Lower-limb exoskeletons often use torque control to manipulate energy flow and ensure human safety. The accuracy of the applied torque greatly affects how well the motion is assisted and therefore improving it is always of interest. Feed-forward iterative learning, which is similar to predictive stride-wise integral control, has proven an effective compensation to feedback control for torque tracking in exoskeletons with complicated dynamics during human walking. Although the intention of iterative learning was initially to benefit average tracking performance over multiple strides, we found that, after proper gain tuning, it can also help improve real-time torque tracking. We used theoretical analysis to predict an optimal iterative-learning gain as the inverse of the passive actuator stiffness. Walking experiments resulted in an optimum gain equal to 0.99 ± 0.38 times the predicted value, confirming our hypothesis. The results of this study provide guidance for the design of torque controllers in robotic legged locomotion systems and will help improve the performance of robots that assist gait. Being able to reduce interface impedance, increase the reactiveness of robotic systems and thus improve human safety and comfort  was used to sample sensory data at 5 kHz, filter them at 200 Hz, and then compute control outputs in terms of desired motor velocity. The actuation unit included a low-inertia 1.6 kW AC servo motor, a 5:1 gear, and a motor driver configured to velocity control mode . A digital encoder  was used to measure motor position.−1 effective stiffness (in terms of ankle position) was attached at the end of the rope to realize series elastic actuation.A uni-directional Bowden cable was used to transmit forces from the motor side to the exoskeleton side. The cable was made of a coiled-steel conduit  and a 0.003 m diameter synthetic rope. A spring  of a 190 N·m·radThe exoskeleton frame applied an plantarflflexion torque to human ankle. Torque was measured using strain gauges , and amplified using a 1 kHz signal conditioner . Ankle angle was measured using a digital encoder .We made the following assumptions in generating a simplified model of the system for the purpose of theoretical analysis:F.Both static and dynamic frictions in Bowden cable transmission were zero. Thus, cable tension at the motor output pulley side was always the same as the exoskeletons side. This tension was denoted as The Bowden cable transmission and the series spring together behaved as a linear spring, i.e.,Kc was the combined effective stiffness of the Bowden cable and series spring; θp and θe were the pulley and exoskeleton joint positions relative to where the Bowden cable first started to go slack; rp and ra were the pulley radius and the lever arm at the ankle joint.where ra was fixed. Therefore, torque applied to human body by the exoskeleton was alwaysExoskeleton joint rotated in a small range. Thus, the cable tension lever arm at exoskeleton joint side was almost constant, i.e., Denoting the aspect ratio of transmission asand combining it with Equations (1) and (2), the torque applied by the exoskeleton to the human ankle wasKt was the transmission stiffness which related applied torque to exoskeleton joint. It was defined asWhere Prior work in the field found that in real-time torque tracking of lower-lime exoskeletons during walking, the combination of model-free, integral-action-free feedback control and iterative learning were most effective controller with complicated and time-varying dynamics of human-robot interactive systems  and the desired torque was set as Equation (7).n − 1 and time index i the learning controller has reached stabilization with perfect torque tracking, i.e.,In this study, we were interested in the real-time torque tracking performance under gait variations after the stabilization of iterative learning, i.e., disturbance rejection performance of the controller. Therefore, we assumed that at stride The dynamic changes of the desired and generated torque in the next strides due to human gait variations are then investigated hereinafter.n − 1 to n at index i ofAssuming an ankle kinematics change from stride then the desired torque changes byBased on the iterative learning rule in Equation (9) and the assumption of perfect motor position tracking in Equation (8), current desired and actual motor position aren and index i isTherefore, combining Equation (12) with Equation (4), the measured torque at stride and the desired torque isn and index i, the torque error isCombining Equations (20) and (21), for stride n − 1, the torque error of stride n is minimized when the desired and passive stiffness match. This agrees with a previous study on the optimization of passive stiffness for torque tracking  isThis means with perfect torque tracking in stride n to n + 1 at time index i, i.e., θe = θe, the desired torque values have τdes = τdes. The actual torque at current stride and index isIn case there is no change in ankle kinematics from stride n + 1 and index i, isTherefore, the latest torque error, i.e., that of stride Kt·Kl·R = 1, i.e., if and balance is restored.n to n + 1 at index i, i.e.,On the other hand, if there exists ankle kinematics change from stride we havei, n + 1) isand the actual torque at  isCombining Equations (20) and (21), the torque error at ,Then, the error at  then hasThe error at  was involved. In all experiment sessions, the subject walked with a self-paced frequency on a treadmill running at 1.25 m/s while wearing the ankle exoskeleton on the right foot. All experimental protocols were approved by Carnegie Mellon University IRB.The experiments of this study was conducted not to quantify human reactions but the performance of various torque control conditions. Therefore, only one healthy subject  was set as 50 ms.In presentation of all experimental methods and results, this paper uses meter, Newton-meter and degree as the units for distance, torque and angle. The testbed system had an aspect ratio e,0 = −2(deg), in which θe = 0 was defined by the neutral standing position. The tested desired stiffness Kdes spans (Three different desired quasi-stiffnesses in the form of Equation (7) were implemented to test the hypothesis. In all cases, θes spans  with a mWith every desired stiffness value defined by a torque-angle curve, we tested it in combination with three different passive transmission stiffness values by changing the series spring in the ankle exoskeleton . Two werKt, with different spring configurations were experimentally evaluated using passive walking where motor position was locked. The process went like this: under each set-up, motor position was fixed where forces began to be generated when the participant stood in a neutral position. Then the participant walked while wearing the exoskeleton on the treadmill for more than one hundred steady strides. Such sessions were done multiple times for each stiffness configurations. For one hundred steady strides of each walking session, the instantaneous passive stiffness value was calculated and plotted against the torque values. The values of effective passive stiffness, The list of springs used and their corresponding properties and their actual values of passive stiffness as calculated with methods discussed above are listed in For each desired stiffness and passive stiffness combination, ten iterative learning gains that span a range with a 20-times difference between the maximum and minimum around the predicted optimal value per Equation (24) . TherefoThe theory-predicted optimal iterative learning gain values according to Hypothesis 1 were also listed in the table as a comparison.Four different indicators of torque tracking performance were calculated for each combination. The first as we call an “absolute error” was defined as the mean of stride-wise root-mean-squared torque errors over the one hundred stable strides. The second one was a “relative error” defined by the absolute error divided by the mean of peak desired torque of the one hundred stable strides. Besides, the normalized instantaneous torque error value of a specific time index within the stride was defined by dividing the original torque error by the standard deviation of a centered 1 × 100 ankle position array of the same time index over all one hundred strides. A centered ankle position array of one particular time index of the stride was defined as the difference between the original array and filtered array as demonstrated in For each unique combination of desired and passive stiffness values, we investigated the relationship between the defined error indices and the corresponding learning gains to test the hypothesis.The mean of root-mean-squared torque errors of various learning gains were presented against the ratio of actual learning gains to the theory predicted one in logarithmic scale, i.e., lKl,opt) . For eacKl value at which the value of the polynomial was minimized as the experimental optimum of iterative learning gain and label it as Kl,opt,exp. The We defined the Kl = Kl,opt, which agreed with Hypothesis 1. Furthermore, among all four torque error indices, the absolute error normalized to the standard deviation of centered ankle position trajectories showed experimental optimum closest to theory prediction. The average of experimental optimal gains under nine different combinations of passive and desired stiffness values was 0.9929 ± 0.3846 times the predicted optimum .Due to the presence of the non-linear, complex, and time-varying system dynamics and the employment of a highly simplified model, many features were not reflected in the theoretical hypothesis, which led to imperfection in the alignment between theory and experiment results. One issue that contributed was the non-linearity of the passive stiffness coming from the slow stretching of the synthetic rope as demonstrated by Regardless of the imperfection of system modeling, the torque tracking errors did arrive at a minimum at the neighborhood of the hypothesized optimal iterative learning gain. The shallow slope of changes around the optimum value also suggested a rather relaxed learning gain tuning process. When the iterative learning gain spans a range of [50% 200%] relative to the theoretical optimum, the average increase in torque error is expected to be <5% of that at the optimal gain. Considering a relative torque error of only 2–8% of desired torque at the optimum, a 5% increase on top is rather insignificant and will not harm exoskeleton performance or accuracy of experiments.This study is the last part of a series of three studies focusing on improved torque tracking for lower-limb exoskeletons during walking. The first one compared a group of prominent controllers used in this type of devices, and identified model-free, integral-control-free feedback combined with feedforward iterative learning as the most effective controller structure, with the potential to reduce torque tracking error to around 1% of peak desired torque (Zhang et al., A lot of complications, such as frictions and other non-linearities, uncertainties, time-varying dynamics were not discussed or featured in the analysis of this study. The reason was that previous studies (Zhang et al., Besides, stability of iterative learning was also solved in previous studies (Zhang et al., This study is the last part of a trilogy of studies on accurate torque tracking for lower-limb exoskeletons during human walking, followed by the identification of an effective control structure (Zhang et al., The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Carnegie Mellon University IRB. The patients/participants provided their written informed consent to participate in this study.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.


Furthermore, it has overlap with four potent CD4+ T-cell epitopes. Structure-based B-cell epitope prediction has identified ORF9b61-68 to be immunodominant, which partially overlaps with one of the linear B-cell epitopes (ORF9b65-69). ORF3c CD4+ T-cell epitopes  and linear B-cell epitope (ORF3c14-22) have also been identified as the candidate epitopes. Similarly, M.ext and 7a.iORF1 (overlap with M and ORF7a) proteins have promising immunogenic regions. By considering the level of antigen expression, four ORF9b and five M.ext epitopes are finally shortlisted as potent epitopes. Mutation analysis has further revealed that the shortlisted potent uORF epitopes are resistant to recurrent mutations. Additionally, four N-protein  epitopes are found to be potent. Thus, SARS-CoV-2 uORF B-cell and T-cell epitopes identified here along with canonical ORF epitopes may aid in the design of a promising epitope-based polyvalent vaccine (when connected through appropriate linkers) against SARS-CoV-2. Such a vaccine can act as a bulwark against SARS-CoV-2, especially in the scenario of emergence of variants with recurring mutations in the spike protein.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kills thousands of people worldwide every day, thus necessitating rapid development of countermeasures. Immunoinformatics analyses carried out here in search of immunodominant regions in recently identified SARS-CoV-2 unannotated open reading frames (uORFs) have identified eight linear B-cell, one conformational B-cell, 10 CD4+ T-cell, and 12 CD8+ T-cell promising epitopes. Among them, ORF9b B-cell and T-cell epitopes are the most promising followed by M.ext and ORF3c epitopes. ORF9b Although spike glycoprotein-based vaccines have been developed in a fast-track mode to combat SARS-CoV-2  and are capable of inducing a broad immune response . Experimviz., less than 15 amino acids length)  . Thus, 1 length) , and it The published SARS-CoV-2 uORF sequences  were useCD8+ T-cell (MHC I/HLA I) epitope prediction was done against the 78 HLA class I alleles  and supen.html) ] were alFor linear (continuous) B-cell epitope prediction, protein sequences were examined for putative B-cell epitopes using the Bepipred 2.0 server  by applyTo further evaluate the epitopes based on their sequence conservation, 775,392 SARS-CoV-2 whole genome sequences  were subjected to nucleotide and amino acid mutation analyses. For this, the gene sequences corresponding to the uORFs were translated using the reference sequences  with thewww.iedb.org)  , is empl–www.iedb.org).By applying the above criteria , have the B-cell linear epitopes .EDKA (residue numbers 61–68) conformational epitope has partial overlap with one of the linear B-cell epitopes .Since the structure of ORF9b protein (PDB ID: 6Z4U) alone is known among the uORF proteins, the conformational epitopes present in the ORF9b protein are alone investigated. A recent SARS-CoV-2 immunoglobulin G (IgG) epitope profiling study has shown that along with the spike protein and N protein, ORF9b protein also elicits IgG-specific SARS-CoV-2 response . The pre50 values. Notably, one of the promising ORF9b epitopes, “KVYPIILRL,” has an overlap with the SARS-CoV tor2 strain epitopes (Source: IEDB) (www.iedb.org). Most interestingly, another ORF9b promising epitope  predicted here has also been identified in a recent study through IgG epitope profiling  epitopes (which have percentile rank ≤1) are predicted from 21 uORF proteins  (rofiling . ). Refer Fourteen uORF proteins , 3a.iORF2, M.ext, 6.iORF, 7a.iORF1, 7b.iORF1, N.iORF1 (ORF9b), N.iORF2, 10.iORF, and 7a.iORF3) are predicted to have 140 CD4+ T-cell epitopes with a percentile rank of ≤1 . It also has an overlap with three other ORF9b potent CD4+ T-cell epitopes, namely, “PIILRLGSPLSLNMA,” “YPIILRLGSPLSLNM,” and “VYPIILRLGSPLSLN” .Furthermore, 17 potent CD4+ T-cell epitopes are predicted to have individual population coverage between 10.54% and 37.64%. The ORF9b epitope “To further investigate the binding specificity or flexibility of the CD8+ T-cell epitopes to HLA class I alleles, the allele-binding coverage of the predicted promiscuous epitopes is analyzed by considering seven HLA class I supergroups  and 10 H–To investigate the conservation of the predicted SARS-CoV-2 uORF conformational B-cell, linear B-cell, CD8+ T-cell (HLA I), and CD4+ T-cell (HLA II) epitopes, mutational analysis is carried out for the proteins expressed by uORFs. The results reveal that uORF proteins are having 5, 4, and 2,642 high, moderate, and low recurrent mutations, respectively (Even 18 months after the coronavirus disease 2019 (COVID-19) outbreak, treating and preventing SARS-CoV-2 infection are still a big challenge. Especially, the emergence of variants with recurring mutations in the spike protein poses major challenges in treating the SARS-CoV-2 infection. An immunoinformatics approach is employed here to facilitate the multi-epitope vaccine design, which may aid in overcoming the challenges in the traditional vaccine design. Although several studies have been carried out in this regard –35, theywww.iedb.org)  . Based o61-68)] and four linear B-cell epitopes  can be considered for the vaccine design. However, “EDKAF” (ORF9b65-69) is excluded for the vaccine design as it overlaps (underlined) with the conformational B-cell epitope “LNSLEDKA” (ORF9b61-68). Thus, a total of four B-cell epitopes are considered for the vaccine design.From the pool of 31 potent epitopes, 13 epitopes are proposed to be highly suitable for the multi-epitope SARS-CoV-2 vaccine design as discussed below. Since ORF9b followed by M.ext have better expression level among the uORF proteins, epitopes from these proteins can be considered for the vaccine design. The only conformational uORF B-cell epitope . Furthermore, “KVYPIILRLGSPLSL” (ORF9b40-54) overlaps (underlined) with the ORF9b CD8+ T-cell epitope “KVYPIILRL” (ORF9b40-48) that covers six out of seven HLA class I supergroups  is the utmost promising CD4+ T-cell epitope and is considered for the vaccine design. Similarly, “RTLSYYKLGASQRVA” (M.ext187-201) is considered to be a potential epitope. Although some of the uORF proteins CD8+ T-cell epitopes have good world population coverage, they are not considered for the vaccine design due to the low expression level of the corresponding protein  and “IIFWFSLEL”  epitopes.Similarly, the following CD4+ T-cell epitopes can be promising vaccine candidates: “KVYPIILRLGSPLSL” (ORF9b protein . Such ex40-54) is reported in SARS-CoV Tor 2 strain [Source: IEDB] (www.iedb.org). The epitopes “SELVIGAVI” (M.ext149-157) (19-28) (213-230) (187-201) (87-95)  , GTITVEE 19-28) , “RIGNYK213-230) , “RTLSYY-28 , “RI (87-95)  are repoMethods). In the case of promiscuous epitopes shortlisted from canonical proteins, only 17% of them bind to HLA class I alleles that fall into the same HLA supergroup (50)  and “QIGYYRRATRRIRGG” (N protein83-97) are predicted to be potent CD8+ and CD4+ T-cell epitopes, respectively. Notably, the predicted “LSPRWYFYY” (N protein104-112) epitope [covers three HLA class I supergroups ] (83-97) has partial overlap with the previously reported epitope (257-265) and “RGPEQTQGNF” (N protein277-286) are considered to be potent epitopes for the vaccine design. Among them, “KPRQKRTAT” (N protein257-265) has partial overlap with the conformational B-cell epitope of the N protein and has been reported in previous immunoinformatics studies  are considered, as it tops among the canonical and uORF proteins in terms of the relative translational level . “LSPRWYand S6)] , 32, 33. epitope , 32, 34.138-146, M.ext89-97), “GTITVEELK” , “LSPRWYFYY” (N104-112), “LPDEFVVVT” (ORF9b87-95), “RTLSYYKLGASQRVA” , “KVYPIILRLGSPLSL (ORF9b40-54),” “QIGYYRRATRRIRGG” (N83-97), “LNSLEDKA” (ORF9b61-68), “AVGRDQNNVGP” (ORF9b29-39), “ASQRVAG”  “RIGNYKLNTDHSSSSDNI” , “KPRQKRTAT” (N257-265), and “RGPEQTQGNF” (N277-286) epitopes predicted in this study can be used for multi-epitope-based SARS-CoV-2 vaccine design, wherein each epitope is connected through a suitable linker region (Aforementioned potent epitopes identified from N protein (four epitopes), M.ext/M protein (five epitopes), and ORF9b protein (four epitopes) may be more appropriate for the vaccine design, since these proteins occupy the top 3 positions among the SARS-CoV-2 proteins in terms of the relative translational level  . ConsideThus, the potent epitopes from the proteins expressed by the canonical ORFs and uORFs of SARS-CoV-2 can be used in the design of multi-epitope vaccine against SARS-CoV-2.40-48] CD8+ T-cell (MHC I/HLA class I) epitope is the most promising epitope not only based on the antigenicity, immunogenicity, and IC50 but also based on its highest HLA class I allele coverage, viz., it covers six out of seven HLA class I supergroups. Furthermore, this region has an overlap with the four potent CD4+ T-cell (MHC II/HLA class II) epitopes. Among the shortlisted uORF epitopes, eight linear B-cell, one conformational B-cell, 10 CD4+ T-cell, and 12 CD8+ T-cell epitopes are finally suggested as potent epitopes based on the mutational analysis. Similar immunoinformatics analysis is also extended for 26 canonical ORF proteins. Considering the high expression level of N protein , M/M.ext protein , and ORF9b protein (encoded by uORF), 13 potent epitopes from these proteins are finally considered for the proposed multivalent vaccine design.To facilitate the design of a multi-epitope vaccine against SARS-CoV-2, an immunoinformatics analysis has been carried out here to identify the potential linear B-cell, conformational B-cell, CD4+ T-cell, and CD8+ T-cell epitopes present in the 23 uORF proteins. Using stringent criteria, nine linear B-cell, one conformational B-cell, 17 CD4+ T-cell, and 13 CD8+ T-cell uORF epitopes are shortlisted. Notably, the current study has identified ORF9b epitopes as promising candidates for the multi-epitope vaccine design. “KVYPIILRL” [ORF9bThe original contributions presented in the study are included in the article/PU carried out the immunoinformatics analysis. LP wrote codes for mutation data analysis and plotting. CS wrote scripts to generate the plots and did plotting. LP, CS, and PU analyzed the data. PU and TR wrote the manuscript. PU independently devised the immunoinformatics analysis protocol. TR designed and supervised the project. All authors contributed to the article and approved the submitted version.LP and CS thank MHRD for fellowship. PU thanks CSIR for fellowship.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 claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Specifications TableIn the finite elements method (FEM), the analytical behavior of a solid can be approximated by a discrete behavior. The discrete solution is usually obtained by nodal displacements, while the continuous problem solution can be found by interpolating the nodal displacements using interpolating (shape) functions Therefore, in geometric nonlinear analysis, to converge to the analytical behavior, a refined discretization or the use of a high-order finite element with an increase in the order of the polynomial of the basis functions is necessary. However, when the shape functions are obtained from the differential equation solution of the problem, the frame member discretization could be reduced The differential equation that describes a linear problem is obtained from the equilibrium of an infinitesimal element in an undeformed configuration, leading to the Hermitian interpolation functions. However, for geometric nonlinear or second-order analysis, the equilibrium of an infinitesimal element in the deformed configuration should be considered, and the differential equation considers the nonlinearity caused by the axial load in the deformed infinitesimal element Furthermore, to implement the unified method proposed by Rodrigues et al. In addition, to reach the expressions presented in this paper, an updated Lagrangian kinematic description considering spatial elements is used. Besides, the stiffness matrix is adjusted to consider finite rotation effects. Furthermore, the formulation employs the Green strain tensor considering all higher-order terms, which leads to accurate results and reduces the buckling load of spatial frame structures, showing that not considering these effects can overestimate the resistance of a structure.The presented method combines three types of nonlinearities, namely, higher-order terms in the Green strain tensor, higher-order terms in the finite rotations, and the nonlinearity induced by the equilibrium of the infinitesimal element in the deformed configuration considering the axial effect. One main difference of the present formulation is that the interpolation functions for displacements and rotations in an element are directly obtained from the solution of differential equations that consider the equilibrium in the deformed configuration. As a consequence, the resulting interpolation functions depend on the axial force in the element. In addition, the present formulation considers the shear deformation influence, and expressions are developed in 3D. These considerations extend the classical tangent stiffness matrix for geometric nonlinear analysis , as explThe manuscript provides explicit expressions for the proposed interpolation functions and tangent stiffness matrix. Since this formulation involves trigonometric and hyperbolic functions, for the sake of computational efficiency the tangent stiffness matrix can be conveniently rewritten in polynomial functions using a Taylor series expansion. The manuscript shows expressions for expansions using two, three, and four terms. The paper in which the formulation is deduced In a second-order elastic analysis, the structure behavior can be stated according to the equation The conventional formulation of the tangent stiffness matrix is obtained from the virtual work principle considering an updated Lagrangian description using cubic (Hermitian) functions and disregard higher-order terms in the strain tensor and shear deformations (Euler-Bernoulli beam theory). However, in a geometric nonlinear analysis context, this discrete solution introduces simplifications to the mathematical idealization for the structure behavior, since these interpolation functions are not compatible with the mathematical idealization for the response of the continuous medium. Additionally, according to Therefore, the method presented in this paper is for geometric nonlinear analysis and consists of calculating the tangent stiffness matrix of a 3D frame finite element, P > 0), and trigonometric expressions in cases of compressive forces, negative axial loads (P < 0). The separation between tension and compression can be an additional conditional statement that can influence the efficiency of the code. This separation is not necessary if the implementation works directly with exponential functions [1– Construction of elastic component of tangent stiffness matrix: 2–Construction of geometric component of tangent stiffness matrix: 3– Construction of axial and torsion interaction matrix: 4– Tangent stiffness matrix calculation: Theoretical concepts of the matrix formulated are developed in unctions ,17. The Despite Elastic component - P > 0), the elastic component of the tangent stiffness matrix can be written in terms of hyperbolic functions, and the coefficients become:For a tensile force (P < 0), the elastic component of the tangent stiffness matrix can be written in terms of trigonometric functions, and the coefficients become:For a compressive force (Geometric component - P > 0), the geometric component of the tangent stiffness matrix can be written in terms of hyperbolic functions, and the coefficients become:For a tensile force (P < 0), the geometric component of the tangent stiffness matrix can be written in terms of trigonometric functions, and the coefficients become:For a compressive force , the matrix that considers the interaction between the axial load and the torsion is given according:For a tensile force (The coefficients are written in terms of hyperbolic functions:P < 0), the interaction between the axial load and the torsion is given according:For a compressive force (The coefficients are written in terms of trigonometric functions:Taylor Series ExpansionThe tangent stiffness matrix developed using complete expressions with hyperbolic and trigonometric functions (TBT_Large_complete) provides accurate results for nonlinear analysis and for buckling load prediction to spatial structures. However, the hyperbolic and trigonometric functions might be computationally inefficient. In addition, in some cases, mainly for initials loads steps in a nonlinear code and for reduced axial loads, the algorithm can present numerical instability. To improve computation efficiency and avoid numerical problems, a Taylor series expansion is proposed, which generates polynomial functions. Furthermore, the user can select the number of terms in the Taylor expansion according to:TBT_Large_2tr – Timoshenko beam theory, 2 terms in tangent stiffness matrix (1 elastic + 1 geometric). The geometric term corresponds to factor that multiplies the coefficient P. Conventional formulation TBT_Large_3tr – Timoshenko beam theory, 3 terms in tangent stiffness matrix (1 elastic + 2 geometric). The geometric term corresponds to factor that multiplies the coefficients P and P².TBT_Large_4tr – Timoshenko beam theory, 4 terms in tangent stiffness matrix (1 elastic + 3 geometric). The geometric term corresponds to factor that multiplies the coefficients P, P² and P³.The coefficients of the complete tangent stiffness matrix of a 3D frame element, including the elastic components and the geometric components with all terms of the Taylor expansion, are shown in When the constant odrigues  and the L = 1 m, Young's modulus E = 107 kN/m2, a section form factor To verify the developed tangent stiffness matrix, the buckling of spatial frames was studied, adopting just one element per member, and with a moderate slenderness ratio .In order to check the performance of the stiffness matrices presented in this paper, a convergence study was implemented. The presented method can be used to perform a nonlinear geometric analysis of spatial structures using minimal or a reduced discretization, whereas, while the usual tangent stiffness matrix would require a more refined mesh to predict the structure behavior. The method can be implemented in any structural analysis software. Matrix coefficients use hyperbolic and trigonometric expressions. Alternatively, polynomial expressions for geometric stiffness coefficients using a Taylor series expansion with 4 or 3 terms are proposed, and all necessary equations are available online in open source.The presented tangent stiffness was calculated by using interpolation functions for displacements and rotations in a frame element obtained directly from the solution of differential equations that consider the equilibrium in the deformed configuration. As a consequence, these interpolation functions depend on the axial force in the element, and also on the parameters of the Timoshenko beam theory. Moreover, the interpolation functions deduced in the deformed configuration can be used to improve visualization in a graphics program and the computation of the local P-delta effect using just one element per member.P > 0), the interpolation functions for displacements and rotations are written in terms of hyperbolic functions, according to:For a tensile force (P < 0), the interpolation functions for displacements and rotations are written in terms of trigonometric functions:For a compressive force (Rodrigues P = 0), conventional cubic interpolation functions are identical to the ones developed in this work. As the axial load increases, analytical interpolations functions present a distinct behavior, as expected. This influence becomes more evident for elements with Analytical interpolation functions plots are shown in The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Buccal mucosal graft (BMG) urethroplasty is considered as gold standard in the treatment of urethral stricture disease. The successful outcome after BMG urethroplasty varies between 66 and 99%. One of the possible causes for failure is BMG contracture. Primary BMG contracture rate is poorly understood and unreported. The present study aimed to evaluate the extent of contracture of buccal mucosa immediately after harvesting. t-test.  This was a prospective observational study conducted in the Department of Urology at our institute between January 2016 and December 2019. All patients with urethral stricture disease undergoing BMG urethroplasty for the first time were enrolled in the study after obtaining informed consent. Demographic and patient clinical profile was noted. Based on the intraoperative urethral stricture size, the preharvest graft was marked on the buccal mucosa and the size was calculated. Postharvest unstretched size of the graft was measured immediately after graft removal from the oral cavity. Alteration in BMG size was analysed using paired  Forty-four patients were included in the study. Mean age of the patient was 53.6 years. Mean stricture length was 7.45 cm (range 4–12 cm). Mean pre- and postharvest BMG size was 8.3 × 1.5 cm and 7.6 × 1.3 cm, respectively. There was a 8.4% decrease in length and 9.5% decrease in width of the buccal mucosal graft.  Primary buccal mucosal graft contracture is around 8.4% in length and 9.5% in width. It would be better to mark wider than necessary while harvesting buccal mucosa so that tension-free anastomosis is performed. Buccal mucosal graft (BMG) urethroplasty is considered as the treatment of choice for long segment anterior urethral stricture disease . Easy avThe failure of BMG urethroplasty has been attributed to various reasons such as progression of primary disease and loss of vascularity due to over mobilisation of urethra, recurrent infections, and possible contracture of graft.Graft contracture can be primary or secondary. Primary graft contracture happens immediately after harvest from the donor site is a well-described phenomenon in skin grafts . It can A prospective observational study was conducted in the Department of Urology at a tertiary institute from January 2016 to December 2019 after obtaining the institute's ethical committee approval (project no. 20/128). Patients planned for augmentation urethroplasty using BMG were included in the study, after obtaining consent. Patients' demographic and clinical findings were noted. All cases were operated by a team of three urologists alternating between the buccal graft harvesting and the urethroplasty . Stricture characteristics were recorded. A thorough oral cavity examination was performed. Patients who had undergone previous oral surgeries or radiation were excluded from the study.BMG urethroplasty was performed under general anaesthesia with nasal intubation. After intubation, the patient was in lithotomy position, preliminary urethro-cystoscopy was done using 6/7.5 Fr semirigid ureteroscope, and findings were noted. Through a midline perineal incision, the urethra was identified, isolated, and stricture segment was laid open. The stricture length was measured using a measuring ruler.BMG harvesting was done in rose position with a sandbag under the shoulder blade and neck extended. Dingman retractor was used to retract the jaws apart. The graft that needs to be harvested was outlined with a marker pen for one centimeter more than the measurement of stricture Figures , taking t-test. Descriptive data were presented in the form of mean, range, percentage, and standard deviation. Statistical analysis was completed using SPSS software, version 21.0 . Statistical significance was kept below 0.05. The confidence interval was set at 95%.Pre- and postharvest size of BMG was compared using a paired Forty four patients who fulfilled the inclusion and exclusion criteria were included in this study. Demographic data are presented in Forty patients (91%) underwent dorsolateral onlay (Kulkarni technique) , and 4 (p < 0.001) in length. Mean pre- and postharvest BMG width was 1.5 and 1.3 cm, respectively (p < 0.001) in width.Mean preharvest and postharvest BMG length was 8.3 and 7.6 cm, respectively  which shectively  which alIn patients aged more than 55 years, the mean pre- and postharvest BMG dimensions were 8.9 × 1.5 and 8.2 × 1.3 cm, respectively. While in patients aged less than 55 years, the mean pre- and postharvest BMG dimensions were 8.1 × 1.5 and 7.5 × 1.3 cm, respectively. However, the differences in the contracture between the age groups were not statistically significant . TobaccoThe complications in the donor site are listed in Outcome-related variables in augmentation urethroplasty can be listed as surgical technique, graft quality, stricture characteristics, and patient-related factors. Nontransection of urethra during urethroplasty had better results than transecting urethroplasty . KulkarnType of graft and graft contracture are other outcome-related variables in augmentation urethroplasty. Skin, bladder, and intestinal and oral mucosa  grafts are the preferred sites of donors for the reconstruction of the urethra . The proThe incidence of graft contracture in BMG urethroplasty varies from 3 to 22.5% in various studies –18. OralGraft contracture leads to suboptimal results in BMG urethroplasty. Primary graft contracture, if not accounted for, can lead to overstretching of the graft. This graft overstretching can widen the urethral lumen initially, but in long term, the graft can shrink rapidly, especially after the removal of the catheter. Prior knowledge of the extent of primary graft contracture can give an estimate of extra size of the graft needed. To some extent, primary graft contracture can be dealt by meshing, spreading, and suture fixation of the graft. However, this can be challenging if the size of the urethral defect is large. Surprisingly, no study has focused on this issue in BMG urethroplasty. In our study, we found 8.4% decrease in length and 9.5% decrease in the width of the BMG. As we took one centimeter extrasize graft, tension-free anastomosis was possible. Loss of elastic recoil is the reason for primary graft contracture . The extLarger size graft creates larger raw area in the donor site. It leads to inability to close the donor site. Muruganandham et al.  reportedPrevious studies have reported oral cavity bleeding after BMG harvest in 5–21% , 27. ThePersistent oral numbness (9.1%) and restricted mouth opening (2.3%) are the common long-term sequelae following BMG harvest in our study. Behura et al.  in theirSalivatory problems such as excessive salivation or decreased salivation have been reported following BMG harvest , 27. TheThe relatively small sample size was one of the limitations of this study. We did not measure the thickness of the graft and hence could not relate the bearing of graft thickness with the shrinkage rate. Since all the donor sites were left open for healing, we could not compare the morbidity with the donor sites which could have been sutured.Primary graft contracture in buccal mucosa is around 8.4% in length and 9.5% in width. It would be better to mark wider than necessary while harvesting buccal mucosa to obtain a tension-free and successful BMG urethroplasty. Donor site morbidity is reasonable if it is left open and allowed to heal by secondary intention.


Southern African Journal of Infectious Diseases by the editors, mostly to describe changes that have been made to the journal. These have also been taken as an opportunity to chronicle the history of the journal so that we do not forget where the journal has come from and appreciate where we are headed in the future. This editorial will not deviate much from that pattern. The title of one of the previous editorials was ‘The journal – past, present, future’; therefore, rather than using the same title, I ruminated on whether I should call it ‘the good, the bad and the ugly’, but eventually settled on ‘yesterday, today and tomorrow’.There have been a number of editorials written over the years for the A number of changes have occurred in the journal since I took over in 2003 as the editor-in-chief. These ‘yesterday’ events included making changes to the name and look of the journal, changes to the editorial board and bringing the journal into the world of ‘open access’. The latter is not only the way that most journals are going, but is also an important consideration as many of the big funders of research require study findings to be published immediately in open access journals. It is easy to understand the reason why much of this research funding comes from taxpayers’ money and therefore the funders believe, quite rightly, that the study outcomes should be placed in the public domain as soon as possible after study completion.One of the most significant ‘today’ events is that Prof. Hendrik Koornhof elected to retire as the emeritus editor of the journal after a very long association with the journal, including a stint of approximately 15 years as editor-in-chief of the journal. The total contribution that Prof. Koornhof has made, not only to the journal but also to infectious diseases in South Africa, is enormous and cannot be adequately expressed in words that would do it justice. We are all indebted to him and owe him our gratitude.Other changes listed as ‘today’ events in this editorial include the fact that we have changed publishers. We have moved from Medpharm Publications to AOSIS. The reasons for this are purely financial and logistic, rather than any concerns about expertise, professionalism or interpersonal relationships. The editorial committee have enjoyed being with, and interacting with, Medpharm Publications and their staff over all these years. The journal owes a considerable ‘thank you’ to Medpharm Publications, which carried our journal for many years, despite tough economic times, and kept us afloat.It is a harsh reality that the greatest challenge facing journals worldwide is financial viability. No longer can journals rely on the income generated from advertising revenue. This is particularly true in the field of infectious diseases, a speciality in which one of the most commonly prescribed agents is an antibiotic, many of which are genericised, and not terribly expensive. This has impacted significantly on our ability to generate income through advertising. It is for this reason that the other bit of ‘today’ changes we have made is to start charging ‘page fees’/‘article processing charges’ for manuscripts submitted to, and accepted, in the journal from the beginning of 2019. Fortunately many of the universities in South Africa, and elsewhere, have set up measures for supporting open access publishing and many of the funders also allow applicants to build publication costs into their funding applications. It is pleasing to see that despite the introduction of page charges, new submissions to our journal do not seem to be tailoring off. The rate of articles submitted from sub-Saharan Africa, and even outside Africa is also still on the increase.The most important ‘tomorrow’ issue that we need to concentrate on is getting the journal indexed in PubMed. Fortunately, AOSIS is highly experienced in doing this and has taken a number of journals through the indexing process. This is our most immediate and urgent action in the near future.


International students in China are facing difficulties while adapting their Chinese culture, and their life is influenced by the widespread of the Coronavirus Disease 2019 (COVID-19), and caring for their mental health is currently challenging. As a result, our aim is to explore the current mental health care of this minority in China and to provide useful suggestions for future research and institutes. We used the systematic review method, and it was conducted on 11 existing pieces of literature. Our results confirm the unsatisfying psychological situation of international students and the lack of research in this area. We focus on the causes and symptoms of mental problems and explore the effectiveness of methods. As China’s international education is progressing, this country has been a hotspot for international students, and the number of international students who study in China is constantly increasing. In 2018, there were 492.185 international students from 196 countries in China, among which 59.95% from Asia, 16.57% from Africa, 14.96% from Europe, 7.26% from America and 1.27% from Oceania . InternaIn this article, we use the systematic review method to summarize the current process of studying the international students’ mental health situation in China. We did not adopt the meta-analysis method for the diversity of mental problems and personal differences among international students in China.We adopt the systematic review method to present studies on the mental health of international students in China. We only included articles that are written in English. Considering the novelty of this theme, we set no time filter, and the earliest article adopted was published in April, 2014. The database review was completed in August, 2021. Pubmed, the Web of Science Core Collection, Medline and Google Scholar are included in the searching process. We choose keywords as follows when proceeding the database research: international student  in China and mental health . Finally, we found 90 articles. Among them, 86 were found on PubMed, 2 were found on Google Scholar and 2 were found on Web of Science. After removing duplicates, 86 articles were retained. Other study material, for example, the consequences brought by acculturative stress, were collected from PubMed.n = 46) and local students in Chinese universities (n = 22). Further, articles are excluded if they: (1) explore new methods to conduct education without referring to psychological aspects (n = 3); (2) focus on physical problems (n = 1); (3) focus on international students in other countries (n = 2); (4) are letters to the Editor (n = 1). Finally, 11 articles were included. In total, 86 articles were found. Initially, we removes articles that focused on Chinese international students  had significant and negative influences on loneliness, and loneliness  had significant and positive influence on smartphone use. Smartphone use  and loneliness  showed significant and positive effects on smartphone addiction. Therefore, they came to the conclusion that lower individualism leads to higher loneliness, and loneliness contributes to mobile phone addiction. They also discussed advice and implications for further research.Jiang et al.  investigp < 0.01, F = 143.12, p < 0.001). Their results also indicated that low cultural competence, homesickness and low self-confidence were significantly and positively associated with depression, and they emphasized the importance of self-confidence in the role of preventing depression.Liu et al.  highlighp = 0.037). This study also indicated that over half (60.4%) of the studied population suffer from high stress. As a result, it is necessary to provide support according to other studies, including religion, mental health and cultural background. Their conclusion was that high level stress leads to menstrual disorders, and menstrual disorders rate is high in international students in China.Ansong et al.  addresseJiang et al.  aimed toShan et al.  investigHu et al.  used theWang et al.  conductep < 0.001), and the interaction between social support and self-esteem had a significant effect on attachment closeness . The effect of social support on depression was significant , and the effect of attachment closeness on depression was also significant . Their conclusion was that in the relationship between social support and depression, self-esteem plays a moderating role in the mediating effect of attachment closeness.Li et al.  studied Gu et al.  investigLi et al.  studied We can conclude from the literature included that international students in China have a relatively high rate of depression compared to normal Chinese people, whose depression rate is 2.1% . AccordiAcculturative stress is defined as the process of confronting challenges in cross-cultural exchange settings , which iChina mainly adopts quarantine measures in order to control the spread of COVID-19, which have proven to be effective . During We can summarize from the analysis presented above that international students’ mental health situation is not satisfying, so it is urgent to do more research and improve coping strategies. Two studies ,16 emphaAs for the Chinese government, better policies should be introduced. International students who stay in the mainland may enjoy a discount when buying psychotropic drugs, or the cost may be included in their medical insurance. Moreover, a great many hospitals in China nowadays have international medical centers that provide special services for foreigners, and some hospitals include psychological counseling in these centers. These institutions can be introduced to more hospitals and may cooperate with international centers in colleges. They may promote their services to foreign students at a reasonable price. The mental health care of international students in China is not satisfying, and the attention paid to this minority is insufficient. Under COVID-19, variation occurs in their life, and they need to adapt. As can be seen from the results, there is little research in this area. Despite abundant research of depression and anxiety, other diseases are not included. Researches in each aspect are not comprehensive enough. Chinese universities should provide better services for international students. The cooperation between the Chinese government and universities is indispensable for the improvement of international students’ mental health. Further research is needed on the interaction among different mental diseases and on more effective measures to improve their mental health. There is still a lot of room for this issue to be improved.


Level of Difficulty: Advanced.)An 82-year-old man with a HeartMate II left ventricular assist device presented with low-flow alarms and cardiogenic shock secondary to left ventricular assist device outflow graft obstruction. Given high risk for redo sternotomy, the heart team decided on percutaneous intervention with peripheral stents, a procedure that is currently limited to case reports. (  Admission vital signs were a pulse of 100 beats/min, blood pressure 84 mm Hg by Doppler, respiratory rate 22 breaths/min, 90% oxygen saturation on room air, and 22-lb weight gain. Physical examination was significant for LVAD hum, bibasilar crackles, and 2+ pitting edema of the lower extremities. The 24-h LVAD readings showed pump speed of 9200 RPM, flow of 3.6 l/min (range: 2.6 to 5), PI 5.0, and power 5.0 W. During his stay, alarm frequency increased to 42 red heart alarms the night before intervention.Additional medical history included atrial fibrillation and dual-chamber pacemaker implantation. Home medications included warfarin, aspirin, sildenafil, hydralazine, digoxin, and amiodarone. Diuretic agents had been discontinued 1 month previously because of recurrent pre-syncope.The differential diagnosis for low-flow alarms includes hypovolemia, acute blood loss, right-sided heart failure, inflow graft obstruction, or outflow graft obstruction (OGO) . OGOs ar2 and mixed venous saturation of 44%. Computed tomography confirmed OGO  2.8), stable creatinine (1.2 mg/dL), stable anemia (hemoglobin 9.6 g/dL), and mildly elevated lactate dehydrogenase . Echocardiography showed severe left ventricular dilation, increased from 6 months previously. Right heart catheterization showed a cardiac index of 1.54 l/min/mrmed OGO  and 2.Fi.8, stabl2 , final access-site angiography showed no evidence of bleeding or aneurysm  via right radial access and Viabahn covered stents  in our initial procedure because our patient’s LVAD OGO embolization risk was uncertain. Transaxillary access was chosen because of the use of short-shaft balloons, which would not reach the outflow graft via femoral access, and the need for 8-F access, which would preclude a radial approach. Using a micropuncture needle under ultrasound guidance, the left axillary artery was cannulated and an 8-F Pinnacle sheath  was placed after pre-closing the artery. Next, the obstruction was evaluated on intravascular ultrasound (IVUS), which demonstrated material between the graft and outflow sleeve causing obstruction. The minimal luminal area measured 44.6 mm2 . A Viaba2 , necessi2 . Final Ianeurysm . Unfortuaneurysm .Figure 3OGO should be suspected if LVAD low-flow alarms occur associated with outflow graft velocity change, left ventricular dilation, or worsening mitral regurgitation. Proposed mechanisms for nonthrombus stenosis include neointimal hyperplasia, kinking of the sleeve, or blood-derived proteinaceous materials between the graft and outflow sleeve causing compression, which surgeons have observed on LVAD explants .In particular, proteinaceous material has been observed between the outflow graft and bend relief in most cases . It is bStenting of OGO is the preferred treatment, followed by corrective surgery if unsuccessful . A systeCovered stents are necessary if there is thrombus or material within the outflow graft at risk for embolization . HoweverIVUS also helped identify propagation of the material intraprocedurally, which ultimately led to stenting of the entire outflow graft. On this basis, we believe the longest stent possible should be used to limit propagation of the obstruction, particularly during post-dilation, and IVUS should be performed to verify that all material is covered.Finally, case reports have shown recurrent obstruction, with 2 prior reported incidents 76 and 556 days post-stenting . To our Our patient remains well since his second procedure and has had no further alarms. Follow-up imaging 3.5 months later confirmed that no further OGO had occurred.LVADs are used with increased frequency in patients with advanced heart failure. Understanding device complications and management options will be vital to their long-term management. We present our experiences in percutaneous intervention on LVAD OGO and key points we learned that may assist others in optimizing their results with this procedure.The authors have reported that they have no relationships relevant to the contents of this paper to disclose.


Interestingly, previous studies showed that two of our selected epitopes have strong immune response against WNV. Therefore, selected epitopes could be strong vaccine candidates to prevent WNV infections in human. However, further in vitro and in vivo investigations could be strengthening the validation of the vaccine candidate against WNV.West Nile Virus (WNV) is a life threatening flavivirus that causes significant morbidity and mortality worldwide. No preventive therapeutics including vaccines against WNV are available for human use. In this study, immunoinformatics approach was performed to design a multi epitope-based subunit vaccine against this deadly pathogen. Human (HLA) and Mice (H-2) allele specific potential T-cell and B-cell epitopes were shortlisted through a stringent procedure. Molecular docking showed selected epitopes that have stronger binding affinity with human TLR-4. Molecular dynamics simulation confirmed the stable nature of the docked complex. Furthermore,  Flavivirus) [West Nile virus (WNV) is a single-stranded RNA virus  ,2 and itvivirus) . Generalvivirus) ,5. The vvivirus) ,7. Sincevivirus) ,8. Betwevivirus) . WNV infvivirus) . Since lvivirus) , Kerala vivirus) , West Bevivirus) , Tamil Nvivirus) , Madhya vivirus) , Andhra vivirus) , and Punvivirus) . The ecovivirus) ,19. Conshttp://www.violinet.org/). Some of those vaccine candidates are under pre-clinical studies and a very few are under clinical trials; however, none of the WNV vaccines has yet been approved to be used in human host. Currently, only the YFV-based vaccine (ChimeriVax-WN02) has been evaluated in Phase II trial [The genome of WNV virus is approximately 11kb that encodes three structural proteins: Capsid protein (C), Pre-membrane/membrane protein (prM/M), and envelope protein (E). WNV virus also encodes seven non-structural proteins ; however their role yet to be understood . The hetII trial . Other hII trial –27. Nonein vitro and in vivo murine model and thereby reducing time, costs and repetition of error trails of traditional vaccine development. In this study, by utilizing immunoinformatics and reverse vaccinology approaches, we design a candidate multi-epitope chimeric vaccine against the WNV virus that is likely to be safe and immunogenic against the WNV infection.The growing advances in the field of bioinformatics open a new era to design next generation vaccines through the knowledge of immunoinformatics; the detailed protocol and immune responses have been widely reviewed in published literatures –31. In rhttp://www.uniprot.org/uniprot). Further ProtParam tool at Expasy server (http://expasy.org/cgi-bin/protpraram) was used to characterize the functional physiochemical parameters of all the proteins of reference strain [In this study, we adopted computational methods to predict the effective vaccine candidates against WNV Virus where UniProt ID- Q9Q6P4 was considered as the reference strain. All the proteins of this strain including structural  and non-structural  viral proteins were retrieved in FASTA format from UniProt  database (e strain .http://crdd.osdd.net/raghava/abcpred/) [http://tools.iedb.org/bcell/) [http://tools.iedb.org/bcell/) [http://tools.iedb.org/bcell/) [http://tools.iedb.org/bcell/) [Linear B cell epitopes were predicted by combining several B cell epitope prediction server including ABCpred (bcpred/) , Bepipre/bcell/) , Emini S/bcell/) , Karplus/bcell/)  and Park/bcell/) . CombinaABCpred is a recurrent neural network (ANN) algorithm for predicting B-cell epitopes (random peptides) of maximum length of 20 residues. The server was trained on 700 B-cell epitopes and 700 non B-cell epitopes for predicting B-cell lymphomas. The predicted accuracy achieved by this server is 65.93% using recurrent neural network. We set the default threshold of 0.51 in ABCpred server for BCL prediction. The BepiPred-2.0 server, a Random Forest algorithm predicts B-cell epitopes by comparing a set of trained epitopes and non-epitope from crystal structures. Epitopes with threshold value >0.5 are considered and colored in yellow on the graph. Emini Surface Accessibility tool calculates the surface accessible epitopes by using a formula Sn = (n+4+i) (0.37)-6 where n is the value of fractional surface probability, Sn refers the surface probability and values of i varies from 1 to 6. Peptide sequence with Sn greater than 1.0 indicates the probability of being as surface accessible epitopes. The output of Karplus and Schulz flexibility tool provides seven amino acids window lengths of epitope based on three scales for flexibility calculation. Parker Hydrophilicity Prediction tool predicts the hydrophilicity of peptides by calculating the retention times of peptides during high-performance liquid chromatography (HPLC) on a reversed-phase column. Hydrophilic epitope regions were predicted by a window of seven residues score. Again, predicted B cell epitopes were also screened through VaxiJen, AllerTOP, ToxinPred and TMHMM webserver for final candidate selection.http://www.cbs.dtu.dk/services/NetCTL/) [T cell epitopes were identified by using a non-linear artificial neural networks-based server, NetCTL 1.2 server (NetCTL/)  where HLhttp://www.ddg-pharmfac.net/vaxijen/) was used to analyze the antigenicity of the proteins [http://tools.iedb.org/immunogenicity/) [http://www.cbs.dtu.dk/services/TMHMM/) [http://www.cbs.dtu.dk/services/NetMHC/) was used for cross checking with the selected epitopes for mouse allele (H-2) binding [VaxiJen server (proteins . Epitopeproteins . Additioproteins . Immunognicity/) . Finally/TMHMM/) . After a binding ,59. Epithttp://tools.iedb.org/mhcii/) [IEDB MHC II binding database (/mhcii/)  was usedhttp://crdd.osdd.net/raghava/ifnepitope/) [Further these epitopes were screened through VaxiJen, AllerTOP, ToxinPred and TMHMM webserver. After cross checking with these servers final selected epitopes were then screened through IFNepitope webserver (pitope/) . This sein silico vaccinology approach, conservancy analysis is used to determine the degree of epitope distribution in the homologous protein set. We used the epitope conservancy analysis tool (http://tools.iedb.org/conservancy/) at the IEDB (Immune Epitope Database) for the analysis of conservancy pattern of the desired epitopes. The sequence identity threshold was set at 100%, and a set of 100 homologous sequences of the selected 10 antigenic protein were retrieved from the NCBI  database with BLASTp tool against the WNV Virus, for using as homologous proteins set in the conservancy tool [In case of ncy tool .However, in South East Asia, Europe and USA closely related flaviviruses are circulating. These viruses does not protect each other; there can be a possibility of cross protective immunity as all these viruses shares the same family. Furthermore, as a rule in flaviviruses, virus neutralizing antibodies is necessary. That’s why the conserved B cell epitopes of WNV virus were then again ran in the conservancy analysis tool to look for homology in the Dengue Virus  and Japanese encephalitis viruses reference polyprotein to check if any of the B cell epitope is conserved and similar in those polyproteins or not. If our predicted epitopes are matched in these polyproteins, there may be a cross reaction possibility can be occurred.http://tools.iedb.org/population/) [For effective vaccination, a vaccine molecule must provide a broad-spectrum protection against different populations around the world. However extreme polymorphic behavior of MHC molecules (Near around 6000) causes different MHC derived pool/frequencies in individuals of different ethnicities/country. Thus, selecting multiple peptides with different HLA binding capacities can increase coverage of population around the world. To address this issue, IEDB population coverage tool (lation/)  was util90) were calculated. [To do that, all parameters in IEDB population coverage interface kept at default (1), the projected population coverage (2), the average number of HLA combinations recognized by the populations (3), and minimum number of HLA combinations recognized by 90% of the population  to stimulate robust immune-reaction [For the construction of the final vaccine protein, predicted B cell epitopes and T cell epitopes were joined by suitable fusion protein linkers. Then Universal Pan HLA DR sequence (PADRE) sequence was attached to overcome the problems caused by highly polymorphic HLA class-2 alleles in the final vaccine construct . C-termireaction . Beta-dereaction . Thus utreaction .http://expasy.org/cgi-bin/protpraram) [Eschericia coli (E.coli) proteins from the experimental solubility dataset and vice versa [http://crdd.osdd.net/raghava/algpred/) was used for prediction of allergenicity of the vaccine construct [ProtParam, a tool available on ExPASy server (tpraram)  was usedtpraram) . This sece versa . Additioonstruct .Secondary structure is the general two-dimensional form of local segments of a protein or nucleic acid sequence. In proteins, secondary structure can be defined by the hydrogen bond patterns between backbone amino and carboxyl groups of proteins which is found in an atomic-resolution structure.http://bioinf.cs.ucl.ac.uk/psipred/) [PSIPRED (sipred/)  an artifsipred/) .http://robetta.bakerlab.org/), a protein structure prediction online server. Robetta is continually evaluated through CAMEO  for tertiary structure prediction. Also, Robetta server utilizes custom sequence alignments for homology modeling, constraints, local fragments and so on. However, it uses the PDB100 template database and a co-evolution based model database (MDB) which is updated weekly.The tertiary structure of multi-epitope vaccine subunit vaccine was predicted by using Robetta (https://zhanglab.ccmb.med.umich.edu/ModRefiner/) was used to enhance the accuracy of the predicted 3D modeled structure [https://servicesn.mbi.ucla.edu/PROCHECK/) [On the other side, protein model structure produced by different protein structure prediction strategies mostly relies on similarity between the input and available template structure of PDB. So, the whole protein structure needs to be refined for improving the template-based protein model structure beyond the precision level. Therefore, ModRefiner server (tructure . FurtherOCHECK/)  was usedDiscontinuous or conformational epitopes are those that consist of multiple, distinct segments from the primary amino-acid sequence. Conformational epitopes residues are distantly placed in the sequence and brought together by physicochemical folding of the Protein’s tertiary structure. . Most ofhttp://tools.iedb.org/ellipro/) [ElliPro (llipro/)  a disconin silico approach that can evaluate the binding correlation between a ligand and a receptor molecule [https://bioinfo3d.cs.tau.ac.il/PatchDock/) for analyzing protein-protein docking interaction to determine the binding affinity of our designed peptide vaccine with immune receptor TLR-3, 4, 7 and 8  [Molecular docking is an molecule . We assectively) . PDB filMolecular dynamics simulation is recognized as an effective method for molecular analysis of biological systems ,78,79. IThe simulation of docked vaccine was performed to study interaction pattern of vaccine and changes in the complex after molecular docking with respective ligand. Additionally, the molecular dynamics simulation was carried out for 10ns long.We analyzed the RMSD (Root Mean Square Deviation of the atomic position), RMSF (Root Mean Square Fluctuations) and Rg (Radius Gyration) of the vaccine-receptor complex. The RMSD, RMSF and Rg can be used to obtain information about our Biosystems. The RMSD includes the calculation concerning the reference structure (backbone) of the average atom location in a molecule. This method is used to calculate the average changes in atom displacement for evaluating the conformational shifts and Biosystems stability. The average deviation of a particle  from the reference location  over time is calculated with root-mean-square fluctuation (RMSF). RMSF therefore analyzes the structural portions which fluctuate more (or less) from their mean structure. The gyration radius (Rg) represents the compactness of the structure. The lower degree of fluctuation with its simulation stability indicates the higher compactness and rigidity of the device.Homo sapiens expression system. Four restriction sites  were avoided from the optimized cDNA sequence. GC content, CAI values were again evaluated, and only highly efficient cDNA sequence was inserted in pAdTrack-CMV shuttle vector [BglII and EcoRV restriction site under strong CMV promoter through SnapGene v5.1 [To construct an adenovirus based vaccine, Kozak consensus sequence containing codon for methionine was added at the N-terminal end of vaccine protein. Codon optimization was done in GenScript server for e vector  in betweene v5.1  restrictene v5.1 .The amino acid sequences of structural and non-structural proteins of WNV virus (UniProt ID- Q9Q6P4) were retrieved from UniProt Database. The physiochemical properties  of each protein were analyzed using ExPASy ProtParam tools .Linear B cell epitopes were screened only from the three structural proteins. Several B cell epitopes prediction server including ABCpred, Bepipred, and Emini Surface Accessibility Prediction tool of IEDB were used for identification of potential B cell epitopes from WNV structural proteins. Linear B cell epitopes of 16-mer predicted through ABCpred server were cross-checked with BepiPred 2.0 and Emini Surface Accessibility Prediction tool of IEDB server and overlapped epitopes were shortlisted . MoreoveNetCTL 1.2 server was used to predict the T cell epitopes of corresponding WNV proteins . Full screening of epitope is provided in Supplementary After CD8+ T cell prediction, IDEB MHC II binding database predicted a total of 497 epitopes from all 10 proteins of WNV that can bind at least one mouse H-2-I allele. Among these epitopes, only 124 were selected that predicted to be antigenic, non-allergenic, non-toxic, and showed outside topology. Furthermore, IFNepitope server predicted 77 epitopes to be IFN-γ positive. Therefore, these 77 epitopes were also capable of inducing Th1 humoral responses. However, we found no IFN-γ inducing epitope from NS1 protein. Full screening is provided in supplementary Conserved epitopes against multiple serovars are prerequisite to design a universal broad-spectrum vaccine. Hence, each of the CTL, HTL, and BCL epitopes predicted from previous pipelines were screened with respective 100 homologous protein sequences of all other serovars of WNV. IEDB conservation analysis tool was used for conservancy analysis of epitopes against other serovars of WNV. At 90% sequence identity threshold, all the CTL, HTL, and BCL epitopes showed 100% conservancy –S3 FilesTo investigate whether the conserved B cell epitopes  have similar sequence homology IEDB conservancy analysis tool was applied and compared against the reference polyproteins of Dengue and Japanese encephalitis viruses . Similarity in epitopes indicate possible cross-reaction. None of these epitopes showed conservancy against the reference proteome of both Dengue and Japanese encephalitis viruses ; indicatMHC-I and MHC-II prediction server at IEDB predicted the corresponding allele for each of the proposed nine CD8+ (CTL) and nine CD4+ (HTL) epitopes. The output was given in units of IC50 nM. Peptides with IC50 values of <50, <500 and <5000 nM are considered to have high, intermediate and low affinity respectively. Here, alleles with IC50 value <500 were selected as optimum binders for the population coverage analysis.Population coverage was calculated with corresponding HLA alleles (IC50 <500 nm) of selected nine CTL and nine HTL epitopes (one epitope from each protein) through population coverage tool at IEDB interface. We observed 98.55% (CTLs) and 98.99% (HTLs) of world population coverage. A strong immune response requires activation of both CD4+ and CD8+ T cells. Population coverage analysis of the combined MHC-I and MHC-II epitopes revealed an average of 99.99% coverage of the population worldwide. Whereas, the Europe, England, North America, United States, and West Africa showed the highest (100%) population coverage of the combined MHC-I and MHC-II epitopes .Yersinia was added at the C-terminal end of the construct which was separated from the BCL epitope by an EGGE linker. These linkers would ensure maximal immunity and efficient presentation of epitopes in the body while PADRE sequence would induce CTL responses and ensure maximal MHC-II allele coverage. The final construct of multi-subunit vaccine comprises 488 amino acid residues and is represented in The chimeric vaccine was constructed with final nine CTL, nine HTL, and six B cell epitopes (BCL). Epitopes were joined based on genomic arrangement of their corresponding proteins in virus. GGGS linkers were added to separate each CTL epitope from other CTL epitopes, whereas GPGPG linkers were used to separate each HTL epitope from other HTL epitopes. KK linker was added to separate each BCL epitope from other BCL epitopes. Beta defensin-3 (GIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCCRRKK), a TLR4 agonist, was added at N-terminal end of the construct followed by EAAAK linker and PADRE sequence (AKFVAAWTLKAAA). GGGS linker was used to separate PADRE sequence from CTL. The C-terminal invasin sequence (TAKSKKFPSYTATYQF) of in vitro), more than 20 hours in case of yeast cell (in vivo) and over 10 hours if expressed in E. coli (in vivo). Molecular weight (50.914 kDa) of the construct was in favorable range. The recombinant vaccine protein was also predicted to be soluble (0.551) in E. coli heterologous expression system. Furthermore, AlgPred predicted the construct to be non-allergenic in nature  in nature. Physiochemical analysis revealed that the construct was highly stable (Instability index 29.55), basic in nature , and highly thermostable . The estimated half-life was found 30 hours in mammalian reticulocytes , 145 sheet (29%), and 240 (50%) coil regions.ROBETTA server generated 5 models (3D) of vaccine protein using comparative modeling. Among those, pre-refinement quality analysis through PROCHECK tool revealed model no. 5 as the best candidate as it had highest number of residues in most favored region (83.1%). To improve the standard of predicted 3D modeled structure beyond the accuracy, refinement was performed using ModRefiner. Refined structure showed RMSD value of 1.268 and TM-score 0.9755 to initial model (model no. 5). Post-refinement quality analysis through PROCHECK tool revealed improved Ramachandran plot as 88.1% residues are in most favored regions, 11.1% in additionally allowed region, 0.5% in generously allowed region, and only 0.3% in disallowed region .ElliPro at IEDB was utilized for identifying the discontinuous B-cell epitopes. The PDB structure of the vaccine protein was used as an input for the prediction of conformational epitopes in the tertiary structure of the vaccine construct. ElliPro identified seven epitopes on the three-dimensional structure of the vaccine construct where highest predicted score was 0.735 with residual position of 359–386 .Vaccine construct was docked with human TLR- 3, 4, 7, and 8 receptors . PatchDoThe molecular dynamics simulation was performed for 10ns timescale and RMSD, RMSF, and Rg plots were drawn to check the flexibility and stability of the vaccine-receptor complex. The RMSD plot showed significant results highlighting the stability of the vaccine-receptor complex . The RMSRoot means square fluctuations (RMSF) helps to explain the protein area that fluctuate throughout the simulation. The flexibility of each residue is therefore measured in order to provide a deeper insight into the degree to which protein binding influences complex’s flexibility. The RMSF plot showed very less fluctuations of the atoms in the complex ; suggestAs the radius of gyration shows the compactness of the complex structure, there should be fewer movements or deviation of the positions of the residues of the complex in the simulation trajectory in order to deem the complex to be stable. The Rg plot  showed lThe molecular dynamics simulation results showed significant structural flexibility and stability of the vaccine-receptor complex; thus implies a positive response by the immune receptor.https://www.genscript.com/) was utilized to generate reversely transcribed nucleotide sequences of recombinant vaccine protein for human cell expression system. GC content of obtained cDNA (1467 bp) was 62.25% whereas CAI value was calculated as 0.92. These values above indicate efficient expression of optimized codon in human host cell. This sequence was inserted as GOI under strong CMV promoter in pAdTrack-CMV vector through SnapGene v5.1 software. BglII restriction site was added at the 5’ end followed by Kozak sequence which ensures maximal expression of the desired gene. Stop codon TAA was added at 3’ end of the optimized codon followed by EcoRV restriction site. Stop codon would ensure termination of gene translation , showed potential antigenicity, surface accessibility, non-toxicity, and non-allergenicity. These epitopes may induce production of WNV specific antibodies in human. Neutralizing antibodies (IgM and IgG) appear to terminate viremia by controlling hematogenous spread of the WNV virus to Central Nervous System [302-309, E303-333, E333-365, and E389-391 [383-398: YIVVGRGEQQINHHWH) for vaccine construction contains experimentally proved amino acid residues capable of inducing neutralizing antibodies. Likewise E antigens, PrM antigens incorporated in ChimeriVax-WN02 vaccine also induced neutralizing antibodies and untill now are thought to be the most promising candidate as a human vaccine for WNV [480-488: SIALTFLAV, PrM 153–161: VVFVVLLLL, Capsid C51-59: LAFFRFTAI, NS1120-128: KSILFAPEL, NS2A9-17: FQLGLLVVF, NS2B13-21: LMFAIVGGL, NS3358-366: KTVWFVPSV, NS4A63-71: MTMGVFFLL, NS5486-494: LEFEALGFL) were immunogenic, non-toxic, and non-allergenic. These were predicted to bind with human alleles and at least one mouse allele. From published studies, we found an epitope (E480-488: SIALTFLAV) of E protein overlapped with experimentally validated MHC class I binding epitope (E481-488: IALTFLAV) in mouse model [in vivo.In this study, Linear B cell epitopes were screened only from structural proteins  of WNV. Six epitopes, two from each protein  were used in vaccine construct since IFN-γ induces MHC class II expression and activation of macrophages [CD4+ T cell (HTL) is another key immune cell type that acquires Th1 or Th2 phenotypes and stimulates immune responses ,124. CD8rophages . It is wrophages .in vivo separation of individual epitopes effectively in natural environment [E. coli heterologous expression system [in vivo expression of stable mRNA [Conservancy analysis of epitopes in different WNV serovars suggested universal candidacy of the designed vaccine. Also, population coverage calculated for conserved MHC class I and II epitopes showed >99.99% worldwide coverage in a combined manner. EAAAK linker used in the constructed vaccine would ensure ironment ,80. As Gironment  in epitoironment  and ensuironment ,132. Theironment –136. Theironment . Finallyble mRNA  and may ble mRNA . In receble mRNA . Mammalible mRNA ,141. VLPble mRNA . On the ble mRNA ,144. Adeble mRNA .Adenovirus based vaccines showed promising results in clinical trials against several infectious diseases such as HIV, Malaria, Hepatitis C virus, Ebola virus, Tuberculosis, Rotavirus and also cancers such as Prostate cancer, Lymphoma, melanoma, and others . It is nin silico cloning approaches of the vaccine construct ensure high expression within microbial system.West Nile virus (WNV) is continuously spreading across Europe and other continents, i.e. North and South America, Asia, and many other regions of the world. Using immunoinformatics approach, we designed a multi epitope-based subunit vaccine against WNV. Human (HLA) and Mice (H-2) allele specific immunogenic epitopes including Cytotoxic T-lymphocytes (CTL), Helper T-lymphocytes (HTL), and B cell lymphomas (BCL) were screened and selected for vaccine design. Final vaccine constructs consist of B and T-cell epitopes, Universal pan-HLA DR or PADRE (13 mer) sequence, β-defensin-3 adjuvant (45 mer), and Invasin molecule at N-terminal of the construct with fusion protein linkers. Whereas, Invasin molecule (16 mer) was added at the C-terminal end to elicit strong immune responses. Conservancy and population coverage analysis of the promiscuous epitopes revealed the robust immune response against multiple serovars of the WNV and various ethnicities. Molecular docking revealed stronger binding affinity with human TLR-4; later confirmed by molecular dynamics simulation. Our computational analysis suggests that the constructed vaccine was highly immunogenic, safe, non-toxic, and stable in nature. Eventually, S1 FigThe Highest predicted score was obtained 0.735.(TIFF)Click here for additional data file.S2 FigBgl II and EcoRV restriction sites (red marked). Recombinant plasmid consists of 10688 base pairs. b) Predicted secondary structure of mRNA for vaccine expressed through pAdVTrack-CMV. The 5’ end of predicted mRNA structure does not contain any pseudoknot or hairpin.a) Gene of Interest was inserted between (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.S3 File(XLSX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.S5 File(DOCX)Click here for additional data file.S6 File(XLSX)Click here for additional data file.S1 Graphical abstract(TIF)Click here for additional data file.


Leendert Pieter van Ofwegen passed away on 25 June 2021 at the age of 68 years. He had been diagnosed with lung cancer in late 2020, which in June this year appeared to be in complete remission. Tragically, there was a relapse. His health suddenly worsened while he was on holiday in Marbella (Spain) and the disease rapidly led to his death. With his passing, the scientific community loses a great and devoted octocoral taxonomist who was a good friend to his colleagues and a dedicated mentor to his students.Leendert (“Leen”) was born in Leiden on 22 April 1953. From 1959 to 1965 he attended primary school (“Rozenlaanschool”) in Boskoop, a town in the province of South Holland, where he lived with his parents and elder brother while he grew up. Boskoop is famous for its woody plant and perennial nurseries, which employed many local people, including Leen’s father. Leen attended secondary school in Alphen aan den Rijn (“Christelijk Lyceum”) from 1965 to 1970, after which he studied biology at Leiden University from 1970 until 1980. His study was interrupted by military service, which was compulsory in the Netherlands at that time.RMNH, presently Naturalis Biodiversity Center), which at that time was a centre of systematic research on Cnidaria. He worked under supervision of Koos den Hartog (curator of “Coelenterata”), who proposed that Leen could work on the taxonomy of octocorals from the Saba Bank, which were collected during an expedition to the former Netherlands Antilles, currently the Dutch Caribbean . That collection belonged to the Invertebrate Zoology department of the Smithsonian Institution in Washington, D.C. (USNM), and was on loan to the RMNH to make it available for research by octocoral expert Dr. Jacob Verseveldt. Verseveldt, however, was mostly interested in studies of fleshy octocorals . At a later stage, he also became involved in molecular analyses applied to the taxonomy of octocorals, as shown by co-authored publications with colleagues in Leiden and from abroad  at Dona Paula, Goa, India , which resulted in the establishment of Chromonephthea and many new species in that genus s Figure , and Dr.Octocoral material in museum collections is either stored in ethanol or dried, both of which cause specimens to lose their original shape and often their colour. In order to learn about the natural habitus of soft corals, Leen decided that he had to learn diving with the use of SCUBA. This enabled him to participate in fieldwork sampling during surveys and large-scale expeditions in the company of his colleagues of the Naturalis marine research team , even before he got a position as a curator Figures , 3, 4: iMost of these expeditions were in Indonesia and in this way Leen established a close collaboration with two octocoral scientists from the Research Centre for Oceanography, Indonesian Institute of Sciences, Jakarta: M.I.Y.T. Hermanlimianto (Yosephine Tuti), specialised in gorgonians, and Anna E.W. Manuputty, specialised in soft corals. Both of them also visited Leen and his collection; Yosephine did so several times Figure . All in th International Conference on Coelenterate Biology, Noordwijkerhout, The Netherlands (1996); the 9th International Coral Reef Symposium Bali, Indonesia (2000); the 2nd International Workshop on Octocoral Taxonomy, Darwin, Australia (2002); the 7th International Conference on Coelenterate Biology, Lawrence, Kansas, USA (2003); the 10th International Coral Reef Symposium, Okinawa, Japan (2004); the 1st Asia Pacific Coral Reef Symposium, Hong Kong (2006); the 2nd Asia Pacific Coral Reef Symposium, Phuket Thailand (2010); and the 8th International Conference on Coelenterate Biology, Eilat, Israel (2013). He acted as an instructor with other colleagues during two octocoral taxonomy workshops: in Trivandrum, Kerala, India (2005) and in Phuket, Thailand (2007).In order to strengthen his international contacts, Leen attended several international conferences and workshops to meet with other octocoral workers Figure , such asNephtheididae was also the subject of his PhD from Leiden University, obtained on 6 November 2007 . After his retirement, Leen stopped his editorial tasks, but he continued publishing papers until his death. His dedication and expertise in octocoral taxonomy is demonstrated by an impressive series of publications and a large collection of specimens. In honour of Leen’s octocoral expertise one species was named after him, Fascicliaofwegeni Janes, 2008, based on type material collected by Leen in the Seychelles . In the same year he also became editor of corallia , with whychelles .Chromonephtheaegmondi, C.franseni, C.goudi, C.hoeksemai, C.slieringsi, Dendronephthyavervoorti, Lignellahartogi, Sinulariavanderlandi), and several others were named after octocoral specialists . Having known Leen for more than 35 years, it is beyond doubt that he will be missed by his colleagues and students, who will remember him not only for his life-time dedication to octocoral taxonomy and his vast knowledge on that topic, but also for his characteristic smile (Figures Leen described many new taxa himself or with co-authors, including seven families, 25 genera, and 214 species, ranging in publication years from 1987 until the present (Appendix


PANDB was synthesized in the presence of dodecylbenzenesulfonic acid (DBSA), which functioned as a dopant and surfactant. The electrical conductivity of the conducting PANDB/χ-Al2O3 core-shell nanocomposite was approximately 1.7 × 10−1 S/cm when the aniline/χ-Al2O3 (AN/χ-Al2O3) weight ratio was 1.5. The transmission electron microscopy (TEM) results indicated that the χ-Al2O3 nanoflakes were thoroughly coated by PANDB to form the core-shell (χ-Al2O3-PANDB) structure. The TEM and field-emission scanning electron microscopy (FE-SEM) images of the conducting PANDB/χ-Al2O3 core-shell nanocomposites also indicated that the thickness of the PANDB layer (shell) could be increased as the weight ratio of AN/χ-Al2O3 was increased. In this study, the optimum weight ratio of AN/χ-Al2O3 was identified as 1.5. The conducting PANDB/χ-Al2O3 core-shell nanocomposite was then blended with water-based polyurethane (WPU) to form a conducting WPU/PANDB/χ-Al2O3 blend film. The resulting blend film has promising antistatic and electrostatic discharge (ESD) properties.Polyaniline doped with dodecylbenzenesulfonic acid/χ-aluminum oxide (PANDB/χ-Al Polyaniline (PANI) is an important member of the intrinsically conducting polymer (ICP) family. Because of its unique electrochemical properties and environmental stability, PANI has been extensively studied by numerous researchers ,3,4,5,6 In recent years, encapsulating inorganic materials inside a PANI shell has become the most popular and interesting aspect of composites. The combination of inorganic components with conducting PANI has attracted considerable attention because of the resulting composites’ novel physical and chemical properties and potential applications. These core-shell conducting composites can provide new synergistic properties that cannot be attained from the individual materials alone.2O3 [xFe3−xO4 [2 [2 [2 [Preparing conducting core-shell PANI/inorganic composites via conventional mixing or blending in melt or solution form is difficult because PANI is insoluble, infusible, and possesses poor mechanical properties. One of the most promising methods to prepare conducting core-shell PANI/inorganic composites is through an in situ chemical polymerization of a monomer in the presence of inorganic components. Many groups have reported conducting PANI/inorganic core-shell composites such as PANI/Y2O3 , PANI/ZnxFe3−xO4 , PANI/T-xFe3−xO4 , PANI/Ti3−xO4 [2 , PANI/MnO4 [2 [2 , PANI/Zr[2 [2 [2 , PANI/al[2 [2 [2 ,22,23, P[2 [2 [2 , and PAN[2 [2 [2 .However, PANI doped with strong protonic acids is intractable. There have been a number of attempts towards making conducting PANI processable by doping with various dopants. The most attractive and promising system is PANI doped with dodecylbenzenesulfonic acid (DBSA) (PANDB). This is because DBSA containing a polar head and a long nonpolar chain which can function both as a surfactant and dopant. This advantage allows PANDB to not only become soluble in various organic solvents  but also to have better compatibility with hydrophobic polymers. Therefore, conducting PANI doped with DBSA (PANDB) was adopted in this study.2O3 nanoflake is one of the metastable polymorphs of transition alumina with a relatively high aspect ratio, thermal stability, porosity, and specific surface area. Due to its flake structure and thickness of around 20~50 nm, the χ-Al2O3 nanoflake can be used as an excellent reinforcement to enhance both the strength and stiffness of semicrystalline and glassy thermoplastics, elastomers, and epoxy resins.The χ-Al2O3 core-shell nanocomposites through an in situ polymerization process in the presence of DBSA as the dopant. The goal of this study is to improve the electrical conductivity of the χ-Al2O3 nanoflake and let the χ-Al2O3 nanoflake coated with conducting PANDB form the conducting PANDB/χ-Al2O3 core-shell nanocomposites. In addition, the optimum weight ratio of aniline/χ-Al2O3 (AN/χ-Al2O3) to synthesize the conducting PANDB/χ-Al2O3 core-shell nanocomposites is also determined. Moreover, we also discuss the influences of the weight ratio of AN/χ-Al2O3 on the electrical conductivity, chemical structure, and morphology of the conducting PANDB/χ-Al2O3 core-shell nanocomposites. Finally, the synthesized product will be blended with water-based polyurethane (WPU) to form a conducting WPU/PANDB/χ-Al2O3 blend film. The antistatic tests of the surface of the pure WPU film and the conducting WPU/PANDB/χ-Al2O3 blend film will be examined and compared.To the best of our knowledge, no studies have reported the preparation for conducting PANDB/χ-Al2O3 nanoflake powder, which has a high porosity and high specific surface area, was supplied by the Particulate Research Center at the National Cheng-Kung University, Tainan City, Taiwan. The sectional diameter of one χ-Al2O3 nanoflake was in the range of 150 to 500 μm, and the thickness of one χ-Al2O3 nanoflake was in the range of 20 to 50 nm. Meanwhile, the specific surface area of the χ-Al2O3 nanoflake was in the range of 130 to 150 m2/g. The nonionic WPU dispersion, tackiness, and curing agents for WPU were supplied by Hailsun Chemical Co., Ltd. .Aniline (AN) and ammonium persulfate (APS) were purchased from Merck KGaA . Dodecylbenzene sulfonic acid (DBSA) was purchased from Tokyo Kasei Kogyo Co., Ltd. . The porous χ-AlPure PANDB was directly prepared by a chemical oxidation polymerization according to the modified procedure described in our previous study ,27. 8 g 2O3 core-shell nanocomposite was synthesized via an in situ chemical oxidation polymerization. 2 g of χ-Al2O3 was dispersed in 100 mL of an aqueous solution that contained 1 g of aniline. The resulting dispersion was then stirred at room temperature for 10 min. The AN/χ-Al2O3 weight ratio was set as 0.5. Then, 50 mL of an aqueous DBSA solution was added to the solution. After 10 min, 30 mL of an aqueous APS solution was added dropwise to the dispersion with constant stirring. The aniline:DBSA:APS molar ratio was 1: 0.8: 1. The resulting mixture was allowed to react for 2 h at room temperature. Subsequently, the product was washed with deionized water until the filtrate became colorless; the product was then dried in a vacuum oven at 60 °C for 24 h. To determine the effect of the weight ratio of AN/χ-Al2O3 on the properties of the conducting PANDB/χ-Al2O3 core-shell nanocomposites, products with weight ratios of 1.0, 1.5, and 2.0 were also prepared. 2O3 core-shell nanocomposite via an in situ polymerization process.The conducting PANDB/χ-Al2O3 core-shell nanocomposite was added to the WPU dispersion. The WPU/20wt% (PANDB/χ-Al2O3) mixture was then thoroughly mixed. Next, a tackiness agent was added to the mixture under mechanical stirring until the mixture viscosity reached around 7500 cps. A curing agent of 1.5 g was added to the mixture sequentially and was completely and well mixed. The resulting mixture was degassed with a vacuum pump to eliminate air bubbles. Finally, the air bubble-free mixture was cast on a polyethylene terephthalate (PET) substrate and heated to 120 °C for 2 min, then 150 °C for 2 min to complete the curing reaction of WPU. For a comparison, the pure WPU film was also made.WPU dispersion was placed into a beaker equipped with a mechanical stirrer. 20wt% of the synthesized conducting PANDB/χ-Al5 psi for 2 min at room temperature. The electrical conductivity σ (S/cm) of the sample was examined by a four-point probe meter  at room temperature. Each sample was first compressed into a sample with a diameter of 10 mm. Then, 10 different locations on the sample surface were measured and averaged. The obtained results were averaged and reported. The surface resistance (Ω/sq) of the pure WPU film and conducting WPU/20wt%(PANDB/χ-Al2O3) blend films was examined by the same equipment. 10 different locations on the sample surface were measured and averaged.In total, 0.1 g of sample was weighed and then pressed under 3.0 × 10−1 at 32 scan/s. The powdered samples and potassium bromide (KBr) (weight ratio of approximately 1:99) were grounded together into fine powders, and the homogeneous mixture was then pressed into a pellet for analysis.The chemical structure of the sample was determined by Fourier-transform infrared spectroscopy (FTIR)  over the wavenumber range 400–4000 cmThe sample was dispersed in absolute alcohol under ultrasonic agitation for 1 h at room temperature. An ultraviolet-visible spectrophotometer  was used to measure the optical absorbance of the sample solution at a wavelength range of 300 to 900 nm.X-ray diffraction (XRD) patterns of the synthesized sample were obtained at room temperature using an X-ray diffractometer  equipped with a Cu-Kα radiation source (λ = 0.154 nm). The diffraction patterns were recorded at a scan rate of 2°/min over the 2θ range from 5° to 80°.The sample was examined by transmission electron microscopy  by casting a diluted dispersion of the sample onto a carbon-coated copper grid. The TEM was operated at an accelerating voltage of 80 kV.The sample was dried after filtration. The sample was then fractured, and each sample was coated with a gold-palladium film. The surface morphologies of the sample were examined using a field-emission scanning electron microscope .2O3 core-shell nanocomposites. The electrical conductivity of the pure PANDB is approximately 6.3 × 10−1 S/cm. 2O3 core-shell nanocomposites is increased as the weight ratio of AN/χ-Al2O3 is increased. When the weight ratio of AN/χ-Al2O3 is equal to 1.5, the conductivity of the conducting PANDB/χ-Al2O3 core-shell nanocomposite is approximately 1.7 × 10−1 S/cm. This result implies that when the weight ratio of AN/χ-Al2O3 is higher, the amount of PANDB generated is higher. Therefore, the surface of χ-Al2O3 can be covered more completely. As a result, the conducting path of the product increases, and the electrical conductivity increases. However, when the weight ratio of AN/χ-Al2O3 is increased to 2.0, the conductivity of the conducting PANDB/χ-Al2O3 core-shell nanocomposite remains at approximately 1.7 × 10−1 S/cm. This result indicates that the optimum weight ratio of AN/χ-Al2O3 is equal to 1.5 in this study.2O3 core-shell nanocomposites. The absorption peak of the −CH2− stretching vibration (resulting from the DBSA molecules) was observed at 2926 cm−1 for all of the samples. For the pure PANDB . Because these absorption peaks were all located in the range of 1000–1200 cm−1, a broad peak was observed instead of each single peak in the FTIR spectrum. The peaks at 700–800 cm−1 are attributed to a characteristic feature of the B–NH–Q bond or the B–NH–B bond and to the out-of-plane bending vibration of benzenoid or quinonoid –C–H and –N–H bonds. Additionally, the peaks at 2900–2950 and 3267 cm−1 are due to the stretching vibration mode of the –C–H (–CH3 or –CH2–) and –N–H bonds, respectively. These characteristic peaks are in good agreement with the data reported in the literature [terature ,29.2O3 core-shell nanocomposites , while the characteristic peaks of χ-Al2O3 decrease . This is because when the weight ratio of AN/χ-Al2O3 increases, more PANDB is synthesized and there is more PAND coating on the surface of the χ-Al2O3 nanoflake.ectively . It can 2O3 core-shell nanocomposites. The morphology of the pure PANDB is fiber-like. Its diameter is approximately 0.2 μm, and its length is approximately 1–3 μm  blend films are around 6.0 × 1010 and 1.5 × 104 Ω/sq., respectively. It can be found that adding the 20wt% (PANDB/χ-Al2O3) conducting nanocomposite into the WPU matrice can significantly reduce the surface resistance of the WPU film from 6.0 × 1010 to 1.5 × 104 Ω/sq. This is due to the formation of conducting paths through the WPU film by containing 20wt% conducting PANDB/χ-Al2O3 nanocomposite. 2O3) blend film (2O3) blend film. This phenomenon indicates that the WPU/20wt% (PANDB/χ-Al2O3) conducting blend film can function as a promising antistatic or electrostatic discharge material.The surface resistances of pure WPU film and WPU/20wt%  conducting nanocomposite into the WPU matrix can significantly reduce the surface resistance of the WPU film from 6.0 × 1010 to 1.5 × 104 Ω/sq. The result indicates that the WPU/20wt% (PANDB/χ-Al2O3) conducting blend film can function as a promising antistatic or electrostatic discharge material. In future work, the synthesized conducting PANDB/χ-Al2O3 core-shell nanocomposite can be used as a novel and multiple functional reinforcement and be blended with semicrystalline and glassy thermoplastics, elastomers, or epoxy resins. Thus, PANDB is expected to provide electrical conductivity, and χ-Al2O3 nanoflakes are expected to enhance the mechanical properties of the polymer matrix. Meanwhile, the synthesized product can be used as antistatic and static discharge material and shows good potential for use in chemical sensors, biosensors, and anticorrosion.In this study, an in situ polymerization method was used to synthesize conducting PANDB/χ-Al