Our SOAP kernel-basedmetrics allows us to compare, discriminate, and correlate differentFFs at different model resolutions in an unbiased, high-dimensionalway. This also captures differences between FFs in modeling nonaverageevents , for example, the liquid-to-gelphase transition in dipalmitoylphosphatidylcholine (DPPC) bilayers,for which our metrics allows us to identify nucleation centers forthe phase transition, highlighting some intrinsic resolution limitationsin implicit versus explicit solvent FFs.Molecular dynamics simulations of all-atom and coarse-grained lipidbilayer models are increasingly used to obtain useful insights forunderstanding the structural dynamics of these assemblies. In thiscontext, one crucial point concerns the comparison of the performanceand accuracy of classical force fields (FFs), which sometimes remainselusive. To date, the assessments performed on different classicalpotentials are mostly based on the comparison with experimental observables,which typically regard average properties. However, local differencesof the structure and dynamics, which are poorly captured by averagemeasurements, can make a difference, but these are nontrivial to catch.Here, we propose an agnostic way to compare different FFs at differentresolutions , by meansof a high-dimensional similarity metrics built on the framework ofSmooth Overlap of Atomic Position (SOAP). We compare and classifya set of 13 FFs, modeling 1-palmitoyl-2-oleoyl- A plethora of experimental techniques such as NMR,5 calorimetry,6 SANS,7 and SAXS8 have beenapplied to lipid bilayers to obtain average structural and dynamicinformation at different resolutions. This large amount of experimentaldata paved the way to the creation and the cross-validation of reliablemodels that can be simulated by means of molecular dynamics (MD).The use of all-atom (AA) provides, in principle, an atomic-resolutioncomputational microscope for investigating the dynamic evolution ofmembranes in great detail.9 Despite thetremendous advance in computational capabilities observed in the lastdecades, classical MD at atomistic resolution is still unable to coverall the time scales of biological interest.10 For this reason, starting from the beginning of the 90's, variousmodels with a reduced number of degrees of freedom were proposed,from united atom (UA) representations, where the aliphatic hydrogenatoms are removed and their mass is added to the bound heavy atom,11 to coarse-grained (CG), where a single “CGbead” is formed by usually two to five heavy atoms,12 to super-CG models, where a single lipid canbe represented by three to four larger CG beads.13 The reduction of the number of degrees of freedom providesa dramatic speed up in the simulations, which is nonetheless accompaniedby an unavoidable loss of entropic contribution (and thus accuracy),typically compensated by properly adjusting the enthalpic contributions.10 The evaluation of the precision and the performanceof a force field (FF) (at any level of resolution) is in general obtainedby comparing average equilibrium observables computed from simulationsof the bilayer models to the experimentally available ones. A largebody of work has addressed the problem of comparing state-of-the-artlipid FFs and their accuracy with respect to increasingly preciseexperimental data.17 However, the general assessment of the performance of an FF remainsa difficult problem which concerns multiple parameters at the sametime. Moreover, comparing various FFs becomes particularly awkwardwhen different representations/resolutions in the modeling of thechemical system are employed and when the internal dynamic organizationof the membrane, its uniformity/non-uniformity, and  dynamicfluctuations become important.Lipid membranes are ubiquitous in biological systems, and theirchemical and mechanical characteristics directly impact the regulationof the cell machinery.sn-glycero-3-phosphocholine , three UA models , and three wet CG , two polarizable CG (Martini 2.2p29 and Martini 2.3p30), and two implicit-solventCG models . Our analysis demonstrates thatstate-of-the-art FF potentials exhibit differences in terms of widelystudied equilibrium and dynamic average observables  vectors35 are extremely useful to provide a high-dimensional,agnostic, and rich description of molecular environments in molecularsystems. SOAP has been successfully applied in exploring the conformationallandscape of single molecules,36 nucleationphenomena,37 molecular assembly classification,38 and the formation, stability, and intrinsicdynamic complexity of soft supramolecular assemblies,40 providing a rich structural/dynamical characterization of complexmolecular systems that is not easy to obtain with human-based approaches.In this work, we consider 1-palmitoyl-2-oleoyl-ne POPC, 1 bilayerervables 2, making41), finding a substantial agreement between the two.Herein, we employ a distance between the average SOAP representationsto compare and classify the different data ensembles obtained fromthe MD simulations using the different FFs. The analyzed FFs havesome internal differences in their organization that can be detectedwith SOAP: this allow us to classify the FFs based on a global scoreof similarity. Our results with the SOAP metrics are validated bycomparing them with an analogous analysis based on a well-known distributionmetrics . We investigated the liquid-to-gel phase transition ina pure bilayer of dipalmitoylphosphatidylcholine (DPPC) lipids simulatedat different temperatures: in the gel phase, mixed gel–liquid,and liquid phase. By computing individual SOAP vectors for each lipidmolecule in the bilayer across MD simulations at different temperaturesand clustering these with Probabilistic Analysis of Molecular Motifs(PAMM),42 we could clearly identify nucleationcenters underpinning the phase transition, comparing how well CG FFsat different scales (wet and dry) reproduce the features of the system.Finally, to provide further evidence of the local chemical–physicalorigins of differences between the FFs that the SOAP environment representationallows us to elucidate, we focused on the comparison between two widelyemployed explicit-solvent and implicit-solvent CG models   and projectingit onto a basis of orthogonal radial functions gn(r) and spherical harmonics Ylm, which for the i-th site can be expressed asj index runs overall the sites of the species α in the cutoff. The coefficient cnlmj,α displays a dependenceon Γ to underline that it changes as a function of the global3D configuration of the lipid bilayer system. Furthermore, in thisspecific case, the SOAP power spectrum calculation is carried outby expanding the local bead density ρi(α), which accounts for the 3D displacement of all beads ofthe lipids in the bilayer at each considered MD snapshot, within acutoff from the center of each SOAP spectrum . We anticipate that in our case, rcut is at 3 nm: in this way, the SOAP spectracentered in all CG phosphate groups account for all CG particles relatedto lipid heads, the other phosphate groups, and the tails in the lipidbilayer at a given snapshot of the MD trajectory, which reflect levelsof order/disorder, displacement of particles, and so forth in thelipid bilayers. It is worth noting that ρi(α) is multicomponent .In practice, 43 package. Additional details and explanation can be foundin Supporting Information, but for extensivemathematical derivations, we refer the reader to the original articleon the SOAP method.35The SOAPe in ref ). Equati35A similaritymeasure between two environments centered in two sites can be formallydefined by building a linear kernel of their density representations.Such a kernel can be analytically computed, and it can be reducedto the dot product of the two sites’ SOAP power spectra.KSOAP goes from 0 for completely different to 1 for matchingenvironments.pi is the i-th center’s power spectrum.Both kernel and distance representation give a bounded measure ofhow similar two sites are .The power spectra and xy dimensions using nmax = 8 radial basis function and Lmax = 8 maximum degree of spherical harmonics (a Gaussian density smoothingσ = 0.1 nm was used). The SOAP descriptors were centered onthe center of mass of the phosphate groups of each lipid in the simulatedbilayer models (consistent with the Martini 2.2 beads representation),including also the polar organic group atoms of the hydrophilic head and the two tail terminal beads in the environment, thus considering also theenvironment of the lipid tails inside the membrane. It is worth tounderline that while the SOAP vectors are centered on the phosphategroup of each lipid in the bilayer models, the SOAP analysis takesinto account all the different types of beads involved in the lipidrepresentation , four beads per lipid in totalin the system. Besides the parameters for the harmonic and radialexpansions, the last arbitrary-free parameter to select for SOAP calculationwas the cutoff distance. We selected a value of 3 nm on the basisof two considerations: (i) the cutoff needs to be large enough toinclude all particles intended to be characterized by SOAP descriptors;the most demanding criteria are for the four-beads system for whichthe cutoff needs to be smaller than the width of the membrane to excludethe opposite surface beads, thus smaller than about 3.7 nm, but largeenough to include the tail beads, located in the mid-part of the membrane,thus larger than about 2 nm. (ii) Ideally, the descriptors shouldprovide an optimal cutoff range beyond which any further refinementdoes not add information . Preliminary tests showed that the effective rankof the distance kernel matrix as a function of the SOAP cutoff distance,for the set of systems under analysis, plateaus for cutoffs ≥3nm . This indicated thatin this case, 3 nm is a good cutoff for our SOAP–PAMM analysis.In detail, a shorter cutoff does not allow us to discriminate in asatisfactory way between the relevant states in the systems . On the other hand, usinga cutoff larger than 3 nm was observed not to add much to the analysis,while at the same time making it computationally heavier. All theSOAP calculations were carried out using the DScribe43 python package. The complete set of input files and scriptsused to analyze the trajectories is available on GitHub at https://github.com/GMPavanLab/lipids-comparison.For all systems,the SOAP descriptors were calculated under periodic boundary conditionsalong the 44 and with FCIalgorithm,45 obtaining, respectively, anestimation of 24 and 25 dimensions, using a 3 nm cutoff. The firstpoint was to make the gridding computationally feasible .In particular, we started from the AA systems, as these have an intrinsicallymore complex behavior than UA or CG systems. In order to reduce thedimensionality of the data set or the AA systems (given the high complexityof the AA systems), we selected a sample of spectra from the globalSOAP spectra obtained during the analysis  on which we employed PCA. This allowed us to reachthe best compromise between the quantity of information retained inthe data set and the computational cost/feasibility of the analysis.We then calculated the density using the PaK algorithm on each systemseparately.46 PaK is a nonparametric densityestimation method that exploits the exponential distribution of thedistance between the first two neighboring points to define a commondistribution, in the hypothesis of a locally constant and sufficientlysmooth density .SOAP descriptors are rotational invariantembedding of the environment surrounding a given particle. In orderto compare the conformational space originated by the different FFsvia MD simulations, one can consider to (i) use a similarity kernelon the average SOAP descriptors calculated over the whole trajectory,as we explained in the sections above, or (ii) estimate the probabilitydensities resulting from the ensemble of the environments’SOAP power spectra over a reduced representation and calculate a distributionaldistance over such densities. To obtain a comparison of type (ii),we took the original SOAP spectra and we evaluated its intrinsic dimensionvia the TwoNN algorithm42 is a density-based clustering technique developed withthe core idea of partitioning the Probability Distribution Function(PDF) of data sampled from molecular simulations to identify structuralmotifs. The input required is a set of vectors that completely characterizethe molecular environment of the system under analysis . From these high-dimensional data, PAMM workflowstarts from an iterative kernel density estimation of the data. Oncea stable estimation of the PDF is obtained, a density-based clusteringis performed, identifying the different local maxima in the PDF, whichdefine cluster centroids. Each cluster is thus fit using a Gaussianmixture model that ultimately gives the so-called probabilistic motifidentifiers42 that translate in the system-specificfingerprint. The clustering analyses for our systems were performedusing the original PAMM code42 (availableonline at https://github.com/cosmo-epfl/pamm) as a baseline, along with a tailored Python3 code wrapper to handlethe different steps in the analysis workflow and the data postprocessing.The SOAP–PAMM procedure used in this phase is consistent withthat recently used to characterize the complex internal dynamics andthe emergence/resorption of defects in supramolecular polymers—alldetails are available in refs (PAMM in refs  and 40)42 is a dhttps://github.com/GMPavanLab/lipids-comparison.All the DPPC lipid bilayerswere simulated for a total of 1 μs of simulation time at theselected temperatures. SOAP local descriptors were calculated fromthe production trajectories, considering one snapshot every 10 ns. We initially merged all the datacoming from the same FF simulations, performing a dimensionality reductionperformed with a linear PCA, limiting the estimation to the firstfive eigenvectors. In this way, all the temperatures are in the sameparameter space and can be directly compared. We thus performed thePAMM clustering on this low-dimensional space, and we obtain the clusterseparation and interconversion matrices shown in 48 which have been then minimized and equilibrated according to well-establishedprotocols . For the UA FFs, we obtained the input files for Berger lipidsfrom the internet50 and for Gromos-CKP and Gromos43a1-s3from Lipidbook,51 following also in thiscase the minimization and equilibration protocols indicated by CHARMM-GUI.We performed 1 μs of MD simulation using GROMACS 2018.652 patched with PLUMED54 (details on the simulation parameters used for each system are availablein Supporting Information). For Sirah,we employed AMBERTools using the simulation parameters given on thehome page of the FF . Alsoin this case, we performed a 1 μs-long MD simulation for dataproduction.We startedby simulating a bilayer formed by 128 POPC molecules (64 per leaflet)at 303 K using three different FF resolutions 1: AA (CHDHH), and phosphate group density profiles foreach case . Regarding the DHH (top center in 58) can, for example,make the thermal vibrations larger/smaller, broadening/narrowing thedensity distributions. A great variability is present in membranecompressibility , bilayer thickness . Finally, averaging these on all the equilibriumMD snapshots considered in the analysis ,we finally obtain a global insight into the complexity of the lipidbilayer in terms of the structural and dynamic features of the localenvironment surrounding, on average, the lipid centers in the systemin the equilibrium MD regime. Breaking down the SOAP analysis in thisway has three main advantages. (i) The SOAP calculations for the systemsare computationally manageable. (ii) This helps in focusing the analysison the supramolecular dynamics of the lipid bilayers , reducing noise that is intrinsically present in the MDtrajectories. Finally, (iii) this guarantees to compare the differentmodels studied herein using a common metrics. Assuming that we aresampling well the conformational space in the equilibrium MD regimefor each simulated system, we therefore obtained 13 average SOAP spectrathat are informative on the structural dynamics of POPC lipid bilayersat 303 K.While such parametersmay be useful to compare between the different FFs, it is not easyto obtain a complete, exhaustive picture from such a low-dimensionalanalysis. A more general, high-dimensional analysis would be desirablefor a more rigorous comparison. To define a metrics that can be appliedto compare lipid models having a different resolution , we started from considering a unified lipid representation wellsuited for such a comparison. We chose four sites (beads) in the lipidstructure that are in common between all considered lipid models andthat we use for our SOAP analysis: one bead for the lipid chargedhead group, one bead for the phosphate group, and one bead for eachof the two alkylic lipid tails. In particular, one SOAP vector iscentered in the phosphate bead of each lipid molecule in the system,and it considers in the analysis all other beads in the atomic (particle)environment that surrounds each lipid (phosphate) center in the membrane.The choice of the phosphate group as the SOAP centers is due to theircentral position, both in geometric terms in the lipids structureand in chemical terms, as this group is at the interface between thehydrophobic and hydrophilic parts of the lipid molecules. As demonstratedalso in similar studies on the internal dynamics of assembled supramolecularpolymers,Supporting Information), which provides a distancematrix which is indicative of the similarity (in the SOAP space) betweenthe FFs  of the lipid bilayers. This is reasonableand could be expected to some extent, as SOAP is in fact a high-dimensionalway to represent the spatial displacement of atoms/beads along theMD trajectories .41 between the probability distribution sampledvia MD simulations for all FF pairs (details in the methods sectionin Supporting Information). This test hasbeen carried out to verify the degree of agreement between the twoapproaches. A correlation plot between the SOAP distance and the Jensen–Shannondivergence computed between the average SOAP spectra is availablein Supporting Information .The distance metrics calculated between the average SOAP spectra thenallowed us to discriminate and compare between different FFs for POPCat a temperature of 303 K, for which all systems are in the liquidphase.Such a use of distance measurements between the average SOAP environmentshas been validated by calculating the Jensen–Shannon divergence58 However,all these analyses and comparisons provide evidence that is limitedto the average characterization of the bilayers, while, on the otherhand, it has been shown that the behavior of complex supramolecularassemblies  may be strongly controlledby local events, or fluctuations, that cannot be captured with averageevaluations.61While this approach allows us to compare the FFsbetween them in a rather comprehensive way —we learn, for example, that some CG FFs are closer toUA and AA FFs compared to other ones—it is not straightforwardto link such information extracted from high-dimensional analysesto human-comprehensible data. At the same time, the comparison witha few experimental data 2a does n37 where typical ice nucleationsites were probed in a QM-based liquid water model by means of a SOAPanalysis, we investigated the transition of a lipid bilayer betweenthe liquid phase and the gel phase. In particular, we were interestedin testing the efficiency of a high-dimensional SOAP-based approachto detect and characterize local nucleation processes underpinningthe formation a new rearrangement of the bilayer during a phase transition. In a similar way, in the absence of identification of any nucleationevent, such an analysis can be useful in identifying limitations inthe FF representation of the lipid assembly. The experimental meltingtemperature Tm for POPC is approximately273 K,63 which is inconvenient for workingwith classical MD. We thus decided to turn to another well-studiedlipid, DPPC, whose transition from the gel to liquid phase occursat a temperature of ∼315 K.64 Forthe wet Martini FF, Tmelting has beenreported in semiquantitative agreement at ∼295 ± 5 K,65 while for Dry Martini, Tmelting has been estimated at ∼333 K.66 We built a bilayer model composed of 1152 DPPC molecules(576 per leaflet), parametrized using the Martini 2.2 and Dry MartiniCG FFs . Both CG models were minimized andequilibrated using the same MD procedure as in the POPC case (see Supporting Information for details). In particular,the lipid bilayers were simulated at 273, 293, 323, 333, and 353 Kfor both wet Martini 2.2 and Dry Martini CG models. All the productionMD simulations were 1 μs-long.To move our investigation to a deeper level, similarly toa recent study42 to classify the lipidsbased on their surrounding environment, their local levels of orderand disorder, and the fluctuation/persistence of the latter alongthe equilibrium MD trajectories. This allowed us to classify the lipidsin the bilayer during the simulation based on their SOAP spectra andto discriminate those belonging to the gel or to the liquid phase.The clustering procedure was performed on both the FFs merging allthe spectra obtained from the five different temperatures (detailsin the Methods section) to identify the same clusters along all thetemperatures. In particular, from those lipids that dynamically changethe cluster during the equilibrium MD, we could obtain a rather comprehensivedynamic picture of the equilibrium between gel and liquid phases atthe different temperatures and to compare how these are reproducedin an explicit-solvent or in an implicit-solvent CG FF. The resultsof the PAMM analysis at different temperatures for the Martini 2.2and Dry Martini DPPC bilayers are reported in We used again four beads per lipid for the SOAP analysis , and we set thephosphate bead as the SOAP center for calculating each spectrum. Thisrepresentation is a compromise between the computational cost andcomprehensiveness of the analysis, and it is particularly well suitedfor studying phase transitions, as the tail arrangement is indicativeof the phase transition in a lipid bilayer. In this analysis, we computedthe SOAP spectra independently for each single lipid in the bilayerwithout averaging them. We thus obtained 1152 spectra per analyzedframe (one every 10 ns of MD). We then used an unsupervised clusteringmethod, the PAMM,T < Tmelting to T > Tmelting . This suggests that the SOAP–PAMM analysis of Figure S5 may, to some extent, distortthe information obtained from such an analysis, which may be relevantespecially in the case of, for example, large, deformable bilayerassemblies that are capable to bend . This is intrinsically better handled by the moreabstract SOAP–PAMM analysis of While the phase transition and its local origin appear to be well-reproducedby the explicit-solvent Martini 2.2 FF, the implicit-solvent Dry MartiniFF provides a different picture  orto the disordered (liquid) phases in the bilayers, and we can reconstructthe equilibrium structural dynamics inside the bilayers, as well asthe dynamic nucleation of gel/liquid phases across the temperaturesin a comprehensive way. Comparing explicit-solvent and implicit-solventCG FFs (Martini 2.2 and Dry Martini), our results clearly demonstratethat while the Martini 2.2 models well the phase transition, the implicit-solventDry Martini FF does not model well transitions that are strongly triggeredby local events. A next possible step to further deepen the investigationexploiting this SOAP metrics could be the specific comparison betweenAA/UA FFs, for example, considering a larger number of beads in thelipids molecules as particles accounted in the SOAP analysis. Furthermore,we envisage that such data-driven metrics can be adapted to scoreand compare a variety of FFs, not restricted to lipids, and that thiswill become particularly useful for analyzing molecular models ofcomplex interacting  systems.In conclusion, we presented a data-driven dimensionality reductionapproach based on a metrics coming from the SOAP framework whichis able to quantify the similarity between FFs at different levelsof resolution. We applied this to compare how various FFs model lipidbilayers at different levels of resolution . UsingPOPC lipids as a reference case, our analysis highlights the goodagreement between state-of-the-art AA and UA FFs. Regarding wet CGFFs, we observed a substantial equivalence in the wet Martini familyand a significant improvement in the global representation of POPCbilayers in the 2016 version of Dry Martini compared to the originalDry Martini version (2014), which is found closer to the other wetCG FF analyzed, Sirah 2.1, and to the fine-grained FF studied. Theseanalyses offer great opportunities to obtain detailed insights intothe different representations of the model bilayers by the differentFFs which are poorly captured by conventional, average analyses. Ourlocal SOAP–PAMM analysis, for example, allows us to identifyin an unbiased and unsupervised way the different states for the lipidsin a DPPC bilayer at different temperatures, below and above the transitiontemperature


Panax ginseng, Alpinia galanga, Vitis vinifera and Moringa oleifera, the extracts of V. vinifera seed, A. galanga rhizome and M. oleifera leaf reversibly inhibited TYR diphenolase activity with IC50 values of 94.8 ± 0.2 µg/mL, 105.4 ± 0.2 µg/mL and 121.3 ± 0.4 µg/mL, respectively. Under the same conditions, the IC50 values of the representative TYR inhibitors of ascorbic acid and kojic acid were found at 235.7 ± 1.0 and 192.3 ± 0.4 µg/mL, respectively. An inhibition kinetics study demonstrated mixed-type inhibition of TYR diphenolase by A. galanga and V. vinifera, whereas a rare uncompetitive inhibition pattern was found from M. oleifera with an inhibition constant of iiK 73 µg/mL. Phytochemical investigation by HPLC-MS proposed luteolin as a specific TYR diphenolase ES complex inhibitor, which was confirmed by the inhibition kinetics of luteolin. The results clearly showed that studying TYR inhibition kinetics with plant extract mixtures can be utilized for the screening of specific TYR inhibitors.Tyrosinase (TYR) is a type III copper oxidase present in fungi, plants and animals. The inhibitor of human TYR plays a vital role in pharmaceutical and cosmetic fields by preventing synthesis of melanin in the skin. To search for an effective TYR inhibitor from various plant extracts, a kinetic study of TYR inhibition was performed with mushroom TYR. Among  The second reaction, diphenolase, oxidizes l-DOPA to l-dopaquinone  and maxV (maximum reaction rate) when compared to the TYR activity without an inhibitor, and a high concentration of substrate cannot overcome the inhibition [While synthetic TYR inhibitors developed based on the catalytic mechanism can exhibit strong inhibition, these compounds also often establish cytotoxic and mutagenic effects against mammalian cells . At the hibition ,8,9.Inhibitors from natural resources, such as plant extracts, often suffer from low and non-specific inhibition. Besides, the vagueness of the inhibition mechanism of the extracts greatly hinders practical application, regardless of many advantages, such as low cost, direct production with less formulation, and safety . RegardlPanax ginseng (ginseng rhizomes and leaves), Alpinia galanga  Vitis vinifera (grape seeds) and Moringa oleifera (moringa leaves), were selected. Along with total phenolic contents, TYR inhibition and inhibition kinetics were studied to determine the TYR inhibition type, and the bioactive compound in the extract of the uncompetitive inhibitor was further investigated by HPLC-MS and detailed inhibition kinetics.Therefore, we have investigated the inhibition of TYR by selected plant extracts by adopting a TYR inhibition kinetic study. In detail, high phenolic plants consumed by Asian people in their daily diet, including A. galanga, 95% ethanol was instead used, due to the high concentration of hydrophobic composition. A. galanga rhizome BL (651.9 ± 4.2 mg), followed by M. oleifera leaf (472.4 ± 2.8 mg) and A. galanga leaf (448.1 ± 2.3 mg). P. ginseng leaf showed lowest phenolic content among the tested plants.Polyphenolics are a group of plant secondary metabolites synthesized by shikimate and polyketide pathways. Many of these compounds with catechol or resorcinol functional groups, such as flavonoids, were known to inhibit TYR activity , and are50  correlation. The result showed moderate correlation , which means there is a good negative correlation between TPC and IC50 values  and 1.01 ± 0.24 OD492/min, respectively, for the diphenolase activity of mushroom TYR used for the experiments. The mK value obtained from our assay system was similar to the reported value [y-intercepts in the Lineweaver–Burk plot obtained in the presence of various concentrations of the inhibitor were used to determine the inhibition constants of iK and iiK  for the inhibition of TYR diphenolase activity by luteolin . Apparently, the extract of M. oleifera contains other bioactive compounds that may interfere with TYR catalysis. Furthermore, the extract contained only 1.6% of luteolin. Therefore, we propose the glycosides of luteolin, such as luteolin 7-methyl glucuronide and luteolin 7--glucoside), can also be uncompetitive TYR diphenolase inhibitors, based on the HPLC-MS analysis.The phytochemical composition of the nalysis, , luteolinalysis,  and was stigated . The inhluteolin . The detf 103 μM , but lowActivity-guided isolation of bioactive compounds from natural resources has been a popular method for the development of value-added products, including medicines, functional foods, and cosmetics. In this study, we aimed to search an alternative methodology in the screening of TYR diphenolase inhibitors from the widely consumed plant extracts. Aas long as effective uncompetitive inhibition is screened, investigation of the enzyme inhibition kinetics of plant mixtures can be an efficient approach for the development of specific TYR inhibitors due to the guaranteed safety of plant extracts. M. oleifera, V. vinifera and A. galanga exhibited strong inhibition against TYR diphenolase, even compared with ascorbic acid and kojic acid, which are renowned TYR inhibitors. Further inhibition kinetics investigation was applied to elucidate the inhibition mechanism of the plant extracts. The observed mixed-type inhibition by V. vinifera and A. galanga implied that the compounds in the extract act non-specifically on both the free enzyme and ES complex at low concentrations. Interestingly, the uncompetitive modes of inhibition by M. oleifera extract strongly suggested specific ES complex inhibition by unidentified bioactive compounds. HPLC-MS analysis provided the phytochemical composition of M. oleifera, and luteolin and its glycosides were proposed as uncompetitive inhibitors. The inhibition by luteolin and M. oleifera in the presence of non-ionic detergent, triton X-100, was also performed to confirm that these are not Pan-Assay Interfering Substances (PAINS) [l-DOPA and oxy-TYR [l-DOPA complex during the catalytic reaction. The results showed that  (PAINS) . The res oxy-TYR ; hence, l-dihydroxylphenylalanine (l-DOPA) were purchased from Sigma-Aldrich, St. Louis, MO, USA.Dimethyl sulfoxide (DMSO), Folin–Ciocalteau reagent, and gallic acid were purchased from Fluka, Switzerland. Mushroom tyrosinase (2587 units/mg), luteolin, and M. oleifera, A. galanga and P. ginseng were collected from local gardens in Khon Kaen, Thailand, during October 2017, and V. vinifera seeds were provided by Visootha (Nikki) Lohinavy, Gran Monte Asoke Valley in Pak Chong, Nakorn Rachasima, Thailand. Clean plant materials were dried by hot air oven at 65 °C  and powdered. All plant samples were extracted with 70% ethanol in a 1:4 w/v ratio, except A. galanga rhizomes which was extracted by 95% ethanol in the same ratio of sample to solvent. The mixture was stirred for 8 h at room temperature, and then filtrated by Whatman filter paper no. 1. All extracts were concentrated by rotary evaporator . A. galanga rhizomes extract resulted in two layers, an oily yellow upper layer (YL) and brown lower layer (BL). All extracts were stored at 2 °C.Leaves and rhizomes of Total phenolic content (TPC) was measured by the Folin–Ciocalteu method . Gallic r = correlation coefficient, xi = TPC, xi, yi = IC50, and yi.Pearson coefficient (bivariate) correlation was determined by using the following equation,l-DOPA conversion into the colored dopachrome according to the published method [l-DOPA (15 mM in 0.1 M PBS pH 6.8) was added to each well and incubated at 37 °C for 30 min in a dark place. Absorbance was recorded by microplate reader spectrophotometer  at 492 nm. Kinetic parameters of mK and maxV for TYR diphenolase activity with l-DOPA were determined by using the Lineweaver–Burk equation:TYR inhibition was detected by the inhibition of the d method . Briefly50. All studies were conducted in triplicates and compared statistically. Kojic acid and ascorbic acid were used as the reference for TYR inhibitors.The concentration of plant extract which inhibited 50% enzyme activity was determined as ICA. galanga rhizome BL , V. vinifera , M. oleifera , as well as luteolin , were selected to determine the type of inhibition by Lineweaver–Burk plots. Each extract was added to the reaction mixture of 0.5–5.0 mg/mL of l-DOPA and 100 μL of 0.1 mM PBS (pH 6.8). The reaction was initiated by adding 50 μL of mushroom TYR at 37 °C. After incubation for 30 min, the absorbance was recorded by microplate reader spectrophotometer at 492 nm. The kinetic parameters of mK and maxV were assessed from the original simulation dataset, using a Lineweaver–Burk plot of data obtained from different l-DOPA concentrations. The inhibition constant of iK (inhibition constant for free enzyme) was determined from the concentrations of the extract and the slopes of the Lineweaver–Burk plot by using the equation: iiK (inhibition constant for ES complex) was determined from the concentrations of the extract and the y-intercepts of the Lineweaver–Burk plot by using the equation: The extracts of M. oleifera leaf (100 mg/mL) in 50% MeOH was ultra-sonicated for 20 min and centrifuged  for 10 min. The supernatant was filtrated through the 0.2 µm nylon membrane and the filtrate was injected into a liquid chromatograph-quadrupole time-of-flight mass spectrometer (LC-QTOF MS), . HPLC was equipped with a Zorbax Eclipse Plus column . Formic acid (0.1%) in water (solvent A) and acetonitrile (solvent B) were used as the mobile phase and the gradient elution was as follows: 98% A, 0–2 min; 90% A, 2–25 min; 85% A, 25–40 min; 80% A, 40–48 min; 75% A, 48-68 min; 70% A, 68–80 min; 50% A, 80–85 min; 0% A, 85–90 min.; 98% A, 90–100 min. Peaks were detected at the wavelengths of 254 nm and 280 nm. The MS spectra were acquired in both positive and negative ion modes with auto MS/MS. Full-scan mode from m/z 100 to 1700 was applied. The HPLC peaks were identified by using the spectrum database for organic compounds in the METLIN database.The extract of


Ventricular tachycardia arising from the papillary muscles and other endocavitary structures are preferably ablated under intracardiac echocardiographic (ICE) guidance whenever feasible. However, the availability, need of trained operators, and the expenses involved restrict the routine use of ICE in many cath labs. Point density exclusion (PDX) mapping is a simple technique that doesn't demand any additional expense or tool apart from the routine electroanatomical mapping and thus can be widely applied in mapping of arrhythmias arising from endocavitary structures. The following report describes such a case and explains the method of performing PDX mapping However, this is not freely available in most of the electrophysiological (EP) labs globally and significantly adds to the procedural cost thus hampering its utility. Point density exclusion (PDX) mapping, a recently described simple technique, helps to visualize the endocavitary structures like the papillary muscle (PM), needs no additional tools apart from high density geometry and activation mapping, and can be done using any electroanatomical mapping (EAM) system.2A 48‐year‐old gentleman with mitral valve prolapse and moderate mitral regurgitation had recurrent episodes of VT with one of the episodes needing electrical cardioversion. His cardiac magnetic resonance imaging showed no myocardial enhancement and normal biventricular function. He was planned for electrophysiological study and ablation for the VT. The electrocardiogram suggested a VT arising from the posteromedial PM and the procedure was done using the Ensite Precision system  for EAM. The basal intracardiac intervals were normal, and the tachycardia was consistently inducible by burst pacing from the right ventricular apex but not with the extra‐stimulus protocol. The left ventricular (LV) geometry was created using the Advisor HD grid mapping catheter  and the Ensite Precision system, using the retroaortic approach. The activation map revealed a focal mechanism with a centrifugal spread of activation from the site of the earliest activation in the mid‐level of the posteroseptal LV aspect. The local electrogram here had high frequency pre‐potentials that preceded the surface QRS by 30 ms, and there was an excellent (92%) match during pace‐mapping from this region Figure . The Adv3The PDX mapping has several advantages over ICE‐guided imaging in EP procedures. Firstly, the acquisition of the geometrical points and activation mapping from the endocavitary structure is simultaneous and from the same catheter unlike in ICE. Secondly, there is no need of any additional hardware/software for employing this technique apart from the conventional EAM, thus improving the feasibility and reducing the procedural cost so that it can potentially be applied in any EP lab with EAM facility. Thirdly, the procedural time is less than when using ICE for imaging/image integration. Fourthly, the technique is platform neutral and can be used with any of the EAM systems.The use of a contact force sensing catheter for ensuring a good contact with the endocavitary structure, though preferable, is not mandatory as illustrated in the current case. The near‐perpendicular bending of the electrode grid and the shaft of the Advisor HD grid catheter indicates good tissue contact, and only such sites were used to derive the PDX mapping in our case.However, there are also a few potential concerns while using PDX mapping as a replacement for ICE guided ablation. The catheter stability needs to be ensured as the PDX mapping will not be able to detect catheter movements and the resultant inaccuracy. The utility of ICE for visualizing lesion formation, and as a guide for visualizing structures like the interatrial septum, pulmonary veins, and detecting pericardial effusion obviously cannot be replaced by PDX mapping. Also, PDX mapping is a post‐acquisition processing technique that is applied after obtaining the entire geometrical and the activation points and is heavily dependent on a skillful technical assistant.4In the standard EAM, the inner geometrical points are automatically excluded as more outer points are added as the mapping catheter roves. Thus, endocavitary structures would be excluded from the final representation of the geometry. In PDX mapping, these internal points and the corresponding activation timing are manually included after the acquisition of a high‐density geometry and thus the visualization of the endocavitary structures is made possible. For ensuring accuracy, including only the inner points with a contact force of at least 8 gms is recommended though other surrogates may be used as in the current case.After acquisition of a high‐density EAM, the surface fill is reduced to 10% so that the “negative space”, which represents the inner points originally excluded from the map, is seen Figure . This isNone of the authors have any competing interests or conflicts of interests to declare.


This results in site-selective cross-coupling of dichloroarenes with fluoroarenes and fluoroheteroarenes, with selectivity controlled by catalyst structure. This study demonstrates that Pd-catalyzed C–H activation can be used productively in combination with a non-covalently-directed mode of catalysis, with important implications in both fields.Systems incorporating catalyst–substrate non-covalent interactions are emerging as a versatile approach to address site-selectivity challenges in remote functionalization reactions. Given the achievements that have been made in this regard using metals such as iridium, manganese and rhodium, it is surprising that non-covalent catalyst direction has not been utilized in reactions incorporating palladium-catalyzed C–H activation steps, despite palladium being arguably the most versatile metal for C–H activation. Herein, we demonstrate that electrostatically directed, site-selective C–Cl oxidative addition is compatible with a subsequent C–H activation step, proceeding  Electrostatically-directed oxidative addition is compatible with a subsequent C–H activation step, enabling site-selective coupling of remote chlorides with fluoroarenes and fluoroheteroarenes. However, there is relatively little precedent for non-covalent catalyst direction being used in tandem with palladium-catalyzed C–H activation. An interesting recent example from Crimmin and co-workers suggested dispersion interactions can influence regioselectivity in the palladium-catalyzed C–H alumination of toluene.16 If compatibility could be more broadly demonstrated it would pave the way for new and innovative design strategies incorporating non-covalent design elements for controlling site-selectivity in Pd-catalyzed C–H activation reactions.In recent years, non-covalent interactions have been increasingly explored as a powerful tool for modulating positional selectivity in transition-metal catalyzed reactions.4sSPhos is a commercially available, water-soluble phosphine that we repurposed such that the sulfonate group engages in a non-covalent interaction with the substrate.17 Experiments provided support for a scenario wherein the potassium cation of the deprotonated substrate interacts with the sulfonate group of the ligand, leading to oxidative addition being directed to the C–Cl bond at the substrate meta position (upper pathway). Following this, transmetalation or amine coordination/deprotonation was followed by reductive elimination to typically give a single regioisomer as product. At the outset of this work we questioned whether it might be possible to replace the transmetalation step with a CMD step to enable C–H bond activation to occur on the coupling partner. This would not only increase the efficiency of the C–C bond formation by avoiding prefunctionalization of one reactant, but more importantly would demonstrate proof-of-concept that CMD is compatible with non-covalent catalyst direction, in this case to control site-selectivity in oxidative addition to the C–Cl bond. Proof that this is viable may have broader implications for palladium-catalyzed C–H activation as it could act as a stepping-stone to non-covalent catalysis being applied for control of site-selectivity in the challenging C–H activation step of a catalytic cycle (vide supra). To test our proposal, we sought to examine fluoroarenes and fluoroheteroarenes as coupling partners (lower pathway). In addition to being well suited to CMD with Pd-catalysis,18 success with this substrate class would also provide complementarity to our previous work. We had attempted to use polyfluorophenyl boronic acids, trifluoroborate salts and MIDA-boronates as coupling partners under our previous conditions but observed no conversion in all cases.19 This was also the case when using the sSPhos G2 pre-catalyst  (iPrOAc but still observed no product formation (entry 2).18 Addition of substoichiometric amounts of pivalic acid was found to be beneficial, in accordance with previous observations.20 Good yield was obtained with 60 mol% PivOH and, importantly, excellent levels of site-selectivity for coupling of the chloride at the meta position was obtained . The yield could be improved by switching from the typical sodium salt of sSPhos to the more soluble tetrabutylammonium salt, sSPhos(NBu4) (entry 5). Finally, an evaluation of Pd sources showed that [(Cinnamyl)PdCl]2 gave improved yield (entry 6). When switching the ligand to standard SPhos the site-selectivity dropped to 1.2 : 1, in line with the hypothesis that the sulfonate group on the ligand is crucial for this outcome (entry 7). To probe the effect of having an arene sulfonate moiety present but detached from the ligand structure, we carried out the reaction using 20 mol% SPhos with 20 mol% potassium 2,4-dimethoxybenzenesulfonate as an additive (entry 8). This showed similarly low site-selectivity as observed when using SPhos. The equivalents of pentafluorobenzene could be reduced to three equivalents before significant effect on yield was observed (entries 9 and 10). However, for the purposes of scope exploration, nine equivalents were retained in the remainder of the studies, due to anticipated lower reactivity of some lesser fluorinated arenes.When approaching the optimization of the proposed reaction, our primary concern was whether the conditions required for effective C–H activation might interfere with the electrostatic interaction presumed to be responsible for the site-selectivity in the oxidative addition step. We commenced our investigations with triflate-protected dichlorobenzylamine ene (2a) . Under tTo provide support for the proposed electrostatic interaction, in which the potassium cation is thought to play a crucial role, we carried out experiments wherein stoichiometric amounts of various crown ethers are added under the optimized reaction conditions . These s1a with pentafluorobenzene, we next evaluated the scope of the perfluoroarene component (3b and 3c). 1,3,5-Trifluorobenzene represented the limit of reactivity (3d); whilst selectivity was high, conversion was moderate and such a drop-off of reactivity is precedented.18 Tetrafluorobenzenes bearing various substituents reacted smoothly and the scope encompassed methoxy (3e), trifluoromethyl (3f), alkynyl (3g), ester (3h) and acetamide (3i) groups. A further perfluoroarene could be incorporated without issue (3j). It is interesting to note that the site-selectivity, whilst typically >10 : 1, does vary to some degree with the fluoroarene partner, despite the selectivity-determining step occurring in the oxidative addition. We attribute this small variation to a solvent effect – as the reactions are relatively concentrated (0.5 M) and nine equivalents of arene are being used, this typically means a ∼1 : 1 ratio by volume of fluoroarene : solvent. It is therefore foreseeable that changes in the fluoroarene component could be manifested in minor fluctuations in oxidative addition regioselectivity.Having optimized conditions for the site-selective coupling of omponent . Undesir5a). Two different constitutional isomers of trifluorinated pyridine also reacted well, with C–H activation viable at either the C4- (5b) or C3- (5c) position relative to the pyridine nitrogen, dependant on the nature of the fluorination. Even 3,5-difluoropyridine reacted, albeit with low conversion (5d). In this case presumably the inductive withdrawal of the pyridine nitrogen compensates for the low degree of fluorination, still permitting some reactivity to be obtained, and representing the current limit of reactivity according to the present protocol.We next sought to determine whether the reaction would be compatible with fluorinated heteroarenes and so evaluated several variously fluorinated pyridines . Pleasin5e) and phenylpropylamine (5f) derivatives was tolerated, with reduced but still synthetically useful levels of site-selectivity. We found that benzylic substitution poses no problem for the catalyst control with both methyl (5g) and phenyl (5h) groups at that position giving excellent results.We next explored the scope of the aryl chloride component . Extendi11 This sulfamate salt 1b undergoes highly site-selective coupling with tetrafluoropyridine (13 : 1). The product was subjected to acid-promoted sulfamate cleavage followed by trifluoroacetylation, to enable isolation as amide 5i. As found previously with cross-coupling, a sulfonate as well accommodated as an electrostatic directing group in this C–H activation reaction, giving excellent selectivity for coupling at Clm . Herein, we also demonstrate that a mono-basic benzyl phosphonate also results in excellent site-selectivity, with the coupled product isolated after methylation as 5k. Perhaps the most commonly encountered Brønsted acidic functional group, the carboxylic acid, functions as an excellent electrostatic directing group, tolerating a variety of chain lengths. Chlorinated phenyl acetic acid (5m), hydrocinnamic acid (5n) and phenylbutyric acid (5o) all resulted in excellent site-selectivity. Furthermore, an alkene could be tolerated in the chain without side reactions and with no erosion of site-selectivity (5p). We next sought to evaluate the ability of our ligand to override a substrate's innate selectivity. As anticipated, coupling of 3,4-dichlorobenzoic acid with tetrafluoropyridine using SPhos as ligand gave very high selectivity for the para-chloride (6l), presumably due to substrate electronics rendering this the most electron deficient of the two C–Cl bonds. In contrast, simply switching the ligand to sSPhos rendered a switch in site-selectivity giving a remarkable 4 : 1 ratio of coupling at Clmvs. Clp, and 5l as major product.Whilst the triflyl-protected benzylamines can be manipulated post-reaction, obtaining the free amine is challenging. We demonstrate that the parent benzylamine can be readily converted to the corresponding potassium sulfamate salt by treatment with chlorosulfonic acid followed by potassium hydroxide.N-oxides and pyrazine N-oxides are viable C–H activation partners to be used in place of the fluoroarenes (21 These were demonstrated on two different substrate classes of aryl chloride, triflamide 1a and sulfonate 1c. Although the conversions were only moderate under the conditions optimized for the perfluoroarenes, importantly site-selectivity was excellent in both cases and these promising results demonstrate the broader potential of this strategy.Finally, we disclose that thiazole roarenes .21 TheseIn summary, we have demonstrated that electrostatically-directed palladium catalysis using sulfonylated phosphine ligands is compatible with CMD-type C–H activation in a single catalytic cycle. This results in a highly site-selective fluoroarylation of a number of substrates bearing two undifferentiated C–Cl bonds, with anionic handles for the electrostatic interaction encompassing triflamide, carboxylate, phosphonate, sulfonate and sulfamate. From a practical viewpoint, this work expands the scope of coupling partners able to engage in this site-selective coupling, since fluoroaryl boronic acids were incompatible with the cross-coupling protocol. More importantly though, this is a powerful demonstration that non-covalently-directed catalysis for control of site-selectivity can be used in conjunction with C–H activation. This paves the way for future, related developments in which site-selectivity may be controlled in the C–H activation step itself.There are no conflicts to declare.SC-011-D0SC00105H-s001


Subcortical screw placement is currently performed using frontal view fluoroscopy or intraoperative O-arm navigation system. The emergence of a novel technique for spinal navigation based on individual navigation templates created using 3D printing technology determines the need to study their safety and effectiveness in subcortical implantation.The aim of the study was to evaluate and compare the efficacy of subcortical implantation of pedicle screws in the lumbar spine using individual navigation templates versus intraoperative fluoroscopy.The study was based on the analysis of treatment results in 39 patients who underwent surgery with subcortical implantation of 130 screws using the MidLIF technique. In group 1, navigation templates were used, in group 2 — intraoperative fluoroscopic control. Comparative analysis of implantation correctness and time, the total operation time, and radiation load was performed.The mean distance between the screw and the cortical plate recorded in the groups ranged within 1.20–3.97 mm, without statistically significant difference (p>0.05). The mean time of pedicle screw implantation was 137.0  s in group 1 and 314.0  s in group 2. The total operation time was reduced from 173.0  min in group 2 to 119.0  min in group 1. The average of 1.0  X-ray image was performed to place one screw in group 1, while it was 12.0  in group 2. The differences between the groups in terms of implantation time and radiation load were statistically significant (p<0.05).Compared with intraoperative fluoroscopy, the use of individual navigation templates for subcortical implantation of pedicle screws provides their correct positioning with a significant reduction in both operation time and radiation load at similar safety. The main advantages of the subcortical trajectory for placement of pedicle screws, as compared to the classical one, are stronger fixation and a more medial location of the insertion point, which makes it possible to reduce the dissection area and make the access smaller and less traumatic versus the classical midline approach. It is believed that stronger fixation is achieved due to the proximity of the screw to the cortical bone throughout its entire length –7. SubcoOne of the directions of using 3D printing in spinal surgery is application of individual navigation templates (INT), which allow pedicle screw implantation with high accuracy and safety. Some authors –10 have The aim of the study was to evaluate the efficacy of subcortical implantation of pedicle screws in the lumbar spine using 3D printed individual navigation templates versus intraoperative fluoroscopy.The study involved analyzing the results of implantation of 130 pedicle screws placed in 39 patients aged 37 to 71 years during decompression and stabilization surgery in the lumbosacral spine using MidLIF technology. The study complies with the Declaration of Helsinki (2013) and was performed following approval by the Ethics Committee of Pavlov First Saint Petersburg State Medical University (Russia). Written informed consent was obtained from each patient.The screws were placed on the subcortical trajectory: from the inferomedial to the superolateral direction of the pedicle .Two groups of patients were formed by randomization method using random numbers: group 1 — implantation using INT (n=19), 66 screws; group 2 — implantation with intraoperative fluoroscopic control (n=20), 64 screws.A channel in the vertebral pedicle was formed using a drill 2 mm in diameter or a high-speed drill with a 1–2 mm bur. A polyaxial screw 4.35 mm in diameter and 35–40 mm in length was implanted. When using INT, the trajectory was planned according to the principle of the maximum proximity of the screw to the cortical bone of the pedicle, placing the screw tip under the endplate in its lateral part.In group 1, monolateral single-level INT were used in 5 cases, while bilateral single-level INT were used in 14 cases. A part of the vertebral arch was used as a support platform . The innTo assess the correctness of the screw position, there was developed a system of analysis based on the following criteria:the minimum distance to the medial border of the pedicle at the insertion point and the distance from the lateral edge of the vertebral body to the screw tip were calculated for each screw in the axial plane;the distance from the screw to the lower edge of the pedicle at the insertion point and the distance from the upper edge of the vertebral body to the screw tip were calculated in the sagittal plane .Evaluation criteria in the compared groups included implantation safety based on the disruption of cortical layer continuity, the frequency of complications, the number of revision interventions, the distance from the screw to the cortical layer, deviation degree when using INT, the number of X-rays taken and the time spent on both implantation and the entire operation.The time of implantation was recorded from the moment of completing the operative access to complete placement of all screws. In cases when after application of the template it was necessary to perform additional skeletonization of the surface due to unsatisfactory contact, this period was also counted as time spent on implantation.st and 3rd quartiles. Differences between the groups were assessed using the Mann–Whitney U-test and the Kruskal–Wallis test for two independent samples; the differences were considered statistically significant at p<0.05.The data obtained were processed using the Statistica 10.0 software. All samples used were represented by non-normal distributions, which was confirmed by graphical methods as well as using the Kolmogorov–Smirnov and Shapiro–Wilk tests. In this regard, the data were presented as a median, 1According to implantation safety analysis, in group 1 (INT), there were four cases of cortical bone perforation, one case of cranial pedicle perforation, two cases of perforation of the lateral wall of the vertebral body by the screw tip, and one case of endplate perforation by the screw tip. According to CT data, one patient underwent revision with reimplantation (replacement) of a screw . During caudal migration at the pedicle level, the patient had no radicular symptoms after surgery, therefore the screw was not replaced.In group 2 (fluoroscopy), 5 cases of perforation were revealed. Three perforations of the cortical layer were noticed at the level of the pedicle: cranial, caudal, and lateral. In two cases, the screw tip exceeded the bone at the level of the vertebral body: disruption of the endplate continuity and lateral exit of the screw. The differences between the groups in the number of perforations were not statistically significant. Repeated operations in order to replace the screws were not performed in group 2. Differences in the number of reoperations due to incorrect implantation were not statistically significant, p>0.05.No damage to neural structures was recorded in any of the groups.Assessment of the distance between the screw and the cortical bone at the control points is presented in the Statistically significant differences in the distance with a lower value were obtained when using INT in the axial plane in the area of the screw tip see the , point 2Deviation analysis at all control points showed the average deviation from the planned trajectory within 0.9–6.3 mm, which can be regarded as an acceptable indicator taking into account the morphometric parameters of lumbar spine pedicles. At the insertion point, the deviation was mostly registered in the sagittal plane, at the end point — in the axial plane; the difference between the groups was not statistically significant (p>0.05).The average time of pedicle screw placement was 137.0  s in group 1 and 314.0  s in group 2. The average operation time was 119.0  min in group 1 and 173.0  min in group 2 (p<0.05).When installing one screw, the average of 1.0  X-ray images were performed in group 1 and 12.0  images in group 2 (p<0.05).Subcortical implantation of pedicle screws in the lumbar spine is becoming more and more popular due to its minimal invasiveness and greater strength compared to traditional transpedicular fixation , 11, 12.At present, the most common technique is screw placement using intraoperative fluoroscopy in frontal and lateral projections or CT navigation . HoweverThe actively developing technique of INT manufactured using 3D printing technologies has become the subject of multiple studies devoted to its application in all parts of the spine –21. The In this regard, the vector of INT application for the lumbar spine was reoriented to the use of non-classical trajectories. For example, Shao et al.  have proAlong with these studies, there is a promising direction of research devoted to the use of a subcortical trajectory, in which the pressure of the paravertebral muscles is minimized and, as a consequence, the correctness of INT application increases.Kaito et al.  have conIn 2018, Kim et al.  presenteMarengo et al.  demonstrAccording to our study, the use of INT for screw implantation in the lumbar spine was not accompanied by an increase in implantation safety and did not affect the number of reoperations associated with incorrect screw placement as compared to the use of intraoperative fluoroscopy. Screw placement with the aid of navigation templates contributes to the achievement of a more correct trajectory and a closer position of the screw to the cortical bone, which, hypothetically, should have a favorable effect on the stability of the structure and reduce the risk of subsequent bone resorption around the screw. Our experience has shown that when using fluoroscopic control (even in the case of biplanar fluoroscopy) it is extremely difficult to form an implantation trajectory ensuring the screw tip to be located in close proximity to the cortical bone in two planes. Consequently, implantation correctness should be higher than with intraoperative CT navigation, which allows assessing the insertion trajectory in three planes before the start of insertion.When planning the trajectory, the surgeon has to choose between three different options:A);a more medial location of the insertion point with the most correct implantation axes in conjunction with the removing a part of the spinous process (B);a more medial location of the insertion point without resection of the spinous process, combined with a decrease in the axial angle of implantation, i.e. more “straight” insertion of the screw with a greater distance from the cortical bone at the end point (C).lateral displacement of the insertion point in observance of implantation correctness at other control points (Studying the possibilities of using INT, we tested all three implantation variants, of which the latter was preferred since the resection of the spinous process could serve as an unfavorable biomechanical factor due to the removal of the ligamentous apparatus, while a more straight trajectory is associated with a greater distance from the cortical bone in the end point.The use of INT for transpedicular fixation along the subcortical trajectory in the lumbar spine is also accompanied by a statistically significant decrease in the time of implantation and radiation exposure and promotes achievement of a more correct trajectory of the screw.Compared with intraoperative fluoroscopy, the use of individual navigation templates for subcortical implantation of pedicle screws provides their correct positioning with a significant reduction in both operation time and radiation load at similar safety.


It is an effective expansion of the research on the Board of Directors to do the research based on different board meeting forms and their effects sampling A-share companies listed in 2007–2017, the article empirically tests the impact of the times of board meetings, the proportion of on-site board meetings on listed companies’ over-investment. Consequently and significantly, the times of board meetings is positively correlated with over-investment, while the proportion of on-site board meetings is negatively correlated with over-investment. That is, the on-site meeting for the Board decision-making will better inhibit the enterprises’ over-investment behaviors. Further research shows that when there is a controlling shareholder in the company or in a dual position, the on-site board meeting no longer has a significant inhibitory effect on over-investment. By research on the independence of the Board of Directors, it is found that when selecting on-site board decision-making, the existence of independent directors has an over-investment suppression effect, and the higher the proportion of independent directors, the more obvious the inhibitory effect is. The samples are divided into state-owned enterprises and private enterprises, the study found that when choosing on-site board meetings, state-owned enterprises have a greater inhibitory effect on over-investment than private enterprises. The findings of this study will enrich the research of the board meeting and provide a new testing method for the relevant research of the Board of Directors. Sina.com., Dongfang Eastmoney.com., and China Financial Information. Many listed companies are increasingly inclined to choose the form of the communication meetings when a board meeting is held in 2007–2018  are widely used in the board meeting of the listed companies. When choosing the meeting form of the Board of Directors in the company law of China, there is no clear requirement for the meeting form. As a more convenient form of decision-making at the board meetings, the communication voting mode of the Board of Directors is more and more favored by the company. According to the statistics of the CCER database and the data of relevant websites such as The Board of Directors is the core of corporate governance which is a coordinator of conflicts of interest between the company owners and managers, and plays a decisive role in the company’s business strategy . It makeThe Board of Directors is the very important internal oversight and control mechanism in the corporate governance . Board mThe contributions of this research: (1) Most of the previous literatures have focused on the board structure and directors’ characteristics, such as the characteristics of independent directors , Female The Board of Directors exercises its functions and powers through the board meetings. The board meetings are a very specific and key issue in the modern corporate management and corporate governance. It is an important way to ensure directors’ participation in decision-making and understand the strategies of company . It’s alThe separation of ownership and control makes a large number of agency problems unavoidable in modern companies. Managers may not make decisions in the interests of shareholders. According to Principal-Agent Theory and Information Asymmetry Theory, managers are more likely to use information advantage. Jensen and Meckling  have fouBased on the above analysis, we formulate the following:Hypothesis 1a: There is a positive relationship between the number of board meetings and the over-investment.Hypothesis 1b: There is a positive relationship between the number of communication meetings and the over-investment.Hypothesis 1c: There is a positive relationship between the number of on-site meetings and the over-investment.The Board of Directors is the core of corporate governance. The purpose of corporate governance is not only to build the effective governance structure, but also to implement scientific decisions through the effective governance mechanisms. Board decision making is a special form of the group decision making . The perBased on the theory of information asymmetry, for a group decision-making organization, an important feature of making effective decisions is the ability to integrate the relevant information mastered by decision-making members and apply it to the final decision-making. That is, when some important information is mastered by different members, an effective group decision-making organization needs communication and information sharing among members, So that information can be effectively mastered by all members , 27. BoaModern organizational theory holds that the Board of Directors is an organization with strategic decision-making as its core. To participate in strategic decision-making, supervise managers, and control the CEO is the key function of the Board of Directors . The on-Based on the above analysis, we formulate the following:Hypothesis 2: There is a negatively relationship between the proportion of the on-site meetings and the over-investment.Our samples are initially composed of all listed companies on the Shanghai Stock Exchange (SHSE) and Shenzhen Stock Exchange (SZSE) from 2007 to 2018. The data of this study were derived from the CSMAR and CCER database. The relevant data of the board meetings come from CSMAR’s the governance structure research database of China listed company and CCER database; the relevant data for calculating over-investment are from the CSMAR database. In order to ensure the quality of the data, the effective samples are determined based on the general principles of domestic research. The following samples are excluded: (1)The financial and insurance listed companies; (2)The companies whose data cannot be obtained during the sample study; (3)The number of board meetings data below two times; (4)The companies’ asset-liability ratio more than 1; (5)The companies with special transaction status such as ST or * ST; (6)The samples missing financial data. Finally, we obtain valid 23159 samples. In order to overcome the possible effects of extreme values, we performed 1% and 99% Winsorize shrinking on the continuous variables. Data selection and sorting were performed using Excel 2010, and regression analysis was performed by Stata 15.1.We mainly explore the impact of the number of board meetings and the proportion of the number of on-site meetings on the over-investment of listed companies in China. According to the needs of this research, the dependent variable uses the related variables of over-investment, and the independent variables use the number of board meetings, the number of board communication meetings, the number of board on-site meetings and the proportion of the number of board on-site meetings.In order to ensure the robustness of the research results, we adopted two methods to measure the over-investment. The first method is to refer to the research of Richardson , we use Invest: The cash paid for the purchase and construction of fixed assets, intangible assets and other long-term assets minus the cash recovered from the disposal of fixed assets, intangible assets and other long-term assets is higher than the total assets at the beginning of the previous year.Size: The size of the company, measured by the logarithm of the company’s total assets.Lev: The asset-liability ratio.Cash: Cash holdings, calculated using the sum of total cash at the end of the year and cash equivalents divided by total assets.Tq: Tobin’s Q value.Listage: The age of company list. Calculated by subtracting the company’s listing year from the inspection year.Return: Annual return on stocks.Ind and Year: Industry and annual dummy variables.The second method is to take the opposite of the net cash flow of investment activities in the cash flow statement and divide it by the total assets of the current year to measure the company’s total investment. The measured value exceeds the annual industry average indicates over-investment.The number of board meetings in various forms. There are two kinds of meeting held by the Board of Directors: Regular meeting  and Ad hoc meetings . According to the provisions of the company law in China, each listed company must convene at least two board meetings during one year. There are no specific requirements for the form of board meetings, and there are two main methods used in practice, on-site meeting, and communications meeting. The number of board meetings intuitively reflects the strength of the board’s behavior . Board mProportion of on-site meetings: It is defined as the proportion of the number of board meetings held by the company in the form of on-site meetings in a year to the total number of board meetings in the year. Different meeting forms directly determine the quality of decision-making, which affects the survival and development of the company. We believe that different meeting forms will have different effects on the company’s investment behavior.In order to ensure the accuracy and validity of the hypothesis test results, referring to previous research, the following control variables were selected: Year, Indus, Age, Size, Leve, Growth, Eps, difout, state, Frshr, Bsize, Outra, Comp, dual, Commeete, Dshr .In order to test the impact of the number of board meetings, the number of on-site meetings, the number of communication meetings on the over-investment (Hypothesis 1). The following models are used:i = a0+a1Mti+a2Difouti+a3Epsi+a4Bsizei+a5Outrai+a6Compi+a7Duali+a8Commeetei+a9Dshri+a10Frshri+a11Sizei+a12Levei+a13Growthi+a14Agei+a15Statei +∑Ind+∑Year+εOverinvi = a0+a1Temti+a2Difouti+a3Epsi+a4Bsizei+a5Outrai+a6Compi+a7Duali+a8Commeetei+a9Dshri+a10Frshri+a11Sizei+a12Levei+a13Growthi+a14Agei+a15Statei +∑Ind+∑Year+εOverinvi = a0+a1Osmti+a2Difouti+a3Epsi+a4Bsizei+a5Outrai+a6Compi+a7Duali+a8Commeetei+a9Dshri+a10Frshri+a11Sizei+a12Levei+a13Growthi+a14Agei+a15Statei +∑Ind+∑Year+εOverinvIn order to test the impact of the proportion of the number of on-site meetings on the over-investment (Hypothesis 2). The following model is used:i = a0+a1Osmtrai+a2Difouti+a3Epsi+a4Bsizei+a5Outrai+a6Compi+a7Duali+a8Commeetei+a9Dshri+a10Frshri+a11Sizei+a12Levei+a13Growthi+a14Agei+a15Statei +∑Ind+∑Year+εOverinvIn order to test the relationship between the number of board meetings and over-investment, we conduct regression tests on the number of board meetings, the number of communication meetings, the number of on-site meetings, the proportion of on-site meetings and over-investment. Test their relationship based on different meeting forms.Hypothesis 1a, 1b, 1c, hypothesis 2 test.According to whether the company has a controlling shareholder, the data is divided into two groups for statistical analysis.The OLS regression results as follows:Regarding the regression of the number of on-site meetings of the Board of Directors and the over-investment, the results show that the number of on-site meetings and over-investment are positively correlated regardless of the company has a controlling shareholder or has not a controlling shareholder. It is consistent with the results of assumption 1.Regarding the regression of the proportion of on-site meetings and the over-investment, the results show that the regression results of the proportion of on-site meetings and the over-investment are not significant overall when the company has a controlling shareholder, the increase in the proportion of on-site meetings will weaken the over-investment. It is mainly because the controlling shareholder can easily use his position to control the Board of Directors and the Board of Supervisors, thereby achieving superior control in corporate governance. It is difficult to form an effective internal control and check-and-balance mechanism in the case of highly concentrated equity. When the company does not have a controlling shareholder, the OLS regression results are overall significant. The proportion of on-site meetings and the over-investment are significantly negatively correlated at a 5% level. The increase in the proportion of on-site meetings can inhibit the over-investment. This is mainly because when the company does not have a controlling shareholder, the Board of Directors will not appear to be a dominant phenomenon, and the directors can communicate more fully at the board meeting. Thereby the on-site meetings can inhibit the company’s over-investment.According to the situation that one manager holds both the position of general manager and the position of president of Board of Directors , the data is divided into two groups for statistical analysis.The OLS regression results are as follows:Regarding the regression of the number of on-site meetings and the over-investment, the results show that the number of on-site meetings and the over-investment are positively correlated regardless of duality or no. It is consistent with the results of Hypothesis 1. However, when the two positions are separated is weaker than when the two positions are held concurrently. The results indicate that when the company in the state which the two positions are separated, the effect of on-site meetings is stronger than when the two positions are held concurrently.Regarding the regression of the proportion on-site meetings and over-investment, the results show that when the company is duality, the regression results are not significant overall. It shows that when the proportion of on-site meetings increases when the company is not duality, the inhibitory effect on excessive investment will be weakened. This may be due to the increase in the degree of entrusted agency when the company is duality, the general manager supervising himself, and excessive concentration of power. When holding a board meeting, the general manager is likely to use his power to influence the voting results of the board meeting, and the profit-seeking nature of managers will make them tend to overinvest. When the company is not duality, the proportion on-site meetings of the Board of Directors and over-investment OLS regression results are overall significant and significantly negatively correlated at the 5% level. When the company implements the separation of the two positions, the proportion of on-site meetings have an inhibitory effect on over-investment. This may be due to the fact that when the company is not duality, the influence of the manager on the Board of Directors is relatively small, and the independence of the Board of Directors increases. The on-site meeting can communicate more fully, and the directors can better supervise the managers to inhibit the managers from investing in projects with negative net cash flow to maximize their own interests.According to different proportion of independent directors where the ratio of independent directors of the board is below 0.5 and above 0.5. The data is divided into two groups for statistical analysis.The OLS regression results are as follows:Regarding the regression of the number of on-site meetings and the over-investment, the results show that when the proportion of independent directors is below 0.5, the number of on-site meetings and the over-investment are significantly positively correlated at the 1% level. It is negatively correlated, but not significant when the proportion of independent directors is above 0.5. This shows that independent directors can effectively curb the over-investment behavior of the company.Regarding the regression of the proportion board on-site meetings and the over-investment, the results show that the number of on-site meetings and the over-investment are significantly negatively correlated at the 5% level when the proportion of independent directors is below 0.5 or When the proportion of independent directors is 0.5 or above. This shows that due to the externality of independent directors, the managers can be more effectively supervised. When on-site meetings are used, independent directors have an inhibitory effect on excessive investment, and the higher the proportion of independent directors, the more significant the inhibitory effect.According to The different nature of company’s equity, the data is divided into two groups: the state-owned enterprises and the private enterprises.The OLS regression results are as follows:Regarding the regression of the number of on-site meetings and the over-investment, the results show that the number of on-site meetings and the over-investment are significantly positively correlated at the 1% level when the company is privately held. The number of on-site meetings is positively correlated with and the over-investment when the company is state-owned, but not significant. Compared with private companies, state-owned companies are more cautious in investment behavior, even if there are more board meetings, there will be no more over-investment.Regarding the regression of the proportion on-site meetings and the over-investment, the results show that the proportion on-site meetings and the over-investment are not significantly correlated when the company is private. The proportion on-site meetings and the over-investment are significantly negatively correlated at 10% when the company is state-owned. It shows that when the company is a state-owned holding company, the effect of on-site meetings is better than when the company is a private holding company. On site meeting is more effective in state-owned company’ decision-making. This is due to the more effective supervision mechanism of state-owned companies. At the same time, it also reflects that there are more factors influencing the investment behavior of private companies.In order to ensure the robustness of the empirical results, we conducted a robustness test in two ways.Change the variable calculation method. This paper uses Richardson model to calculate the measurement method used by enterprises when they overinvest. Tobin Q is replaced by the growth rate of sales revenue, and then overinv2 is obtained to replace overinv2 for robustness test. The overall results showed no difference, which verified the hypothesis of the research again.The samples was changed for robustness test, and the samples from 2008 to 2017 were selected for empirical test. This paper studies the relationship between the number of board meetings, the proportion of on-site meetings and the over-investment. Based on the data of listed companies from 2007 to 2017, take the number of board meetings, on-site meetings, communication meetings, and the proportion on-site meetings of the Board of Directors as independent variable, and the company’s over-investment is the dependent variable. The analysis found that the decision of the on-site board meeting of the Board of Directors will inhibit the over-investment to a certain extent. Further research found that when the company has a controlling shareholder and the duality, the on-site meetings’ inhibitory effect on over-investment will be weakened. The presence of independent directors has a certain effect on suppressing over-investment. The higher the percentage of independent directors, the more obvious inhibitory effect is. The comparison between the state-owned companies and the private companies found that the on-site meeting’s inhibitory effect on over-investment is more obvious in state-owned enterprises.Board meetings are an important way for the Board of Directors to make decisions and exercise supervisory power over company matters. The quality of board meetings directly affects the quality of decision-making and the effectiveness of supervision. The conclusion of this article shows that the on-site meeting of the Board of Directors is conducive to making more favorable decisions and better supervision for the company. Based on the conclusions of this article, the following suggestions are made:First, regarding the choice of the form of board meetings, for listed companies, it is necessary to recognize the importance of board meetings, establish and improve a more complete board meeting operating system, make different regulations for different matters. For decisions on major matters of the company, especially proposals requiring full communication between directors, we recommend that a board meeting be held in the form of an on-site meeting; For some procedural votes, in order to save costs, the form of communication meeting can be used appropriately, but the information required for the meeting must be sent to the directors, especially the external directors, in advance, so that they can fully understand the matters to be voted and allow the board meeting can play its due role. For the relevant regulatory authorities, they can set up a corresponding system to set a minimum requirement for the proportion of board meetings of listed companies. Regular board meetings should be stipulated as on-site meetings. Temporary meetings can be freely chosen according to the matters discussed form.Secondly, in the internal governance structure of the company, we should strengthen the focus on independent directors, increase the status of independent directors, appropriately increase the proportion of independent directors in the Board of Directors, and provide independent directors or external directors with company-related business information in a timely manner. Reduce the occurrence of information asymmetry, so that independent directors can really play a role in the decision-making of board meetings; combined with the current split share structure reform, change the ownership structure and the dominant ownership structure, improve related laws and regulations and the articles of association of listed companies, strengthen the supervision of large shareholders, reduce the control of large shareholders on the Board of Directors, prevent them from infringing the interests of small and medium shareholders for their own interests; Increase the independence of the Board of Directors, strengthen the supervision of the Board of Directors on management, In particular, the general manager should be strictly supervised by the Board of Directors, rather than self-monitoring; Private enterprises may have fewer supervision procedures when making investment decisions than state-owned enterprises, and it may be easier to make decisions to increase investment. Therefore, the Board of Directors of private enterprises should be more cautious when making decisions and should pay more attention to on-site meeting.Thirdly, during the COVID-19 period, most of the world is using online meetings. This, of course, include board meetings. Online meetings seem to be becoming a trend. In such a situation, on the one hand, the enterprise should also do a good job in the communication meeting, increase the information communication before, during and after the board meeting, and realize the optimal realization of the meeting information communication. On the other hand, the enterprise should also provide more information communication channels and face-to-face on-site communication opportunities for the directors of the board, and should try their best to use on-site meeting for major decision-making matters.In short, the choice of the format of the board meeting should be determined according to the importance of the company’ss decision-making matters, and should not be affected by the structure of the Board of Directors, the control of the major shareholders, the management, the nature of equity, etc., so that the board meeting becomes a booster for the company’s development.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.