Activity-based protein profiling technology reveals malate dehydrogenase as the target protein of cinnamaldehyde against Aspergillus niger.

Cinnamaldehyde displays strong antifungal activity against fungi such as Aspergillus niger, but its precise molecular mechanisms of antifungal action remain inadequately understood. In this investigation, we applied chemoproteomics and bioinformatic analysis to unveil the target proteins of cinnamaldehyde in Aspergillus niger cells. Additionally, our study encompassed the examination of cinnamaldehyde's effects on cell membranes, mitochondrial malate dehydrogenase activity, and intracellular ATP levels in Aspergillus niger cells. Our findings suggest that malate dehydrogenase could potentially serve as an inhibitory target of cinnamaldehyde in Aspergillus niger cells. By disrupting the activity of malate dehydrogenase, cinnamaldehyde interferes with the mitochondrial tricarboxylic acid (TCA) cycle, leading to a significant decrease in intracellular ATP levels. Following treatment with cinnamaldehyde at a concentration of 1 MIC, the inhibition rate of MDH activity was 74.90 %, accompanied by an 84.5 % decrease in intracellular ATP content. Furthermore, cinnamaldehyde disrupts cell membrane integrity, resulting in the release of cellular contents and subsequent cell demise. This study endeavors to unveil the molecular-level antifungal mechanism of cinnamaldehyde via a chemoproteomics approach, thereby offering valuable insights for further development and utilization of cinnamaldehyde in preventing and mitigating food spoilage.

The relationship between the microclimate and efflorescence of revealed mural paintings and the later protection strategy.

Throughout history, there were many fine mural paintings concealed within ancient buildings, hidden beneath layers of plaster, wall or other structure. In recent decades, research and practical efforts had primarily focused on nondestructive diagnosis of these hidden murals and the removal of their surface coverings. However, limited attention had been given to the consequences of overlay removal on mural preservation. This study aims to address this gap by examining the revealed mural paintings in the Prince Shi's Palace as a case study, employing an analysis of mural ontology, investigation into the preservation environment, and simulation experiments to comprehensively analyze the factors contributing to the rapid deterioration of these revealed murals. The findings indicated that certain carbonated lime particles adhered to the mural surface, and in a humid environment, these particles absorbed sulfur dioxide from the atmosphere and converted into calcium sulfate. The strong hygroscopic property of calcium sulfate enhanced the humidity on the surface of murals, thereby increasing their susceptibility to sulfur dioxide absorption. Over time, the murals suffered more sulphate and moisture damage. Additionally, historically inaccurate interventions such as non-breathable shading curtains installation and organic coatings reinforcement exacerbated this deterioration process. Therefore, safeguarding the integrity of these exposed concealed murals necessitated prioritizing the preservation of environmental humidity and acid gas levels.

A portable microfluidic device integrated with electrochemical sensing platform for detection of multiple binders in ancient wall paintings.

The aging of the proteinaceous binders will cause the cultural relics to suffer from diseases such as flaking, cracks, and even peeling. Identifying the type of binders in a timely manner is conducive to restore diseased cultural relics. High-throughput and portable detection system are of great significance for researching cultural relic materials on the archaeological site. Therefore, in this work, a portable electrochemical microfluidic device for the simultaneous detection of casein, ovalbumin, and peach gum binders was developed. The proposed electrochemical immunosensor technology integrated with microfluidic device achieve the goals of miniaturization, portability and reagent-saving. For casein, ovalbumin and peach gum, excellent performance was obtained in terms of their limits of detection (LOD) at 0.237, 0.507, and 0.403 ng mL-1 (S/N = 3), respectively. In addition, the microfluidic sensing platform exhibited acceptable anti-interference ability, stability, and storage capacity. In order to evaluate the practical application value, the proposed microfluidic sensing device was applied for detecting eight archaeological samples from different historic sites. This work demonstrates great potential for high-throughput, portable detection of cultural relic proteinaceous binder materials.

Microbiome Diversity and Cellulose Decomposition Processes by Microorganisms on the Ancient Wooden Seawall of Qiantang River of Hangzhou, China.

Archaeological wood, also known as wooden cultural relics, refers to ancient wood that has been worked by humans. Further insights into the decomposition mechanism of archaeological wood are needed for its preventive conservation. In this study, we assessed the microbiome diversity and cellulose decomposition processes on a 200-year-old ancient wooden seawall - the Qiantang River of Hangzhou, China. We used high-throughput sequencing (HTS) to deduce the metagenomic functions, particularly the cellulose-decomposing pathway of the microbial communities, through bioinformatical approaches. The predominant cellulose-decomposing microorganisms were then verified with traditional isolation, culture, and identification method. The results showed that the excavation of archaeological wood significantly altered the environment, accelerating the deterioration process of the archaeological wood through the carbohydrate metabolism and the xenobiotic biodegradation and metabolism pathways, under the comprehensive metabolism of complex ecosystem formed by bacteria, archaea, fungi, microfauna, plants, and algae. Bacteroidetes, Proteobacteria, Firmicutes, and Actinobacteria were found to be the predominant source of bacterial cellulose-decomposing enzymes. Accordingly, we suggest relocating the wooden seawall to an indoor environment with controllable conditions to better preserve it. In addition, these results provide further evidence for our viewpoints that HTS techniques, combined with rational bioinformatical data interpretation approaches, can serve as powerful tools for the preventive protection of cultural heritage.

Dynamics of microbial community composition during degradation of silks in burial environment.

The silk residues in the soil formed the unique niche, termed "silksphere." Here, we proposed a hypothesis that silksphere microbiota have great potential as a biomarker for unraveling the degradation of the ancient silk textiles with great archaeological and conservation values. To test our hypothesis, in this study, we monitored the dynamics of microbial community composition during silk degradation via both indoor soil microcosmos model and outdoor environment with amplicon sequencing against 16S and ITS gene. Microbial community divergence was evaluated with Welch two sample t-test, PCoA, negative binomial generalized log-linear model and clustering, etc. Community assembly mechanisms differences between silksphere and bulk soil microbiota were compared with dissimilarity-overlap curve (DOC) model, Neutral model and Null model. A well-established machine learning algorithm, random forest, was also applied to the screening of potential biomarkers of silk degradation. The results illustrated the ecological and microbial variability during the microbial degradation of silk. Vast majority of microbes populating the silksphere microbiota strongly diverged from those in bulk soil. Certain microbial flora can serve as an indicator of silk degradation, which would lead to a novel perspective to perform identification of archaeological silk residues in the field. To sum up, this study provides a new perspective to perform the identification of archaeological silk residue through the dynamics of microbial communities.

Fabrication of a novel electrochemical immunosensor for the sensitive detection of carcinoembryonic antigen using a double signal attenuation strategy.

Carcinoembryonic antigen (CEA), an acidic protein, is a characteristic antigen produced by the tumor of various cancers (eg, breast, cervical, rectal, lung, etc.). Therefore, the detection of cancer antigens is very important for the early diagnosis and treatment of cancer. In this study, a novel of "signal off" strategy for electrochemical immunosensor was developed to detect CEA. To this end, Prussian blue nanoparticles (PB NPs), an electroactive substance, were used as the immunological platform. In addition, CuO2@SiO2 nanocomposites, which release Cu2+ and H2O2 under acidic conditions, were synthesized. The generated Cu2+ can replace the high spin iron (FeIII) in PB NPs, which in turn reduces the oxidation peak current of PB NPs. Due to the peroxidase-like nature of PB NPs, they can react with self-generated H2O2 to generate hydroxyl radicals (·OH), which can further convert 4-chloro-1 naphthol (4-CN) into a non-conductive polymer that accumulates on the electrode surface, this leads to a further reduction in the electrical signal of the PB NPs. Moreover, the self-generated Cu2+ and H2O2 can reduce the introduction of exogenous substances and improve the detection accuracy. Square wave voltammetry (SWV) revealed that the electrical signal of PB NPs gradually decreased with increasing CEA concentration. In addition, the electrical signal of PB NPs exhibited a good linearity in the range from 0.01 pg mL-1 to 80 ng mL-1, where in the logarithm of CEA concentration and the detection limit was as low as 0.0032 pg mL-1.

Phosphonium-based ionic liquids as antifungal agents for conservation of heritage sandstone.

With a view to preventing fungal deterioration of historical stone artworks, we report the use of phosphonium-based ionic liquids (ILs) as potent antifungal agents against dematiaceous fungi commonly found on heritage stones. Three ILs: tributyldodecylphosphonium polyoxometalate [P44412][POM], tributyltetradecylphosphonium polyoxometalate [P44414][POM], and trihexyltetradecylphosphonium polyoxometalate [P66614][POM] were prepared and their thermal stabilities and in vitro antifungal activities were evaluated. From the ramped temperature thermogravimetric analysis and antifungal experiments it can be clearly observed that the alkyl chain length of the tetraalkylphosponium cation has a significant influence on the thermal and antifungal properties. The thermal stability and antifungal activity decreased as the number of carbon atoms of the alkyl substituents increased and, thus, followed the order [P44412][POM] > [P44414][POM] > [P66614][POM]. In addition, inoculation of four fungal species on IL-coated sandstone surfaces showed significant inhibition of fungal growth, endowing the materials with potential applications in heritage sandstone conservation.

An electrochemical immunosensor for the detection of carcinoembryonic antigen based on Au/g-C3N4 NSs-modified electrode and CuCo/CNC as signal tag.

Carcinoembryonic antigen levels in the human body reflect the conditions associated with a variety of tumors and can be used for the identification, development, monitoring, and prognosis of lung cancer, colorectal cancer, and breast cancer. In this study, an amperometric immunosensor with CuCo/carbon nanocubes (CuCo/CNC) as the signal label is constructed. The bimetal-doped carbon skeleton structure has a high specific surface area and exhibits good electrocatalytic activity. In addition, Au/g-C3N4 nanosheets (Au/g-C3N4 NSs) are used to modify the substrate platform, facilitating the loading of more capture antibodies. The reaction mechanism was explored through electrochemical methods, X-ray powder diffraction, X-ray photoelectron spectroscopy, and other methods. Kinetic studies have shown that CuCo/CNC have good peroxidase-like activity. In addition, the electrocatalytic reduction ability of CuCo/CNC on hydrogen peroxide can be monitored using amperometric i-t curve (- 0.2 V, vs. SCE), and the response current value is positively correlated with the CEA antigen concentration. The prepared electrochemical immunosensor has good selectivity, precision, and stability. The dynamic range of the sensor was 0.0001-80 ng/mL, and the detection limit was 0.031 pg/mL. In addition, the recovery and relative standard deviation in real serum samples were 97.7-103 % and 3.25-4.13 %, respectively. The results show that the sensor has good analytical capabilities and can provide a new method for the clinical monitoring of CEA.

The electrochemical immunosensor of the "signal on" strategy that activates MMoO4 (M = Co, Ni) peroxidase with Cu2+ to achieve ultrasensitive detection of CEA.

A new type of ultrasensitive electrochemical immunosensor with "signal on" strategy was designed for quantitative detection of CEA. The sensing strategy design is based on the following principles: We use HMSNs-Cu2+@HA as the signal probe, the structure of HA is destroyed under acidic conditions, and the released Cu2+ activates the substrate material MMoO4 (M = Co, Ni) Peroxidase activity initiates the reaction of catalytic H2O2 and realizes the "signal on" condition of electrical signals. This strategy has the following advantages: (1) HA coating of HMSNs-Cu2+ can prevent Cu2+ leakage, has good biocompatibility and can be connected with more antibodies. (2) The prepared sensor has the characteristics of high sensitivity and a low detection limit. When the electrode substrate was CoMoO4, the detection range of the immunosensor was 0.01 pg/mL-40 ng/mL, and the detection limit was 0.0035 pg/mL (S/N = 3). This work innovatively applies the catalytic activity of metal ion-activated nanozymes in the detection of CEA, providing a new perspective for the monitoring and analysis of cancer markers.

Polyoxometalate-Ionic Liquids (ILs) and Polyvinyl Alcohol/Chitosan/ILs Hydrogels for Inhibiting Bacteria Colonising Wall Paintings.

Historical monuments are increasingly being threatened by unexpected microbial colonizers, leading to their subsequent deterioration. Here, two tetraalkylphosphonium polyoxometalate ionic liquids (Q14-IL and Q16-IL) were successfully synthesized, which showed excellent antibacterial activity against four bacteria colonising wall paintings. Notably, Q14-IL exhibited superior antibacterial efficacy compared to longer alkyl Q16-IL. Additionally, polyvinyl alcohol/chitosan (PVA-CS) hydrogels containing two ILs were prepared, and the morphology, thermal stability, swelling ratio and antibacterial activity were systematically evaluated. The results suggest that higher CS content resulted in more uniform micropores and increased the swelling ratio. However, fewer antibacterial ILs were released and diffused over time from the matrix. Hydrogels with 5% CS content exhibited the highest antibacterial activity, which was mainly attributed to the synergetic antibacterial activity of positively charged ammonium (-NH3+) groups of CS and quaternary phosphonium cation of ILs. This study may provide an alternative strategy for fighting against bacterial communities colonising ancient artworks.

Hydrophobicity of Polyacrylate Emulsion Film Enhanced by Introduction of Nano-SiO2 and Fluorine.

This study proposes to utilize modified Nano-SiO2/fluorinated polyacrylate emulsion that was synthesized with a semi-continuous starved seed emulsion polymerization to improve the hydrophobicity, thermal stability, and UV-Vis absorption of polyacrylate emulsion film. To verify the proposed method, a series inspection had been conducted to investigate the features of the emulsion film. The morphological analysis indicated that Nano-SiO2 was surrounded by a silane molecule after modification, which can efficiently prevent silica nanoparticles from aggregating. Fourier transform infrared spectra confirmed that modified SiO2 and dodecafluoroheptyl methacrylate (DFMA) were successfully introduced to the copolymer latex. The particle size of latex increased with the introduction of modified Nano-SiO2 and DFMA. UV-Vis absorption spectra revealed that modified silicon nanoparticles can improve the ultraviolet shielding effect obviously. X-ray photoelectron spectroscopy illustrated that the film⁻air interface was richer in fluorine than film section and the glass side. The contact angle of modified Nano-SiO2/fluorinated polyacrylate emulsion containing 3 wt % DFMA was 112°, slightly lower than double that of polyacrylate emulsion, indicating composite emulsion films possess better hydrophobicity. These results suggest that introducing modified Nano-SiO2 and fluorine into polyacrylate emulsion can significantly enhance the thermal stability of emulsion films.

Immunological Methods for the Detection of Binders in Ancient Tibetan Murals.

Tibetan mural samples from the Jiazhaer mountain cave were studied using enzyme-linked immunosorbent assays (ELISA) and immunofluorescence microscopy (IFM). Samples containing protein binders were first identified using ELISA, and then IFM was used to determine the location of protein binders. Using these methods, we discovered gelatin and casein in samples from wall murals, distributed in both red and black pigments. We excluded the possibility of contamination by conducting further experiments where simulated samples were spiked with milk. We conclude that both gelatin and casein were used as binders in the pigments of the Tibetan Buddhist murals in the Jiazhaer (Transliteration from Tibetan) mountain cave. This is the first evidence of casein being used as a binder in Chinese mural pigments.

An Adaptive Prediction Target Search Algorithm for Multi-AUVs in an Unknown 3D Environment.

For a target search of autonomous underwater vehicles (AUVs) in a completely unknown three-dimensional (3D) underwater environment, a multi-AUV collaborative target search algorithm based on adaptive prediction is proposed in this paper. The environmental information sensed by the forward-looking sonar is used to judge the current state of view, and the AUV system uses this environmental information to perform the target search task. If there is no target in the field of view, the AUV system will judge whether all sub-regions of the current layer have been searched or not. The next sub-region for searching is determined by the evaluation function and the task assignment strategy. If there are targets in the field of view, the evaluation function and the estimation function of the adaptive predictive optimization algorithm is used to estimate the location of the unknown target. At the same time, the algorithm also can reduce the positioning error caused by the noise of the sonar sensor. In this paper, the simulation results show that the proposed algorithm can not only deal with static targets and random dynamic interference target search tasks, but it can also perform target search tasks under some random AUV failure conditions. In this process, the underwater communication limits are also considered. Finally, simulation experiments indicate the high efficiency and great adaptability of the proposed algorithm.

Biomineralization Induced by Colletotrichum acutatum: A Potential Strategy for Cultural Relic Bioprotection.

Colletotrichum acutatum is a fungus capable of biomineralization reported in our previous study. In this paper, we compared the ability of this fungus to induce mineralization under different calcium sources, pH levels, and differing carbon availability. Here we found that organic acids, the alkalinity of the environment, and low carbon conditions were major factors influencing calcium carbonate precipitation. High performance liquid chromatography showed that citric acid was a metabolite produced by C. acutatum, and that other organic acids including formic, propionic, α-ketoglutaric, lactic, and succinic acids can be used by this fungus to promote CaCO3 formation. Based on these findings, the mechanism of the biomineralization induced by C. acutatum should be divided into three processes: secreting organic acid to dissolve limestone, utilizing the acid to increase the alkalinity of the microenvironment, and chelating free calcium ions with extracellular polymeric substances or the cell surface to provide a nucleation site. Interestingly, we found that hydroxyapatite rather than calcium carbonate could be produced by this fungus in the presence of phosphate. We also found that the presence of acetic acid could inhibit the transformation of vaterite to calcite. Further, we evaluated whether the proliferation of C. acutatum could influence the deterioration of stone relics. We found that low carbon conditions protected calcium carbonate from dissolution, indicating that the risk of degradation of limestone substrates caused by C. acutatum could be controlled if the fungi were used to consolidate or restore stone monuments. These results suggest that C. acutatum-induced biomineralization may be a useful treatment for deteriorated stone relics.

Role of Fungi in the Formation of Patinas on Feilaifeng Limestone, China.

Feilaifeng is a cultural heritage site that contains unique Buddhist statues which date back to the Five Dynasties period (907 AD-960 AD). The site was inscribed on world heritage list by UNESCO in 2011. Various patinas, which may be caused by fungi, have covered the surface of the limestone and have severely diminished the esthetic value of the statues and altered the limestone structure. Culture-dependent method was used to isolate and identify the fungi. After incubation on modified B4 medium, the calcifying fungi were identified by optical microscopy and scanning electron microscopy combined with X-ray energy-dispersive analysis. Aspergillus, Penicillium, and Colletotrichum were observed as the biomineralizing fungi. X-ray diffraction showed that the patina consisted of calcite (CaCO3), but the crystals synthesized by the identified fungi were whewellite (CaC2O4·H2O) for Aspergillus and Penicillium, and vaterite (CaCO3) for Colletotrichum. In addition, the metabolites of Colletotrichum suppressed the transformation of vaterite to calcite, but Mg2+ could inhibit the function of the metabolites. The different crystal form between the patina and the products of fungi may suggest two different pathways of patina formation and provide important reference data for studies of the mechanisms of biomineralization, cleaning of the patina, and protection of the Feilaifeng statues.

Deterioration-Associated Microbiome of Stone Monuments: Structure, Variation, and Assembly.

Research on the microbial communities that colonize stone monuments may provide a new understanding of stone biodeterioration and microbe-induced carbonate precipitation. This work investigated the seasonal variation of microbial communities in 2016 and 2017, as well as its effects on stone monuments. We determined the bacterial and fungal compositions of 12 samples from four well-separated geographic locations by using 16S rRNA and internal transcribed spacer gene amplicon sequencing. Cyanobacteria and Ascomycota were the predominant bacterial and fungal phyla, respectively, and differences in species abundance among our 12 samples and 2 years showed no consistent temporal or spatial trends. Alpha diversity, estimated by Shannon and Simpson indices, revealed that an increase or decrease in bacterial diversity corresponded to a decrease or increase in the fungal community from 2016 to 2017. Large-scale association analysis identified potential bacteria and fungi correlated with stone deterioration. Functional prediction revealed specific pathways and microbiota associated with stone deterioration. Moreover, a culture-dependent technique was used to identify microbial isolates involved in biodeterioration and carbonatogenesis; 64% of 85 bacterial isolates caused precipitation of carbonates in biomineralization assays. Imaging techniques including scanning electron microscopy with energy-dispersive spectroscopy, X-ray diffraction, and fluorescence imaging identified CaCO3 crystals as calcite and vaterite. Although CaCO3 precipitation induced by bacteria often has esthetically deleterious impacts on stone monuments, this process may potentially serve as a novel, environmentally friendly bacterial self-inoculation approach to the conservation of stone.IMPORTANCE Comprehensive analyses of the microbiomes associated with the deterioration of stone monuments may contribute to the understanding of mechanisms of deterioration, as well as to the identification of potentially beneficial or undesirable microbial communities and their genomic pathways. In our study, we demonstrated that Cyanobacteria was the predominant bacterial phylum and exhibited an increase from 2016 to 2017, while Proteobacteria showed a decreasing trend. Apart from esthetic deterioration caused by cyanobacteria and fungi, white plaque, which is composed mainly of CaCO3 and is probably induced by Crossiella and Cyanobacteria, was also considered to be another threat to stone monuments. We showed that there was no significant correlation between microbial population variation and geographic location. Specific functional genes and pathways were also enriched in particular bacterial species. The CaCO3 precipitation induced by an indigenous community of carbonatogenic bacteria also provides a self-inoculation approach for the conservation of stone.

Distribution and Diversity of Bacteria and Fungi Colonization in Stone Monuments Analyzed by High-Throughput Sequencing.

The historical and cultural heritage of Qingxing palace and Lingyin and Kaihua temple, located in Hangzhou of China, include a large number of exquisite Buddhist statues and ancient stone sculptures which date back to the Northern Song (960-1219 A.D.) and Qing dynasties (1636-1912 A.D.) and are considered to be some of the best examples of ancient stone sculpting techniques. They were added to the World Heritage List in 2011 because of their unique craftsmanship and importance to the study of ancient Chinese Buddhist culture. However, biodeterioration of the surface of the ancient Buddhist statues and white marble pillars not only severely impairs their aesthetic value but also alters their material structure and thermo-hygric properties. In this study, high-throughput sequencing was utilized to identify the microbial communities colonizing the stone monuments. The diversity and distribution of the microbial communities in six samples collected from three different environmental conditions with signs of deterioration were analyzed by means of bioinformatics software and diversity indices. In addition, the impact of environmental factors, including temperature, light intensity, air humidity, and the concentration of NO2 and SO2, on the microbial communities' diversity and distribution was evaluated. The results indicate that the presence of predominantly phototrophic microorganisms was correlated with light and humidity, while nitrifying bacteria and Thiobacillus were associated with NO2 and SO2 from air pollution.

Effects of electric field on micro-scale flame properties of biobutanol fuel.

With the increasing need of smaller power sources for satellites, energy systems and engine equipment, microcombustion pose a potential as alternative power source to conventional batteries. As the substitute fuel source for gasoline, biobutanol shows more promising characteristics than ethanol. In this study, the diffusion microflame of liquid biobutanol under electric field have been examined through in-lab experiment and numerical simulation. It is found that traditional gas jet diffusion flame theory shows significant inconsistency with the experimental results of micro scale flame in electric field. The results suggest that with the increase of electric field intensity, the quenching flow rate decrease first and increase after it reach its minimum, while the flame height and highest flame temperature increase first and drop after its peak value. In addition, it was also observed that the flame height and highest temperature for smaller tube can reach its maximum faster. Therefore, the interaction between microscale effect and electric field plays a significant role on understanding the microcombustion of liquid fuel. Therefore, FLUENT simulation was adopted to understand and measure the impacts of microflame characteristic parameters. The final numerical results are consistent with the experimental data and show a high reliability.

Microchemical Study of Pigments and Binders in Polychrome Relics from Maiji Mountain Grottoes in Northwestern China.

In this study, an integrated analytical method was developed to investigate the composition of both the inorganic pigments and organic binders of polychrome relics in Maiji Mountain Grottoes in northwestern China. Cross-sections of each sample were prepared at the beginning of the study, and all experiments were carried out on these cross-sections. Polychromic structures were revealed by optical microscopy and scanning electron microscopy-backscattered electron imaging. Inorganic materials were determined by using SEM coupled with an energy dispersive spectrometer and µ-Raman spectrometer, whereas organic materials were identified by staining techniques and highly sensitive and specific immunofluorescence microscopy. Data showed that the red colors are attributed to one or two pigments of red ochre, cinnabar, and minium; the blue pigment is natural lazurite; the green pigment is ascribed to atacamite; the white color is attributed to potassium feldspar; and the black surface is formed by the discoloration of minium to plattnerite under the influence of environmental factors. Regarding organic binders used in painting and preparation layers, mammalian animal glue and chicken egg white were both found alone or in mixture. Finally, the conclusion is made that the Secco technique is employed in polychrome relics from Maiji Mountain Grottoes.

[The Analysis of Traditional Lime Mortars from Zhejiang Province, China].

The components of ancient mortars have always been an important research field in historic building conservation. It has been well known that using traditional mortars in conservation projects have many advantages, such as compatibility and stability. So, developing new binding materials based on traditional mortar has become an international study hotspot. With China's economic development, the protection of ancient buildings also began to put on the agenda, but the understanding on Chinese traditional mortar is limited, and rare literatures are reported. In the present work, the authors investigate seven ancient city wall sites in Zhejiang Province in situ, and subsequently laboratory analysis were carried out on collected mortar samples. The characterizations of mortar samples were made by multi-density gauge, XRD, FTIR, TG-DSC and wet chemical analysis. The experimental results showed that: the main component of masonry mortars is calcium carbonate, the content between 75% - 90%, and they should be made from relatively pure lime mortar. The raw materials of mortar samples were mainly calcareous quick lime, and sample from Taizhou city also contained magnesium quick lime. There are four city walls were built by sticky-rice mortars. It suggests that the technology of adding the sticky rice soup into mortar was universal in the Ming Dynasties. These mortars have lower density between 1.2 and 1.9 g x cm(-3); this outcome should be the result of long-term natural erosion. We have also analyzed other chemical and physical characteristics of these masonry mortars. The results can afford the basic data for the future repairmen programs, development of new protective materials, and comparative study of mortars.