A new environmentally friendly cemented oil shale residue–steel slag–ground granulated blast furnace slag backfill (COSGB) was prepared using oil shale residue (OSR), steel slag (SS) and ground granulated blast furnace slag (GGBS) as constituent materials. Based on univariate analysis and the Box–Behnken design (BBD) response surface method, the three responses of the 28 days unconfined compressive strength (UCS), slump and cost were used to optimize the mix ratio. Using a combination of scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD) and mercury intrusion porosimetry (MIP), the reaction products, microscopic morphology and pore structure of the specimens with the optimal mix ratio at different curing ages were analyzed. The influence of heavy metal ions from the raw materials and the COSGB mixtures on the groundwater environment was studied by leaching tests. The research demonstrates that the optimal mix ratio is GGBS mixing amount 4.85%, mass ratio of SS to OSR 0.82, and solid mass concentration 67.69%. At shorter curing age, the hydration products are mainly calcium alumino silicate hydrate (C-A-S-H) and calcium silicate hydrate (C-S-H) gels. With the increase of curing age, ettringite (AFt) and C-S-H gels become the main source of the UCS. Meanwhile, the porosity of the filler decreases continuously. The leaching concentration of heavy metal ions from the COSGB mixtures is all lower than the leaching concentration of raw materials and meet the requirements of the Chinese groundwater quality standard (GB/T 14848-2017). Therefore, this new COSGB cannot pollute the groundwater environment and meets backfill requirements. The proposed technology is a reliable and environmentally friendly alternative for recycling OSR and SS while simultaneously supporting cemented paste backfill (CPB). CPB can control mining subsidence and utilize solid waste, so this material has become an important means of green mining . On the missions . Partialmissions . OSR andmissions . If OSR missions .Many scholars have carried out research and development on new CPB materials and the mechanical properties of CPB materials. Mangane et al.  used 20%To analyse the micro hydration mechanism of CPB, Yan et al.  performeIn terms of the environmental effect of CPB, Hu et al.  studied These research results have greatly contributed to the understanding of the material development, macromechanical behaviors, microstructure and environmental impacts of CPB. But no research on the preparation of CPB paste using OSR, SS and GGBS as constituent materials has been reported. Moreover, the micro hydration mechanism and the impact of this new CPB paste on the groundwater environment are unknown.In this paper, OSR, SS and GGBS are used as constituent materials to prepare a new environmentally friendly COSGB. The response surface method was used to optimize the mix ratio for COSGB. The specimens with the optimum mix ratio were also analyzed for their hydration products, micromorphology and pore structure. Contaminants in COSGB were studied by leaching tests.The OSR in the mixture was taken from the Fushun West Open-pit Coal Mine . This material is solid waste associated with coal and used after crushing. The SS in the experiment was taken from the Anshan Iron and Steel Group . The GGBS is S95 grade slag produced by Shandong Kangjing New Material Technology Co., Ltd. . Tap water was used for mixing. Photographs of the OSR, SS and GGBS raw materials are shown in 2, and the mold was removed after 24 h of curing [First, OSR, SS and GGBS were poured into a mixer and stirred for 3 min to ensure that the materials in the mixer were homogeneous. After that, pre-weighed water was poured into the mortar mixer and mixed for 4 min. The slump test was performed immediately after mixing. The COSGB mixture was poured into cylindrical molds with dimensions of Φ50 × 100 mmf curing . These cCOSGB is a mixture of OSR, SS, GGBS and water. The solid mass concentration is a factor used to determine the mass percentages of solids and liquids. GGBS mixing amount is considered separately as a factor due to its relatively high cost. The mass ratio of SS to OSR is used as a factor because only the qualities of SS and OSR are uncertain. Thus, three factors, GGBS mixing amount, mass ratio of SS to OSR and solid mass concentration, were selected for the mix design. Based on a large number of preliminary experiments, single-factor analysis was performed, and the appropriate ranges for the three factors were determined in turn. The design of single-factor experiments is shown in 3) and three solid materials (kg/m3) was calculated according to the mix ratio. Finally, the unit cost of COSGB (USD/m3) was calculated based on the unit prices of raw materials.GGBS cost is the purchase price from Shandong Kangjing New Material Technology Co., Ltd., China. As solid wastes, OSR and SS only calculate the processing cost. The unit prices of raw materials are shown in Response surface methodology (RSM) is an optimization method that integrates experimental design and mathematical modelling. Based on the BBD test results, a response surface model was developed . The forxi and xj represent the independent variables, y represents the design response value, and a0, ai, aii, and aij are the constant, linear, quadratic, and interaction coefficients.In Equation (1), To obtain the optimal mix ratio of COSGB materials, the desirability function method was used .di.First, we established a single-response desirability function, di denotes the desirability function of the i-th response. Yi denotes the i-th response. Li is the lower limit of the i-th response value. Ui is the upper limit of the i-th response value. Ti is the target value of the i-th response. Equation (2) is applicable to the response variable in which desirability increases with the increase in the response value. Equation (3) is applicable to the response variable with an optimal target value, and the desirability closest to the set target value is greater; Equation (4) is applicable to the response variable in which desirability increases with the decrease in the response value.In Equations (2)–(4), di values is the overall desirability function D (Equation (5)). ∑si = 1 in Equation (5), the higher si value means that the target value is more important.Second, the geometric mean of all responding single expected target D. The optimal mix ratio was selected for the highest D value.Using a single-response function as a constraint, nonlinear regression was performed for the overall desirability function ARD) [Pre) and the design experimental (Exp) values. The ARD reflects the accuracy of response surface model predictions. When the ARD is less than 5%, it indicates that the prediction accuracy is higher and the reference ability is greater.Finally, Equation (6) was used to calculate the absolute relative deviation (ARD)  between The UCS of the COSGB was tested according to the ASTM C39/C39M-15a  specificA XRD-6100 X-ray diffractometer  was used for phase analysis of the specimens. A JSM-7500F scanning electron microscope  was used to analyse the surface morphology changes. A FYFS-2002E energy dispersive spectroscopy (EDS) detector  was used for the elemental analysis of hydration products. An IRPrestige-21  Fourier transform infrared (FTIR) spectrometer was used to characterize the molecular structure and chemical bonds of the specimens. An Autopore IV 9500  mercury porosimeter was used to measure the total porosity and pore size distribution of the specimens.The toxic leaching of the backfill materials was analysed by the horizontal oscillation method  ,32. Firs2/kg. The SS is light grey, it is mainly composed of particles and powders with a particle size of less than 1.20 mm, and it has a specific surface area of 519 m2/kg. The GGBS is a milky white powder with a uniform particle size of 0.01 mm and a specific surface area of 1570 m2/kg. The particle size curves of the three raw materials are shown in The OSR is brown, with a particle size is mostly concentrated between 0.6–1 mm, and it is irregular and angular in shape, with a specific surface area of 549 mThe mass ratio of SS to OSR was fixed at 1:1, and the solid mass concentration was 65%. The effects of the GGBS mixing amount on the 28 days UCS, slump and cost of the COSGB were obtained by varying the GGBS mixing amount, as shown in When the mass ratio of SS to OSR was fixed at 1:1 and the GGBS mixing amount was 5%, the solid mass concentration was adjusted, and the effects of the solid mass concentration on the UCS, slump and cost of the COSGB were obtained, as shown in The effects of the SS to OSR mass ratio on the 28 days UCS, slump and cost of the COSGB were obtained by adjusting the SS to OSR mass ratio at a solid mass concentration 65% and GGBS mixing amount 5%, as shown in Based on the analysis results of the above single-factor analysis, Based on the BBD method, 17 sets of mix ratio (including 5 center point replicates) were designed and carried out, and 28 days UCS and slump tests as well as cost calculations were performed for different mix ratio. The mix ratios and response values are shown in According to the test results obtained in The formula used to fit response 1 (28 days UCS) is shown in Equation (7):The formula used to fit response 2 (slump) is shown in Equation (8):The formula used to fit response 3 (cost) is shown in Equation (9):The results of the analysis of variance (ANOVA) obtained by conducting significance tests are shown in p-value is the main consideration. If p < 0.05, the factor is significant in the model. Otherwise, it is not significant. When p < 0.01, the factor is highly significant in the model [p-values of the regression models for Y1, Y2 and Y3 were all less than 0.01, indicating that these mathematical models were statistically significant. Moreover, the R2 values of the correlation coefficients for each model fit were above 0.90. Y1, Y2 and Y3 were 0.9875, 0.9834 and 0.9994, respectively. The closer the value of the correlation coefficient (R2) was to 1, the more accurate the fit was. The above results fully show that the measured values of compressive strength, slump and cost of the COSGB are in good agreement with the predicted values. Thus, Equations (7)–(9) fit the experimental results well and are able to accurately predict the 28 days UCS, slump and cost within the given range.For each significant factor in the model, the he model . The p-vThe 3D response surface provides a more intuitive description of the relationship between the interaction of the two factors and the response value, so that the effect of changes in the levels of the factors on the response value can be generalized. The greater the curvature of the response surface is, the more significant the effect of factor interactions is. Conversely, the impact of the factor is not significant .P-value for AC is 0.0464 (<0.05), indicating that the interaction between AC is more obvious than the other interactions. The three-dimensional response surface for the AC interaction is shown in A and C, the curvature of the 28 days UCS response surface (28 days UCS growth rate) increases. This suggests that the decrease in total solids is accompanied by an attenuation of the 28 days UCS. The increase in total solids means a decrease in the total water in the COSGB. On the one hand, a reduction in water leads to a lower initial porosity of COSGB and tighter bonding of the COSGB matrix. On the other hand, the lower water content in the COSGB indirectly enhances the alkali concentration in the contact environment and promotes the hydration process of the mixture.Based on the experimental results and the variance analysis of the 28 d UCS, the p < 0.01 for BC indicates the highly significant interaction of BC. The 3D response surface of the BC interaction is shown in B and C factors. As B decreases and C increases, the curvature of the slump response surface (the growth rate of the slump) increases. The increase in the ratio of SS to OSR significantly increases the slump value, because OSR is composed of loose, irregularly shaped and angular fine particles with very rough and water-absorbent surfaces. The high friction between the mixture and SS particles reduces the fluidity of the COSGB mixture, resulting in a lower slump value of COSGB mixture. In addition, the low density of OSR results in a large amount of OSR at the same mass, which is also an important factor affecting the solid mass concentration. The increase in OSR indirectly affects the slump of the COSGB mixture.For response 2 (slump), as can be seen in the ANOVA results , p < 0.0p-values obtained for the interaction terms of BC and AC are less than 0.05, showing that they all have a significant impact on the cost. The AC interaction with a relatively smaller p-value is given as an example in Regarding the response values of cost, the wti = 1 is chosen in this study. In this study, the 28 days UCS is selected and has the same importance as slump and cost [s1 = s2 = s3 = 1/3).The COSGB is designed to maximize the strength of the backfill and minimizing the cost per cubic meter of COSGB material, while meeting the flow requirements needed for backfill. Therefore, the individual desirability functions used for the COSGB optimization are set as follows: the maximum value is chosen for the 28 days UCS (Equation (2)), the slump target is 200 mm  (Equatioand cost  (s1 = s2D (Equation (5)). The optimal mixture ratio was selected for the highest D value. The final results are as follows: the GGBS mixing amount is 4.72%, the mix ratio of SS to the OSR is 0.82, and the solid mass concentration is 67.69%. And the predicted (Pre) response values are as follows: 28days UCS of 2.10 MPa; slump of 200 mm; cost of 5.21 USD. The experimental (Exp) values of COSGB obtained by the optimal mixture ratio test are as follows: 28 days UCS of 2.12 MPa; slump of 205 mm; cost of 5.17 USD.Based on using a single-response function as a constraint, nonlinear regression was performed for the overall desirability function ARDs for the 28 days UCS, slump and cost are 0.94%, 2.44% and −0.77%, respectively. All of the errors are less than 5%, which indicates that the prediction accuracy is high and the prediction has a strong reference value for the optimization results of COSGB.According to Equation (6), the In the meantime, the variation in the strength of the COSGB with curing age for the optimal mix ratio is shown in 2 emissions. While OSR, SS and GGBS used in COSGB are all solid wastes. Therefore, COSGB is more environmentally friendly than traditional CPB.The change in the UCS of COSGB with the curing age is shown in The optimal COSGB mix ratio was used. At different curing ages, the hydration mechanism of the three raw materials was analyzed with a combination of XRD, FTIR, SEM-EDS and MIP.The XRD patterns of OSR, SS, GGBS and COSGB at different curing ages are shown in θ range of 20–45°, which indicates the coexistence of amorphous silicate gels [3) and quartz (SiO2) are present in the COSGB after mixing and curing. These phases are derived from unreacted SS and OSR particles. Under longer curing ages, internal kyanite (Al2SiO5), a type of mullite, is exposed with the dissolution of the surface crust of OSR particles [4)(H2O)2) and calcium silicate hydrate (C-S-H) are detected in the cured matrix.As shown in ate gels . Crystalarticles . In the 4) and ferric oxide (Fe3O4) that are originally present in OSR and the magnesium hydroxide (Mg(OH)2) and calcium hydroxide (Ca(OH)2) that are originally present in SS, disappeared [2 produced by hydrolysis and the Ca(OH)2 in SS make the hydration environment gradually alkaline. This creates conditions for the dispersion and dissolution of GGBS vitreous, and promotes the continuous hydration reaction of SiO2 and A12O3 in the mixture. Additionally, the Al2O3 and SiO2 presenting in OSR and GGBS, and the Ca(OH)2 of SS, can generate AFt with CaSO4 by hydration reaction. AFt is one of the sources enhancing the UCS of the COSGB [2 to form C-S-H gels [As shown in appeared . This shhe COSGB . The mixS-H gels . Therefo2Si4O9(OH)2) only appeared at the 1d curing age, and it was not found during the rest of the curing ages. Because the chemical composition and short-term structure of C-S-H gels are similar to those of natural zeolites. C-S-H gels with zeolite-like structures may appear at short curing ages. This is consistent with the conclusion of Hanjitsuwan et al. [It should be noted that there are two more special changes in n et al. . The othn et al.  also fouThe FTIR spectra of the COSGB at different curing ages are given in −1 [−1 and 550–560 cm−1 are related to the presence of mullite . This is consistent with the results obtained from the XRD analysis [−1 is the vibrational band of –OH and H2O, and the peak at 1464 cm−1 is the vibrational band of –OCO–, representing the formation of carbonates. Combined with XRD analysis, it can be concluded that the product is CaCO3.As shown in −1 . A serieanalysis . The pea−1 correspond to Al–O and Ca–O vibrational bands respectively, and the absorption peak of crystalline water appears at 1651 cm−1, which indicate the formation of AFt during hydration action. From the curves in −1 overlaps due to the asymmetric stretching vibration of Si–O–T (Si or Al) in the C (A)-S-H gels. The vibration results in the produce of a broad and intense band in the abovementioned interval [−1 in The peaks at 550 and 870 cminterval . Additioal −1 in ,47.−1 to 1032 cm−1) with increasing curing age. Criado et al. [It is noteworthy from o et al.  suggest In summary, the main hydration products of the COSGB are a mixture of AFt and C-S-H gel, which is consistent with the XRD analysis.To investigate the microstructure of COSGB, SEM and EDS analyses were performed on specimens with different curing ages .The internal structure and the changes in the hydration reaction products of the COSGB were characterized by SEM-EDS. As shown in At 1 day curing age, a C-(A)-S-H gel forms at the beginning of the hydration reaction after mixing. Then more gel products form, not only filling the existing voids but also binding the remaining solid particles together. As shown in The test results of the pore structure of the specimens at different curing ages are analyzed using the MIP test method (as shown in The MIP test results show that the cumulative pore volume (CPV) increases with decreasing pore diameter. During the test, continuous pores in the COSGB specimen, including “ink-bottle” pores, can be detected from the intrusion curves . The totCombined with the graphs, the total pore area, median pore diameter and porosity of COSGB specimens all decrease to varying degrees with increasing curing age. This result verifies that the hydration reaction continues after 3 days, and performed reaction products fill the original pores, promoting the conversion of large pores to small pores. It is worth noting that the same results were observed as for Portland cement paste, where the pore size and porosity generally decreased with hydration ,55. It fFilling materials such as OSR, SS and GGBS may carry heavy metals and other pollutants. Whether the formation of filled paste poses a threat to the groundwater environment needs to be measured and analyzed.2+, Mn2+ (acid leaching) and Cr6+ contents in the leachate of OSR and SS exceed the standard, and the Fe2+ content in the leachate of GGBS exceeds the standard. As shown in 2+, which slightly exceeds the standard. Fe2+ content of the original backfill materials, the SS and OSR, are high, which results in the slight overrun of Fe2+ leaching after backfilling and mixing. The leaching concentrations of all ions except Fe2+ are below 0.1 mg/L. Comparing the water leaching with acid leaching conditions, the leaching concentration of heavy metals is greater under acid leaching conditions than under water leaching conditions, which is consistent with the leaching of raw materials. From the curing age of the COSGB, the concentration of Cr(VI) fluctuates with increasing curing age in the order 1 day > 3 days > 14 days > 7 days > 28 days, and the leaching concentration of other heavy metal ions keeps decreasing. With the increase in curing age, the pore size of the hydrated gel decreases, the permeability decreases, the contaminants are encapsulated, and the migration characteristics are greatly reduced. The pH of the leachate is alkaline and fluctuates between 10.7 and 11.4 as the curing age increases. The alkaline chemical reaction also inhibits the migration of many heavy metals. From another point of view, acid mine water can be neutralized, but this is unfavourable for neutral or alkaline mine water. In general, the concentration of contaminants leached from the developed COSGB meets the requirements of the Chinese groundwater quality standard, and thus, it is safe for application in backfill.The red line in (1)3.A new cemented oil shale residue-steel slag-ground granulated blast furnace slag backfill (COSGB) without additives was developed. By optimizing the mix ratio, the GGBS mixing amount is 4.85%, the mass ratio of SS to OSR is 0.82, and the solid mass concentration is 67.69%. For the optimal mix ratio of COSGB, the 28 days UCS value is 2.12 MPa, the slump value is 205 mm, and the cost value is 5.17 USD/m(2)2 from SS and the hydration products of OSR, which dissolves the GGBS vitreous and promotes the hydration reaction inside the COSGB mixture. At shorter curing age, the hydration products are mainly C-A-S-H gels and C-S-H gels. With increasing curing age, the amount of hydration products increases rapidly. At the same time, the needle-like AFt crystals are combined with the flocculated C-S-H gel to make the COSGB structure more compact and improve the overall stability.As the curing age increases, the connection porosity of the COSGB material decreases significantly, and the UCS gradually increases, reaching 82% of the day 28 strength at 7 days, and slowly increasing in the later period. The alkaline environment is produced by the Ca(OH)(3)The pH of the leachate of the three raw materials is alkaline. This is why the strength increases without the addition of an alkali exciter to the COSGB material. The leaching concentrations of the heavy metal ions of COSGB meet the requirements of the Chinese groundwater quality standard (GB/T 14848-2017).(4)Using OSR, SS and GGBS as raw materials, a safe, environmentally friendly and economical COSGB was prepared. The new COSGB material replaces traditional OPC, controls carbon emissions and reduces the cost of CPB materials. The technology has achieved the maximum utilization of industrial solid wastes , and avoided the damage to the ecological environment due to the accumulation of OSR and SS. It is an effective way for cleaner production in the mining industry. When applied to backfill, the leaching process of COSGB cannot cause harm to water environment. This provides a broader application prospects for COSGB materials.(5)This study focuses on only one area of OSR and SS. In the future, the effects of OSR and SS from different areas on the preparation of COSGB should be further studied. However, due to the complexity, variability, high requirements and harsh implementation environment of the mining industry, the paper does not fully consider the evolution of the mechanical properties of COSGB in complex environments and its impact on the water environment. In the next step, it is necessary to study the influences of environmental factors such as groundwater pH, salt corrosion, temperature and stress conditions on the mechanical properties and toxic leaching of COSGB.This study explored the possibility of using CPB, OSR, SS and GGBS without the addition of any chemical reagents as solid waste cementation filling. The optimal mix ratio was found through single-factor analysis and the BBD response surface method. In addition, a variety of microstructural characterization techniques were used to gain insight into the hydration mechanism. The environmental impact of COSGB based on the optimal ratio was evaluated. The main conclusions summarized from the experimental results are as follows.


This research aims to expand the knowledge on the level of development of segmental flexibility, to girls aged 7–14 years, who practice synchronized swimming. The study includes 112 girls aged between 7 and 14 years, divided into groups on age, every two years, and on the period of synchronized swimming between 6 months and 42 months. The study focused on three body segments, namely: torso, hip, and shoulder. Segmental flexibility was assessed using 5 tests: standing trunk flexion, shoulder flexibility, Hip-split legs sideways, Hip-split antero-posterior with the right foot forward, and Hip-split antero-posterior with the left foot forward, performed in the gym. The statistical analysis was performed using the SPSS-24 software aiming at the following parameters: arithmetic means (X), standard deviation (SD), minimum (Min), maximum (Max), CI–95% Confidence Interval for Mean with the two lower and upper marks, Kolmogorov-Smirnov test for testing the normality of data distribution and a multifactor ANOVA analysis, using the F test. The most significant improvements highlighted by the differences between initial and final were for: the shoulder flexibility test in the 13–14 years’ groups; flexibility of the spine registered the biggest difference between the 9–10 years’ group; for hip-split legs sideways the biggest difference was between 9–10 years’ group and 13–14 years and 9–10 years, too. The hip-split antero-posterior tests with the left and also, for right foot forward, showed the biggest differences between tests for 13–14 age groups. The development of joint flexibility has an upward evolution, being conditioned by the age of the practitioners and by the operating methodology specific to synchronized swimming. The longer the training period, the greater the premises for the development of segmental flexibility. Synchronized swimming is a sport discipline, which requires the development of specific technical elements, called mandatory figures, according to some models adopted, requiring a high performance, within the competition ,2.The higher level of technicality specific to competitive synchronous swimming requires the performance of complex and synchronous figures with high parameters of technique, shape, and amplitude and which are based on the special abilities of practitioners in terms of functional physical capacity .The specificity of synchronized swimming involves precise synchronized movements and high-risk acrobatic maneuvers, mostly performed underwater, requiring flexibility, kinesthetic awareness, and aerobic and anaerobic conditioning ,5.For the realization with a high technicality of the imposed elements, an important factor is the level of development of the motor qualities. Segmental flexibility is an essential condition that contributes to the increase of spectacularity from the point of view of artistic appreciation, being responsible for the accuracy and precision of the shape of the elements performed, from a technical point of view . FlexibiAccording to FINA, the technical evaluation takes into account Visible scales of deviation of the angle, from 1 to 15 degrees from a small, from 16–30 degrees for a medium, and over 30 degrees for large deviation, which implies a process of continuous training of efficient and concrete segmental flexibility, both at the level of the lower limbs, the hip joint as well as the scapula-humeral center and the spine. The technical evaluation representing 30% of the final grade in different powers depending on the work formation on execution and synchronization, aims at the following aspects: the design representing the degree of compliance with those positions and movements specified in the description of the figure reported to the accuracy of all body positions and transitions; the control manifested by the level of performance achieved by the control factors aiming at the use of force and coordination to demonstrate mastery of figure execution aiming for example: maintains stable correct positions, specific transient body movements accurately and effortlessly, a general impression of ease in achieving performance, the direction of execution of movements, as well as ease and synchronization in performing specific figures .The evaluation of the technical level in synchronized swimming focuses on the appreciation of the level of performance achieved on a series of imposed figures, a level largely influenced by the degree of flexibility and amplitude of the movements. In this sense, a study on flexibility in girls aged 12.7 ± 1.1 years, who practiced swimming, showed that in the training process there is no special emphasis on the development of this quality, concluding that additional research is needed in other sports in the aquatic environment, such as synchronized swimming .Several studies show that the system of training synchronized swimmers should aim at complex approaches to motor capacity, through the systematic and scientific evaluation of the somatic and motor parameters involved in achieving performance goals ,10,11,12In aquatic sports activities, physical training involves training in both the aquatic environment and in gymnasiums. Synchronized swimming requires athletes a higher level of development of motor qualities such as strength, endurance, and especially flexibility, which can be improved more effectively by exercising in various environments . StudiesTaking into account the main positions specific to synchronized swimming, we made correspondence between the specific positions, the joints involved in the movement, and the required segmental flexibility  The imagStudies on the aspects of flexibility in synchronized swimming, on different age categories are relatively few and focus mainly on classic mobility tests. We believe that studies should aim at a complex assessment of flexibility involving both classical tests and the degree of flexibility in performing figures specific to synchronized swimming in relation to the particularities of age and level of training.The novelty of our study consists in evaluating the developed level of segmental flexibility in girls, by age categories for 7–14 years in relation to the level of sports training in synchronized swimming. This study can provide a concrete orientation of the evolution of the values of segmental flexibility in girls who practice synchronized swimming necessary for specialists in the field in selecting the design and implementation of training methodologies, as well as in their orientation in the initial and secondary sports selection.This study aims to evaluate the effects of applying a program to a specific exercise program on the segmental flexibility of synchronous swimming athletes aged 7–14 years, divided into groups of 2 years each.The interventional study used the devices to measure the anthropometric parameters: thalliometer for investigating height (cm); Tanita BC 730 scale for monitoring body composition parameters, metal anthropometric band for arm span, centimeter, and flexometer for segmental flexibility assessment.The present study was conducted in accordance with the guidelines of the Declaration of Helsinki, and it was approved by the Review Board of the Physical Education and Sports program, date of approval 3 May 2021. For this article, all authors contributed equally; also for this manuscript, all authors have an equal contribution with the first author.-SS1—group 7–8 years: initial testing 1–3 April 2021; implementation of the training program 5 April–3 June; final testing 5–7 June;-SS1—group 9–10 years: initial testing 5–7 April 2021; implementation of the training program 8 April–7 June 2021; final testing 8–10 June 2021;-SS1—group 11–12 years: initial testing 7–9 April 2021; implementation of the training program 12 April–10 June 2021; final testing 12–14 June 2021;-SS1—group 13–14 years: initial testing 12–14 April 2021; implementation of the training program 15 April–14 June 2021, final testing 15–17 June 2021Periodization of the study:All the tests and training programs were applied and practiced at the gym. All test data were collected by a trained team in a single session, which included anthropometric measurements followed by evaluation of physical parameters. Anthropometric measurements were performed before the start of the physical warm-up, at the beginning of the evaluation session. The physical tests were performed after a specific warm-up program lasting 20 min.Anthropometric measurements included: Waist, Body Weight, and Span Arms, under similar conditions for all subjects . The Spa-Flexibility of the spine: standing, with legs, close together and the tips at the edge of the test apparatus , subjects make a maximum forward bending of the spine while maintaining this position for three seconds, record centimeters displayed digitally on the Flexometer;-Flexibility of the hip and lower limb joints consisted of 3 tests: the maximum lateral distance of the legs  in the horizontal plane, measuring centimeters from the pubis to the ground; the maximum antero-posterior distance in the horizontal plane of the legs (breaking) with the two variants: the right leg forward or the left leg forward, it is measured how many centimeters are from the pubis to the ground-Shoulder flexibility: from a standing position with arms outstretched and crossed back, keep your arms crossed as far as possible and measure the distance between the middle fingers of the two palms.Segmental flexibility was assessed using 5 tests:The physical training specific to synchronized swimming consists of two workouts performed in the aquatic environment and a workout in the gym, for all groups of girls. The flexibility development program was performed during gym workouts, including exercises in acrobatic gymnastics, rhythmic gymnastics, and basic gymnastics, with an emphasis on improving the range of motion . To the This is an interventional study with a convenience sample, consisting of 112 girls aged between 7 and 14, who practice synchronized swimming at the Torpy sports club in Targu Mures, Romania. The subjects included in the study were divided into groups according to age, every two years, and the period of synchronized swimming. Parents or legal tutors, as well as the participant in the study, provided informed consent to participate in this study. The anthropometric features are presented in -SS1 group consisting of 32 girls aged 7–8 years, who practice synchronized swimming for 6–8 months, subjects participate in national competitions only as spectators;-SS2 group consisting of 42 girls, aged between 9–10 years, with a sports experience of 12–18 months and who participates once a year in national competitions;-SS3 group consisting of 28 girls, aged between 11–12 years, with a sports experience of 18–24 months and who participates twice a year in national competitions, as well as twice a year in interclub competitions;-SS4 group consists of 30 girls, aged 13–14 years, with a sports experience of 24–42 months and who participate twice a year in national competitions and 2–3 international competitions per year.Sampling includes:Anthropometric results reveal a uniform development and within the limits of normality of all subjects of the experiment.In this study we used SPSS 24 software to calculate the following statistical parameters: arithmetic mean (X), standard deviation (SD), minimum (Min), maximum (Max), effect size, the statistical power of the sample (SP), Confidence Interval for the mean (CI–95%) with the two benchmarks lower and upper, Also, we calculate the Kolmogorov-Smirnov test to test the normality of data distribution.p < 0.05 for all analyses.For Cohen’s effect size interpretation, we used: over 0.20 means small effect size, over 0.50 means medium effect size, over 0.80 means large effect size. For this study, the significance was set at The evolution of the development of segmental flexibility compared to the 4 age stages considered for the study was ascending with no periods of regression in terms of all segmental flexibility tests. The most significant confidentiality intervals of the initial test, per category of ages, were recorded at the Hip—split antero-posterior with the right foot forward for the 7–8-year-old group CI95% = 5.21–17.24; for the shoulder flexibility in the group of 9–10 years CI95% = 24.08–34.58; for the shoulder flexibility in the 11–12 age group CI95% = 24.93–37.95; for the hip—split antero-posterior with the right foot forward in the group of 13–14 years CI95% = 3.26–14.79. The most significant confidentiality intervals of the final test, per category of ages, were recorded at the shoulder flexibility for the 7–8-year-old group CI95% = 26.51–33.61; for the shoulder flexibility in the group of 9–10 years CI95% = 24.08–34.58; shoulder flexibility for the 11–12-year-old group CI95% = 24.35–35.07; for the hip—split antero-posterior with the right foot forward in the group of 13–14 years CI95% = 3.64–15.02.The analysis of the results of the study in all tests and for all age categories reflects the fact that the values of the Cohen parameter were between 0.13 and 0.46 which reflects a small size of effect size. The values of statistical power were between 0.80 and 0.87, which shows that the values of the study are statistically significant compared to the threshold of 0.80 .The processing of the results showed that the segmental mobility varies depending on age, the segment involved in the movement, and the training period . For a bOur study complements the level of general knowledge about synchronized swimming, and in particular, brings a scientific contribution on the impact of the level of training on the development of segmental flexibility, on 4 age categories at the junior level. The results of our study show that flexibility varies depending on age and implicitly depending on the level of sports experience. In synchronized swimming, sports training is differentiated on levels of training that aim at the minimum acquisition of a certain number of specific technical elements that require a high degree of flexibility and level of technicality. Flexibility has a determining role in optimizing the technical and performance level in synchronous swimming, and the comparison of the four age groups of junior athletes allowed us to identify the differences in evolution in developing the flexibility of different body joints involved in performing specific technical figures. The results of the study show a small size effect in all samples and for all age groups, which we consider to be the result of the limited period of time of only 3 months in which they had their specific intervention program to develop segmental flexibility.The specific flexibility of synchronized swimming is very little studied, and previous research is mainly aimed at elite athletes, older than our subjects. On this premise, we will analyze the results of our study on flexibility compared to the results of other studies considering age. Flexibility is generally considered to be one of the most important motor skills, it involves the range of possibilities of movement of a single joint or a series of joints ,18.In the field of sports, several technical elements require a high degree of flexibility from athletes in various joints. Studies have shown that the higher the level of segmental flexibility, the better the level of performance in water sports, and synchronized swimming falls into this category ,20.The results of our tests are in line with the development trends of segmental flexibility identified in previous studies, so a study conducted in 2008 on passive and active balance flexibility on elite athletes with an average age of 20.6 ± 2.5 in the split legs sideways test, recorded the following results of the arithmetic mean of 2.82 (2.18), passive split and 1.77 (4.38) in an active split .The flexibility of the spine favors the posture and the amplitude of the movement of the other body segments, being essential in performing the specific figures of synchronized swimming ,23. Our A comparative study was conducted on two age categories, aged 11–12 (n = 65) and 13–14 years (n = 70), female, aiming at the flexibility of the spine, between swimmers—experiment group and those who do not practice swimming, the results were the following: in the category 11–12 years 6.24 ± 4.19 in the control group, respectively 6.39 ± 4.93, and in the category 13–14 years the control group had 9.27 ± 10.04 and the swimmers 3.29 ± 14.3 . These rSegmental gymnastics is an effective means of developing flexibility, at all ages, as evidenced by several studies ,27,28,29Numerous studies highlight the interrelationship between sports experience and the content and duration of specialized training for the development of flexibility in relation to the vast range of practitioners ,33,34,35Limits and strengths of the study. After completing the study, we were able to identify some relevant limitations. The study included only subjects aged 7 to 14 years. Extending the study to other age groups would help identify particular issues depending on the subject of the current study. In the study we considered only samples of girls; a relevant limitation of the current study was that the results of our study could not be compared with previous studies because their number is very limited and covers only some of the research aspects of us, which led us to refer to complementary sports such as artistic gymnastics and swimming. Another limitation would be the relatively small number of subjects included in the study, but this fact is due to the small number of junior girls’ practitioners of synchronized swimming in Romania. The flexibility tests of the spine also involve those of the hips and hamstrings, in our study we did not aim to evaluate the “Surface Arch Position” test, this fact we consider to be a limitation because not all tests were performed in immersion. We did not aim to compare the results of the research samples with control samples, because the number of practitioners on the vast categories studied is insufficient to correspond to the inclusion criteria.The strengths of the study can be summarized as follows: the number of segmental flexibility parameters targeting the main segmental joints involved in performing technical elements in synchronized swimming, the complexity of specific tests to assess segmental mobility applied to junior synchronized swimmers; also the analysis on 4 age samples divided into groups every two years, framed in 7–14 years, the way of evaluating the impact of the specific training of synchronized swimmingThe analysis of the research results between the age groups highlights the fact that between the initial and the final testing, the highest progress was registered for the flexible shoulder in the 13–14 year’s group, and the lowest progress at 7–8 year’s group; for the flexibility of spine the best result was obtained at 9–10 year’s group and the lower progress at 11–12 year’s group; for the hip-split legs sideways the best result was obtained at 9–10 and 13–14 year’s groups and the lower progress at 11–12 year’s group; for the hip-split antero-posterior with the right food forward the best result was obtained at 13–14 year’s group and the lower progress at 11–12 year’s group; for the hip-split antero-posterior with the left food forward the best result was obtained at 13–14 year’s group and the lower progress at 7–8 year’s group.Following the analysis of the results, we can see an upward evolution of the development of joint flexibility for all age groups, and all joints considered in the test were in accordance with the particularities of growth and physical training development. The results of our study agree with previous studies on the level of development of segmental flexibility which is dependent on several factors including the age of onset of specialized training, the content of the training program that must be specialized for developing segmental flexibility in correlation with the specifics and particularities of the sport practiced and with the execution technique. The results of our study complete the level of knowledge on the development of segmental flexibility of synchronized swimming practitioners at the junior level, which may have a major future impact on the training methodology of athletes and implicitly on the level of development of the execution technique.


The position calibration of inertial measurement units (IMUs) is an important part of human motion capture, especially in wearable systems. In realistic applications, static calibration is quickly invalid during the motions for IMUs loosely mounted on the body. In this paper, we propose a dynamic position calibration algorithm for IMUs mounted on the waist, upper leg, lower leg, and foot based on joint constraints. To solve the problem of IMUs’ position displacement, we introduce the Gauss–Newton (GN) method based on the Jacobian matrix, the dynamic weight particle swarm optimization (DWPSO), and the grey wolf optimizer (GWO) to realize IMUs’ position calibration. Furthermore, we establish the coordinate system of human lower limbs to estimate each joint angle and use the fusion algorithm in the field of quaternions to improve the attitude calibration performance of a single IMU. The performances of these three algorithms are analyzed and evaluated by gait tests on the human body and comparisons with a high-precision IMU-Mocap reference device. The simulation results show that the three algorithms can effectively calibrate the IMU’s position for human lower limbs. Additionally, when the degree of freedom (DOF) of a certain dimension is limited, the performances of the DWPSO and GWO may be better than GN, when the joint changes sufficiently, the performances of the three are close. The results confirm that the dynamic calibration algorithm based on joint constraints can effectively reduce the position offset errors of IMUs on upper or lower limbs in practical applications. In recent years, inertial measurement units (IMUs) have attracted increasing interest in the field of human motion analysis. The wearable sensor motion capture system is less costly, more flexible, and more portable than optical camera-based motion capture devices ,2. By moRegarding IMU position displacement, there are currently two types of IMU position calibration, divided into static calibration, e.g., quiet standing, and dynamic calibration, e.g., knee flexion. For static calibration, the work  realizedThe limitations of the above calibration methods can be summarized as follows:(1) The wearing position of the sensor needs to be fixed or special tools are required ,10,11.(2) The limbs are required to perform specific motions, but it is difficult for volunteers with damaged joints to complete them. Even volunteers with normal physical activities need to be guided by professionals ,13.(3) The direction of IMUs must be estimated in advance to complete the calibration, and it is easy to make mistakes, especially when using a magnetometer, which is vulnerable to the interference of magnetic field ,15,21.(4) When the joint rotates, it cannot be fully rotated in all specified directions, resulting in the decline of calibration accuracy ,19.Considering the limitations of previous calibration algorithms, and based on the joint constraints proposed in , in this(1) The four IMUs are bound to the waist, upper leg, lower leg, and foot, respectively, for the gait experiment, and the data of accelerometers and gyroscopes of each IMU are collected. High-precision IMU-Mocap equipment is bound on the lower limbs for synchronous data acquisition with IMUs. High-precision motion capture equipment is only for reference. In addition, we place IMUs in two different positions, and three subjects are tested in two positions, including one female of height 165 cm, and two males of height 175 cm and 180 cm respectively.(2) The collected data are substituted into the GN, DWPSO, and GWO for position estimation to obtain the position information of IMUs relative to the limbs.(3) Using the calibrated IMU position information, we establish the human lower limb coordinate system to calculate the angles of the hip, knee, and ankle joint in each DOF. Additionally, to improve the performance of attitude calibration, the quaternion fusion algorithm is used to fuse the data of the accelerometer and gyroscope of single IMU.(4) The performance of the three algorithms is evaluated by comparing them with the high-precision IMU-Mocap reference device.The following of the paper is organized into 5 parts. In In this study, we focus on the calibration of IMUs’ positions relative to the lower limb joints. According to the international society of biomechanics (ISB) standard  and the S, For the IMU position estimation, human lower limbs can be simplified as rigid segments connected by joints. t  The spherical jointThe spherical joint is analyzed on the hip joint as an example and is applicable for the ankle joint. Assuming A and B are connected through the joint fined by .The IMU position displacement will cause the errors of Equation , and the(2) The hinge jointThe knee joint is a hinge joint, and the model of the knee joint rotation axis estimation proposed in  is definEquation (5)rJK,SEquation  to be unEquation .(6)eJK Randomly generate initial values of (2) Calculate the deviation vector Equation .(3) Calculate the Jacobian matrix Equation , and theEquation (10)[wS](4) Update Equation  and retuFor Equation , the opt(1) Randomly generate initial values of (2) Calculate Equation (3) Calculate the deviation vector Equation .(4) Calculate the Jacobian matrix Equation  and calc(5) Update Equation  and retuAccording to the definition of the DH coordinate system in , the thrEquation .By analyzing the algorithm, the limitations of the GN are as follows:(1) In the process of using the GN, the Jacobi matrix theoretically needs to be positive definite; however, the calculation may not be of full rank. When people walk, the motion of the knee joint is mainly flexion and extension, i.e., there is a significant change in only one DOF. When in other DOF, such as internal/external rotation of the knee joint, quations –10), th, thwx=wy(2) In the process of practical calculation, the GN is iterated along with one or two of the matrix entries, which causes the high complexity of the Both the population algorithm and genetic algorithm simulate the adaptability of the individual population on the basis of natural characteristics and use some transformation rules to solve the optimal solution through the search space. However, individual variation will occur in the process of the genetic algorithm, which cannot completely solve the constraints in the optimization problem . TherefoUnder the same constraints as , we intrN is the number of particles in the population, and At time t, let Equation . The vecEquation .(17)xJHFor Equations  and 6),,6), the (1) Initialization (2) Substitute Equation  to obtaiEquation  to obtai(3) According to Equations  and 20)20) to ca(4) Update (5) Repeat steps 2∼4 until N is small, the performance of dynamic weight is better than the fixed weight. It is suitable for some scenes that require high timeliness. When the N is large, the performance of dynamic weight is similar to fixed weight.In step 4 of the algorithm, Equation .(24)μ˜=Equation . When thTo verify the influence of different population algorithms on IMUs’ position calibration performance, we also introduce the grey wolf optimizer (GWO)  for posip is a different individual in the population and The parameter vector containing the IMUs position is expressed by Equation . The vecIn the GWO iteration, the position of each gray wolf represents a feasible solution in the solution space. Let the wolf with the best position be (1) Encircling Preyfpα=h1·xJ(2) Huntingquations  and 28)(28)xJi,p·ψ·r2−ψ,xJi,pεγ+1·ψ·r2−ψ,xJi,pεγ+1(3) Attacking PreyDuring the iteration, when The algorithm steps at time t are as follows:(1) Initialization (2) Calculate the individual fitness of the population by substituting Equation  and xJK,Equation . Select (3) Calculate the position vectors of Equation . Update quations  and 29)pε relati(4) If the maximum number of iterations is reached, go to step 6. Otherwise, go to step 5.(5) Reorder to determine the position of the gray wolf, and go to step 2.(6) Output current optimal solution by Equation .To calculate the joint angle, the coordinate system of each limb needs to be constructed first. The previous method of establishing the limb coordinate system is to let the subject stand in a standard standing posture. The limitations of this method have been analyzed in In human kinematic analysis, it is crucial to determine the spatial relationship between two adjacent limbs. The establishment of the spatial relationship between two limbs depends on the coordinate frame fixed on each limb, i.e., the coordinate system attached to a limb. The commonly used method in domestic and overseas is to establish the coordinate frame on the axis of the proximal or posterior joints of each limb . In thisAs shown in In The attitude transformation between two adjacent sensors is defined by Equation .(30)RBAAccording to the analysis in Equation .(31)x+xEquation .(32)a+xThe normal line of Equation .(33)rxaAccording to Equations  and 33)33), VBN Equation .(34)zL2The attitude transformation matrix from Equation .(35)RBLSimilarly, we can obtain Equation .(36)RCLAt initial stage, c is cos and s is sin. Euler angle can be calculated by Equations RL2Equation .(39)RLiquations –42).wh.whβ, whi, j, k is an imaginary unit, i, j and k.To improve the accuracy of attitude acquisition by single IMU, we need to fuse the attitude rotation matrix in Equation . The qua(1) Quaternion initializationAt time t, the quaternion of attitude change is Equation .(44)q0=(2) Correction of angular velocity errorBased on the definition of cosine matrix and Euler angles in , the gragtOs=ROsOEquation .(46)gxOIn the process of IMU attitude rotation, the gravity vector measured by the accelerometer is Equation .(47)ξx=(3) Data fusionThe cross product error is adjusted by proportional-integral (PI) controller  to corre(1) At time t, integrate the cross product error by Equation .(48)ξx+(2) The fused gyro measurements are defined by Equation .(49)wxOEquation  increase(4) Update quaternion(5) Convert the updated quaternions into matrix forms by Equation , and theIn Considering that different gait characteristics of different people may affect the experimental results and avoid the randomness of the experimental results, in the data acquisition phase, the data of the accelerometer and gyroscope are acquired by three subjects, including one female at a height of 165 cm (subject 1) and two males at heights of 175 cm (subject 2) and 180 cm (subject 3), respectively. Among them, the data of motion capture device are only used for reference, which is different from the data collected by IMUs and they do not affect each other. As shown in To analyze the accuracy of the algorithm, we compared the deviation between the three algorithms and the reference value. The angle calculated by the GN, DWPSO, and GWO are estimated values, and the angle calculated by motion capture equipment are reference values. The Root Mean Squared Error (RMSE) between the estimate values and the reference value for each DOF is calculated by Equation .(51)RMSCombined with the analyses in To more intuitively evaluate the consistency between the results of the three calibration algorithms and the reference, we selected the angle values of Through the above analysis, it shows that in the IMUs position calibration of three joints of human lower limbs, the three algorithms have achieved good calibration results, and the calibration accuracy of the population algorithm is better than GN. When the joint changes sufficiently in a certain DOF, the results of the three algorithms are close. When the joint changes are insufficient, the calibration accuracy of the population algorithm is obviously better than GN. For two different population algorithms the DWPSO and GWO, different choices can be made according to practical applications.In this work, we introduce the DWPSO, GWO, and GN algorithms to realize the dynamic calibration of IMUs’ positions based on human lower limb joint constraints. The performance of the algorithm is evaluated by gait experiments. The results show that the three algorithms have achieved IMU position calibration and are suitable for estimating the angles of the hip, knee, and ankle of humans during free walking. The simulation results show that the DWPSO has the best calibration performance, followed by the GWO and GN. When the joint rotation is sufficient or the joint is in the main motion, the performances of the three algorithms are close. When the joint rotation is insufficient, the performances of the DWPSO and the GWO are significantly better than the GN.At present, our work has achieved an IMU position calibration of human lower limbs. However, when applied for a whole-body calibration, a large amount of data may cause the decline of the searchability of the DWPSO and GWO. In future work, we need to conduct further experiments.Another route of future work is that when the offset error of IMUs position drifts slowly over time in the short term, an accelerometer and gyroscope can be combined to estimate the joint axis of the knee joint, and further improve the position calibration accuracy.


In addition to primary pollutants CO and NOx emitted from mobile sources, PM2.5 (primary and secondary) and secondary Ozone (O3) during the lockdown period will also be analyzed using both statistical methods on air quality data and the modelling method with emission and meteorological data input to an air quality model. By estimating the decrease in traffic volume in the Sydney region, the corresponding decrease in emission input to the Weather Research and Forecasting—Community Multiscale Air Quality Modelling System (WRF-CMAQ) air quality model is then used to estimate the effect of lockdown on the air quality especially CO, NO2, O3, and PM2.5 in the Greater Metropolitan Region (GMR) of Sydney. The results from both statistical and modelling methods show that NO2, CO, and PM2.5 levels decreased during the lockdown, but O3 instead increased. However, the change in the concentration levels are small considering the large reduction of ~30% in traffic volume.In early 2020 from April to early June, the metropolitan area of Sydney as well as the rest of New South Wales  experienced a period of lockdown to prevent the spread of COVID-19 virus in the community. The effect of reducing anthropogenic activities including transportation had an impact on the urban environment in terms of air quality which is shown to have improved for a number of pollutants, such as Nitrogen Dioxides (NO The COVID-19 pandemic in 2020 has caused death and economic misery in all countries of the world. The disease was named as Coronavirus Diseases 2019 (COVID-19) by WHO and is caused by a type of coronavirus strain called Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)  uet al. 20 [21] use. (2020)  also use. (2020)  used WRF. (2020)  in the s. (2020)  with satThe main reason for the reduction during the lockdown period is the reduced anthropogenic activities which cause the emission of air pollutants and hence, degrade air quality in the environment. The transportation sector, which is still much reliant on combustion type technology, is the main contributor to the emission of pollutants. Kaskaoutis et al. (2021)  have shoA significant reduction in traffic volume and hence, congestion on the Sydney road since the lockdown began in late March 2020 is shown in x and CO as well as for particulate matters such as PM10 and PM2.5, will reduce during the lockdown period. In Singapore, Li and Tartarini  u. (2020)  also use. (2020)  used theThe results from the use of WRF-CMAQ air quality model in our study to predict the effect of COVID-19 lockdown on air quality in GMR of Sydney confirm and are consistent with the results from the statistical and diurnal analysis.2, CO, and PM2.5. However, this also caused an increase in the secondary pollutant O3 median and average levels. The simulation using the air quality model on the effect of emission change on air quality also confirmed the above results derived from the statistical and diurnal analysis of air quality monitoring data. Even though the change in air quality during the lockdown period is small, it is detectable.The reduction of emission mainly from the transport sector during the COVID-19 lockdown period in Sydney and NSW in April to June has resulted in an improvement of air quality in the GMR in terms of the reduction of the pollutants of NOThe lesson from the COVID-19 lockdown showed that the improvement in air quality due to the reduction of transport emission is small but measurable. In addition, it highlights the importance of not only the transition from the current dominant combustion-technology vehicle fleet to the electric or non-combustion technology, but also the effort in emission reduction from other sources  including biogenic sources such as dust to make a significant impact on air quality improvement in the urban areas.


In materials science and engineering, a significant amount of research has been carried out using indentation techniques in order to characterize the mechanical properties and microstructure of a broad range of natural and engineered materials. However, there are many unresearched or partly researched areas, such as, for example, the investigation of the shape of the indentation load–displacement curve, the associated mechanism in porous materials with clastic texture, and the influence of the texture on the constitutive behavior of the materials. In the present study, nanoindentation is employed in the analysis of the mechanical behavior of a benchmark material composed of plaster of Paris, which represents a brand of highly porous-clastic materials with a complex structure; such materials may find many applications in medicine, production industry, and energy sectors. The focus of the study is directed at the examination of the influence of the porous structure on the load–displacement response in loading and unloading phases based on nanoindentation experiments, as well as the variation with repeating the indentation in already indented locations. Events such as pop-in in the loading phase and bowing out and elbowing in the unloading phase of a given nanoindentation test are studied. Modulus, hardness, and the elastic stiffness values were additionally examined. The repeated indentation tests provided validations of various mechanisms in the loading and unloading phases of the indentation tests. The results from this study provide some fundamental insights into the interpretation of the nanoindentation behavior and the viscoelastic nature of porous-clastic materials. Some insights on the influence of indentation spacing to depth ratio were also obtained, providing scope for further studies. Porous materials are encountered in a large number of engineering applications, such as, for example, in petroleum/energy engineering, space exploration, biomedical, industrial, and civil engineering. Understanding of the mechanical performance of materials and the development of constitutive modeling necessitates the use of nano-to-micromechanical-based approaches as they offer insights into the relationship between bulk behavior and microstructural characteristics. Examples of this may refer to structural/cementitious materials ,2,3,4, sIn the present study, the nanoindentation technique was employed to investigate the relationship between the (constitutive) stress–strain response, as observed from the indentation tests, with the structural characteristics of a highly porous material composed of plaster of Paris (PP). This material has a clastic nature and a network of pores of various sizes; thus, it can be used as a benchmark to understand the fundamental behavior of highly complex materials used in various applications, such as the construction industry ,13 and tThis article is structured as follows: H) and elastic modulus (mE) of a given material are directly dependent on the accuracy in the estimation of the elastic contact stiffness (S) from the load–displacement curves. As shown in Equation (1), the reduced elastic modulus (rE) (described in Equation (2)) is directly related to the elastic stiffness and the area (cA) with a geometric factor β (after King [maxF).The primary mechanical characteristics derived from indentation tests are the hardness and elastic modulus of a given material. Reliable values of hardness (ter King ) used foter King . Additioϑ represents the Poison’s ratio, E represents the elastic modulus, and the subscripts m and I represent the tested material and the indenter, respectively.In Equation (2), The unloading stiffness (or elastic stiffness) is computed assuming the unloading curve as a linear function, which was upgraded by Oliver and Pharr , proposiFrom the nanoindentation observations, mostly of brittle materials, phenomena such as pop-in (or chipping), elbowing, and pop-out were generally observed in the load–displacement curves, which define the material characteristics and their transformations during the test ,26,27,28Shales are one of the most prominently tested porous geological materials to explore their mechanical properties through nanoindentation tests ,32,33,34The contact stresses developed during the indentation test are critical to estimate the possible factors leading to irregularities in the load–displacement curves, hence the variation in the hardness and modulus values. Juliano et al.  used thej represents a given data point. The stress (σ) is a simple ratio of indentation load (F) to the contact area (cA). The contact area, in turn, depends on the displacement corresponding to the indenter impression (cd) and correction factors for an imperfect indenter tip . The elastic displacements (sd) governing the unloading curve are dependent on the load and the unloading stiffness (S) based on the linear approximation of the unloading curve based on Sneddon [In Equations (4)–(8), the subscript  Sneddon . These fz-axis (loading axis) of the nanoindentation apparatus, air indents were performed before each session regulating the electrostatic force–displacement variation of the indenter. Additionally, a few trial indentation tests were performed on a standard material  to compare the range of mechanical properties estimated with the standard values. Plaster of Paris (PP) is a gypsum plaster (CaSO4. 1/2H2O), an ultrafine white powder that crystallizes quickly when mixed with water. The hemihydrate powder was mixed with water at a water-to-plaster (w/p) ratio of 0.8 (by weight), and consecutively, the mixture was cast in molds to form small blocks feasible to be tested in the nanoindentation apparatus. The influence of the w/p ratio on the porosity of gypsum was studied by Isern and Messing [w/p ratio for a given mixing time. With a w/p ratio of 0.8, the resultant PP block is a brittle-to-ductile material with a highly porous structure. 3 . As a procedure of calibration of the  Messing , and it 3 (after ) and thePA) and also the density of the pores (PD). The density of the pores is defined as the total surface area of all pores in a given surface material area of 100 μm × 100 μm. The surface area of the pores was calculated using an image processing tool (ImageJ), where the 2D images obtained from the optical surface profiler of a given scanned area were processed to threshold the 8-bit type image , and only 25% of the pores had values in the top one order of magnitude, signifying a greater number of pores being very small with an area lower than 1 μm2. However, these micro-scale surface pores also can greatly affect the nanoindentation results depending on the targeted indentation load and displacement. Energy-dispersive X-ray Spectroscopy (EDS) was also performed to quantify the elements present in the PP blocks (without Ag coating) tested in this study. A representative EDS spectrum is shown in 2 for each measurement, as shown in the SEM image in 2. Moreover, the porous structure and the pore sizes and shapes in the plaster blocks are extremely complex  was used to obtain the surface profiles of the plaster blocks, with an objective to observe the surface pore distribution. The vertical scan interferometry technique was adopted to obtain the surface profiles using white light. The pores in the material originating from the surface can be identified in the surface profile as large and sudden depressions compared to the rest of the surface. The surface profile of a representative plaster block is shown in pe image c of the pe image b. A binape image d. For a The nanoindentation tests in the present study were conducted in a grid format with 25 indents spaced at 50 μm in both lateral directions, hence covering a 200 μm × 200 μm area on the block. The grid format is considered highly important, particularly in the indentation studies related to the chemical composition of the material to cover the surface of the material in a uniform and standard way . BesideThe indentation hardness and elastic modulus of plaster block were calculated based on the discussions presented in maxF). The maximum indentation displacements (maxd) ranged between 88.6 nm and 447.2 nm (5 times), with the elastic fractions of the displacements  ranging between 2.4% and 66.3% for cycle 1 of indentation. In cycle 2, the maxd range reduced to 81.6–314.5 nm (3.8 times), with the e,fd values ranging between 18.9% and 89.3%. e,fd against maxd, indicating a decreasing trend of elastic displacement fraction with increasing maximum indentation displacement for both indentation cycles, and the corresponding ranges of values can be observed.Besides the elastic stiffness, both these primary mechanical properties, hardness, and elastic modulus, are also strongly dependent on the displacements during the indentation test for the given maximum indentation load , as the development of micro-cracks and dislocation during cycle 2 in the densified zones leads to more pop-in events with smaller lengths than soft deformations.The second mode with moderate abruptness in the displacement had a softer behavior during the pop-in event and was less frequent in a given test with a maximum pop-in length of 15.8 nm. The extreme mode of pop-in events is associated with significantly large deformations (more than 100 nm) with an insignificant increase in the indentation load before the hardening part of the loading curve. However, these large deformations are not abrupt during the indentation test, and only one such event was observed in a given test. By attributing the pop-in events to the fracture development and the dislocations in the material during indentation, the micro and moderate modes of pop-in events can be correlated to the brittle nature of material deformation. A higher density of micropores and relatively denser interlocking of needle-shaped grains to form a continuous micro-structural fabric could lead to pop-in events with short lengths. On the other hand, regions on the plaster surface with weaker interlocks developed during the crystallization process led to soft deformations of the indenter during the loading phase. A representative set of such zones are highlighted in the SEM image produced before the indentation test b. These In As discussed in The loading phase of the stress–displacement curves with a plateau or a decreasing trend releases the stored strain energy as the unloading phase begins. The unloading curves, as discussed in Oliver et al.  reviewedNose effect (or bowing out) was observed in a limited number of tests (6 out of 50 tests: 12%) and only in cycle 1 of loading a. These maxF , then the compressive stresses generated during the loading phase would be very limited. Delayed elbow event in unloading indicates that the compressive stresses are gradually released, and the change in the slope (elbow) occurs due to the sudden upliftment of the material associated with the crack opening. In the present study, the indentation tests in cycle 1 tests showed elbow events at the end of the unloading phase and not before the lower quarter (125 μN) during unloading.From In cycle 2, 90% of the tests (45 out of 50 tests) showed elbowing events in their unloading curves; the elbowing events also occurred earlier in the unloading phase compared with cycle 1 . However, better conformity of the hardness values was shown in cycle 2 with significantly reduced coefficient of variation values;The test data showed lower indentation displacements and correspondingly higher indentation hardness values in cycle 2 tests compared to cycle 1, which suggests densification of the material resulting from the pore collapse under the indenter during the first cycle. It was interpreted, due to the highly porous and brittle nature of the PP blocks, that cycle 1 of indentation led to a pop-in phenomenon and significant soft deformations occurred in the loading phase, causing possible pore collapse;Regions of the PP blocks with weaker interlocks led to a softer and ductile response, whereas pop-in behavior was majorly of brittle nature and occurred in regions with the denser interlocking of the microparticles;Major observed modes of behavior of the unloading phase could be classified as (i) elbowing , and (ii) bowing out  and they are related with the relaxation of the material upon unloading. Additionally, significant increases in pop-in events in the loading phase and elbowing events in the unloading phase of cycle 2 tests reconfirmed the densifications of the indent zones during cycle 1 test;Unlike most minerals whose indentation tests revealed their dominant material behavior to be ductile, brittle, or viscoelastic, the present material (PP), which has applications ranging from the construction industry to medicine, showed a highly complex behavior with a mix of ductile deformations, brittle breakage, and often viscoelastic deformations;Some indications of indentation spacing to depth ratio influence on the mechanical properties were observed, despite the ratio being greater than 100, suggesting a need for further studies on the aspect of indentation spacing to depth ratio influence on highly porous and clastic materials. Additionally, given the highly complex porous structure of the PP blocks, a quantitative correlation between the pore attributes and indentation tests using grid-based tests can provide a much deeper understanding of their deformation mechanisms.Nanoindentation tests were conducted on blocks of plaster of Paris (PP) in the present study, employing a grid format with 25 indents spaced at 50 μm in both lateral directions, covering, in this way, a 200 μm × 200 μm area on the block. Emphasis was placed on understanding the influence of previous loading history on the stress–strain response of the material, which loading history causes (due to pre-application of indentation) damage on the surface of the PP block, thus influencing the consecutive nanoindentation results. For this purpose, two loading–unloading cycles were applied for each contact zone of the predefined grid. The major conclusions from the study are summarized as follows:


With an estimated 1 billion people affected across the globe, influenza is one of the most serious health concerns worldwide. Therapeutic treatments have encompassed a number of key functional viral proteins, mainly focused on the M2 proton channel and neuraminidase. This review highlights the efforts spent in targeting the M2 proton channel, which mediates the proton transport toward the interior of the viral particle as a preliminary step leading to the release of the fusion peptide in hemagglutinin and the fusion of the viral and endosomal membranes. Besides the structural and mechanistic aspects of the M2 proton channel, attention is paid to the challenges posed by the development of efficient small molecule inhibitors and the evolution toward novel ligands and scaffolds motivated by the emergence of resistant strains. Influenza A virus is an important pathogen that still causes the death of 290,000–650,000 people in seasonal outbreaks worldwide . It pertWhile infections in humans directly from animals are unusual, sporadic infections and outbreaks caused by influenza A viruses have occurred in the past. The most severe pandemic was the “Spanish Flu” of 1918–1919, which caused 20–50 million deaths by influenza A H1N1 virus strain. The subsequent health challenges were the “Asian flu” in 1957 and the “Hong Kong flu” in 1968, which were caused respectively by H2N2 and H3N2 strains, and more recently the 2009 swine influenza pandemics, which pertains to the H1N1 strain . The latThis review aims to discuss experimental and computational efforts invested in elucidating the structural basis of the proton conducting activity of the M2 protein channel and the discovery of antiviral compounds targeting the M2 transmembrane domain. To this end, the manuscript first describes the role of the M2 proton channel in the replicative life cycle of the influenza A virus. Then, attention is focused on the structure of the proton channel, which is directly implicated in the proton transfer across the viral membrane as a preliminary event leading to the fusion of viral and host membranes and the subsequent release of the viral genetic material. Emphasis is made on the molecular mechanisms that underlie the proton conduction through the lumen of the M2 proton channel. The discussion also highlights the role played by key residues located in the transmembrane region, and especially the efforts spent in the determination of the pKa of the imidazole rings present in the His37 tetrad. Finally, the manuscript examines the mechanism of action of selected inhibitors, and the evolution followed in the design of novel compounds introduced to increase the inhibition potency and to mitigate the emergence of resistance to current treatments.in vitro models, the virus can also affect different cells that contain targets decorated with sialic acid moieties  protein located in the viral envelope of the influenza A virus. It is a selective, pH-dependent channel that regulates the acidification of the interior of the virion, leading to dissociation of the viral RNA from its bound matrix proteins and release of the viral genetic material for replication .From a structural point of view, the M2 channel is a homotetramer, each monomer consisting of 97 amino acids and can be divided into four main regions: 1) the highly conserved, unstructured N-terminal domain (residues 1–21) located in the viral exterior, which assists the incorporation of the M2 channel into the virion, 2) the TM domain (residues 22–46) , which aThe TM region contains the binding site for M2 inhibitors in the interior of the pore, filling a pocket located between the tetrads formed by residues Val27, Ser31, Gly34, and His37 . This po+ and K+, through an antiporter-like mechanism, carrying out the metal cation efflux in conjunction to proton influx, but the permeability to protons has been estimated to be 106–107 higher than the conduction of alkaline cations , which is lost upon return to alkaline conditions (pH 8.5). However, whereas no currents were observed for mutated channels containing Phe41, Cys41, or Ala41 prior to acidification, outward H+ currents were detected by changing acidic conditions to alkaline solution.The functional relevance of His37 and Trp41 is revealed by the drastic effects triggered by mutations in these positions, which cause a reduction in the pH sensitivity of the channel, and a disruption in the proton conduction . Thus, mThe specific role played by His37 and Trp41 in assisting the proton flux through the pore of the M2 channel has been the subject of intense efforts, mainly focused on the TM domain, which reproduces most of the biophysical, site-directed mutagenesis and electrophysiological features of the full-length protein. These studies, further assisted by X-ray crystallography of structures solved in membrane-mimetic solvents at different pH and temperature, solution NMR, solid-state NMR (ss-NMR) spectroscopy (see below), in conjunction with computational simulations, have crystallized in several models of proton conduction.An early mechanistic hypothesis was the water wire model, also known as the shutter mechanism , which aIn contrast to the shutter model, the currently accepted proton conduction mechanism assumes a proton relay model . AccordiDeeper knowledge into the molecular events implicated in proton conduction has been gained from ss-NMR studies. Hu et al. refined the proton relay model suggesting that proton conduction is concomitant with His37 conformational changes  Hu et a. At high−1, which would justify the temperature dependence of proton transport, though possibly this value may contain a contribution arising from the barrier for proton transfer  simulations and hybrid quantum mechanics/molecular mechanics (QM/MM) methods. MD simulations were performed using methylammonium cation to examine the response of water molecules lying in the pore to an incoming positively charged moiety for the +2 state of the M2 channel . The res−1) for His37 deprotonation to the viral interior, leading to a predicted conductance well below the experimental value, suggesting that the ion channel is inactive at high pH conditions. In the +2 state, however, although the electrostatic repulsion between the incoming proton increases the barrier from viral exterior to His37, deprotonation of His37 is facilitated by the increased electrostatic repulsion between the leaving proton and the His37 tetrad, this process being further assisted by larger exposure to water molecules and increased flexibility of Trp41residues. Thus, the His37 deprotonation barrier is decreased to ∼12 kcal mol−1, and the predicted conductance  is in agreement with the measured values , Okada et al. reported that the proton channel is activated by low pH with a transition midpoint at pH 5.7 . Using tThe differences found in the pKas reported in Computational studies have also been performed with the aim to estimate the pKa of the His37 tetrad, while providing atomistic details of the pH-dependent protonation process. In this regard, Dong et al. performed QM/MM calculations to characterize the structural features of the +3 state and its subsequent deprotonation to the +2 species . CalculaChen et al. have used constant pH replica-exchange MD simulations to determine the pKa values using a simulation model consisting of the TM helix . In thiset al. have used CpHMD simulations performed using explicit solvent and the multisite λ-dynamics to estimate the pKa values of the His37 tetrad  and its ethyl analog, rimantadine (Rmt), were patented in 1961 and 1963, respectively , but the13C-labeled M2TM and perdeuterated Amt in ss-NMR studies performed in a lipid bilayer  determined from free energy calculations  approach  and freeup and down, respectively). Binding of Amt would follow a sequential mechanism that would involve trapping of the protonated Amt by the negative electrostatic field created by the tetrad of Asp24, followed by the adoption of a transiently populated intermediate with Amt in the up state, and finally the reorientation in the interior of the pore to the down state, which is the thermodynamically favored orientation  is reasonable, since the theoretical estimate omits the free energy contribution due to the release of Amt to the aqueous solvent, and the fact that the apparent Ki might not properly correspond to an equilibrium measurement of the inhibitory constant -spiro decan]-4-amine) shown in 1 occupies approximately the same position as the ammonium group of Amt in complex with the wild type channel (PDB entry 6BKK)  . This is4, which has an IC50 value of 18.7 μM against the wild type channel and of 0.3 μM against the V27A species. MD simulations showed that the ammonium group occupies the lower aqueous site forming solvent-mediated hydrogen bonds with the His37 residues in both wild type and V27A, but in this latter case the compound was slight shifted toward the N-terminus due to the extra space afforded by Ala27.A combination of computational techniques, biophysical studies, functional assays and classical medicinal chemistry approaches has been used also to design potent inhibitors of V27A. This mutation imposes a reduction in the steric constraint found in the wild type channel, suggesting the compounds with a size-expanded hydrophobic cage would be better suited to fit the larger volume of the pocket in the V27A channel . An exam5 reported by Vázquez and coworkers  of 16°. Nevertheless, in the V27A complex 5 adopted the down orientation, but was also found in a up-like arrangement, presumably facilitated not only by the larger volume in the pore, but also by the widening of the helices at the location of Ala27, as the cross-diagonal distance between Cα atoms at position 27 was enlarged by 1.1 Å compared to the wild type channel. The effect of including other polycyclic scaffolds as the hydrophobic cage have led to analogs 6 and 7 (50 ∼ 2 μM), but a lower inhibition in the V27A mutant   , suggestThe resistance of the S31N channel, which is the most prevalent in currently circulating influenza viruses, to inhibition by Amt is reflected in the more variable motion and orientation of the ligand in the mutated channel. Thus, whereas Amt occupies a stable position in the wild type channel, it showed larger positional fluctuations in the pore of the mutated channel. This trait is reflected in a decreased energetic stability of the ligand in the proximity to the Asn31 side chains, as reflected in potential of mean force calculations. Finally, this destabilization was accompanied by an increase in the density of water molecules around and above Amt in the S31N channel compared to the wild type species, enabling water molecules to fill laterally the available space in the channel pore . Further2 -N-{methyl}tricyclo decan-1-aminium), also denoted M2WJ332, 50 of 16 μM, being however much less effective against the wild type ion channel -N-[(5-bromothiophen-2-yl)methyl]tricyclo decan-1-aminium; 50 values of 4.6 and 1.8 μM, respectively). The chemical scaffold of compound 3 combines an adamantane moiety with a bromothiophene unit. However, the most remarkable structural feature is that 3 adopts completely flipped arrangements in the wild type and mutated channels , and the effect of cholesterol, paying particular attention to the location of channel blockers and water structure, the presence of chloride anions, structural properties of the helical bundle, and the binding site of cholesterol. While this study deserves practical interest for the choice of parameters in simulations of the M2 proton channel (and related viroporins), we limit ourselves to highlight three findings. First, the results obtained for M2 complexes with Amt at pH 8 revealed that chloride anions approached Arg45 and occupied a position located between Trp41 and Arg45 (at a distance close to 15.5–17.5 Å from the Amt nitrogen atom) presence of chloride anions stabilized in the interior of the pore. The anions approached in the C-terminus. The anions approached Arg45. Second, the simulations with CHARMM36 preserved the structure of the amphipatic helices, providing a correct description for the wedge shape geometry that might be required to modulate the saddle-shape curvatuve of the membrane for the release of virions (As a final remark, it is worth noting a very recent study by Kolokouris and coworkers where a systematic analysis of setup conditions and methodological details, covering from docking calculations to MD simulations, is presented for the M2 channel . This st virions . Finally virions , paving Resistance to current pharmacological treatments is a severe health challenge, as illustrated by the emergence of viral strains resistant to drugs interfering with the M2 proton channel, such as Amt and Rmt, but also to antiviral agents targeting neuraminidase, such as oseltamivir, as noticed in a resistant strain associated to the His275Tyr mutation in this protein . CurrentFinding new active antiviral compounds is an urgent need, which may be explored resorting to the application of a combinatorial therapy that should potentiate the effect of two or three drugs endowed with distinct mechanisms of action, hopefully being less susceptible to inactivating mutations . A long-This approach is illustrated by the research efforts invested in the M2 proton channel. In spite of the apparent simplicity posed by the membrane-embedded four-helix bundle, the flow of protons reflects a complex synergy between different molecular events, involving the acidity of the His37 residues, the structural coupling between His37 and the gating Trp41 residue, the structure of the hydrating water molecules in the pore, the pH-dependent conformational arrangement of the TM helices, the electrostatic funnel created by acidic residues in the N-terminus of the channel, and the influence of the amphiphilic helix and cytoplasmic tail on the flexibility of the TM domain, in addition to the potential influence exerted by other factors such as the nature of the lipid environment in the viral capside.3 is encouraging. This scientific background defines the framework to address novel questions about the mechanisms of drug resistance and the guidance to novel antiviral and treatment approaches, such as the potential dependence between drug resistance and the nature of the viral strain or the infected cell type, which have been the subject of recent studies (Although a full understanding of the interplay between these factors, which encompass a diverse range of spatial and time processes, has not yet been achieved, the progress consolidated in the last decades through the combined use of structural, biophysical, physiological and computational techniques is impressive, and the successful development of compound  studies . Hopeful