tissue corresponds to a fish sausage , suction performance is required to be at least 50 mm3/sec and a device with a 2 or 3 mm tip is suitable. In addition, a maximum rotation speed of 30,000 rpm is satisfactory. If finer resection is necessary, a device with a 1 mm tip also can be a candidate. When the target is a very high viscosity tissues; i.e. the process is similar to mixing meat , in order to suppress the flow volume through the device and reduce the resistance being applied to the screw, for example, the intermittent operation of the cutting and suction may be necessary.Considering use of the proposed device for removal of diseased tissue and assuming it would be used to remove a target tissue which is about 50 cc Click here for additional data file.


Ingredients of aloe vera leaves are being increasingly used for their anti-inflammatory and slimming properties. However, serious allergic reactions are very rare and only occasionally reported. Here, we present an eleven year old girl with anaphylaxis within minutes after topical administration of aloe vera to a skin lesion. On admission, the girl showed no signs of respiratory or cardiovascular compromise after having been stabilized by her GP. Apart from mild allergic rhinitis symptoms during the birch pollen season, she had no other atopic disease. The family history revealed respiratory allergies in the mother and anaphylaxis to insect stings in the father.Allergy tests showed elevated total IgE levels (67.8 kU/l) in the ImmunoCAP® as well as allergen-specific IgE to rPhlp 1, nPhl p 4 and rPhl p 5, nAmb a 1 , rBet v 1 and cross-reactive sensitization to nuts and to the CCD marker nMUXF3 in the ISAC®. Sera from our patient and from seven additional allergic individuals were tested by ELISA for IgE-binding to aloe vera protein extract, Bet v 1 and two glycoproteins, HRP and Api g 5 (HRP-horse radish peroxidase and Api g 5 in celery). Six out of seven control sera were sensitized to Bet v 1 and one was allergic to peanut and sensitized to cross-reactive carbohydrate determinants (CCDs). Additionally, aloe vera protein-induced mediator release was tested in all patients using a rat basophil leukemia cell(RBL) assay. Whereas all birch pollen allergics showed strong IgE-binding to Bet v 1, IgE- binding to aloe vera was only observed in our patient and in two other serum samples, one from a birch pollen- and one from a peanut-allergic patient. Interestingly, all three patients showed much stronger binding to Api g 5 and HRP, as compared to the aloe vera extract. RBL assay showed a strong and dose-dependent mediator release of 60% in our patient and of 40% and 20% in the two patients with IgE positivity to Aloe vera.In summary, we believe that IgE antibodies to CCDs or Bet v 1 homologues in aloe vera may induce serious allergic reactions in atopic individuals.


Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ.170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c), which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA.In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c), a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues SerM. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain –pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes.Together, these results describe novel signaling mechanisms in  Mycobacterium ulcerans is a slow growing pathogen, which is prevalent in many tropical and sub-tropical countries. M. ulcerans possesses unique signaling pathways with only 13 STPK containing genes. This is strikingly different from its closest homolog Mycobacterium marinum and surprisingly closer to the human pathogen, Mycobacterium tuberculosis. PknQ, MupFHA and MupDivIVA are regulatory proteins encoded by the virulence determining plasmid pMUM001 of M. ulcerans. In addition to characterizing the STPK, we focused on deciphering the basis of interaction between the three partner proteins leading to the identification of critical residues. Present study describes the newly identified phosphoserine-based interactions, which is unique amongst the FHA-domain containing proteins. We confirmed our results using structural analysis via specific mutants and their interaction profiles. Importantly, these data highlight the significance of FHA domains and their role in understanding cellular signaling. This work will encourage further studies to elucidate role of M. ulcerans signaling systems. It will also raise questions like how less studied tropical bacterial pathogens acquire eukaryotic-like Ser/Thr protein kinase and exhibit unusual mechanisms to interact with its partner domains. Mycobacterium ulceransM. ulcerans evolved from an Mycobacterium marinum ancestor through reductive evolution and acquired a large virulence determinant plasmid (pMUM001) M. ulcerans pathogenesis Buruli ulcer is a disease of skin and soft tissues caused by the bacteria Mycobacterium tuberculosis, suggesting that STPKs may regulate multiple cellular processes M. tuberculosis and the majority of them have been shown to be involved in pathogenesis and drug resistance Pathogenic species of mycobacteria require stringent control on cell division for survival in their host, and thus are likely to acquire specialized mechanisms through evolution to achieve this control. Signaling proteins that sense environmental changes and mediate cell response are important for regulating cell division. For instance, bacterial Serine/Threonine Protein Kinases (STPKs) are known to regulate cell division by sensing and responding to specific signals in the host environment M. tuberculosis are phosphorylated by STPKs ForkHead-Associated (FHA) domain containing proteins are the key interacting partners of STPKs that mediate the signals inside the cells emanating from the cognate kinases M. tuberculosis physiology and virulence, we explored the molecular transducers and their associated FHA domains in the M. ulcerans genome. We identified 13 STPK-encoding genes in silico and focused on a novel STPK- PknQ, encoded by the virulence-associated plasmid pMUM001. We identified MupFHA (Mup018c) and MupDivIVA (Mup012c) as the substrates of PknQ and characterized the interaction of PknQ and MupDivIVA with MupFHA. MupFHA contains one FHA domain that interacts with PknQ, as well as with phosphorylated MupDivIVA. Importantly, we found that the interactions of these three M. ulcerans proteins encoded by the virulence-associated plasmid are dependent on phosphorylation of serine and threonine residues.Based on the importance of STPKs in http://blast.ncbi.nlm.nih.gov/Blast.cgi) with proteomes of M. ulcerans and M. marinum as target and desired protein sequence as query. The sequences of M. ulcerans STPKs were extracted using NCBI. These sequences were used for multiple sequence alignments, performed using ClustalW . Phylogenetic analysis was performed using the sequences of STPKs in PHYLIP and phylogenetic tree was constructed http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml).BLAST search was performed using the NCBI-BLASTp (M. tuberculosis PknB (PDB ID: 1MRU) that shows 42% identity with the PknQ sequence, was chosen as template. We focused on the predicted catalytic domain of PknQ (1 to 280 aa). The three-dimensional models for PknQ were generated using MODELLER version 9v1. General features were evaluated on the basis of the MODELLER's energy and DOPE scores while detailed reliability indices were obtained by the PROCHECK program. The PROSA Z-score was calculated using PROSA-II. Best models were chosen and further refinements were carried out. Disordered activation loop region was identified and loop refinement procedure was applied using the automatic loop refinement method provided in MODELLER 9v1 auto-model class protocol. In order to model the loop reliably, 500 loop models were sampled followed by model validation using DOPE scores and Verify 3D. PTM-Viena server was used to modify the protein and phosphate group was added at 2 different positions- Ser170 and Thr174. Ab initio loop modeling was further used to minimize the post-translation modification effects. For this purpose, loop modeling protocol of standalone version of Rosetta 3.4 modeling suite was used and 50 outputs were taken To generate the structure of PknQ, intracellular kinase domain of Similarly, structural modeling of the FHA domain was performed with MODELLER 9v1. MupFHA protein sequence spanning the residues 10 to 100 was modeled (containing the FHA domain and the interaction motif). In order to use the maximum benefits of sequence identities and to model the maximum part of sequence contacting FHA domain, multi-template approach provided by MODELLER 9v1 was utilized and the two templates of Rv0020c FHA domain  were used to predict the three dimensional homology model of MupFHA. The 3D models for native FHA domain and mutants were generated, on the basis of previously generated structures of Rv0020c and pThr peptide 170 and pThr174. First 10 solutions were analyzed for each docking and best docked poses were chosen. A short minimization was performed with Gromacs to minimize the complexes. Intermolecular interactions and docking scores were analyzed.Protein-protein dockings were performed using HEX 6.3 molecular docking program, correlation type were chosen as shape and 2000 solutions were chosen for final evaluation. Other parameters were chosen as default. Structures of the wild type FHA domain and mutants were used for docking with PknQ containing pSer170 and mutation at pThr174 to alanine (PknQ-pSer170/Thr174Ala), (3) phosphorylation at Thr174 and mutation at pSer170 to alanine (PknQ-Ser170Ala/pThr174). All the simulation studies were performed on GROMACS 4.5.5 using ffG43a1p force field provided by Gromacs official website (http://www.gromacs.org/Downloads/User_contributions/Force_fields) and uploaded by Graham Smith, which included extended parameters for modified residues Three complexes were prepared using molecular dynamics simulation- (1) a double phosphorylated PknQ-FHA complex, (2) mutant complex having single phosphorylation on Ser+ ions were added. A short energy minimization step was performed on the solvated system with steepest descent algorithm for 50000 steps.The protein complex was dissolved at the center of a cubic box, solvated with single point charge water molecules. Solute-box distances of 1.0 nm were specified. To simulate the protein-protein complex system and in order to solve the issue of surface effects, periodic boundary conditions were applied. To neutralize the net charge on the protein, NaTwo phases of equilibration were conducted for 100 ps each- under constant Number of particles, Volume and Temperature (NVT) followed by constant Number of particles, Pressure and Temperature (NPT). Positional restraints were applied on the protein to allow solvent molecules to relax around the structure. In the second stage, positional restraints were lifted and the system was coupled to a heat bath at 300 K using the Berendsen thermostat and allowed to equilibrate for 100 ps The production run was performed with suitable parameters for 10 ns at a temperature and pressure of 300 K and 1 atm, respectively. Coordinate sampling was performed at every 2 fs time interval. Bond-lengths were constrained using the Linear Constraint Solver (LINCS) algorithm Escherichia coli strain DH5α (Novagen) was used for cloning and BL21 (DE3) (Stratagene) for the expression of recombinant proteins. E. coli cells were grown and maintained with constant shaking (200 rpm) at 37°C in LB broth supplemented with appropriate antibiotic (100 µg/ml ampicillin and/or 12.5 µg/ml chloramphenicol), when needed.flpknQ , its catalytic kinase domain kdpknQ (1–344 aa), mup018c (1–362 aa) and mup012c (1–87 aa), the respective genes were amplified by PCR from M. ulcerans Agy99 plasmid pMUM001 using specific primers (Rv0020c was PCR amplified from M. tuberculosis H37Rv genomic DNA and cloned in pProEx-HTc vector. The plasmid derivatives were confirmed with restriction digestion and DNA sequencing (Invitrogen) (M. tuberculosis PstP (Rv0018c) and Bacillus anthracis PrkD/PrkDS162A were cloned as described earlier For cloning of  primers . The resitrogen) . M. tubeTo generate the specific site mutants  of PknQ,E. coli BL21 (DE3). The recombinant GST-tagged fusion proteins were affinity purified with glutathione sepharose column. The His6-tagged proteins were purified by Ni2+-NTA affinity chromatography. For both purifications, similar procedures were followed as described before The plasmids were transformed and proteins were over-expressed in MBP-tagged MupDivIVA was purified using amylose resin as described before In vitro kinase assays of PknQ or PknQK41M (1 µg each) were carried out in kinase buffer  containing 2 µCi [γ-32P]ATP  followed by incubation at 25°C for 0–30 minutes, as described previously kd), unless specified. Myelin Basic protein (MyBP) (5 µg) was used as an artificial substrate for PknQ in a time-dependent in vitro kinase assay. Substrates MupFHA and MupDivIVA (5 µg each) were added with PknQ (1 µg) for phosphotransfer reactions carried out in kinase buffer containing 2 µCi [γ-32P]ATP followed by incubation at 25°C for 30 minutes. To determine the ionic requirements of PknQ, in vitro kinase assays were performed in the kinase buffer containing 20 mM HEPES [pH 7.2] with various concentrations of divalent cations  were included additionally, as indicated. Inhibition assays with ammonium [iron-III] citrate and hemin (Sigma) were performed in the presence of 10 mM Mn2+ and Mg2+ each. Reactions were terminated by 5× Laemmli sample buffer followed by boiling at 100°C for 5 minutes. Proteins were resolved by 10% SDS-PAGE and analyzed by Personal Molecular Imager . Quantification of radioactive bands was done by Quantity One 1-D Analysis Software (Bio-Rad).32P-PknQkd was separated by SDS-PAGE after in vitro phosphorylation and electroblotted onto Immobilon PVDF membrane (Millipore). PAA analysis by two-dimensional thin layer electrophoresis (2D-TLE) was performed as described earlier 32P-MupFHA and 32P-MupDivIVA (phosphorylated by PknQkd) were analyzed similarly.Autophosphorylated 6-PknQkd (5 µg) was autophosphorylated in vitro using 1 mM cold ATP, in the presence or absence of MupFHA (20 µg). For identification of phosphosites in MupFHA and MupDivIVA, kinase assays were performed with GST-MupFHA (5 µg) and MBP-MupDivIVA (5 µg) in the presence of 1 mM cold ATP and PknQkd (2 µg). Samples were resolved on 10% SDS-PAGE and gels were stained with coomassie brilliant blue stain. The stained bands corresponding to desired protein sizes were cut-out and used for mass-spectrometry as described earlier In order to identify the sites of autophosphorylation in PknQ, HisProteins were resolved by SDS-PAGE and transferred onto nitrocellulose membrane. Membrane was then blocked with 3% bovine serum albumin (Sigma) in phosphate-buffered saline containing 0.1% Tween-20 (PBST) overnight at 4°C. After blocking, the blot was washed thrice with PBST followed by incubation with primary antibodies at 1∶10,000 dilution for 1 h at room temperature. Subsequently, after five washes, the blot was incubated in secondary antibodies  at 1∶10,000 dilution for 1 h at room temperature. The blots were developed using SuperSignal West Pico Chemiluminescent Substrate kit (Pierce Protein Research Products) according to manufacturer's instructions.6-PknQ was over-expressed in E. coli BL-21 (DE3) cells and whole cell lysate was allowed to bind to the resin. The resin was washed to remove the non-specific unbound proteins. The lysates of E. coli BL-21 (DE3) cells were prepared separately containing over-expressed GST-tagged MupFHAwt (wild type), MupFHAR41A and MupFHAS55A. These three lysates were incubated with immobilized His6-PknQ for 1 h at 4°C. The resin was washed again followed by elution. The eluted fractions were analyzed by SDS-PAGE and immunoblotted with anti-GST antibodies (Abcam).For affinity pull-down assays, Pierce Pull-Down PolyHis Protein∶Protein Interaction Kit was used . Briefly, Hismup018c cloned in pGEX-5X-3 or mup012c cloned in pMAL-c2x were co-expressed in E. coli BL-21 (DE3) cells with pACYC-PknQ. PknQK41M was used as a negative control to generate unphosphorylated proteins. MupDivIVA or MupFHA were thus purified as MBP-tagged and GST-tagged proteins, respectively. Phosphorylation status of these proteins was analyzed by Pro-Q Diamond phospho-protein gel staining of SDS-PAGE gels followed by SYPRO Ruby protein gel stain, as described before 6-tagged kinase (PknQ and its mutants or PrkD/PrkDS162A mutant) or MBP-MupDivIVA (and its mutants) were dissolved in coating buffer (carbonate-bicarbonate buffer [pH 9.2]) at a concentration of 10 µg/ml and adsorbed (1 µg/well) on the surface of a 96-well ELISA plate  for 2 h at room temperature. After rinsing the wells five times with PBST, the reactive sites were blocked (3% BSA in PBST) overnight at 4°C. The adsorbed proteins were challenged with varying concentrations of soluble GST-tagged proteins (MupFHA and its mutants or Rv0020c) dissolved in blocking buffer for 1 h at room temperature. After five washes with PBST, the wells were treated with HRP-conjugated monoclonal antibody against GST (Abcam) at 1∶10,000 dilution for 1 h at room temperature. Followed by five washes, the chromogenic substrate o-phenylenediamine dihydrochloride  and H2O2 were used to measure the interaction. After addition of stop solution (2.5 M H2SO4) the absorbance was read at 490 nm. The experiments were performed 3 times with freshly purified proteins along with their mutants.Sandwich ELISA was performed as described earlier S55A (150 nM each) was adsorbed on the 96-well ELISA plate. After washing, 100 nM of pThr , pSer  or a random unphosphorylated peptide  was added. The interaction was measured as described above except the use of anti-His antibody (Abcam) at 1∶10,000 dilution. The resulting values were normalized to MupFHAS55A interaction, which was used as a negative control.For interaction study with peptides, GST-tagged MupFHA or MupFHAM. ulcerans, we performed a BLASTp search using the sequence of the catalytic domain of M. tuberculosis PknB (1–331 aa), corresponding to the most conserved mycobacterial STPK M. ulcerans STPKs showed that they possess unique kinase modules that are divergent from its close homolog, M. marinum. Out of the 24 putative STPK encoding genes present in M. marinum, only 13 are retained in M. ulcerans. This finding suggests that although M. ulcerans has evolved from M. marinum, it has retained only those STPKs that are necessary for its survival in humans while excluding those that may confer adaptation to M. marinum in fish. STPKs are broadly classified on the basis of conserved catalytic Arg/Asp (RD) residues present between subdomains VIa and VIb M. ulcerans belong to the RD kinase family, with PknG being the only non-RD kinase. The domain architecture of these kinases show a modular organization in which the kinase domain is located at the N-terminus  harbors all 12 conserved Hank's subdomains present in eukaryotic STPK counterparts kd and subsequent structural analysis revealed that the kinase domain consists of two lobes joined by a hinge segment. Catalysis occurs at the interface of the two lobes, where the catalytic amino acid residues interact with both ATP and the protein substrate , which is separated from the extracellular C-terminal domain harboring a FepB-like iron transporter module through a transmembrane region . PknQkd ubstrate . Based okd (1–344 aa) was cloned, over-expressed and the His6-tag fusion protein was purified from E. coli . As shown in K41M was inactive. The phospho-transfer potential of PknQ was also assessed in a time dependent manner and phosphorylation of MyBP was quantified  on PknQresidues . Mass spresidues , Fig. 4B164 and Thr166 sites, since their homologous residues are known to be the most conserved phosphorylated residues in the activation loop of STPKs in vitro kinase assays with [γ-32P]ATP, resolved on SDS-PAGE, and analyzed by autoradiography. In the quantitative analysis, there was no significant loss in phosphorylation observed in the PknQT164A single mutant, while PknQT166A and PknQT164/166A showed significant loss in signal intensity . In M. tuberculosis, several STPK-FHA domain containing protein partners are broadly conserved at the same genetic loci, such as PknB-Rv0019c, PknF-Rv1747 or PknH-EmbR In order to regulate and amplify signals, protein kinases associate and interact with a number of proteins within the cell. Most of the bacterial protein kinases are known to exhibit synteny with their substrates. In M. ulcerans, we performed a BLASTp search using Rv0020c of M. tuberculosis as a query M. marinum, which has ten FHA domain containing proteins, including the one encoded by the pMUM003 plasmid in M. marinum DL240490). A domain analysis and homology search showed that all of the proteins have functional homologs in M. tuberculosis except MupFHA  is present in the vicinity of pknQ (mup011), suggesting that the two proteins encoded by these genes may interact with each other. In order to confirm this hypothesis, we first performed multiple sequence alignment of MupFHA and other known FHA-domain containing proteins. The alignment shows that the five most conserved residues of FHA-domain  are also present in MupFHA , Thr166 and Thr260 do not make close contact with MupFHA  and PknQ-pThr174 (Tpo174) in stabilizing the complex formed with MupFHA  with the FHA domain   . Apart fmodeling .Bacillus anthracis kinase PrkD that autophosphorylates on Ser162 residue, in addition to several threonine and tyrosine residues S162A where the interaction was found to be reduced after mutation of Ser162  and the second expressing MupFHA (pGEX-5X-3). Affinity purified MupFHA that was co-expressed with PknQ (renamed as MupFHA-P) showed an intense phosphorylation signal, while no phosphorylation was found in MupFHA co-expressed with PknQK41M (MupFHA-UP) (FHA domain containing proteins are known to be phosphorylated by their neighboring kinases PknQK41M . To furtpFHA-UP) .T166A, PknQS170A and PknQT174A compared to the wild-type and PknQT164A , which confirmed the results obtained from the PAA analysis , while no signal was observed on the spots corresponding to pSer and pTyr . We subsanalysis , Table 38A/T214A . No loss41 and Ser55, were mutated to alanine and the effect on PknQ-mediated phosphorylation of MupFHA was subsequently analyzed. We found that the MupFHAR41A and MupFHAS55A mutants had an approximately 40% and 80% loss of phosphorylation signal compared to wild-type protein, respectively  affects the kinase activity of PknH. Although EmbR2 is not phosphorylated by PknH, it inhibits the kinase autophosphorylation and inactivates the protein M. tuberculosis, regulates the cell wall regulator pseudokinase MviN in vitro kinase assay, indicating that their interaction may have a negative impact on the kinase activity  and act as a principal controller of this signaling scheme. However, MupFHA may only act as an additional regulator of the kinase activity, while Ser/Thr phosphatase (Mul_0022) encoded in M. tuberculosis, PknA and PknB regulate Wag31, which is a DivIVA domain containing protein that regulates growth, morphology, polar cell wall synthesis, and peptidoglycan synthesis mup011 (pknQ) and mup012c . Phosphorylation-specific ProQ Diamond staining revealed that MupDivIVA was phosphorylated only when co-expressed with the catalytically active kinase (MupDivIVA-P) and not with the kinase-inactive mutant (MupDivIVA-UP)  . PAA anavIVA-UP) . Mass spvIVA-UP) . IncidenvIVA-UP) . Compariproteins . Such strylation .M. tuberculosis Rv0020c was used to compare pThr specificity  or MupDivIVA-UP (unphosphorylated) and GST-tagged MupFHA were used in a sandwich ELISA. We found that MupDivIVA phosphorylation increased its affinity for MupFHA . Furthercificity . This anM. ulcerans, which is the causative agent of Buruli ulcer. M. ulcerans is a slow growing bacterium (slower than M. marinum and M. tuberculosis) in silico analysis, we identified 13 STPKs in the M. ulcerans genome that are distinct from its close relative M. marinum that has 24 STPKs M. tuberculosis have been classified in five clades M. ulcerans also has individual STPKs related to all five clades with an over-representation of the PknF/PknI/PknJ clade. Analyses of STPKs and FHA domain encoding genes confirmed that M. ulcerans underwent reductive evolution compared to M. marinum.In this study we analyzed the STPK-mediated signaling system of M. ulcerans kinome exclusive than M. tuberculosis and other characterized bacterial kinomes Mycobacterium species, including pMUM001 (M. ulcerans Agy 99), pMUM002 (M. liflandii 128FXT), and pMUM003 (M. marinum DL240490) mup011] to the FHA domain-containing protein [mup018c]), although pMUM003 is slightly different, most likely due to a frameshift mutation B. anthracis, Y. pestis, and ShigellaY. pestis, the virulence-associated plasmid pLB1 encodes a STPK YpkA that acts as a direct inducer of cell death by promoting apoptosis and actin depolymerization during infection M. ulcerans has previously been described M. ulcerans.The presence of PknQ on the virulence-associated plasmid pMUM001 makes the M. ulcerans, we elucidated the biochemical characteristics of this kinase. Our biochemical characterization identified both common and novel features. PknQ kinase activity is dependent on cofactors such as Fe2+, Mn2+, Mg2+, and Zn2+. Interestingly, the kinase is activated by Fe2+, while its activity is inhibited by Fe3+. This inhibition occurs in the presence of hemin, indicating that PknQ activity is regulated by iron and its redox state in the cellular milieu. The role of iron in the regulation of its kinase activity is justified by the presence of a FepB-like iron transporter domain at the C-terminus of PknQ. However, further studies are required to validate this aspect of PknQ signaling.To understand the significance of PknQ in M. tuberculosis STPKs, activity is regulated by two threonine residues present in the activation loop. Analogous residues (Thr164 and Thr166) are also phosphorylated in PknQ, but only Thr166 is critical for autophosphorylation activity. Furthermore, a novel serine residue, Ser170, was found to regulate the autokinase and substrate phosphorylation activities of PknQ. In fact, phosphorylation of activation loop residues is known to be required for stabilization of kinases as it can induce specific conformational changes important in substrate binding To understand the activation mechanism of PknQ, several mutants were generated. We found that the autophosphorylation of serine and threonine residues in the activation loop region regulates PknQ activity. In most We also identified the FHA-domain containing protein MupFHA as an interacting partner of PknQ. To understand how MupFHA and PknQ interact, we generated structural models to establish the basis of MupFHA and PknQ interaction. Interestingly, the PknQ and MupFHA interaction highlighted several novel aspects. The structural analyses suggest that PknQ-MupFHA interaction follows the canonical binding mode of pThr interaction with specific arginine and serine residues of FHA-domain. However, MupFHA additionally interacts with a pSer residue present in the activation loop of PknQ. The structural models were validated by experimental analysis using ELISA and affinity pull-down assays, thus establishing the structure-activity relationship for PknQ. Together, these findings highlighted the unconventional molecular recognition patterns of kinase∶FHA interactions.M. tuberculosis CDC1551 strain. Nevertheless, MupFHA signaling is unique, as PknH does not phosphorylate EmbR2, while MupFHA is a substrate of PknQ. This regulation of kinase activity by FHA domains could have important implications in the spatio-temporal regulation of cellular signaling. It is important to note that the critical activation loop residues were phosphorylated even in the presence of MupFHA, thus indicating that MupFHA may only regulate the secondary phosphorylation sites and substrate binding of PknQ. To understand this inhibition further, we applied mass spectrometry and found a significant reduction in number of phosphorylation sites. However, the mass spectrometry analysis did not quantitate the phosphorylation stoichiometry of each site and we cannot rule out the possibility that the phosphorylation of key activation loop residues is also inhibited.The second unique aspect of the MupFHA∶PknQ interaction is the role of MupFHA as a negative regulator of PknQ. To the best of our knowledge, this is only the second report of any bacterial FHA-mediated regulation of the cognate kinase activity. It has been previously reported that EmbR2 inhibits PknH phosphorylation M. tuberculosis Wag31, another DivIVA domain-containing protein M. tuberculosis is already known to be phosphorylated by PknB and PknA and this phosphorylation is critical for cell growth and peptidoglycan synthesis The third aspect of MupFHA signaling is the ability of both PknQ and MupFHA to interact with another phosphorylated protein, MupDivIVA. Our results indicate that PknQ phosphorylates MupDivIVA, which is a homolog of M. ulcerans and has revealed many novel aspects of signaling systems among Mycobacterium species. Our results indicate that PknQ could be an important sensor of extracellular cues in M. ulcerans and can propagate the signals to MupFHA and MupDivIVA. Moreover, iron and MupFHA may act as quenchers in this phosphorylation cascade. Taken together, our data underscore the importance of structure-activity studies in unraveling the PknQ-MupFHA signaling axis in M. ulcerans and provide an interesting starting point to work towards understanding this pathogen.In conclusion, our study is the first analysis of signaling pathways in Figure S1Domain analysis of M. ulcerans STPKs. Since the STPKs are not characterized, we used SMART domain analysis to predict the possible domains present in these STPKs on the basis of their conserved protein sequences. PknQ possesses N-terminal catalytic domain and C-terminal extracellular region containing a periplasmic-binding domain, which also contains sequences conserved for FepB (such as iron transporter domain).(TIF)Click here for additional data file.Figure S2PknQ domain organization. Multiple sequence alignment showing conserved Hank's subdomains present in PknQ. The alignment was done with M. tuberculosis STPKs PknB and PknJ. The conserved subdomain sequences have been highlighted (yellow) and the corresponding domains have been marked.(TIF)Click here for additional data file.Figure S3Relative phosphorylation efficiencies of PknQ and its mutants. (A) Histogram shows relative phosphorylation considering the intensity of PknQWT as 100%. Corresponding autoradiogram (top) and SDS-PAGE image (lower) is shown. (B) Histogram shows relative phosphorylation of PknQ phosphorylation site mutants, considering the intensity of PknQ as 100%. The experiment was repeated three times and error bars represent S.D. of three values.(TIF)Click here for additional data file.Figure S4Multiple sequence alignment of MupFHA. Multiple sequence alignment of amino acid sequences of M. ulcerans MupFHA, M. tuberculosis Rv0020c and Human enzymes Rad53-FHA1, Rad53-FHA2 and polynucleotide kinase (PNK). MupFHA Residues corresponding to the five most conserved FHA-domain residues are colored red. Additional MupFHA residues that are involved in binding with PknQ are colored green.(TIF)Click here for additional data file.Figure S5Docking analysis of MupFHA with PknQ activation loop. The structure of PknQ phosphorylated at Ser170 and Thr174 was docked with MupFHA. As clearly evident, Thr166 does not interact with MupFHA. Figure A shows the whole complex and B shows enlarged section of interaction locus.(TIF)Click here for additional data file.Figure S6Interaction of MupFHA and PknQ in the presence of phospho-peptides. Treatment of MupFHA with pThr or pSer peptides saturates the phosphoprotein binding sites of the FHA domain. Therefore, PknQ and MupFHA show decreased interaction after the phospho-peptide treatment as most of the phosphoprotein binding sites are already saturated. Relative interaction values were calculated considering 100% interaction in absence of any peptide. Addition of pSer or pThr peptides leads to decrease in interaction and thus proves phospho-specific affinity of MupFHA.(TIF)Click here for additional data file.Figure S7S162A.Relative interaction of MupFHA with PrkD and its mutant PrkD ELISA was used to demonstrate the interaction of these proteins and relative interaction was calculated considering the interaction of PrkDwt as 100%. PrkDS162A shows considerably less interaction with MupFHA.(TIF)Click here for additional data file.Figure S8Role of Ser/Thr phosphatase. (A) Multiple sequence alignment  of M. ulcerans Ser/Thr phosphatase (Mul_pstP) with M. tuberculosis phosphatase (Mtb_pstP). The alignment shows >90% sequence identity. (B) Dephosphorylation of PknQ and MupFHA by M. tuberculosis PstP. Autoradiogram (lower panel) shows loss in phosphorylation signal in presence of PstP. Corresponding SDS-PAGE image is shown in upper panel.(TIF)Click here for additional data file.Figure S9Multiple sequence alignment  of M. ulcerans MupDivIVA with M. tuberculosis Wag31. The phosphorylated residues are highlighted in both the sequences. As shown in the alignment, the 3 phosphorylated serine residues of MupDivIVA are localized in the same region as that of the only phosphorylation site of Wag31.(TIF)Click here for additional data file.Table S1Primers and clones used in the study.(DOCX)Click here for additional data file.Movie S1Movie of the PknQ-pSer170/pThr174 and FHA domain complex simulation for 10 ns (stick representation).(MP4)Click here for additional data file.Movie S2Movie of the PknQ-pSer170/Thr174Ala and FHA domain complex simulation for 10 ns (stick representation).(MP4)Click here for additional data file.Movie S3Movie of the PknQ-Ser170Ala/pThr174 and FHA domain complex simulation for 10 ns (stick representation).(MP4)Click here for additional data file.


Two models with different treatment of the disordered Fe site are presented.The compound with the idealized composition Ba 2CaFeAl2F14, dibarium calcium iron(II) dialuminium tetradecafluoride, were obtained serendipitously by reacting a mixture of the binary fluorides BaF2, CaF2 and AlF3 in a leaky steel reactor. The compound crystallizes in the usovite structure type (Ba2CaMgAl2F14), with Fe2+ cations replacing the Mg2+ cations. The principal building units are distorted [CaF8] square-antiprisms (point group symmetry 2), [FeF6] octahedra (point group symmetry -1) and [AlF6] octahedra that are condensed into undulating 2∞[CaFeAl2F14]4− layers parallel (100). The Ba2+ cations separate the layers and exhibit a coordination number of 12. Two crystal structure models with a different treatment of the disordered Fe site , are discussed, leading to different refined formulae Ba2Ca1.310 (15)Fe0.690 (15)Al2F14 [model (I)] and Ba2CaFe0.90 (1)Al2F14 [model (II)].Crystals of the title compound, with the idealized composition Ba Most of the usovite-type representatives known so far were prepared and structurally determined by Babel, Tressaud and co-workers over the last three decades F8] sqare-antiprisms (point group symmetry 2) and [(MII2)F6] octahedra (point group symmetry MIII3)F6] octahedra. The [(MII2)F6] and [(MIII3)F6] octahedra are connected by corner-sharing into infinite crosslinked double chains 1∞[(MII2)F2F4/2(MIII3)2F8F4/2] extending parallel to [010] F8] square-antiprisms into undulating (100) layers with composition 2∞[(MII1)(MII2)(MIII3)2F14)]4−, with the Ba2+ cations separating the individual layers  Åa Table 1. Wheras M—F bond lengths of the [(MII2)F6] octahedra was also observed for a series of other usovite-type structures and was associated with an occupational disorder of the MII2 site. For these models, either a mutual substitution of Ca2+ (on the MII1 site) with corresponding divalent transition metal ions on the MII2 site, or partial replacement of the divalent transition metal ions by Ca2+ at the MII2 site was considered, resulting in stoichiometric compounds and Ca-richer compounds, respectively :0.345 (7) for this site  and converged with the same reliability factors and remaining electron densities as the model without an incorporation of Ca2+ and underoccupation of the Fe2+ site only . The refined formula for this model is Ba2Ca2Fe0.90 (1)Al2F14. Bond lengths and angles of the two models are the same within the corresponding standard uncertainties ] and consequently requires the presence of an element in a higher oxidation state (here most probably Fe3+) to compensate the negative charge of −2 of the [Ba2CaAl2F14] framework. Although in this case rather a decrease of MII2—F bond lengths should be expected (contrary to the findings of the current study), it cannot competely ruled out that Fe3+ ions are present at this site. As a matter of fact, based on diffraction data alone, there is a clear tendency towards model (I) but no definite answer whether Fe is partly replaced by Ca on the MII2 site [model (I)] or is statistically occupied by Fe2+ and small amounts of Fe3+ [model (II)]. Complementary analytical techniques like Mössbauer spectroscopy will be needed in future to shed some light on this problem.On the other hand, an 3 , CaF2  and BaF2  were mixed in the stoichiometric ratio 1:1:1 and thoroughly ground in a ball mill, pressed into tablets and placed in a carbon tool steel container shielded with a molybdenum foil. NH4F·HF  were added to the mixture to increase the HF pressure, to expel the remaining oxygen and to adjust a slightly reducing atmosphere during the reaction. The reactor was then closed and heated to 1173 K in the course of 20 h, kept at that temperature for 24 h, and then cooled slowly to 973 K at a rate of 10 K h−1, kept at this temperature for 24 h and finally cooled to room temperature overnight. After opening the reactor it became evident that parts of the molybdenum foil were torn apart accompanied by a severe attack of the inner container wall. Single crystals of the title compound were separated from the obtained colourless to light-green bulk material. X-ray powder diffraction of the bulk revealed the formation of α-BaCaAlF7 as the main phase and the title compound as a minority phase. Some additional reflections were also present that could not be assigned to any known phases.The binary fluorides AlFet al., 1980R1 = 0.034 and wR2 = 0.089. However, negative residual electron density at the Fe atom pointed to an underoccupation and/or a statistical disorder of the MII2 site with a lighter element present. In fact, free refinement of the site occupation factor for this site resulted in only 90% occupancy and significant better reliability factors , the site occupation factor of the Fe site was refined freely without contribution of Ca2+ at this site. The remaining electron densities  modelI, modelII, global. DOI: 10.1107/S2056989015009251/br2250modelIsup2.hklStructure factors: contains datablock(s) modelI. DOI: 10.1107/S2056989015009251/br2250modelIIsup3.hklStructure factors: contains datablock(s) modelII. DOI: 1401194, 1401194, 1401195CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Attentional capture is usually stronger for task-relevant than irrelevant stimuli, whereas irrelevant stimuli can trigger equal or even stronger amounts of inhibition than relevant stimuli. Capture and inhibition, however, are typically assessed in separate trials, leaving it open whether or not inhibition of irrelevant stimuli is a consequence of preceding attentional capture by the same stimuli or whether inhibition is the only response to these stimuli. Here, we tested the relationship between capture and inhibition in a setup allowing for estimates of the capture and inhibition based on the very same trials. We recorded saccadic inhibition after relevant and irrelevant stimuli. At the same time, we recorded the N2pc, an event-related potential, reflecting initial capture of attention. We found attentional capture not only for, relevant but importantly also for irrelevant stimuli, although the N2pc was stronger for relevant than irrelevant stimuli. In addition, inhibition of saccades was the same for relevant and irrelevant stimuli. We conclude with a discussion of the mechanisms that are responsible for these effects. Visual attention is the selection of visual information for purposes such as in-depth processing, perception, or action control. Because we have to select information at all times, understanding attention is a key to an understanding of almost any form of cognition. To date, however, the mechanisms by which attention operates are not fully understood.One persistent debate in this area concerns the role of inhibition of irrelevant stimuli as one form of top-down control over attention. Whereas some researchers believe that inhibition of attention is a response to initial capture of attention and, thus, follows preceding attentional capture by an irrelevant stimulus , other rTo start with the first proposition, many researchers argued that salient objects capture attention in a bottom-up way cf. , 4). Acc. Acc4]). delayed relative to responses to probes presented at the location of the shape target. This delay evidently reflected active inhibition of the color distractor that developed over time. Like Theeuwes et al. ).strategy , 41. In strategy –45, but strategy , 47). Instrategy . Second,A further point that needs discussion is the relation between capture and IOR. Originally, IOR was regarded as the reflection of preceding capture . Under tThe present results also show that IOR can be induced by color singletons. Previous studies such as Gibson and Amelio  failed tIn conclusion, in line with the late inhibition or disengagement theory, we have shown that the irrelevant and the relevant distractor first both captured attention (reflected in their N2pcs) before they were actively inhibited (reflected in saccadic IOR). This lack of proactive inhibition was also found if only the fastest responses were analyzed. However, we found little indication that IOR was stronger after irrelevant than relevant cues. Therefore, it is not likely that disengagement was the only responsible process. Early inhibition (among the slower responses) or contingent capture must have also contributed to the N2pc differences between relevant and irrelevant cues.


Morus multicaulis Perr.) was isolated using a macroporous resin column. The primary component, which was purified by semi-preparative high-performance liquid chromatography diode array detection (HPLC-DAD), was identified as mulberroside A (MA) by liquid chromatograph-mass spectrometer (LC-MS), 1H and 13C nuclear magnetic resonance (NMR) spectra. In total, 4.12 g MA was efficiently extracted from one kilogram of mulberry bark. The enzymatic analysis showed that MA inhibited the generation of dopachrome by affecting the activities of monophenolase and diphenolase of tyrosinase in vitro. This analysis indicated that MA and oxyresveratrol (OR), which is the the aglycone of mulberroside A, exhibited strong inhibition of the monophenolase activity with IC50 values of 1.29 µmol/L and 0.12 µmol/L, respectively. However, the former showed weaker inhibitory activity than the latter for diphenolase. For the monophenolase activity, the inhibitory activity of MA and OR was reversible and showed mixed type 1 inhibition. Additionally, the inhibition constant KI (the inhibition constant of the effectors on tyrosinase) values were 0.385 µmol/L and 0.926 µmol/L, respectively, and the KIS (the inhibition constants of the enzyme-substrate complex) values were 0.177 µmol/L and 0.662 µmol/L, respectively. However, MA showed competitive inhibition of diphenolase activity, and IK was 4.36 µmol/L. In contrast, OR showed noncompetitive inhibition and IK = ISK = 2.95 µmol/L. Taken together, these results provide important information concerning the inhibitory mechanism of MA on melanin synthesis, which is widely used in whitening cosmetics.A bioactive ingredient in an ethanol extract from the branch bark of cultivated mulberry Husang-32 ( Tyrosinase, which is a polyphenol oxidase, is the key enzyme in melanin synthesis and possesses monophenolase and diphenolase activity. This enzyme can catalyse two distinct reactions: the oxidation of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA) (first reaction) and the oxidation of L-DOPA to dopaquinone (second reaction). Then, dopaquinone, through a non-enzyme-catalysed process, is transformed into leukodopachrome (third reaction). Leukodopachrome is oxidised into dopachrome by a dopaquinone (fourth reaction), which is an extremely fast and non-enzyme-catalysed process. Then, dopachrome is transformed to melanin through a series of chemical- and enzyme-catalysed reactions. The process of tyrosinase catalysing L-tyrosine to dopachrome shows that dopachrome synthesis can be suppressed when any of the steps are inhibited. Additionally, not all substances that can inhibit the formation of dopachrome are tyrosinase inhibitors, for example, thymol Cortex Mori albae) and branch were health food especially the root was widely used in the folk prescription of traditional Chinese medicine. Modern pharmacological study proves that the aqueous or methanol extracts of Cortex Mori have anti-inflammatory, hypoglycemic, hypotensive, anticarcinogenic and anti-microbial activities; and the chloroform and alkali extracts have the obvious anti-inflammation and anti-asthmatic activities.Mulberry (Morus) trees, belongs to the family of Moraceae, have been widely cultivated in several sericulture countries . In fact, early in 2002 the Chinese Ministry of Health declared that the mulberry leaf and mulberry (fruit) were not only food, but also drugs; and the root bark  is an oxyresveratrol diglycopyranoside, where 4-OH and 3′-OH are substituted by two glucose molecules . MA has Ramulus mori of the cultivated mulberry (Husang No. 32) Morus multicaulis Perr. Using a macroporous resin column for adsorption and for separation and then purification by reversed-phase high-performance liquid chromatography (RP-HPLC), we obtained much MA monomer. Next, we systemically studied and analysed the mechanism of the inhibition activity of MA on the mushroom tyrosinase.The above-mentioned research concerning the separation, purification, and identification of MA and its tyrosinase inhibition were primarily identified from the mulberry root. There is only rare systematic research and analyses regarding the extraction and separation of mulberry branches, which are often used as agricultural waste or as firewood. Thus far, there is a lack of systemically studies and analyses concerning the mechanism of the strong inhibitory action of MA on the monophenolase and diphenolase activities of the mushroom tyrosinase. In this study, we extracted an alcohol solution from Morus multicaulis Perr. were collected from the mulberry field of Soochow University in November 2010.Fresh one-year-old branches of cultivated mulberry (Husang No. 32) from The ethanol extract of mulberry branch bark was prepared by a previously described 2O, 0%, 30%, and 100%). The fractions were concentrated and lyophilised into powders.A weighed amount of the branch bark powder was dispersed in water, added to a D101 macroporous resin chromatography column  and eluted with a gradient elution of ethanol-water  was used to analyse the samples. All the sample used was precisely formulated into a solution of 0.5 mg/ml. Mobile phases A (0.4% aqueous acetic acid) and B (acetonitrile) were prepared according to the following procedure: initial 0-20 min, linear change from A/B = 95:5 (v/v) to A/B = 85:15 (v/v); 20–30 min, linear change from A/B = 85:15 (v/v) to A/B = 80:20 (v/v); 30–35 min, linear change from A/B = 80:20 (v/v) to A/B = 45:55 (v/v) and then held for 5 min. The mobile phase flow-rate was set at 1.0 mL/min, and aliquots of 10 µl were injected for analysis. The detection wavelength was 324 nm, scanning from 200 nm to 600 nm. All analyses were performed at room temperature.y) and with the standard injection volume (µg) as the abscissa (x).The preparation of MA standard curve was as follows. In total, 10 mg of MA standard was dissolved in water and then formulated into a solution of 1.0 mg/mL. Samples of 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, and 14.0 µL of MA standard solution were injected into the HPLC system. A standard curve was drawn, with the peak area as the vertical axis (The sample preparation was performed using a Semi-Preparative HPLC (Shimadzu LC-6A) equipped with a DAD detector (SPDM20A). The semi-preparative column consisted of a Shim-pack Prep-ODS (H) column (250×20 mm) fitted with a guard module (Shim-pack G-ODS). Mobile phase A was 0.4% aqueous acetic acid, and mobile phase B was acetonitrile . The detection wavelength was 324 nm, scanning from 200 nm to 600 nm. All preparations were performed at room temperature. The gradient program used was as follows: 0–30 min, linear change from A/B = 93:7 (v/v) to A/B = 80:20 (v/v); 30–40 min, linear change from A/B = 80:20 (v/v) to A/B = 50:50 (v/v) and then held for 10 min. The flow rate was 5.0 mL/min, and aliquots of 5.0 mL were injected. The peak corresponding compound was collected at the retention time of 33.148 min and then concentrated and lyophilised into powders.(+)) scanning, 380°C; DP: 25; CUR: 30; G1: 40; G2: 35; EP: 10; CE: 10.The liquid chromatograph-mass spectrometer (LC-MS) conditions were as follows: a Q-Trap 2000 LC/MS/MS electro-spray ionisation source (ESI source) from ABI Company; an Agilent 1100 LC system; a Shimadzu Shim-Pack VP-ODS  chromatographic column, a column temperature of 18°C; mobile phase: 10% acetic acid aqueous solution:acetonitrile  = 60:40 (v/v); Enhanced Mass Spectrometry (EMS 3OD) and 0.5 mL deuterated dimethyl sulphoxide (CD3SOCD3), respectively. The carbon spectrum (13C-NMR) and hydrogen spectrum (1H-NMR) were measured using a 300 MHZ superconductive nuclear magnetic resonance (NMR) spectrometer.The lyophilized powder of the sample was dissolved using 0.5 mL deuterated methanol  on the catalysis of L-tyrosine to dopachrome was determined using the 2PO4-Na2HPO4 temporary buffer solution (pH 6.8), and the volume was set to 100 mL. In total, 0.25 mg/mL enzyme solution and a certain concentration of the sample solution were prepared. All the solutions were filtered using a syringe filter with an aperture of 0.22 µm. The components of the reaction system were as follows: 80 µL N-acetyl-L-tyrosine was mixed with 10 µL sample solution and incubated at 30°C for 5 min, and then 10 µL enzyme solution was added. During the period from 0–60 min, 20 µL mixed solution was injected into the HPLC system every 10 min to detect changes in N-acetyl-L-tyrosine. The HPLC conditions were as follows: a Shimadzu HPLC pump equipped with a Shimadzu 9082652 VP-ODS column (250 mm×4.6 mm). The detection wavelength was 275 nm. The injection volume was 10 µL, and the flow-rate was 1.0 mL/min. The mobile phases were A (water) and B (acetonitrile), which were prepared according to the following linear procedure: 0–8 min, A/B = 85:15 (v/v). All analyses were performed at room temperature.The effect of the enzyme-catalysed process was determined using a previously described The effect of the non-enzyme-catalysed process was determined by a previously described 2PO4-Na2HPO4 temporary buffer solution (pH 6.8), with a substrate concentration of 0.5 mmol/L and with an enzyme solution concentration of 1.0 mg/mL. The samples were dissolved in DMSO. First, 100 µL sample solution was added, then 2.8 mL substrate solution, which was pre-incubated at 30°C, was added, and last, 100 µL enzyme solution was added and immediately mixed. The curve was drawn using real-time tracking of the optical density change over time at 475 nm. The measuring instrument was a Hitachi U3000 UV-visible spectrophotometer. The enzyme activity was obtained from the slope of the line. The sample concentration-response curve was plotted as the inhibition rate of samples to the enzyme against the concentrations of effectors. The IC50 value, which represented the concentration of sample at which 50% of the tyrosinase activity was inhibited, was obtained from the curve.The measurement of the sample's inhibitory activity on monophenolase activity was based on 0.5 mmol/L L-tyrosine as the substrate The measurement of the sample's inhibitory activity on diphenolase activity was based on 0.5 mmol/L L-DOPA as the substrate. The experimental procedure and calculations were identical to those above-mentioned procedures and calculations.2PO4-Na2HPO4 temporary buffer solution (pH 6.8); however, the amount of enzyme was changed to determine the effects of different concentrations of sample on the reaction of mushroom tyrosinase catalysing L-tyrosine or L-DOPA as substrates. Mapping the reaction rate in terms of the amount of enzyme determined whether the inhibition was a reversible reaction. If the lines all passed through the origin, then reversible inhibition was demonstrated. However, if the lines were parallel, then irreversible inhibition was demonstrated.The concentration of the substrate was fixed in 3.0 mL of 50 mmol/L NaH2PO4-Na2HPO4 temporary buffer solution (pH 6.8); however, the amount of substrate (L-tyrosine or L-DOPA) was changed to determine the effects of different concentrations of effectors on the reaction of mushroom tyrosinase catalysing the substrate. Using the Lineweaver-Burk y-axis, then competitive inhibition was indicated. If the lines intersected on the x-axis, then noncompetitive inhibition was indicated. If the lines intersected in the first or fourth quadrant, then this inhibition was mixed type 1 or 2. If the lines were parallel, then anticompetitive inhibition was indicated. The inhibition constant of the effectors on tyrosinase (IK) and the inhibition constants of the enzyme-substrate complex (ISK) were obtained by a secondary plot of the concentration of the effectors against the slope of the line and the y-axis intercept, respectively.And the inhibition type and inhibition constant were determined as follows. The concentration of the enzyme solution was fixed in 3.0 mL of 50 mmol/L NaHIn total, 2.0 kg powder as treated using the previously described Using RP-HPLC to analyse E30, the analysis of the 3D chromatogram  demonstrThere were no other interfering peaks, except at the retention time of 21.05 min in the 3D chromatogram , which sThe compound obtained was identified using MS and NMR.+, 569.2 [M+H]+, 406.9 [M-162]+, 245.0 [M-162-162+H]+, and showed that the molecular weight of the compound was 568.2.MS spectra showed the following positive ion ESI-MS m/z values: 591.2 [M+Na]1H-NMR  δ: 7.34 , 7.22 , 6.84 , 6.68 , 6.54 , 6.53 , 6.49 , 6.36 , 4.79 H-1″), 4.79 H-1′″), and 3.85–3.21 . Anomeric H signals in the 3.85–3.21  are overlapping signals. The original NMR spectrum has been added as 13C-NMR  δ: 120.2 (C-1), 157.1 (C-2), 104.8 (C-3), 160.4 (C-4), 109.3 (C-5), 128.4 (C-6), 127.6 (β-C), 124.9 (α-C), 141.9 (C-1′), 107.2 (C-2′), 159.5 (C-3′), 103.9 (C-4′), 159.5 (C-5′), 108.1 (C-6′), 102.0 (C-4(glc)C-1″), 74.8 (C-2″), 77.9 (C-3″), 71.3 (C-4″), 78.1 (C-5″), 62.4 (C-6″), 102.3 (C-3′ (glc)C-1′″), 74.9 (C-2′″), 77.9 (C-3′″), 71.3 (C-4′″), 78.1 (C-5′″), and 62.5 (C-6′″). The original NMR spectrum has been added as 1H-NMR  δ: 9.80 , 9.38 ,7.44 , 7.22 , 6.94 , 6.64 , 6.58 , 6.52 , 6.55 , 6.35 , 4.79 H-1″), 4.79 H-1′″), 5.24 , 5.10 , 4.51 , 3.70 , 3.50 , and 3.18–3.30 . Both anomeric H signals in the 3.50  and 3.18–3.30  are overlapping signals. The original NMR spectrum has been added as 1H-NMR (CD3OD), 13C-NMR and 1H-NMR (CD3SOCD3) data with the literature 9, 16, the compound was identified as oxyresveratrol-4-O-β-D-glucopyranosyl-3′-O-β-D-glucopyranoside or mulberroside A (MA). Its molecular formula is C26H32O14, and the structure is shown in By comparing the When no inhibitor was added, tyrosinase continuously catalysed L-tyrosine to dopaquinone and to dopachrome during the period from 0–12 min ; thus, the contents of dopaquinone and dopachrome in the reaction system rose . AdditioN-acetyl-L-tyrosine was used instead of L-tyrosine as the substrate for mushroom tyrosinase  because p-benzoquinone to substitute leukodopachrome and dopaquinone to simulate the non-enzymatic process. L-DOPA and p-benzoquinone were converted to dopaquinone and to hydroquinone by a redox reaction. HPLC was used to detect the changes in p-benzoquinone and hydroquinone contents over time in the reaction system, and the results are shown in p-benzoquinone and hydroquinone were at 245 nm and 280 nm, respectively, and the retention times were 7.76 min and 4.86 min in the Shimadzu VP-ODS column (250 mm×4.6 mm), respectively. p-benzoquinone when no samples were added to the system. Within 10 min, p-benzoquinone decreased by 75%, and at 20 min, p-benzoquinone decreased by 90.62%. p-benzoquinone content did not change and was not suppressed by the addition of MA and OR. As shown in p-benzoquinone were not influenced by MA and OR. Therefore, MA and OR do not play a role in the conversion of dopaquinone to dopachrome, which further illustrates that MA is a real tyrosinase inhibitor, similar to OR, and has inhibitory activity on both monophenolase and diphenolase.To measure whether the samples affected the second stage of the dopachrome synthesis, we used L-DOPA and 50 values of MA and OR were 1.29 µmol/L and 0.12 µmol/L, respectively. These results indicated that MA and OR strongly inhibited the monophenolase activity of the mushroom tyrosinase. The inhibitory activity of OR was approximately 10-fold stronger than that of MA, which indicated that the two glucose molecules added to OR had some effect on the inhibition of the monophenolase activity of the mushroom tyrosinase.The samples that inhibited the monophenolase activity of tyrosinase were measured using L-tyrosine as the substrate. In the reaction system, OR was included for comparison. By adding different concentrations of samples to the reaction system, when the concentration of L-tyrosine was unchanged, but the amount of enzyme added was changed, the reaction velocity (V) of the enzyme-catalysed oxidation of the substrate was measured by the change in the amount of enzyme ([E]). OR was the control in this test, which measured the mechanism by which MA inhibits the monophenolase activity of tyrosinase. The selected sample concentrations of MA were 0, 0.22 mmol/L, 0.44 mmol/L, 0.88 mmol/L and 1.76 mmol/L; the sample concentrations of OR were 0, 0.21 mmol/L, 0.42 mmol/L, 0.84 mmol/L and 1.68 mmol/L; and the enzyme concentrations were 0.8 mg/mL, 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL and 2.5 mg/mL. The results are shown in x-axis, and the reciprocal of the reaction rate 1/V was the y-axis. The results are shown in maxV) of the enzymatic reaction but also to an increase in the Michaelis-Menten constant (mK). These results showed that MA inhibited the monophenolase of the mushroom tyrosinase by a mixed-type inhibitory pattern, which was identical to OR. The inhibition constants of MA and OR on free enzyme (IK) and on the enzyme-substrate complex (ISK) were obtained by plotting the slope and the y-axis intercept against the sample concentration  and the amount of enzyme ([E]) was measured after adding different sample concentrations. The concentrations of MA were 0, 2.0 µmol/L, 4.0 µmol/L and 8.0 µmol/L; the concentrations of OR were 0, 0.1 µmol/L, 0.2 µmol/L and 0.4 µmol/L; and the enzyme concentrations were 0.2 mg/mL, 0.4 mg/mL, 0.8 mg/mL and 1.0 mg/mL. A plot of absorbance change against the amount of enzyme  showed t6. The reciprocal of the substrate concentration 1/[S] was the x-axis, and the reciprocal of the reaction rate 1/V was the y-axis. The results are shown in y-axis was obtained in the plot of MA, which indicated that the maximum reaction velocity (maxV) did not change when the sample concentration increased; however, the Michaelis-Menten constant (mK) increased. This result indicated typical competitive inhibition. The lines obtained by the double reciprocal plot of OR intersected on the x-axis, which indicated that the Michaelis-Menten constant (mK) was not affected by increased sample concentrations but led to decreased maximum reaction velocity (maxV). This result indicated non-competitive inhibition. Secondary plots of the slope and y-axis intercept of each line against sample concentration were obtained to determine the inhibition constants of MA on the free enzyme (IK = 4.36 µmol/L) and OR on the enzyme-substrate complex (ISK = IK = 2.95 µmol/L)  .Tyrosinase is a polyphenol oxidase and is not only the key enzyme in the food browning reaction 50 values only had 10-fold differences. However, both of these hydroxyls had strong inhibition that is a mixed inhibition type. However, the presence of the two glucose molecules had a significant effect on the inhibition of the diphenolase activity of the tyrosinase. The IC50 value of MA was only 7.45 µmol/L and was almost 20 times that of OR. The inhibition types of MA and OR on the diphenolase activity were different. These findings illustrated that the presence of the two glucose molecules seriously affected the diphenolase inhibitory activity.Concerning the molecular structure analysis, the hydrogen of the two phenolic hydroxyls at the 4′ and 3′ position in OR were substituted by two glucose molecules, which slightly affected the activity of MA in inhibiting the monophenolase activity of tyrosinase, and the IC50 value of OR was 0.46∼1.10 µmol/L, depending on the assay conditions 50 value of OR extracted from the mulberry twigs was 0.943 µmol/L (0.23 µg/L), which was reported by Lee et al. 50 value. The IC50 values of OR, which inhibited the monophenolase and diphenolase activities, were approximately 4-fold and 30-fold lower than that of our experiment, respectively. Their inhibition mechanism and type were also identical 50 value of MA for the inhibition of the monophenolase activity was almost 40-fold lower than our results, and the inhibition type was different. Kim's results showed competitive inhibition, whereas our results showed mixed-type inhibition. In addition, our results showed that MA had apparent inhibition of the diphenolase, which differed from their reports Our results were both similar and different when compared with the previous findings reported by others. The results concerning the effect of OR on the inhibition of the mushroom tyrosinase were similar to our results. The strong tyrosinase inhibitory activity of OR was previously confirmed in other studies, which found that the ICO-β-D-glucoside, oxyresveratrol-3′-O-β-D-glucoside and OR. This result indicated that MA was metabolised before being absorbed in vivo. This conversion into OR can increase its permeability and then can easily be absorbed by cells, which can use a variety of physiological activities of OR. Thus, the pharmacological effects of MA should be studied together with OR to obtain in-depth knowledge concerning these compounds.MA has a strong inhibitory activity on tyrosinase. However, compared with OR, which has a similar structure, there are few reports concerning the biological activity and function of MA. MA also has poor permeability Although Wu In this study, only D101 macroporous resin and a semi-preparative HPLC system were used to efficiently extract MA from mulberry twigs. The purity of MA was as high as 98.3%. The enzymatic analysis showed that MA and OR had strong inhibitory activities on the mushroom tyrosinase. In vitro, MA inhibited the synthesis of dopachrome primarily by inhibiting the two steps of the first process, which inhibited tyrosinase activity, and had weaker inhibitory activity on diphenolase. However, MA had no effect on the non-enzymatic process.50 value was 1.29 µmol/L, which had a significant difference compared with the inhibitory activity of OR (IC50: 0.12 µmol/L). Compared with OR (IC50: 0.39 µmol/L), MA showed weaker inhibition of the diphenolase of the mushroom tyrosinase, and the IC50 value was 7.45 µmol/L. The analyses of the inhibition mechanism of the monophenolase and diphenolase of the tyrosinase indicated that MA showed reversible inhibition, which was similar to OR. The inhibition activities of MA and of OR on the monophenolase of tyrosine was mixed-type 1. Additionally, the inhibition constants IK were 0.385 µmol/L and 0.926 µmol/L, respectively, and ISK values were 0.177 µmol/L and 0.662 µmol/L, respectively. For diphenolase activity, there were some differences between MA and OR. MA showed competitive inhibition, and KI was 4.36 µmol/L; however, OR showed noncompetitive inhibition, and I = KIS = K2.95 µmol/L.Tyrosinase inhibitory activity analyses showed that MA resulted in strong inhibition of the monophenolase activity of the mushroom tyrosinase. Additionally, the ICFigure S11H-NMR  spectrum of the MA.(PDF)Click here for additional data file.Figure S213C-NMR  spectrum of the MA.(PDF)Click here for additional data file.Figure S31H-NMR  spectrum of the MA.(PDF)Click here for additional data file.