Multiaxis three-dimensional (3D) continuous basalt fiber/cementitious concretes were manufactured. The novelty of the study was that the non-interlace preform structures were multiaxially created by placing all continious filamentary bundles in the in-plane direction of the preform via developed flat winding-molding method to improve the fracture toughness of the concrete composite. Principle and off-axis flexural properties of multiaxis three-dimensional (3D) continuous basalt fiber/cementitious concretes were experimentally studied. It was identified that the principle and off-axis flexural load-bearing, flexural strength and the toughness properties of the multiaxis 3D basalt concrete were extraordinarily affected by the continuous basalt filament bundle orientations and placement in the pristine concrete. The principle and off-axis flexural strength and energy absorption performance of the uniaxial (B-1D-(0°)), biaxial ((B-2D-(0°), B-2D-(90°) and B-2D-(+45°)), and multiaxial (B-4D-(0°), B-4D-(+45°) and B-4D-(−45°)) concrete composites were considerably greater compared to those of pristine concrete. Fractured four directional basalt concretes had regional breakages of the brittle cementitious matrix and broom-like damage features on the filaments, fiber-matrix debonding, intrafilament bundle splitting, and minor filament entanglement. Multiaxis 3D basalt concrete, particularly in the B-4D structure, controlled the crack phenomena and it was recognized as a more damage-tolerant material than the neat concrete. Two-dimensional 2D) fabrics and three-dimensional 3D) preforms are used with cementitious matrix to make textile-reinforced concrete (TRC), thus, multiple continuous filamentary TOWs, which is an untwisted bundle of continuous filaments, are incorporated in designed architecture considering the special concrete topology. Numerous research studies on TRC, especially 2D planar fabric-reinforced concrete, were conducted by several researchers ,2,3,4. TD fabricsD preformThe most commonly used types of fibers in fiber-reinforced concretes are alkali-resistant glass fiber, high-modulus high-strength para-aramid fiber and high-modulus high-strength polyacrylonitrile (PAN) carbon fiber as well as high-strength polyethylene and PVA fibers. On the other hand, basalt fiber has emerged recently due to its cost effectiveness, comparable strength, high temperature resistance, freeze and thaw performances ,26,27,28It has been reported that axial-reinforced helical circular basalt bar concrete enhanced the bending strength and ductility of neat concrete ,36. It wPeled and Bentur stated that the weave pattern in the flexural capacity of concrete enhanced the bonding and strain-hardening properties by using low modulus polyethylene and polypropylene yarns ,53. SingIt was indicated that the fine diameter filament TOW in concrete displays greater load-carrying capacity compared to that of large diameter fiber in textile-reinforced concrete. The reason was largely due to the strong interfacial bonding between the cementitious matrix and high-modulus filament bundle with finer diameter. The finer diameter filament TOWs, owing to their larger surface area, are in contact with the matrix materials more precisely than the large-diameter filament TOWs ,70. OtheThe mechanical properties and electromagnetic interference (EMI) shielding effectiveness of nanoconcrete is improved by addition of the multiwall carbon nanotubes (MWCNT), primarily due to the bridging effect, pore filling and dense calcium silicate hydrate (or C-S-H) material ,75. It wAn artificial neural network (ANN) method was used to model the flexural strength of textile-reinforced concrete (TRC) . AnotherThere have been many types of research on discontinuous high-strength and high-modulus fiber-reinforced concretes, including two-dimensional biaxial fabric-reinforced concretes being made. However, very few studies have been performed on the mechanical properties of multiaxis 3D preform concrete reinforced by continuous filament TOWs with the angular architecture of sparse array. The novelty of this research is that the non-interlace preform structures were multiaxially developed by placing all continious filamentary bundles in the in-plane direction of the preform via developed flat winding-molding method to improve the flexural fracture toughness of the concrete composite. Therefore, the aim of this research was to examine the principle and off-axis flexure properties of multiaxis 3D basalt fiber/cement concrete composites experimentally.3, respectively.Basalt fiber  was used to design the multiaxis three-dimensional (3D) preforms for concrete composite. The properties of the basalt fibers are presented in 2)-based silica sand in accordance with AS 114131. The density and melting point of silica sand were 2.65 g/cm3 and 1750 °C, respectively. A polycarboxylate-type superplasticizer complying with the standard (ASTM C 494 type F) was utilized to improve the rheology and workability of the cementitious matrix. In addition, superplasticizer kept the water/cement ratio low and also attributed to the uniform dispersing of the constituents in the cementitious mixture.Multiaxis 3D basalt preform and multiaxis 3D basalt concrete fabrication steps were broadly explained in the reference ,89. HoweMultiaxis three-dimensional (3D) preform was considered to eliminate crimp exchange and crimp extension (or displacement) in the biaxial fabric. This perhaps caused the stress concentration and led to the ineffective stress transfer in the interlacement region in the biaxial fabric, where the strength performance and fracture mechanism were probably affected ,91,92. PThe “winding and molding method” formed the multiaxis 3D preform, in that this method was proposed by Dr. Bilisik’s continued research efforts since 2000 ,89. The 4, [90°/0°]2 and [90°/±45°/0°]1, respectively  were developed. They were basically made up of four-layered uniaxial (0°), biaxial (0°/90°) and four-direction (90°/±45°/0°) concrete composites. Four-layered basalt fiber concretes were denoted as B-1D, B-2D and B-4D, and their layer sequences were [0°]ectively . B-1D, B3, 34.51 wt.%) and siliceous fine sand  were weighted and blended at 100 rpm for a minute, as shown in 3; 17.12 wt.%) and superplasticizer  into the dry mixture, they were again blended at a speed of 150 rpm for one minute [The basalt preforms were consolidated using traditional hand lay-up technique in order to make multiaxis 3D basalt concrete. The manufacturing steps of multiaxis 3D basalt concrete are displayed in e minute . Then, i® software  with a 100 kN loading cell. The speed of the cross-head motion was 0.9 mm/min. The flexure tests in the principle and ± bias directions were repeated 5 times and 3 times, respectively. The flexural test was conducted at the standard laboratory atmosphere temperature of 23 ± 2°C and relative humidity of 50 ± 10%. The ASTM C642-13 standard is generally considered to identify the density, absorption and void contents in the multiaxis 3D basalt TOWs concrete [psσ) and initial stiffness (sI) were calculated using Equations (1) and (2), respectively.b is sample width (mm); d is sample thickness (mm); L is support span length (mm); P is maximum applied load (N); psσ is flexural strength (MPa); sI is initial stiffness (N/mm); fcP is first crack load (N) and fcδ is first crack displacement (mm).The flexural strength of the multiaxis 3D preform concrete was identified by using a simple beam structure with four-point loading. Though ASTM C78/C78M-18  and ASTMconcrete . The ASTconcrete . The ASTconcrete . Thus, tThe multiaxis 3D basalt fiber concrete samples were examined by a scanning electron microscope  before and after the off-axis and principle flexure test. The macro-scale images of the flexure samples were defined via a digital camera  . A steel ruler was used to measure the crack lengths in the fractured flexure samples. A digital caliper  was employed to find the crack widths in the fractured samples after the flexure test. In addition, a digital ruler was utilized to measure the crack distance on the multiaxis 3D fiber concrete sample under four-point flexure load . Off-axi® software. Therefore, statistical computations on the raw data including flexural first crack load and displacement, flexural maximum load and displacement, flexural first crack strength and flexural maximum strength, as well as flexural stiffness, were carried out and analyzed. They are introduced in Flexural data were achieved for the pristine (CC) and basalt concrete (B-1D-(0°), B-1D-(90°), B-1D-(+45°), B-2D-(0°), B-2D-(90°), B-2D-(+45°), B-4D-(0°), B-4D-(90°), B-4D-(+45°) and B-4D-(−45°)). After the flexural test data obtained from the Shimadzu AG-× 100 (Japan) testing machine, they were transferred to a Microsoft (MS) Excel 2013 spreadsheet via TrapeziumIn In In As depicted in the load-displacement graph of B-1D, B-2D and B-4D basalt TOW concretes , fiber oFor the flexural maximum load in principle directions (0° and 90°), B-1D-(0°) was 58.92% higher than B-2D-(0°) and 38.61% higher than for B-4D-(0°), and B-4D-(0°) was 14.65% higher than B-2D-(0°). However, B-4D-(90°) was 29.18% and 14.74% lower than B-2D-(90°) and B-1D-(90°), respectively. B-2D-(90°) was 20.38% higher than B-1D-(90°). For the flexural maximum load in the bias directions (+45° and −45°), B-4D-(+45°) was 51.66% and 82.83% greater than B-2D-(+45°) and B-1D-(+45°), respectively. B-2D-(+45°) was 2.32% greater than B-1D-(+45°). Moreover, B-4D-(+45°) was 5.88% higher than B-4D-(−45°). It was found that flexural load carrying performance in various developed basalt fiber concrete composites was proportional to the fiber orientation and amount of fiber in a particular direction. In addition, B-1D-(0°), B-4D-(0° and ±45°) and B-2D- concrete composites were from 12.20% to 337.95% greater in comparison with the pristine concrete, respectively. It was identified that the basalt fiber placement in the control concrete and the orientation of basalt fiber affected the flexural capacity of the concretes.In the first crack load, as depicted in As depicted in Off-axis flexure strength, first crack strength and initial stiffness of all multiaxis 3D basalt concretes are shown in Regarding flexural maximum strength in the principal directions (0° and 90°), B-1D-(0°) was 58.92% higher than B-2D-(0°) and 38.61% higher than B-4D-(0°), and B-2D-(0°) concrete composite was 12.78% higher than B-4D-(0°). However, B-4D-(90°) concrete was 29.21% and 14.73% greater than B-2D-(90°) and B-1D-(90°), respectively. B-2D-(90°) concrete was 20.45% higher than B-1D-(90°). Regarding the flexural maximum strength in the bias direction (+45° and -45°), B-4D-(+45°) was 51.74% greater than B-2D-(+45°) and 82.86% greater than B-1D-(+45°), and B-2D-(+45°) was 20.51% greater than B-1D-(+45°). In addition, B-4D-(+45°) composite was 5.94% greater than B-4D-(−45°). It was realized that the flexural strength performance of various developed basalt fiber concrete composites was proportional to the fiber orientation and amount of fiber in a particular direction, depending upon the structural fiber architecture. In addition, B-1D-(0°), B-2D-(0° and 90° and +45°) and B-4D-(0° and ±45°) concrete composites had results higher 338.13% and 12.28% compared to that of the control concrete, respectively. Therefore, the basalt fiber placement in the control concrete and the orientation of basalt fiber influenced the flexural strength capacity of the concrete. In first crack strength, as demonstrated in As demonstrated in Effc is flexural first crack energy corresponding to the load-displacement in area X0X1X2; Efml is flexural maximum load energy corresponding to load-displacement in area X0X1X3X4; Efsh is flexural strain-hardening energy corresponding to load-displacement in area X2X1X3X4; Efss is flexural strain-softening energy corresponding to load-displacement in area X4X3X5X6 and Eft is flexural total energy corresponding to load-displacement in area X0X1X3X5X6.Average flexure energy data for the multiaxis 3D basalt fiber concretes are demonstrated in As exhibited in As exhibited in The flexural strain-hardening energy of the basalt fiber concretes  extended from 0.51 to 10.70 J. Regarding flexural strain-hardening energy in the principal directions (0° and 90°), B-1D-(0°) concrete was 184.80% higher than B-2D-(0°) and 185.12% higher than B-4D-(0°), and B-2D-(0°) composite was almost equal to B-4D-(0°). However, B-2D-(90°) concrete was extraordinarily high (7.45 times) compared to B-4D-(90°). B-1D-(90° and +45°) had no strain-hardening energy stage. Regarding flexural strain-hardening energy in the bias directions (+45° and −45°), B-4D-(+45°) was 15.33% lower than B-4D-(−45°). However, B-4D-(+45°) was 312.88% higher than B-2D-(+45°) concrete composite. It was found that flexural strain-hardening energy absorption performance on various developed basalt fiber concrete composites was proportional to fiber orientation and amount of fiber in a particular direction, depending upon the structural fiber architecture. In addition, B-1D-(0°), B-2D- and B-4D-(0° and ±45°) concretes were extraordinarily greater compared to the pristine concrete.Fourier transform infrared (FTIR) spectra of the CC (pristine concrete), basalt fiber, B-1D, B-2D and B-4D basalt fiber concrete composites are shown in −1 was assigned. Concerning the other parts of the spectrum, the band at 1083 cm−1 may be attributed to Si-O asymmetric stretching vibrations [−1 can be attributed to the -C-C-, -C-H- groups [−1 was the absorption peak of the Si–OH and –OH groups. Basalt fiber shows characteristic bands assigned as stretching vibration of Si–O–Si at 890 cm−1 [-1, corresponding to characteristic -Ca-O, Mg-O and Fe-O single bond peaks in the basalt fiber structure [−1 range a group of bands with small intensities was detected. These bands can be attributed to the stretching vibrations of the Me\O\H bonds, where “Me” stands for metals which are constituents of basalt fiber [In the FTIR spectrum of the pristine concrete (CC), as demonstrated in brations . The ban- groups ,102,103.890 cm−1 ,105. A gtructure . In the lt fiber .−1 bands may be attributed to the deformation vibrations of H2O, polycarboxylate, -Si-OH and -Ca-OH groups [−1 band spacing, the deformation vibrations of -CH2-CH-  groups were found. On the other hand, the signal between 2300 and 2200 cm−1 corresponded to −C≡N (nitryl) group. The signals between 1600 and 1300 cm−1 were attributed to stretching vibrations of the -C=C-, -C=O and -C=N groups. The signal observed between 1100 and 800 cm−1 was assigned to the Si-O single-bond stretching vibrations.In the FTIR spectrum of the B-1D (warp cross-section), B-2D (filling cross-section) and B-4D (warp cross-section) basalt/cementitious concrete composites , a commoH groups . In the 2O, polycarboxylate, -Si-OH and -Ca-OH groups were detected between 3500 and 3000 cm−1 bands. The deformation vibrations of the -CH2-CH-  groups were observed between 3000 and 2850 cm−1 band spacing. The signals between 2300 and 2200 cm−1 were attributed to the −C≡N (nitryl) group. The signal observed between 1600 and 1300 cm−1 was assigned to stretching vibrations of the -C=C-, -C=O and -C=N groups. The Si-O single-bond stretching vibrations were found between 1100 and 800 cm−1. It was considered that fiber placement and fiber orientation in the concrete did not affect their FTIR spectrum. From the FTIR analysis, we comparatively identified a strong physical interaction between cementitious components and basalt fiber (filament TOW). These findings were supported by fractured sample SEM images . Hence, their directional flexural properties were homogeneously distributed throughout the concrete structure. As a result, the B-4D structure controlled the number of cracks, crack width and crack distance and enhanced the flexural energy absorption capacity of the concrete structure. However, more study is required, especially cement–fiber interlaminar regions in the multiaxis 3D fiber concretes.In the FTIR spectrum of the B-4D (-bias cross-section), B-4D (+bias cross-section), B-4D (filling cross-section) and B-4D (warp cross-section), as illustrated in Flexure fracture results are exhibited in As displayed in Regarding flexural crack width in principle directions (0° and 90°), B-1D-(0°) concrete was 11.23% higher and 38.99% lower than B-2D-(0°) and the B-4D-(0°) composites, respectively. In addition, B-2D-(0°) was 45.15% lower than B-4D-(0°). On the contrary, the crack width in B-2D-(90°) was 36.27% greater than that of B-4D-(90°), which was near catastrophic failure and B-1D-(90°), which exhibited complete catastrophic failure. Regarding flexural crack width in the bias direction (+45° and −45°), B-4D-(+45°) concrete was 183.42% and 258.63% greater than B-2D-(+45°) and B-1D-(+45°) composites, respectively. The crack width of B-2D-(+45°) was 41.01% greater than that of B-1D-(+45°). However, B-4D-(+45°) was 159.42% higher than B-4D-(−45°). The in-plane crack length in all the developed 3D basalt fiber concrete composites was equal (75 mm). Crack spacing in B-4D- was approximately small compared to those of B-2D-(0° and 90°) and B-1D-(0°) due to the number of the cracks. However, the numbers of cracks in B-4D, and to some degree in B-2D, were greater and multiaxially distributed compared to B-1D. It was deduced that multiaxis 3D basalt fiber concretes, in particular the B-4D structure, managed the number of cracks, crack width, and crack distance during four-point flexure loading. Nonetheless, additional research efforts are expected to identify the plane and sectional crack initiation and propagation under off-axis loading.Various fractured multiaxis 3D basalt fiber concretes, including B-1D-, B-2D-, and B-4D- as well as neat concretes on the top, bottom and cross-sections, and their failure modes after the off-axis flexure test, are exhibited in As displayed in The fracture mechanism probably comprised regional matrix fracture, matrix-filament bundle delamination for each basalt TOW set in the concrete, filament TOW bridging because of tensile strengthening by means of principle  fibers, partly complex shearing via particularly off-axis yarns  and shortly after, outer filament pull-out  and stick-slip in the outer filament-matrix regions, which led to interfacial shear strength between fiber and matrix or core  filament–filament frictions, which led to complicated interfacial frictional strength, including telescopic movement of filament bundles upon filament-to-filament breakages. Thus, the warp, filling and ± bias yarns carried the flexure load via a complex load distribution of the matrix. Nonetheless, additional studies are required to understand the complicated crack initiation-propagation, considering some types of stress concentration or delaying micro-crack in the through-the-thickness for each fiber set during flexure loading.Multiaxis 3D fiber/matrix concretes can find several applications subjected to aggressive environments. For instance, they can be used as prefabricated panels, bridge deck, industrial floors, seawall construction, substation reactor bases and airport runways . In addiMultiaxis 3D basalt fiber/matrix concretes were made. Their flexural properties were empirically investigated and compared to the pristine concrete. Failed interior areal morphologies of basalt fiber concretes were examined by image processing techniques.The non-interlace preform structures were multiaxially created by placing all continuous filamentary bundles in the in-plane direction of the preform via developed flat winding-molding method.Four distinct phases of the load-displacement graph in multiaxis basalt concretes were identified: elastic phase, first crack phase, strain-hardening and entire failure phase.It was discovered that the placement and orientations of the continuous basalt fiber in the pristine concrete exceptionally affected the load-bearing capacity of multiaxis 3D concrete composites . The fleThe flexural energy absorption performance of the B-1D-(0°), B-2D-(0° and 90°) and B-4D- concretes were outstandingly higher compared to the control concrete.Plain concrete exhibited fatal brittle crack. The fracture mechanisms in the four directional basalt concretes (B-4D) were recognized as regional brittle matrix fracture and broom-like damage features on the filaments, filament bundle-matrix debonding for each yarn set and filament bundle splitting along with minor filament entanglement near the core of the B-4D, fiber bridging in the fiber TOWs (maybe particle bridging in the matrix) because of tensile strengthening and immediately after outer filament pull-out and stick-slip between matrix-outer filament or core filament-filament friction were observed. This probably induced multiple micro-cracks in B-4D concretes. However, the numbers of cracks in B-4D and B-2D concretes were greater and multiaxially distributed compared to B-1D concrete composite.The multiaxis 3D basalt fiber concrete, particularly in the B-4D structure, controlled the number of cracks, crack width and crack distance during four-point flexure loading. Thus, the multiaxis 3D basalt fiber concretes can be regarded as “damage-tolerant materials” compared to the control concrete.However, additional research studies are required to understand the crack initiation and propagation in the multiaxis 3D concrete volume under complex loading.


Non-destructive testing, with non-contact from a remote location, to detect and visualize internal defects in composite materials such as a concrete is desired. Therefore, a noncontact acoustic inspection method has been studied. In this method, the measurement surface is forced to vibrate by powerful aerial sound waves from a remote sound source, and the vibration state is measured by a laser Doppler vibrometer. The distribution of acoustic feature quantities  is analyzed to statistically identify and evaluate healthy parts of concrete. If healthy parts in the measuring plane can be identified, the other part is considered to be internal defects or an abnormal measurement point. As a result, internal defects are detected. Spectral entropy (SE) was used to distinguish between defective parts and healthy parts. Furthermore, in order to distinguish between the resonance of a laser head and the resonance of the defective part of the concrete, spatial spectral entropy (SSE) was also used. SSE is an extension of the concept of SE to a two-dimensional measuring space. That is, based on the concept of SE, SSE is calculated, at each frequency, for spatial distribution of vibration velocity spectrum in the measuring plane. However, these two entropy values were used in unnormalized expressions. Therefore, although relative evaluation within the same measurement surface was possible, there was the issue that changes in the entropy value could not be evaluated in a unified manner in measurements under different conditions and environments. Therefore, this study verified whether it is possible to perform a unified evaluation for different defective parts of concrete specimen by using normalized SE and normalized SSE. From the experimental results using cavity defects and peeling defects, the detection and visualization of internal defects in concrete can be effectively carried out by the following two analysis methods. The first is using both the normalized SE and the evaluation of a healthy part of concrete. The second is the normalized SSE analysis that detects resonance frequency band of internal defects. Spectral entropy is often used in the time domain in recent years in the field of speech ,2 and elIn recent years, collapse accidents due to the aging deterioration of concrete structures  have occurred all over the world. At the inspection site of concrete structures, most of the on-site inspections are tap inspections and visual inspections. The tapping inspection largely depends on the skill and experience of the inspector, and there is a problem that it is expensive to install scaffolding in a high place, so it is expected to develop a method that can measure from a long distance without contact.In order to solve this problem, a noncontact acoustic inspection method has been studying using acoustic irradiation induced vibration ,8. In thConventionally, these two entropy amounts have been used without normalization. For that reason, although relative evaluation within the same measurement plane was possible, there was the issue that changes in the entropy value could not be evaluated in a unified manner in measurements under different conditions and environments. Therefore, in this paper, it is verified whether it is possible to perform a unified evaluation of different defective parts of concrete specimen using normalized SE and normalized SSE.After performing “Time–Frequency gate” processing  to reducThere are two methods to detect internal defects for noncontact acoustic inspection. One is a method to draw a scatter plot using two acoustic feature quantities , and to identify a healthy part, a defective part, or an abnormal measurement point by the measured point’s existence area. The other is a method to detect the resonance frequency bands observed on the measurement surface by using spatial spectral entropy. These two methods have been applied individually but can be combined to perform internal defect detection in concrete.Spectral entropy (SE) is a feature quantity that expresses the whiteness of a signal. It has a high value for a signal with a uniform spectrum, such as white noise, and a low value for a signal with a non-uniform spectrum, such as an audio signal. The spectral entropy  previousIn the previous paper, Normalized spectral entropy has been reported for signal analysis in the time domain ,14. For Here, i and j are the grid point numbers for the vertical and horizontal direction, respectively. m by n gives the number of all measurement points.From the difference between spectral entropy and normalized spectral entropy, it is explained why it is necessary to normalize spectral entropy. Using the result of concrete specimen, it is explained how general defect detection is performed and how a healthy part of concrete is identified, and it becomes clear why the normalization of spectral entropy is necessary in the detection of internal defects of concrete.In general, the analysis frequency band is determined by the frequency band of the emitted sound waves, and the frequency band of the emitted sound waves is selected to include the resonance frequency band of internal defects. In order to compare data under different measurement conditions, it is necessary to eliminate the influence of the difference in the number of samplings due to different analysis frequency bands. The solution is to normalize.If normalized spectral entropy is used, it is possible to evaluate on the same scale. (a) and (b) in As shown in In general, there are the following tendencies: the healthy part has low vibration energy and high spectral entropy; the defective part has high vibration energy and low spectral entropy; and an abnormal measurement point has high vibration energy and high spectral entropy. This is summarized in In defect detection, it is necessary to identify a healthy part of concrete in the measurement plane. If a healthy part can be identified, the other parts are the defective part or an abnormal measurement point.For one measurement point, two acoustic feature quantities  are calculated from its vibration velocity spectrum. It has been found that each distribution of two acoustic feature quantities in a healthy part of concrete follow the normal distribution. Taking advantage of this fact, measuring points in a healthy part can be found by statistical methods. Using the healthy part extraction algorithm , a healtIn defect detection, a defective part and a healthy part are discriminated based on the distribution of a healthy part of concrete. So, defect detection will be robust if the distribution of spectral entropy at a healthy part shows the same level value without depending on the measuring conditions. It is possible to compare the measurement results of different actual concrete structures with a unified standard.In Therefore, in Equation (3), the SSE is normalized to eliminate the influence of the number of data, that is, the number of measurement grid points. In The effect of normalization was investigated for two types of defects, such as a cavity and a peeling as an internal defect of concrete.The concrete wall specimen has a size of 2000 mm × 2000 mm × 300 mm and a weight of approximately 1200 kg. The shape and embedded depth of a circular cavity defect are as shown in The concrete wall with a cavity defect  or a peeling defect  was installed about 5.0 m from an LRAD of sound source. The distance from a laser Doppler vibrometer to the measurement surface is approximately 7.7 m for the cavity defect , 8.0 m for the peeling defect . The notation  indicates that the diameter of a circular defect is 200 mm, and the embedded depth is 60 mm. As an emitted waveform, a single-tone burst wave   when calculating the vibration energy ratio. As the energy reference value of the vibration energy ratio, the minimum value of vibration energy in the circular cavity defect  was adopted.Since spectral entropy is normalized, a cavity defect and a peel defect can be compared on the same scale. A healthy part of concrete can be evaluated by normalized spectral entropy and vibrational energy ratio on the same scale. The value of spectral entropy in a healthy part of concrete is distributed in the range of 0.93–0.95. The value of vibration energy ratio in a healthy part of concrete is distributed in the range of 0–3 dB.In a cavity defect, the measurement points of the defective part are scattered in the lower right direction from the distribution of a healthy part of concrete. On the other hand, in a peeling defect, since the vibration energy of the defective part is about the same as that of a healthy part of concrete, the measurement points are scattered below a healthy part of concrete. In order to distinguish between a healthy part and the defective part of peeling, it is necessary to evaluate by normalized spectral entropy.Comparing (a) and (b) in Therefore, in order to distinguish between a healthy part and the defective parts, it is important to evaluate a healthy part on the second axes called spectral entropy. In order to cause resonance and efficiently vibrate the defective parts, the optimum emitted waveform is selected depending on the type, size, and buried depth of internal defects, so the measurement conditions differ. In particular, the frequency range of the emitted waveform affects the spectral entropy value. By normalizing spectral entropy, this problem can be resolved. It is possible to accumulate and utilize data on a healthy part of concrete on the same scale even if the measurement conditions are different.It is identified statistically whether each measured point is a healthy part of concrete or not using the healthy part extraction algorithm. In In this way, robust defect detection can be performed by evaluating the distribution of a healthy part of concrete using normalized spectral entropy and vibrational energy ratio. Moreover, it is also applicable to actual concrete structures in various environments.For a circular defect , a vibration velocity signal was observed at each measurement grid point by a scanning laser Doppler vibrometer (SLDV). After Time-Frequency gate processing, those signals were transformed to vibration velocity spectrum by FFT. Using 121 spectra obtained at each measurement grid point, SSE analysis was performed using Equation (3).As a result of SSE analysis, the SSE value in a healthy part of concrete shows fluctuation in a certain range with respect to the median value of the distribution of SSE values. By normalizing the SSE, different internal defects can be evaluated on the same scale.Suppose you have glasses that can see a specific frequency, such as infrared rays. When looking at the measurement surface, a local peak can be seen on the measurement surface. In that case, the SSE value decreases at that frequency. In other words, in SSE analysis, it is possible to detect a local peak on the measurement surface in the frequency domain. In particular, when a resonance peak due to an internal defect appears on the measurement plane, the SSE value decreases at that resonance frequency. When it has a uniform distribution or a normal distribution on the measurement surface, such as the case of resonance of a laser head, the SSE value rises according to the nature of entropy. The detected resonance frequency of a laser head is excluded from the calculation of the vibrational energy ratio.By SSE analysis, both the resonance frequency bands of a cavity defect and a peeling defect were detected. As shown in The black dotted circle in If the resonance frequency band of the internal defect is known by SSE analysis, a clear acoustic image of the internal defect can be obtained simply by calculating the vibrational energy ratio at the range of the resonance frequency band.−13 [m/s]2) when calculating the vibration energy ratio. As an energy reference value of the vibration energy ratio, the minimum value of vibration energy in the circular cavity defect  was adopted.Vibration energy is calculated within the range of frequency band where resonance occurs, so the vibration energy at measurement points where the internal defect exist becomes large, and the defect image becomes clear.Normalization is defined for two types of entropy used in noncontact acoustic inspection method and experimental and analysis results are showed for internal defect such as a cavity or a peeling. One is spectral entropy (SE) used to detect the internal defects of concrete in a non-destructive test. SE can also be used to evaluate a healthy part of concrete. The other is SSE , which can detect the resonance frequency band of internal defects in the measuring plane. Through two kinds of entropy, the effective detection and visualization of internal defects in concrete was verified. Each entropy can be used separately or together, depending on a situation of concrete structures.


Paramagnetic metallohost systems can bind guest molecules and find application as biomimetic catalysts. Due to the presence of the paramagnetic metal center, rigorous characterization of these systems by NMR spectroscopy can be very difficult. We report here that metallohost-guest systems can be studied by using the paramagnetic relaxation enhancement (PRE) effect. Manganese(III) porphyrin cage compounds are shown through their PRE to thread and bind viologen guests, including a polymeric one. The binding constants and dethreading activation parameters are lower than those of the metal-free porphyrin cage compounds, which is proposed to be a result of charge repulsion of the trivalent metal center and dicationic viologen guest. The threading rate of the manganese(III) porphyrin cage onto the polymer is more than 10 times faster than that of the non-metallated one, which is ascribed to initial binding of the cage to the polymer chain prior to threading, and to an entron effect. Paramagnetic metallohost systems are difficult to characterize. Here the authors report that the paramagnetic relaxation enhancement effect can be used to prove by nuclear magnetic resonance experiments that Mn(III) porphyrin cage compounds can bind and thread low molecular weight and polymeric guests. Diamagnetic systems are the simplest to study since they display sharp lines, allowing for a multitude of 1D and multidimensional techniques to be applied. Paramagnetic systems pose complications for studying host–guest interactions since chemical shift and nuclear relaxation can be greatly influenced by the presence of unpaired electrons. However, even these challenging properties can provide opportunities for answering chemical questions and be utilized to gain insight into exchange and binding processes. Here, we show that metallohost–guest binding and exchange in a paramagnetic manganese(III) porphyrin macrocycle can be studied by NMR spectroscopy by exploiting the paramagnetic properties of the metal center, i.e., by using the paramagnetic relaxation enhancement (PRE) effect. So far, PRE has been utilized by various NMR methods studying paramagnetic systems such as characterization by means of hyperfine shifts8, measurement of protein–ligand kinetics via dissolution dynamic nuclear hyperpolarization (DNP)9, studying Xe host-guest complexes via paramagnetic hyper-CEST11, paramagnetic GEST (host–guest variant of CEST) combining MRI and NMR12, and diffusion NMR (DOSY)14. Furthermore, a toolbox using standard NMR techniques has been reported in the characterization of paramagnetic compounds15. In biochemistry, PRE has mainly been applied for the study of enzyme mechanisms18, protein structure characterization22, drug discovery27, and protein dynamics and exchange reactions33. To the best of our knowledge PRE NMR has not been used to study association and dissociation phenomena in organic and supramolecular chemistry.NMR spectroscopy is a powerful tool in supramolecular chemistry, not only for characterizing host-guest systems and performing binding studies, but also for investigating the kinetics of host–guest exchange at equilibrium35. In previous work, we studied the mechanism of exchange of small N,N′-dialkyl-4,4′-bipyridinium (viologen) guests as well as polymers containing a viologen moiety in diamagnetic  porphyrin macrocycles through UV–Vis, fluorescence, and EXSY NMR spectroscopy36. Unfortunately, spectroscopic studies on the actual catalytic system, i.e., the Mn(III)-porphyrin macrocycle, have not been possible, and as a result the effect of the Mn center in the porphyrin on exchange and binding has not yet been determined. This is due to the lack of fluorescent properties of Mn porphyrins, inconclusive UV–Vis behavior, and short T2s, which cause unmanageably broad peaks in the 1H NMR spectra, making EXSY studies impossible to perform. Hence, we decided to study the dynamics of the actual host–guest system by measuring the PRE on an excess of free viologen guests, including both low molecular weight and polymeric ones  porphyrin cage compound Mn1 can thread and bind viologen guests, including a polymeric one. The binding constants and de-threading activation parameters unexpectedly are lower than those of the metal-free porphyrin cage compound H21. Furthermore, the threading rate of Mn1 onto the polymer is much faster than that of H21, which is ascribed to initial binding of the cage to the polymer chain prior to threading, and to an entron effect.The studies presented here are part of an overarching project aimed at the development of manganese(III) porphyrin macrocyclic catalysts that can thread onto a polybutadiene chain, and while moving along it, convert the polymer double bonds into epoxidesR1,obs (s−1)) and transverse ) relaxation rate of a given nucleus can be observed. This observed relaxation rate is defined as the sum of the diamagnetic (R0) and paramagnetic (Rp) relaxation rates  and T2 (s) values of a specific signal in a sample containing both the paramagnetic host and diamagnetic guest compound in solution. The terms R1,0 and R2,0 can be obtained by measuring the T1 and T2 values of this signal in a sample containing only the diamagnetic guest compound in solution. Exchange parameters can be calculated from Rp, which, however, is dependent on the regime of exchange.In the presence of a paramagnetic center, an increase in the longitudinal  fast exchange of solvent inside an empty host to determine the association constant of viologen guest binding, and (2) slow, reversible dissociation of viologen guests to determine dissociation rate constants.1H NMR spectrum of the diamagnetic metal-free porphyrin macrocycle H21 gives well-defined and isolated sets of signals , all host signals broaden dueThe r−6 distance component. In order to accurately calculate the exchange parameters of the host–guest systems, the population of bound host should be determined for each measured system, which can be calculated through the respective binding constants. Due to the difficulty of measuring binding through routinely-used spectroscopic techniques40, we resorted to biochemical NMR spectroscopy for inspiration, as our hosts mimic to some extent properties of metalloproteins and metalloenzymes. Claridge et al. have taken advantage of the change in R1,obs of the bulk solvent water when it has access to a paramagnetic metal center buried inside a protein to determine the binding constant of a second, more competitive ligand to this metal center41. As soon as the competitive ligand binds to the metal center completely, the water will only be affected by the weaker outer-sphere effects, resulting in a smaller R1,obs.to the paramagnetic effect of the metal center on the cage compound Fig. , bottom.H21 to reside inside the cavity of the host and thus is an ideal reporter solvent42. Acetonitrile was also tested as a reporter solvent, but upon binding it displayed an overall smaller change in the relaxation rate, reducing its reliability. A binding curve was constructed by measuring the relaxation rate of bulk chloroform while varying the guest concentration and keeping the Mn1 concentration constant. From these PRE binding titrations, the binding constants of the Mn1/V1, Mn1/V2, and Mn1/VP complexes could be determined  of the paramagnetic host–guest complex Mn1/V1 (3.68 ± 0.16) is significantly lower, i.e., by three orders of magnitude, than that of the diamagnetic host–guest complex H21/V1 (7.29 ± 0.26). This indicates that H21/V1 forms a much more stable complex than Mn1/V1. This decrease in binding affinity after manganese insertion may be explained by repulsion between the positively charged dicationic viologen guest molecule, and the trivalent metal center which will destabilize the binding of V1 inside the cavity. In this connection it is worth mentioning that the electronic structures of manganese(III) porphyrins, e.g., Mn(III)TPP Cl, have been widely studied in the literature. A partial charge build-up on the metal center was found to occur as a result of the relatively labile Mn−Cl bond45.As shown in Table Mn1/V2 (log(Ka) = 4.20 ± 0.17) was lower than that of the diamagnetic combination H21/V2 (log(Ka) = 5.78 ± 0.30)39. However, V2 binds more strongly inside Mn1 than V1, whereas the opposite was true for the diamagnetic host H21. This could be caused by the decrease in sterics when the bulkier methyl cyclohexyl groups on either side of the viologen moiety in V1 are replaced by methyl groups, allowing the resulting guest V2 to be positioned more favorably inside the cavity of Mn1. This may result in a slight reduction of repulsive interactions between the metal-center and V2, in contrast to Mn1/V1 where there is less freedom of movement.As expected, the binding constant for Mn1/VP (log(Ka) = 3.23 ± 0.18) was found to be similar in magnitude to Mn1/V1 (log(Ka) = 3.68 ± 0.16). This is consistent with our previous research where binding constants were determined for H21 and poly-THF substituted viologen guests with variable numbers of monomer units (34–88)47. The obtained binding constants (log(Ka) ~7.4) were very similar to that of the small, symmetric viologen guest V1 with H21 (log(Ka) = 7.29 ± 0.26), indicating that similar (de)stabilizing effects are present. A range of differently metallated porphyrin cages is currently under investigation. A step-by-step how-to guide for determining association constants and dissociation parameters is included in the Supplementary information.Lastly, the binding constant for 1H-NMR experiments, such as 1D EXSY, on host-guest complexes involving Mn1 are not possible due to the excessive broadening of the relevant host resonances. Upon adding ten equivalents of a viologen guest V1 to Mn1, we can observe a similar paramagnetic-induced broadening of the ortho (8.85 ppm) and meta (8.45 ppm) free guest proton resonances compared to the resonances in the diamagnetic host-free spectrum  in the presence of Mn1 (0.5 mM) compared to the host-free condition  and a paramagnetic contribution (Rp). Rp can be further defined as the sum of inner-sphere   is the mean-residence time of the guest bound to the host, and TM (s) is the relaxation time constant the guest experiences while bound. By taking the inverse of τM, the observed dissociation rate constants can be determined and thereby their corresponding energy barriers. When the system falls into a slow-to-intermediate exchange regime with respect to relaxation, τM can dominate Rp under certain conditions and therefore the TM term can be neglected, simplifying the equation to Eq. Rp is dominated by τM, and therefore suitable for measuring the rate of dissociation of the guest from the host, three requirements have to be met: 1) Rp should increase at higher temperatures  Rp is independent of the field strength  R1,p should approximately be equal to R2,p  for V1 could be calculated using Eqs. kd), the observed dissociation rate constant has to be corrected for the host and guest dependence on the residence time of the ligand near or on the metal (τM). If both the host and guest concentration have no influence on τM, kd,obs will be equal to kd. However, if there is an additional host or guest dependence on the residence time, the kd,obs will have to be corrected for the order of the dependence. We determined the host and guest dependence on the exchange by measuring the residence time of the guest in the V1/Mn1 complex at different V1 and Mn1 concentrations  were extracted from the Eyring plots  than that of the diamagnetic host–guest complex H21/V1 . The difference of ~4.5 kcal mol−1 in ΔΔG‡ mirrors the ~5 kcal mol−1 difference in ΔΔG (Table In order to assess the influence of Mn(III) Cl insertion into the porphyrin ring we may compare the activation energy barriers for dissociation of the Mn1 and viologen polymer VP. This guest molecule has been studied previously with H21, where it was confirmed that threading and exchange do occur36. We previously had expected and assumed that polymers would thread through Mn148, but PRE NMR studies now offer the possibility to definitively prove whether this hypothesis holds true. For the Mn1/VP system Robs should be compared to Ros. Here, Robs is measured by a sample containing Mn1 and VP, and for Ros a sample containing MnRot and VP. If Robs ≈ Ros then the inner-sphere contribution is near-zero and there will be  no exchange and, consequently, possibly no threading. If the observed relaxation rate is much larger than the outer-sphere contribution (Robs ≫ Ros), then this supports that exchange and hence threading occurs. We performed these PRE NMR experiments and found that the observed relaxation rates  indeed are significantly larger than the outer-sphere contributions , i.e., by approximately a factor of nine, which is in a similar order of magnitude as found for Mn1/V1. These experiments, therefore, demonstrate that Mn1 indeed threads VP.The PRE experiments were repeated with Mn1/VP  compared to Mn1/V1 . The notion that the destabilized binding of V1 in the cavity of Mn1 results in facile dissociation and therefore faster exchange is further confirmed by the measured binding constants of Mn1/VP (Ka = 1692 ± 295 M−1) and Mn1/V1 (Ka = 4796 ± 769 M−1), which are in the same order of magnitude. Furthermore, since chloroform exposure to the cavity was the observable feature in the binding studies, these experiments directly point to complete dissociation of the polymer as opposed to a slipping process in which the viologen moiety slips in and out of the cavity without full dissociation of the polymer-chain.From the Eyring plot Fig.  the actiH‡ = 3.17 ± 0.33 kcal mol−1, ΔS‡ = −36.89 ± 3.29 cal K−1 mol−1) it is evident that for the Mn1/VP system the dissociation is almost entirely governed by entropy. Possible reasons for the high entropy contribution to the dissociation of the polymer could be linked to the notion that a significant extent of order should be achieved for complete dissociation of a molecule with this length. It could also be influenced by the resolvation of the polymer chain after dissociation or possible wrapping of the polymer-chain around the porphyrin host37. The significantly lower activation enthalpy  found for Mn1/VP compared to Mn1/V1  may be attributed to the possibility that the viologen-moiety of VP is threaded, but not deeply bound inside the cavity of Mn1, as opposed to Mn1/V1, resulting in a lower dissociation activation enthalpy for Mn1/VP. Another possibility is the difference in sterics between V1 and VP. The observed higher ΔH‡ of the former compound may be a result of the presence of the bulkier methyl cyclohexyl substituents on either side of its viologen moiety compared to the unbranched carbon chains attached to that of VP, possibly complicating dissociation. To test the latter theory and to see if the activation enthalpy would be lower, the PRE NMR measurements were repeated with methyl viologen V2 (see Supplementary Information p 35–40). Here, similar ΔH‡ values for the dissociation of Mn1/V2  compared to that of Mn1/V1  were found, rejecting the hypothesis that less bulky substituents on the viologen guest result in lower activation enthalpies. Hence, we attribute the appreciable difference in dissociation ΔH‡ values measured for Mn1/V1 and Mn1/VP to an increase in statistical host positions on the polymer chain, as opposed to Mn1/V1 where the viologen moiety is deeply bound inside the cavity of its host.From the measured energy barriers for exchange  can be calculated from Mn1/VP remains relatively insensitive to the temperature  × 105 M−1 s−1) than combinations of VP with the metal-free host (kon = (1.9–3.6) × 104 M−1 s−1)46. Analysis of the Eyring parameters reveals that the difference between the threading of VP through Mn1 and through H21 is solely enthalpic in nature, as the entropy values are very similar. We tentatively ascribe this to a pre-binding effect, which has been proposed in earlier work49. It involves a preassociation of the porphyrin cage with the polymer and diffusion along its chain, allowing the polymer end to loop back into the cavity of the host (effective molarity effect), aiding the threading process. We suggest that this process is more effective for Mn1/VP than for H21/VP because of the positive charge residing on the Mn1 center, interacting stronger with the oxygen atoms in the polymer chain. Furthermore, the interaction of the polymer end with the cavity of the cage, which initiates the threading process (entron effect)47, will be stronger for Mn1 than for H21. The above mechanism explains why the ΔS‡ values for Mn1/VP and H21/VP are similar, but the ΔH‡ value is lower for Mn1.Lastly, from the now known Mn1/V1 displays lower energy barriers for guest dissociation compared to H21/V1. The combined results of lower binding constants and lower dissociation barriers for Mn1/V1 compared to H21/V1 indicate that for the paramagnetic system ground-state effects play an important factor in the decrease of the activation barriers for dissociation. These destabilizing effects may be caused by repulsive interactions between the positively charged metal center and the dicationic viologen guest. Furthermore, decreasing the bulkiness on the substituents of the guest compound (compare Mn1/V2 versus Mn1/V1) resulted in slightly stronger binding and faster dissociation, which likely is a result of the increased freedom of movement of the guest inside the cavity of the host in combination with decreased steric interactions.We have shown that paramagnetic relaxation enhancement experiments can be utilized to investigate chemical exchange binding strength in the field of metallohost–guest chemistry for systems displaying both slow to medium exchange rates (utilized to measure dissociation) and fast exchange rates (utilized to perform association titrations), even if the resonances corresponding to the bound species are not directly visible. Binding titration studies revealed that a paramagnetic host–guest complex, involving a manganese(III) metal in the porphyrin ring of the cage compound, displays significantly lower binding affinities for viologen guests compared to those of the non-metallated porphyrin cage with the same guest compounds. Furthermore, Mn1/VP, which involves a polymer chain to which a viologen moiety is attached. The much larger value observed for the relaxation rates  compared to the outer-sphere contributions  presents irrefutable evidence that Mn1 is capable of threading VP, confirming a long-standing hypothesis. Surprisingly, the threading rate of Mn1 onto VP was found to be considerably faster than that of the non-metallated porphyrin cage H21 onto VP. We attribute this to a pre-association of the positively charged Mn1 complex to the outside of the oxygen-containing poly-THF chain, allowing the polymer end to loop back and find the cavity of Mn1 more easily (effective molarity effect). The methodology presented in this paper provides a blueprint for how binding and dynamics in other paramagnetic systems, especially catalytic ones, which are extensively studied in the field of organic chemistry, could be thoroughly investigated.The PRE studies could also be performed on the host–guest complex 51 (Carr-Purcell-Meiboom-Gill) pulse sequence and the PROJECT-CPMG52 (Periodic Refocusing of J Evolution by Coherence Transfer CPMG) sequence on a Bruker 500 MHz Avance III spectrometer equipped with a Prodigy BB cryoprobe or a Bruker 300 MHz Avance III HD nanobay spectrometer equipped with a BBFO probe. For all VT experiments, the temperature was calibrated using pure ethylene glycol for temperatures of 20 °C and higher. Methanol was used to calibrate the temperatures lower than 20 °C.Longitudinal relaxation rates were measured through inverse recovery experiments and transverse relaxation rates were measured via by utilizing the CPMG2CO3 and acetonitrile was distilled and dried over CaCl2. For all titration studies, which were carried out at 22 °C, the solutions were irradiated with light of 419 nm.Fluorescence quenching titrations were performed on a JASCO FP-8300ST Spectrofluorometer in a quartz cuvette with 1 cm path length. Samples for titrations consisted of anhydrous chloroform and anhydrous acetonitrile . Anhydrous chloroform (>99%) and acetonitrile were obtained from Sigma-Aldrich. Before use, the chloroform was filtered over dry KSupplementary InformationPeer Review File


To assess (i) the pattern of antibiotic prescribing in Danish general practice, (ii) the use of diagnostic tests [point-of-care (POC) and tests analysed at the hospital laboratory (laboratory tests)], and (iii) the frequency of diagnostic testing in relation to antibiotic prescriptions.Retrospective cross-sectional register-based study.General practice in a geographical area of Denmark covering 455,956 inhabitants.We studied redeemed antibiotic prescriptions and performed diagnostic tests in general practice from 2013 to 2017 among inhabitants in nine selected municipalities.Frequency of antibiotic courses. Frequency and type of diagnostic testing performed in relation to types of antibiotics.A total of 783,252 antibiotic courses were redeemed from general practice with an overall decrease of 19% during 2013–2017. Diagnostic testing increased by 6% during this period. POC tests comprised the majority of performed diagnostic tests (83%) with C-reactive protein (CRP) as the most frequently used test. A 27% increase in the use of laboratory tests was observed. Tests were performed in relation to 43% of all antibiotic courses; most in relation to prescriptions for sulphonamide and trimethoprim (57%) and rarely when prescribing tetracyclines (10%). Conflicting with national guidelines, Danish GPs prescribed fluoroquinolones without performing any kind of diagnostic testing in 48% of the cases.Diagnostic tests , can increase diagnostic certainty and lead to a reduction in antibiotic use in general practice.A decrease in antibiotic courses in general practice in Denmark was observed during 2013–2017, while the use of diagnostic tests increased.A diagnostic test was performed in relation to 43% of antibiotic courses.Only 52% of prescribed fluoroquinolones was related to a diagnostic test, conflicting with national guidelines.This study provides an overview of the use of diagnostic tests in relation to antibiotics and creates basis for further research into the variability between types of antibiotics. The study indicates that there is room for improvement to use diagnostic tests as an aid to promote prudent antibiotic use. The World Health Organisation (WHO) considers antimicrobial resistance as one of the largest threats to public health . To reduIn Denmark, about 90% of antibiotics are prescribed in the primary health care sector , with arAcute infections are common reasons for consulting the general practitioner (GP). Although most infections are of viral origin or non-severe bacterial infections, many of them are treated with antibiotics In Denmark, antibiotics are mainly prescribed for urinary tract infections and acute respiratory tract infections .Diagnostic tests are valuable tools for increasing diagnostic certainty to support a prescription of antibiotics or to refrain from this ,12. GPs A previous Danish study reported variability in the use of tests between types of antibiotics and between practices . To our The aim of this study was to explore (i) the antibiotic prescribing pattern in Danish general practice, (ii) the use of diagnostic tests (POC or laboratory tests), and (iii) the frequency of use of diagnostic testing in relation to prescribing in total and by type of antibiotic from 2013 to 2017.This retrospective cross-sectional study aimed at providing a descriptive overview of antibiotic prescribing and the use of diagnostic tests in Danish general practice between 2013 to 2017.Most Danish citizens are registered with a GP, and services are tax-funded with a mixed capitation and fee-for-service system . The outThis study used national registers as described below. The study sample included redeemed prescriptions for systemic antibacterial drugs in the nine municipalities.Based on the registers mentioned below, we created a study base consisting of all individuals residing in nine selected municipalities, who at any point between 2003 and 2017 redeemed an antibiotic prescription. As of 1 January 2017, the study base consisted of 405,989 individuals, comprising 89.0% of the total population in the nine municipalities . The remaining 11% of the population who were not included comprised patients who did not at any point between 2003 and 2017 redeem an antibiotic prescription.The Danish National Prescription Registry: This database contains complete information on all prescriptions redeemed by residents at outpatient pharmacies. All systemic antibiotics are available only by prescription, and the register is reported as having high validity and completeness [leteness . InformaService Provider Register: This database contains information on all health service providers and specialty codes. We used specialty codes linked to prescriptions to ensure the inclusion of antibiotic prescriptions and tests exclusively from general practice and out-of-hours services (OOHS).The Danish National Health Service Register: This database contains information about activity codes used for reimbursement of services in general practice. Type of service  and time of registration were used in the study [he study .The Department of Clinical Microbiology database, Odense University Hospital (DCMO): This database contains information on laboratory tests performed in general practice and analysed at the hospital laboratory for the population in the nine municipalities. The database is used for the registration of laboratory test results and for communication to the clinician. The DCMO provided us with information about the type of test , Chlamydia trachomatis) and the date the test material was received at the DCMO.The Danish Civil Registration System: This system contains socio-demographic information about all residents in Denmark. We used information about residency.Statistikbanken.dk: A publicly available service from Statistics Denmark, which was accessed to determine population size in the nine municipalities under study for reference use.Data were obtained from the following registers and linked through encrypted unique patient and provider identifiers:For the first and third aims, we created a subsample including all redeemed antibiotic prescriptions from 2013 to 2017 for this population. We restricted the dataset to prescriptions issued exclusively from general practice using the Service Provider Register.) (referred to as laboratory tests). For the second aim (use of diagnostic tests) the dataset included all patients in the study base. Based on The Danish National Health Service Register we included POC tests from general practice from 2013 to 2017. Moreover, we included laboratory tests based on data from The Department of Clinical Microbiology, Odense University Hospital (DCMOThe antibiotic prescribing pattern was described by computing the redeemed antibiotic courses for each type of antibiotic (ATC level 4) per year and proportions were calculated. If a prescription was preceded by another prescription with the same ATC code within 14 days, it was interpreted as the continuation of a current antibiotic course, and the second prescription was excluded. For the second aim, we computed the frequency of each type of diagnostic test per year and the proportion each type comprised. The third aim (exploring tests performed in relation to the types of antibiotic) was assessed by using the information on performed POC tests and laboratory testing in relation to redeemed antibiotic courses from 2013 to 2017 grouped by ATC codes. Since reimbursements are registered weekly, we linked tests to the Wednesday in the week of registration. A test was determined as linked to an antibiotic prescription if the test was registered from 7 days before to 7 days after an antibiotic prescription was redeemed to account for the fact that diagnostic tests may be used in a variety of ways  and since there can be a delay between the time of test performance and time of registration.A total of 783,252 antibiotic courses were redeemed during the study period (2013–2017).A total of 2,526,634 diagnostic tests were performed during the five years . POC tesChlamydia trachomatis. Cultures comprise the vast majority of laboratory tests compared to PCR tests. However, PCR tests for respiratory tract bacteria have more than tripled .Laboratory tests overall increased by 28,5%. Urine culture was by far the most frequently performed test, followed by testing for Overall, diagnostic tests were performed in relation to 43.4% of the antibiotic prescriptions .The most frequently used tests performed in relation to antibiotic prescriptions were CRP (17.1% of redeemed prescriptions) and POC urine tests (16.9%). The CRP test was mainly used in relation to the prescribing of penicillins with beta-lactamase inhibitors (37.1%) followed by macrolides (25.1%). The use of POC urine tests analysed in practice or urine tests sent to the hospital laboratory were frequently related to prescribing of penicillins with extended-spectrum, sulphonamides and trimethoprim, fluoroquinolones, and the group of ‘other antibacterials’.Sulphonamides and trimethoprim were the antibiotics most often issued in relation to a diagnostic test (56.8% of prescriptions). Penicillins with extended-spectrum followed, with diagnostic tests used in 55.6% of prescribing cases. For fluoroquinolones a test was performed in relation to 51.8% of all prescriptions, with a laboratory test in 23.9% of all the cases. Tetracyclines on the other hand were related to testing in 9.5% of the cases.Antibiotic prescribing decreased from 2013 to 2017 for all types of antibiotics except for penicillins with an extended spectrum. Meanwhile, the use of diagnostic tests increased, especially the use of CRP tests, all types of cultures, and PCR for respiratory tract bacteria. This development could be explained by national awareness campaigns from 2016 to 2020 which may influence both GPs and patient expectations. In the same period accreditation in general practice has been mandatory bringing focus on quality in diagnosis and treatment and encouraging comparison of own practice figures (including antibiotic prescribing) with other practices [The study shows that less than half of antibiotic courses had a related diagnostic test, even though Danish GPs have full access to POC tests and laboratory tests and are reimbursed for using these. A large variation was seen between groups of antibiotics with sulphonamides and trimethoprim related to a test in 57% of prescribing cases whereas tetracycline was related to testing in 10% of the cases.The study covers an established geographical area with more than 450,000 inhabitants. The study is based on registers that are known to be of very high validity . The DanWe restricted analyses to descriptive statistics on the aggregated level to create an overview of how often and in relation to which type of antibiotic, tests were performed, rather than conducting an in-depth analysis of how the diagnostic tests are used. We found descriptive statistics sufficient to answer our three research questions. An association study on the individual level would provide insights into what patient and GP factors are associated with antibiotics prescribing and testing. This will be a natural next step to pursue. Stratification by type of infection could reveal diagnostic strategies for different types of infection. The databases used for this study provide information about the diagnosis stated on the prescription by the prescriber. However, nearly half of prescriptions lack a disease-specific diagnose , and thePrescriptions from general practice and out-of-hours service (OOHS) cannot be distinguished in the registers. Differences in diagnostic approaches in the two settings must be expected, since fewer diagnostic tools are available in the OOHS, and typically the GP does not know the patient beforehand. GPs in the OOHS had access to a few diagnostic methods  but not to laboratory tests. If the patients are disabled or too ill to go to the GP, the GP will make home visits and have even fewer diagnostic tools.The study base comprises patients who redeemed at least one antibiotic prescription between 2003 and 2017 (corresponding to 89% of the population in the study area). We do therefore not have information about patients who did not at any point between 2003 and 2017 redeem an antibiotic prescription and cannot report whether they had any tests performed. We cannot test for differences, but individuals with no redeemed antibiotic prescriptions in the 15 years may differ from the rest of the population in terms of being more healthy or less likely to attend the GP. However, we consider that the large study coverage may provide results representative for the population of interest.The study shows a temporal link between the use of a diagnostic test and antibiotic prescription, but the study cannot confirm that the diagnostic test and antibiotic prescription concern the same health problem. It would be relevant to study the use of tests related to indications for prescriptions but the completeness of specific clinical indications in the registers are only around 68% [Previous studies show that the use of CRP and urine tests can improve diagnostic accuracy and risk classification ,22–26. TBordetella pertussis and Mycoplasma pneumonia [PCR tests for respiratory tract infections are not commonly used. However, the prevalence has more than tripled over the study period. Reasons behind this could be increased focus on not prescribing macrolides without diagnostic testing and epidemics of neumonia .We assessed the use of diagnostic tests from 2013 to 2017 and found higher proportions of prescriptions related to a test compared to a Danish study by Haldrup et al. exploring the same topic also using national registers but from 2004 to 2013 . This maClinical uncertainties are inherent in medicine but more pronounced in primary care, and clinicians may opt for diagnostic testing for (1) a more certain diagnosis; (2) monitoring and susceptibility patterns to ensure correct treatment . HoweverWe found a decreasing use of antibiotics concurrent with an increase in diagnostic testing. This could indicate an increasing awareness of antimicrobial resistance among GPs and efforts to avoid unnecessary antibiotic use.We found that for sulphonamide and trimethoprim, tests were performed in relation to just 57% of all prescriptions. This group of antibiotics is almost exclusively used for urinary tract infections in Denmark in which guidelines advise active use of diagnostic tests. This could indicate room for improvement since tests are easily available, the GPs are reimbursed for using them, and the tests can assist the GPs in identifying patients who would benefit from antibiotic treatment and, equally important, who would be better off without. In a broader perspective, increased use of these tests may increase diagnostic certainty and reduce the use of antibiotics.Importantly, access to diagnostic tests is not the same as making good use of them. We do not know the correct cut-off between antibiotic prescription and the use of diagnostic testing. In general, tests are intended to increase diagnostic certainty, provide susceptibility patterns, or to monitor treatment (including wait-and-see) effects. Importantly, many cases of antibiotic prescribing do not necessarily include diagnostic testing.This study provides knowledge on the extent to which laboratory tests are used. The study cannot determine whether the use of tests is associated with a higher quality of care. To investigate this further, it would be relevant to explore the variation in the use of tests among GPs and for which patients and indications they are used. Furthermore, it is relevant to study if the testing actually makes a difference in the use of antibiotics.


LRRK2/PARK8 gene with G2019S mutation. We previously established a fly model of PD carrying the LRRK2-G2019S mutation that exhibited the parkinsonism-like phenotypes. An herbal medicine, Gastrodia elata Blume (GE), has been reported to have neuroprotective effects in toxin-induced PD models. However, the underpinning molecular mechanisms of GE beneficiary to G2019S-induced PD remain unclear. Here, we show that these G2019S flies treated with water extracts of GE (WGE) and its bioactive compounds, gastrodin and 4-HBA, displayed locomotion improvement and dopaminergic neuron protection. WGE suppressed the accumulation and hyperactivation of G2019S proteins in dopaminergic neurons and activated the antioxidation and detoxification factor Nrf2 mostly in the astrocyte-like and ensheathing glia. Glial activation of Nrf2 antagonizes G2019S-induced Mad/Smad signaling. Moreover, we treated LRRK2-G2019S transgenic mice with WGE and found that the locomotion declines, the loss of dopaminergic neurons, and the number of hyperactive microglia were restored. WGE also suppressed the hyperactivation of G2019S proteins and regulated the Smad2/3 pathways in the mice brains. We conclude that WGE prevents locomotion defects and the neuronal loss induced by G2019S mutation via glial Nrf2/Mad signaling, unveiling a potential therapeutic avenue for PD.The most frequent missense mutations in familial Parkinson’s disease (PD) occur in the highly conserved  Parkinson’s disease is a brain disorder that leads to tremors and difficulties with balance and coordination. These symptoms are caused by the loss of neurons which release a chemical messenger that is needed to regulate movement called dopamine. Most treatments for this disease work by boosting levels of dopamine in the brain, but this can lead to severe side effects and these drugs often become less effective over time.Gastrodia elata Blume (or GE for short) has previously been reported to relieve symptoms of Parkinson’s disease in both human and animal studies when administered as a decoction or formula. However, it is unclear how GE protects dopamine-producing neurons and if this mechanism involves another type of brain cell known as glia that has also been linked to Parkinson’s disease.A traditional Chinese medicine called To investigate, Lin et al. studied fruit flies and mice that carry a genetic mutation that produces the symptoms and molecular characteristics of Parkinson’s disease. The experiments showed that when the flies and mice were fed food containing water extracts of GE, they experienced less difficulties moving and had a higher number of intact dopamine-producing neurons.Lin et al. found that GE switched on a protein in glial cells located near dopamine-producing neurons. Activation of this protein, called Nrf2, inhibited a signaling pathway in degenerating neurons that leads to the disease state. As a result, less dopamine-producing neurons were damaged and the animals’ coordination and balance were maintained.These findings suggest that GE could potentially provide an alternative or complementary therapy for Parkinson’s disease, although it still needs to be studied further in humans. If the same effect is observed, the specific compounds in GE that have this protective effect could be isolated and analyzed to see if they could be used for treatment. Parkinson’s disease (PD) is a highly prevalent neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra projecting to the striatum of the basal ganglion, representing a circuit involved in motor planning and coordination. As a consequence, PD is associated with motor abnormalities, bradykinesia, hypokinesia, rigidity, and resting tremor. Currently, the most frequently applied pharmacological treatment, levodopa (L-DOPA), exerts limited motor improvement and elicits negative side effects . Hence, PARK genes have been genetically linked to PD, a number that is increasing , induce characteristic PD symptoms and pathologies such as loss of dopaminergic neurons and the appearance of Lewy bodies  has been used to treat neurological disorders for centuries  is often used as an alternative or dietary treatment for human diseases, including PD . Althougenturies . GE has enturies . The majenturies . Additioenturies , highligDrosophila G2019S model. We identified two distinct pathways induced by WGE in the model, that is, suppression of Lrrk2 protein accumulation and hyperphosphorylation in neurons, and activation of the Nrf2 pathway in glia, particularly in astrocyte-like and ensheathing glia. We show that WGE-induced Nrf2 activation antagonizes the Gbb-activated Mad signaling in glia, contributing to neuronal protection. WGE also suppressed the hyperactivation of G2019S proteins and antagonized Smad2/3 signaling in a LRRK2-G2019S mouse model, which restored locomotion, protected dopaminergic neurons, and regulated the microglia hyperactivation. Conservation of the pathways impacted by WGE treatment in both the Drosophila and mouse G2019S models implies the beneficial effects of GE and represent a reliable and effective complementary therapy for PD.In the present study, we treated G2019S animals with water extract of GE (WGE), and its bioactive compounds, gastrodin and 4-HBA. We have investigated the impact of WGE treatment on PD in restoring locomotion and protecting dopaminergic neurons in the GAL4-UAS system to express the human G2019S mutant of Lrrk2 by the Ddc-GAL4 driver (Ddc>G2019S) in dopaminergic neurons and then assessed the anti-geotactic climbing activity of adult flies. We observed that locomotion of Ddc>G2019S flies was affected significantly relative to control flies expressing human wild-type Lrrk2 (Ddc>Lrrk2) . Both Ddc>Lrrk2 and Ddc>mCD8-GFP flies showed comparable climbing activities in the first six weeks, and a significant number of Ddc>mCD8-GFP flies were still able to climb above the 8 cm mark at week 7. Therefore, we used the Ddc>Lrrk2 line as a control for Ddc>G2019S flies in subsequent experiments to dissect the specific mode of pathogenicity of the G2019S mutation.We employed the c>Lrrk2) . At weekould not . We alsoDdc>G2019S flies at all three doses elicited a significant improvement in their climbing ability  in weeks 5 and 6. The higher doses of WGE (0.5 and 1.0%) still exerted beneficial effects at weeks 3 and 4, albeit not as significantly as the 0.1% dose, but had no beneficial effect in weeks 5 and 6. As 0.1% is the lowest among the three doses tested, we lowered the dose of WGE to 0.02% and found that 0.02% WGE was less effective than 0.1% starting at week 3 till week 6, suggesting that 0.1% is the optimal dose in restoring the climbing activity of Ddc>G2019S flies . WGE treatment of  ability . Strikin9S flies . We alsolar food . The effDdc>G2019S flies with food containing gastrodin (0.1 mM) equivalent to the amount in 0.1% WGE also restored locomotion of Ddc>G2019S flies in weeks 2–4, though its impact diminished to nonsignificant levels at weeks 5 and 6. However, increasing the gastrodin dose 10-fold (1.0 mM) resulted in improved climbing activity at weeks 5 and 6  or G2019S mutant protein (elav>G2019S)  . Howeverav>Lrrk2 . This ouav>Lrrk2 , consist 2 and 4 . However0.1% WGE . In the rylation . We furtrylation . We obse feeding . Hence, 505 in 3-day-old adult fly head extracts and found reduced levels of pAkt in elav>G2019S flies relative to elav>Lrrk2 controls  in Drosophila. Intriguingly, neither overexpression (UAS-cncC-FL2) nor RNAi knockdown (UAS-cncTRiP) of Nrf2 in dopaminergic neurons had an impact on the climbing ability of Ddc>G2019S flies  to levels comparable to WGE-fed control flies  without Nrf2 knockdown  and used the GAL4 driver to manipulate Nrf2/CncC activity in glia (repo>cncC-FL2 for overexpression and repo>cnc-RNAi for knockdown). As a first step, we validated the age-dependent locomotion decline of Ddc-LexA>G2019S flies that was severer than Ddc-LexA>Lrrk2 and could be rescued by 0.1% WGE feeding . Significantly, the Ddc-LexA>G2019S; repo>cncC-FL2 flies performed better in the climbing assay than Ddc-LexA>G2019S; repo-GAL4 without Nrf2 overexpression , which had little impact on the already declined climbing activity in Ddc-LexA>G2019S; repo-GAL4  . Thus, glial overexpression of Nrf2 partially restores locomotion of Ddc-LexA>G2019S flies, and glial depletion of Nrf2 abolishes the ability of WGE to rescue impaired locomotion.Next, we investigated if WGE-induced Nrf2 activity in glia is effective in promoting locomotion in G2019S flies. To this end, we employed the  feeding . We thenpression . We alsoepo-GAL4 . ImportaDdc-LexA>G2019S; repo-GAL4 flies compared to Ddc-LexA>Lrrk2; repo-GAL4 controls . We observed comparable levels of Lrrk2 and pLrrk2 in the control and Nrf2 overexpression lines  or ensheathing glia  abolished the improved locomotion elicited by WGE treatment, recapitulating the effect of pan-glial Nrf2 knockdown  (cnc in cortex (np2222), perineurial (np6293), or subperineurial (moody) glia. We confirmed the involvement of astrocyte-like and ensheathing glia by means of Nrf2 overexpression in astrocyte-like  or ensheathing  glia, with both treatments improving the climbing activity of Ddc-LexA>G2019S flies not subjected to WGE feeding (Five types of glia with different morphologies and functions have been identified in the fly brain . We decinc-RNAi) . This ou feeding . These aARE-GFP expression in astrocyte-like and ensheathing glia of flies expressing G2019S mutant protein and subjected to WGE treatment. We focused on the astrocyte-like and ensheathing cells located adjacent to dopaminergic neurons. In control flies expressing wild-type Lrrk2, we detected basal levels of GFP signal in mCherry-positive astrocyte-like  or ensheathing glia (R56F03)  . We dete(R56F03) . Thus, oMad that encodes the pathway’s downstream effector to restore locomotion in Ddc>G2019S flies  or constitutively active type I receptor Tkv (Q253DUAS-tkv) was sufficient to impair locomotion, even without expressing G2019S mutant protein in neurons. However, impaired locomotion was rescued in both cases by 0.1% WGE feeding (Mad or Q253Dtkv and they were restored by 0.1% WGE treatment (G2019S mutant protein in dopaminergic neurons has been shown previously to enhance the expression of the proprotein convertase Furin 1 (Fur1) that processes the BMP signaling molecule Gbb for maturation and release, leading to activation of the BMP signaling pathway in glia . We conf9S flies . To addrreatment . Similarreatment , glial o feeding . In addireatment . TogetheDdc-LexA>G2019S flies  partially rescued the locomotory impairment caused by G2019S mutation  further enhanced this effect , suggesting that Nrf2 functions mainly to antagonize Mad activity  , even in the presence of WGE treatment, maintained high pMad levels in glia  . Depleti in glia . Thus, mMad modulates expression of the Nrf2 target ARE-GFP. We observed diminished GFP signal in PPL1-surrounding glia of Ddc-LexA>G2019S adult fly brains relative to the Ddc-LexA>Lrrk2 control. Heterozygosity of Mad in Ddc-LexA>G2019S flies  restored the level of GFP signal, implying that Mad modulates targeted gene expression induced by Nrf2 activity  were increased in the substantia nigra of LRRK2-G2019S mice, but this increase was suppressed by WGE treatment  cause PPL1 dopaminergic neuron degeneration, accelerating impaired age-associated climbing ability -projection dopaminergic neurons in the PPL1 cluster inhibits climbing performance . Mutatio ability . Dopamin ability . In cont ability . A signilt flies . We postDrosophila, Rab10 and Lrrk2-G2019S synergistically affect the activity of dopaminergic neurons, mediating deficits in movement  were UAS-mCD8-GFP (#5137), elac-GAL4 (#8760), repo-GAL4 (#7215), Ddc-LexA (#54218), UAS-cncTRIP (#25984), tsTub-GAL80 (#7108), alrm-GAL4 (#67032), R56F03-GAL4 (#39157), Q253DUAS-tkv (#36536), and K00237Mad (#10474). NP2222-GAL4 (#112830) was from the Drosophila Genomics Resource Center and NP6293-GAL4 (#105188) was from the Kyoto Stock Center. Other stocks include UAS-Flag-LRRK2-WT  for subcloning into LexAop plasmids (Addgene), which were for microinjection . The transgenes were site landed at an attP site on the 2nd chromosome (25C6). For temperature-shift assay of tsGAL80 flies, parental flies were maintained at 19°C and allowed to mate, before collecting the F1 adults and shifting them to 29°C to inactivate GAL80.All fly stocks were maintained on standard cornmeal-based food medium at 25°C. LRRK2-WT , UAS-Fla2-G2019S , Ddc>GALDdc>GAL4 , ARE-GFP ; rabbit anti-Akt ; rabbit anti-Drosophila phospho-Akt Ser505 ; rabbit anti-Nrf2 ; rabbit anti-phospho-Nrf2 (Ser40) ; mouse anti-HO-1-1 ; rabbit anti-GAPDH ; and rabbit anti-alpha tubulin , followed by blotting with secondary antibodies peroxidase-conjugated goat anti-rabbit IgG  and peroxidase-conjugated goat anti-mouse IgG .Adult brain extracts were prepared according to a previously described protocol . In brieARE)-GFP reporter assay was a modification of the protocol from a previous study  and maintained at the animal center of the National Taiwan University Hospital (NTUH). Non-transgenic (nTg) and heterozygous transgenic LRRK2-G2019S mice were obtained by crossing heterozygous LRRK2-G2019S mice with wild-type FVB/NJ mice (JAX stock #001800). Mice at 8.5 months old were assigned to one of three groups (5–6 mice per group): nTg, LRRK2-G2019S, and LRRK2-G2019S fed with WGE (0.5 g/kg body weight per day) (Transgenic per day)  for 3 moWe employed two behavioral tests to assay mouse motor function, that is, an open-field assay to assess spontaneous locomotor activity and CatWalk XT gait analysis to assay coordination. Behavioral experiments were conducted blind to genotype, as described previously .After 3 months of WGE treatment, mice were sacrificed at the age of 11.5 months. The substantia nigra and striatum were dissected out. The substantia nigra was subjected to immunostaining, as described previously . Anti-ty1292, Abcam, ab203181, 1:1000), anti-Smad2 , anti-phospho-Smad2 , anti-Smad3 , anti-phospho-Smad3 , and anti-beta actin . After washing, peroxidase-conjugated goat anti-rabbit IgG  or peroxidase-conjugated goat anti-mouse IgG  were used as secondary antibodies.Frozen nigrostriatal brain tissues were homogenized and mixed with lysis buffer to determine protein content and for immunoblotting, as described previously . The memt-test or one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. p<0.05 were considered indicative of significance. For exact n numbers, p-values, F-values, t-values, and degrees of freedom of each statistical test, please see the statistical information in All statistical analyses were carried out in GraphPad Prism 6 . Data are presented as mean ± standard error (SEM). Statistical analysis was performed using either Student  Your study elucidates the molecular mechanisms by which a Chinese traditional medicine compound, GE, confers neuroprotection in mouse and fly models of LRRK2 G2019S-induced Parkinson's disease and provides an avenue for a potential therapeutic treatment for patients. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:Gastrodia elata Blume in Lrrk2-G2019S Parkinson's disease" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Suzanne Pfeffer as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Fengwei Yu (Reviewer #2).Thank you for submitting your article "Glial Nrf2 signaling mediates the neuroprotection exerted by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Please address the issues raised by the reviewers with textual changes.Congratulations on a nice piece of compelling scientific work!Reviewer #2 (Recommendations for the authors):Can WGE treatment of 3-4-week-old G2019S flies also alleviate locomotive defects despite the onset of PD progression? If the authors have the data available, it would be nice to include in the revised version. However, this experiment is not required for the publication of the manuscript.In a previous study by the same lab, they show that Nrf2 pathway is upregulated in neurons upon Lovastatin treatment, suggesting a neuronal function of Nrf2. Nrf2 pathway was also recently reported to regulate developmental neurodegeneration (i.e. dendrite pruning) in a cell-autonomous manner. It could be interesting to examine if Lovastatin treatment upregulates Nrf2 activity in neurons or glia in PD flies. The authors may discuss this part or investigate in their future study. Reviewer #2 (Recommendations for the authors):Can WGE treatment of 3-4-week-old G2019S flies also alleviate locomotive defects despite the onset of PD progression? If the authors have the data available, it would be nice to include in the revised version. However, this experiment is not required for the publication of the manuscript.Ddc>G2019S flies was started on the first day of the fourth week and climbing activity was assessed at the end of the fourth week. Interestingly, 0.1% WGE is also effective to significantly improve locomotion of Ddc>G2019S flies. The protective effect was reduced at week 5 and diminished at week 6 with continuation of WGE feeding. The result was included in the revised manuscript as Figure 1—figure supplement 2. We also revised the descriptions in Results (Lines: 134-140) and Figure legends of Figure 1—figure supplement 2 (Lines: 944-950) as below.We thank the reviewer for the comment. We had investigated the therapeutic effect of WGE in the PD fly model. In the experiment, WGE feeding of Lines: 134-140Ddc>G2019S flies that were fed with regular food without WGE for 3 weeks. At week 4, these Ddc>G2019S flies also showed a significant improvement in their climbing ability, compared to the age-matched Ddc>G2019S flies fed continuously on regular food . The effect of improving climbing activity in the WGE-fed Ddc>G2019S flies was reduced at week 5 and diminished at week 6, suggesting that WGE feeding starting at earlier stages is important for long-term locomotion improvement.”“We also tested the WGE effect on the Lines: 944-950Ddc>G2019S flies starting WGE feeding at week 4.“Figure 1—figure supplement 2. Locomotion improvement of Ddc>Lrrk2, Ddc>G2019S, and Ddc>G2019S with 0.1% WGE feeding at week 4 were assessed at weeks 3, 4, 5 and 6. Bar graphs show success rates  of flies climbing over 8 cm height in 10 sec. One-way ANOVA and Tukey’s post-hoc multiple comparison test were performed and statistical significance was shown as *** for p < 0.001, * for p < 0.05 and ns for no significance.”The climbing activities of In a previous study by the same lab, they show that Nrf2 pathway is upregulated in neurons upon Lovastatin treatment, suggesting a neuronal function of Nrf2. Nrf2 pathway was also recently reported to regulate developmental neurodegeneration (i.e. dendrite pruning) in a cell-autonomous manner. It could be interesting to examine if Lovastatin treatment upregulates Nrf2 activity in neurons or glia in PD flies. The authors may discuss this part or investigate in their future study.We thank the reviewer for the comment. We added a paragraph to address the neuronal function of Nrf2 and lovastatin-related future works in Discussion (Lines: 437-441), as below.Lines: 437-441“…In a previous study, lovastatin treatment provides neuroprotection in the G2019S-induced PD model, also through the Akt/Nrf2 pathway . As activation of neuronal Nrf2 plays a non-conventional role in promoting developmental dendrite pruning , it remains interesting to further study the cell types that mediate the action of lovastatin.Newly included references:Cell rep, 36(5), 109466. https://doi.org/10.1016/j.celrep.2021.109466Chew, L. Y., Zhang, H., He, J., and Yu, F. (2021). The Nrf2-Keap1 pathway is activated by steroid hormone signaling to govern neuronal remodeling.


Despite extensive research studies, recycled aggregates and worn-out tyres of motor vehicles are still not fully reused and are hence disposed of in ways that are damaging to the environment. Several studies have been carried out on recycled aggregate and rubberized concrete, but very limited studies are conducted on rubber recycled aggregate concrete. This study focuses on the workability, mechanical properties and durability performance of concrete made with 100% recycled aggregates and crumb rubber at different replacement level . The first stage of the study covers the effect of incorporating crumb rubber at different concentration on the workability and mechanical properties of recycled aggregate concrete. The results revealed that the workability and mechanical properties of the recycled aggregate concrete can be used for structural applications when 5% of crumb rubber are used to replace recycled aggregates. The 28-days compressive strength of the rubberized recycled aggregate concrete with 5% crumb rubber concentration is reduced by 21.1% and 32.8% when compared to recycled aggregate concrete and control concrete, respectively. The second stage of the study assesses the durability performance of the recycled aggregate concrete with 5% crumb rubber concentration. The 5% crumb rubber content for durability tests was considered because the ultrasonic pulse velocity tests revealed that the quality of the recycled aggregate concrete is questionable if the concentration of crumb rubber particles is beyond 5%. The durability performance using the surface resistivity test also shows that the chloride ion penetration of recycled aggregates concrete with 5% crumb rubber replacement is moderate using air dried curing technique and high using the water bath curing method. Hence the study suggests the use of rubber recycled aggregate concrete for applications were the exposure condition is not extreme. Waste tyre disposal is currently causing serious environmental issues all over the world. Every year, approximately I billion waste tyres are generated globally, with 1.6 billion new tyres being produced . StudiesThe performance of recycled aggregate concrete generally decreases ,10,11,12The research results so far also indicate that incorporating crumb rubber in concrete decreases the resulting concretes’ compressive and tensile strengths. This is attributed to the lower strength of rubber particles, and their weak bonding with cement paste ,22,23,24The current study assesses the performance of concrete made with recycled aggregate and crumb rubbers at different replacement levels. Different studies have been conducted on concrete performance either made with recycled aggregates or crumb rubber, but very limited studies utilize both recycled materials to make concrete ,7,8. Fur80 (recycled aggregates obtained from concrete products with 20% impurities). The water absorption rates and the densities of both the recycled and natural aggregates are shown in CEM 11/B-V 32.5N Portland fly ash cement complying with  was usedThe grading of the natural aggregates, recycled aggregates are shown in This study designed a reference concrete of 40 MPa cylindrical compressive strength as shown in The recycled aggregates and fine sand were incorporated into the concrete mixer and mixed for 60 s followed by crumb rubber and 50% of water for another 60 s. Cement was then added for another 30 s and finally the remaining quantity of water was added in 120 s to attain a uniform concrete mix.For regular reinforced concrete structures using recycled aggregate concrete, durability is a particular concern ,11,39 beIn this study, the concentration of crumb rubber particles in recycled aggregate concrete is limited to 5% in order to preserve its mechanical properties. Hence, the durability assessment of the recycled aggregate concrete with 5% of crumb rubber concentration will be conducted.The surface resistivity test was used in this study to measure the durability performance (chloride ion penetration) of the recycled aggregate concrete with crumb rubber. The resistivity tests were conducted on cylindrical specimen of size ϕ100 × 200 mm based on the specifications presented in . The fouFor each mix, slump tests were carried out and results presented to ascertain the workability of the concrete. The workability of all mixes was assessed by means of slump test according to . The conThe workability of the recycled aggregate concrete was enhanced to level comparable to the reference concrete (165 mm) by adding superplasticizer (1% of the cement weight). The added superplasticizer compensates for the water absorbed by the recycled aggregates during mixing process ,44,45. HFor each mix, three concrete cubes were tested as per specifications made in  and the The results of the ultrasonic pulse velocity in The failure mode of the concrete mixes without crumb rubber is brittle. However, addition of crumb rubber enhances the ductility of the recycled concrete mixes. This due to the higher deformability of the crumb rubber particles used in the replacing the recycled aggregates.Three split tensile strength tests were carried out for each mix based on the specifications in  and the The samples for the concrete resistivity tests were cured for 28 days prior to testing. Two curing techniques were adopted in this study; some samples are fully submerged in tap water at a temperature of (20 ± 2) °C and relative humidity ≥95% while others are wrapped with damp cloth at a temperature of (20 ± 5) °C. The samples cured in tap water were tested under surface saturation condition. All the samples are marked at four locations equally spaced at 90 degrees as shown in The surface resistivity of concrete mixes cured for 7 and 28 days are shown in However, under air dried state the durability performance of the recycled concrete with 5% crumb rubber concentration is moderate based on the The effect of crumb rubber on the strength and penetration of chloride ion in recycled aggregate concrete was investigated using the surface resistivity test method. Different concentrations  of crumb rubber in recycled aggregate concrete were considered in the study. The results clearly shown that addition of crumb rubber reduces the compressive strength of the recycled aggregate concrete moderately when its concentration is limited to 5%. The study also observes the resistivity of chloride ions for recycled aggregate concrete with 5% crumb rubber concentration. Based on the ultrasonic pulse velocity results, the use of recycled aggregate concrete with crumb rubber content beyond 5% is questionable.The results of the surface resistivity tests shows that the resistivity of the recycled aggregate concrete to chloride ion penetration is moderate under the air dried curing technique. However, using the water bath technique, the resistivity was found to be low based on the specifications listed in Furthermore, the ductility of recycled concrete with crumb rubber is found to be greater than that of recycled concrete without crumb rubber. As a result, using rubberized concrete for structural members subject to seismic loads is a promising potential application. Rubberized recycled concrete is an alternative solution for meeting sustainability targets and reducing embodied carbon in the construction industry. However, its limitations are the reduction in strength and the willingness of contractors to use the concrete product on new projects due to the previously mentioned associated drawbacks.Further research on the durability performance of the recycled concrete with crumb rubber concentration using different mix ratios is needed. Pre-treatment of the crumb rubber particles could also be considered when assessing the durability of the recycled concrete.