Larix decidua Mill.) wood, and to use reinforced polylactic acid (PLA) fiberglass (20 wt. %) filament for 3D printing this connector using AM with fused filament fabrication (FFF) technology. The design of the connector, the possibility of using this type of material, and the deposition method of filament were investigated in this research. For this purpose, several evaluation methods were applied: microscopic investigation with 50×, 100×, and 200× magnifications, both of the filament and of the 3D printed connector; mechanical testing of corner joint formed with the help of connector between chair leg and the two stretchers; and a microscopic investigation of the connectors’ defects that occurred after applying the compression and tensile loads on the diagonal direction of the L-type joint. The microscopic investigation of the composite filament revealed the agglomerations of glass fibers into the core matrix and areas where the distribution of the reinforcements was poor. The heterogeneous structure of the filament and the defects highlighted in the 3D printed connectors by the microscopic investigation contributed to the mechanical behavior of L-type connecting joints. The bending moments resulting from compression and tensile tests of the 3D printed connectors were compared to the results recorded after testing, under the same conditions, the normal mortise–tenon joint used to assemble the abovementioned chair components. The larch wood strength influenced the mechanical results and the conclusions of the microscopic investigations, as well as the analysis of the broken connectors after testing recommended the change of connector design and filament deposition direction.Additive manufacturing (AM) is used in many fields and is a method used to replace wood components or wood-jointed furniture components in the furniture industry. Replacing wood joints by 3D printed connectors would be an advantage, considering the fact that during the process of assembling furniture, the execution technology of the joints is difficult, time-consuming, and labor-intensive. Advanced technology of AM applied in furniture manufacturing helps the designers to create new concepts of product design, with no limits of shape, number of joints, color, or size. The diversity of 3D printers and AM technologies provides the selection of materials in relation with the applicability of the 3D printed object. In this respect, the objective of the present research is to design a 3D printed connector to be used for jointing three chair components, namely the leg and two stretchers made from larch ( Additive manufacturing (AM) is a constantly expanding field  which waOne of the materials used in FFF technology is polylactic acid (PLA). In order to improve its physical and mechanical properties, solutions have been found to reinforce it with various fibers  ,12,13,14Fiberglass was found to be used as a reinforcing material in the composition of polyamide-based material for printing an entire piece of furniture with SLS technology . ReinforTwo methods are applied in the literature to test the stiffness of joints: diagonal compression and diagonal tensile test ,23,24,25The present research has an objective to replace the basic glued mortise–tenon joints used for assembling the chair leg and stretchers with a 3D printed connector in which the three chair components are introduced. This replacement brings the advantage of easing the assembly process and reducing the production time, with operations such as processing and gluing the tenon–mortise joints not being necessary in this case. Considering that glass fiber-reinforced PLA composites develop enhanced mechanical properties ,28,29,30Larix decidua Mill.) wood for comparing the results.The mechanical strength of the 3D printed connecting joint was investigated on an L-type joint subjected both to diagonal compression and tensile tests. In parallel, the same tests were conducted on mortise–tenon corner joints made from larch . The printer has the following characteristics: usable filament diameter of 2.85 mm, nozzle temperature up to 260 °C, open filament system, maximum printing speed of 200 mm/s, build area of 250 mm × 300 mm × 520 mm, layer height between 0.05 mm and 0.5 mm, resolution of 0.05 mm. The designed connector is presented in The main printing parameters used for 3D printing were: print speed of 50 mm/s; printing temperature of 250 °C; layer height of 0.2 mm; 100% fill density. The 3D printing parameters were selected according to the recommendations made by the filament manufacturer and following the preliminary results obtained by the 3D printed parts in terms of mechanical strength for different printing parameters. The designed connector was intended to replace the normal mortise–tenon joint , commonlLarix decidua Mill.). Larch wood is a resinous wood with good stability and small moisture movement. It is appreciated for its fine structure, its toughness, and high density compared to other resinous woods. It is strong and sturdy and is often used for yacht building, joinery, stairs, and floors, as well as for external cladding projects and outdoor furniture, due to its good resistance to weathering. In the furniture industry larch wood is mainly used for chairs, benches, and tables and has a large availability on the market at a low cost. The sizes were 210 mm × 35 mm × 19 mm for the chair stretchers and 50 mm × 35 mm × 35 mm for the piece of the leg. The wood moisture content of the larch wood was 9.2%. The tolerances of wooden component sizes were selected for a tight fit with the connector.The wooden components (leg and stretchers) were made from larch wood  manufactured by Filaticum, Miskolc, Hungary.The characteristics of the filament are presented in The complete technical characteristics of the filament are presented in their respective technical data sheets.The initial filament deposit has the role to create the base for the next layers of deposition and to define a closed contour which will be referred to as perimeter (P) of the 3D printed connector. The layer-by-layer deposition in FFF printing technology is done at an inclination angle of 45° related to the perimeter direction, and perpendicular to the previous layer .Microscopic investigation of filament and 3D printed specimens was carried out using an optical microscope from Nikon OmniMet-Buehler . Three magnifications were used  for optical microscopy of three specimens of reinforced PLA fiberglass filament and three 3D printed samples prepared for mechanical tests. Longitudinal and transversal cuts of the parts and filament were embedded in Dentacryl technical resin for technical uses in mechanical engineering, and subjected to optical microscopy .Digital microscope INSIZE ISN PM200SB  with 10X magnification was used to investigate the defects which occurred after 3D printed connectors were subjected to compression and tensile loads in L-type joints structures. The digital microscope was used with its minimal magnification power and resolution of 1600 × 1200. The objective of microscopy was to better visualize the defects which occurred as a result of failure under tensile and compression loads. Three connectors were investigated before and after compression and tensile tests.L-type joints were subjected both to diagonal compression and tensile tests, according to the model provided by other research works ,24. The tM) was calculated according to Equation (1), in N·m.L is the moment arm, or the length of the corner joint, in m; F is the maximum failure load, in N.The bending moment in the tensile testing (cM) was calculated according to Equation (2), in N·m.L is the moment arm, or the length of the corner joint, in m; F is the maximum failure load, in N.The bending moment in the compression testing (Both mortise–tenon L-type joints and 3D printed connecting joints were subjected to compression and tensile testing and the results were afterwards compared.Microscopic investigation of filaments and 3D printed samples before testing aimed to anticipate the mechanical behavior of corner-joints and to explain the results of the tests. The microscopic investigation of the tested connectors will complete the image and further explain the causes of the samples’ failures.The microscopy of the cross-cut section of filament is shown in In The microscopy of the longitudinal section of the filament is shown in The microscopy of the cross-cut section of the 3D printed samples is shown for two samples in In The defect marked with 4 in The microscopy of the longitudinal section for two 3D printed connector specimens are shown in In In Both mortise–tenon joints and 3D printed connecting joints were subjected to tensile and compression loads in the same conditions of the tests. The calculated values of bending moments for tensile and compression testing of investigated joints are presented in The results presented in The joints made with 3D printed connectors have been tested as shown in The results from The marked areas on the connectors were investigated by 10× magnification to more clearly observe the occurred cracks.The detail of the area marked with 1 b top shoThe same delamination between filament layers was also observed on the 3D printed connector after being subjected to tensile test, as seen in the details from The microscopic investigation of the fiberglass-reinforced PLA filament has shown random distribution of glass fiber into the matrix, with areas where the presence of the reinforcement was not detected and areas where fiberglass bundles agglomerated the matrix, especially in the core, where the adherence between these fibers and matrix was poor. These defects could affect the filament strength in certain zones.On the other hand, the microscopic investigation of the samples cut from 3D printed connectors revealed that defects occurred as a result of printing parameters variation during the printing process. The low mechanical strength of 3D printed connecting joints and delamination between filament layers is explained by the direction of filament layers deposition, which is oriented parallel to the length of the stretcher with one component of the force acting perpendicular to this direction and tending to detach the layers.From the research conducted we can say that the application of this technology in the furniture field is possible and beneficial with large perspective, especially for chairs with complex joints, involving more stretchers and various jointing angles. Three-dimensional printed connectors are also a benefit to product design, with the potential to combine colors and structures.Further research work will be focused on changing the direction of filament deposition and adjusting the printing parameters, so to avoid the occurrence of major defects in 3D printing.


Due to the demand for increasing trainload and enhancing some existing heavy-haul railways, the low reserve value of bearing capacity is a problem for the 32 m-span simply supported beam. The fatigue behavior of prestressed concrete beams in a heavy-haul railway loaded by 33 t and larger axle weight of trains was experimentally investigated. The experimental results of the fatigue behaviors, including fatigue deformation, crack propagation behavior, and strains of classical materials were obtained and analyzed. A fatigue behavior assessment model was established to investigate the residual stiffness and yield point degradation of the beams loaded by the trainload. The effects of train fatigue cycles and prestress loss on the residual stiffness and yield point degradation models of the beams were analyzed. The results indicated that the crack development process had three stages during the fatigue process: the derivative stage, gradual development stage, and fatigue failure stage. Trainload was the main external factor influencing the fatigue behavior of prestressed concrete beams. The increase in trainload accelerated the degradation rate of the residual stiffness of the beams and yield point, reducing the fatigue life. The prestressing strand was primarily used to delay the concrete cracking in the tension zone. When the beam was not cracked, the prestressed concrete beam showed good fatigue performance, and the degree of prestressing did not affect the fatigue life of the beams. When the maximum fatigue load exceeded the cracking load of the beam, prestress loss in beams became a critical issue that accelerated the degradation rate of fatigue strength and reduced fatigue life. The higher the fatigue damage degree, the more pronounced the effect of prestress loss on the fatigue strength of the beams. The fatigue failure of prestressed concrete beams occurred in the bottom tensile steel bar. Therefore, when the trainload of a heavy-haul railway is greater than the cracking load of the beam, it is recommended to strengthen the beam by prestressing and strictly control the trainload to avoid yield failure. Prestressed concrete structures are widely used in essential facilities such as buildings, bridges, and offshore platforms due to the advantages of a lightweight, high stiffness, and good crack resistance. In China, fully prestressed concrete beams are widely used in heavy-haul railway bridges . For exaSeveral studies have been performed on the fatigue performance of prestressed concrete beams. Castilho et al. ,12,13,14Compared with general highway and railway bridges, the applied load of heavy-haul railway bridges has the characteristics of large load, large-amplitude vibration, high frequency, etc., which makes the cumulative fatigue damage and cracking problems more prominent. Yu et al. ,21 studiThe existing literature only presented limited information on the fatigue performance of heavy-haul railway prestressed concrete beams. According to the demand for increasing trainload and enhancing some existing heavy-haul railways, the low reserve value of bearing capacity is a problem for the 32 m-span simply supported beam. In this study, standard 32 m prestressed concrete beams in the heavy-haul railway were tested under fatigue loading, and the fatigue performance of prestressed concrete beams under 33 t or higher trainload was examined. A numerical model based on the fatigue properties of materials and Vehicle-Bridge coupling vibration was proposed to assess the fatigue behavior of the field beams. The fatigue performance assessment model of the field beams was verified with the experimental results. The presented model was then employed to study the effect of fatigue load range and prestress loss on fatigue life, fatigue strength, stiffness, and post-fatigue ultimate capacity. s5 steel strands  were selected as prestressing steel bars with an ultimate tensile strength value of fpk = 1860 MPa and parabolic shape. The steel strands were tensioned at one end, and the concrete age had exceeded 28 days. The beam ends were equipped with reinforcement mesh, an embedded steel plate, and spiral reinforcement to withstand the local pressure. The anchor end of the model beam as shown in σcon = 1395 MPa). The initial prestress was about 77% of the ultimate tensile strength. Standard 32 m prestressed concrete beams in heavy-haul railway were taken as the prototype, and five scale test model beams were designed. The geometry and reinforcement of the model beams are shown in The test setup for the model beam is shown in Pmin was determined according to the dead load of heavy-haul railway bridges. Then, under the action of gravity and Pmin, the tensile steel stress of the model beam was the same as that of the field beam under the dead load. The maximum fatigue load Pmax of the C1 beam was determined according to the stress amplitude of the tensile reinforcement of 32 m prestressed concrete beam analyzed in Pmin and Pmax was consistent with that of the original beam. The C2–C4 beams were used to investigate the fatigue behavior of prestressed concrete beams when the trainload continues to increase to exceed the cracking load. The loading conditions of the test beams are shown in The tests were performed on a PMS-500 electo-hydraulic servocontrolled fatigue testing machine. The loading frequency of the testing machine is 0–5 Hz. For the fatigue test, the test beams were loaded to the maximum fatigue load first, and then the applied load was reduced to the mean value. After that, a sine wave load cycle was applied to the test beams, and the loading frequency was 3 Hz. The fatigue tests were completed at the predetermined number of cycles, and a static loading test was performed to obtain the displacements and the strains.The tensile steel bar of the test beam S1 under static loading yielded first. Then, the concrete in the compression zone began to crush. The collapse of the mode beam was governed by concrete crushing, and the failure mode is shown in It should be noted that after the prestressing strands were tensioned, the strain of the prestressing strand in the midspan of the beam is about 4762 με, the strain of the tensile steel bars at the bottom of the beam is about −342 με. Then, the high-pressure grouting pump is used to grout and seal from the reserved grouting port. Due to the reasons for construction, some strain gauges of steel bar and prestressing strand were damaged before or at the beginning of the test. Therefore, in the result analysis, the undamaged strain data in the flexural segment were selected as far as possible. The strain of the concrete compression zone was given by strain gauge 1 illustrated in The model beams test showed that when the fatigue load level was lower than the pre-compression stress of concrete in the tension zone, the test beams exhibited excellent fatigue performance. With the increase in fatigue load level, the fatigue damage characteristics of the test beams were similar to those of partially prestressed concrete beams with a high prestressing degree . The fat4) and main crack width (unit: mm). For instance, 249.61 (0.09) indicates that the main crack (red crack) of the test beam C3 width is 0.09 mm after 249.61 × 104 fatigue cycles, and the main crack width in the gradual development stage was about 0.06–0.10 mm. When the maximum crack width of the test beams C2, C3, and C4 reached 0.10 mm, the corresponding fatigue cycles were 4.17 million cycles, 2.6 million cycles, and 0.81 million cycles, respectively. Throughout the whole fatigue test, the damage of the test beams accumulated, and the cracks expanded due to cyclic creep. When the stress of the bottom tensile steel bar reached the yield point, the displacement of the beams and the width of the main crack increased rapidly with the fatigue cycles, which eventually led to the fatigue failure. Compared with partially prestressed concrete beams [te beams , fully py/n (unit: ×10−6), where y/n is the ratio of midspan displacement y (unit: mm) and the number of cycles n. The fatigue crack limit state is a critical indicator of prestressed concrete structures. It can be seen from The relationship between the number of fatigue cycles and midspan displacement of the beams is shown in The load-strain curves of concrete in the compression zone, prestressing strand, and tensile steel bar of the beam are shown in σ is the fatigue stress amplitude, N is the number of constant-amplitude fatigue failure cycles, m is the material constant, and C is the fatigue detail constant of the tensile steel bar.The relationship between the stress amplitude of the tensile steel bar and the number of fatigue failure cycles (fatigue life) is defined by the S-N curve (see Equation (1)).It can be seen from the above analysis that the fatigue failure of prestressed concrete beams occurs in the outermost tensile steel bars. fr and fpt are the stress amplitude and the ultimate tensile strength, respectively.For the fatigue life of prestressing strand, Naaman  conducteAs(p). When the tensile steel bars or prestressing strand reach the fatigue limit state after fatigue loading N cycles, the effective bearing area is As(p)(N). The force in the steel bars before and after fatigue loading should satisfy the following formula:fys(p) is the yield strength of the tensile steel bar and prestressing strand.The fatigue damage process of the steel bar is a crack propagation process and reduces the effective bearing area . This pan cycles can be expressed as:It is assumed that the effective bearing area of the steel bars under constant-amplitude fatigue stress conforms to the Miner linear cumulative damage criterion . The effThe tensile stress of the tensile steel bar is greater than the yield stress, and the tensile stress of prestressing strand is greater than the ultimate tensile strength.σ is the stress tensor; E0 is the initial elastic stiffness tensor of concrete; D is the concrete damage tensor; ε is the concrete strain tensor; εe and εp are the concrete elastic strain tensor and plastic strain tensor, respectively; “:” is the double dot product.A1+, A2+, P1, P2, c, x1, and x2 are parameters related to d+, A1−, A2−, P3, and x0 are parameters related to d−. These parameter values can be obtained from the literature [A previous study  proposedtion (9) :(9)d+=A1terature .n fatigue recycles, and n fatigue recycles, which is calculated according to Equation (11):n fatigue recycles, calculated according to Equation (12). Nth repeated loading, respectively, and calculated according to Equation (9), where n and Based on the damage characteristics of concrete under static loading and the influence of repeated loading on the evolution of concrete damage, the static beam-chain model was extended to the fatigue beam-chain model in the literature  to expreterature .(13)logThe irrecoverable fatigue damage variable given as : (14)di,The irrecoverable fatigue damage variable given as :(16)di,n−5 m/s2, which is less than 0.01 m/s2, indicating that the balanced state of the train finite element model meets the requirements.3. The elastic modulus of the sleeper is 32.5 MPa; Poisson’s ratio is 0.27; and the density is 2.5 g/cm3. The elastic modulus of the track-bed is 140 MPa; Poisson’s ratio is 0.27; and the density is 2.0 g/cm3. The parameters of concrete, tensile steel bars, and prestressing strand are consistent with those of the test beams.The spatial vibration analysis model of the track-bridge, including rail, fastener, sleeper, beam, support, and pier, is shown in (1)The finite element model of the bridge was established in the finite element analysis software; the vehicle system dynamics model was established in SIMPACK .(2)The modal analysis and substructure analysis of the bridge finite element model were performed, and the flexible body FBI files were generated and imported into SIMPACK .(3)Spring damping was used to set up the track layers connection, and the primary and secondary suspension system of the train; the wheel-rail contact was used to realize the coupling effect of the track and bridge.(4)The normal time-history force of wheel-rail contact point under the excitation of track irregularity was analyzed for trains with different axle weights. The expected values of wheel-rail force were calculated and applied to the finite element model in A 32 m standard height prestressed concrete beam bridge of a heavy-haul railway was selected for a field test to verify the rationality of numerical calculation. When the C80 freight car (axle weight of 25 t) passed on the bridge at a speed of 60 km/h, the vertical amplitude and vertical acceleration response in the midspan of the beam were measured. The layout of field test sensors is shown in 2 to 0.6921 m/s2 and −0.65 m/s2 to 0.6349 m/s2, respectively. The vertical acceleration simulation value was slightly different from the measured value. However, it was still also within the fluctuation range, indicating that the dynamic response of the bridge in the proposed vehicle-bridge coupling model can adequately reflect the actual dynamic characteristics of the structure.The wheel-rail force time-history curves under different axle weights and operating speeds can be obtained through the vehicle-bridge coupling simulation calculation. Taking the second wheelset of the front bogie of the first train as an example, the vertical time-history force of wheel-rail is shown in The finite element model of prestressed concrete beams is shown in The residual stiffness and yield point degradation of the beams loaded by 33 t and larger axle weight of trains were investigated, and analyzed the effects of train fatigue cycles and prestress loss on the residual stiffness and yield point degradation models of the beams.The stress state of prestressed concrete beams before fatigue loading was calculated:σlI) occurs due to the friction between the prestressing strand and the pipeline, the anchor deformation, and the elastic shrinkage of concrete when the prestressed steel strand is tensioned in batches. After the prestressing strand is released, the concrete and non-prestressed steel bars experience pre-compression stress. The second batch of prestress loss of the prestressing strand (σlII) occurs due to the relaxation of the steel strands and the shrinkage and creep of the concrete, which include [During the steel strand tensioning, the first batch of prestress loss ( include :(17)σlI=σl1 is the prestress loss caused by the friction between the prestressing strand and the pipeline. σl2 is the prestress loss caused by the anchor deformation and the shrinkage of the prestressing strand. σl4 is the prestress loss caused by the elastic shrinkage of concrete when the prestressed steel strand is tensioned in batches. σl5 is the prestress loss caused by the relaxation of the prestressing strand, and σl6 is the prestress loss caused by concrete shrinkage and creep. The prestress loss can be calculated according to the formulas given in the code [In the above equation, the code . The cal(1)The finite element model of prestressed concrete beams was established. The material parameters of concrete, prestressing strand, tensile steel bars, and stirrups were input. The material parameters were consistent with the test beams.(2)The effective prestress of the prestressing strand before fatigue loading was calculated according to Equations (17) and (18). The initial prestress was applied to the prestressing strand by the cooling method.(3)The stress amplitude of tensile steel bars, prestressing strand, and concrete in the compression zone under fatigue loading was determined. The fatigue life was calculated according to Equations (2), (3) and (13).(4)n fatigue cycles, the fatigue elastic modulus damage of concrete in the compression zone, the effective bearing area of the prestressing strand, and the tensile steel bars was calculated according to Equations (5) and (11). Then, the material properties were redefined.After (5)n fatigue cycles, the maximum stress of concrete in the compression zone, prestressing strand, and tensile steel bars of beam midspan and loading point section was calculated.After (6)The damage of prestressed concrete beam was assessed by whether the concrete in the compression area, prestressing strand, and tensile steel bars reached the ultimate strength, i.e.,N = n.Yes: the fatigue life of prestressed concrete beams n = n + Δn, return to step (4) and continue calculation.No: The finite element analysis method for evaluating the fatigue behavior of prestressed concrete beams is shown in In the simulation calculation, the outermost tensile steel bars of prestressed concrete beams reached the ultimate tensile state first. When the tensile steel bars reached the ultimate tensile state, the prestressed concrete beams were regarded as damaged. The failure mode of the beam was consistent with that of the test beam. The tensile reinforcement fracture governs the collapse of the assessment beam. Hence, the relationship between the fatigue life of the prediction model and the stress amplitude of the tensile steel bars conforms to Equation (2). The simulation results and test results of the beams S1 and C3 are compared in A fatigue behavior analysis was conducted for the fatigue behavior assessment model by considering that the prestress loss was 0–30% and cracking coefficient was 0.85–1.23 during the fatigue process. The fatigue behaviors included fatigue life, stiffness, and yield capacity.The effect of the fatigue load level on the fatigue life of the beams is shown in fn−f0)/f0, f0 is the midspan displacement before fatigue loading, and fn is the midspan displacement after n fatigue cycles. When prestress loss was 0%, the cracking coefficient was 0.9, and the stiffness degradation degrees of the beams were 30%, 40%, and 50%, the corresponding fatigue cycles were 2.54 million cycles, 3.86 million cycles, and 5.10 million cycles, respectively. When the cracking coefficient increased from 0.9 to 1.03, the corresponding fatigue cycles were 1.73 million cycles, 2.44 million cycles, and 3.07 million cycles, respectively. When the cracking coefficient increased from 0.9 to 1.03 and prestress loss increased by 20%, the corresponding fatigue cycles were 0.36 million cycles, 0.64 million cycles, and 0.86 million cycles, respectively. The results show that the increase in the fatigue load level of the beams accelerated the stiffness degradation. The higher the degree of stiffness degradation, the more pronounced the influence of the fatigue load level on the stiffness. The concrete in the tension zone and tension steel bar gradually withdraw from work because of the accumulative fatigue damage. The fatigue capacity of the beams was borne mainly by the prestressing strand and the concrete in the compression zone. Prestress loss in the beams accelerated the degradation rate of the fatigue strength and reduced the fatigue life. The higher the fatigue damage degree, the more pronounced the effect of prestress loss on the fatigue life of the beams.The effects of the fatigue load levels and prestress loss on stiffness degradation of the beams are shown in y = 1−(P0−Pn)/P0, where P0 is the yield capacity of the beams before fatigue loading and Pn is the yield capacity after the beam is loaded for n times. It can be seen from the figure that the yield capacity degradation law of the beams was the same as that of the stiffness degradation. When prestress loss was 0%, the cracking coefficient was 0.9, and the yield capacity degradation values of the beams were 5%, 10%, and 15%, the corresponding fatigue cycles were 0.54 million, 1.90 million, and 3.45 million, respectively. When the cracking coefficient increased from 0.9 to 1.03, the corresponding fatigue cycles were 0.40 million, 1.59 million, and 2.76 million, respectively. When the cracking coefficient increased from 0.9 to 1.03 and prestress loss increased by 20%, the corresponding fatigue cycles were 0.40 million, 1.14 million, and 2.16 million, respectively. The results show that the increase in the fatigue load level and prestress loss in the beams accelerated the degradation rate of yield capacity. The higher the degree of yield capacity degradation, the more pronounced influence of the fatigue load level and prestress loss on the yield capacity. The impact of the fatigue load level on the yield capacity degradation of the beams is shown in (1)When the trainload was lower than the cracking load and the beam was not cracked yet, the beams exhibited excellent fatigue performance, and the degree of prestressing will not affect the fatigue life. When the trainload exceeded the cracking load of the beam, the crack resistance of the prestressed concrete beam was poor, and the fatigue life of the beam decreased significantly with the increase in the trainload and prestress loss. The fatigue failure of prestressed concrete beams occurred in the bottom tensile steel bar. Therefore, it is reasonable to take the number of fatigue fracture cycles of tensile steel bars as the fatigue life of the beams.(2)The crack development process of prestressed concrete beams displayed three stages: derivative stage, gradual development stage, and fatigue failure stage. Compared with partially prestressed concrete beams, fully prestressed concrete beams were more sensitive to cracks. Once cracked, the bottom tensile steel bar of the beams can easily undergo fatigue failure. Hence, the cracking of fully prestressed concrete beams should be strictly controlled.(3)The static strength of prestressed concrete beams after fatigue loading was related to the fatigue damage degree. When the trainload was lower than the cracking load and the beam was not cracked yet, the degree of prestressing had little effect on the stiffness of the beam. When the beam was cracked with the accumulated fatigue damage or the trainload was greater than the cracking load of the beam, the concrete in the tension zone will gradually withdraw from work, and the force of the prestressed tendons will increase. Therefore, with the increase in the number of trainload fatigue cycles, the effect of prestress loss on the stiffness of the beam was more significant. With the increase in the trainload, the greater the fatigue damage degree of the prestressed concrete beam under the same fatigue cycles, and the greater the influence of prestress loss on the stiffness of the beam. Therefore, when the axle weight of the heavy-haul train is greater than the cracking load of beam, it is suggested to strengthen the beam by increasing prestress level.(4)The increase in train load and prestress loss in the beams accelerates the degradation rate of yield-bearing capacity. The higher the degree of yield capacity degradation, the more pronounced the influence of fatigue load level and prestress loss on the yield capacity. When the trainload was lower than cracking load (cracking coefficient was 0.9) and the number of fatigue cycles was 3.45 million, the yield capacity of the beam decreased by 15%. Under the same degradation degree of yield capacity, when train load was greater than cracking load and the cracking coefficient increased from 0.9 to 1.03, the number of fatigue cycles of heavy-haul train decreased by 20%. The number of fatigue cycles decreased by 37.4% when the cracking coefficient increased from 0.9 to 1.03, and prestress loss increased by 20%. Therefore, when the prestressed concrete beam reaches a certain degree of fatigue damage, the axle weight of heavy-haul train should be strictly controlled to avoid yield failure of the beam.Due to the demand for increasing trainload and enhancing some existing heavy-haul railways, the low reserve value of bearing capacity is a problem for the 32 m-span simply supported beams. In this study, experiments were performed to investigate the fatigue behavior of prestressed concrete beams in a heavy-haul railway under 33 t and higher train loads, fatigue behaviors including fatigue deformation, fatigue life, crack propagation, and strains of classical materials. A fatigue behavior assessment model was established based on the experimental results to investigate the residual stiffness of the beams and yield point degradation under different prestress losses and load levels. The following conclusions can be drawn:


The molecular dynamics method was used to study the structure and properties of dendrigrafts of the first and second generations in methanol–water mixtures with various volume fractions of methanol. At a small volume fraction of methanol, the size and other properties of both dendrigrafts are very similar to those in pure water. A decrease in the dielectric constant of the mixed solvent with an increase in the methanol fraction leads to the penetration of counterions into the dendrigrafts and a reduction of the effective charge. This leads to a gradual collapse of dendrigrafts: a decrease in their size, and an increase in the internal density and the number of intramolecular hydrogen bonds inside them. At the same time, the number of solvent molecules inside the dendrigraft and the number of hydrogen bonds between the dendrigraft and the solvent decrease. At small fractions of methanol in the mixture, the dominant secondary structure in both dendrigrafts is an elongated polyproline II (PPII) helix. At intermediate volume fractions of methanol, the proportion of the PPII helix decreases, while the proportion of another elongated  Ideal dendrimers are regular, spherical, hyperbranched molecules emanating from a single center and consisting of branched fork-shaped repeating units with one input handle and two (or less often) three output prongs. For this reason, the number of elements in each next generation doubles (or triples) and dendrimers have a large number of terminal groups available for functionalization. The first dendrimers were synthesized more than 40 years ago ,2,3,4,5.At the same time, the synthesis of dendrimers, in general, and lysine dendrimers, in particular, involves many protection and deprotection steps, which makes it rather slow and expensive. That is why, in the early 2000s, the new methods for the synthesis of slightly less regular lysine dendrigraft molecules were elaborated . In thesLysine dendrigrafts in mixed solvents are also of interest for creating new amphiphilic block copolymers similar to amphiphilic linear–dendron block copolymers and dendron–dendron block copolymers (Janus dendrimers). In recent years, we have simulated both lysine/peptide dendrimers and lysine dendrigrafts, as well as lysine hybrid linear-dendron molecules , and molThere are very few publications in the literature devoted to modeling dendrigrafts in water, and there are practically no publications describing their behaviors in other solvents beside DMF ,57 and tSnapshots of DGL1 and DGL2 dendrigrafts at the end of the molecular dynamic simulation of DGL1  and DGL2ameter α ,100,101 i-th atom, correspondingly, and i-th atom to the center of mass of the dendrigraft. We used several options for estimating the size of the dendrigraft. The first option is the mean-squared gyration radius Unlike the dendrimer, which has a single central core, the dendrigraft has a linear core consisting of eight lysine amino acid residues. Therefore, the size of the lysine dendrigraft can vary much more strongly depending on the core conformation than the size of the dendrimer. It is known that the size of a second-generation dendrigraft in water is approximately 2.2 times larger than in dimethylformamide . Thus, tDGL2 see a, while Another way to evaluate the size of a dendrigraft is to obtain its hydrodynamic radius. In this work, to estimate the hydrodynamic radius, we used the Kirkwood approximation ,103:(2)RIt is easy to see that the ratio in Rh/Rg : for theReff see  is an adThe distribution function P(Rg)  of the rfor DGL1 a at ϕ = For DGL2 b, the wir is the radial distance from the center of mass of the dendrigraft, i-th atom to the center of mass of the dendrigraft.The local structure of the dendrimer around its center of mass (COM) can be estimated using the radial distribution function of the local densityr = 0.8–1.0 nm .We calculated the integral characteristic of the total charge  “coA further increase in the methanol fraction from 0.7 to 0.9 leads to an increase in the fraction of compact conformations from the “ing plot  demonstrIn this paper, model dendrigrafts of the first/second generations were considered. The first generation dendrigraft (DGL1) consists of eight linearly linked L-lysine monomers. The symmetrized model of the second-generation dendrigraft (DGL2) includes the same backbone as in DGL1 and eight lysine side chains of five lysines each. Thus, DGL2 consists of 48 lysine residues and its mass and charge are six times greater than those of DGL1 see .Dendrigrafts were simulated in a water–methanol mixture at different volume fractions of methanol. It is well known that methanol contains the same OH group as water and the hydrophobic CH3 group, which makes methanol molecules amphiphilic. Water and methanol also have different compressibility κT ,117,118.For all simulations of DGL1 and DHGL2, in this paper, the AMBER-99SB-ildn force field  and the tion see , the proy κT see  were useWe analyzed the simulation results using programs and procedures that were described in our previous papers on the simulation of linear polymers, polyelectrolytes, peptides, polymer brushes, dendrimers, and hyperbranched polymers ,129,130.In this work, dendrigrafts of the first and second generations in water–methanol mixtures were studied using the molecular dynamics method. Different volume fractions of methanol were considered. Both dendrigrafts retained their spherical shapes at all methanol fractions. Their sizes and conformational microstructures almost did not change with the increase in the methanol fraction from 0.2 to 0.6. However, a further increase in the methanol fraction from 0.7 to 0.9 led to a decrease in the dendrigraft size and a transition from the extended secondary structures (polyproline II and delivery ,132,133.


Acoustic cavitation caused by the ultrasound-activated impurities and the induced heterogeneous nucleation and supercooled nucleation in the groove melt was the main cause of the α-Al refinement, the eutectic Si modification and the improvement in the mechanical properties.Ultrasonic treatment was applied to an A356 aluminum melt with different modifiers, and the effects of ultrasonic treatment on the structure and properties of the A356 alloy were studied. The results showed that α-Al was effectively refined with different ultrasonic modification treatments. In particular, ultrasonic treatment showed the most obvious refinement with macroscopic grains of unmodified alloy and optimized the refinement of secondary dendrite arm spacings in the Sr/Ce synergistic alloys. The eutectic Si of the unmodified A356 alloy had no obvious change after the ultrasonic treatment, but the branch diameter of the eutectic Si reduced in the Sr and Sr/Ce modification alloys after the ultrasonic treatment. The ultrasonic treatment significantly improved the ultimate tensile strength and elongation of the as-cast A356 alloy with the unmodified material, which was due to refinement of the α-Al grains by the ultrasonic treatment. After the T6 heat treatment, the ultimate tensile strength values of the alloys showed no obvious change due to the ultrasonic treatment, but the plasticity of the alloy was significantly improved. Mg The A356 alloy is widely used in lightweight automotive materials, electronic communications and other applications, owing to its excellent casting properties, corrosion resistance and high toughness ,2,3. LimStructural refinement is an effective method for improving the toughness of the castings . In receIn recent years, researchers have tried to refine and modify A356 alloys with synergistic modifiers. Qiu et al. reported that Al-6Sr-7La reduced the secondary dendrite arm spacing (SDAS) of the matrix and also modified the morphology of eutectic Si from plates to fibrous and granular. The ultimate tensile strength (UTS), yield strength (YS), elongation (EL) and hardness of the alloy were increased by 13.8%, 7.7%, 52.4% and 6.4%, respectively, with addition of 0.5 wt.% Al-6Sr-7La . Wu adde5FeSi phase in the Al-8Si-0.55Fe alloy from 18 μm to 12 μm, and the corresponding uniformity of the distribution was improved [2; however, the mechanism of modification was not clear [Based on the cavitation and acoustic flow effects of ultrasonication, ultrasonic treatment accelerates melt flow by the periodic growth and explosion of air bubbles, thus changing the pressure and temperature field of the melt ,21,22,23improved . Puga etot clear . ResearcIn this study, A356 alloys with different modifiers were subjected to ultrasound. The effects of ultrasonic treatment on the microstructure and mechanical properties of A356 alloys were studied, and the strengthening mechanism is discussed in detail.3) preheated to 260 °C, and an ingot was obtained by air cooling.The experimental materials consisted of commercial pure Al, Mg, Al-20Si, Al-5Sr and Al-5Sr-5Ce master alloys. The preparation process was as follows: first, approximately 2 kg of raw material  was melted at 750 °C, held for 30 min and then cooled to 700–720 °C by furnace cooling. Second, Mg and modifier were added to the melt by using titanium tools, approximately 10 g of refining agent was added to the melt after 5–15 min and the melt was stirred slowly with titanium tools. Third, the melt was held for 30 min, and then the slag was removed. In the fourth step, the acoustic radiator of the ultrasonic vibration equipment  was preheated for 2 min and inserted into the melt to a depth of approximately 1 cm , and the results are listed in 2 and 2.25 mm2, respectively. Tensile tests (GB/T228.1-2010) were carried out at room temperature by a material testing machine  with a strain rate of 2 mm/min. The average values of the three groups were taken as the tensile properties of the alloy, and the morphology of tensile fracture was observed by scanning electron microscopy. The solidification curve of the alloy was measured by power-compensated DSC equipment The microstructures and phase compositions of the alloys were analyzed by optical microscopy (LEICA DMI3000 M), scanning electron microscopy  and energy dispersive spectrometry . Image Pro-Plus 6.0 software was used to analyze the morphological characteristics of the microstructures, including the SDAS, area and aspect ratio of the second phase. The statistics data of the originated from 30 metallographic photographs. The multiple of images used to count the morphological characteristics of α-Al and the eutectic silicon were 50 and 1000, respectively, and the total analysis area were about 90 mmThe effects of the different treatment conditions on the structure of the as-cast A356 alloy are shown in 2, which was 7% lower than the area seen without the ultrasonic treatment. The average areas of the eutectic Si in the A356SU and A356SCU alloys were approximately 6.34 μm2 and 6.28 μm2, which were 15% and 3% lower than those seen without the ultrasonic treatment, respectively. To improve the accuracy of the measurement, the T6 heat treatment was implemented before characterizing the morphology of the eutectic Si, and the statistical results are shown in The tensile results for the samples in the as-cast and T6 states are shown in To investigate the fracture mechanism, fracture morphologies were observed by SEM, as shown in Heterogeneous nucleation theory  indicateUltrasonic treatment has a significant refining effect in different alloy modifications, and its mechanism is mainly related to shock waves and microjets generated when ultrasonic bubbles collapse . First, According to the model of impurity-induced twinning (IIT) , the atoUltrasonic vibration activated the oxide inclusions , Sr and The strengthening mechanism of the A356 alloy mainly includes fine grain strengthening, second phase strengthening and solution strengthening ,37.The strengthening coefficient T of the alloy can be calculated as:d) is the grain diameter. From Equation (2), the diameter (d) of the strength grains in polycrystals is on the (−1⁄2) order of magnitude, i.e., the finer the alloy grains, the higher the strength.According to the Halle–Petch equation , the strAccording to the Orowan mechanism  for the 2, b is the mode of Park’s vector, which is simplified to Δτ ≈ f) is the volume fraction of particles. As the ionic radius (r) or particle spacing (d) decreases, the strengthening effect increases. The smaller the radii of eutectic silicon particles and the smaller the particle space, the higher is the strengthening of the alloy.The critical tangential stress of the dislocation line depends on the minimum radius of curvature (2Si plays a decisive role in strengthening the A356 alloy during the T6 heat treatment [Mgreatment ,41, whicThe sharp edge of the eutectic Si in alloy A356 easily caused stress concentration , which eUltrasonic treatment refined α-Al grains under different modification treatments. After the ultrasonic treatment, the coarse columnar and equiaxed Al grains were transformed into evenly distributed equiaxed grains with different modifiers. The SDAS of the A356 alloy with Sr modification was reduced by 8.5% compared with the alloy with no ultrasonic treatment.The eutectic Si of the unmodified A356 alloy had no obvious change by the ultrasonic treatment, but the branch diameter of the eutectic Si becomes smaller in the Sr and Sr/Ce modification alloys after the ultrasonic treatment.The UTS and EL of the as-cast alloys with different modifiers were improved by the ultrasonic treatment. In particular, the strength and elongation of the unmodified alloy were increased by 9.8% and 11.3%, respectively.After the T6 treatment, the UTS values for the alloys with different modifications showed no obvious change due to the ultrasonic treatment, but the ultrasonic treatment significantly improved the plasticity of the alloy with the Sr/Ce modification.The improvement in UTS in the as-cast alloys was caused by the refinement of α-Al, while the improvement in the plasticity in the T6 state was caused by the modification of the eutectic Si.The effects of the ultrasonic treatment on the microstructure and tensile properties of the A356 alloys under different modification treatments were investigated, and the main conclusions are as follows:


Cow hair, a by-product of tannery waste, is usually stockpiled on a large scale as waste, which leads to serious environmental impacts. In this paper, cow hair was used as a reinforcement fiber to improve the mechanical strength of iron tailing-based foam concrete. The effects of the amount of cow hair fiber on the apparent density, compressive strength, and flexural strength of foam concrete were investigated by a series of characterization methods. Meanwhile, Image-Pro Plus software was used to analyze the porosity, average pore size, roundness, and other parameters of the specimens with different amounts of cow hair fiber. Results revealed that a proper amount of cow hair fiber can form a stable three-dimensional network structure inside the foam concrete and promote a uniform distribution and size of the pore structure inside the test piece. This could effectively improve the compressive strength, flexural strength, and crack resistance of the foam concrete, and when the fiber content was 0.2 wt%, the foam concrete exhibited the best mechanical properties, with a compressive strength of 11.19 MPa and a flexural strength of 3.58 MPa. The present work was in agreement with the strategic objective of resource recycling and solid waste utilization, which was conducive to the development of the circular and green economy. However, the structural characteristics of light weight and porosity have also led to the generally low strength and toughness of foamed concrete [Foamed concrete (FC) is a cement-based composite material with a low density . In previous studies, the introduction of iron tailings with relatively low pozzolan activity and a certain hardness as fine aggregates into foam concrete systems can effectively improve the mechanical properties of the materials ,35,36. T2O2 and water to the above mixture and stir quickly for 5 min; then, pour the slurry into a 40 mm × 40 mm × 160 mm mold. After standard curing for 24 h, demold the sample and place it in the curing box for standard curing . In this study, the influence of the amount of CHF on the performance of foam concrete was explored. As shown in 0, N0.05, N0.1, N0.15, N0.2, N0.3, and N0.4, respectively.The preparation process of the cow hair fiber foamed concrete involved in this experiment can be divided into three steps . The speρ of the sample was calculated using the formula ρ = M/V, where M represents the mass of the sample after drying, and V represents the sample volume.The density of the specimen was tested according to the standard JC/T 1062-2007. The apparent density of the sample needs to be tested after placing sample with a curing age of 28 days in an oven at 110 ± 5 °C to a constant weight. The apparent density R of the sample can be determined according to the formula R = F/A, where F represents the breaking load of the sample, and A represents the compressed area of the sample.The compressive strength of the specimen was tested according to the standard GB/T50107. The compressive strength of the sample was tested using an electro-hydraulic pressure testing machine. The compressive strength f of the test piece can be determined according to the formula f = 3 FL/2) × 100%, where ρ represents the block density of the sample.To calculate the porosity of the sample, it is necessary to first test its true density. The sample, cured for 28 days and dried to a constant weight, was ground and tested using the T0352-2000 mineral powder density test method (Li’s bottle method) to obtain the true density of the sample as For the test of apparent density, compressive strength, flexural strength, and porosity of the foam cement, each sample was tested five times, and the average value was taken.The uniformly dispersed fibers can form a chaotically distributed three-dimensional network structure inside the foam concrete system, effectively arresting growth and widening of cracks in the material and improving the material’s mechanical properties. The fiber’s aspect ratio and tensile strength are the keys to its function in foam concrete. The specific performance indices of the CHF used in this experiment are shown in 2), calcite (CaCO3), hematite (Fe2O3), and dolomite (CaMg(CO3)2). The specific XRD spectrum is shown in The particle size of the iron tailing sand used in this experiment was mainly distributed in the range of 70–110 μm; the ignition loss was about 2.13%, and its chemical composition is shown in Foam concrete is a cement-based composite material with a large number of pores on the inside. The pore structure inside foam concrete is one of the crucial parameters of FC, for it is closely related to the strength and porosity of the foam concrete ,38,39. T(1)Overview of Pore StructureA high-definition camera was employed to observe the pore structure of the material. Before shooting, 180-mesh, 400-mesh, and 1200-mesh sandpapers were used to polish the sections of the specimens, and the parts were smooth and visible to the naked eye. After testing the CHF, a soft brush was used to brush off the dust blocked in the air holes as much as possible to avoid affecting the test results. After the shooting, PS software was employed to cut each picture into a size of 5 cm × 5 cm, as shown in As shown in (2)PorosityThe effect of CHF content on the porosity of foamed concrete is shown in (3)Average pore sizeThe effect of CHF content on the average pore size of foam concrete is shown in (4)Roundness valueThe roundness value, the degree of the pores close to a circle, is a characteristic parameter used to characterize the geometric shape of the pores in foam concrete . When thThe effect of CHF content on the roundness value of the foam concrete is plotted in The microscopic morphology of the FC composite is shown in 3 to 1057 kg/m3), and this result was consistent with the change in porosity mentioned above. This phenomenon was also caused by the CHF three-dimensional network structure inside the concrete slurry, which limited the generation of large-sized pores. Additionally, adding CHF will affect the fluidity of the slurry, and the decreased fluidity will lead to the defoaming phenomenon inside the foamed concrete.The compressive strength of the foam concrete is plotted in However, with the increase in CHF content from 0.15 wt% to 0.4 wt%, under the condition of constant cement content, the amount of cementitious material used for the fiber–concrete bonding interface gradually increased with the increase in CHF content, resulting in defects in the pore wall of the material; the influence of excessively added fibers on the crystallinity of cement hydration products is also one of the reasons for the reduction in the flexural strength of the specimens.The ratio of the flexural strength and compressive strength (F/C) is an important parameter of concrete to explore its crack resistance. A large F/C value means that the material exhibits strong flexural resistance. As shown in The change in the porosity of foamed concrete will influence its strength. The relationship between the density and the compressive strength and flexural strength of the cow hair fiber-reinforced foam concretes was linear when the addition of CHF was between 0 and 0.2 wt%, and this result had good agreement with the findings given elsewhere . This shIn this paper, the CHF content was used as a single-factor variable, and the apparent density, compressive strength, flexural strength, and pore structure  of the foam concrete were used as experimental parameters to explore the effect of CHF content on foam concrete. Considering the impact on each performance, the specific conclusions are as follows.Compared with the blank control group, all foamed concretes incorporating CHF showed higher compressive strength, flexural strength, and crack resistance, proving that CHF can improve the mechanical properties of foamed concrete. The compressive strength of the specimen reached a maximum value of 11.19 MPa when the CHF content was 0.2 wt%; the flexural strength reached a maximum value of 3.58 MPa when the CHF content was 0.15 wt%; and the fold-to-compression ratio reached a maximum value of 0.335 when the CHF content was 0.2 wt%.With the gradual increase in CHF content, the porosity of the foamed concrete continued to decrease. When the CHF content was 0.2 wt%, the average pore diameter and pore roundness of the specimens decreased to minimum values of 1.92 mm and 1.08, respectively.After a comprehensive analysis of the mechanical properties of foam concrete, it was found that, when the CHF content was 0.2 wt%, the foam concrete had the best mechanical properties; the compressive strength of the specimen was 11.19 MPa (an increase of 68.37%), the flexural strength was 3.36 MPa (increased by 50%), and the fold-to-compression ratio was 0.335 (increased by 8.06%). At the same time, after a comprehensive analysis of the characteristic parameters of the pore structure of the foamed concrete, it was found that the specimen had the most stable pore structure in the range of CHF contents ranging from 0.2 wt% to 0.3 wt%. Within this dosage range, the pores in the foam concrete were evenly distributed and uniform in size, and the stable pore wall structure also provided good mechanical properties of the specimen.


In VE, 3 out of 12 subjects showed the same results for respiratory metabolic analysis and the AT by the V-slope method. In VCO2, 3 out of 12 subjects showed the same results for the respiratory metabolic analysis and AT by the V-slope method in VCO2. (4) Conclusions: Strucchange was more objective and significant in identifying the AT than the V-slope method.(1) Background: When measuring anaerobic work threshold (AT), the conventional V-slope method includes the subjectivity of the examiner, which cannot be eliminated completely. Therefore, we implemented an engineering method using strucchange to objectively search for the inflection point of AT. (2) Methods: Seventeen subjects (15 men and 2 women) were included in the study. The subjects rode an ergometer and performed a ramp load test for 18 min and 30 s. (3) Results: In VE (Ventilation), 11 out of 12 subjects had the same results with 95% confidence intervals for the AT by the strucchange and respiratory metabolic apparatus. In VCO Many studies on exercise therapy that use an appropriate exercise load index have been conducted on the elderly and cancer patients [2) against oxygen uptake (VO2) in exhaled air. However, the extraction of structural change points [2 (ml/kg/min); (3) nonlinear increase in respiratory quotient RQ; (4) increase in oxygen equivalent VE/VO2 without a change in carbon dioxide equivalent VE/VCO2, and (5) increase in end-expiratory oxygen concentration (PETO2: mmHg) without a change in end-expiratory carbon dioxide concentration (PETCO2: mmHg).In recent years, the average life expectancy in Japan has been nearing 100 years. Therefore, many researchers are focusing on the relationship between long life expectancy and good health throughout one’s life. In other words, studies on maintaining good health without compromising the quality of life (QOL) have been gaining popularity. However, good health and QOL are directly related to individual awareness. Although the current coronavirus pandemic is forcing people to restrict their daily activities, awareness regarding the importance of individual health is being raised by making people aware of the importance of wearing masks, hand sanitization, social distancing, vaccination, and other measures. The risks of lifestyle-related diseases, such as cancer, diabetes, cardiovascular diseases, as well as age-related decline in physical functions, are known to be reduced with appropriate physical activity, especially indoor exercises to relieve the stress caused by the lack of physical activity. Toshima et al. . Wannamepatients ,7. Tanebpatients  reportedpatients . Howeverpatients  describepatients  proposedpatients  found thpatients ,15, a coe points  includese points  for the e points , the AT e points . An explA total of 17 subjects , 15 healthy men and 2 healthy women, engaged in regular daily exercise were included in the study. Informed consent was obtained from all the subjects involved in the study, which was conducted according to the guidelines of the Helsinki Declaration. This study was approved by the ethical review of the Nagaoka University of Technology . The experiments were conducted in the sports engineering laboratory of the Nagaoka University of Technology and the laboratory of the Seiryou Rehabilitation Institute. The experimental protocol and apparatus are presented in Each ventilation index was recorded on a PC, converted to A/D, averaged for 10 s, and smoothed with a simple moving average (3 points). The smoothed data were saved on a USB drive and analyzed using the strucchange package in R language on another PC. The AT of each subject was calculated using the analysis function (V-slope method) of the respiratory metabolic measurement device (Vmax 29 s) and reflected as a solid vertical line in the graph (time axis) of each ventilation index. Furthermore, the AT identification by the V-slope method, which is an analog method, was performed by two examiners: one of the authors (examiner A) and a clinical laboratory technician with more than 20 years of clinical experience (examiner B). Each examiner performed the V-slope method twice, and the mean value (converted to W) was used. Interclass correlation coefficients (ICCs)  were calIn The strucchange results for the VE of subject K are shown in The results of the strucchange results of VE (subject K) after smoothing are shown in 2 of subject K are shown in The results of strucchange in the VCO2 (subject K) after smoothing are shown in The results of strucchange results of VCO2 of subject K are shown in The number of breakpoints (RQ). The results of strucchange in the VCOThe results of strucchange results of RQ (subject K) after smoothing are shown in 2, and RQ associated with a gradual increase in exercise load, and the points of change in all 17 subjects (A–Q) were detected. This is a result of the structural change extraction by strucchange. The background of the extraction of the structural change point is considered to be the process of the increase in CO2 storage in the tissue [3− against the increase of CO2 in the blood during the gradual increase in exercise load. One hundred watt AT detection from 2, the results showed a significant decrease in the number of people. The respiratory quotient, RQ, is expressed as VCO2/VO2 and is defined as a measure of carbon dioxide emissions divided by oxygen uptake. As described previously, VCO2 or VO2 can be analyzed separately; however, when they are analyzed as a combined parameter, the results differ significantly due to the inclusion of errors from the analysis of each parameter. The cause of this difference has not yet been identified. Therefore, the RQ results require further investigation. There are two reasons why the respiratory metabolic analysis system could not detect the change points. One reason is that the subject could not continue the experiment and became exhausted. The other reason is the consideration of individual physiological effects on the subject. In the latter case, it is possible that the conventional V-slope method [2 was shifted to the right in the V-slope method during gradual exercise, and the AT could not be detected. This may be due to the state in which increased CO2 in the blood is not sufficiently discharged from the alveoli during exercise load escalation; that is, the chemoreceptors are not functioning. In other words, CO2 gas is stored in the alveoli and is not sufficiently expelled from the alveoli. Therefore, it is possible that the CO2 that is no longer expelled is absorbed by the alveoli and circulated back into the bloodstream, causing a rightward shift  showed the same results in respiratory metabolic analysis  and the AT by the V-slope method. In this study, we performed a progressive loading test using a bicycle ergometer, measured and analyzed each ventilation parameter using a respiratory metabolic device, searched for the change point of the ventilation parameter by strucchange, and conducted an anaerobic threshold (AT) search. This study is summarized as follows.(1)2).It was possible to detect the change point by strucchange in each ventilation index 2. The RQ results differed significantly from the two aforementioned results due to possible errors in the analysis.We confirmed that the results of respiratory metabolic analysis and the confidence intervals by strucchange were almost identical in VE and VCO(3)The V-slope method was objectively evaluated using inter-examiner reliability (ICC), but the results were unreliable.However, the study also realized several methods and ways to improve the experimental procedure and the conclusion of the results. The first is to improve the quality of judgments by eliminating as many subjective factors as possible, such as the number of examiners in the V-slope method. The second is to make the AT detection system more versatile by considering past exercise habits and the presence or absence of personal medical history because of individual differences among subjects. Third, there is an error in analyzing RQ. It is necessary to fix the cause of the error and the analysis program. In the future, we would like to develop a medical-engineering collaboration system for diagnosis based on analysis using engineering methods and physiological ventilation indices.


Arabidopsis thaliana suspension cells carrying a site-specific integration of target genes. Two types of templates were used to obtain knock-ins, differing in the presence or absence of flanking DNA homologous to the target site in the genome. For the targeted insertion, we selected the region of the histone H3.3 gene with a very high constitutive level of expression. Our studies showed that all five obtained knock-in cell lines have rearrangements at the borders of the integrated sequence. Significant rearrangements, about 100 or more bp from the side of the right flank, were found in all five plant lines. Reorganizations from the left flank at more than 17 bp were found in three out of five lines. The fact that rearrangements were detected for both variants of the knock-in template (with and without flanks) indicates that the presence of flanks does not affect the occurrence of mutations.Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We studied the presence and extent of DNA rearrangements at the junction of plant and transgenic DNA in five lines of  The development and improvement in methods of molecular biology and genetic engineering, as well as the rapid development of genomic editing methods using CRISPR/Cas9, allows researchers to set themselves the task of targeted delivery of target genes to almost any selected constitutively transcribed genome regions. This will make it possible in the future to abandon the laborious screening of a large number of transgenic lines and directly obtain highly productive lines producing recombinant proteins with target genes delivered to transcriptionally active regions. Site-specific endonucleases, the best known of which is the Cas9 endonuclease, are capable of producing DNA double-strand breaks (DSBs) in a given region of the genome, which can then be repaired by one of two main eukaryotic mechanisms: non-homologous end joining (NHEJ) or homologous directed repair (HDR). In all eukaryotes, with the exception of yeast, repair mainly occurs according to one of the varieties of the NHEJ mechanism ,2,3. WheArabidopsis thaliana line with knockouts of the NHEJ components should lead to the emergence of plants resistant to T-DNA integration [A. thaliana plant genome during genetic transformation [The study of regions of integration of DNA inserts (knock-ins) using CRISPR/Cas9 technology is of great interest from the point of view of revealing the mechanisms of integration of transgenic DNA into the plant genome and establishing the features of the functioning of cell repair systems. The integration of the delivered fragment into the region of the DNA DSB, carried out by the Cas9 nuclease, occurs using the same repair mechanisms as in the integration of exogenous DNA fragments during modification of the plant genome using genetic engineering methods. It was established that in the case of knockout genes encoding key repair proteins by the NHEJ mechanism, the integration of exogenous DNA (T-DNA) occurred due to an alternative mechanism  . Accordiegration . A wide ormation  and currormation ,14. The A. thaliana plants [Zea mays [A. thaliana suspension cell lines carrying knock-ins in the region of the histone H3.3 gene were created. The template for knock-in carried two genes—A dIFN gene encoding deltaferon (dIFN) as the target gene and a nptII gene giving the resistance to kanamycin as a marker. After a series of biolistic transformations, three lines were obtained using a knock-in template carrying flanking DNA homologous to the insertion site in the plant genome and presumably integrated into the genome by the HDR mechanism. Two lines were obtained using a knock-in template without flanking DNA and presumably integrated into the genome by the NHEJ mechanism [Most of the genetic constructs intended for the delivery of transgenes to the target region using the CRISPR/Cas9 genomic editing technology include flanking DNA regions homologous to the insertion site ,16,17,18a plants  and Zea echanism .A. thaliana suspension cell lines carrying site-specifically integrated target genes in the region of the histone H3.3 gene and to show the presence or absence of mutations at the junction of plant and transgenic DNA as well as to evaluate the differences in frequency and extent of the mutation arising during insertion through the mechanisms of HDR and NHEJ.The aim of our work was to study five A. thaliana histone superfamily protein (AT4G40040) from one side and identity with vector DNA  from the other side. Additionally, control PCR was performed with primers located inside the construct  .2 and 20 kanamycin resistant calli with construct pIFN (H3.3).3. To confirm the integration of the target gene into the target region of the histone H3.3 gene, PCR was used with primers, one of which was located inside the transferred construct, and the other on the plant DNA region outside the flanking sequence present in the construct  .Not all kanamycin-resistant lines turned out to be knock-ins; most of the resulting lines were the result of random insertion of the target construct into the Arabidopsis genome. Two genetic constructs differed in the delivery efficiency of target genes in a target region. After 10 biolistic transformations, the largest number of knock-ins in the target site, six lines, were obtained using the pIFN (H3.3).3 genetic construct, while only three were obtained using the pIFN (H3.3).2 construct. The production of lines with knock-in is described in more detail in our previous work .A. thaliana is shown in After biolistic transformation, calli cultivated on a culture medium with kanamycin represent a heterogeneous mass of cells, including both cells with knock-in events in the target region and cells with the integration of target genes into random regions of the genome. Unfortunately, the procedures for the analysis and cultivation of callus, which involve the separation of heterogeneous material into parts, led to the loss of part of the knock-ins identified at the early stages of selection. Thus, as a result, 5 stable monoclonal lines of suspension cell cultures with knock-in were finally obtained: two (I1 and I6) using the pIFN (H3.3).2 genetic construct, which was inserted into the plant genome presumably by the NHEJ mechanism, and three  using the pIFN (H3.3).3 construct, its insertion into plant genome, presumably occurred by the HDR mechanism, due to the presence in the construct of flanking sequences homologous to the insertion site. The general scheme for obtaining lines with knock-in into the target region of the histone H3.3 gene of A. thaliana suspension cell lines with knock-ins are presented in The results for the analysis of the alignment of fragments characterizing the right and left junctions of plant and transgenic DNA for all five obtained nptII gene), then came the P-NOS sequence of the nptII gene promoter, which should be located in this place, but in line 29 this promoter lacked the first 46 bp.A total of 10 fragments were obtained for the right flank (RF) and 2 fragments for the left flank (LF). Multiple alignment of the fragments adjacent to the RF revealed small areas of homology with the GST tag sequence located in front of the RF. Two isolated fragments, when aligned with the sequences of the constructs used to obtain knock-ins, also revealed the insertion of two P-NOS promoter regions in a row, 98 bp long in forward and 69 bp long in reverse orientation, between the GST tag and plant DNA, immediately followed by the sequence of flanking plant DNA without the first 4 bp. At the LF, insertions of parts of the template for knock-in were also observed, immediately after the end of the sequence of the LF, there were 34 bp that do not have homology in the database, then there was an 83 bp sequence homologous to the DNA region before the T-NOS terminator .3 plasmid sequence lying immediately after the RF sequence, in sections of 150–160 bp in forward and reverse orientation and interspersed with smaller sections of plasmid DNA of unknown origin, also forming hairpins. Alignment of the sequence obtained for the LF of the construct showed minimal mutation at the insertion site, namely, the presence of two additional cytosine residues at the junction of the A. thaliana genomic DNA and the DNA of the target insertion.Most of the sequenced fragments (five fragments) in the search for homology in BLAST showed a match with the sequences of the A. thaliana genome, but not directly related to our insertion site. The other two fragments were also homologous to each other and had homology with a region from the pIFN (H3.3).3 plasmid used to create knock-ins, located in the plasmid near the LF, outside the sequence of the knock-in template. Alignment of the sequence obtained for the LF of the knock-in showed the insertion of a small additional sequence of 24 bp, which, when searched for homology in the database using BLAST, showed homology with a region of the 4 th chromosome of A. thaliana. The same insertion occurred at the junction of plant and plasmid DNA in line I1. The sequence of plant DNA from the LF side was completely preserved.In total, seven DNA fragments were obtained for the RF of the knock-in in line 4-1. Two homologous fragments did not align either with the insertion site or with the plasmids used to create knock-ins, and when searching for homology in BLAST, they showed a match with the sequences of the In the study of line I1, sequence alignment of seven PCR fragments for the RF, obtained by various methods, made it possible to conclude that there were large rearrangements in the region of integration of the right side of our template into the plant genome. Rearrangements began 15 nucleotides from the end of the GST sequence, i.e., from the end of our construct; alignment with the sequences of the plasmids used to create the knock-in showed that after the target knock-in sequence, the insertion of 400 nucleotides also occurred, which had homology with the sequence of the Cas9 gene from the Cas9H33 plasmid. The methods we used did not allow us to find the beginning of the plant DNA sequence and determine the exact length of the rearrangement. Alignment of the sequences obtained for the LF of the insert for line I1 showed the presence of an insertion of 17 bp identical to the insert at the left border for line 4-1. At the same time, it should be noted that the insertion in these two lines presumably occurred according to different mechanisms—4-1 by the HDR mechanism, I1 by the NHEJ mechanism. No other rearrangements were found in plant or transgenic DNA at the LF.A. thaliana and the DNA of the transgenic insertion, as well as in line 38.For line I6, sequence alignment of 6 PCR fragments for the RF with the DNA sequence of our insert revealed the presence of an insertion of 2 nucleotides (AC) after the end of the GST gene sequence. At the same time, instead of plant DNA, after the end of the insertion, there were three insertions that showed homology with different regions of the pIFN (H3.3).2 and Cas9H33 plasmids—170 bp, 260 bp in forward and reverse orientation. The fragments were homologous to the regions that match in both plasmid vectors that were used in this study, so it was not possible to correctly determine the source of insertion of these fragments into plant DNA. We failed to identify the beginning of plant DNA. Alignment of the sequences obtained for the LF of the construct showed minimal disturbances at the insertion site—the presence of two additional cytosine residues at the junction of the genomic DNA of Site-specific gene integration via CRISPR/Cas9 relies on endogenous DNA repair machinery, which is used to repair genomic DNA damage due to DSBs created by Cas9 . DSBs acIn a natural system, the template used in the repair process via HDR is the sister chromatid of the corresponding damaged region. This repair mechanism is less prone to error than NHEJ since it uses an identical unbroken DNA sequence as a template for the repair . HowevernptII and its terminator were inserted, immediately after the sequence of the left flanking sequence of plant DNA, and the first 46 nucleotides of the nptII promoter was deleted. It should also be noted that this line showed reduced resistance to kanamycin, which is apparently associated with rearrangements in the promoter region of the kanamycin resistance gene (nptII). When using a combination of T-DNA and Cas9, rearrangements are also observed on both sides of the integrated construct in work on rice. Overall, the 5′ end of the T-DNAs were relatively well conserved (0–37 bp deletion) compared to the 3′ ends (1–926 bp deletion). The five T-DNAs integrated at the target site in a site –specific manner had larger deletions than the randomly integrated T-DNAs at the left borders: 42–926 bp vs. 1–10 bp [Our study has shown that rearrangements can occur both with knock-in template carrying flanking DNA homologous to the insertion site in the plant genome and presumably integrated into the genome by the HDR mechanism and with a knock-in template without flanking DNA and presumably integrated into the genome by the NHEJ mechanism. Based on the accounts of flanking genomic DNA sequences following site-specific integration, it can be said that rearrangements at the junction of plant DNA and transgenic construct are rather a normal phenomenon. For example, a maize deletion of approximately 600 bp of the ZMUbi intron of the donor sequence was observed in progeny from two T0 plants, while three T0 plants contained an approximately 400-bp duplication of the right flank homology sequence at the 3′ end of the donor template . Large d 1–10 bp . Clarke  1–10 bp  showed t 1–10 bp .Perhaps more promising for obtaining knock-ins is the system CRISPR/Cas12a, also known as CRISPR/Cpf1. Cas12a has two different features that seem to be beneficial for GT, compared to Cas9. First, the induced DSB site is close to one end of the protospacer, far away from the PAM sequence. This feature possibly lets the target be cleaved multiple times, thus increasing the chances of GT, without being blocked by mutations induced by NHEJ. Second, Cas12a produces 5′ protruding ends following DSB induction. Utilizing CRISPR/Cas12a in rice also achieved high GT efficiency of up to 8% and all independently identified targeted insertions resulted in precise genomic site integration events, leaving intact junction regions between genomic DNA and upstream homology arm DNA, and downstream homology arm and corresponding genomic DNA .The uneven distribution of rearrangements observed by us on the right and left sides of the side construction can be explained due to a combination of HR and NHEJ or by the fact that in our case, the integration occurred according to the one-sided invasion model of recombination , which sUnfortunately, we were not able to fully establish the pattern of rearrangements on the right side of plant DNA in four of our five lines. We believe that one reason for difficulties in characterizing this rearrangement is the relatively large size of the affected sequence. In three out of five lines , DNA sequences homologous to both vectors used to create knock-ins and lying outside both the T-DNA and the template for knock-ins were identified. However, a detailed analysis of the alignment allows us to assert that in line 38, the inserted additional parts of the plasmid DNA sequence originate from plasmid pIFN (H3.3).3, and not from Cas9H33. In plasmids pIFN (H3.3).2 and pIFN (H3.3).3, knock-in templates are surrounded by Cas9 recognition sites. Such sites are necessary for excising the knock-in template from the plasmid and its linearization. The presence of this sites significantly increases the frequency of knock-ins ,18,19. HThe high number of DNA rearrangements at the insertion site could potentially be explained by the high level of genetic variability in the suspension cell culture. The level of genetic variability of cultivated plant cells depends on the duration of their cultivation in vitro. Young cell cultures in the first months of cultivation are characterized by an increase in heterogeneity ,41. LongA. thaliana suspension cell lines carrying a site-specific insertion of target genes, none of the lines was without rearrangements in integrated DNA. Apparently, in order to obtain a knock-in with intact plant and plasmid DNA junctions, it is necessary to carefully plan the composition of genetic constructs used to obtain the knock-in, avoiding repetitive elements whenever possible. Finally, the sequences of the flanking regions of loci modified by knock-in should be investigated and reported, to prove that both newly formed junctions arose via HDR at the target locus and are intact.Despite the fact that we successfully obtained a series of A. thaliana (L.) Heynh  was used as the initial plant material. This cell line deposited in the All-Russian collection of higher plant cell cultures ) under No. 85 and designated as NFC-0 was kindly provided by Ph.D. Nosov A.V.  [The fast-growing cell line of  Russia) . The cel Russia)  with theGlycine max cells were donated by R. Stupar [A. thaliana U6 promoter was used as an intermediate vector for inserting selected spacer regions into the sgRNA sequence.The plasmids pBlu/gRNA (no. 59188) for an intermediate cloning step and Cas9 MDC123 (no. 59184) with the Cas9 endonuclease gene under the control of the 2_35S CaMV promoter optimized for expression in . Stupar  from theA. thaliana was used as a template for the amplification of sequences flanking the integration site of the target gene. The signal peptide sequence directing the synthesized protein to the apoplast was amplified on a Daucus carota genomic DNA template. The plasmid pGEX4T-1 was the source of the GST (glutathione S-transferase) gene encoding the tag for affinity purification of the target protein. For the amplification of the nptII gene sequences (neomycin phosphotransferase II) and the CaMV35S promoter of the cauliflower mosaic virus, the pBi121 plasmid was used as a template. The dIFN (deltaferon) gene, a recombinant analog of human interferon gamma for the plant cells expression, was amplified from the pIFN--trp2-D plasmid [Nucleotide sequences for creating genetic constructs were obtained by PCR using appropriate oligonucleotides and templates. The genomic DNA of  plasmid .http://crispor.tefor.net, Santa Cruz, CA, USA) was used to select the guide RNA for knock-in into the region of the HTR5 gene. The sequence for knock-in was located between the locus of histone H3.3 gene HTR5 (At4g40040) and the adjacent gene encoding the 12 kDa subunit of microsomal signal peptidase (At4g40042), preceding the coding region of the HTR5 gene.The region of the histone H3.3 gene HTR5 was chosen as the region for the integration of the target gene. The CRISPOR software (The selected sequences that determine the specificity of the guide sgRNA were transferred into the Cas9 MDC123 plasmid using the pBlu/gRNA intermediate plasmid using the corresponding oligonucleotides : for thedIFN gene (encodes human γ-interferon) and the selective nptII gene (provides resistance of transformed cells to kanamycin), two types of genetic constructs pIFN (H3.3).2 and pIFN (H3.3).3 were used, the schemes of which are shown in For delivery to the H3.3 histone gene region of the target A. thaliana was carried out using biolistic transformation with the immobilization of plasmids pIFN (H3.3).2 or pIFN (H3.3).3 together with the plasmid Cas9H33 on the gold particles. In total, 10 biolistic transformations were performed for each construct.Delivery of the target dIFN gene to the region of the HTR5 gene of Callus transformation was performed using a PDS1000/He gene gun  with the following biolistic parameters: particle size, 0.6 µm; membrane burst pressure, 1100 pci; the vacuum pressure in the chamber was 27 mm Hg, and the distance to the explant was 6 cm. Immobilization of an equimolar mixture of plasmids onto gold particles, one of which included the plasmid with the target gene, and the other with the Cas9H33 plasmid, was carried out according to the method of the gene gun manufacturer. For each transformation, 6 Petri dishes with prepared calli were used, and each callus was fired twice.Three days after the biolistics procedure, the calli were transferred to selective SH medium of the same composition with kanamycin at a concentration of 100 mg/L and cultivated in light with an intensity of 20 thousand lux at a photoperiod of 18/6 h (day/night). Passages to fresh media of the same composition were carried out weekly. The resulting transgenic, antibiotic-resistant callus was transferred into cell suspensions. Suspensions were cultivated in the dark on an orbital shaker with a stirring intensity of 160 rpm. Subsequently, monoclones were obtained from these cultures by plating the original cell suspensions. To select cell clones, the principle of maximum dilution of cell suspensions of each of the obtained cell lines was used with alternating passages on liquid and agar media. A 4-day-old suspension culture of plant cells was filtered through a sieve with a pore size of 0.9 mm, 1 mL of the filtrate passed through the sieve was obtained, diluted 3–4 times with SH medium, and 1 mL of the diluted filtrate was applied to the surface of a Petri dish with agar medium SH containing 100 mg/L kanamycin. The cells were spread evenly over the surface of the agar with a Drygalski spatula. After 2 weeks, separately grown plant cell clones of each of the lines were passaged onto a fresh SH medium of the same composition. Ten monoclonal calluses were selected for each of the original cell lines; one of these calluses, the most promising in terms of cytological characteristics, was introduced in a suspension culture for further study.DNA was isolated from the calli and PCR analysis was performed using the appropriate primers. Genomic DNA was isolated using CTAB buffer according to the Allen protocol . To confThe target gene insertion sites in the plant genome were analyzed in two ways. To identify the left border of the insert, PCR was used with primers Lo_plan3 and Up_H3.3 for knock-ins obtained with the construct pIFN (H3.3).2 (without plant flanks in the construct) and Lo_plan3 and Up_H3.3_1 for knock-ins obtained with the construct pIFN (H3.3).3 (carrying flanks homologous to plant DNA)  and Lo11345 (F), the second PCR, for which a 100-fold diluted mixture obtained in the first PCR was used, with primers UpINS9368 (B) and Lo11187 (E), or UpINS9525 (C) and Lo11187 (E), or UpINS9368 (B) and Lo10714 (D)  were treated with MfeI or AsuII restriction enzymes, after which the preparations were treated with ligase and, using the resulting mixture as a template, two consecutive PCRs were performed, the first with BclI1 and BclI2 primers, the second with BclI3 and BclI4 primers .In this work, we have shown that site-specific insertion of foreign DNA successfully occurs with constructs oriented both to insertion by the mechanism of homologous recombination, carrying flanking regions of DNA homologous to the insertion site, and to insertion by the mechanism of non-homologous end joining, without flanking DNA. At the same time, rearrangements at the junctions of plant and transgenic DNA were detected in the lines obtained with both constructs. Thus, both mechanisms cannot guarantee accurate, error-free site-specific insertion of DNA upon knock-in. The size of the rearrangements can be quite significant up to several hundred nucleotides, and more complex rearrangements were found for the right flank of the insert than for the left. In this case, the rearrangements can consist not only of additional plasmid DNA, but also the elements of the template for knock-in on both sides of the construct can be rearranged. When planning an experiment, this fact must be taken into account, and it may be justified to add additional “ballast” DNA regions at both ends of the knock-in template to protect key structural elements from rearrangements occurring during site-specific integration.