Humans can physically interact with other humans adeptly. Some overground interaction tasks, such as guiding a partner across a room, occur without visual and verbal communication, which suggests that the information exchanges occur through sensing movements and forces. To understand the process of motor communication during overground physical interaction, we hypothesized that humans modulate the mechanical properties of their arms for increased awareness and sensitivity to ongoing interaction. For this, we used an overground interactive robot to guide a human partner across one of three randomly chosen paths while occasionally providing force perturbations to measure the arm stiffness. We observed that the arm stiffness was lower at instants when the robot’s upcoming trajectory was unknown compared to instants when it was predicable - the first evidence of arm stiffness modulation for better motor communication during overground physical interaction. Even without explicitly shared goals, two human partners can physically interact with each other to perform collaborative tasks. Prior work suggests that sophisticated motor communication strategies exist in physically interacting human dyads that can lead to improved performance of the joint task6, distinction of skill levels8 or roles9, or even motor learning and adaptation to the task13. Understanding this biomechanical strategy of physical interaction may be the first step in facilitating better physical human-human interaction (pHHI) and ultimately in developing safe and effective physical human-robot interaction (pHRI)14.Humans can physically interact with one another without verbal communication15. During physical interaction, human arms are used as a medium for two-way motor communication17. Since interactions through arms can be characterized by their movements and the interaction forces, the causal relationship between these two physical quantities, which is mechanical impedance, may reveal human motor control strategies. Indeed, previous works investigated arm stiffness (the spring-like component of impedance) to study motor control in humans for stability tasks20 or in seated reaching tasks25.A promising focal point for observing such communication during pHHI or pHRI may be human arm impedance26. This implies that during practical overground interaction tasks, such as walking assistance, humans may modulate their arm stiffness for increased sensitivity depending on the ongoing interaction dynamics, particularly in the absence of verbal communication or visual feedback. This further motivates the investigation of the arm stiffness during pHRI or pHHI to uncover the underlying motor communication strategy.Moreover, it was recently suggested that the arm stiffness may affect perception of small forces and/or movements, which is a crucial factor for effective motor communication28, was used in an overground pHRI experiment. The task was designed to simulate a guided walking task such that the human subjects were compelled to focus on the movement of the robot to understand its movement intention. We applied perturbations to measure arm stiffness at two importantly different time points during the trials: either at the point where three potential robot trajectories diverge such that the subjects are least certain of their path, or near the end of the trial when the subjects are most certain that the robot always continues straight forward. By comparing the human arm stiffness in these two conditions that require different levels sensitivity of motor communication, we present the first evidence of human arm stiffness modulation for effective overground pHRI.To this end, this work presents the first measurement and analysis of human arm stiffness during overground physical interaction between a robot leader and a human follower. The recently developed overground interactive robot, OphrieSubjects walked with an interactive robot while closing their eyes and holding the handle on the robot arm. The robot moved on one of three trajectories that involved straight and deviated segments. When asked to guess which trajectory the robot moved on, the subjects were able to respond correctly in 386 out of 400 total trials, implying that the subjects were paying close attention to the movement of the robot throughout the experiment. The average interaction force among all the subjects and all trials due to background stiffness right before the perturbation onset was observed to be a pushing force of 2.215±0.976N. That is, on average, the subjects were pushing the robot towards their front-right direction and the resulting reaction force from the robot was in the opposite direction. This can be seen in Fig. 22. The mean arm stiffness and the standard deviation for the individual subjects based on the perturbation direction  is shown in Fig. The interaction dynamics were estimated using linear regression, as discussed in the ‘data acquisition and analysis’ section. Out of 400 trials, 28 trials were excluded from analysis due to the estimated stiffness being outliers. The average stiffness across all subjects and conditions is 106.819±63.741 N/m. The stiffness values for different conditions and perturbation direction can be found in the Table R2 values between the estimated and measured interaction forces of all 40 trials were 0.568±0.108 (subject 1), 0.567±0.087 (subject 2), 0.533±0.078 (subject 3), 0.578±0.084 (subject 4), 0.528±0.047 (subject 5), 0.559±0.062 (subject 6), 0.539±0.072 (subject 7), 0.516±0.040 (subject 8), 0.520±0.071 (subject 9), and 0.582±0.175 (subject 10).The acceptability of the linear regression result was evidenced by the low positive estimates of endpoint inertia (0.207±0.275 kg) and damping (9.114±8.321 Ns/m) parameters, which were expected of relaxed human arms and with the low intrinsic mechanical impedance of the robot arm. The mean equilibrium position of the hand was estimated to be at 0.027±0.045 m  away from the workspace center towards the robot. To further address the reliability of the interaction dynamics estimation, the interaction forces were estimated using the estimated inertia, damping, stiffness, and the measured hand trajectory. This was then compared against the actual interaction force. The mean and standard deviation of the 29. Therefore, parametric tests can be utilized for analysis. The stiffness data between the two curved trajectories . Hence, stiffness measurements from all curved trajectory trials were grouped and labeled as a single ‘after deviation’ condition. All stiffness measurements from the straight trajectory trials were labeled as ‘at deviation’ condition.The skewness and the excess kurtosis of the estimated stiffness values were 1.275 and 1.472, which indicated that the distribution cannot be considered substantially non-normalp>0.090). Likewise, there is no significant interaction between the subjects and the conditions , which implies that the trend in variation of the arm stiffness in lateral to forward direction is not consistent among subjects . This suggests that the arm stiffness in different directions varies differently based on when it was measured—that is, before or after the deviation in Fig. We performed a three-way ANOVA with the following factors: subject—10 levels, conditions—2 levels (‘at deviation’ and ‘after deviation’), and perturbation direction—2 levels . The omnibus test Table  revealedsee Fig. . Likewisp<0.0001). That is, the arm stiffness was lower when the robot trajectories may or may not make a curve  showing strong inter-subject variability. The main effect of the perturbation direction was also significant (p<0.0001), consistent with the well-known elliptical shape of 2-D stiffness reported in the literature30.The main effect of the conditions on stiffness estimates was significant. The stiffness of the arm was significantly higher in the ‘after deviation’ condition than in the ‘at deviation’ condition  when the subjects would be compelled to pay attention to the haptic cues from the robot and (b) when they are not compelled to. The former is implemented as the ‘at deviation’ trials where the subjects were perturbed at the exact location at which the robot turns in 50% of the trials . This indicates that humans modulate their arm stiffness during overground physical interaction tasks. The modulation was related to whether they were compelled to listen to haptic information (i.e. ‘at deviation’ condition) or not (i.e. ‘after deviation’ condition). Note that the arm stiffness modulation was completely voluntary - the subjects were free to choose how stiff or compliant their arm could be during the entire experiment. The results imply that the subjects may have lowered their arm stiffness in the forward direction due to the need for more sensitivity in haptic communication ‘at deviation’. When less sensitivity was required ‘after deviation’, their forward arm stiffness was higher. In the lateral direction, this modulation did not occur, which may imply that the need for communication was less in that direction.We observed that the arm stiffness was significantly different in ‘at deviation’ condition trials compared to ‘after deviation’ condition trials  was decided freely by the subject as needs arose. This study implies that the said ‘need’ could be the need for higher sensitivity during times when the robot’s movement was more unpredictable.Our result is consistent with the hypothesis in our prior studyThe modulation of the stiffness in the forward direction was significant, whereas in the lateral direction, the modulation was not significant Fig. . This ma30. The shape and size of the 2-D stiffness ellipse depends on the posture22, where the ellipse eccentricity is higher as the hand gets further away from the chest. In our study, the forward stiffness is higher than the lateral stiffness in the ‘after deviation’ condition, indicating high eccentricity and suggesting that the hand may have been further away from the chest than it was during the ‘at deviation’ condition. The modulation of stiffness with posture (instead of muscle activity) is more likely given the low stiffness values that are comparable to the stiffness of the relaxed arms in22. Future experiments with arm kinematics and electromyography may address this hypothesis.Another possibility is that the modulation of stiffness may have been a result of the change in the arm postureThe main effect of the subjects was significant, which was expected because every individual is different and might respond to similar perturbation differently. It is not uncommon to have higher inter-subject variability in experiments involving humans, especially with regards to bio-physiological variables such as arm stiffness. More variance could have been added due to the nature of the experiment having high degrees of freedom as an overground experiment . Factors such as the subjects’ walking habits (posture), height, or arm muscle cross-section all would contribute to this variability.While the robot’s velocity setpoint did not differ by trials in this experiment, future usage of Ophrie may include applications with varying velocity profiles including the maximum speed. Our observations suggest that the human user’s arm stiffness would be lower when predictability is lower with the robot partner. The robot’s speed, by itself, does not increase or decrease predictability and therefore may not affect the arm stiffness of the user. However, the rate at which the robot reaches its setpoint speed (fast or slow) may motivate the human user to change their arm stiffness values, since higher acceleration may be perceived as lower predictability. For example, an overall slower robot with frequent periods of high acceleration and deceleration may be perceived as less predictable than a faster robot with a slow rate of change of speed.31. It may be argued that the robot partner would feel more human-like to the user if the robot arm’s mechanical impedance was similar to that of a typical human. In this view, the low impedance of the robot arm may provide an impression of attentiveness to the user’s intent. The robot may also selectively lower the stiffness in specific directions to communicate its movement intent or role-taking to the user12.The arm stiffness modulation observed in this study has further implications in the design and control of robots for overground pHRIA total of 10 healthy young adults  without any self-reported neurological disorder took part in the study. Subjects signed a written informed consent form before participating in the experiment. The research protocol was approved by the Institutional Review Board of the University of Missouri System, and was performed in accordance with relevant guidelines and regulations.28.A guided walking experiment was designed wherein the human participants were asked to walk alongside the robot Ophrie—that acted as a leader—while holding the robot’s handle. Ophrie was specially designed for the overground pHRI experiments. Its manipulator possesses unique characteristics that are critical to overground physical interaction experiments involving humans, such as low inherent endpoint impedance, the ability to apply and measure small force perturbations (<10 N), and the capability of endpoint movement speed of 0.845m/s assuming cyclic motion of 2 Hz within the workspacex0.15 m2 2-dimensional space parallel to the ground plane  is the location of the end point with respect to the center of the workspace , K is the scalar level of background stiffness, and  to capture the directional differences in the arm stiffness. The choice between +x and -x or between +y and -y could be made arbitrarily since the model used in this study and in the literature15 assumes linearity. It is well known that the 2D-constrained arm presents a 2D stiffness ellipse15. Since arm stiffness is affected by the arm configuration30, the measurement of stiffness ellipse requires that perturbation is provided at the same arm configuration across multiple trials. This was possible for seated experiments such as22 where the subject’s trunk location was fixed with respect to the robot. However, this is not possible for overground pHRI experiments where the trunk location with respect to the robot must be allowed to vary . Thus the two directional arm stiffness were measured and reported as individual scalar values. The number of directions was limited to two to avoid excessively long experiments that could result from having three or more perturbation directions. The varying arm configuration in overground pHRI experiments was also why force perturbation was chosen over position perturbation in this study, since the latter requires a knowledge of the expected position or trajectory of the arm movement at the time of perturbation32.The arm stiffness measurements were made in two orthogonal directions  and the direction of force perturbation (2 levels). The experiment was designed as a Randomized Complete Block (RCB) design. Each block consisted of eight randomly distributed trials where there were two ABDEF, two ABGHI, and four ABC trajectories. The ABC trajectory constituted the ‘at deviation’ condition and the two deviated trajectories constituted the ‘after deviation’ condition. There were the same number of trials for straight versus deviated trajectories such that the same number of measurements could be made for ‘at deviation’ and ‘after deviation’ conditions. Each of these groups had an equal number of forward and lateral perturbations. There were 5 blocks totaling the number of trials per subject to 40. Each experiment on one subject took approximately 2.5 hours.36.f is the interaction force measured after the onset of force perturbation, x, m, b, and k are the inertia, damping, and stiffness from the interaction dynamics, respectively. The interaction dynamics is the function of arm movement and its derivative. A widely used simplified model was adopted in this study where we assume the interaction dynamics in task space as passive, linear, and time-invariant second-order dynamics27. The interaction forces were measured using the mini45 force/torque sensor , which is lodged between the endpoint of the robotic arm and the interaction handle. All data were recorded with a sampling rate of 1 KHz. After recording, the hand position and force data were filtered in Matlab  using a Butterworth low-pass zero-lag filter with 40 Hz cutoff frequency.The hand position with respect to the robot can be calculated using the motors’ encoder positions and forward kinematics. The motor shaft position was measured with the single-turn absolute Biss sine encoder (2048 LPR) embedded in the servo motors . The kinematic details of the robot arm can be found inm, b, and k in Equation k) is the main outcome of interest, whereas the inertia (m) and the damping (b) were only used to check the validity of the estimation.From the measured hand position data during perturbation, we used the earlier portion of the arm movement response that shows the characteristics of a passive second-order system. First, the hand velocity versus time graph was plotted in Matlab and the time instant of the second peak  was manually identified, since the general trend of the arm movement up to this instant were similar across subjects and also resembled the second-order response. Then, the time interval from the perturbation onset to the time of second peak was selected as the period of data for analysis—which was on average 438.5±113.7 milliseconds. The perturbation force, hand position, and hand velocity during the 800 ms perturbation for a representative subject can be seen in Fig. th or 75th quantiles—were screened out using JMP statistical software . ANOVA was performed using JMP on the remaining 372 stiffness values. A significance level of p=0.05 was used.Each trial from the experiment fetched one scalar stiffness value in either forward or lateral direction. It can be considered as the arm’s resistance to an external perturbation towards the axis of the perturbation. Out of 400 trials performed by 10 different subjects, 17 were discarded because the stiffness estimates were negative. From the remaining 383 trials, 11 outliers—defined as values more than 3 times the interquantile range past the 25


Starch is an inexpensive, easily accessible, and widespread natural polymer. Due to its properties and availability, this polysaccharide is an attractive precursor for sustainable products. Considering its exploitation in adhesives and coatings, the major drawback of starch is its high affinity towards water. This study aims to explain the influence of the silane-starch coating on the hydrophobic properties of paper. The analysis of the organosilicon modified starch properties showed an enhanced hydrophobic behavior, suggesting higher durability for the coatings. Molecules of silanes with short aliphatic carbon chains were easily embedded in the starch structure. Longer side chains of silanes were primarily localized on the surface of the starch structure. The best hydrophobic properties were obtained for the paper coated with the composition based on starch and methyltrimethoxysilane. This coating also improved the bursting resistance and compressive strength of the tested paper. A static contact angle higher than 115° was achieved. PDA analysis confirmed the examined material exhibited high barrier properties towards water. The results extend the knowledge of the interaction of silane compositions in the presence of starch. Starch, along with cellulose and lignin, is perceived as one of the most attractive biopolymers due to its properties, availability, and low price. Starch is a polysaccharide of plant origin. Sources include roots , tubers , stems , cereal grains , and legumes , among others . Starch However, available methods of starch usage in the industry do not guarantee sufficient hydrophobization of paper products used in high moisture conditions, especially transport packaging . SpiridoThe use of the starch properties to create colloidal solutions in hot water and its film-forming properties to evenly distribute compounds with hydrophobic characteristics, such as organosilicon compounds on the surface of biodegradable materials, appears to be a sustainable method of hydrophobization ,10,11. TSurface sizing and creating a film on a cellulose carrier is one of the most common operations used in the paper industry ,21. In tThere are known methods of paper hydrophobization using alkoxysilanes with substituents  with Energy Dispersive X-Ray (EDX) analysis and hydrophobic properties  between starch and organosilicon compounds in their application on a paper as a cellulose matrix. This study aims to explain the influence of the structure of the silane-starch coating on the hydrophobic properties of paper.The liquid–paper interaction is a very complex phenomenon. This process can be divided into two main stages: wetting and penetration. During the wetting stage, the water contact with the paper surface is hampered due to an air film adhering to the surface and air presence inside the surface pores. After the wetting stage, water finally begins to enter the paper structure and displaces air from the pores. The internal specific surface area of paper and its thickness increase, and micro-bubbles of air are observed within the adsorbed water. These air micro-bubbles are scattering centers for ultrasonic radiation; thus, a lower intensity signal is transmitted through the sample. The ultrasonic signal decreases faster as the liquid penetrates faster into the paper. The results of the measurements of the dynamics of water penetration into the structure of the paper samples with the coatings applied and the reference paper (without the coating) are presented in The results presented in the table are confirmed by the curves presented in N-(2-Aminoethyl)-3-aminopropyltrimethoxy silane (AATMS) is much less effective (63°). These results  or containing amino groups (AATMS), regardless of their solubility in water, formed a compact structure on the surface of the aqueous solution of starch. Notably, the production of a similar coating on the starch surface was not tantamount to giving the hydrophobic properties of cellulose` materials. The accumulation of starch-containing NTES sparingly soluble in water on the surface of the paper created a water barrier, which was confirmed by the results of the high contact angle and PDA analysis. The use of AATMS, which is soluble in water under the same conditions, did not increase the hydrophobic properties of the material.w/w) to reduce the temperature of starch gelatinization. It also allowed the starch structure to loosen, as reported by other authors [w/w). Organosilicons used in the current study are shown in Commercial wheat starch was used in the presented research . The amylose content in the starch was 31.2 ± 0.16 wt%. The lipid content was 0.25 ± 0.02 wt% and the protein content was 0.30 ± 0.01 wt%. An aqueous solution of wheat starch (5% concentration) was stirred at 70 ± 5 °C in the presence of sodium hydroxide -3-aminopropyltrimethoxy silane (AATMS), and tetraethoxysilane (TES).Organosilicons used in the presented research have carbon chains of different lengths: methyltrimethoxysilane (MTMS), n-octyltriethoxysilane (NTES), 2 for cellulose paper (Whatman cellulose no. 3), by dipping it in the organosilicon solution (5 s) and letting it dry at room temperature for 48 h. Morphological and chemical characterization was performed to understand the hydrophobization mechanism.These four formulations were applied as a coating, using 100 g/mWater penetration dynamics for paper samples coated with the starch-silane coated cellulose was measured using the PDA apparatus, Module S 05 (Emtec Electronic GmbH). The hand sheets were cut into three samples, each with a size of 3 cm × 7 cm. Measurements were performed separately for each sample in demineralized water at 20 °C, following the standard procedure described by the manufacturer . The folA PGX+ Goniometer from Testing Machines Inc.  was used for the contact angle measurements. The tests were carried out according to the TAPPI T 458 standard method. The water drop volume was 4 μL.Water uptake test was conducted according to ISO 535:2014 . Time of the test was 60 s.Compressive strength (SCT test) was conducted according to ISO 9895:2008 standard . Bursting strength was conducted according to ISO 2758:2001 .The surface morphology of the samples was examined by the SEM method with cryogenic preparation system (Cryo-SEM), which enables direct investigation of the sample in the vitrified state. Cryo-scanning electron microscopy effectively observes wet samples without causing drying artifacts. It preserves the “natural” internal structure of the sample. This is particularly true for samples in the life sciences, where structures will retain their original conformation only in their fully hydrated state. Images were taken using the JEOL JSM-7001F TTLS  scanning electron microscope equipped with the PP3000T cryo-SEM preparation system, which allows the cryo specimen to be prepared, processed, and transferred into the SEM chamber. The samples were cryo-fixed by plunging them into sub-cooled nitrogen  and transferred by the vacuum cryo-transfer shuttle to the preparation chamber mounted onto the SEM. Inside the preparation chamber, at −185 °C, the specimen was fractured to expose a fresh surface. Then, it was sublimated and coated with a thin platinum layer. Finally, the sample was loaded under vacuum into the SEM chamber, where it remained frozen during imaging on the cold-stage, cooled by a nitrogen carrier (−190 °C). The images of all samples were taken by applying an accelerating voltage of 10 kV and using a secondary electron (SEI) detector. Elemental analysis and cryo-SEM elemental mapping were performed using the energy dispersive microanalysis (EDX) mode of an X-ray-equipped SEM that also applied a voltage of 10 kV.The interaction of water and cellulose coated with starch modified with organosilicon compounds depends primarily on the physical properties and structure of the organosilicon compound itself. Based on the research, it can be concluded that the distribution of silicon in the gelatinized starch network depends primarily on the length of the organic chain in the organosilane molecule. MTMS molecules, which have very short side chains (methyl group), were easily embedded in the starch structure. Silanes containing longer side chains  were localized mainly on the surface of the starch structure. The distribution of silanes resulting from their size does not translate unequivocally into making the coated materials hydrophobic. The physical properties of the organosilicon compounds primarily determine these properties. Poorly soluble substances such as NTES increase the hydrophobic properties, and water-soluble substances such as AATMS do not improve these properties. An even distribution of silane in starch and the developed permanent MTMS-starch bond indicate that this composition may be considered as a new type of hydrophobic agent for papermaking purposes. Static contact angle analysis and PDA analysis confirmed the high barrier properties of the examined material towards water. NTES containing an octa-carbon side chain in its structure produced a thin film on the starch surface, which also increased the barrier property of the coated paper. Despite producing a thin film on the starch surface, silane containing hydrophilic amine groups in its side chain did not provide barrier properties to the paper.


In silico methods for immune epitope prediction have become essential for vaccine and therapeutic design, but manual intra-species comparison of putative epitopes remains challenging and subject to human error. Created initially for analyzing SARS-CoV-2 variants of concern, comparative analysis of variant epitope sequences (CAVES) is a novel tool designed to carry out rapid comparative analyses of epitopes amongst closely related pathogens, substantially reducing the required time and user workload. CAVES applies a two-level analysis approach. The Level-one (L1) analysis compares two epitope prediction files, and the Level-two (L2) analysis incorporates search results from the IEDB database of experimentally confirmed epitopes. Both L1 and L2 analyses sort epitopes into categories of exact matches, partial matches, or novel epitopes based on the degree to which they match with peptides from the compared file. Furthermore, CAVES uses positional sequence data to improve its accuracy and speed, taking only a fraction of the time required by manual analyses and minimizing human error. CAVES is widely applicable for evolutionary analyses and antigenic comparisons of any closely related pathogen species. CAVES is open-source software that runs through a graphical user interface on Windows operating systems, making it widely accessible regardless of coding expertise. The CAVES source code and test dataset presented here are publicly available on the CAVES GitHub page. An epitope, also referred to as an antigenic determinant, is a part of an antigen recognizable by the immune system and elicits specific immune responses. In silico approaches for epitope prediction have become essential for vaccine development and the examination of antigenic evolution in pathogens of interest, especially as the Coronavirus Disease 2019 (COVID-19) pandemic progresses and the world fights to contain emerging SARS-CoV-2 variants of concern (VOCs). Exemplified by the Immune Epitope Database (IEDB), in silico epitope analysis tools are widely applied in research and development settings, attributable to their high time efficiency and cost-effectiveness in addition to their constantly improving accuracy .,17.16,17In multiple occurrences, epitopes from the same originating sequence were predicted as overlapping 15-mer epitopes spanning a larger cumulative region. This was seen throughout the reference and alpha VOC epitope predictions, as these larger overlapping peptides were present in all L1 categories . The S receptor-binding domain  and the receptor-binding motif  are of particular interest due to their interactions with the host ACE2 receptor . The L1 CAVES ran the required indel search, L1, and L2 analyses on the SARS-CoV S epitope sequences, producing results in approximately 3.6 seconds. The numbers of epitopes sorted into each L2 category are detailed in L1E_L2E contained L1 epitopes that were exact matches between the reference and the alpha VOC, additionally finding an exact match to a pre-existing database epitope in L2. This category contained 82 putative epitopes , spanning both the S1 and S2 subunits of the S gene . Thirty-L1E_L2P contained epitopes that were exact matches in L1 but found only partial matches with database epitopes in L2. Consequently, this category was large, with 444 partial matches spanning the full length of the S gene . These wL1E_L2N contained 15 L1E epitopes that did not have any degree of match with the database epitopes in L2 . This inL1P_L2E reported 85 putative epitopes from L1P that had exact matches to database epitopes in L2 . Of these, 58 were within the S1 subunit, with 39 in the RBD and 11 of these covering the RBM ,18. The L1P_L2P contained 462 epitopes from L1P that had partial matches to database epitopes in L2 . Match lengths spanned from 1–14 amino acids, with 12 as the most common match length and matches covering the full length of the S gene .L1P_L1N contained partially matching epitopes from L1 that did not find a match with any database epitope in L2. This included 16 epitopes, 7 from the reference and 9 from the alpha VOC, with 13 located in the S1 subunit and 3 in the S2 subunit. L1P_L2N included the reference sequence epitope covering the A570D region described in the L1 results above .L1N_L1E contained unique putative epitopes from L1N that had exact matches to database epitopes in L2. This included six matches, all of which involved L1N epitopes derived from the reference sequence. Three matches were within the S1 subunit, and three were in the S2 subunit . These eL1N_L2P reported 49 L1N epitopes  that found partial matches to database epitopes in L2. Match lengths spanned from 1–14 amino acids with overlaps of 1, 6, 9, 10, and 14 tied for the most common length. Thirty-one matches were within the S1 subunit, with eight of these in the RBM and five covering the Furin cleavage site ,18. EighL1N_L2N contained three epitopes from L1N that were unable to match to any database epitopes in L2. These epitopes were all from the alpha VOC and covered the del69–70, D614G, and S982A mutations, as described in Env epitope prediction with the IEDB-AR Tepitool produced 679 epitopes for subtype B and 653 epitopes for subtype C. As the panel of the 27 most frequent A and B alleles was used during both TepiTool runs, duplicate epitopes existed in each file with different HLA alleles and percentile scores. Without duplicates, the subtype B and C files contained 242 and 229 unique epitopes, respectively. The Los Alamos database list of best-described CTL/CD8 + epitopes contained 42 epitopes within the Env region that were used in CAVES L2 analysis.CAVES ran the required indel search, L1, and L2 analyses on the HIV-1 Env epitope sequences, producing results in approximately 5.8 seconds. All epitopes fulfilled the criteria for the L1 and L2 categories they were sorted into, and no epitopes were misplaced. All mismatched positions were successfully reported by CAVES to be in the L1N category and corresponded to mutations previously identified  between the subtype B and C sequences. The number of epitopes sorted into each CAVES results category is detailed in Pairwise comparison of highly similar sequences from pathogen sub-variants, directly based on their predicted epitope profiles, allows for analysis of their antigenic evolution, primarily driven by host immunity. By comparing putative epitopes of the spike proteins from two SARS-CoV-2 lineages, we present CAVES as a novel tool and demonstrate its usage to address the challenges in comparative epitope analysis of two closely related viral variants. This work is further supported by an additional comparative analysis of putative epitopes from the envelope proteins of two HIV-1 subtype references. CAVES fills a gap in the current public toolset as, to the best of our knowledge, no other program currently exists to compare variant epitope sequences while accommodating substitutions, insertions, and deletions that alter the amino acid sequence. We showed that CAVES L1 analysis successfully compared all putative epitopes in both the SARS-CoV-2 and HIV-1 analyses and accurately binned them into the exact match (L1E), partial match (L1P), or novel (L1N) categories. Furthermore, CAVES L2 comparison using the database search results will quickly distinguish, for all L1 epitope match categories, which epitopes have or have not been experimentally validated, effectively allowing users to identify novel areas of study and accelerate global research efforts. CAVES allows users to run only one of its two levels of comparison or both as a bundle to satisfy varied application needs.From an evolutionary perspective, these data are very informative in defining the evolutionary trend of SARS-CoV-2. For instance, the protein regions where the L1E and L1P epitopes reside could be considered relatively conservative. In contrast, the L1N epitopes present only in the earlier reference strain were lost when the virus evolved into the alpha lineage. Conversely, the L1N epitopes found only in the alpha VOC were gained when the virus mutated. This hypothesis is well-supported by the observation that all signature mutations in the alpha strain are associated with the novel epitopes identified by CAVES ,17. It iWhile initially designed to study SARS-CoV-2 VOCs, CAVES will undoubtedly be applicable to any emerging or re-emerging SARS-CoV-2 variants, which require continued monitoring of epitope changes to better understand how the virus evolves antigenically as the pandemic continues. Apart from SARS-CoV-2, CAVES is also broadly applicable to any other viral or bacterial pathogens with sub-lineages or variants of similar genomic/proteomic sequences, as demonstrated here by the additional analysis of HIV-1 epitopes. Likewise, the CAVES methods presented here in the context of SARS-CoV-2 evolution could be applied for evolutionary analysis of any similar pathogens so long as the compared sequences are similar enough to allow for meaningful comparison. Moreover, CAVES simplifies the workflow of such comparative analyses and significantly improves time efficiency, making in silico methods much more feasible for a broader range of studies.The IEDB-AR was used for its exemplary epitope prediction tools, and the resulting output formats were defined as the standard input for CAVES. Notably, CAVES could be easily adapted to compare the output from any epitope prediction tools for in-depth comparison or computational validation. There are many computational tools available for T cell epitope prediction, and the IEDB-AR B cell linear epitope prediction tool itself hosts seven different methods to choose from . OftentiIt is important to note that the sequence positions for any IEDB database-derived epitopes used in CAVES L2 are not relative to the given input sequence used to query the IEDB but instead to the source protein that the epitope was cataloged under in the database. The IEDB utilizes NCBI taxonomy and accessions to provide standardized nomenclature for protein data wherever possible and provides these with each epitope record . Using tCAVES identifies unique and matching epitopes, given input data derived from epitope prediction tools and IEDB epitope database inquiry. Consequently, the ability of CAVES to find biologically relevant matches is limited by the epitope prediction data it is provided with, and unsuitable input data will not yield meaningful results. For instance, the quality and application value of CAVES L1 results would depend on the epitope prediction tool used for CAVES input data generation. Similarly, the accuracy and breadth of the L2 results would be determined by the comprehensiveness of the IEDB database of confirmed epitopes at any given time. As such, the results demonstrated here will only be reproducible while the IEDB-AR epitope prediction tools and the IEDB database entries remain unchanged or when pre-made files from the provided sample dataset are used.It is acknowledged that both CAVES L1 and L2 comparisons render a large amount of data in the partial match categories, which can be tedious to sort through. With epitopes in these categories, we prioritized the comprehensiveness of CAVES over the simplicity of its results and sorted all matches that did not fulfill the exact match or novel criteria into this middle category. While it is justifiable to focus only on the exact match and novel epitopes with CAVES, we believe that partial matches may still contain meaningful information. For example, if comparing two epitopes that both have a length of 15 amino acids, it is plausible that a match of 14 or 13 amino acids may still be useful for the study purpose. In contrast, a match of only one or two amino acids may be negligible for the user. Rather than setting a length threshold for defining what constitutes a partial match, we leave it to the user to decide how much overlap is required to be relevant. As CAVES results are produced in Excel spreadsheets, we strongly suggest taking advantage of the Excel filter function to aid in this analysis.CAVES was designed for the comparative analysis of closely related but genetically distinct sequences, and thus, any mutations, including in-frame indels, contained within putative epitopes could be of significance for studying antigenic evolution in mutated regions of the genome. By reading the start positions of each epitope and knowing where any indels occur, CAVES will reposition the sequence locations accordingly to accommodate shifted sequences during the matching process. Using sequence positions also allows CAVES to speed up its search process by only comparing epitopes from overlapping regions. While CAVES accounts for in-frame indels, it will not accommodate frameshift nucleotide mutations that lead to fundamentally new, unrelated proteins that are thus unsuitable for CAVES comparison. Recognizably, this inaugural version of CAVES only accommodates the analysis of datasets derived from two original protein sequences for each run, which limits its application if working with large numbers of sequences.In conclusion, here, we present a novel bioinformatics tool that automates the comparative analysis of putative epitope profiles derived from relevant sequences with high similarity. While initially designed to compare SARS-CoV-2 variants, CAVES is applicable for antigenic evolutionary analyses of any pathogens with sub-lineages or sub-variants. This freely available software represents a new addition to the public toolbox that addresses the increasing need for in-depth immune epitope analysis.


Arenga pinnata palm starch (APPS), glycerol and benzoyl peroxide simultaneously in the twin-screw extruder. Morphology analysis of TPS revealed that the starch granules were damaged and gelatinized in the extrusion process. No phase separation is observed in TPS, which exhibits that starch granules with and without benzoyl peroxide were uniformly dispersed in the matrix. The addition of benzoyl peroxide resulted in increased density of TPS from 1.37 to 1.39 g·cm−3, tensile strength from 7.19 to 8.61 MPa and viscosity from 2482.19 to 2604.60 Pa.s. However, it decreased the elongation at break of TPS from 33.95 to 30.16%, melt flow rate from 7.13 to 5.73 gr/10 min and glass transition temperature from 65 to 52 °C. In addition, the thermal analysis showed that the addition of benzoyl peroxide increased the thermal stability of TPS and extended the temperature range of thermal degradation.Thermoplastic starch (TPS) has three main disadvantages, i.e., poor mechanical properties, low thermal stability and water sensibility. To overcome these disadvantages, TPS properties can be improved by starch modification, adding reinforcements and blending with other polymers. In this research, to prepare modified TPS, starch modification was carried out by in situ modification. The modified TPS was prepared by adding  Arenga pinnata palm starch (APPS) is also considered an agro-industrial residue in the agricultural industry [A. pinnata palm starch can be obtained from the core of an unproductive (in terms of sugar and fruit) A. pinnata palm tree’s trunk [A. pinnata tree grows in more humid parts of subtropical and tropical areas [A. pinnata can produce about 50–100 kg of APPS [−3 [A. pinnata palm starch has been used as a material for the preparation of TPS [−3, 4.8 MPa and 38.10%, respectively [−3 [Thermoplastic starch (TPS) is considered one of the most promising alternatives to fossil-based ones for disposable packaging material applications , mainly industry . A. pinn’s trunk ,7. A. pial areas . It is wal areas  and Taiwal areas ,7 and soal areas . Therefo of APPS ,10. The  of APPS  with 36. of APPS  amylose APPS [−3 . The gelAPPS [−3 . In addin of TPS ,10,11. TTPS can be prepared by starch into TPS  under heHowever, compared to conventional plastics, TPS has three main drawbacks, i.e., low mechanical properties, low thermal stability as well as water and humidity sensibility ,17,20. TIn the oxidized starch, the purpose of oxidation is to generate more functional groups, i.e., carbonyl and carboxyl, thereby increasing the functionality and reactivity of native starch. The preferred oxidants are hydrogen peroxide, sodium hypochlorite, potassium permanganate, chromic acid, nitrogen dioxide , ozone aModifying starch by oxidation to obtain modified TPS has disadvantages, i.e., it requires time and cost and the process steps become longer because the starch is modified first and then the oxidized starch produced is used as raw material for TPS. The objective of this study is to overcome these disadvantages, in particular shortening the modified TPS preparation time. Therefore, in this study, starch modification was carried out via an in situ process simultaneously with the preparation of TPS with the extrusion process in the twin-screw extruder. Arenga pinnata palm starch (APPS) with an amylose content of 23.19% was obtained from the local industry located at Klaten, Central Java, Indonesia. Glycerol (CAS 56-61-5) and benzoyl peroxide (CAS 94-36-0) were purchased from Merck, Darmstadt, Germany.In this research, The preparation of thermoplastic starch (TPS) was conducted by a twin-screw extruder ,29,30,31−1.The functional groups of the APPS, TPS and TPSB were obtained using Fourier-Transform Infrared Spectroscopy . Thus, 32 scans were recorded for each sample while it was set in attenuated total reflectance (ATR) mode with a diamond ATR crystal at a wave number range between 500 and 4000 cmThe density of the APPS, TPS and TPSB was measured (five replicates) in accordance with the ASTM D 792 standard at 23 °C and 50% relative humidity.The surface morphology of APPS and the cross-section morphology of TPS and TPSB were observed using scanning electron microscopy  at 3 kV for APPS and 10 kV for TPS and TPSB. After submersion in liquid nitrogen, the TPS and TPSB samples was broken. The surfaces had a thin gold layer applied to the sample before analysis.The crystallinity of APPS, TPS and TPSB was studied by X-ray diffractometer . The X-ray diffraction pattern observed the 1.54 wavelength, 40 kV voltage and 15 mA filament emission. The radiation reflection of APPS, TPS and TPSB was measured at the 2θ angle of 5–50°.The mechanical properties of TPS and TPSB were determined using an electronic universal testing machine (UTM) , in accordance with ASTM D 638-14 standard, at 21 °C and relative humidity around 50%. Thermo Scientific Haake MiniJet Pro is used to prepare a dumbbell (dogbone) tensile test sample specimen according to ASTM D 638-14 type V. Five specimens of each type of TPS and TPSB film were calculated at tensile rates of 10 mm/min. Each sample was randomly measured three times at various locations using a digital thickness gauge  to determine the film thickness.The rheological properties of TPS and TPSB were studied using the Melt Flow Index (MFI) Instrument Ceast Model 7026, in accordance with ASTM E 1238. Measurement of melt flow rate (MFR), viscosity and shear rate value was carried out at 190 °C with load 2.16 kg.The thermal properties of APPS, TPS and TPSB were characterized using differential scanning calorimetry (DSC) . Each sample was heated until 200 °C with a 10 °C/min heating rate under a nitrogen atmosphere (flow rate = 20 mL/min).The stability of APPS, TPS and TPSB was studied using a thermogravimetric analyzer . A crucible pan containing 5 mg of sample was heated from 25 °C to 600 °C at a rate of 10 °C/min. A flow of 20 mL/min was used to purge the nitrogen gas.−1, associated with the hydroxyl group from APPS and glycerol, while the absorption peak at a wave number of about 2921 and 2884 cm−1 was associated with the C–H group in starch [−1 is the H–O–H vibration of water molecules in the amorphous region, which may be absorbed in the samples [−1 are associated with CH2 bending and wagging (out of plane bending) of CH2. The absorption peaks between 1500 cm−1 and 1200 cm−1 overlap each other between C–H stretching and O-H bending, making it difficult to distinguish the difference in the absorption peaks in this part of the spectrum [−1 and 900 cm−1 is the vibration of a functional group of C–O, C–C and C–O–H. Similar absorption peaks of APPS were also reported by previous studies [The FTIR spectrum of APPS, TPS and TPSB are shown in n starch ,26. In a samples ,18. The spectrum . Fuente spectrum  reported studies , while t studies . Zhang e studies  reported studies . In addi−3, respectively. The presence of glycerol as a plasticizer destroys and weakens the inter- and intra-molecular hydrogen bonding between starch molecules, thereby increasing the free volume and mobility between molecular chains and decreasing the density of TPS [−3 [−3 [−3 to 1.39 g·cm−3. The higher density value of TPSB compared to TPS is, presumably, because the TPSB molecules are arranged more neatly and orderly so that the density value is greater than the TPS density value. The extrusion process is expected to produce thermoplastic materials with more neatly and consistently ordered molecules [The density of APPS, TPS and TPSB is shown in y of TPS ,11,25. Polecules . In addiThe morphology of APPS, TPS and TPSB was examined by SEM. The samples were fractured in liquid nitrogen before testing. The X-ray diffraction pattern of APPS, TPS and TPSB shows the semi-crystalline  characteristic, as can be observed in H, VA or EH crystallinity, which is formed during the thermo-mechanical process [In addition, at TPS and TPSB, new diffraction peaks also appeared at 2θ 13.39°, 18.13° and 20.68°. These peaks are characteristic crystallinities type VH, which is formed during thermo-mechanical processing in the twin-screw extruder [ process . The extThe mechanical properties in the form of tensile strength, elongation and elastic modulus of TPS and TPSB are shown in The stiffer TPSB results in limited mobility of the molecular chain in TPSB, thereby reducing flexibility, which results in a decrease in elongation at break. In addition, in the extrusion process, depolymerization also occurs, which facilitates the tendency of molecular reassociation, with a greater potential for interaction . Therefo−1 to 7.63 s−1.The melt flow rate (MFR), shear rate and viscosity of TPS and TPSB are shown in The strong hydrogen bonds between oxidized starch with hydroxyl groups of starch will result in a decrease in chain mobility of TPSB, an increase in stiffness, an increase in viscosity and a decrease in shear rate, thereby reducing the melt flow rate of TPSB. The relationship between viscosity and shear rate has also been revealed : when thThe DSC curve and the glass transition temperature (Tg) APPS, TPS and TPSB are shown in Thermal stability of APPS, TPS and TPSB was studied by TGA. The thermal degradation curve of TPS also has two stages of thermal degradation, which are similar to those of APPS. The initial thermal degradation is also related to the evaporation of water. However, the initial thermal degradation of TPS has a different pattern with APPS and the thermal degradation temperature range in the early stages is higher, up to 180 °C. In addition to that, the peak of thermal degradation in the early stages of TPS occurs at a temperature of 150 °C. The main thermal degradation of TPS occurs in a temperature range of 260–400 °C and shows two main stages of thermal degradation. The first major thermal degradation is at a temperature of 260–310 °C, with a peak of thermal degradation at 290 °C, associated with the decomposition of the glycerol-rich phase, while the second major thermal degradation occurs in a temperature range of 310–400 °C, with a peak thermal degradation at a temperature of 330 °C, associated with the degradation of amylose and amylopectin starch. This result has good accordance with a previous study ,26. The Arenga pinnata palm starch was successfully carried out. Modified TPS was prepared by adding palm starch, glycerol and benzoyl peroxide simultaneously with the twin-screw extruder. Morphology analysis of TPS showed that the starch granules were damaged and gelatinized in the extrusion process. No phase separation is observed in TPSB, which exhibits that starch granules with and without benzoyl peroxide are uniformly dispersed in the matrix. The use of benzoyl peroxide in the preparation of TPS increases the density, tensile strength, viscosity and thermal stability as well as extending the thermal degradation temperature range of TPS. However, it reduces elongation at break, elastic modulus, melt flow rate, shear rate and glass transition temperature. The results of this study are expected to provide insight into the potential of A. pinnata as a new starch source for the development of biodegradable materials. In addition, in situ modification for the preparation of modified TPS with the extrusion process in the twin-screw extruder can reduce TPS preparation time because it only requires one preparation stage, meaning there is no need to modify the starch first. The characteristics of modified TPS produced in this study are expected to contribute to the further development of biodegradable materials.In this research, in situ modification for TPS preparation based on


In 2022 the International Brazilian Journal of Urology received the highest impact factor of his history and this fact was possible because the serious peer review process of our Journal . In thisThanks a lot!!!!!


Sympotthastia remained unstable. Our study revealed that the mitogenomes of Diamesinae are highly conserved, and they are practically useful for phylogenetic inference.Mitogenomes have been widely used for phylogenetic reconstruction of various Dipteran groups, but specifically for chironomid, they have not been carried out to resolve the relationships. Diamesinae (Diptera: Chironomidae) are important bioindicators for freshwater ecosystem monitoring, but its evolutionary history remains uncertain for lack of information. Here, coupled with one previously published and 30 new mitogenomes of Diamesinae, we carried out comparative mitogenomic analysis and phylogenetic analysis. Mitogenomes of Diamesinae were conserved in structure, and all genes arranged in the same order as the ancestral insect mitogenome. All protein‐coding genes in Diamesinae were under stronger purifying selection than those of other nonbiting midge species, which may exhibit signs of adaptation to life at cold living conditions. Phylogenetic analyses strongly supported the monophyly of Diamesinae, with Boreheptagyiini deeply nested within Diamesini. In addition, phylogenetic relationship of selected six genera was resolved, except  Here, coupled with one previously published and 30 new mitogenomes of Diamesinae, we carried out comparative mitogenomic analysis and phylogenetic analysis. Mitogenomes of Diamesinae were conserved in structure, and all genes arranged in the same order as the ancestral insect mitogenome. Phylogenetic analyses strongly supported the monophyly of Diamesinae, with Boreheptagyiini deeply nested within Diamesini. Since iIn this study, we provide 30 newly sequenced (nearly) complete mitogenomes from 30 species representing Boreheptagyiini (four species of one genus) and Diamesini (26 species of five genera) using next‐generation sequencing. We analyzed the genomic structure, base composition, substitution, and evolutionary rates among Diamesinae, expanding our knowledge of its diversity of mitogenomes. Coupled with published data, we carried out phylogenetic analysis of Boreheptagyiini and Diamesini based on 31 mitogenomes.22.1Field collection of 30 species were conducted in China during 2014–2020, using classical insect collection techniques such light traps, sweep traps, Malaise traps, and D‐nets. Specimens were preserved in ethanol , and stored at dark at −20°C before morphological and molecular analyses. The total genomic DNA was extracted from thorax of adult and middle larval bodies using a Qiagen DNA Blood and Tissue Kit (Qiagen) following the manufacturer's protocol. After DNA extraction, the cleared exoskeleton of thorax was mounted in Euparal on microscopy slides together with the corresponding wings, legs, and antennae following the procedures outlined by Sæther . The DNABoreoheptagyia species) and 26 species of Diamesini . Since mitogenomes of another four tribes were not available for current molecular study, we could not reconstruct the phylogeny of the whole subfamily Diamesinae. Therefore, by integrating one public Potthastia species (GenBank accession: MW373523), a total of 31 species of Boreheptagyiini and Diamesini were selected as in‐groups. In addition, we selected one Prodiamesinae species  and one Orthocladiinae species  as outgroups for phylogenetic analyses. Detailed information could be found in Table Thirty mitogenomes were newly sequenced in this study, representing four species of Boreheptagyiini . The raw sequencing reads were trimmed with Trimmomatic  barcode sequence for each species was obtained by Sanger sequencing herein and from previous study . Ribosomal RNA (rRNA) genes and protein‐coding genes (PCGs) were annotated by aligning with homologous genes of Potthastia sp. in Geneious. Newly sequenced mitogenomes were submitted to GenBank (accession numbers: pending). The mitogenome maps were drawn by the CG View server V 1.0   Figure . The sta) Figure .Boreoheptagyia, ND1 and ND4L showed a lower Ka/Ks value in Boreoheptagyia and in Pagastia, and the remaining ten showed a lower Ka/Ks value in Diamesa and in Pagastia  of all the 13 PCGs is less than 0.35, and the ATP8 exhibits the largest Ka/Ks value while the COI has the lowest Ka/Ks value Figure . To betta Figure . We alsoEach mitogenome of Diamesinae contains 22 typical tRNA genes, with A+T content ranging from 74.0% to 77%. The nucleotide skew of tRNA genes among Diamesinae is consistent, exhibiting positive AT‐skew and negative GC‐skew Figure . Both 123.2Sympotthastia was unstable  + (Diamesa + (Pagastia +Pseudodiamesa)))) was inferred from the PCG12 and PCG12R datasets; (ii) (Potthastia + (Boreoheptagyia + (Sympotthastia + (Diamesa + (Pagastia +Pseudodiamesa))))) was inferred from the AA dataset. The topology inferred from the AA dataset had the strongest nodal support. Based on three different datasets, Boreheptagyiini was deeply nested within Diamesini. The monophyly of Boreoheptagyia, Diamesa, Pagastia, Potthastia, and Pseudodiamesa was well supported by mitogenomes.Generally, six phylogenetic trees constructed by BI and ML analyses are similar in topology, only with the position of e Figure . The mone Figure . Within 44.1A total of 31 mitogenomes of Diamesinae are included in this study, of which 10 mitogenomes have incomplete control region by the highly gene duplication Cameron, . The len4.2Stenochironomus  + (Pseudokiefferiella + (Parapotthastia + (Onychodiamesa + (Diamesa +Lappodiamesa)))). Willassen  are sister to the remaining Diamesini. In our study, Potthastia is placed as the oldest of all Diamesinae genera studied here. Our result corresponds very well by Willassen ; Investigation ; Methodology ; Software ; Writing – original draft (lead). Zheng Liu: Funding acquisition (lead); Writing – review & editing . Li‐Ping Yan: Investigation ; Methodology ; Writing – review & editing . Xin Duan: Formal analysis ; Software . Wen‐Jun Bu: Supervision ; Validation . Xin‐Hua Wang: Investigation ; Supervision ; Validation . Chen‐Guang Zheng: Formal analysis ; Investigation ; Writing – original draft ; Writing – review & editing .The authors declare that they have no conflicts of interest.Supplementary MaterialClick here for additional data file.


Leptospira spp., there are five sphingomyelinases exclusively expressed by pathogenic leptospires, in which Sph2 is expressed during natural infections, cytotoxic, and implicated in the leptospirosis hemorrhagic complications. Considering this and the lack of information about associations between Sph2 and leptospirosis severity, we use a combination of immunoinformatics approaches to identify its B-cell epitopes, evaluate their reactivity against samples from leptospirosis patients, and investigate the role of antibodies anti-Sph2 in protection against severe leptospirosis. Two B-cell epitopes, Sph2(176-191) and Sph2(446-459), were predicted in Sph2 from L. interrogans serovar Lai, presenting different levels of identity when compared with other pathogenic leptospires. These epitopes were recognized by about 40% of studied patients with a prevalence of IgG antibodies against both Sph2(176-191) and Sph2(446-459). Remarkably, just individuals with low reactivity to Sph2(176-191) presented clinical complications, while high responders had only mild symptoms. Therefore, we identified two B-cell linear epitopes, recognized by antibodies of patients with leptospirosis, that could be further explored in the development of multi-epitope vaccines against leptospirosis.Sphingomyelin is a major constituent of eukaryotic cell membranes, and if degraded by bacteria sphingomyelinases may contribute to the pathogenesis of infection. Among  Leptospirosis is a tropical and neglected emerging zoonotic disease that afflicts humans and other animals . It is cLeptospira, which can infect humans and almost all mammals, as well as reptiles and amphibians  and other bacteria including Listeria ivanovii (SmcLUniprot ID: Q9RLV9), Bacillus cereus , Staphylococcus aureus (Uniprot ID: A0A7U4AUV1), and Pseudomonas spp. strain TK4 (Uniprot ID: Q93HR5) were conducted by MAFFT [Sequence alignment and identification of conserved patterns among sphingomyelinases from by MAFFT .Sequences predicted as antigenic linear B-cell epitopes were synthesized using fluorenylmethoxycarbonyl (F-moc) solid-phase chemistry ,42 , resulting in 51 leptospirosis patients (MAT positive), reactive in MAT, and 36 negative controls (MAT negative). The study was reviewed and approved by the Oswaldo Cruz Foundation Ethical Committee and the National Ethical Committee of Brazil (number CAAE: 31405820.8.0000.5262).Samples of confirmed leptospirosis cases and the control group were screened for the presence of naturally acquired antibodies against the synthetic peptides via ELISA as previously described .Briefly, MaxiSorp 96-well plates  were coated with 100 μg/mL of a peptide. After overnight incubation at 4 °C, plates were washed with phosphate-buffered saline (PBS) and blocked with PBS-containing 5% non-fat dry milk (PBS-M) for 1 h at 37 °C. Individual serum samples diluted 1:100 on PBS-M were added in duplicate wells, and the plates were incubated at 37 °C for 1.5 h. After three washes with PBS-Tween20 (0.05%), bound antibodies were detected with peroxidase-conjugated goat anti-human IgG  or goat anti-human IgM , diluted at 1:1000 (in PBS-M), and incubated for 1 h at 37 °C, followed by TMB . The reaction was stopped by the addition of HCl (1N), and the absorbance was read at 450 nm using an xMark™ microplate absorbance spectrophotometer . The results for total IgG and IgM were expressed as the reactivity index (RI)—the ratio between the mean optical density (OD) of tested samples and the mean OD of 44 control group samples plus 2.5 standard deviations (SD). Subjects were considered IgG responders to a particular antigen if the RI was higher than 1.p-value < 0.05 was considered significant.The obtained data were analyzed using GraphPad Prism 8.0 . First, to determine if a variable was normally distributed, the one-sample Kolmogorov–Smirnoff test was used. Differences in frequencies of IgG and IgM responders to synthetic peptides were evaluated using Fisher’s exact test, while the reactivity indices against synthetic peptides between responders to each epitope were compared using the Mann–Whitney test. A two-sided L. interrogans serovar Lai Sph2. In this way, considering that each amino acid residue was predicted as inserted in a linear B-cell epitope by at least five algorithms, we identified two sequences as continuous B-cell epitopes in the studied protein: GHDERAKRISKSDYVK (Sph2(176-191)) and TPTKSGHKKKYDQV (Sph2(446-459)). As shown in In this study, we used a combination of nine algorithms to predict linear B-cell epitopes from (176-191) and Sph2(446-459), were predicted as antigenic, presenting Vaxijen scores of 1.173 and 1.763, respectively.The protein Sph2 was considered a protective antigen, obtaining a score of 10.458 in the VaxiJen algorithm. Regarding predicted B-cell linear epitopes, both sequences, Sph2Leptospira spp. sphingomyelinases. Moreover, when compared with sphingomyelinases of other bacteria, Sph2 presented identities ranging from 42% to 49%  and Sph2(446-459) were considered non-conserved among microorganisms not belonging to the genus Leptospira, because they presented an E-value greater than one on BlastP. Interestingly, compared with Sph2 from other pathogenic leptospires and other sphingomyelinases, we observed a different profile of conservation between both epitopes. Sph2(176-191) presented more than 70% of identity with pathogenic leptospires, highlighting 100% of identity in 20% of studied pathogenic leptospires . This epitope also presented 100% of identity when compared with L. interrogans serovar Lai Sph4, and presented a mean identity of 42.2% when compared with sphingomyelinases from other bacteria. On the other hand, Sph2(446-459) was highly conserved among all studied leptospires SPh2, presenting more than 70% of identity. Interestingly, the epitope Sph2(446-459) was also highly conserved in other Leptospira interrogans serovar Lai sphingomyelinases, presenting identities that ranged from 71.4% to 100%, but presented low conservation when compared with other studied bacteria sphingomyelinases, presenting identities less than 37.5%  and five in the central metal-binding site . Considering this, when we compared aligned amino acids in Sph2 from L. interrogans serovar Lai with other pathogenic leptospires, the histidine at position 293 (H293) was the unique amino acid conserved in all studied leptospires. Moreover, the number of modified amino acids in the catalytic or metal-binding sites ranged from one to six among the studied Sph2. In this context, while Sph2 from L. interrogans serovar Bataviae; L. alstonii serovars Pingchang and Sichuan; L. borgpetersenii serovars Pomona and Hardjo-bovis; L. noguchii serovars Panama and Autumnalis; and L. santarosai serovar Arenal presented no modifications in amino acid residues in the catalytic or metal-binding sites, L. interrogans serovar Australis presented six (67%) different residues. Additionally, there were eight surface-exposed amino acids described as being associated with the interaction with the host cell membrane . Interestingly, these amino acids seemed to be less conserved among pathogenic leptospires, only one out of the three known serovars  presented no changed amino acids in the alignment, while the other studied proteins presented from three to six changed amino acids. Additionally, when compared with sphingomyelinases from other bacteria, Sph2 presented differences only in amino acids involved with the interaction with host cells, while all amino acids of the catalytic site and metal-binding sites remained unaltered  was inserted in Sphingomyelinase C domain (Sph2(155-440)). Finally, aiming to allow the visualization of predicted epitopes, catalytic and metal-binding sites, and amino acids associated with the interaction with the host cell membrane in the Sph2 3D structure, we highlight these structures in the Alphafold predicted model (ID: AF-P59116-F1)  .Leptospira spp. (MAT positive), presenting antibody titers on MAT ranging from 1:800 to 1:12800 (mean: 1:2839 ± 2407), and were grouped as reactive against Leptospira spp. (RL), while 36 patients were non-reactive against Leptospira spp. (NRL).The studied population was composed of 87 Brazilian febrile suspected leptospirosis cases based on their contact with rodents, floods, or other risk factors. Among them, 51 patients were reactive to p = 0.134). Moreover, in both groups, RL and NRL, about 11.5% of deaths cases were reported. As shown in The studied population presented was 35.7 (±17.5) mean years old, and clinical and epidemiological data were obtained between 1 and 74 days after the beginning of symptoms (mean 12.5 ± 12). Our studied group was mostly composed of men (85%), but we did not observe statistical differences in the frequency of men in RL (90%) and NRL (78%) (p = 0.0159), calf pain , renal insufficiency , and pulmonary hemorrhage  were statistically more frequent in RL patients.Regarding reported symptoms, fever (84%), myalgia (77%), and headache (63%) were the most prevalent symptoms among studied patients. However, comparing the frequencies of symptoms among groups, only jaundice , while the samples of 14 individuals (27%) cross-reacted with two serovars, one individual sample (2%) cross-reacted with serovars Australis, Hebdomadis, and Autumnalis, and another (2%) recognized the serovars Copenhageni, Canicola, Icterohaemorrhagiae, and Tarassovi  and Sph2(446-459) in plasma samples from 51 confirmed leptospirosis patients (RL: MAT reactive to Leptospira spp.) and 36 non-confirmed cases (NRL: non-reactive to Leptospira spp.). First, using the defined cut-off, the predicted epitopes were specifically recognized by serum samples of the patients from the RL group, as the NRL samples were non-responders against the predicted epitopes ((176-191) and Sph2(446-459) were specifically recognized by 33.3% (n = 17) and 19.6% of RL individuals, respectively, with a prevalence of IgG immune response to both epitopes. As shown in (176-191) and Sph2(446-459), while only one (2%) and two (3.9%) patients presented IgM antibodies, respectively ((446-459), while the other two IgM responders also presented IgG antibodies. Moreover, among 20 individuals that presented antibodies against at least one Sph2 epitope, ten individuals only presented antibodies against Sph2(176-191), three against Sph2(446-459), and seven individuals presented antibodies against both epitopes. Regarding the magnitude response, there were no statistical differences between IgG reactivity indexes against each peptide, which ranged from 1.05 to 3.23 (median: 1.27) against Sph2(176-191) and ranged from 1.07 to 1.81 (median: 1.19) against Sph2(446-459)  c.(176-191) in patients reactive to serovars: Tarassovi (n = 6), Copenhageni (n = 3), Canicola (n = 1), Copenhageni/ Icterohaemorrhagiae (n = 3), Copenhageni/Tarassovi (n = 2), Tarassovi/Icterohaemorrhagiae (n = 1), and Copenhageni/Hebdomadis (n = 1). Furthermore, specific antibodies against Sph2(446-459) were detected in patients reactive to serovars: Tarassovi (n = 4), Copenhageni (n = 2), Grippotyphosa (n = 1), Copenhageni/Icterohaemorrhagiae (n = 2), and Copenhageni/Tarassovi (n = 1).Regarding the detection of antibodies in patients infected by different serovars, we detected specific antibodies against Sph2(176-191) and Sph2(446-459). In this context, considering that pulmonary hemorrhage was a symptom exclusively observed among RL patients, we highlight that its frequency was statistically higher among responders to Sph2(176-191) (29.4%) than among non-responders . However, this difference was not observed among responders (20%) and non-responders (12.2%) to Sph2(446-459) (p = 0.6116).Based on the observed differences in the frequencies of symptoms between RL and NRL patients, we further explored the frequencies of cases presenting jaundice, renal insufficiency, pulmonary hemorrhage, calf pain, and deaths by comparing the frequencies of these symptoms between individuals whose sera recognized or not the epitopes Sph2(176-191) (23.5%) and Sph2(446-459) (30%) were not statistically different when compared with non-responders to Sph2(176-191)  and Sph2(446-459) , and neither when compared with the observed frequency of death cases in the NRL group . Regarding other symptoms, we did not observe statistical differences between responders and non-responders to synthetic peptides. Remarkably, the frequency of renal insufficiency cases among non-responders to Sph2(446-459) (41.5%) was statistically higher than the frequency observed in NRL , while the frequency among responders to Sph2(446-459) (30%) was statistically similar to both non-responders (p = 0.7208) and NRL (p = 0.3442). Additionally, responders and non-responders to Sph2(176-191) presented similar frequencies of renal insufficiency , both higher than the NRL group . Additionally, we observed higher frequencies of jaundices among responders to Sph2(176-191)  and non-responders to Sph2(446-459)  than among NRL (11.1%), and observed higher frequencies of calf pain among responders to Sph2(176-191) (76.5%) than NRL . Remarkably, clinical complications, such as pulmonary hemorrhage, were only observed in individuals with low or no-reactivity against Sph2(176-191), while individuals with high reactivity indexes to this epitope presented only mild symptoms.As shown in Leptospira spp. [Leptospira, sphingomyelinases play an important role in their survival in the mammalian host by mediating the lysis and release of essential nutrients from the host cells [L. interrogans serovar Lai, Sph2 and SphH are proven to have cytotoxic properties and are expressed during infection [L. interrogans serovar Lai Sph2 and to explore their association with the clinical data of naturally infected patients.Sphingomyelin (SM) is a major constituent of eukaryotic cell membranes and the ability to degrade this phospholipid by bacteria may consequently contribute to the pathogenesis of infection. Sphingomyelinases are a group of hemolysins, present in both eukaryotes and prokaryotes, which are related to phospholipid metabolism in the former and that frequently act as toxins in the latter , that arira spp. ,44. In pst cells , but arest cells ,46. Amonnfection ,47. HoweL. interrogans serovar Lai Sph2. This approach had been used and improved by our group in recent years to predict epitopes in viruses [(176-191)) and TPTKSGHKKKYDQV (Sph2(446-459)), which did not present similar epitopes described in BLASTP, in the Immune Epitope Database http://www.iedb.org/home_v3.php (accessed on 20 June 2020) (data not shown), and also did not present similar sequences described in humans and mice, or in other databases from PeptideAtlas http://www.peptideatlas.org/map/ (accessed on 20 June 2020). Therefore, predicted epitopes were considered non-conserved among hosts and other bacteria, suggesting them as antibody targets specific against Leptospira spp. In this context, when compared with other sphingomyelinases from L. interrogans serovar Lai, we observed that Sph2 is highly similar to Sph4, presenting more than 99% of identity, while other sphingomyelinases presented identities from 53% to 66.4% (L. interrogans serovar Lai and serovar Bataviae (identity of 90%), Sph2 from serovar Lai was more similar to L. noguchii serovars Panama and Autumnalis (identities of 89.6%) than to other serovars of L. interrogans , corroborating studies that recognized the great numbers of serovars as an obstacle that has hampered the development of a universal vaccine for leptospirosis [First, we used a combination of algorithms to predict linear B-cell epitopes in  viruses ,26, bact viruses , and pro viruses ,48,49, rto 66.4% . Moreovespirosis .L. interrogans serovar Lai and Sph2 from other pathogenic leptospires. Remarkably, the epitope Sph2(446-459) was highly conserved among Sph2 from pathogenic leptospires and among sphingomyelinase from L. interrogans serovar Lai, presenting more than 71.4% of identity when compared with studied sequences. However, Sph2(176-191) was highly conserved in only three studied Sph2  and in Sph4, presenting 100% of identity with these proteins, while presenting less than 56.3% of identity when compared with other studied proteins. From our point of view, the high conservation of Sph2(446-459) among pathogenic leptospires suggests that this epitope may be inserted in a region under low selective pressure by the host immune response. In line with this assumption, according to the prediction of algorithms InterPro family, TIGRFAMs, and nSMase (data not shown), this epitope is located out of the Sph2 Sphingomyelinase C domain (Sph2(155-440)), supporting the hypothesis of low selective pressure given that all amino acids involved in Sph2 activities are located on the Sphingomyelinase C domain [Based on these data and aiming for the future constructions of multi-epitope vaccines, we explored the conservation of predicted epitopes among other sphingomyelinases from C domain , a commoC domain .L. interrogans serovar Lai presents all amino acids described in the catalytic and metal-binding sites highly conserved (100% of identity) when compared with sphingomyelinases from other bacteria, such as Listeria ivanovii and Bacillus cerus, which are considered membrane-damaging virulence factors that induce hemolysis, a reduction in phagocytosis [L. interrogans serovar Lai [L. interrogans serovar Copenhageni Fiocruz L1-130 to lyse sheep erythrocytes [(176-191) was closely located to the catalytic site, metal-binding site, and the region related to interaction with the host membrane. This finding suggests that antibodies against Sph2(176-191) could hamper the functionality of Sph2 by blocking the interaction with the host, or even the ligation to cofactor or substrate. However, studies aiming to identify the active sites of Sph2 from Leptospira spp. and to evaluate their activity and the role of antibodies against their epitopes remain a lack in the literature.In the same way, we evaluated the variability of amino acids in the catalytic site, metal-binding site, and the region of interaction with host membranes . First, ocytosis , and escocytosis . Additioocytosis , which iovar Lai , and prohrocytes . Based o(176-191) and Sph2(446-459) in blood samples from 51 Brazilian patients with leptospirosis, which presented reactivity (or cross-reactivity) to 14 Leptospira spp. serovars, with the prevalence of Tarassovi and Copenhageni. Interestingly, this observation is in disagreement with the recent revision of Browne and collaborators, which reported the serovars Icterohaemorrhagiae, Canicola, and Pomona as the most prevalent in human leptospirosis in the Americas from 1930 to 2017, with Pomona and Canicola being the most prevalent in Brazil [Until now, antibodies against Sph2 were detected in the blood of mares following leptospiral abortion, but not in horses immunized with bacterins , and in n Brazil . From ouLeptospira spp. were able to recognize at least one of these epitopes, with a prevalence of IgG antibodies among responders to both Sph2(176-191) and Sph2(446-459). Both epitopes Sph2(176-191) and Sph2(446-459) were recognized by patients’ samples reactive to at least one of the most prevalent serovars , while Sph2(176-191) was also recognized by patients’ samples reactive to serovars Canicola and Hebdomadis, and Sph2(446-459) was also detected in samples reactive to serovar Grippotyphosa. Remarkably, among the serovars detected in our studied population, only samples reactive to serovars Wolffi (n = 1), Pomona (n = 1), Wolffi/Sejroe (n = 1), and Australis/Hebdomadis/Autumnalis (n = 1) presented no reactivity to at least one of predicted epitopes. However, considering the low level of identity shared by L. interrogans serovar Lai Sph2(176-191) and other pathogenic species, we cannot discard a higher frequency of responder and reactivity index investigating the epitopes from other important serovars. Therefore, the real cross-reactivity of these epitopes needs to be explored more in serological and epidemiological studies, since the absence of reactivity could be related to the limited number of studied patients and the expression of Sph2, which is markedly upregulated by changes in osmolarity and temperature [In this context, thinking about reactivity and cross-reactivity to predicted antibodies, first, we confirmed that the predicted epitopes were naturally immunogenic, since about 40% of serum samples from patients naturally infected by perature , determiL. interrogans serovar Copenhageni did not present protection when challenged with a virulent strain of L. interrogans serovar Pomona; however, the authors propose that this can be related to a lack of correct folding of the recombinant proteins [(176-191) ((176-191) presented only mild symptoms. Based on this data and on the position of Sph2(176-191) close to the catalytic site and the region of interaction of Sph2 with the host membrane, we conjecture that this epitope should be better explored as a target of protective antibodies against leptospirosis; however, more studies are necessary to prove this hypothesis. On the other hand, we also hypothesized that different levels of Sph2 expression related to Leptospira species and host factors can also be related to reactivity against Sph2 epitopes and could be associated with leptospirosis hemorrhagic symptoms, reinforcing the necessity of more studies to prove Sph2 and its epitopes as vaccine candidates.Moreover, the protective potential of antibodies against Sph2 is still unknown. Regarding its potential as a vaccine antigen, hamsters immunized with the recombinant Sph2 from proteins . Unfortu176-191) . We beliLeptospira elicit a serovar-dependent immunity [Leptospira antigens experimentally validated remains scarce, and justifies our study, since the identification of protective epitopes is the key to the design of an effective and universal multi-epitope vaccine for human leptospirosis.Up to now, the main vaccines proposed against immunity . Though,immunity . Moreoveimmunity , supportimmunity ,59,60,61L. interrogans serovar Lai and to assess their natural immunogenicity in human leptospirosis. However, further studies using patients from different regions and additional Leptospira spp. serovars are necessary to investigate the real protective role of antibodies against epitopes Sph2(176-191) and Sph2(446-459) in leptospirosis. Moreover, studies in animal models are essential to prove their potential as a vaccine antigen, singly and combined with other identified epitopes.In brief, we used immunoinformatics to predict linear B-cell epitopes in Sph2 from L. interrogans serovar Lai. Our data corroborate the association of Sph2 with hemorrhagic complications from leptospirosis. Moreover, based on its location and associations with clinical data and specific immune response, we suggest that Sph2(176-191) can be further explored in multi-epitope vaccines to prove its protective potential in animal models.Our study was the first to identify antibody targets in Sphingomyelinase 2 from


IntroductionUltrasound elastography is a method of measuring soft tissue stiffness to detect the presence of pathology. There are several ultrasound elastography devices on the market. The aim of this study was twofold. Firstly, to determine the validity of three different ultrasound systems used to measure tissue stiffness. Secondly, to determine the operator reliability and repeatability when using these three systems.Materials and methodsTwo observers undertook multiple stiffness measurements from a phantom model using three different ultrasound systems; the LOGIQ E9, the Aixplorer, and the Acuson S2000. The phantom model had four cylindrical-shaped inclusions (Type 1-4) of increasing stiffness values and diameter embedded within. The background phantom stiffness was fixed. The mean, standard deviation, and coefficient of variation (CV) were calculated from measured stiffness readings per diameter per inclusion. Intra-observer variability was assessed. The validity of the measured stiffness value was assessed by calculating the difference between the measured elasticities and actual phantom elasticities. Bland-Altman plots with limits of agreement were used to display the inter-observer agreement. The intraclass correlation coefficients (ICC) were used to measure intra-observer, inter-observer, and inter-system reliability.ResultsEach observer undertook 1020 measurements. All three systems generally underestimated the stiffness values for the inclusions; the higher the actual stiffness value, the more significant the underestimation. The percentage difference between measured stiffness and actual stiffness varied from -79.1% to 12.7%. The intra-observer variability was generally less than 5% for observers using the LOGIQ E9 and the Aixplorer systems but more than 10% over the stiffer inclusions (Types 3 and 4) for the Acuson system. There was 'almost perfect' intra-observer reliability and repeatability for both the LOGIQ E9 and the Aixplorer systems; this was 'moderate' for the Acuson system over specific inclusions. For all systems, there was 'almost perfect' inter-observer reliability and repeatability between Observer A and Observer B. The inter-system reliability and repeatability were 'almost perfect' between the LOGIQ E9 system and the Aixplorer system but 'poor' and 'moderate' when the Acuson system was matched with the LOGIQ E9 system and the Aixplorer system, respectively.ConclusionThis study has demonstrated that the Acuson, LOGIQ E9, and Aixplorer ultrasound systems have low variability, high reproducibility, and good intra-observer and inter-observer reliability when used to measure tissue stiffness. However, they all underestimated the stiffness values during this in vitro study. This study also revealed that not all ultrasound systems are comparable when measuring tissue stiffness, with some having better inter-system reliability than others. Ongoing standardization of technology is required at the manufacturer level. Ultrasound elastography or shear wave elastography (SWE) is an ultrasound-applied imaging technique used to determine tissue stiffness due to underlying disease. This non-invasive technology allows healthcare professionals to observe and quantify signs of abnormal tissue stiffness in deeper structures . Tissue SWE technology employs the use of stimulated shear waves to determine Young's modulus (stress to strain ratio) of tissues in kilopascals (kPa): the higher the value, the higher the stiffness -4. An ulThere are several ultrasound systems on the market. Much of the technology and mechanisms behind SWE are patented and, therefore, inaccessible to researchers and clinicians . HoweverTherefore, the aim of this study was twofold. Firstly, to determine the validity of three different ultrasound systems by comparing measured and actual stiffness values. Secondly, to determine operator reliability by calculating the ultrasound systems' intra-observer, inter-observer, and inter-system reliability.The ultrasound systemsThe three ultrasound systems used for this study were the Acuson S2000 , the Aixplorer , and the LOGIQ E9 . The Aixplorer and LOGIQ E9 systems use two-dimensional SWE technology to measure kilopascals' stiffness (kPa). The Acuson S2000 system uses point SWE technology and measures the shear wave velocity in meters per second (m/s): a conversion factor was used to change this to kPa. The manufacturer-recommended brightness mode (B-mode) and elastography settings were used to ensure readings' consistency and methodology standardization. All three systems had similar linear probes with similar frequency bandwidths to allow comparison between the systems.The phantomA phantom model was used to represent human tissue to obtain stiffness values. The Elasticity QA phantom  was loaned from Computerized Imaging Reference Systems Company  for this study Figure . This phStudy designObservers A and B, blinded to each other's readings, undertook a set of measurements on the elastography phantoms using a single linear probe. Measurements were done on five partitioned sites on each of the four inclusions, totaling 20 sites. Each observer was instructed to do 20 repeat measurements per site, equating to 400 readings per ultrasound system used. Therefore, 1200 readings per observer were expected after using the three systems. In order to standardize the method, the ROI for each diameter was predetermined and remained the same for the Aixplorer and LOGIQ E9 ultrasound systems. However, a rectangular fixed ROI was used for the Acuson S2000 system. Each reading was taken after two frame rates after the probe was positioned, and the elastography function was selected and recorded.Statistical analysisStatistical analyses were performed using IBM SPSS Statistics for Windows version 22.0 . Each set of 20 measured stiffness readings attained per diameter per cylindrical inclusion was averaged to calculate the mean, standard deviation (SD), and coefficient of variation (CV). Intra-observer variability was assessed by the coefficient of variation (CV). The CV was determined by dividing the standard deviation by the mean value: the lower the CV, the lower the variability. A CV less than 0.1 10%) was considered low variability. The validity of the measured stiffness value was assessed by calculating the mean difference (and percentage mean difference) between the measured stiffness values and actual phantom stiffness values. Bland-Altman plots with limits of agreement  were used to display inter-observer agreement and reliability and the difference between observers against the mean to display systematic variation % was con.A total of 2080 stiffness measurements were taken for this study, 1040 per observer. Each observer took 400 measurements of the phantom using the LOGIQ E9 system, 400 using the Aixplorer system, and 240 using the Acuson S2000 system. The Acuson system could not obtain stiffness measurements from the two smallest diameters (4.1 mm and 2.5 mm), so these were omitted. The Acuson system could not obtain measurements accurately at the 3.0 cm depth, the standardized parameter across the three systems. Therefore, a depth of 2.7 cm (reaching the three largest diameters) was used for the Acuson system.Table Intra-observer variability, validity, and accuracyThe intra-observer variability was generally low for both observers, indicated by a CV range of 0.01-0.05. The lowest variability was noted while using the Aixplorer ultrasound system, and the highest (CV more than 0.1) was noted while using the Acuson system, especially when measuring over the Type 3 and Type 4 inclusions.Both observers reported an extensive range in the mean differences across all systems, indicating significant measurement biases and low validity and accuracy. The percentage difference between measured stiffness and actual stiffness varied from -79.1% to 12.7%. When using the LOGIQ E9 and Aixplorer systems to measure the Type 1 inclusion, observers overestimated the stiffness values. Stiffness values were underestimated using all three systems over all other ROI; the higher the actual stiffness value of the inclusion, the greater the underestimation. The Acuson system underestimated the stiffness value at every ROI and had the largest percentage differences between measured and actual stiffness values as the inclusion stiffness increased from Type 1 to Type 4.Inter-observer agreementInter-observer agreement was evaluated using Bland Altman plots and the 95% limits of agreement. Figure Intra-observer reliabilityICC was calculated to determine intra-observer reliability. Table Inter-observer reliabilityTable Inter-system reliabilityThe ICC was used to determine inter-system reliability and repeatability. Table Ultrasound elastography has been widely introduced into the clinical environment. Studies and clinical use have extended to carotid lesions, breast lesions, pancreatic cysts, myocardial stiffness, liver lesions, testicular torsion, and malignant thyroid nodules, to name a few -18. ThusThis study aimed to investigate the validity and operator validity across three different ultrasound systems. Two independent observers (Observer A and Observer B) measured stiffness/stiffness levels of four different inclusions in a single phantom model using the LOGIQ E9 system, the Aixplorer system, and the Acuson system. Within the phantom were four inclusions ; each inclusion had five partitions with increasing diameter. The LOGIQ E9 and the Aixplorer systems slightly overestimated the stiffness values for the softest inclusion (Type 1) at its smallest diameter; the Acuson system failed to get measurements over the two smallest diameter sections of all inclusions. All three systems underestimated the stiffness values for the inclusions at all other ROIs when used by both observers; the higher the actual stiffness value of the inclusion, the greater the underestimation. The Acuson system underestimated the stiffness value by the greatest degree, by up to 80% for the stiffest (Type 4) inclusion. Observer A consistently had slightly higher stiffness values than Observer B for each measurement. The Acuson system could not get a reading for the two smallest diameters on the softest inclusion (Type 1). The intra-observer variability was generally low for both observers, especially when using the LOGIQ E9 system and the Aixplorer system, with CV less than 5%. At the same time, the variability was higher with the Acuson system , thus overestimating the stiffness value of the softest inclusion. A similar explanation would account for the marked underestimation of stiffness values for all the stiffer inclusions, especially those with actual stiffness values greater than the background (phantom stiffness). Previous research has questioned the validity of the phantoms being used for such studies .The stiffness readings from the Acuson system were the least accurate of the three systems, with the greatest underestimation and highest variability. These inaccuracies have been highlighted in other studies using this system ,6,9,19. This study demonstrated high intra-observer and inter-observer reliability/agreement. Previous phantom studies have also reported comparable high intra-observer and inter-observer agreement ,7,20. ThThis result has profound clinical importance, as they highlight that not all ultrasound systems are equivalent when measuring tissue stiffness. Ultrasound elastography is widely employed for diagnostic purposes; therefore, treatment, response, and prognosis decisions are based on such platforms. The results may differ if patients move between healthcare institutions employing alternative ultrasound systems; therefore, clinical decision-makers must consider this during clinical practice. Conversion formulae have been suggested to adapt the measurements between ultrasound systems to aid comparability .Study limitationsThe probes used in this study could not read at 6 cm depth as originally intended for this study, and therefore, depth analysis was excluded. All probes used were linear in design, but their frequency range was not identical; their frequencies varied from 2-10 MHz, which may have introduced some bias into the stiffness readings. However, the frequencies for the Aixplorer, LOGIQ E9, and the Acuson systems are comparable at 10 MHz, 9 MHz, and 9 MHz, respectively, so the effect of frequency may be less profound. To standardize the method, the ROI for each diameter was predetermined, and the readings were recorded after two frame rates. However, in practice, this was only partially possible. The ROI was easily specified to Aixplorer before readings, but for the LOGIQ E9, the ROI had to be manually circumscribed for every reading. The ROI was rectangular by default on the SA system. Therefore, these manipulations may have added errors or bias to the readings. Tissue-mimicking phantoms do not possess the viscoelastic properties of actual tissue. Therefore the results obtained do not necessarily fully reflect the stiffness readings in vivo in the clinical environment .Further researchThe phantom's stiffness may affect the inclusions' stiffness readings. The validity of the phantoms used in studies could be evaluated by employing multiple phantoms and observers and comparing more stiffness readings. Few studies have compared the reliability and accuracy of results obtained at different depths by ultrasound systems in a phantom. Therefore, depth analysis in vivo and in vitro is recommended for future research. More in vivo studies are required to determine actual tissue stiffness at different sites so accurate phantom can be developed for repeatability and variability studies of stiffness reading.This study has demonstrated that the Acuson, LOGIQ E9, and Aixplorer ultrasound systems have low variability, high reproducibility, and good intra-observer and inter-observer reliability when measuring tissue stiffness. However, they all underestimate the true stiffness values during in vitro studies, requiring a correction formula. This study also revealed that not all ultrasound systems are comparable when used to measure tissue stiffness, with some having better inter-system reliability than others. Therefore, applying the readings from one machine to another in the clinical environment may not be accurate. So ultrasound systems require standardization between manufacturers to propel them into absolute clinical integration. We hope this study not only contributes to the literature but also encourages further research into this exciting topic.