X1 direction for the RP-H-RP, which are the overall response mode at low equivalent explosive masses, transitional response mode at medium equivalent explosive masses, and local response mode at large equivalent explosive masses, respectively. Meanwhile, the honeycombs exhibit two deformation modes in the X2 direction, i.e., the expansion mode at low equivalent explosive masses and local inner concave mode at large equivalent explosive masses, respectively. Interestingly, a counter-intuitive phenomenon is observed on the loaded boundary of the H-RP. Besides, the energy distribution and buffering capacity of different parts on the honeycomb models are discussed. In a unit cell, most of the energy is absorbed by the edges with an edge thickness of 0.04 mm while little energy is absorbed by the other bilateral edges. For the buffering capacity, the honeycomb in the X1 direction behaves better than that in the X2 direction.Lightweight aluminum honeycomb is a buffering and energy-absorbed structure against dynamic impact and explosion. Direct and indirect explosions with different equivalent explosive masses are applied to investigate the in-plane deformation characteristics and energy-absorbing distribution of aluminum honeycombs. Two finite element models of honeycombs, i.e., rigid plate-honeycomb-rigid plate (RP-H-RP) and honeycomb-rigid plate (H-RP) are created. The models indicate that there are three deformation modes in the  Aluminum honeycomb is a promising lightweight structure—a typical bionic structure ,2,3. DueNumerical simulation is an effective way to study the deformation properties of honeycombs because it is difficult to observe the deformation modes of honeycombs experimentally. Therefore, there are some researchers who have conducted their work by numerical simulations ,11,12,13In engineering applications, such as explosion-proof tank, weapon protection engineering, it is possible that honeycomb is subjected to explosive loading. Li et al.  investigAccording to the literature above, deformation properties of honeycomb under dynamic impact or indirect explosive loading have mostly been analyzed, but it is rare to study in-plane deformation characteristics of aluminum honeycomb structure core impacted by direct blast load. There are two typical applications that led to this work. One is as a passive explosive wave energy sensor, which has the characteristics of a low-cost, no electricity measurement and shock wave energy measurement under bad environment, while the other is as a shock absorber in the spaceflight separator, which can effectively reduce the shock to protect the electronic equipment on the test platform. However, deformation characteristics and energy distribution of honeycomb structures subjected to direct explosive loads are unclear.This work focuses on the direct interaction between the honeycomb structure and spherical blast wave. The layout of the rest of the paper is arranged as follows. In X1 direction. The vertical direction of X1 in the in-plane is referred to as the X2 direction, and X3 is along the out-of-plane direction. The in-plane dimension of the aluminum honeycomb sample (X1-X2) is 47.6 mm × 46.0 mm with a height of 4 mm, based on an experimental honeycomb sample, and the number of repeated unit cells in the X1 and X2 directions is 14 and 15, respectively. The sketch of a unit cell is shown in X1 direction, which is 0.08 mm, is twice that of the other directions.An orthotropic hexagonal honeycomb structure a is consX1 direction and the X2 direction, respectively, for each model. Besides, the height of the burst in this work is fixed as 50 cm, which describes the distance between the explosive and the ceiling of the model.The present work creates two types of models under air blast, which are the honeycomb-rigid panel (H-RP) and the rigid panel-honeycomb-rigid (RP-H-RP), respectively, as sketched in X1 and X2, respectively, to research the effect of different loading surface shapes on the deformation characteristics.Numerical simulation is conducted by ANSYS/LS-DYNA (Version 971), a popular software to simulate the dynamic impact or detonation. Based on two types of models in Here, the keyword LOAD_BLAST is used to define an air blast function for the application of pressure loads to explosives in conventional weapons, in which trinitrotoluene (TNT) is used as the explosive to simulate the detonation in the keyword file. Related parameters of TNT are taken from reference . BesidesX3 direction. It is noted that additional components should be installed to prevent the out-of-plane deformation in actual applications. As for contacts, the automatic double-sided face is employed between parts of the models. Self-contact is used for the bucking contact of the same part. Hourglass energy and energy dissipation are computed and included in the energy balance to make the simulation accuracy. The scale factor for the computed time step is 0.9 and the output time step of energy and force is 5 μs. The shell elements between the different parts of the honeycomb structure are connected by common nodes. The main variable in the work is the equivalent mass of the TNT controlled by LOAD_BLAST. Three typical cases with equivalent explosive masses of 10 g, 20 g, and 40 g are employed in the work.In these models, all finite elements are quadrilateral 2D grids, where the thickness of the SE is 0.04 mm, the thickness of the DE is 0.08 mm, and the type of these elements is the Belytschko-Tsay Shell 163 with five through-thickness integration points to provide simulation accuracy. All nodes limit the freedom of translation in the In addition, the effect of different grid densities is analyzed. As a result, the elements with 0.125 mm are created in the following discussion. Besides, the mesh size also fits well with the numerical simulation results in reference . Howeverε.In order to analyze the effect of grid density, three sizes of grids are selected, 0.5 mm, 0.25 mm, and 0.125 mm, respectively. Taking the 10 g TNT explosive as an example, the deformation characteristics of the different mesh sizes at four typical moments are obtained, as shown in X1 direction, the overall response mode in the small equivalent explosive masses case, the transitional response mode in the medium equivalent explosive masses case, and the local response mode in the large equivalent explosive masses case, respectively, as shown in With the increase of equivalent explosive masses , there are three deformation modes in the The overall response mode means all parts in the model take part in the process of deformation. In the deformation mode, the honeycomb sample is compressed by the upper panel, but the compact state of the honeycomb is not achieved. In detail, at 550 μs, the shock wave begins to propagate into the honeycomb structure. At the moment of 1000 μs, the compressive strain of the honeycomb structure is up to 0.14. A “C” shape folding interface appears near the impact section. Then, an “X” folding interface (or double “C” folding interface back to back) occurs near the upper plate at 1360 μs and a “C” interface appears near the bottom plate. At the moment, the honeycomb structure is compressed to the maximum deformation which is 0.18. After that, the upper panel starts to rebound with a constant speed of 5 m/s at 1800 μs because of the elastic potential energy stored in the honeycomb structure and the compression strain decreases to 0.15. Besides, an interesting phenomenon occurs that some cells near the central axis keep the regular hexagonal configuration in the whole process, and it seems that the cells never received any load.In the transitional response mode, the honeycomb sample can be exactly compact. The blast wave reaches the honeycomb structure at 455 μs. At 800 μs, the compression strain of the whole honeycomb structure is 0.25, which has exceeded the maximum deformation in the overall response mode. At this moment, the clear deformation interface in the honeycomb structure is a mixed buckling region of “I” and “C” shape, while the area near the upper panel shows the local compaction state and presents the “I” shape. Then, the shape of “C” becomes plain gradually and becomes the “K” mode at the moment of 1200 μs. Once the folding degree of the cells close to the upper panel at the end of the impact section increased to 0.62 at 1800 μs, a larger span of “K” shape occurs. At last, the upper panel bounces back at 6.7 m/s. In addition, the “regular cells” mentioned in the overall response mode disappear in the transitional response mode.The local response mode is similar to the high-speed loading of deformation proposed by Hu . At 375 X2 direction is perpendicular to the X1 direction in the in-plane region. In order to compare the deformation difference with the X1-RP-H-RP, the explosion load is also applied in the X2-RP-H-RP. Two types of deformation modes occur, expansion mode in the small equivalent explosive masses case and local inner concave mode in the large equivalent explosive masses case, respectively, as illustrated in The X1-RP-H-RP, both of which are 550 μs, because they have the same explosive yield and blast height. At 890 μs  are obtained with the above two models. In order to obtain the effect of the direct explosive load on the honeycomb and analyze the influence of the shape of the explosive load surface on the deformation characteristics of honeycomb, H-RP is studied in this work. Taking 10 g X1-H-RP in X2-H-RP in The similarity is shown in the internal deformation characteristics of the honeycomb structure. In Different from the deformation characteristics of the RP-H-RP, a counter-intuitive deformation phenomenon appears near the explosive load surface in the H-RP. In this phenomenon, the shape of the loading wave is a concave face while the deformation surface of the honeycomb structure is a convex face in in-plane directions, as shown in Due to the low density of the honeycomb structure, the variation of the honeycomb structure deformation characteristics is essentially caused by the influence of the upper and lower panels on the propagation of the blast wave in these models. The propagation process of the shock wave in the honeycomb structure is shown in v1 is the velocity after the action of the upward convex shock wave, v2 is the velocity after the action of the reflected wave at the upper interface, and v0 is the velocity of the upper panel. The directions of these speeds is shown in v1 and v2 is opposite, so both particle 1 and particle 2 have moved downward at the velocity of v0, which is consistent with the deformation state in v1 and v2 are moving in the same direction, so Particle 1 is moving up at (v1 + v2), and Particle 2 is moving up at v4, which is the vertical component of (v1 + v2). Therefore, Particle 1 is above particle 2 because v4 is less than (v1 + v2). In addition, v3, the horizontal component of (v1 + v2), causes Particle 2 to shift in the horizontal direction, so the upper interface will expand to both sides.Two typical locations are selected from the honeycomb to analyze the honeycomb deformation distribution. Particle 1 is a particle in the middle of the upper interface of the honeycomb, indicated by a red circle. Particle 2 is a particle in the right area of the interface on the honeycomb, represented by a green circle, as shown in ζ is defined as follows,Epart represents one or more of the five components mentioned above, Etotal is the total energy of the model.For RP-H-RP, the total energy is divided into five parts at different moments, internal energy of SE, internal energy of DE, kinetic energy of rigid plate (marked as RP), kinetic energy of SE, and kinetic energy of DE, respectively. In order to compare the energy distribution of each part during the compression process, the energy ratio X1-RP-H-RP  and honeycomb-rigid plate (H-RP) are created to investigate the in-plane deformation properties of honeycombs subjected to air blast. Conclusions can be drawn as follows,X1 direction, i.e., the overall response mode in the small equivalent explosive masses case in which there is a “C” or multi “C” folding shape, transitional response mode in the middle equivalent explosive masses case, in which there is an “I” and “C” folding shape, and the local response mode in the large equivalent explosive masses case in which there is only an “I” folding interface. There are mainly two deformation modes in the X2 direction, expansion mode and local inner concave mode. In the expansion mode, the shape of the convex cells is changed into concave and some “I” shape folding interfaces form “(1) At the different equivalent explosive masses, there are three deformation modes in the (2) The mechanism of deformation characteristics under different explosive equivalent conditions is given by wave propagation, and the counterintuitive phenomenon is explained due to the reflection of circle shock wave by the bottom plate but there is no secondary reflection on the ceiling plate in the H-RP.(3) The honeycomb structure can absorb most of the energy carried by a shock wave. In the energy absorption process, it is mainly the deformation energy of the cell single edges with 0.04 mm thickness (marked as SE) that plays a major role. However, there is a low proportion of the internal energy of the cell double edges with 0.08 mm thickness (marked as DE). It provides a new way to improve the energy-absorbed capacity of honeycombs.X1 direction is better than that in the X2 direction.(4) The honeycomb structure can effectively attenuate the stress peak of a shock wave. Besides, the buffering effect in the The model presented is not yet validated experimentally; therefore, it represents a preliminary numerical approach of the deformation behavior of honeycomb structures under direct and indirect explosion.


Paracoccus denitrificans is a strictly respiring bacterium with a core respiratory chain similar to that of mammalian mitochondria. As such, it continuously produces and has to cope with superoxide and other reactive oxygen species. In this work, the effects of artificially imposed superoxide stress on electron transport were examined. Exposure of aerobically growing cells to paraquat resulted in decreased activities of NADH dehydrogenase, succinate dehydrogenase, and N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD) oxidase. Concomitantly, the total NAD(H) pool size in cells was approximately halved, but the NADH/NAD+ ratio increased twofold, thus partly compensating for inactivation losses of the dehydrogenase. The inactivation of respiratory dehydrogenases, but not of TMPD oxidase, also took place upon treatment of the membrane fraction with xanthine/xanthine oxidase. The decrease in dehydrogenase activities could be fully rescued by anaerobic incubation of membranes in a mixture containing 2-mercaptoethanol, sulfide and ferrous iron, which suggests iron–sulfur clusters as targets for superoxide. By using cyanide titration, a stress-sensitive contribution to the total TMPD oxidase activity was identified and attributed to the cbb3-type terminal oxidase. This response (measured by both enzymatic activity and mRNA level) was abolished in a mutant defective for the FnrP transcription factor. Therefore, our results provide evidence of oxidative stress perception by FnrP. Respiratory chains of mitochondria, bacteria and archaea are a series of membrane-associated enzymes, called respiratory complexes, that perform electron transfer from reduced substrates such as NADH or succinate to dioxygen, which is converted to two molecules of water in a four-electron process catalyzed by the terminal oxidase. Energy derived from these redox reactions is conserved into a protonmotive force across the membrane and then utilized by ATP synthase to generate ATP. However, dioxygen can also undergo stepwise reduction, electron by electron, at other sites of the chain. Such uncontrolled side reactions lead to the formation of harmful superoxide and other reactive oxygen species (ROS) that compromise cell viability by interacting with DNA, RNA, lipids and proteins.c reductase and cytochrome c oxidase . Succinate dehydrogenase was measured at 578 nm (ε = 20.5 mM−1 cm−1) using 20 mM succinate as electron donor, 0.05 mM 2,6-dichlorophenolindophenol (DCIP) as electron acceptor and 0.05 mM phenazine methosulfate (PMS) as mediator. 2.5 mM NaCN was added to both dehydrogenase assays to inhibit terminal oxidases. N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) oxidase was measured by monitoring the formation of the Wurster’s blue (WB) cation-radical at 612 nm (ε = 12.7 mM−1 cm−1) nm in the presence of 0.3 mM TMPD. Oxygen uptake due to NADH and succinate oxidation was followed amperometrically in a closed magnetically stirred vessel of 3 mL total volume, tempered to 30 °C and fitted with a Clark-type oxygen sensor. The reaction mixture contained 0.2 mM NADH or 5 mM succinate in 0.1 M sodium phosphate (pH 7.3). Data were obtained with an EmStat potentiostat operated by the PalmSensLite software . All activity measurements were conducted in triplicate and error bars represent the standard deviation (SD).Spectrophotometric assays were carried out at 30 °C on an UltroSpec 2000 spectrophotometer  in 1 cm cuvettes containing 2 mL of 0.1 M sodium phosphate buffer of pH 7.3. NADH dehydrogenase was measured at 340 nm  were incubated with xanthine (0.1 mM) and xanthine oxidase (10.4 mU) at 25 °C in a 0.6 mL volume of 0.1 M sodium phosphate buffer (pH 7.3). The mixture was stirred in a beaker on a magnetic stirrer and 10 µL samples were periodically withdrawn for activity measurement. Control samples without membranes were also analyzed in order to exclude possible background reactions. The involvement of superoxide was confirmed by addition of superoxide dismutase (130 U).2S and 0.1 M sodium phosphate buffer (pH 7.3) in which membranes were suspended at the final concentration of 0.5 mg protein per mL. After bubbling with argon for 10 min to remove the dissolved oxygen, (NH4)2Fe(SO4)2 was added up to 0.25 mM and the mixture was incubated on ice for 60 min. Finally, the membranes were separated by ultracentrifugation  and resuspended in argon-saturated pure buffer. In a control experiment, (NH4)2Fe(SO4)2 was omitted from the restoration buffer.The procedure used is a slight modification of previously described protocols ,25. A so+ and NADH concentrations in control and stressed cells were determined by adapting an enzymatic cycling assay described previously /KM)/(1+([NADH]/KM)+ ([NAD+]/Ki)) was higher in paraquat treated cells compared to untreated controls (42% vs. 31%).Having identified the respiratory NADH dehydrogenase as a target of oxidative insult, we next analyzed the actual changes in NADH/NADvesicles . The Micc to two terminal oxidases (aa3- and cbb3-type). To dissect the contributions coming from individual enzymes, a titration with cyanide was conducted , theiewed in . The dehP. denitrificans via the tricarboxylic acid cycle in combination with malic enzyme and pyruvate dehydrogenase producing acetyl-CoA necessary for the cycle operation 2+ cluster-containing monomers. The superoxide formed by oxygen reduction is believed to be rapidly recycled back to oxygen by the combined actions of superoxide dismutase and catalase [2+ cluster compared to the reaction with O2. The dimer/monomer ratio can be further shifted towards monomer by superoxide-mediated degradation of the [2Fe–2S]2+ cluster, yielding a cluster-less apo-form of FNR [cbb3-type terminal oxidase is due to FnrP inactivation by superoxide. Since the cbb3-type terminal oxidases are characterized by their high affinity for O2 [2+ cluster by superoxide could thus be an alternative to another mechanism of superoxide sensing which is mediated by another transcription factor (SoxR) and relies on reversible oxidation of a [2Fe–2S]+ cluster .P. denitrificans. In vivo, the inactivation defect is partly compensated by decreased product inhibition due to a marked lowering of NAD+ concentration in superoxide-stressed cells. We also demonstrate that electron transfer in the branched terminal part of the respiratory chain is modulated by superoxide at a transcriptional level. The common denominator of all these superoxide effects appears to be oxidative damage to the iron–sulfur centers of the involved proteins.In summary, these data identify respiratory NADH dehydrogenase as the main target for direct superoxide attack among the respiratory complexes of


Constructing two-dimensional (2D) polymers with complex tessellation patterns via synthetic chemistry makes a significant contribution not only to the understanding of the emergence of complex hierarchical systems in living organisms, but also to the fabrication of advanced hierarchical materials. However, to achieve such tasks is a great challenge. In this communication we report a facile and general approach to tessellate 2D covalent organic frameworks (COFs) by three or four geometric shapes/sizes, which affords 2D COFs bearing three or four different kinds of pores and increases structural complexity in tessellations of 2D polymers to a much higher level. The complex tessellation patterns of the COFs are elucidated by powder X-ray diffraction studies, theoretical simulations and high-resolution TEM. Constructing two-dimensional (2D) polymers with complex tessellation patterns is important for the fabrication of advanced materials but achieving such complexity is a great challenge. Here the authors report a tessellated 2D covalent organic framework with increased structural complexity as well as different pore structures. With an aim to mimic natural materials in functions as well as in structures, fabrication of materials with hierarchical structures has drawn considerable attention and great progress has been achieved over the past decades5. However, so far the researches have mainly focused on zero-dimensional (0D), one-dimensional (1D), and three-dimensional (3D) hierarchical materials10. In terms of two-dimensional (2D) hierarchical structures, they are closely related to an intriguing subject in mathematics, namely tessellation, which refers to completely tiling a plane by one or more geometric shapes without overlaps and gaps11. Tessellation patterns are not only widely observed in the nature, but also frequently used in art, architecture and manufacturing. However, the development of tessellations in 2D materials is just in the bud. As the archetype of 2D materials, graphene exhibits a simple hexagonal tilling13. Similarly, simple tessellation patterns are also observed for the other inorganic 2D materials such as MoS2, h-BN and phosphorene15, due to the limitation of their structural motifs. On the other hand, 2D polymers, which are composed entirely of organic building blocks, provide tremendous structural variabilities. In this context, 2D covalent organic frameworks (COFs), an emerging class of crystalline porous materials precisely integrated with periodical organic units through covalent bonds in a plane and interlayer stacking to form layered 3D crystals via non-covalent interactions17, have been widely recognized as general 2D polymers, while a stricter definition of 2D polymers refers to those isolated as monolayers19.The existence of complex hierarchical structures widely found in biological materials is not only one of the most miraculous phenomena in the nature, but also a key foundation for biomaterials to achieve functions25, catalysis30, drug delivery32, sensing35, proton conduction37, to optoelectronic devices44. However, despite the progress achieved, the topological structures of 2D COFs are still limited to simple tessellation patterns, which are mainly tiled by only one geometric shape , leading to COFs with homogeneous porosities47. In 2014, we reported a Kagome-type 2D COF in which hexagonal mesopores and triangular micropores alternately and periodically distribute48. Following this dual-pore COF, triple-pore COFs were successful achieved in 2016, which increased the complexity in the tessellations of 2D COFs to a higher level49. Such heteropore COFs possess hierarchical structures and exhibit heterogeneous porosities, which benefit for exploiting new properties, functions and applications57. For example, making use of the advantage that the different kinds of pores can be independently modified and functionalized, distinct functions can be introduced into different types of pores and thus advanced materials with integrated functions can be fabricated. Moreover, different guest molecules, even those incompatible with each other, might be encapsulated in the different types of pores of one COF due to their different pore environments. On the other hand, hierarchically porous materials have already been found to exhibit some advantages over homogeneous ones, such as minimizing diffusion barriers, improving mass transport, and increasing distribution of active sites4. Indeed, the inimitable advantages of COFs with hierarchical porosity were demonstrated in recent research led by Ma, which clearly showed that a Kagome-shaped dual-pore COF outperformed the COF with uniform porosity in enzyme catalytic performance by loading the enzyme in the large hexagonal pores while making the small triangular pores free to transport reactants and products58. Despite the great potential applications, COFs with complex tessellation patterns have remained very difficult to achieve.Thanks to the advantages of their conjugated skeletons and porosities, 2D COFs have been extensively exploited to be functional materials with versatile applications ranging from gas separation and storageWe herein report a facile and general approach to fabricate 2D COFs which bear three or four different kinds of pores in one framework. To the best of our knowledge, the latter is the highest level of hierarchy and complexity held by a 2D COF reported so far, demonstrating a successful step further to challenge the structural complexity in the tessellations of 2D polymers.59 and desymmetrization60 strategies simultaneously. As illustrated in Fig. 3-symmetric double-linking-site building block leads to a monomer with C2-symmetry. The condensation of it with linear monomers is expected to afford COFs bearing three different kinds of pores  confirmed formation of imine bonds in the polymers, as evidenced by the appearance of vibration bands of C=N units in their spectra , (ii) staggered stacking , based on which the simulated powder X-ray diffraction (PXRD) patterns were obtained  and transmission electron microscopy (TEM) were further conducted to investigate the morphologies and interior structures of the as-obtained COFs. SEM images showed spherical morphologies for all the four COFs  or (1 0 0) reflection of the predicted triple-pore and tetrad-pore COF models with AA stacking , indicating the validity of the predicted COF structures.To verify the reliability of the structural identifications of the COFs, which are based on the PXRD results above, detailed TEM study was carried out. Firstly, the fine structures of the lattice fringes in the TEM images were further examined. As shown in Fig. a or c derived from the Pawley refinements of the PXRD data, indicating that the observed crystal structures of the four COFs match well with the theoretical structure models along the b-axis. The images inserted in the upper right corners are the corresponding diffraction patterns of the four COFs obtained through fast Fourier transformation (FFT), further indicating single-crystal characteristics of these 2D COF flakes. The TEM investigation again corroborates that the as-prepared COFs hold the predicted triple-pore and tetrad-pore framework structures.Furthermore, as shown in Figs. 2 g−1 for Tri-COF-DAB, Tri-COF-BZ, Tetra-COF-DAB, and Tetra-COF-BZ, respectively  were estimated to be 1.02, 1.15, 0.34, and 1.07 cm3 g−1. Derived from their theoretical N2 adsorption isotherms  analysis because no suitable model could be applied to these highly complexed COFs. Taking Tetra-COF-BZ as an example, PSD analysis was carried out by trying all the kernels/pore models available from the instrument. However, not an appropriate model was found to be suitable for the COF, as the PSD profiles obtained did not have a definite meaning to the COFs  and 1,4-diaminobenzene  were added into a mixture of 1,4-dioxane (0.75 mL), mesitylene (0.75 mL), and acetic acid  in a glass ampoule. The ampoule was sealed under vacuum after three freeze-pump-thaw cycles. Then the mixture was heated at 120 °C without disturbance for 72 h to afford a yellow solid at the bottom of the tube. After being cooled to room temperature, the solvent was decanted and the solid was washed with anhydrous 1,4-dioxane and DCM for three times, respectively, and then dried under dynamic vacuum at 120 °C for 4 h to afford a yellow powder , which was insoluble in common organic solvents such as acetone, ethanol, and N, N-dimethylformamide. Anal. Calcd. For C82H51N7: C, 86.82; H, 4.53; N, 8.64. Found: C, 81.26; H, 4.98; N, 7.50.TPM  and benzidine  were added into a mixture of 1, 4-dioxane (1 mL), mesitylene (1 mL) and acetic acid  in a glass ampoule. The ampoule was sealed under vacuum after three freeze-pump-thaw cycles. Then the mixture was heated at 120 °C without disturbance for 72 h to afford a yellow solid at the bottom of the tube. After being cooled to room temperature, the solvent was decanted and the solid was washed with anhydrous 1,4-dioxane and DCM for three times, respectively, and then dried under dynamic vacuum at 120 °C for 4 h to afford a yellow powder , which was insoluble in common organic solvents such as acetone, ethanol, and N, N-dimethylformamide. Anal. Calcd. For C76H47N7: C, 86.26; H, 4.48; N, 9.27. Found: C, 80.05; H, 5.13; N, 7.02.FPM  and DAB  were added into a mixture of 1,4-dioxane (0.25 mL), mesitylene (0.75 mL) and acetic acid  in a glass ampoule. The ampoule was sealed under vacuum after three freeze-pump-thaw cycles. Then the mixture was heated at 120 °C without disturbance for 72 h to afford a yellow solid at the bottom of the tube. After being cooled to room temperature, the solvent was decanted and the solid was washed with anhydrous 1,4-dioxane and DCM for three times, respectively, and then dried under dynamic vacuum at 120 °C for 4 h to afford a yellow powder , which was insoluble in common organic solvents such as acetone, ethanol, and N, N-dimethylformamide. Anal. Calcd. For C94H59N7: C, 87.76; H, 4.62; N, 7.62. Found: C, 84.79; H, 5.14; N, 7.25.FPM  and BZ  were added into a mixture of 1,4-dioxane (1 mL), mesitylene (1 mL) and acetic acid  in a glass ampoule. The ampoule was sealed under vacuum after three freeze-pump-thaw cycles. Then the mixture was heated at 120 °C without disturbance for 72 h to afford a yellow solid at the bottom of the tube. After being cooled to room temperature, the solvent was decanted and the solid was washed with anhydrous 1,4-dioxane and DCM for three times, respectively, and then dried under dynamic vacuum at 120 °C for 4 h to afford a yellow powder , which was insoluble in common organic solvents such as acetone, ethanol, and N, N-dimethylformamide. Anal. Calcd. For CSupplementary Information


Pin1 is unique and only catalyzes the phosphorylated Serine/Threonine-Proline (S/T-P) motifs of a subset of proteins. Since the discovery of Pin1 as a key protein in cell cycle regulation, it has been implicated in numerous diseases, ranging from cancer to neurodegenerative diseases. The main features of Pin1 lies in its two main domains: the WW (two conserved tryptophan) domain and the PPIase domain. Despite extensive studies trying to understand the mechanisms of Pin1 functions, how these two domains contribute to the biological roles of Pin1 in cellular signaling requires more investigations. The WW domain of Pin1 is known to have a higher affinity to its substrate than that of the PPIase domain. Yet, the WW domain seems to prefer the trans configuration of phosphorylated S/T-P motif, while the PPIase catalyzes the cis to trans isomerasion. Such contradicting information has generated much confusion as to the actual mechanism of Pin1 function. In addition, dynamic allostery has been suggested to be important for Pin1 function. Henceforth, in this review, we will be looking at the progress made in understanding the function of Pin1, and how these understandings can aid us in overcoming the diseases implicated by Pin1 such as cancer during drug development.Pin1 belongs to the family of the peptidyl-prolyl  This allows the cells to exert more biological functions with lesser number of proteins. There exist many types of PTMs, of which, the reversible phosphorylation is widely studied for its role in regulating many signaling cascade . Initial) domain . The WW ) domain –5. This outcomes , 6. Thisoutcomes , 8.So far, Pin1 has been extensively studied for its role in various cellular functions, particularly in cell cycle regulation , 6, 9–11cis/trans isomeric activity. This would then trigger its intended downstream cellular signals and effects. Many models have been introduced to explain the molecular mechanism of how Pin1 exert its catalytic activity. However, there has not been a model in agreement to truly explain how Pin1 acts on its biological substrates. Therefore, in this review, we will highlight the progress of Pin1 research in elucidating the actual mechanism of Pin1, with an emphasis on the structural importance of Pin1 on its function, and how the perturbation to this fundamental structure could explain for its roles in diseases such as cancer. We will also discuss how these structural features could be used in the drug development of Pin1 inhibitors.In addition, there have been many structural and protein dynamic studies to understand how Pin1 could target its biological substrates to exert its intrinsic cis/trans isomerase activity in Pin1 , and part of the PPIase domain (S138 to R142). This domain interface has been suggested to play an important role in interdomain communication that regulates the function of Pin1 upon substrate binding –62. Thesin vitro as compared to the PPIase domain  protein and splicing factor, proline-, and glutamine-rich (SFPQ) protein for its activity. As such, there seems to be two types of Pin1 interactors that requires either the binding of both WW and PPIase domains, or just the WW/PPIase domain alone.Indeed, previous studies have shown that the PPIase domain of Pin1 is able to bind to pS/T-P motif instead of just its WW domain , 58, 65.l Figure . In theitrans over cis configuration. As such, the PPIase isomerization would not have occurred with this sequential binding.These proposed four models of Pin1 interaction with its biological substrates for functional regulation might suggest two aspects. Firstly, there is still lack of concrete evidences to highlight which model(s) Pin1 may deploy for its interaction with its biological substrates. Secondly, these models demonstrate the potentially diverse interaction that Pin1 has on its already diverse biological interactors to play a role in the regulation of various biological functions. All these models do have their merits as well as potential doubts as to its suitability. For instant, the sequential binding model does fulfill the characteristics of the WW domain having a higher affinity of peptide binding to the pS/T-P motif than the PPIase domain. In proteins with multiple sites of pS/T-P motifs, this model could explain the use of the WW domain to localize the concentration of the PPIase domain for isomerization. However, for substrate with a single pS/T-P motif, the potential release of the WW domain from this motif, followed by the binding of the PPIase domain for catalytic activity does not seem to be energy favored. This is also highlighted by studies that we have mention previously , 68–70, via the WW domain of Pin1, the increased local concentration of Pin1 would allow the binding to its biological substrate in the vicinity to elicit its downstream biological processes. Furthermore, the pS/T-P motif of the kinase could be in a trans configuration state to allow for WW domain binding. In addition, the catalysis-first binding model could also be associated with the multimeric binding model where Pin1, bound to the active kinase via the WW domain, is brought to its target substrate by the active kinase. Subsequently, the active kinase would phosphorylate the Pin1 substrate, allowing for the PPIase domain of the Pin1 to bind and catalyze the cis-trans conformational change. Furthermore, the suggested simultaneous binding model could also fit the multimeric binding model, although this would occur on a single protein, instead of the proposed protein complex in the multimeric binding. Of interest, the binding of the WW domain in the trans configured pS/T-P motif could lead to conformational changes to the PPIase domain, increasing its binding capacity to the cis configured p-S/T-P motif and subsequently its catalytic efficiency. The potential role of interdomain communications between the WW domain and the PPIase domain of Pin1 has indeed been studied in recent years as discussed in the next section.For multimeric binding to an active kinase in vivo, suggesting the presence of interdomain communication. In addition, studies conducted on Pin1 and tau interactions highlighted the need of WW domain interaction with the pS/T-P motif for the effective function of the PPIase activity  studies . As highg et al. , 87, Namg et al.  found thvia a hinge-like movement, an observation echoed by a more recent study done by Campitelli et al.  in Saccharomyces cerevisiae from death  of 72.3 nM, which is 100× less than the IC50 of the first Pin1 inhibitor juglone. API-1 was found to bind to the PPIase domain of Pin1. The authors went on to show that API-1 was able to suppress the proliferation of hepatocellular carcinoma cells as well as inhibiting the tumor growth of xenografted mice. These evidences demonstrated the therapeutic value of API-1 as a cancer drug candidate. Another study by Cui et al. (via the synthesis of various pyrimidine derivatives. The authors were able to identify four potential Pin1 inhibitors with IC50 of <3 μM. Furthermore, this type of drug is only the second covalent binding Pin1 inhibitors to the phosphate-binding loop in the PPIase domain besides juglone. These studies clearly highlight the potential therapeutic value of Pin1 as a cancer drug target. Unfortunately, no drugs thus far has reached clinical trials (The development of Pin1 inhibitors has remained a challenge in the field of cancer studies as many known inhibitors of Pin1 remains unspecific enough to only block the effects of Pin1 despite a number of Pin1 inhibitors already found , 98, 99.i et al.  demonstrl trials , 98.in vivo binding affinity to Pin1. The authors went on to improve the permeability of the benzothiophene class of drug to eventually yield dihydrothiazoles as a potent Pin1 inhibitor with low micromolar inhibitory activity toward cancer cell proliferation (Of interest, most drugs identified thus far are mainly targeting the PPIase domain, especially the catalytic domain, with few drug candidates found to target the WW domain  as summaferation . Howevercis configured pS/T-P motif (Of importance, these studies only made use of the PPIase domain as the main consideration in designing of their structure-based Pin1 inhibitor. The authors did highlight some important points supporting intra-structure dynamics of the whole Pin1. Firstly, they noticed that despite the rich hydrogen bonding potential within the phosphate-binding loop in the PPIase domain of Pin1, hydrophobic interactions were central to benzothiophene binding to Pin1. Secondly, they also highlighted the presence of H-bond formation beyond the phosphate-binding loop that could be important for the Pin1 inhibitor binding infinity. Lastly, they highlighted the high degree of flexibility in Pin1 active site interaction with the inhibitors. All these observations do align with our previous discussion where interdomain communications between WW and PPIase domains could affect the side chain dynamics on the conserved hydrophobic conduit region and alters PPIase domain binding affinity , 91, 92.-P motif . Hencefoin vivo circumstances are yet to be determined and could be explored in future drug development for Pin1 inhibitor.As suggested from all the studies on the two domains of Pin1 covered in this review, the WW domain binding displays a potentially important role in driving the signaling cascade triggered by Pin1 catalytic function. To advance our development of promising Pin1 inhibitors for treatment of diseases such as cancer, there are important considerations to be taken note of. Much interest has been put forth on identifying Pin1 inhibitors that target the PPIase domain as well as its active site. As suggested in this review, the binding affinity of Pin1 inhibitors could be affected by the substrate that is bound to the WW domain. Therefore, binding affinity studies for Pin1 inhibitors screening should consider the substrate effect upon its binding to the WW domain, and how this effect could alter the Pin1 inhibitors binding to the PPIase domain or its active site. Of interest, a very recent study by Momin et al.  using MDThis potential substrate-dependent Pin1 activity could be important in future studies. This is highlighted in the lack of depth in the field of Pin1 research thus far. Most studies done on Pin1 mechanism focuses on just a few biological substrates of Pin1. As suggested in this review, the vast repertoire of Pin1 functional substrates and the different biological processes it could affect do point to the fact that Pin1 might behave differently in the presence of different substrate. This difference in behavior could affect its structural dynamics that makes identifying a functional Pin1 inhibitor difficult for different types of diseases. Therefore, to advance the research of the role of Pin1 in the pathogenesis of various diseases, more emphasis on the diversity of Pin1 substrates being tested must be done. As mentioned previously, Pin1 inhibitor screening could be done in the presence of its effector substrate to evaluate the efficacy of the Pin1 inhibitor in affecting the phenotype of the disease being studied. In addition, studies looking into how Pin1 cooperation with kinase in exerting its isomeric activity could be conducted to investigate if the proposed multimeric model holds true, and how the subsequent findings could affect the considerations when developing Pin1 inhibitors. Indeed, studies thus far do suggest that Pin1 is not a lone ranger, and it always needs a helping hand to exert its function. To advance the development of drugs to highlight the impact of Pin1 as a cancer therapy target, both domains should be considered during the drug development process for the field to realize the potential of Pin1 as a cancer therapeutic target.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


All cells rely on lipids for key functions. Lipid transfer proteins allow lipids to exit the hydrophobic environment of bilayers, and cross aqueous spaces. One lipid transfer domain fold present in almost all eukaryotes is the TUbular LIPid binding (TULIP) domain. Three TULIP families have been identified in bacteria , but their homology to eukaryotic proteins is too low to specify a common origin. Another recently described eukaryotic lipid transfer domain in VPS13 and ATG2 is Chorein-N, which has no known bacterial homologues. There has been no systematic search for bacterial TULIPs or Chorein-N domains.Remote homology predictions for bacterial TULIP domains using HHsearch identified four new TULIP domains in three bacterial families. DUF4403 is a full length pseudo-dimeric TULIP with a 6 strand β-meander dimer interface like eukaryotic TULIPs. A similar sheet is also present in YceB, suggesting it homo-dimerizes. TULIP domains were also found in DUF2140 and in the C-terminus DUF2993. Remote homology predictions for bacterial Chorein-N domains identified strong hits in the N-termini of AsmA and TamB in diderm bacteria, which are related to Mdm31p in eukaryotic mitochondria. The N-terminus of DUF2993 has a Chorein-N domain adjacent to its TULIP domain.TULIP lipid transfer domains are widespread in bacteria. Chorein-N domains are also found in bacteria, at the N-terminus of multiple proteins in the intermembrane space of diderms  and in Mdm31p, a protein that is likely to have evolved from an AsmA/TamB-like protein in the endosymbiotic mitochondrial ancestor. This indicates that both TULIP and Chorein-N lipid transfer domains may have originated in bacteria. Both eukaryotic and bacterial cells require lipids to be distributed away from the site of their synthesis by lipid transfer proteins , 2. TherE. coli protein YceB and its homologues have the same fold as eukaryotic TULIPs [TUbular LIPid binding (TULIP) lipid transfer domains were discovered 20 years ago in human serum proteins such as bactericidal/permeability increasing protein (BPI) . Two homc TULIPs , 17–19, c TULIPs . OrfX2 aChorein-N is a domain of ~ 110 residues found at the N-terminus of two related, long, universal eukaryotic proteins: VPS13 (3000–7000 residues) and ATG2 (1000–2000 residues) , 21, 22.Here bacterial TULIP-like proteins and eukaryotic Chorein-N domains have been used in remote homology searches for bacterial homologues. Among three new TULIP families, one has precisely the same dimerization interface as found in BPI. Newly described bacterial TULIPs were analysed for likely targeting signals to suggest the parts of bacterial cells where they bind and transfer lipids. Chorein-N domains were found in bacteria at the N-terminus of the closely related intermembrane space proteins AsmA and TamB, and also in Mdm31p in fungal mitochondria, which is presumed to have evolved from a common ancestor with AsmA/TamB. An additional Chorein-N domain is at the N-terminus of one of the bacterial TULIPs. All of the bacterial TULIPs and Chorein-N proteins are therefore implicated to be lipid transfer proteins. Finding both domains in bacteria and their derivatives indicates that they originated in bacteria before eukaryote evolution.The unit cell of the YceB crystal deposited at PDB (3L6I) contains a head-to-head dimer of TULIP-like domains. However, the dimeric interface includes a polyhistidine tag, which creates a sharp bend in the dimer (114°) Fig. a, comparSS = 100% over 389 residues, e-value from sequence alone = 1e− 50). Searches with YceB produced significant hits to four regions in three bacterial proteins families  of the query with libraries of MSAs covering PDB, Pfam and proteomes of model organisms. P47 and OrfX2 produced no significant hits, except to each other , aligned strongly with DUF4403 . DUF2993 domains are also reported by PFAM in some mosses and algal picoplankton, presumably in plastids derived from an ancestral cyanobacterium. Among the three family members in c is not , 28. TheSS 63% over 167 residues, Fig. Overall, these results suggest that TULIP domains are widespread in bacteria, both monoderms and diderms, most being able to dimerize by the same mechanism as BPI, and one having the same pseudodimeric form as BPI. The degree of homology detectable by HHsearch is low between known eukaryotic TULIPs and the previously known bacterial TULIP domains P47/OrfX2 and YceB  and TamB (translocation and assembly module-B)  and the TULIP domain, there is a α/β region of 100 residues , some of which also express OrfX proteins. DUF2140 is predicted to be N-terminally anchored to the outside of the cell, with an unstructured ~ 20 aa linker before the TULIP. The function of these domains cannot be to transfer lipids between distinct membranes. Instead, they may capture or export specific lipids Fig. a.Fig. 6TULIPs in diderms might be involved in intracellular lipid traffic between the inner and outer membranes. DUF4403 domains are preceded by a hydrophobic region and are predicted to be secreted into the intermembrane space. 66% are predicted to have this region cleaved, leaving the bulk of the protein anchored to the membrane by acylation . Of thesRv0817c is like the majority of DUF2993 proteins, being predicted to be secreted into the intermembrane space and anchored in the inner membrane by a N-terminal transmembrane domain. The Chorein-N domain and TULIP are continuous without breaks and may dimerise like 2 TULIP domains Fig. a. The shSS = 35% over 105 residues), which indicates that even low levels of reported homology may be consistent with common origin. We note that several of the alignments detected between bacterial and eukaryotic TULIPs were stronger that this  and DUF4403 are ≤25 aa, implying maximal extension ≤9 nm, and the domains themselves are not long enough to span the intermembrane space (≥20 nm)  Fig. b. So theHHsearch identified homology between Chorein-N domains and the N-termini of AsmA, TamB and Rv0817c, as well as Mdm31p Fig. c. The soE. coli) anchored by a single N-terminal transmembrane domain inserted in the cytoplasmic membrane  have been reduced to one sequence.Sequences used to seed searches are described in Table A. echinatior F4WUM6); and SMP domain . All ten MSAs were submitted to HHpred which reported the top 1000 hits in PFAM v32.0. Unlike the pairwise searches (above), these searches are not symmetrical [MSAs of ten proteins, 4 eukaryotic and 6 bacterial, were used to seed searches of PFAM. Creation of MSAs from all sequences was as above, with 3 PSI-BLAST iterations. The four eukaryotic domains included were: BPI  and C-terminal domain; arthropod TULIP  with DUF1439  and DUF2140. . Residues are coloured according to the ClustalX scheme. Insertions are shown with the number of residues (e.g. “+ 14”) . Deletions are indicated by a black bar across the missing residues. Most DUF2993s have either a very short second β-strand or it is absent. There are also deletions in the final loop. 109 aa of Rv0817c align with the 127 aa core of YceB, both of which are shorter than DUF4403-C (180 aa) and DUF4403-N (220 aa). In Rv0817c, a further 28 aa align with 38 aa of YceB that forms the β-dimerization interface. The reduction in residues in DUF2993’s TULIP derives from (i) deletions before and in the second β-strand, as in DUF2140; (ii) short loops, especially the final loop; (iii) reduced residues in the final helix (14 fewer), covering the same distance by forming an extended polypeptide.Additional file 2. MSAs for pairwise comparisons in HHpred (see Table Additional file 3. Pairwise comparisons in HHpred, corresponding to results in Figs.


Alphaherpesviruses are a subfamily of herpesviruses that include the significant human pathogens herpes simplex viruses (HSV) and varicella zoster virus (VZV). Glycoprotein K (gK), conserved in all alphaherpesviruses, is a multi-membrane spanning virion glycoprotein essential for virus entry into neuronal axons, virion assembly, and pathogenesis. Despite these critical functions, little is known about which gK domains and residues are most important for maintaining these functions across all alphaherpesviruses. Herein, we employed phylogenetic and structural analyses including the use of a novel model for evolutionary rate variation across residues to predict conserved gK functional domains. We found marked heterogeneity in the evolutionary rate at the level of both individual residues and domains, presumably as a result of varying selective constraints. To clarify the potential role of conserved sequence features, we predicted the structures of several gK orthologs. Congruent with our phylogenetic analysis, slowly evolving residues were identified at potentially structurally significant positions across domains. We found that using a quantitative measure of amino acid rate variation combined with molecular modeling we were able to identify amino acids predicted to be critical for gK protein structure/function. This analysis yields targets for the design of anti-herpesvirus therapeutic strategies across all alphaherpesvirus species that would be absent from more traditional analyses of conservation. Mammal, bird, and reptile herpesviruses constitute the family Herpesviridae which is divided into alpha, beta, or gamma, subfamilies based on their biological properties, and gene content4. There are approximately 40 genes shared among all subfamilies of herpesvirus, referred to as the core herpesvirus genes6. These genes encode structural components of the viral nucleocapsid, as well as proteins with functions related to aspects of the viral life cycle that are common to all members of the Herpesviridae, such as DNA replication. In addition to the core set of herpesvirus genes, there are genes specific to members of each subfamily. These genes are expected to be responsible for the biological properties of each subfamily that distinguish it from other subfamilies7.Herpesviruses are a significant cause of morbidity and mortality in human and animal populations. To date there are greater than 100 species of viruses recognized in the order herpesvirales including 8 human herpesviruses7. After the establishment of latency, these viruses will periodically reactivate and spread to sites of innervation causing clinical symptoms of herpesvirus infection. The ability of alphaherpesviruses to enter cells of the nervous system is central to the pathogenesis of infection by these viruses and to date remains poorly understood. One strategy for identifying genes involved in infection of the nervous system is to focus on those genes unique to alphaherpesviruses. While several of these alphaherpesvirus-specific genes have been implicated in neurovirulence, our understanding of how and which of them facilitate entry, establishment and maintenance of latent infection in cells of the nervous system is incomplete.Alphaherpesviruses are distinguished from the beta and gamma subfamilies by their ability to establish and maintain latency in the central and/or peripheral nervous systems of the host19. Mechanistically, the gK/UL20 protein complex interacts with the viral fusogen gB and this interaction is necessary for gB-mediated membrane fusion during virus entry, as well as virus-induced cell-to-cell fusion and the formation of syncytia21. HSV-1 gK is expressed on virion envelopes and functions in virion entry23. Specifically, we have shown that a 39 amino acid N-terminal deletion of gK inhibits virus entry into neuronal axons in vitro and in vivo, but not other cell types 26.gK and UL20 are highly conserved among neurotropic alphaherpesviruses. gK and UL20 have been shown to interact and these interactions are important for virus entry, membrane fusion, cytoplasmic virion envelopment and egress7. gK and UL20 are both present in the virion envelope27. Previously, we have shown that gK has three extracellular and two intracellular domains separated by four transmembrane domains9. Mutations of gK result in viruses with domain-specific defects in entry, assembly, or egress31. These data suggest the domains of gK are functionally distinct, with some involved in entry and others in assembly and egress. At present, the mechanism by which gK functions to facilitate membrane fusion during virus entry and spread, as well as cytoplasmic virion envelopment, is not well understood and is the focus of ongoing work.HSV-1 is the cause of oral herpes and is the prototypical alphaherpesvirus32. Additional selective pressure may be expected to occur across a virus family interacting with divergent host species, since they may be subject to diversifying selection imposed by co-evolution with hosts33. Extracellular domains  are more likely to be involved in entry as well as be exposed to host antibody binding therefore these domains may be expected to evolve more quickly than intracellular domains (2 and 4). Second, some extramembrane domains may exhibit more structural restraints, while others could be less ordered. Elastic network models allow us to estimate the residue fluctuations, or mobility. We might expect that ordered domains with less fluctuation  should evolve more slowly than those with less well-defined structures and high fluctuation . Lastly, transmembrane domains need to exhibit a stable core that maintains the integrity of a helical bundle, while residues on the sides of the helical bundle need to remain hydrophobic due to their interactions with membrane phospholipids. Transmembrane domains may also interact with other viral proteins 35. Given these constraints, they are generally expected to evolve slowly.Since domains of gK are functionally distinct, we hypothesized that they may exhibit variation in evolutionary rate. Such variation is likely driven by several, interacting factors. First, virion assembly, like DNA replication, may be expected to involve primarily conserved viral protein-protein interactions, while entry is partly the result of interactions between viral entry mediators with -host receptor proteins. Viral protein domains on the surface of virions may evolve faster than internal virion proteinsThe various factors outlined above may result in contrasting predictions about a domain’s expected evolutionary rate. For instance, an intracellular domain of gK is likely to be involved in virion assembly. Given this role, the domain should have experienced a relatively stable set of interactions during the diversification of alphaherpesviruses, and would be expected to evolve slowly in order to maintain the fidelity of these interactions. However, structural predictions may suggest that the same domain also has a less well-defined structure than other parts of gK, requiring less precision in folding to maintain function. In this case, the domain’s primary sequence may evolve relatively quickly. The contrasts between these predictions allow us to elucidate the relative strength of the predictions.Overall, our integrated phylogenetic and structural analyses provide new insights in the location and potential functions of gK protein domains. This approach can be extended to the prediction of functional domains and residues of other proteins and glycoproteins, including domains that can be potential targets for antiviral therapy for multiple evolutionary related pathogens.37.We inferred the phylogenetic relationships among 18 species of alphaherpesviruses using a set of genes orthologous to HSV-1 gK . Since all 17 other gK sequences were predicted to possess the same topology as HSV-1, we combined the two domains predicted for PRV domain 2, yielding five extramembrane domains across all orthologs.To test the hypothesis that domains vary substantially in evolutionary rate based on function, we inferred these rates for each of the 5 extramembrane and four transmembrane domains Fig.  across tWe found that domain 2 had the lowest inferred rate of evolution, while domain 4 had a substantially faster rate than all others  and varicellovirus genera (VZV and PRV) were aligned using clustal omegaTo develop a comprehensive and quantitative understanding of variation in evolutionary rate across individual residues, including identification of those that may be under strong constraints, we employed a novel finite mixture model (FMM) for among-site rate variation. While site rate estimates did vary somewhat as the number of mixture components changed Figs –3, the r6. These analyses are particularly valuable for comparison of gKs from the simplex and varicella genera of alphaherpesviruses that possess little amino acid sequence identity, but which are expected to have functional conservation. Additionally, gK has extensive hydrophobic regions that preclude the use of traditional methods to study structure, such as crystallization and circular dichroism. To determine the degree of structural conservation between representative varicello and simplex herpesvirus, we predicted the structures of HSV-2, VZV and PRV and compared these to our previously published HSV-142 predicted gK structure  is also predicted to be the least ordered ) and the degree of ordering in their structures Figs , 6 appeaSite-by-site variation in evolutionary rate was inferred to be 10-fold greater than variation in average rate across domains and genes Figs , and S4.Despite being structurally naive, the model of sequence evolution that we applied resulted in site-specific rate estimates that are spatially clustered Fig.  and ofte43. These tyrosines may be involved in protein-protein interactions and/or the appropriate localization of gK to facilitate cytoplasmic virion envelopment. In this regard, we recently showed that the HSV-1 UL37 tegument protein interacts with gK via specific tyrosine residues located within conserved UL37 regions35.A general region of potential functional importance was domain 2 of gK, given its overall slow rate. In structural predictions for HSV-2, VZV, and PRV, domain 2 exhibits a highly conserved double alpha helix that extends into the cytoplasm. This structure is consistent with its presumed role in virus assembly, a process conserved across alphaherpesviruses. Of specific interest are a number of conserved tyrosines within gK domain 2. Our molecular model and phylogenetic analyses indicate that these tyrosines may play important roles in how gK regulates gB-mediated membrane fusion. Similarly, cytoplasmic virion maturation is a coordinated process that is both spatially and temporally regulated17. Therefore, while domain 4 had the highest rate of evolution, it may still possess a conserved function. Conversely, the slow rate of evolution for domain 5 is surprising, since we previously showed that deleting this entire domain has little effect on viral replication in vitro17. Although deletion of domain 5 does not adversely affect virus replication in vitro domain 5 may be important for gK function in vivo. Transmembrane and near-transmembrane regions may also have conserved features to facilitate proper localization or protein-protein interactions. For instance, in our template based models of alphaherpesvirus gKs, residues near the ends of transmembrane helices are often conserved  as implemented in prottest version 3.248. All available 120 amino acid substitution models were tested, and in each case the Jones92 model with invariant sites and a gamma distribution was preferred by BIC. All individual gene datasets were then concatenated, resulting in an alignment of approximately 41,543 amino acids from each of 59 genes .Eight previously sequenced HSV strains were selected from Genbank to be included in genome-wide phylogenetic analyses , and those that estimated domain-specific parameters for gK. All substitution model parameters, with the exceptions of topology and branch lengths, were unlinked across data subsets, including relative rates of evolution for each gene or partition. Each analysis included 4 independent runs of 5 million generations with 4 Metropolis-coupled chains per run. Analyses were monitored for convergence by examining trace plots of scalar values in Tracer version 1.650. Trace plots show the values of parameters sampled across MCMC generations. Similarity in these values through time, and across runs, improves confidence that we are accurately estimating the posterior distribution. Topological convergence was assessed using the average standard deviation of split frequencies (ASDSF). All analyses were conducted both with and without missing data (gaps). In all cases, recovered topologies and support values did not exhibit strong conflicts, so we only report results from complete datasets. A majority-rule consensus tree was constructed in MrBayes and plotted using FigTree version 1.4.2 for the gK analyses  were taken from Genbank Table . These s52. We initially explored the use of the DPP, but found that mixing and convergence were difficult to achieve for our data, resulting in site-specific estimates that were not reliable. Instead, we employed a finite mixture model (FMM), where the number of rate categories (k) is fixed a priori, although the assignment of sites to categories is random. Following the notation of Huelsenbeck and Suchard52 the assignment of sites to categories can be given as a vector of mappings n is the number of sites in the alignment and αi is the concentration parameter for category i. All of our analyses employed a flat Dirichlet with 53 and the associated Rev scripts are available as supplementary material (https://github.com/jembrown/Evolution2017/blob/master/Example_FMM.rb).To estimate the relative rates of evolution for each residue in gK, we employed a novel partitioning model, where the assignment of sites to rate categories is not fixed. This model is closely related to the nonparametric method of Huelsenbeck and Suchard, who considered the number of rate categories to be a random variable distributed according to a Dirichlet process prior model (DPP)ded Figs –3. Our p54 by Jalview software55 and visualized using the Jalview Blosum62 color scheme; in this color scheme gaps are colored white. If a residue matches the consensus sequence residue at that position it is colored dark blue. If it does not match the consensus residue but the 2 residues have a positive Blosum62 score56 it is colored light blue. Conservation and sequence logo analysis alignment is generated and visualized by Jalview.Multiple sequence alignment (MSA) between four different strains of alphaherpesviruses HSV-1, HSV-2, PRV, and VZV . The MSA was constructed with Clustal Omega sequence profile alignment57. The extramembrane domains were separated by the transmembrane domains. The locations of the transmembrane domains were determined using tmfoldweb server. Secondary protein structures were predicted using PSIPRED and JPRED. Secondary structure elements were enforced in the modeling process. In order to model the transmembrane domain into one alpha helix bundle rather than separate alpha helices, the best template structure was found using tmfoldweb server. The X-ray crystal structures of E. coli YEDX , MOUSE CLAUDIN-19 (PDB code: 3 × 29), Epstein-Barr virus gH and gL complex  were used as templates for the N-terminal, the transmembrane and the domain 2 for HSV-1, HSV-2, and VZV. In case of modeling the N-terminal domain for PRV, Pichia Pastoris Lysyl Oxidase  was used as the template. All of these templates showed high scores with low E-values. Individual domains were assembled according to the current understanding of each domain; the N-term and TM domains were specifically positioned relative to each other by superimposing separate domain structure models onto MOUSE CLAUDIN-19 protein crystal structure (PDB code: 3 × 29). Full-chain protein structures were remodeled using Modeller to refine loops and gaps in between domains.Template-based modeling of gK proteins from four alphaherpesvirus, HSV-1, HSV-2, PRV, and VZV , were achieved by dividing the protein sequences into domains, modeling each domain separately using Modellerhttp://foldxsuite.crg.eu/); the difference in the free Gibbs energy change (∆∆G) before and after performing each mutation is calculated by FoldX Suite. The numbers for the specific mutations are reported as deviations from the average; a large positive ∆∆G indicates the importance of an amino acid residue for structure stability.Amino acid residues in the gK homology model are mutated into alanines one at a time by AlaScan feature in the protein design package FoldX Suite downloaded in 2018 (Supplementary Information


Here, the structure of the  Mycobacterium tuberculosis is the causative agent of tuberculosis, which is a leading cause of mortality worldwide. The survival of M. tuberculosis in host macrophages through long-lasting periods of persistence depends, in part, on breaking down host cell lipids as a carbon source. The critical role of fatty-acid catabolism in this organism is underscored by the extensive redundancy of the genes implicated in β-oxidation (∼100 genes). In a previous study, the enzymology of the M. tuberculosisl-3-hydroxyacyl-CoA dehydrogenase FadB2 was characterized. Here, the crystal structure of this enzyme in a ligand-free form is reported at 2.1 Å resolution. FadB2 crystallized as a dimer with three unique dimer copies per asymmetric unit. The structure of the monomer reveals a dual Rossmann-fold motif in the N-terminal domain, while the helical C-terminal domain mediates dimer formation. Comparison with the CoA- and NAD+-bound human orthologue mitochondrial hydroxyacyl-CoA dehydrogenase shows extensive conservation of the residues that mediate substrate and cofactor binding. Superposition with the multi-catalytic homologue M. tuberculosis FadB, which forms a trifunctional complex with the thiolase FadA, indicates that FadB has developed structural features that prevent its self-association as a dimer. Conversely, FadB2 is unable to substitute for FadB in the tetrameric FadA–FadB complex as it lacks the N-terminal hydratase domain of FadB. Instead, FadB2 may functionally  associate with the enoyl-CoA hydratase EchA8 and the thiolases FadA2, FadA3, FadA4 or FadA6 as suggested by interrogation of the STRING protein-network database.The intracellular pathogen  Mtb FadB is a multi-catalytic enzyme that catalyses both the hydration and the 3-hydroxy dehydrogenation steps in the β-oxidation cycle  was amplified from Mtb H37Rv chromosomal DNA by PCR and the amplified DNA fragment was ligated into the NdeI and HindIII restriction sites of the pET-28b plasmid vector (Novagen). The resulting expression plasmid was heat-shock transformed into E. coli C43 (DE3) cells, which were grown on LB agar plates, selecting for transformants with 25 µg ml−1 kanamycin. Bacterial colonies were propagated in overnight liquid cultures  and used to inoculate bulk cultures . Bulk cultures were grown to an OD600 of 0.6  and protein expression was induced by adding IPTG . The induced cultures were incubated  and the cells were harvested after 12–14 h by centrifugation . Cleared cell extracts were prepared in lysis buffer  supplemented with EDTA-free protease-inhibitor cocktail (Roche) and were purified using Ni–NTA affinity chromatography. The protein was eluted using a step gradient increasing the imidazole concentration in the lysis buffer to 50 mM, 130 mM, 150 mM and 1 M. Eluted fractions were analysed by SDS–PAGE. The 130 and 150 mM fractions were pooled, dialysed into 25 mM HEPES pH 8, 300 mM NaCl, 10%(v/v) glycerol and concentrated by ultrafiltration  to a final concentration of 10 mg ml−1.The protocol for obtaining His-tagged recombinant FadB2 has been described previously (Taylor 2.2.plus screen (Molecular Dimensions) in a 96-well format and using a liquid-handling robot  to pipet drops of 150 nl protein solution plus 150 nl reservoir solution. Diffraction-quality crystals appeared over a reservoir consisting of 0.2 M ammonium citrate dibasic, 20%(w/v) polyethylene glycol 3350. In preparation for X-ray diffraction experiments, crystals were immersed for a few seconds in reservoir solution supplemented with 10%(v/v) ethylene glycol, mounted in nylon loops and quenched in liquid nitrogen.Crystals were obtained by sitting-drop vapour diffusion using the commercial JCSG-2.3.Phaser  and showed that this enzyme catalyses the oxidation of 3-hydroxyacyl-CoA to acetoacetyl-CoA  and does not include the disordered N-terminal hexahistidine purification tag. The asymmetric unit contained six protomers of FadB2 assembled into three dimers . Given the resolution limit and the number of unique copies, noncrystallographic symmetry restraints were applied during coordinate and temperature-factor refinement.In a previous study, we characterized the enzymology of n Table 1a and dors Fig. 2b. Givena) conforms closely to the paradigm of previously solved monofunctional l-3-hydroxylacyl-CoA dehydrogenases. The enzyme presents a two-domain architecture consisting of an N-terminal domain containing a dual Rossmann fold (residues 3–189) and a smaller C-terminal domain . Helices α2, α3 and strand β3 are spliced in between the two βαβ motifs. Strand β6, strand β7 and  strand β8 complete the central parallel β-sheet, with the latter marking the C-terminal boundary of the N-terminal domain. The C-terminal domain consists of a five-helix bundle . The domain boundary is located in the loop between strand β8 and helix α7 , consistent with the structures of human mitochondrial HAD  of 1.16 Å. There is also a close match with the structure of C. butyricum HAD . Both orthologues also crystallized as dimers.FadB2 is a monofunctional enzyme that mediates 3-hydroxyacyl-CoA dehydrogenase activity . Furthermore, FadB forms a noncovalent, heterotetrameric complex with the dimeric ketoacyl-CoA thiolase FadA , while the HAD-C2 domain of FadB remains without a counterpart. However, when the FadB monomer is superimposed onto the FadB2 dimer, the C-terminal domain of the second monomer matches the helices of the HAD-C2 domain of FadB , suggesting that the HAD-C1 and HAD-C2 domains may have arisen from gene duplication.When FadB2 is aligned with FadB by secondary-structure matching, FadB2 overlaps with the C-terminal HAD segment of ly Fig. 4b, whilei.e. mediated by dimerization of the HAD-C1 domain), although this hypothetical dimer incurs partial steric overlap between the hydratase domains. Nevertheless, a small rotation about the axis of the linker helix between the hydratase and HAD domains would resolve the overlap. Thus, it appears that an evolutionary driver of integrating the HAD-C2 domain within the polypeptide chain of FadB is to prevent the self-association of FadB into dimers, so that the surface of the hydratase domain remains available for binding to the FadA dimer.In the structural context of FadB, the HAD-C2 domain has no obvious role in either catalysis or assembly of the trifunctional FadA–FadB heterotetramer . The largest share (∼75%) of the surface area buried in the interface with FadA is contributed by the hydratase domain of FadB, complemented by the contacts of eight residues in the HAD-C1 domain (∼21% of the surface buried in the interface), while only three residues of the HAD-C2 domain contact FadA. Similarly, the location of the HAD-C2 domain relative to the substrate-binding or cofactor-binding sites does not suggest that the HAD-C2 domain can contact the ligands. Therefore, it is not immediately clear what drove the apparent gene-duplication event that gave rise to the C2 domain. Since the five-helix bundle of the C-terminal domain mediates the dimerization of FadB2, one wonders whether FadB could self-assemble into dimers if the HAD-C2 domain were not present. Indeed, when omitting this domain, it is possible to construct a FadB dimer in analogy to the FadB2 dimer , which has been determined in complex with NAD+ and acetoacetyl-CoA .We compared the structure of b) illustrates a close match of contacting side chains, despite the subtle difference in overall conformation. For instance, Ser122, which is essential for catalysis  and we examined the extent to which these contact residues are conserved in FadB2 Fig. 3. Of the 4.et al., 2010fad gene family. Interrogation of the STRING database (Szklarczyk et al., 2017The structural features of FadB2 revealed in this study are fully consistent with the enzymatic characterization that we have reported previously (Taylor 5.et al. (2018The following references are cited in the supporting information for this article: Webb & Sali 2017 and Zimm al. 2018.10.1107/S2059798318017242/mn5119sup1.pdfSupplementary Figs. S1-S5.. DOI: FadB2, 6hrdPDB reference:


Protein-protein interactions are key factors in executing protein function. These interactions are mediated through different protein domains or modules. An important domain found in many different types of proteins is WW domain. WW domain-containing proteins were shown to be involved in many human diseases including cancer. Some of these proteins function as either tumor suppressor genes or oncogenes, while others show dual identity. Some of these proteins act on their own and alter the function(s) of specific or multiple proteins implicated in cancer, others interact with their partners to compose WW domain modular pathway. In this review, we discuss the role of WW domain-containing proteins in breast tumorigenesis. We give examples of specific WW domain containing proteins that play roles in breast tumorigenesis and explain the mechanisms through which these proteins lead to breast cancer initiation and progression. We discuss also the possibility of using these proteins as biomarkers or therapeutic targets. Protein function is determined by the domains and motifs that it harbors. These domains can be functional domains such as the catalytic domains found in many enzymes, or structural domains that are important for protein-protein interactions or the assembly of multi-protein complexes. Thus, protein's function is determined by its catalytic activity and its partners. One of these important domains found in many proteins is the WW domain. WW domain, is the smallest naturally occurring module. It consists of ~35–40 amino acids, including two highly conserved tryptophans (W), after which the module is named (WW). The two Ws are separated by 20–23 amino acids . Based oWW domain oxidoreductase (WWOX) is a tumor suppressor gene, which is altered in different types of cancer, including breast cancer . In breaIn addition to clinical data that supports WWOX role in breast tumorigenesis, different animal models have proved its function in proper mammary gland development and tumorigenesis. It was shown that WWOX is highly expressed in mammary gland tissue and that its targeted deletion results in mammary gland fibroadenoma , as wellWWOX mediates its functions by interacting with and modulating the activities of different proteins either through its WW domains or in a WW domain independent manner. Nonetheless, WWOX has also an oxidoreductase domain that seems to play a role in tumorigenesis too. WWOX alters different cellular processes involved in tumorigenesis. As discussed below, these cell activities include apoptosis, genomic instability, metabolism and metastasis.in vitro and suppression of breast tumor growth in vivo. These effects are mediated by reducing the expression levels of the anti-apoptotic protein Bcl-2 and inducing the pro-apoptotic protein BCL2-associated X protein (Bax)    . Furtherin (Bax) . This inin (Bax) . WWOX wain (Bax) . Anotherin (Bax)  18). In. Inin viin (Bax) . For WBPin (Bax) .Genomic instability is one of the important cancer hallmarks involved in tumor initiation as well as tumor progression , 22. WWOAnother cancer hallmark connected to the tumor suppressor gene WWOX is cell metabolism. The connection between WWOX and cancer cell metabolism was established after linking WWOX protein to Hypoxia-inducible factor 1-alpha (HIF1α) function . In thisMost of cancer patients die from metastasis and not from primary tumors. WWOX was also found to play a role in breast cancer metastasis. Loss of WWOX expression in breast cancer is significantly associated with the number of metastatic axillary lymph nodes and poor survival . In addiAlthough the compelling evidence that indicates with no doubt that WWOX is a tumor suppressor gene, the presence of WWOX in a fragile locus in the genome raises suspicion about its identity of being a classical tumor suppressor gene. As well, its localization in a fragile sequence raises another question on why such an important gene lies in a damage prone locus? The answer to this question might be that loss of WWOX can act as an early alarm for the cell to respond to different types of insults that can lead to cell stress and DNA damage.in vivo  Figure  50). In. In50). The link between BCSC and the Hippo pathway was initially established in a study that demonstrated that TAZ is required to sustain self-renewal and tumor-initiation capacities in BCSCs . TAZ wasRecently, in breast cancer patient samples, high YAP expression profiles correlated with EMT and stemness gene signature enrichment . Finallyin vivo  increases tumor immunogenecity and thus enhances its destruction by the immune system in vivo  is expressed in almost 70% of breast cancers and promotes estrogen-dependent cancer cell proliferation and tumor progression. Recently, it was shown that Secondary Metabolite Unique Regions Finder 1 (SMURF1), via its HECT domain, interacts with and stabilizes ER, and that depletion of SMURF1 decreases ERα-positive cell proliferation  in vivo . This pr in vivo . SMURF2 ll cycle . Analysill cycle . Anotherll cycle   are addressed in a specific study, which makes it difficult compare and judge controversial results obtained. These controversies and different functions of these proteins can be resolved also by future studies that will be based on more global approaches that will study the proteome or even the interactomes of these proteins in different cell contexts and in relation to different cancer related processes regulated by WW domain-containing proteins. Such studies, in addition to illustrating the mechanism(s) of action of the different WW domain-containing proteins, they will identify other biomarkers involved in the execution of phenotypes related to the different WW domain-containing proteins. In fact the identification of such biomarkers and elucidation of exact mechanism of action can be also beneficial in the prediction of therapeutic outcomes related to the manipulation of WW domain-containing proteins in the future.Another future direction that should be followed in the context of WW domain-containing proteins and breast cancer is the identification of new such proteins. It is predicted that there are many other different WW domain-containing proteins that will have impact on breast tumorigenesis. The fishing for such proteins can be based on pulling down WW proteins using known PY rich domains, or by developing and using different softwares to help predict the presence of WW proteins in the human genome.The use of WW domain-containing proteins as drug targets is still a premature idea that needs intensive future research. In this regard, one can raise the question of what to target in these proteins? Shall a drug target the catalytic activity of these proteins? Or their interactions by targeting their WW domains? Or target even their partners and targets? The answer to these questions will never be easy with our current knowledge about these proteins. However, we can speculate about the complexity in choosing which proposed strategy would be a successful one. For example, upon targeting the catalytic domain of different WW domain-containing proteins, a drug can target different proteins that have antagonistic functions in the context of cancer. For instance, if drugs were developed to target the evolutionary-conserved catalytic HECT domain, these drugs will target for example SMURF1 and SMURF2, which were shown to have antagonistic functions in breast carcinogenesis, and this, theoretically, might make such a strategy a failing one. In choosing to target WW domains in order to disrupt protein-protein interaction is also complex. Although there are different types of WW domains, there is still redundancy regarding the presence of a specific type of WW domain in different proteins, and thus it is expected that upon targeting the interaction of a specific WW domain-containing protein, the interactions of different other WW domain-containing proteins will be affected. In this context, here, another issue is present, which is the presence of usually more than one WW domain in one protein. These domains do not seem to be redundant in their interactions. On the contrary, they seem to be very specific which raises another question; Which domain in a specific protein to target? Regarding the last possibility of targeting WW domain-containing protein partners as means of therapeutic intervention, it might seem to be the best option. Although the fact that different WW domain-containing proteins can partner with common and shared partners, there are different examples for the presence of specific targets for different WW domain-containing proteins. These specific targets might act as biomarkers to predict specific treatment success or even be a therapeutic target.Finally, it is obvious that WW domain-containing proteins are critical factors in breast carcinogenesis and that a better understanding of their roles in breast cancer will likely lead to the identification and development of biomarkers and drug targets for cancer treatment.AJ prepared figures and bibliography. ZS wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Appendectomy is one of the most common emergency surgical operations. Stump appendicitis is a rare complication after appendectomy and is caused by acute inflammation of the remnant part of the appendix. Because of the low index of suspicion owing to a previous history of appendectomy, the diagnosis of stump appendicitis is often delayed.Between January 2008 and December 2017, 6 patients were diagnosed with stump appendicitis with or without perforation at a single institution. They had undergone operative management with laparoscopic approach. The clinical data of these patients were retrospectively analyzed by reviewing the medical records and pathologic reports.Five patients were male, with a mean age of 42.4 years (range 11–77 years). The time interval after initial appendectomy ranged from 2 weeks to 30 years. Three patients underwent laparoscopic completion appendectomy, and the others underwent laparoscopic ileocecectomy. The mean hospital stay was 9 days (range 5–13 days). There were no cases of open conversion.Stump appendicitis is a rare complication after appendectomy. A laparoscopic procedure can be performed for management of stump appendicitis with or without perforation. Because of both the low incidence of stump appendicitis and a low index of suspicion owing to a previous appendectomy, the diagnosis of stump appendicitis is often delayed. Also, delayed diagnosis makes surgery more difficult and increases morbidity. Here, we describe 6 cases of stump appendicitis which were treated through laparoscopic procedures, successfully.An appendectomy is one of the most common emergency surgical operations.2From January 2008 to December 2017, 6 patients were diagnosed as having a stump appendicitis with or without perforation, and they underwent laparoscopic procedures at the Department of Surgery at Chungbuk National University Hospital, Korea. All patients were admitted to the emergency room with right abdominal pain and assessed using contrast-enhanced abdominal and pelvic computed tomography (A-P CT). We retrospectively reviewed the patients’ medical records and radiologic images. Retrospective protocol of this study was approved by the institutional review board (IRB) of Chungbuk National University Hospital, South Korea (IRB No 2019-02-006-001). Informed written consent was obtained from the patients for publication of this case report and accompanying images.3Five patients were male, with a mean age of 42.4 years (range 11–77 years). The mean time from initial appendectomy to presentation was 17.3 months (range 2 weeks–30 years). Four patients had previously undergone an open appendectomy. On A-P CT scans, 3 patients demonstrated perforation around the stump appendix. In cases 1 and 3, there was an appendicolith in the short stump appendix with inflammation around the appendix  presented the guidelines for laparoscopic appendectomy. Subramanian and Liang reported a lower incidence of stump appendicitis in the laparoscopic than in the open appendectomy.Following the introduction of laparoscopic appendectomy, there have been reports comparing the incidence of stump appendicitis in laparoscopic and open approaches.The treatment of choice for stump appendicitis is a completion appendectomy, even when perforation has occurred. However, in cases of severe inflammation with abscesses, an ileocecectomy is needed. A laparoscopic approach can be used for management of stump appendicitis with or without perforation. And we have performed laparoscopic procedures in all our cases without surgical complications.5Although a stump appendicitis is a rare complication after an appendectomy, it can be a cause of right-sided abdominal pain in patients who have had appendectomies previously. Because delayed diagnosis is associated with increased postoperative morbidity and also affects the extent of resection, clinicians should consider the possibility of stump appendicitis as a cause of acute abdominal pain. A laparoscopic approach can be adopted for management of stump appendicitis with or without perforation.www.editage.com) for English language editing.The authors thank Editage (Conceptualization: Hanlim Choi, Dong Hee Ryu.Data curation: Hanlim Choi, Dae Hoon Kim.Investigation: Dae Hoon Kim.Supervision: Jae-Woon Choi, Dong Hee Ryu.Writing – original draft: Hanlim Choi.Writing – review & editing: Young Jin Choi, Taek-Gu Lee, Dong Hee Ryu.