Aspergillus nidulans (MAD1445), a pH regulatory mutant, could grow on concrete plates and promote calcium carbonate precipitation.Concrete is susceptible to cracking owing to drying shrinkage, freeze-thaw cycles, delayed ettringite formation, reinforcement corrosion, creep and fatigue, etc. Continuous inspection and maintenance of concrete infrastructure require onerous labor and high costs. If the damaging cracks can heal by themselves without any human interference or intervention, that could be of great attraction. In this study, a novel self-healing approach is investigated, in which fungi are applied to heal cracks in concrete by promoting calcium carbonate precipitation. The goal of this investigation is to discover the most appropriate species of fungi for the application of biogenic crack repair. Our results showed that, despite the significant pH increase owing to the leaching of calcium hydroxide from concrete,  Sustainable infrastructure is the key to creating a sustainable community, due to its significant impact on energy consumption, land use, and global economy. However, many countries are facing the downfall of progressively aging infrastructure that needs rehabilitation. In particular, concrete infrastructure suffers from serious deterioration owing to the effect of various physical and chemical phenomena, such as drying shrinkage, freeze-thaw cycles, reinforcement corrosion, creep and fatigue, and delayed ettringite formation, all of which could lead to concrete cracking.1. In addition, cementitious grouts, mortars, and concrete are also often used to provide shielding and encapsulation of various radioactive waste materials from military, research, and power generation applications. Some waste isotopes as well as their decay products will become a serious radiation hazard for hundreds of thousands of years, which requests exceptionally durable storage.Cracks themselves may not significantly reduce the load-carrying capacity of concrete in the short run, but they considerably weaken the durability of concrete structures, as they channel in water, oxygen, and carbon dioxide, which could potentially corrode the steel reinforcement. Moreover, cracking may promote severe degradation of the non-mechanical properties of concrete, such as the radiation-shielding properties of concrete elements used in nuclear applications. Nowadays, concrete has been the key construction material for reactor containment and biological shielding structures, which are essential components of the nuclear reactors in service worldwide for power generation23.In view of the remarkable social significance of concrete infrastructures and their exceptional service demands, the maintenance and inspection for concrete structures have come into focus. However, continuous inspection and maintenance usually require onerous labor and high investments, which presents a huge and costly challenge. Fortunately, inspired by the amazing capability of the human body to fix broken bones through mineralization, researchers have conducted numerous investigations to equip concrete structures with self-healing properties and generated many innovative solutions3 precipitation, which have been summarized in a comprehensive review provided by Seifan et al.20. Among them, the biotechnological approach by using mineral-producing microorganisms is most desirable. It has been demonstrated that in the environment of concrete, CaCO3 can be precipitated by certain species of bacteria through biologically induced mineralization processes21. Owing to its sufficient compatibility with concrete compositions, CaCO3 is often regarded as the most appropriate filler for concrete cracks.So far, concrete can fix its own cracks mainly through the following three mechanisms: autogenous healing, embedment of polymeric material, and bacteria-mediated CaCO3 mainly through three different pathways, as summarized below. (1) Certain species of ureolytic bacteria have been explored based on their ability to indirectly cause the formation of calcium precipitates by increasing the local pH via catalyzing the hydrolysis of urea to ammonium5. (2) Microbiologists at Delft University of Technology in the Netherlands13 tested the second pathway, in which aerobic oxidation of organic acids results in production of CO2, leading to CaCO3 production in the environment of concrete. However, in the case of low oxygen concentration, such as for most underground structures, the efficiency of this approach can be limited. (3) The third pathway is known as dissimilatory nitrate reduction16. This approach is less efficient than the other two in terms of the amount of produced CaCO3, but it can be applied in anaerobic zones.Bacteria can precipitate CaCO13, which is far from being practical considering the fact that the lifetime of concrete infrastructure can easily be fifty years or even a century. The detrimental environment of concrete, such as very high pH values, tiny pores, serious moisture deficit, varied temperatures, and limited nutrient availability, dramatically influences the microbial metabolic activities and makes bacteria and their spores susceptible to death. In addition, owing to the limited ability of bacteria to produce large amounts of CaCO3, bacteria can only heal small cracks with crack widths less than 0.8 mm20.Although the research on bacteria-mediated self-healing concrete indeed achieved a certain level of success, it still suffers from serious limitations. So far, the viability of bacterial spores embedded in concrete is generally less than six months3 precipitation25, but those species have never been tested in the environment of concrete. Currently, the investigation of fungi has been mostly focusing on their importance in the degradation of organic matter, and their connection with inorganic constituents is limited to acquisition of mineral nutrition by mycorrhizal fungi as well as mineral weathering of lichens and ectomycorrhizae.To address the above-mentioned problems, further investigation on alternative microorganisms becomes potentially essential. In fact, microorganisms are very diverse, and besides bacteria, they also include Archaea, protists, and fungi. Among them, fungi form a species-rich group of eukaryotic organisms, which exhibits vast biodiversity with more than 100 K described species and approximately 1.5 M unknown species. Common fungi include yeasts, lichen-forming fungi, molds, and mushrooms, etc. Recent studies in geomycology demonstrated that certain species of fungi can play a central role in CaCO30. For example, compared with other microbial groups, filamentous fungi exhibit higher surface-to-volume ratios, and therefore possess a larger fraction of organic substrates available for mineral precipitation.The cells of filamentous fungi grow as threadlike structures called hyphae, which possess multiple nuclei and grow apically with new apices emerging from the formation of lateral branches, creating an intertwined 3D network called mycelium. In fact, the existing literature suggests that filamentous fungi have unique features to be used in various applications of biomineralization-based technologies36. However, it has been concluded that there are two critical factors determining the amount of CaCO3 production, i.e., carbonate alkalinity and Ca2+ concentration24. The metabolic activities of filamentous fungi that can increase carbonate alkalinity typically include water consumption, physicochemical degassing of fungal respired CO2, oxidation of organic acids, nitrate assimilation, and urea mineralization24. In addition to carbonate alkalinity, fungal metabolic activities can also affect calcium concentration. Ca2+ concentration within fungal cells should be strictly controlled, i.e., most of the calcium ions need to be gathered at the apex for apical growth and sharply reduced in subapical regions24. To maintain this sharp gradient, fungi need to effectively regulate Ca2+. Ca2+ in the cytoplasm is kept at sufficiently low concentrations by deliberately pumping it out of the cell or by binding it onto cytoplasmic proteins24.The calcification of fungal filaments is a complex process, and it remains incompletely understood24. The production of calcium oxalates has been regarded as a method to decrease their internal Ca2+ concentration24. Precipitation of CaCO3 may be due to a similar passive mechanism to immobilize excessive Ca2+. Excessive alkalinity could also pose a source of stress and precipitation of CaCO3 may be due to intracellular protection24.In addition, fungi can also influence calcium concentration out of self-protection. Metals that are required for fungal growth and metabolism may become toxic if their concentrations are too high. However, unlike other microbial group, many species of fungi can survive and prosper in regions that are seriously polluted by metals. Precipitation of metal minerals onto hyphae was considered as one of the most important mechanisms to explain the superior metal tolerance in fungi. Concrete is a calcium-rich environment, which represents a stress for fungal cells owing to calcium cytotoxicity and subsequent osmotic pressure37. Chitin is known for its ability to bind Ca2+, as it forms a substrate that could considerably reduce the required activation energies for nuclei formation so that the interfacial energy between the fungus and the mineral crystal becomes significantly lower than the one between the mineral crystal and the solution42. Thus, both living and dead fungal biomass can bind ions onto their cell walls, resulting in nucleation and deposition of mineral phases31. Bound Ca2+ can then interact with the dissolved carbonate, resulting in CaCO3 precipitation on the fungal hyphae.While bacteria promote mineral precipitation only through induced biomineralization processes, fungi can do it through both induced biomineralization and organomineralization processes. The cell walls of many fungi contain chitin, a polymer of N-acetylglucosamine3 production, fungi could be used as self-healing agents. The goal of this investigation is to discover the most appropriate species of fungi for the application of biogenic crack repair. To make fungi-mediated self-healing concrete, the fungal spores alongside their nutrients, will be blended into the concrete before the curing process starts. When cracks appear and water trickles into the concrete, the dormant fungal spores will wake up, grow, consume the nutrient soup, and promote CaCO3 precipitates to fix the cracks in situ. After the cracks are finally healed, the bacteria or fungi will make spores and go dormant once more – ready to start a new cycle of self-healing when cracks form again. For existing concrete infrastructures with cracks, the fungal spores and their nutrients can be injected or sprayed into the cracks.Therefore, thanks to their ability to directly and indirectly promote CaCO44. Fungi make responses to the extracellular pH by means of activation of a dedicated transcription factor, PacC50. Mutants in the pacC regulatory gene are extremely heterogeneous in phenotype43. A major class of mutations are gain-of-function pacCc created by truncating the C-terminal region44. The pacCc mutations bypassing the need for the extracellular pH signal result in permanent activation of alkaline genes and superrepression of acidic genes, which leads to alkalinity mimicry47. Irrespective of ambient pH, fungi exhibiting alkalinity-mimicking mutations believe that they are always at alkaline pH and trigger a gene expression pattern similar to that of the wild-type grown at high pH values, which is exactly what is needed for self-healing concrete.Besides wild-type fungal strains, genetically engineered fungi are also important candidates for self-healing concrete. Since the extremely high pH of the concrete environment can be handled by only a few fungi, pH regulatory mutants were the focus here. It is well known that many microorganisms that are capable of growing over a relatively wide pH range can adjust their gene expression according to the ambient pH levels51, we have found that the spores of a wild-type Trichoderma reesei (ATCC13631) germinated into hyphal mycelium on concrete plates and grew well. However, there exist only a few studies on the pH regulation of T. reesei. In filamentous fungi, the best characterized member for gene regulation by ambient pH is Aspergillus nidulans44. Therefore, gene manipulations are easily achievable in A. nidulans. In this study, three different types of alkalinity-mimicking mutants of A. nidulans, i.e., MAD1445, MAD0305, and MAD0306, will be tested.In our previous investigationRhizopus oryzae (ATCC22961), Phanerochaete chrysosporium (ATCC24725), A. nidulans (ATCC38163), A. terreus (ATCC1012), and A. oryzae (ATCC1011). The genomes of these fungal strains have been sequenced and annotated and are publicly available60. In addition, these species do not exhibit any toxicity and belong to Biosafety Level 1 (BSL-1), which is the lowest risk level.The following five different wild-type strains were purchased from American Type Culture Collection (ATCC): Saccharomyces cerevisiae, also called baker’s yeast, as it has been instrumental to winemaking, baking, and brewing since ancient times. Recently, a series of publications were released on the success of designing synthetic set of S. cerevisiae chromosomes, which could allow S. cerevisiae to rapidly evolve and rearrange its genome, optimizing itself for certain applications63. Thus, in this study, S. cerevisiae will also be tested.The above-mentioned fungi are all filamentous fungi. In addition to filamentous fungi, there are also single-celled fungi, i.e., yeasts, that do not form hyphae. One of the most well-known species of yeast is 65. SEM has been used to visualize fungal precipitates67, and TEM has been used to study fungal-biotite interfaces and weathered alkali feldspars69. SEM will be applied to analyze the morphology and composition of the fungal precipitates, which will complement the very local characterization by TEM.To characterize the fungal precipitates, X-ray diffraction (XRD), scanning electron microscope (SEM), and transmission electron microscope (TEM) were applied in this study. XRD is an established technique to identify unknown crystalline phasesIn this section, the experimental procedures of gene replacement to obtain alkalinity-mimicking mutations, survival test of fungi on concrete plates, as well as phase identification and microscopic characterization of fungal precipitates will be presented. The procedures of the yeast strain identification and preparation of concrete specimens are described in the Supplementary Information.pacC mutations were previously published74.The full genotypes of the strains investigated in the current work are given in Table pacC14 and pacC202 can be described as follows. The parental strains with genotype pabaA1 gatA2 palF15 fwA1  were mutagenized with ultraviolet treatment followed by growth selection on media supplemented with 5 mM γ-amino-n-butyrate in presence of 10 mM ammonium tartrate as nitrogen source77. The pacC14 mutant strain was selected as it mimics the growth at alkaline pH values, downregulates the γ-amino-n-butyrate permease, decreases the γ-amino-n-butyrate level in cells, and thus relieves the γ-amino-n-butyrate-induced cytotoxicity. The pacC202 strain was a spontaneous mutant strain derived from the parental strains with genotype pabaA1 sasA60 , selected as it relieves the toxicity of 10 mM γ-amino-n-butyrate. The pacC genomic sequences in pacC14 and pacC202 strains were then obtained by Polymerase Chain Reaction (PCR) amplification.Briefly, the gene replacement procedure to obtain alleles 3-tagged wild-type pacC allele, designated as pacC900, as well as the engineered allele pacC14900 can be described as follows. A null allele of pacC was first constructed by transforming the orthologous pyr4 (orotidine 5′-phosphate decarboxylase) gene of Neurospora crassa flanked by 750 bp of upstream and 700 bp of downstream pacC genomic sequences into a pyrG89 (resulting in pyrimidine auxotrophy) pacC14 strain. Transformation was carried out using linearized fragments containing the pacC moiety of the plasmid. The long flanking sequence ensures high efficiency of homologous recombination-mediated gene replacement. The transformants displaying pyrimidine prototrophy were then selected and tested by Southern blot analyses to confirm that pacC14 was replaced by pyr4. The pacC null strain was then used as recipient strain and transformed with linearized MYC3-tagged pacC carrying C2437A mutation flanked with genomic sequence 825 bp upstream of pacC start codon and 925 bp downstream of stop codon78. Transformants displaying pacC colony morphology were tested for pyrimidine auxotrophy. The gene replacement was again confirmed using Southern blot analyses. The genotypes in the resulting pacC14900 strain were further confirmed by PCR sequencing. The protein expression was confirmed by Western blot analyses using anti-MYC antibody.The procedure to obtain MYCIn this study, potato dextrose agar (PDA) was chosen as growth medium. To investigate the impact of the extracellular pH on fungal growth, the inert pH buffer 3-(N-morpholino)propanesulfonic acid  was added to some of the growth media. Sterilization of concrete plate was carried out in a steam autoclave at 121 °C under 15 psi of pressure for 30 min. A 5 mm diameter mycelial disc of each fungal strain removed from 7-day-old cultures was aseptically placed at the center of each 60 mm Petri dish containing 10 ml growth medium. In some Petri dishes, there exists a concrete plate of 3 mm thickness at the bottom. Sterile PDA plugs were used as the negative inoculum control. The Petri dishes were incubated at 22 °C and 30 °C, respectively, for 21 days.To summarize, as shown in Fig. S1 in the Supplementary Information, for each type of fungal strain, eight different cases were investigated including PDA30, PDA22, MPDA30, MPDA22, CPDA30, CPDA22, CMPDA30, and CMPDA22. All the cases were investigated independently in triplicates.79 were studied using an optical microscope. pH values were recorded after 21 days of incubation by five independent measurements using an Orion 9110DJWP double junction pH electrode, which is a semi-micro combination pH electrode.Radial growth was measured based on two perpendicular diameters. The fungal samples prepared by the tape touch method65.Phase identification of the fungal precipitates were performed on a Siemens-Bruker D5000 powder diffractometer with Cu-Kα radiation in the θ/θ configuration operating at 40 kV and 30 mA. The samples were analyzed over the range of 10° to 80° 2θ at a scan rate of 1°/min with an increment of 0.02° 2θ. Before the measurements, the solid precipitates were isolated by dissolving the fungal sample in a vat of dilute bleach (NaOCl less than 3% by weight) at room temperature and washing it repeatedly in methanol following the published protocolThe fungal precipitates were studied by a Zeiss Supra 55 VP Field Emission SEM with an EDAX Genesis energy-dispersive X-ray spectrometer (EDS) at accelerating voltages of 5 kV to 20 kV. The collected solids were placed in an oven at 50 °C for two days, and then were sputter-coated with carbon by an Emitech K950X Carbon Vacuum Evaporator in order to ensure electrical conductivity. The chemical microanalysis technique EDS was applied in conjunction with the SEM analysis.TEM analysis was performed by a JEOL JEM-2100F TEM operating at 200 kV. The precipitates were collected by centrifugation  and washed multiple times in methanol to get rid of the growth medium. The particles were dried at room temperature then ground using a set of agate mortar and pestle into fine powders. The powders were then collected on copper grids overlain with a carbon-coated collodion film prior to the examination. Bright-field imaging (BFI), selected area electron diffraction (SAED), and high-resolution transmission electron microscopy (HRTEM) were applied to analyze the calcite phases.80. Images were post-processed by using the ImageJ software81.High-resolution X-ray computed microtomography (μCT) was applied to characterize the pore size distribution inside the cement paste specimen. Images were acquired using UltraTom X-ray μ-CT system equipped with a LaB6 X-ray source and a CCD camera with resolution of 4000 × 2624 pixels. Small cubic samples of 0.4 cm × 0.4 cm × 0.4 cm were used, for which the voxel size of the obtained images will be about 1.2 μm. Samples were firmly fixed on a cylindrical holder mounted on a rotating table using an adhesive paste. Acquisition parameters were set as follows: source 230 kV, voltage 90 kV, intensity 200–300 μA, filament LaB6, frame rate 0.4, and 8 averaging images. To impose small angular increments to cover 360° along the rotation axis, a total of 1664 phase contrast images were collected. The acquisition duration was about 30 min for each sample. Based on the raw data measured by μCT, the reconstruction of inner microstructure was carried out by using the Octopus Imaging softwarepacC and the resulting proteins are summarized in Table pacC14 genotype carries a C2437A allele, causing an early stop codon that results in a C-terminal truncation of PacC protein at 492 amino acid. The pacC202 genotype has a deletion from 2353 bp to 2555 bp, resulting in an open reading frame shift after amino acid 464 and an early stop codon. The resulting pacC14900 strain has a MYC3-tagged pacC carrying the pacC14 mutation at the pacC locus.The mutant sequence changes in S. cerevisiae ex-type culture CBS 1171 (NR_111007) and has the same morphological features of S. cerevisiae. Therefore, it is confirmed as the baker’s yeast, i.e., S. cerevisiae.The yeast strain has 99% ITS sequence similarity to the A. nidulans, i.e., MAD1445, has been found to be able to grow well (Table A. nidulans (MAD1445) did not grow in the cases of CPDA22 or CPDA30. The other eight species grew well in all the cases without concrete but did not grow on any concrete plate. The fungal growth in each case and the associated optical microscopic images are shown in Fig. S1, Fig. S2, and Fig. S3 in the Supplementary Information.The measurement results show that, because of the leaching of calcium hydroxide from concrete, the pH values increased significantly from 6.5 to 13.0 -inoculated media: CMPDA22 and CMPDA30. The particles had regular shapes with well-defined crystalline faces, indicating high crystallinity of the precipitates. The mineral crystals exhibited a range of crystalline morphologies from a single pure bulk crystal to plate-like crystals stacked upon each other. Besides, the crystals confirmed fungal involvement, as many cylindrical holes were found in the crystals, which probably occurred in the space inhabited by fungi. The holes also suggested that fungal filaments functioned as nucleation sites during the biomineralization process. As shown in Fig. 3. Note that EDS data may carry error if the sample is not homogeneous or flat. In our test, EDS data are obtained from several relatively flat sample areas, and the results are consistent. In comparison, the amount of calcium precipitation in the control medium was significantly reduced, as evidenced by the absence of faceted particles -inoculated and T. reesei (ATCC13631)-inoculated media, but the amount of mineral crystals was rather limited. As our future endeavors, the effects of the multiple factors promoting fungal growth and CaCO3 precipitation will be tested, such as concentrations of calcium ions and dissolved inorganic carbon, temperature, exposure to ultraviolet light, growth medium composition, fungal spore concentration, and availability of nucleation sites, etc. For examples, so far, our tests have confirmed that adding 4.0 µg/ml aminobenzoic acid and 0.02 µg/ml biotin to the growth medium significantly promotes the growth of A. nidulans (MAD1445); and adding 5.0 mM CaCl2 to the growth medium can promote calcium carbonate precipitation. In addition, a systematic investigation will be conducted to assess the fungal spore germination. According to Wang et al., whether or not the fungal spores in/on the concrete crack germinate can be measured by the oxygen consumption on the crack surface82. Furthermore, we are also testing urease-positive fungi Neurospora crassa84 and Myrothecium gramineum85 for this specific application.The experiments presented in this study have been repeated three times, confirming that the results are reproducible. However, it is still not clear what are the optimal conditions for fungal growth and CaCOIf the fungal spores are larger than the pore sizes in concrete, if they are directly put into cement paste, most spores will be crushed and lost viability during the hydration process. Our results on μCT characterization of concrete microstructure have been shown in Fig. A. nidulans (MAD1445) could grow on concrete plates. In comparison, A. nidulans (ATCC38163), A. nidulans (MAD0305), A. nidulans (MAD0306), R. oryzae (ATCC22961), P. chrysosporium (ATCC24725), A. terreus (ATCC1012), and A. oryzae (ATCC1011) could not grow in the alkali solution caused by the leaching of calcium hydroxide from concrete. The characterization by XRD, SEM, and TEM confirmed that the precipitates promoted by fungal activities were mainly composed of calcite.Concrete is considered as an extreme environment for fungi mainly due to its high pH values. To use fungi to heal cracks in concrete, the first step, which is also the most critical step, is to find the fungi strains which can survive the harsh environment of concrete and can promote calcium carbonate precipitation. The experimental results of this study showed that, despite the significant pH rise caused by the leaching of calcium hydroxide from concrete, one type of pH regulatory mutants of A. nidulans is usually considered relatively harmless to healthy human beings. However, according to the investigation conducted by Bignnell et al.86 on the role of the PacC-mediated pH adaptation in the pathogenesis of pulmonary aspergillosis, elimination of the PacC protein increases virulence in a murine neutropenic model, indicating that activities under PacC control may be crucial for A. nidulans pathogenicity. Therefore, a complete assessment needs to be performed to examine the possible short-term and long-term adverse outcomes of pacCc mutation of A. nidulans on both environment and human beings before it is used in concrete structures.It is important to note that according to the existing literature, Supplementary Information


These nanomaterials can be added to cement with other reinforcement materials such as steel fibers, glass, rice hull powder and fly ash. Optimal dosages of these materials can improve the compressive, tensile and flexural strength of cement-based materials, as well as their water absorption and workability. The use of these nanomaterials can enhance the performance and life cycle of concrete infrastructures. This review presents recent researches about the main effects on performance of cement-based composites caused by the incorporation of nanomaterials. The nanomaterials could decrease the cement porosity, generating a denser interfacial transition zone. In addition, nanomaterials reinforced cement can allow the construction of high-strength concrete structures with greater durability, which will decrease the maintenance requirements or early replacement. Also, the incorporation of nano-TiO2 and CNTs in cementitious matrices can provide concrete structures with self-cleaning and self-sensing abilities. These advantages could help in the photocatalytic decomposition of pollutants and structural health monitoring of the concrete structures. The nanomaterials have a great potential for applications in smart infrastructure based on high-strength concrete structures. Modern concrete infrastructure requires structural components with higher mechanical strength and greater durability. A solution is the addition of nanomaterials to cement-based materials, which can enhance their mechanical properties. Some such nanomaterials include nano-silica (nano-SiO One alternative solution is the construction of concrete structures with higher mechanical strength and higher durability, which will decrease their maintenance requirements or need for early replacement. Thus, the concrete structures can have thinner sections, which will require less quantity of cement-based composites for their construction. Construction engineering requires materials that enhance the mechanical properties of the cement-based composites for modern concrete infrastructure. For instance, the compressive, tensile and flexural strength of concrete structures need to be improved. For this, nanomaterials can be mixed with cementitious matrices to obtain concrete with high mechanical strength ,7,8,9,10this gas ,28. A ma2), nano-alumina (nano-Al2O3), nano-ferric oxide (nano-Fe2O3), nano-titanium oxide (nano-TiO2), carbon nanotubes (CNTs), graphene and graphene oxide. In recent years, several researchers [3S) [2O3 particles can increase the compressive strength of cement-based materials [2O3 nanofibers with a dosage of 0.25% by cement weight may enhance the compressive strength of cement-based materials by up to 30% [2O3. Optimal values of this nanomaterial improve the compressive strength of concrete specimens [2 nanoparticles can be used to build a photocatalytic concrete with self-cleaning and air-purification characteristics [The cement-based materials can be mixed with nanomaterials such as nano-silica  . In addiers [3S) . Nano-siers [3S) ,57,58,59aterials ,61,62,63p to 30% . Anotherpecimens ,65. The eristics . This coeristics ,67,68. Aeristics ,70,71,72eristics . For inseristics . It repreristics ,76.2, nano-Fe2O3, nano-TiO2, nano-Al2O3, CNTs, graphene and graphene oxide. In addition, these studies include nanomaterials that provide self-cleaning and self-sensing abilities to concrete structures. Also, the main challenges of using nanomaterials in cement-based materials are discussed.This review includes recent studies about the effects on the mechanical strength, durability and workability of cement-based composites due to the incorporation of nanomaterials such as nano-SiONano-silica is a nanomaterial employed for civil engineering applications that can replace micro-silica and silica fume. Nano-silica reacts with lime during the cement hydration process and it generates a C–S–H gel that may improve the mechanical strength and durability of concrete. A good dispersion of nano-silica into cement-based materials can accelerate the hydration process of cement paste, allowing a denser microstructure. On the other hand, an excessive number of nanoparticles can cause agglomeration due to their high surface energy, which will provide a non-uniform dispersion. Flores-Vivian et al.  used Por3) and a water-cement ratio of 0.5. During phase A, mixtures were made with a ceramic powder percentage of 0%, 10%, 15%, 20%, 25%, 30% and 40% of the cement weight using the same proportion of aggregates and water. During phase B, mixtures are made with 0.5% and 1% of nanosilica and different ceramic powder content of 10%, 15%, 20% and 25% of the cement weight. All concrete mixtures are fabricated considering the ASTM C192 standard. The results of the compressive strength tests of the concrete (phase A) were obtained with different curing ages . These results show that the concrete strength proportionally decreases with the amount of ceramic powder added to the concrete. The concrete specimen containing 1% of nano-silica and 10% of ceramic powder improved its compressive strength. The impact on the pozzolanic reaction of nano-SiO2 is more effective at an early age.Heidari and Tavakoli  fabricatSupit and Shaikh  determin2-coated TiO2 (NSCT) to RPC. These nanomaterials were studied by scanning electron microscopy (SEM), thermogravimetry (TG) analysis and powder X-ray diffraction (XRD). The acceleration of cement hydration due to the effect of the nucleus played a dominant role in the first days. The CH crystals particles size registered a reduction when the content of NSCT was increased (see In order to reinforce reactive powder concrete (RPC), Han et al.  added naased see . The fle3) and nano-silica. They used type I Portland cement and fly ash, and silica fume as binding agents. The percentages of nano-silica and nano-limestone by cement weight were of 0.5%, 1.0%, 1.5% and 2.0% and 2.0%, 3.0% and 4.0%, respectively. The mixture workability was reduced with respect the control specimen and it was maintained when the amount of nano-limestone is increased. This is due to the small size of nanoparticles that are found on the surface, leaving less water to contribute towards fluidity. The compressive and tensile strength of concretes including nano-limestone and nano-silica were improved with respect to concretes without any additions. The microstructure with highest values of density and mechanical strength was obtained with content levels of 1% nano-silica and 3% nano-limestone, respectively. The mechanical strength of concrete containing nano-silica and nano-limestone is increased with the reduction of the water-cement ratio.Li et al.  examined3) were fabricated based on II Portland cement at ages of 7, 28 and 90 days. In addition, they used 40 different types of mixtures considering 2%, 4% and 6% of replacement with nano-silica and superplasticizer. In the specimens were measured the wave transmission velocity and compressive strength using the exponential relationship between both parameters. An increment of steel fiber volume above 3% increased the wave pulse transmission velocity in the specimens. The compressive strength and wave pulse transmission velocity increased when the percentage of nano-silica achieved above 4% of cement weight; however, both decreased afterwards.Sadeghi et al.  reportedNajigivi et al.  implemenZhang et al.  investigTavakoli et al.  reportedMastali and Dalvand  reportedMohammed et al.  evaluate2 crystals, filling the voids of the C–S–H structure, leading to a denser microstructure.The incorporation of nano-silica optimal dosage in concrete samples may improve their compressive, tensile and flexural strength. The nano-silica added in cement with other materials such as polypropylene, glass and steel fibers with fixed proportions can increase the mechanical properties of the concrete. Concretes with nano-silica absorbed Ca(OH)2O3 in concrete specimens may improve their compressive strength. In addition, the volume electrical resistance of cement mortars with inclusion of nano-Fe2O3 can be altered through the applied load, allowing the measure of compressive stress [The optimal addition of nano-Fee stress . It can 2O3  at ages of 7, 14 and 28 days. 2O3. When the nano-Fe2O3 content increases then the surface morphology is denser. For all the measurements, the addition of nano-Fe2O3 in cement mortars increased their compressive strength compared to control mortar. Maximum values of the compressive strength of the cement samples were achieved using 10% of nano-Fe2O3 content. For this nano-Fe2O3 dosage at curing ages of 7, 14 and 28 days, the compressive strength of the cement mortar was increased up to 66.81%, 69.76% and 25.20%, respectively.Fang et al.  measured2O3, nano-Al2O3, nano-Fe3O4 and nanoclay on some properties of cement composites. These properties were the mechanical strength, hydration heat, water absorption, workability, setting time and durability. For instance, the inclusion of nano-Fe2O3 in the cementitious matrix decreased the water absorption and heat rate values as well as accelerated the peak times. Moreover, the workability of the composite was reduced when the nano-Fe2O3 content was increased. On the other hand, nano-Fe2O3 (0.5%–5% in concretes and 0.5%–10% in mortars) added into the cementitious matrix improved the compressive strength. Nazari et al. [2O3. For this case, cement was partially substituted with nano-Fe2O3  and a water to binder ratio of 0.4 was employed. The workability of concrete is decreased when the nano-Fe2O3 dosage is increased. In addition, Nazari and Riahi [2O3. Rashad  presentei et al.  also stund Riahi  develope2O3 and superplasticizer. The flexural, compressive and tensile strength, and the water permeability of the concrete specimens were improved with the incorporation of nano-Fe2O3 up to 4% by cement weight. The content of nano-Fe2O3 up to 4wt.% of the concrete specimens increased the coefficient of water absorption. The concrete specimens with nano-Fe2O3 enhanced their hydration heat, workability and the compressive, flexural and tensile strength.Khoshakhlagh et al.  studied 2 in concrete specimens can provide self-cleaning properties to the concrete. The concrete containing these nanoparticles can allow a photocatalytic degradation of pollutants  from industrial and automobile emissions. However, this effect is less efficient with aging due to carbonatation [The addition of nano-TiOnatation ,95. 2 thin film on the concrete surface that can provide active oxygen under UV light present in sunlight. Thus, it catalyzes the degradation of organic matters located at the nano-TiO2 coated concrete surface [2 is the chemical stability and low price in comparison with other materials. Moreover, nano-TiO2 can enhance the resistance to water permeability of cement-based structures [2 [2 under curing temperatures of 0, 5, 10 and 20 °C. They used natural river sand, Portland cement (type I ordinary), and TiO2 nanoparticles with size of 15 nm. In the experimental tests were used nano-TiO2 dosages of 1%, 2%, 3%, 4% and 5% by cement weight, respectively. In the fabrication of the specimens, the nano-TiO2 was dispersed in water through ultrasonication. After, cement and sand are mixed during 1 minute. Then, the well-dispersed nano-TiO2 was added and mixed during 60 seconds and after water was incorporated. In the following stage, the mortars are collocated into molds and cured using different temperatures. For the specimens were used a water to binder ratio of 0.5. 2, at curing age of 28 days under different temperatures. The compressive strength characterization is determined according to ASTM C109 [2 dosage. TiO2 nanoparticles can supply an extra space for the precipitation of hydration products. 2 had fast increment with respect to ordinary mortar until that the nano-TiO2 content achieved up to 2 wt.%. This increase slowed down for TiO2 nanoparticles dosages higher than 2 wt.%. The enhanced strength of mortar samples is caused by TiO2 nanoparticles that facilitated the cement hydration and filled the pores in C–S–H gels [2 nanoparticles form a bond between them self and C–S–H gel that improves their strength [ surface . The conructures . Figure tures [2 . Wang ettures [2  investigSTM C109  employinSTM C109 . This chS–H gels . These nstrength .2 as well as the mechanical properties of the cement pastes. 2 nanoparticles and their selected area electron diffraction. The incorporation of nano-TiO2  in cement paste using a water-cement ratio of 0.4 improved the flexural strength  at 28 days age.Feng et al.  examined2. They used Portland cement that was replaced up to 15% weight of waste ash and up to 5% weight of nano-TiO2. The addition of nano-TiO2 in the concrete improved the consistency and reduced the segregation probability. Considering the water absorption and capillarity, a significant decrease was obtained due to the nano-TiO2.Jalal et al.  studied 2 registered a microstructure more refined, which enhanced the resistance to mechanic failures. Other researchers, Yu et al. [2, which increased its mechanical strength. The TiO2 nanoparticles catalyze the decomposition of harmful gases in the air. In addition, the concrete with nano-TiO2 achieved a maximum compressive strength that was 7% higher in comparison with the non-added nanoparticle concrete. In addition, Yu et al. [The weight losses in concrete samples were caused by the rapid formation of hydrated products. The self-compacting concrete with nano-TiOu et al.  reportedu et al.  investig2. This concrete added with 1% nano-TiO2 improved its mechanical properties. They investigated the effects on the dry shrinkage, carbonation resistance, freeze-thaw resistance and resistance to chloride ingress. The addition of nano-TiO2 in concrete could allow it a self-cleaning and photocatalytic behavior. In addition, the normal concrete containing nano-TiO2 could decrease the capillary porosity.Chunping et al.  investig2O3 can accelerate the formation process of C–S–H gel, especially at early-ages, which enhances the strength of composites [2O3 nanofibers with a content of 0.25% by weight of cementitious materials, which improved the compressive strength up to 200 MPa. This represents an increment of 30% in comparison to material strength with only 1% of silica fume. This high compressive strength was achieved with low amount of silica fume. This improved performance is caused by the nanofibers that act as a seed to generate hydration products and contribute the reinforcement for the C–S–H formations, which decrease the number of micro-cracks. In addition, to reach the maximum mechanical performance of the cement-based materials is necessary a suitable dispersion of the Al2O3 nanofibers. A longer dispersion time could reduce the fibers agglomeration, allowing the enhance of mechanical performance. For instance, the compressive strength at 28 days age achieved higher values for specimens with Al2O3 nanofibers dispersed for 3 h. Nevertheless, higher quantities of Al2O3 nanofibers and supplementary cementitious materials did not increase the mechanical behavior of the cement-based materials. 2O3 nanofibers diluted in cement pastes.The use of nano-Almposites . For insmposites  fabricat2O3 on the chloride-binding capacity of cement paste samples. These samples were prepared with nano-Al2O3 dosages of 0.5%, 1.0%, 3.0% and 5%. The chloride-binding capacity was examined using conventional equilibrium tests, in which the samples were exposed with a NaCl solution at 0.05 mol/L, 0.1 mol/L, 0.3 mol/L, 0.5 mol/L and 1.0 mol/L, respectively. Based on the experimental results, the bound chloride content had an increase of 37.2% at NaCl solution (0.05 mol/L) by adding 5.0% of nano-Al2O3. Thus, an appropriate adding of nano-Al2O3 improved the chloride-binding of cement paste samples. Yang et al.  investig2O3 with 20% RHA at 28 and 90 days. The flexural strength of the mortar samples increased up to 34% and 41% by adding 3% nano-Al2O3 with 10% RHA. This addition of nano-Al2O3 generated a denser microstructure in the mortar samples. Mohseni at al.  studied 2O3 on the microstructural properties of the cement paste hydrated at 7 days age. Ordinary Portland cement is substituted with nano-Al2O3 powder with 2% and 4% by cement weight and the water-cement ratio is fixed to 0.4. In this early-age, they did not note changes at the compressive strength of the cement specimen at early age. Barbhuiya et al.  examined2O3 within 7 days of curing. They reported the generation of dense microstructure with larger crystal of portlandite within the cement matrix due to the nano-Al2O3 addition, as shown in 2O3 in the water absorption and electrical resistivity of cement mortars. They used 1%, 3% and 5% of nano-Al2O3 by cement weight. The water absorption had a small reduction with the addition of 1% and 3% nano-Al2O3. However, the water absorption registered a small increment with the addition of 5% nano-Al2O3. The highest electrical resistivity of the cement mortar is achieved with 5% nano-Al2O3.Based on the XRD analysis, Barbhuiya et al.  did not Recently, several researchers ,122,123 1 and 104 S/m. García-Macías et al.  developeLushnikova and Zaoui  studied Recent studies ,146,147 3) were characterized a loading rate of 1.2 KN/s. On the other hand, the flexural strength tests used TEA-GO reinforced samples (160 × 40 × 40 mm3) under a loading rate of 0.2 KN/s. Hu et al.  fabricatTao et al.  investigHigher magnitudes of compressive and flexural strengths  are reached in the cement mortar with GNP dosage of 0.25% (M1) by cement weight. These values represent increments of 8.3% and 15.6% compared with the compressive and flexural strengths of the cement mortar without GNP. However, the values of both strengths decrease when the GNP dosages exceed 0.05%. Thus, cement hydrates with homogeneous spatial distribution could be obtained with appropriate values of GNPs dispersed in cement matrix. 2 than the rGO composites at ages of 1, 7 and 28 days. In addition, the GO composites showed micropores filled with crystalline compounds and C–S–H gel. For the rGO composites was found random pore filling nature with ettringite elements. Qureshi and Panesar  characteKrystet et al.  studied 3) containing SP@GO and SP@FCNT with different dosages . These specimens were water curing at ages of 7, 14 and 28 days to characterize their mechanical strength. The mechanical tests were done using universal testing machine, applying load at the rate of 3.8 kN/s and 0.5 kN/s, respectively. Kaur and Kothiyal  comparedThe addition of graphene family nanomaterials in cement composites can enhance their mechanical strength. This will allow the construction of lighter concrete components with extended durability, thus, the consumption of concrete components could be decreased. This will help with the reduction of the gas pollutants resulting from concrete production.2 and CNTs can provide self-cleaning and self-sensing properties, respectively, of the products obtained with cement-based materials. To achieve the optimal mechanical properties of the cement-based materials it is very important to mix a suitable dosage of nanomaterial with the cement-based materials. For instance, excessive quantities of nanomaterials added to cement can result in lower compressive, tensile and flexural strength of the cement-based structures. This is caused by nonhomogeneous dispersion of nanomaterials in the cement paste. Thus, the mechanical properties of the nanomaterial reinforced cement-based materials depend of several factors such as the dosage and type of nanomaterial, dispersion method, curing days and curing method. Between these factors, the dispersion method can have a significantly effect on the performance of the nanomaterial reinforced concrete.The nanotechnology has allowed the fabrication of nanomaterials that can be incorporated in cement-based materials to generate higher mechanical properties of the concrete structures. The effect of the nanomaterials on the performance of cement-based materials includes the enhance of their compressive, tensile and flexural strength, reduction of the total porosity , acceleration of C–S–H gel generation and increment of Young modulus. Furthermore, incorporation of nanomaterials such as nano-TiOAn important challenge for the application of nanomaterials in the construction industry is the development of efficient methods for the dispersion of nanomaterials in cement samples. An alternative solution to incorporate the nanomaterial into cement-based materials consists in the dispersion of the nanomaterial in water before of incorporating it to the dry components of the cement-based materials . For thiTo develop the large scale production of these modified cement-based materials it will be necessary to develop an efficient nanomaterial dispersion method that allows a stable and satisfactory dispersion in cement-based materials . However2 nanoparticles may be generated with low cost from hydrothermal solutions generated due the magmatic ore intrusion [The application nanomaterials in cement-based composites is attractive due to that enhances their mechanical properties only using small dosages of nanomaterials. However, the high cost of these nanomaterials is a limitation to achieve their commercial application in cementitious materials. In the case of graphene and graphene oxide, their fabrication scale is small and relatively expensive. In the future, an important challenge is the reduction of fabrication costs of the nanomaterials. For instance, SiOntrusion . Thus, nntrusion .Moreover, another challenge is the optimal dosage determination of the nanomaterial added in the cement paste to obtain the higher mechanical strength and greater durability. To meet this goal, more studies about the effect of different types of nanomaterials on the mechanical properties of cement-based materials are required. These studies must include the effect of the combination of nanomaterials with other cementitious materials. Thus, the optimal quantities of the combination of these materials must be examined to find the best mechanical behavior of the cement-based materials. The main challenge of the use of carbon nanotubes (CNTs)/nanofibers in cement paste is the dispersion due to their strong self-attraction and high hydrophobicity ,172. Thi2 particles. Thus, the nano-TiO2 particles could absorb energy from light and employ it to achieve a photocatalytic degradation of pollutions. In 2019, Cardellicchio [2 particles in the form of anatine, which was detected by this study through chemical analysis. Nonetheless, the self-cleaning properties of the shells with nano-TiO2 are only activated when both the sunlight allows the redox of pollutant and the photo-induced hydrophilicity permits the cleaning of the shells [2 particles incorporated in concrete is affected by the porosity and roughness of the concrete surface. The porosity of the concrete surface may allow the water retention and its roughness may help the adhesion of powder on the surface. The chemical and abrasive characteristics of pozzolanic powder may decrease the efficiency of the self-cleaning of the nano-TiO2 added concrete. Therefore, future buildings that use concrete with self-cleaning properties may be affected by powder of volcanic origins or precipitations incorporating desert dust [In addition, the future applications of nanomaterials in the construction industry will require one to consider the local environmental conditions . These conditions could damage the performance of the concrete, reducing its durability and increasing the cost of maintenance. For instance, The Jubilee Church in Rome (2003) was one of the first buildings that used self-cleaning and reinforced concrete ,179,180.2 could generate inflammation in mammalian cells [More investigations about new nanomaterials to improve the mechanical properties of cement-based composites are required. In addition, to achieve the commercial application of these nanomaterials reinforced cement-based composites is necessary to know the effect of the nanomaterials on the mechanical properties of these composites. Therefore, future researches must include the development of novel theoretical models that can predict the mechanical properties of the cement-based materials as function of dosage level of the nanomaterials. Also, more researches about the impact of the nanomaterials in the public health and environment must be developed. There are few studies about the effects of the nanomaterials used in cement-based materials on the public health and environment. For instance, Lee et al.  reportedan cells . Thus, man cells .2 modified concrete can provide it self-cleaning properties and other benefits to help the environment clean. In addition, nano-TiO2 added in concrete can allow the photocatalytic degradation of pollutants  from automobile and industrial emissions. The CNTs reinforced cement-based composites can have self-sensing abilities for applications of structural health monitoring or damage detection. In addition, graphene and GO added in cement-based materials can increase their mechanical strength and durability, as well as develop self-cleaning surfaces and self-sensing abilities.The incorporation of nanomaterials in concrete can improve their compressive, tensile and flexural strength. Recent investigations had considered nanomaterials such as nano-silica, nano-titania, nano-ferric oxide, nano-alumina, CNT, graphene and GO. The addition of these nanomaterials in concrete can achieved denser microstructures, decreasing the water absorption. The workability of the concrete could be improved by adding these nanomaterials. The nano-TiO2 gas. To address this problem, one solution is the addition of nanomaterials to cement-based composites, which can provide structural components with high mechanical strength and great durability. Thus, the maintenance requirements and replacement frequency of the cement-based structural components can be decreased. These advantages can allow the reduction of the percentage of cement used in the construction industry. This in turn will decrease the CO2 emissions caused by the cement fabrication process. In the construction industry, the fabrication of cement-based composites can generate high levels of COThe application of nanotechnology in cement-based materials is still in a research stage. The results of experimental tests of nanomaterials-reinforced cement specimens have demonstrated that they can enhance the mechanical strength and durability of the resultng concretes. Moreover, these nanomaterials can allow a novel generation of smart cement-based composites with strain-sensing abilities for damage inspection and structural health monitoring.


Service-learning (SL) is a contested field of knowledge and issues of sustainability and scholarship have been raised about it. The South African Higher Education Quality Committee (HEQC) has provided policy documents to guide higher education institutions (HEIs) in the facilitation of SL institutionalisation in their academic programmes. An implementation framework was therefore needed to institutionalise the necessary epistemological shifts advocated in the national SL policy guidelines.This article is based on the findings of a doctoral thesis that aimed at developing an SL implementation framework for the School of Nursing (SoN) at the University of the Western Cape (UWC).Mixed methods were used during the first four phases of the design and development intervention research model developed by Rothman and Thomas.The SL implementation framework that was developed during Phase 3 specified the intervention elements to address the gaps that had been identified by the core findings of Phases 1 and 2. Four intervention elements were specified for the SL implementation framework. The first intervention element focused on the assessment of readiness for SL institutionalisation. The development of SL capacity and SL scholarship was regarded as the pivotal intervention element for three of the elements: the development of a contextual SL definition, an SL pedagogical model, and a monitoring and evaluation system for SL institutionalisation.The SL implementation framework satisfies the goals of SL institutionalisation, namely to develop a common language and a set of principles to guide practice, and to ensure the allocation of resources in order to facilitate the SL teaching methodology. The contextualised SL definition that was formulated for the SoN contributes to the SL operationalisation discourse at the HEI. ButinThe institutionalisation of SL in the academic programmes of South African HEIs is guided by the policy documents of the Higher Education Quality Committee HEQC :9–10 revThe latest institutional audit by the HEQC suggests that the strategic objectives of the UWC in relation to CE and SL implementation strategies need to be reviewed (CHE 2008:19). Likewise, the SoN at the UWC has had an obligation to formalise a framework for institutionalising SL in its nursing programme in order to encapsulate the mission of the UWC as an engaged institution and to facilitate the necessary epistemological amendments Julie :1832 advThe aim was to develop an SL implementation framework for the SoN at the UWC.Engagement: The partnership between the knowledge and resources of a university and the expertise of the public and private sectors enriches scholarship, research and creative activity; enhances the curriculum, teaching and learning; prepares educated, engaged citizens; strengthens democratic values and civic responsibility; addresses critical societal issues, and contributes to the public good  has been conceptualised as an engaged pedagogy that integrates theory with relevant community service projects. The SL assignments and group discussions have been designed to facilitate a more reflective approach towards greater integration of the contents of the psychiatric mental health nursing and gender-based violence modules with social responsiveness within nursing as an overarching discipline  integration of engagement into operations; (2) forging partnerships as the overarching framework; (3) renewing and redefining discovery and scholarship; (4) integrating engagement into teaching and learning; (5) recruiting and supporting new champions, and (6) creating radical institutional change  develop a common language, (2) compile a set of principles to guide practice and (3) ensure the allocation of resources to facilitate the SL teaching methodology HEQC :138. TheThe intervention study met all the prescribed ethical procedures of the UWC and received ethical clearance from the Senate Ethics Committee, project registration number 11/1/37 Julie :1836.Limited success was achieved with the building of an authentic community of practice amongst the SL teaching team during the piloting phase of the SL pedagogical model.The developed implementation framework needs to be implemented and evaluated as the next steps to complete the Design & Development Intervention Research. The SL definition that was developed for the school should be regarded as a work in progress, since it was developed before the 11 nurse educators completed the accredited SL short course in 2013. Therefore, this preliminary SL definition will be further refined by a master's degree nurse educator student who is prepared to take up the challenge.This study addresses the gap identified that most HEIs in South Africa fail to establish a standard practice for SL within the formalised systems of their respective academic programmes. The SL implementation framework of this study specifies the intervention elements (change strategies) needed to bridge the gaps that have been identified by the core findings of Phases 1 and 2. The design phase thus includes change intervention elements aimed at bridging the prevailing theory-practice gap that emanated from the conceptual confusion relating to (1) differentiating between SL and other forms of community engagement curricular activities, (2) addressing the lack of knowledge of the national SL policy guidelines by involving the academic staff and clinical supervisors in SL capacity building and SL scholarship, (3) developing an SL pedagogical model for the SoN by providing concrete implementation guidelines to embed SL pedagogy in undergraduate nursing modules that are amenable to this pedagogy , and (4) formulating SL institutionalisation criteria for the nursing programme at the SoN in accordance with the SL quality indicators of the HEQC.


To explore the feasibility of utilizing recycled aggregate concrete (RAC) in revetment construction of the lower Yellow River, a series of mix proportions with local recycled aggregates (RA) were designed to evaluate its mechanical properties and durability. The morphology and micro-hardness of the interface transition zone (ITZ) were also characterized to explain the performance of RAC. Based on the compressive strength data of 13 groups of mixtures, which is larger than 30 MPa, and with the RA substitution rate not less than 50%, the RAC containing 50% recycled fine aggregate (RFA) (HDX50), 70% RFA (HDX70), and 50% recycled coarse aggregate (RCA) (HDC50) were selected. The experiment results suggest that the mechanical performance, frost resistance, and carbonation resistance of the selected RAC is generally poorer than that of natural aggregate concrete (NAC), but can meet the performance requirement of concrete for the revetment construction of the lower Yellow River. The comprehensive performance of these three mixtures ranks as: HDX50 > HDX70 > HDC50. When considering the RA substitution ratio as a priority, HDX70 would be the best choice and can be applied in the revetment engineering. A number of defects are observed on the surface of RA with old pastes attached. Furthermore, the ITZs formed around RA are loose and with low micro-hardness, which is deemed to be the dominating reasons leading to the weaker performance of RAC than that of NAC. The Yellow River, the second largest river in China, has nurtured generations of Chinese people, but its river flood has also caused great losses to human’s lives and properties for thousands of years. In recent decades, the Yellow River conducted integrated management, and the construction of revetment was one of the most important steps ,2 becausConcrete is believed to be a promising replacement, considering that it is a cheap construction material with excellent properties, and it has been successfully used in some river management projects. The biggest problem is that the aggregates needed for concrete, such as gravels and river sands, are also in shortage, and their exploitation will cause damage to the environment like natural stone masonry. Replacing natural aggregates (NAs) with recycled aggregates (RAs) to produce recycled aggregate concrete (RAC) blocks provides an eco-friendly and cost-effective alternative, especially given that tons of waste concrete are produced every year in cities along the Yellow River due to the demolition of old buildings ,4.RAC has been studied for several decades, and many researches ,6,7,8,9 The frost resistance, carbonation resistance, chloride penetration resistance, and impermeability are often used to evaluate the durability of concrete. Generally, RAC is not recommended to use in harsh conditions  because Similar to the mechanical properties and frost resistance, the carbonation resistance of RAC usually decreases with the increase of RA dosage ,26,27. SFurthermore, according to the previous investigations ,29,30, tBased on the above, it can be found that there have been a number of studies about RAC, and almost all of them indicate that the properties of RAC are generally poorer than that of NAC. A few contradictory findings are mainly caused by the different material properties of RA, which can be further traced back to the sources and locations, mix proportions, impurities, such as red block residues and soil, and the service history. All the differences mentioned above make the microstructure of the interface transition zones (ITZ) between the RA and the new cement paste and the RA and the old mortar differ a lot and are very complicated ,38, whicRAC has been applied to many recent projects ,40, suchThe raw materials used in this study consisted of cement, NA, RA, water, and water reducer. The cement was PO 42.5 (Portland Cement), produced by Shanshui Cement Co., Ltd. , and its chemical composition is shown in RA was from the derelict buildings in Jinan city, Shandong province, and had been processed by Shandong Mingran Renewable Resources Utilization Co., Ltd. The processing was mainly about removing the steel bars and most of the red bricks. The appearance of RA is shown in Three groups of RAC were designed: single mixed with RCA, single mixed with RFA, and double mixed with RCA and RFA. Each series includes 4 mix proportions: The substitution ratio of RA being 30%, 50%, 70%, and 100% of NA by mass, respectively. The label of all mixes and the detailed information are shown in After stirring, concrete paste was poured into molds of different sizes. Specimens for compressive strength and carbonation resistance tests were cast in molds with a size of 100 mm × 100 mm × 100 mm, while specimens for flexural strength and frost resistance tests were cast in molds with a size of 100 mm × 100 mm × 400 mm. After casting, the mold surfaces were sealed with thin film to prevent moisture evaporation. Then specimens were demolded after 24 h and cured at 23 ± 1 °C, at relative humidity of 95%. Specimens for compressive strength were cured for 3 days, 7 days, 28 days, 56 days, and 90 days separately, and all other specimens were cured for 28 days.Note that the slump of all mixtures in casting falls in the range of 150~180 mm. The slump of RAC is slightly lower than that of NAC with the same dosage of water reducer. Additionally, the slump of RFA concrete approximates that of RCA concrete with different RA admixtures, attributed to the fact that the water absorptivity of the former is higher, while the actual dosage of the latter is greater. The slump of concrete with mixed RFA and RFC is a little lower. Therefore, when their dosage is higher than 50%, more water reducer is added to reach similar workability to that of concrete with only RFA or RFC, which aims to alleviate the impact of workability on the performance of different mixtures as much as possible.The compressive strength and flexural strength were tested abiding by the GB/T 50081-2002 standard for test method of mechanical properties on ordinary concrete . As intrAccording to the DG/TJ 08-2018-2007 technical code on the application of recycled aggregate concrete , the froiP is the relative dynamic modulus of specimens after n freezing and thawing cycles; nif is the dynamic modulus of specimens after n freezing and thawing cycles, Hz; oif is the original dynamic modulus of specimens before the test, Hz.niΔW is the mass loss rates of specimens after n freezing and thawing cycles, %; niW is the mass of concrete after n freezing and thawing cycles, g; oiW is the original mass of concrete before the test, g.The frost resistance test was conducted abiding by the GB/T 50082-2009 standard for test methods of long-term performance and durability of ordinary concrete . Specime2 concentration at (20 ± 1)%, relative humidity at (70 ± 5)%, and the temperature at (23 ± 2) °C. It should be noted that the distance between specimens should be larger than 50 mm to guarantee all the specimens have the same boundary conditions. The lab photo of the carbonation resistance test is presented in The carbonation resistance test was conducted abiding by the GB/T 50082-2009 standard for test methods of long-term performance and durability of ordinary concrete . Cubic sk can be calculated.x is the carbonation depth, mm; k is the carbonation rate; t is the carbonation time, d.The carbonation depth was tested with the method below. We sprayed phenolphthalein of 1% concentration on the split surface. Due to the alkaline solution of concrete before carbonation, the uncarbonated area turned to purple, whereas the carbonated area stayed unchanged. We marked 12 equally spaced test points along the boundary between the purple area and unchanged color area and measured the distance between each test point and the end surface of the unchanged color area. The carbonation depth was obtained by averaging the 12 measured distances. The carbonation time and depth was reported to follow the empirical relation described in Equation (3), by which the carbonation rate Cubic samples with side lengths smaller than 10 mm were cut out from specimens exposed to different conditions with caution to make sure that at least one of the cutting faces contained both aggregates and slurry. These cubic samples were dried at 45 °C in a vacuum drying oven for 7 days. The macro-appearance of aggregates and interface were observed with an industrial electronic microscope at magnification of 50~100 times. The microscopic morphology of ITZ was observed by a scanning electronic microscope (Quanta 250) at an accelerating voltage of 20 kV and current of 20 mA.The samples for micro-hardness measurement were prepared in the same way as mentioned in (1)Compressive StrengthMoreover, it can be seen from (2)Flexural StrengthITZ is the weak region in either NAC or RAC, which is especially worse for RAC because the interface is usually attached with old slurry see . Figure As for the inherent defects of RA, (1)Process and Characterization of DamageThe appearance changes of RAC and NAC are basically the same with the increase of freezing and thawing cycles. After 25 cycles, the appearance of specimens generally remains intact, and only a small part of RAC specimens have a few edge damages and a small number of voids. After 50 cycles, numerous voids started to appear on the concrete surface, presenting the onset of a pitted surface. The appearance of H0 and HDX50 remained roughly intact, while only a few voids appeared, whereas the surface of HDC50 and HDX70 had a number of voids, and some of them were even connected, leading to a partial pitted surface. After 75 cycles, a pitted surface appeared on the surface of all concrete specimens, and the appearance damage of HDC50 and HDX70 was more severe with large-scale paste spalling. The detailed damage information of the four mix-proportion concrete specimens is presented in (2)Mass Loss(3)Relative Elasticity ModulusCombining the experiment results of appearance, mass loss, and relative elasticity modulus of specimens, it can be found out that the frost resistance of RAC with RFA (HDX50/HDX70) was not necessarily worse than that of NAC, and its elasticity modulus was even higher. However, the frost resistance of RAC with RCA was significantly poorer than that of NAC. RA itself and ITZ were probably the major factors affecting the frost resistance of RAC.It can be observed from Based on the above, it can be drawn that the frost resistance of RAC with RFA (HDX50/HDX70) was slightly weaker than that of NAC, while that of RAC with RCA was significantly worse than that of NAC. This is consistent with the findings of Salih et al. ,22,23, w(1)Carbonation Depth2 infiltration provided by the micro-cracks and large porosity of RA. The carbonation depth of HDC50 with RCA was the highest, reaching 12.4 mm at 28 days, and that of H0 at 28 days was 7.2 mm, the former of which was 1.7 times higher than the latter, and lower than that the result (2.5 times) of Silva et al. [2 infiltration, leading to a more severe carbonation degree. Moreover, since specimens mixed with RFA possess certain inner curing capacities, the hydration degree was more adequate compared with that of specimens mixed with RCA, and the matrix of RFA was denser, which made CO2 infiltration harder. In addition, RFA contained more pastes, which can consume more CO2 compared with RCA. These two factors lead to the result that the carbonation depth of RFA concrete was lower than that of RCA concrete.The development of carbonation depth of specimens is shown in a et al. . This de(2)Carbonation Rate2 infiltration, mitigating the following carbonation reaction, and thus slowed down the carbonation rate. As is shown in 2. Moreover, the Vickers hardness of the ITZ and matrix zone around RA in HDC50 after carbonation is shown in 2 infiltration, and the weaker the carbonation degree. In addition, the carbonation rate of RAC was significantly higher than that of NAC, with HDC50 being the fastest, followed by HDX70 and HDX50, successively.According to the above analysis, it can be summarized that the carbonation resistance of RAC was weaker than that of NAC. However, the carbonation depth of RAC specimens at 28 was smaller than 20 mm, which fully met the carbonation-resistance requirement for the revetment construction of the lower Yellow River . AdditioAbove all, the chosen mix proportions  of RAC successfully met the performance requirement of concrete for the revetment construction of the lower Yellow River, in terms of compressive strength, flexural strength, frost resistance, and carbonation resistance. Therefore, the chosen RAC can be applied to the actual revetment engineering of the Yellow river. After analyzing and combining all the experiment results, the engineering application performance of the three mix proportions is ranked as: HDX50 > HDX70 > HDC50. If the engineering application performance and economic performance of material are considered, RAC HDX70 is the best and is recommended to be used in the actual revetment engineering of the lower Yellow River. Moreover, the RAC designed in this study with local raw material possesses superior mechanical properties and durability, which are as good as those in other studies. Therefore, it can be wildly applied, except for the bank revetment engineering of the Yellow River.(1)Based on the compressive strength data at 28 days, the RAC of the mix proportions HDX50, HDX70, and HDC50 were selected for compressive strength over 30 Mpa and a RA substitution rate no less than 50%.(2)Considering the combination of the appearance, mass loss, and relative elasticity modulus situations of specimens after freeze-thawing cycles, HDX50 possessed the best frost resistance performance, followed by HDX70 and HDC50, successively. Improvement on the relative elasticity modulus of HDX50/HDX70 was observed, compared to NAC.(3)In view of the carbonation depth and rate of RAC, the carbonation resistance of HDX50 was the best, followed by HDX70 and HDC50. Moreover, carbonation could densify the ITZ of RAC and increase its micro-hardness.(4)RA attached with old pastes had lots of micro-cracks and voids, and the ITZ formed around RA were loose and with low micro-hardness, which were deemed to be the dominating reasons leading to the poorer performance of RAC than that of NAC. In addition, RFA concrete had better performance than RFC concrete, since RFA possessed certain inner curing capacities.(5)The selected HDX50, HDX70, and HDC50 meet the performance requirement of concrete for the revetment construction of the lower Yellow River in terms of mechanical and durability performance. Combing performance and RA utilization rate, HDX70 ranks first and is recommended to be used in actual revetment engineering.


IgG4-related disease (IgG4-RD) is a chronic fibro-inflammatory disorder which is characterized by elevated levels of serum IgG4 and infiltration of IgG4-bearing plasma cells in the involved organs. Primary biliary cirrhosis (PBC) and Primary Sjögren's syndrome (pSS) are both distinct from IgG4-related disease. We herein describe a Chinese patient with IgG4-related RPF overlapping with PBC and pSS.We report a case of 69-year-old male with recurrent lower abdominal pain for 10 months. Laboratory data showed elevated erythrocyte sedimentation rate and hepatobiliary enzymes, renal dysfunction, high titers of antinuclear antibody, anti-SS-A antibody and anti-mitochondrial type 2, high immunoglobulin (Ig) G levels and elevated serum IgG4 (9 g/L). Contrast-enhanced computed tomography and magnetic resonance imaging were suggestive of retroperitoneal fibrosis and unilateral ureteral occlusion. Immunohistochemical staining for IgG4 did not demonstrate infiltration of IgG4-positive plasma cells in the retroperitoneal mass, but revealed significant infiltration of lymphocytoplasma cells as well as fibrosis and fibrin accumulation.The patient was diagnosed with IgG4-related retroperitoneal fibrosis based on the International Consensus Diagnostic Criteria. He was also diagnosed with primary biliary cirrhosis and primary Sjögren's syndrome.250 mg ursodeoxycholic acid was administered twice daily, and prednisolone was initiated at a dose of 40 mg/day and then tapered to 25 mg after 45 days.The size of the retroperitoneal soft tissue mass gradually reduced and the abnormal laboratory parameters were restored to normal.This rare clinical condition has seldom been reported in the literature, which suggests that common immunogenetic factors may be involved in the development of IgG-related RPF, PBC and pSS. Due to the wide availability of sensitive diagnostic methods, the estimated annual incidence of RPF has increased to 1.3/100,000 inhabitants.IgG4-related disease (IgG4-RD) is a chronic fibro-inflammatory disorder which is characterized by elevated levels of serum IgG4 and infiltration of IgG4-bearing plasma cells in the involved organs. The condition essentially affects any organ in the body, including the pancreas, kidneys, lungs, lacrimal glands, salivary glands and retroperitoneal cavity. Since Hamano first reported the complications of IgG4-associated RPF, IgG4-RD has been listed as one of the causes of RPF. Involvement of the latter is referred to as IgG4-related sclerosing cholangitis (IgG4-SC), which can present typical imaging features of a thickened bile duct wall with segmental or diffuse biliary strictures, elevated serum IgG4 levels and classic histological features. Although primary biliary cirrhosis (PBC) and primary Sjögren's syndrome (pSS) are both well-defined autoimmune diseases, they are both distinct from IgG4-RDs such as Mikulicz's disease and IgG4-SC.Salivary glands and bile ducts are frequently involved in multi-organ IgG4-RD. Involvement of the former is referred to as Mikulicz's disease, which is associated with elevated serum IgG4 levels and prominent infiltration of IgG4-positive plasmacytes.In this study, we describe a Chinese patient with IgG4-related RPF overlapping with PBC and pSS. We obtained informed consent from the patient for reporting this case. This rare clinical condition has seldom been reported in the literature.2A 69-year-old male farmer presented to our hospital for evaluation of mild left lower abdominal pain. The onset of symptoms occurred ten months prior to his admission to our hospital. The patient was previously admitted to another provincial hospital and misdiagnosed with abdominal aortic dissection. His symptoms were not improved following the administration of oral antibiotics and proton pump inhibitors.Physical examination was unremarkable except for upper right abdomen and periumbilical mild tenderness. Complete blood counts revealed mild anemia (Hb 10.2 g/dL) with normal leukocyte and platelet counts. Biochemistry showed elevated levels of γ-glutamyltransferase , alkaline phosphatase , renal dysfunction [serum creatinine ], and elevated levels of serum amylase . The erythrocyte sedimentation rate was elevated at 84 mm/h (range: 0–20 mm/h). Total serum IgG levels were extremely high , while IgG4 was approximately 6 times the normal limit . Antinuclear antibody titer was 1:320, and his anti-SS-A antibody and antimitochondrial type 2 (M2) antibody were positive. Urinalysis showed a high level of beta-microglobulin, while proteinuria, occult blood, white blood cells and casts were within normal ranges. Other blood tests, including fecal occult blood, C-reactive protein, carbohydrate antigen, carcinoembryonic antigen, and alpha-fetoprotein, were all within normal ranges. No abnormalities were found in the complement system. The T-SPOT.TB test was negative.Radiographs of the chest were unremarkable. Contrast-enhanced abdominal computed tomography (CT) revealed left hydronephrosis and a periaortic mass Fig. . The mas In addition, the patient had intrahepatic cholestasis as well as the high serum level of antimitochondrial M2, and MRCP revealed neither strictures of the lower common bile duct nor a segmental stricture. Therefore, the presence of IgG4-SC was not determined. The patient was suspected to have underlying PBC.The patient underwent biopsy of the retroperitoneal mass using an autobiopsy gun under CT-guidance for further diagnosis of RPF. Penetrating tissue specimens stained with hematoxylin and eosin revealed significant infiltration of lymphocytoplasma cells, fibrosis and fibrin accumulation Fig. A. HoweveAfter ruling out of other possibilities, the patient was finally diagnosed with IgG4-related RPF and overlapping PBC complicated with pSS. With the patient's consent, 250 mg ursodeoxycholic acid was administered twice daily and 40 mg/d (1 mg/kg) of prednisone was administered for 30 days, which was then reduced to 30 mg/d for 15 days followed by 25 mg/d. At 45 days follow-up, the patient exhibited no signs of abdominal pain. A follow-up contrast-enhanced CT scan confirmed that the retroperitoneal soft tissue lesion was markedly reduced , depends on the activity of the inflammatory process. The present case demonstrated a relatively low level of HU compared with the nonenhanced stage (60.5 vs 51.7), which indicated a late inactive inflammatory phase.IgG4-RD is a recently discovered systemic fibro-inflammatory condition characterized by the infiltration of IgG4-positive plasma cells and the formation of tumefactive lesions in the affected tissues. This disease may involve a single or multiple organs. Retroperitoneal involvement has been identified in several studies, which accounts for 20% to 56% of patients with IgG4-RD. An elevated serum IgG4 concentration has been uesed as the cut-off value for the diagnosis of IgG4-RD in the current diagnostic criteria. According to the Comprehensive Diagnostic Criteria, the upper limit of normal (ULN) for serum IgG4 is accepted to be 135 mg/dL. Previous studies have reported that the sensitivity of IgG4 for the diagnosis of IgG4-RD ranged from 82.1% to 97%, whereas the specificity ranged from 79.6% to 97%.,18–22 Moreover, recent research suggests that an elevation in serum IgG4 over 2 times the ULN contribute to distinguishing between IgG4-RD and non-IgG4-RD diagnoses, predicting multiple-organ involvement and the recurrence of the disease. High specificity is inevitably associated with lower sensitivity. Hence the optimal use of serum IgG4 concentrations as an initial diagnostic test, offers important support of the diagnosis of IgG4-RD if it is ≥2.8 g/L, as well as a significant argument against the diagnosis if it is normal. In the present case, the serum IgG4 concentration was approximately 6 times greater than 1.4 g/L, According to the current diagnostic criteria, a complete diagnosis of IgG4-RD including typical organ involvement, histologically compatible features and increased serum IgG4 level, when only typical organ involvement and elevated serum IgG4 (>135 mg/dL) are present, is defined as possible. The present case showed RPF and unilateral ureteral occlusion, with an elevated serum IgG4 despite the absence of increased IgG4-positive plasma cell infiltration. After ruling out potential diseases caused by parasitic worms, allergy or tumor, which can also lead to high levels of IgG4, we concluded that IgG4-RD was the most likely diagnosis.The lack of pathologic evidence enhanced the value of serum IgG4 in the diagnosis of IgG4-RD. As the least prevalent of the 4 IgG subclasses, IgG4 makes up to 80% of total IgG after chronic exposure to antigen. In the case of IgG4-RD, it can be rised to 50 times the upper limit.,25 It is suggested that a small subset of PBC patients may have IgG4-related RPF. However, the clinical features of overlapping IgG4-RD and PBC are unknown due to its rarity. An underlying common pathogenesis may exist in these 2 diseases. It was reported that Toll-like receptors (TLRs) and natural killer cells play an important role in PBC patients. Stimulation with TLR3 and TLR4 ligands can enhance IgG4 production by peripheral blood mononuclear cells in IgG4-RD patients. Therefore, abnormal innate immune responses against microbial antigens may contribute to the pathogenesis of both IgG4-RD and PBC.Primary biliary cirrhosis is a chronic cholestatic disease with a progressive course, which is characterized by the presence of serum antimitochondrial antibodies and histological nonsuppurative destructive cholangitis. In our patient, the MRCP findings excluded the presence of IgG4-SC, and PBC was diagnosed by elevated serum anti-M2 antibody level, which has high diagnostic sensitivity and specificity for PBC. PBC, an autoimmune liver disease, is distinct from IgG4-SC, as IgG4-SC is a typical biliary manifestation of IgG4-RD, which shows segmental strictures, long strictures with prestenotic dilatation, and strictures of the lower common bile duct. To date, a few cases of overlapping PBC and idiopathic RPF have been reported. and their salivary glands are infiltrated by a large number of IgG4+ plasma cells and IgG4-related sialadenitis symptoms respond promptly to steroids. The presence of exocrine dysfunction including positive Saxon and Schirmer tests, high serum level of immunoglobulin, positive anti-SS-A/Ro autoantibody finding and mononuclear cells infiltration in the labial salivary gland without the presence of IgG4+ plasma cells in our patient strongly suggested pSS. The coexistence of IgG4-RD and pSS has been rarely reported. Only one IgG4-RD case in 2594 pSS cases has been reported in the SICCA registry database. It is considered that the present case exhibited 2 different diseases. The identification of similar cases is required to elucidate the mechanism of IgG4-RD and pSS.Except for pancreas, salivary glands have been reported to be among the most frequent organs involved in IgG4-RD . Interestingly, IgG4-related sialadenitis shares similar clinical manifestations with an autoimmune disease, pSS, both conditions may have sicca symptoms, glandular enlargement, hypergammaglobulinemia, hypocomplementemia, and circulating antinuclear antibodies. However, they are entirely different diseases with different immunological backgrounds, research indicated that the incidence of anti-Ro and anti-La reactivity is infrequently found in patients with IgG4-related sialadenitis,In summary, we report a case of a 69-year-old Chinese man who developed IgG-related RPF, PBC and pSS, who responded promptly to prednisone therapy. The combination of these diseases suggests that common immunogenetic factors may be involved in the development of IgG-related RPF, PBC, and pSS. A nationwide survey of the overlap between these diseases is needed to elucidate the clinical features.Data curation: Meng Li, Yihong Fan.Resources: Xuan Huang.Supervision: Yihong Fan.Writing – original draft: Xuan Huang.Writing – review & editing: Bin Lu, Meng Li, Lu Zhang.


Aconitum carmichaelii Debx. It is rich in alkaloids that display a wide variety of bioactivities, and it has a strong cardiotoxicity and neurotoxicity. In order to discriminate the geographical origin and evaluate the quality of this medicine, a method based on high-performance liquid chromatography (HPLC) was developed for multicomponent quantification and chemical fingerprint analysis. The measured results of 32 batches of Fuzi from three different regions were evaluated by chemometric analysis, including similarity analysis (SA), hierarchical cluster analysis (HCA), principal component analysis (PCA), and linear discriminant analysis (LDA). The content of six representative alkaloids of Fuzi  were varied by geographical origin, and the content ratios of the benzoylmesaconine/mesaconitine and diester-type/monoester-type diterpenoid alkaloids may be potential traits for classifying the geographical origin of the medicine. In the HPLC fingerprint similarity analysis, the Fuzi from Jiangyou, Sichuan, was distinguished from the Fuzi from Butuo, Sichuan, and the Fuzi from Yunnan. Based on the HCA and PCA analyses of the content of the six representative alkaloids, all of the batches were classified into two categories, which were closely related to the plants’ geographical origins. The Fuzi samples from Jiangyou were placed into one category, while the Fuzi samples from Butuo and Yunnan were put into another category. The LDA analysis provided an efficient and satisfactory prediction model for differentiating the Fuzi samples from the above-mentioned three geographical origins. Thus, the content of the six representative alkaloids and the fingerprint similarity values were useful markers for differentiating the geographical origin of the Fuzi samples.Fuzi is a well-known traditional Chinese medicine developed from the lateral roots of  Aconitum carmichaelii Debx. (Ranunculaceae) is an herbaceous flowering plant mainly distributed in China, Korea, and Japan. The roots were first recorded as a medicine in the Chin bamboo slips of Choujiatai in the Qin Dynasty (221 B.C.–207 B.C.). Since the Han Dynasty (202 B.C.–220 A.D.), the lateral roots and the tap roots of the plant have been used separately. The lateral roots of A. carmichaelii, also called “Fuzi”, are extensively used with excellent clinical efficacy; however, the medicine does have a significant toxicity [toxicity ,4. ThereAlkaloids are the major type of active substances in Fuzi ,6. AmongA. carmichaelii is mainly distributed in the south of the Yangtze River in China [A. carmichaelii for more than 1300 years. Traditionally, Fuzi from Jiangyou is considered the best. Because of the increasing demands for the herbal medicine, Butuo County in Sichuan Province, Yulong County and Jianchuan County in Yunnan Province, and other places are cultivating A. carmichaelii. Now, the output of Fuzi in Butuo and Yunnan exceeds that in Jiangyou, which accounts for a very large proportion of the market. The geographical origin of herbal medicines is an important factor influencing the quality and price of the medicinal materials [in China . Sichuanin China . Jiangyoaterials . Few resaterials ,23,24. Saterials . HoweverIn this study, 32 batches of Fuzi were collected, including 20 batches from Sichuan Province (eight from Jiangyou and twelve from Butuo) and 12 batches from Yunnan Province (eight from Yulong and four from Jianchuan). Reversed-phase HPLC accounts for over 90% of all HPLC separations in several fields, including pharmaceutical chemistry and the analysis of the bioactive compounds of plants , so the v/v 1:1 mixed solution of isopropanol-ethyl acetate, dichloromethane, ethyl acetate, and methanol), extraction times , and ratios of material to liquid  were investigated. The results showed that the extraction using the isopropanol-ethyl acetate (v/v 1:1) mixed solution afforded the highest extraction efficiency and the most abundant chromatographic peaks. Ultimately, the best extraction method was the extraction using the isopropanol-ethyl acetate (v/v 1:1) mixed solution, with a solid–liquid ratio of 1:25 for 30 min.The extraction method was optimized so as to obtain more information about the chemical constituents of Fuzi. Different extraction solvents , AlltimaTM C18 , and Kromasil C18 , were tested. The Kromasil C18 column was the most suitable one. Different mobile phases, including the acetonitrile/tetrahydrofuran (v/v 25:15)–0.1 mol/L ammonium acetate aqueous solution, acetonitrile–0.2% acetic acid aqueous solution, methanol–0.1% diethylamine aqueous solution, acetonitrile–0.1% diethylamine aqueous solution, methanol–0.1% ammonia aqueous solution, and acetonitrile–0.1% ammonia aqueous solution, were examined. The results showed that the acetonitrile/tetrahydrofuran (v/v 25:15)–0.1 mol/L ammonium acetate aqueous solution was the most appropriate. In order to achieve a good separation of analytes, the following eluting gradients of the mobile phases  using a mixture of acetonitrile/tetrahydrofuran  and 0.1 mol/L ammonium acetate aqueous solution (B) as the mobile phase; eluting Gradient 4 , and the PC2 scores of the Fuzi from Jiangyou and the Fuzi from Butuo also showed a difference (p < 0.05). Then, the PCA scores were used as the input data for LDA in order to calculate the canonical factor. The unsupervised PCA followed by supervised LDA was found to reduce the chance of over-fitting that may occur with a pure LDA model [HCA and PCA are unsupervised methods without classification labels. LDA builds a model based on a data matrix and the known classified information . AccordiDA model . Two canAll of the above three analytical methods could be applied to discriminate the geographical origin of the Fuzi samples. Most of the samples were classified according to their geographical origin. The Fuzi samples from Jiangyou were quite different from the Fuzi samples from Butuo and Yunnan. This significant difference may be related to the planting environment and climate, planting techniques, and harvesting time. As shown in A. carmichaelii on high mountains, and then planting the plant on the plains has developed in Jiangyou. After a series of characteristic cultivation processes, like removing the top part of the plant and the redundant lateral roots, only two–three large lateral roots are retained and harvested in July [A. carmichaelii planted in this area does not undergo the above-mentioned cultivation process. In addition, because of the cold climate, the harvest period is in September [A. carmichaelii in Yunnan Province belongs to the plateau climate zone. The planting pattern is similar to that found in Butuo, and the plant is harvested in October. This process may contribute to the similarity of Fuzi samples from Butuo and Yunnan.During the plant’s long cultivation history, a traditional pattern of breeding the roots of  in July ,34. Butueptember . The culA total of 32 batches samples were collected in 2017, of which eight batches were from Planting Base of Huarun Sanjiu (Ya’an) Pharmaceutical Co., Ltd. in the Villages of Puzhao, Shuangsheng, Qiaolou, Zhulin, Yueai, and Longmen in Jiangyou County, Sichuan Province; 12 batches were from Villages Ruopu, Taijinai, and Luo’en in Butuo County, Sichuan Province; and 12 batches were from Villages Taiping and Hongyan in Yulong County and Hongxing Village in Jianchuan County, Yunnan Province. The plant identities were verified by Associate Professor Ji-Hai Gao .Mesaconitine (110799-200404) and hypaconitine (110798-200404) were purchased from the National Institute for Control of Biological and Pharmaceutical Products of China . Aconitine (MUST-17022206), benzoylaconine (MUST-15012215), benzoylhypaconine (MUST-15010806), and benzoylmesaconine (MUST-15012216) were purchased from Chengdu Must Bio-technology Co., Ltd. .Acetonitrile and methanol of HPLC grade were obtained from Sigma-Aldrich ; tetrahydrofuran, diethylamine, ammonia solution, and acetic acid of HPLC grade were purchased from Chengdu Kelong Chemicals Co., Ltd. ; and ammonium acetate was purchased from Tianjin Kermel Chemicals Co., Ltd. . HPLC-grade water was prepared with a Millipore Milli-Q system . The other reagents and chemicals of analytical grade were from Chengdu Kelong Chemicals Co., Ltd. .v/v 1:1). Final standard solutions were obtained by diluting to the gradient concentrations with the appropriate amount of isopropanol-ethyl acetate and then stored at 4 °C.The reference standards were accurately weighed and dissolved in isopropanol-ethyl acetate  was added, and the conical flask was weighed with a precision of ±0.01 g. The solution was ultrasonicated for 30 min at a temperature no more than 25 °C. After being cooled to room temperature, the isopropanol–ethyl acetate (v/v 1:1) was added to compensate for the lost weight. Thirty-five mL of the filtrate was measured and concentrated in vacuo under 40 °C. The residue was dissolved in 4 mL isopropanol–dichloromethane (v/v 1:1) and filtered through a 0.22-μm nylon filter membrane for the HPLC analysis.The lateral roots of 18 column  at 30 °C with a flow rate of 1 mL/min of the mobile phase, which consisted of acetonitrile/tetrahydrofuran (v/v 25:15 A) and the 0.1 mol/L ammonium acetate aqueous solution (B). The mobile phase used as the gradient elution of 15–16% A in 0–14 min, 16–18% A in 14–65 min, 18–20% A in 65–90 min, and 20% A in 90–95 min. The detection wavelength was kept at 235 nm and the injection volume was 5 μL.A chromatography analysis was carried out on an Agilent series 1100 HPLC liquid chromatograph with a quaternary pump, a degasser, an autosampler, a column compartment, and a UV-variable wavelength detector . The separation was performed on a Kromasil CEach concentration of the standard solutions was analyzed, and six calibration curves were constructed with the peak areas and the content of the standard in the injection solution. The precision of the instrument was tested by repeatedly injecting the same sample solutions six times. Six different solutions made from the same sample were used to evaluate the repeatability of the method. The stability was assessed by the same sample solution at 0, 5, 10, 14, 20, and 24 h. The precision, repeatability, and stability were all evaluated based on the RSDs of the peak areas. The recovery reflected the accuracy of the method, which was measured by comparing the determined content and nominal content.A SA was carried out on a similarity evaluation system for the chromatographic fingerprint of Traditional Chinese Medicine 2012 vision . The ANOVA, HCA, and LDA were analyzed using SPSS 20.0 . PCA were analyzed using SIMCA-P 13.0 . GraphPad Prism 5  was applied on the content comparison of 32 batches of Fuzi.In this study, 32 batches of Fuzi from the main producing areas in China were collected for quality evaluation. A new HPLC method was established for the quick and accurate determination of the alkaloids. The representative active diterpenoid alkaloids were measured, and the statistical analysis was performed. The content of the six alkaloids showed differences in the Fuzi samples from the three geographical origins . Notably, the content ratios of benzoylmesaconine/mesaconitine and diester-type/monoester-type diterpenoid alkaloids may be two chemical features that can be used to differentiate the origin of Fuzi. HPLC fingerprint chromatography was used to reflect the overall phytochemical characteristics of the Fuzi samples, and to evaluate their quality, HPLC fingerprint chromatography was created. The similarity analysis results were calculated with the six common characteristic peaks, and the similarity values of the 32 batches ranged from 0.637 to 0.992, depending on the region. The overall characteristics of the Fuzi samples from Jiangyou were very different from those of the Fuzi samples from Butuo and Yunnan. A further evaluation was performed using chemometric methods (PCA and HCA), and the 32 samples were classified into four groups. The Fuzi from Jiangyou was easily distinguished from the samples from other origins. LDA is a high resolution and satisfactory prediction model for discriminating the geographical origins of Fuzi. It is conjectured that the main reasons for this significant difference of the Fuzi samples may be related to the planting environment, climate, and techniques. In the future, more Fuzi growing regions should be investigated, and molecular biology and epigenetic methods should be used to evaluate the relationship between the medicinal materials and geographical origins.