Biodegradative properties of the tested microfungi were qualitatively characterized through the use of a set of special agar plates: CaCO3 glucose agar , casein nutrient agar (casein hydrolysis), Czapek-Dox minimal medium (pigment secretion); and Czapek-Dox minimal broth . Most of the tested isolates (71.05%) demonstrated at least one of the degradative properties, with Penicillium bilaiae as the most potent, since it tested positive in all four. The remaining isolates (28.95%) showed no deterioration capabilities and were hence considered unlikely to partake in the complex process of fungal deterioration of murals via the tested mechanisms. The obtained results clearly indicate that utilization of fast and simple plate assays can provide insight into the biodegradative potential of deteriogenic fungi and allow for their separation from allochthonous transients, a prerequisite for precise assessment of the amount of risk posed by a thriving mycobiota to mural paintings.The principal purpose of the study was to evaluate  For example, al fresco wall paintings were prepared with variously coloured mineral powders mixed with lime or pure water and applied to fresh lime plaster on the wall. However, in the case of tempera, paints were made by mixing mineral powders with some natural binders, substances such as oils from seeds , casein, egg yolk, starch, etc., and applied to already formed dry mortar on the wall [aw values are able to degrade all the organic and inorganic components artworks are made of, usually resulting in aesthetically unacceptable changes [All works of art are susceptible to microbially induced decay. From primitive decorations made out of mollusc shells around 75.000 years BC through paintings in caves of the Upper Palaeolithic and works of Gothic and Renaissance art to contemporary art of the 21the wall , are conthe wall . Cosmopo changes , 9, 10.  changes .in vitro the deteriorative potential of fungal isolates initially obtained from painted layers of the wall paintings and surrounding air of the old Church of the Holy Ascension in Veliki Krčimir (Serbia), either via cultivation on a set of unique nutrient media that emulate inorganic (limestone) and organic (casein) components of wall paintings or by testing the ability of isolates to secrete metabolites  with negative effects on the structural and/or aesthetic integrity of mural paintings. The action of “harmful” metabolites and secretion of organic acids that cause considerable acidification of the substrate are among the primary mechanisms through which fungi are involved in the biodeterioration of objects comprising our cultural heritage. The type of produced acid is regulated by available carbon sources and is often dictated by the presence of metal ions in the pigments used [al secco technique [al secco wall paintings, while peptide degradation can lead to pH increase and secondary CaCO3 precipitation [The principal purpose of the present research was to evaluate nts used . Adversents used . Furthernts used , 14. Thints used . The selechnique . Casein echnique . Furtherechnique . In Serbechnique . This ispitation , 20. Finpitation , 12, 21.pitation , 22. Forpitation .Seeing as how the ability of fungi to degrade organic and inorganic components of wall paintings and produce and secrete various metabolites is clearly troublesome, a knowledge of their potential ability to induce symptoms of deterioration, gained through utilization of fast and simple plate assays will allow recognition of deteriogenic fungi, thereby enabling a precise assessment of risk to be made and appropriate conservation treatments to be formulated and implemented.in situ optical and scanning electron microscopy [All 38 micromycetes screened for their biodegradative potentials are original isolates obtained from deteriorated frescoes  and surrcroscopy , 26. Likcroscopy , resulti2 surface of the hemocytometer used for this purpose . The suspensions were adjusted to a final concentration of approximately 1.0 × 105 CFU ml-1 with sterile saline solution, and stored at −20°C. Prior to the experiments, conidial suspensions were cultured on solid MEA to check validity of the inocula and verify the absence of contamination.Conidial suspensions of the tested isolates were prepared by washing conidia from the surface of 7 day old malt extract agar (MEA) slants using sterile saline solution  with 0.1% Tween 20 (Sinex Laboratory). Concentrations of conidia in the suspensions were calculated using the formula given in the manufacturer's guidelines :All biodegradative plate assays were performed in triplicate.3 glucose agar plates of the following composition, per litre of deionized water: calcium carbonate, 5 g; glucose, 10 g; agar, 15 g; and deionized water, 1000 ml. The prepared medium was sterilized at 121°C for 15 min, after which pH was adjusted to 8.0 with 10 N HCl and the mixture was cooled to 45°C with gentle stirring to resuspend CaCO3. After pouring into Petri plates, the plates were then kept in a cool place. Petri plates with agarized CaCO3 glucose agar were inoculated with 10 μl of fungal suspensions and incubated for 21 days at 25 ± 2°C . Positive strains displayed a clear zone around the colony, thus confirming calcite dissolution [The potential ability of the tested microfungi to solubilize calcite was screened using CaCOsolution .To determine the capacity of fungi to affect the pH value of the substrate on which they grow, isolates were cultivated in a liquid minimal medium of the following composition, per litre of deionized water: sodium nitrate, 3 g; dipotassium phosphate, 1 g; magnesium sulphate heptahydrate, 0.5 g; potassium chloride, 0.5 g; iron (II) sulphate heptahydrate, 0.01 g; glucose, 10 g; and deionized water, 1000 ml . TitratiThe proteolytic activity of fungal isolates was determined by cultivation on casein agar (CN) plates of the following composition, per litre of deionized water: sodium chloride, 5 g; peptone, 5 g; yeast extract, 3 g; agar, 15 g; and deionized water, 1000 ml. Skimmed milk (250 ml), fractionally sterilized daily (for 3 days) at 100°C for 30 min, was added to 750 ml of warmed sterilized medium , and homogeneously mixed: after cooling to approximately 45°C, the medium was then poured into Petri plates . The cenThe potential ability of the tested microfungal isolates to produce and secrete organic pigments under nutrient-limited conditions , and consequently to induce alterations in the original colouration of the painted layer, was assayed by cultivation on Czapek-Dox minimal medium of the following composition, per litre of deionized water: sodium nitrate, 2 g; dipotassium phosphate, 1 g; magnesium sulphate heptahydrate, 0.5 g; potassium chloride, 0.5 g; iron (II) sulphate heptahydrate, 0.01 g; glucose, 10 g; agar, 20 g; and deionized water, 1000 ml . The medPenicillium bilaiae testing positive in all four. The remaining 11 fungal isolates (28.95%) showed no deterioration capabilities and hence were considered not to partake in the complex process of fungal-induced deterioration of wall paintings via the tested mechanisms. These fungi likely induce deterioration through mechanical stresses created by hyphal penetration into the painted layers and mortar, or by means of other deterioration mechanisms, i.e., enzymatic degradation, biomineralization, etc.The deterioration potential of fungal isolates obtained from the sacral ambient is summarized in Aspergillus europaeus, A. niger and Penicillium lanosum) and four from the surrounding air . Among the tested Aspergillus species, only 18.18% (2/11) had a transparent zone in the culture, while in the genus Penicillium, solubilization ability was observed for the majority of tested isolates   isolates, three of which were obtained from wall paintings  . In all 3%; 5/7) , most noA. niger (2.81 ± 0.47) and P. bilaiae (2.94 ± 0.33), followed by P. griseofulvum, with pH 5.62 ± 0.23. Growth and metabolism of only two species, Gibberella moniliformis and Epicoccum nigrum, caused an increase in alkalinity of the medium, with measured values in the pH range of 8.31 ± 0.17 to 8.47 ± 0.21. In the remaining 33 isolates (86.84%), only moderate changes of substrate pH values, in the range of one pH unit, were documented   were ablcumented .Aspergillus    . Proteolytic activity was also detected in cultures of Bjerkandera adusta and Phaeospheria avenaria. The greatest diameters of the transparent zone were measured for Cladosporium species, with C. cladosporioides showing the largest zone, indicating the strongest proteolytic potential. Furthermore, in a culture of C. cladosporioides cultivated for one week on CN medium, morphological alteration in the guise of a partial change in colour of the colony, from dull green to shades of yellow and beige, was also observed  , mainly %; 7/11)  and to a7%; 2/7) , Cladosp0%; 3/3) , and Chaobserved .Aspergillus  , while in isolates of other fungal genera, such as Gibberella   and Penibberella , pigment bilaiae  were themanginii , the prom nigrum . ChaetomAspergillus and Penicillium dissolve calcite, which fully corresponds with the results of our study. In both cases, only a small number of fungal isolates, 12.72 and 21.15%, respectively, mainly of these two genera, demonstrated a calcite solubilization ability. Apart from the work of Pangallo et al. [3-enriched MEA inoculated with A. niger was provided by Sayer et al. [Doratomyces sp. and Paecilomyces sp. isolated from variously coloured patinas of the Catacombs of St. Callistus and Priscilla in Rome  were the only fungi able to dissolve calcite when cultured on CaCO3 glucose agar, while all Aspergillus and Penicillium isolates tested negative [3 glucose agar used, fungal carbonate solubilization activity presumably depends not only on environmental factors and origin of the isolates, but on available sources of carbon and nitrogen as well. A similar conclusion was also reached in a study of fungal degradation of apatite, galena and obsidian minerals [2 is used as a carbon source for autotrophy, as CaCO3 is not a redox sensitive compound) [3 from the substrate. According to Sterflinger [3, and strong oxalic acid (C2H2O4) is the main “culprit” in most instances. Since organic acids are synthesized, as a by-product of oxidative metabolism during primary fungal metabolism, it is no wonder that in our study and many others as well [3 dissolution was formed very early, i.e., during the first week of the incubation period. However, only in broth cultures of A. niger and P. bilaiae was acid production documented, via substantial changes in pH of the filtrate. It follows that in cultures of other positive isolates, CaCO3 dissolution either occurred as a result of acid production induced by the presence of Ca ions, or by one of the other mentioned mechanisms. In most cases, to produce acids, an abundant carbon source, i.e., sugars, is needed, since intensive growth results in production of more organic acids than is necessary for normal metabolism, the excess being excreted into the substrate. Cultured in Czapek-Dox minimal broth, an essentially oligotrophic medium, only a very small number of isolates, one of Aspergillus and two of Penicillium, caused considerable acidification of the broth medium, possibly owing to acid production. Many species of these two genera, isolated from artworks are known to synthesize acids, with A. niger being one of the strongest producers, primarily of oxalic, gluconic and citric acids [Penicillium bilaiae is also a strong, producer of oxalic and citric acid, while P. griseofulvum is known to synthesize two acids: genistic and shikimic [Aspergillus, Cladosporium, and Penicillium genera are potent acid producers. However, some of these studies were performed utilizing nutrient media rich in glucose, so carbon source availability and abundance evidently constitutes a very important factor in acid metabolism. Moreover, several fungal isolates known to be acid producers in our experiment did in fact lower pH of the broth medium by values on the order, of one pH unit. However, on the basis of such modest changes in pH, it cannot be definitely concluded that acid synthesis occurred without additional analysis by HPLC.Limestone dissolution induced by fungal metabolites is a well-known and thoroughly studied natural phenomenon in the terrestrial environment , albeit o et al. , 34, expr et al. . On the negative . Comparenegative  showed tminerals .This is ompound) , 39, 40.ompound)  suggesterflinger  and Orterflinger , only fu as well , a transic acids , 42, 43.shikimic , 45. Andshikimic , 48, 49,Gibberella moniliformis and Epicoccum nigrum, increase in the pH level of the medium was documented, possibly as a result of secretion of alkaline metabolites, such as NH3 and some polypeptides. Although species of the genus Fusarium are mainly known as producers of oxalic, fumaric, and succinic acid [Fusarium proliferatum isolate AZhT01, from an ancient stone stela, and indicating an increase of pH when cultured in the same medium, were previously reported by Savković et al. [On the other hand, in cultures of nic acid , resultsć et al. . It is ić et al. .Aspergillus and Cladosporium (and Chaetomium and Penicillium species to a lesser extent), produce casease, which breaks the peptide bonds in casein, a finding that corresponds well with previously published data [Aspergillus, Cladosporium, and Penicillium were similarly characterized as the main producers of casease and as such the primary degraders of proteinaceous substrata of cultural heritage objects. And while the proteolytic activity of several fungi studied in these investigations  is corroborated by our results in the study of Pangallo et al. [C. cladosporioides isolated from wall paintings and surrounding air of the Church of Saint Catherine in Velka Lomnica  lacked any observable transparent zone, which completely deviates from the findings obtained in our study, where it showed the highest proteolytic potential. Moreover, several Aspergillus isolates used in our study lacked a hydrolysis zone, although literature data [A. niger and A. flavus, are very tolerant to a wide range of pH and can thus produce enzymes whatever the acidity. Since casein agar medium has an approximately neutral pH value (6.8 ± 0.2), the absence of casein hydrolysis in known producers such as A. oryzae is not unexpected. As for Chaetomium isolates, the two tested species, Ch. ancistrocladum and Ch. murorum, both hydrolyzed casein, which is in accordance with the generally accepted view that this genus encompasses a large number of species that are strong producers of cellulolytic and proteolytic enzymes [Ch. murorum did not produce casease, this may be the result of changes in morpho-physiological features of the isolate induced by growth on frescoes, which are an extreme type of environment. It is also worth noting that, according to Vermelho et al. [Cladosporium isolates had the largest diameters of the hydrolysis zone, which suggests that they possess the highest proteolytic activity and points to them as the primary degraders of protein components within mortar and painted layers of wall paintings. The present study particularly emphasizes the importance of cooperation between mycologists and conservators in the exchange of knowledge regarding fungal proteolytic capacities and protein binders commonly used in secco painting in a given region, since their use even today is largely the result of an insufficient level of communication. Only in this way, by working together in selecting appropriate binders for restoring damaged wall paintings, can re-colonization of restored surfaces be prevented and long-lasting and effective safeguarding of murals be ensured.The casein hydrolysis assay applied in the present study (CN medium) was originally formulated as a simple and rapid method for screening proteolytic activity of microorganisms, so that adequate microbial strains, producers of important proteases, can be selected and further investigated for various uses in industrial biotechnology . As the hed data , 30, 52.o et al.  C. cladoure data , 30, 52 ure data  state th enzymes . Howevero et al. , diameteo et al.  considerAspergillus, Chaetomium, Cladosporium, Fusarium, and Penicillium species isolated from wall paintings, documents, books, paintings, and photographs [Ch. murorum. Which pigment this is cannot be said without chemical analysis, although it is known that some species of the genus Chaetomium, viz. Ch. cochliodes and Ch. globosum, when cultured on various nutrient media produce a very small amount of the purple pigment cochliodinol (C32H32N2O4) proven to possess strong antimicrobial activity [Micromycetes are known producers of a large variety of organic pigments, each with its own structure, composition, and colour, which are regulated by a number of things such as the available carbon and nitrogen source, metals in the substrate, light (UV), and other environmental factors that limit growth . Many istographs , 28, 30.activity .2O2, H2O2/Cu2+ mixture, UVA radiation, etc. [An additional complication is the fact that produced pigments can be secreted into the substrate or (more commonly) bound within the protoplasm or incorporated into composition of the cell wall of hyphae and spores, which must be ascertained via light microscopy before adequate removal methods can be applied . Coloureon, etc. .Based on all of the above, it can be concluded that the vast majority of filamentous fungi have a very important role in the decay of wall painting components via carbonate dissolution, proteolysis, and acid and alkaline action, usually manifested in cracking, peeling, and loss of microfragments, as well as in impairment of aesthetic appearance by secretion of coloured metabolites. It should be noted, however, that some common constituents of the mycobiota of frescoes are nothing more than harmless allochthonous transients. For these reasons, detection of fungal growth on painted layers or the presence in the surrounding air of propagules belonging to species with a now known biodegradative potential should be treated with particular care and appropriate remedial measures in the form of removal of the detected growth and application of microclimate control. Furthermore, introduction of fast and simple biodegradative plate assays as an integral part of the protocol of a modern system for the protection and conservation of artworks should be mandatory, since insight into the total spectrum of physiological features of fungi, common inhabitants of sacral ambients, is a prerequisite for precise assessment of the amount of risk they pose to mural paintings.The obtained results clearly demonstrate that the ambient air and deteriorated wall paintings of the old Church of the Holy Ascension are contaminated with several fungal strains associated with various biodeteriorative effects that most likely are partially or completely the cause of the documented damage. The present study also emphasizes the importance of knowledge about the biodegradative characteristics of fungal dwellers present on or in the immediate vicinity of artworks, as it represents the basis for precise risk assessment and formulation of a set of recommendations to cultural institutions regarding the implementation of preventive and remedial treatments. When known deteriogenic fungi are detected, measures in the guise of monitoring and frequent cleaning, introduction of adequate ventilation, and indoor climate management, must be carried out in order to prevent their establishment and subsequent deterioration of valuable artworks.


According to the TDS assessment, of the total 25 lakes, there are 14 for freshwater, 7 for brackish water and 4 for saltwater; and the respective range of TA was from 0.98 to 40.52. The relationship between TA and TDS indicated significant linear relationship (R2 = 0.9292) in the HCO3−-Na+-I type lakes in the Songnen Plain. There was a general trend that cell density, genera richness and taxonomic diversity decreased with the increase of saline-alkaline gradient, whereas a contrary trend was observed for the proportion of dominant species. When the TDS values were above 3×103mg/L and the TA values were above 15mg/L, there was a significant reduction in cell density, genera richness and biodiversity, and their corresponding values were respectively below 10×106 (ind/L), 15 and approximately 2.5. Through the partial canonical correspondence analysis (CCA), 10.7% of the genera variation was explained by pure saline-alkaline variables. Cyclotella meneghiniana, Melosira ambigua and Melosira granulate were found to become the dominant species in most of these lakes, which indicated that there may be rather wide saline-alkaline niches for common dominant species. About one-quarters of the genera which have certain tolerance to salinity and alkalinity preferred to live in the regions with relatively higher saline-alkaline levels in this study.Many lakes located in the Songnen Plain of China exhibit a high saline-alkaline level. 25 lakes in the Songnen Plain were selected as research objects in this study. Water samples in these lakes were collected from June to August in 2008. Total Dissolved Solids (TDS) and Total Alkalinity (TA) were measured to assess the saline-alkaline level, and partial canonical correspondence analysis (CCA) was conducted as well. The results show that the majority of these lakes in the study area could be categorized into HCO The two[HCO3−])  and theyBiodiversity, which was expressed by Shannon-Wiener’s index, was calculated with the following equations ,43.H′=−H’ is Shannon-Wiener index, pi = ni/N, ni = the individual number of genera i; N = the total number of genera.To investigate the relationship between phytoplankton and environmental characters especially the saline-alkaline conditions, a partial canonical correspondence analysis (CCA) was performed . It was 3 mg·L-1, 7 lakes’ TDS values are > 1×103 mg·L-1 and < 3×103 mg·L-1, 4 lakes’ TDS values are > 3×103 mg·L-1 and < 10×103 mg·L-1, these lakes are classified into freshwater, brackish water and saltwater respectively. The highest TA (namely 40.52) was observed in Etou Lake, while the lowest was in Bali Lake. The pH values showed weak alkaline (> 8.0) in these study lakes except Cile Lake (namely 7.80) and Bali Lake (7.07). The range of temperature in 25 lakes is from 20.86°C to 29.35°C in summer.The environment characteristics of lakes in the Songnen Plain are showed in +, Na+, Ca2+, Mg2+, Cl-, SO42-, CO32- and HCO3-). According to O. A. Arliekin's classification : 10.7%; genera variation explained by the intersection of the saline-alkaline and the other environmental variables  : 3.6%; genera variation explained by the other environmental variables that was not shared by the saline-alkaline variables [step (4)]: 33.8%; unexplained variation and stochastic fluctuations: 100%−48.2% = 51.8%.The genera variation explained by pure saline-alkaline variables (TDS and TA) is performed by partial CCA, whose value is 10.7%, and the inflation factors are both < 10, which shows there is no strong collinearity among different data . PhytoplCyclotella meneghiniana, Haerao Lake (2), Niuxintaobao Lake (5), Xinmiao Lake (12), Keqin Lake (13) and Dalonghu Lake (17) were dominated by Melosira ambigua, Xinhuang Lake (6) and Talahong Lake (8) were dominated by Anabaenopsis circularise, Lianhuan Lake (15), Lamasi Lake (16), Qijia Lake (18), Qingken Lake (20) and Yamudange Lake (24) were dominated by Melosira granulate. It can be observed that the sample sites with the same dominant species are scattered in In the lakes, Etou Lake (1), Xiaolonghu Lake (19), Sanyong Lake (22) and Kuli Lake (25) were dominated by Navicula, Nitzschia, Scenedesmus, Surirella, Fragilaria, Microcystis, Euglena, Oscillatoria, Crucigenia, Cyclotella, Pinnularia and Spirogyra.The hydrochemical characteristics are attributing to the factors of regional climate, hydrogeology, geomorphology and inappropriate human activities. The climate in the Songnen Plain is characterized dried in spring, heat and concentrated rainfall in summer, cool and larger temperature difference in autumn, and long and cold in winter . Precipi+ dominated the total cation and HCO3- was the main component of anions, the water is alkaline and the hydrochemical type is soda-Na. Due to the difference between supply source and drainage modes and regional environment, the salt components and salt contents of lakes are quite different accordingly [For the vast majority of lakes in this study, Naordingly .In the selected 25 lakes, Haerao Lake, Yangsha Lake, Moon Lake and Dalonghu Lake are now reservoirs, water recharge of Huao Lake is performed by taking the underground water from artesian wells around the lake, water recharge of Etou Lake, Lamasi Lake and Yamudange Lake just relay on precipitation and surface runoff. Cile Lake, Niuxintaobao Lake, Xinhuang Lake, Talahong Lake, Dakuli Lake, Chagan Lake, Xinmiao Lake, Keqin Lake, Nanshan Lake, Lianhuan Lake, Qijia Lake and Xiaolonghu Lake mainly relay on the Songhua River, Nenjiang River and its tributaries for water recharge. Sanyong Lake has become a running water lake since 1999 by the Nenjiang River-to-Anda water diversion project. Qingken Lake, Bali Lake, Laojiangshen Lake and Kuli Lake are located in Daqing detention basin, among them, Bali Lake is a pollutant lake, receiving sewage generated from a nearby factory. Additionally, the lakes which are surveyed in this study in the Songnen Plain are used for pisciculture nowadays except Bali Lake .According to the records of lakes, Bali Lake has been contaminated due to the inflow of industrial wastewater from chemical plant and domestic sewage. The stench of Bali Lake could be smelt during the 2008 summer field survey, simultaneously, the pH (7.07) of Bali Lake was relatively low, which indicates that there may exist the emissions of sulfur-containing substance around the lake, resulting in low water pH, high sulphate content, low TA  and low density of phytoplankton [Among the 25 lakes in this study, there were 14, 7 and 4 lakes classified into freshwater, brackish water and saltwater, respectively. The saline-alkaline level in this study area exist the spatial inhomogeneity due to the environmental differences of small area. In general, the saline-alkaline level in noncontributing areas is higher than that in outflow ones; and the saline-alkaline level in the areas that has hydraulic connection with river is lower than that has no such relationship. Due to the differences of time and space of the water source, there is a stage characteristic of water saline-alkaline level with the change of water quantity and water level of the lakes, generally speaking, that is the water level is high, the water quantity is large and the saline-alkaline level is relatively low of the lakes in wet season, and the saline-alkaline level rises, the lakes shrink and even some lakes dry up in dry season.+ and HCO3- had increased). However, there are relatively fewer researches on hydrochemical analyse of the lakes in the Songnen Plain. + and HCO3- in Huao Lake and Etou Lake had increased significantly. However, Chagan Lake, Moon Lake, Haerao Lake, Keqin Lake and Xinmiao Lake, the freshwater lakes, which had supply relationship with rivers, resulting in no obvious rise of the saline-alkaline level, even appeared the phenomenon of reduction of the saline-alkaline level in some lakes, such as Haerao Lake and Xinmiao Lake.In recent years, the water environment problems of salinization and alkalization of the Songnen Plain have become serious accompanied with population growth and the rapid development of industry, agriculture and economy , 46, 47,The previous researches mentioned in this study were performed before 1999, and most of the data from references are the average values calculated during a certain period. Nevertheless, the samples of the present study are obtained in wet season, which may affect the saline-alkaline level of water. It is noteworthy that there is a phenomenon of concentrated precipitation and artificial water diversion for lakes’ storage in the Songnen Plain, thus there may exist quite differences of the results between different researches.Cyclotella meneghiniana, Melosira ambigua and Melosira granulate, tend to persist and even increased their proportion at higher saline-alkaline levels, owing to these dominant species have certain tolerance to salinity and alkalinity to certain extent. Cyclotella meneghiniana numerically dominate (12.90–53.85%) at different levels of TA (10.40–40.52mmol/L) and TDS (1102.07–3158.88mg/L) in several lakes; Melosira ambigua appeared as the dominant taxon (8.99–36.36%) in the lakes of different levels of TA (2.89–6.79mmol/L) and TDS (181.06–652.09mg/L); Melosira granulate dominate (19.32–28.33%) the assemblages from some lakes of different levels of TA (4.58–30.49mmol/L) and TDS (253.45–3025.60mg/L). As described in Krammer & Lange-Bertalot [Cyclotella meneghiniana is known to be halophilous and basophilous, and the taxa usually can be found in saltwater and brackish water; Melosira ambigua and Melosira granulate are the limnoplankton tend to favour low-salt environment. The results of this research about Cyclotella meneghiniana, Melosira ambigua and Melosira granulate roughly correspond with what Krammer & Lange-Bertalot [Melosira granulate has also been found as the dominant taxon in saltwater (Qingken Lake) and brackish water (Yamudange Lake), suggesting that there may be a wider salinity niche for Melosira granulate, although most occurrences occur in freshwater.There are many factors affect phytoplankton growth and community structure in lakes, including salinity, alkalinity, light, nutrients, temperature and so on , 49–51. Bertalot , CycloteBertalot  mentione3mg/L and the TA values were larger than 15mg/L. Especially, the Shannon—Wiener index reflects the distribution, evenness and dominance of species at a particular site. A relatively lower diversity value indicates that some of the species which have competitive advantage in the specific environment present become dominant, resulting in the decrease of species evenness, then the diversity value is decreased, that’s the reason the distribution trend of diversity values of the lakes and the distribution trend of the proportion of the dominant species of the lakes are roughly opposite.Cell density, genera richness, proportion of dominant species and taxonomic diversity (Shannon—Wiener index) showed no significant correlation with salinity  and alkalinity  in this study. However, there was a significant reduction in cell density, genera richness and biodiversity when the TDS values were more than 3×10Navicula, Nitzschia, Scenedesmus, Surirella, Fragilaria, Microcystis, Euglena, Oscillatoria, Crucigenia, Cyclotella, Pinnularia and Spirogyra . Roughly one-third of this amount can also be predicted by the supplied function of the other environmental variables of the samples (step 1-step 3). This means that the genera and the saline-alkaline data have some fairly similar environmental factors, resulting from the interactions and common development of phytoplankton and environment during the process of lakes and biological evolution.Cyclotella meneghiniana, Melosira ambigua and Melosira granulate as the dominant species of this area are the common species usually living in eutrophication water.More than half of the total variation (51.8%) of the genera matrix remains unexplained, which is due to some factors (biotic or abiotic) weren't obtained in this study or to a large amount of stochastic variation remains unclear. It is worth noting, although there was no detailed investigation for light and nutrients in this study, the growth of phytoplankton and its distribution is also greatly influenced by light and nutrients which are usually crucial to phytoplankton and which may have co-effect on phytoplankton in the natural waters. Light is the prerequisite and guarantee for photosynthesis of phytoplankton, and light output have different effects on different species of phytoplankton, which mainly related to the factors such as photoinhibition of phytoplankton and utilization ratio of light energy. The impacts on the phytoplankton community by nutrients deserve studying. The change of the nutrient concentration, especially the ratio of nitrogen to phosphorus, in determining the composition of phytoplankton community plays an important role, which mainly affects the competition and the succession between phytoplankton to change the phytoplankton community. XIAO Haifeng reported that total nitrogen and total phosphorous in the lakes of Songnen Plain are generally high during June to August in 2008 , corresp3−-Na+-Itype. Among the total 25 lakes, 14 of which are classified as freshwater, 7 as brackish water and 4 as saltwater separately according to the TDS assessment, the range of TA is from 0.98 to 40.52. The relationship between TA and TDS indicate significant linear relationship (R2 = 0.9292) in the HCO3−-Na+-I type lakes in the Songnen Plain. To compare with the previous researches, the level of TDS and the content of Na+ and HCO3- in Huao Lake and Etou Lake had increased due to their no hydraulic connection with rivers, but there is also no obvious increase of the saline-alkaline levels of the lakes which had hydraulic connection with rivers.The hydrochemical characteristics in the Songnen Plain are affected by regional climate, hydrogeology, geomorphology and inappropriate human activities. Salinity and alkalinity which are measured by TDS and TA are the basic characteristics of hydrochemistry of the lakes in the Songnen Plain. Most lakes in this study could be categorized into HCO3mg/L and TA values above 15mg/L results in significantly reduced values of cell density, genera richness and biodiversity.There are 232 taxa of phytoplankton found in the 25 lakes with the characteristics of phytoplankton communities of less taxa and more abundance. Phytoplankton communities of the lakes in the Songnen Plain generally respond to increases in salinity and alkalinity, with decreases in cell density, genera richness and taxonomic diversity and an increase in proportion of dominant species. Increases in TDS values above 3×10Cyclotella meneghiniana, Melosira ambigua and Melosira granulate appeared in the lakes with different saline-alkaline levels as dominant species, owing to there may be rather wide saline-alkaline niches for the species. Furthermore, about one-quarter of the genera preferred to live in the environment of relatively higher saline-alkaline levels in this study, indicating these genera have strong tolerance to salinity and alkalinity.The genera variation explained by pure saline-alkaline variables (TDS and TA) is performed by partial CCA, which value is 10.7%. The analyses of partial CCA demonstrated that S1 Appendix(XLS)Click here for additional data file.S2 Appendix(XLS)Click here for additional data file.S3 Appendix(XLSX)Click here for additional data file.S1 Photo(JPG)Click here for additional data file.S2 Photo(JPG)Click here for additional data file.S3 Photo(JPG)Click here for additional data file.


Populus trichocarpa genome  and a total of 42 PHT genes were identified which formed five clusters: PHT1, PHT2, PHT3, PHT4, and PHO. Among the 42 PHT genes, 41 were localized to 15 Populus chromosomes. Analysis of these genes led to identification of 5–14 transmembrane segments, most of which were conserved within the same cluster. We identified 234 putative cis elements in the 2-kb upstream regions of the 42 PHT genes, many of which are related to development, stress, or hormone. Tissue-specific expression analysis of the 42 PtPHT genes revealed that 25 were highly expressed in the roots of P. tremula, suggesting that most of them might be involved in Pi uptake. Some PtPHT genes were highly expressed in more than six of the twelve investigated tissues of P. tremula, while the expression of a few of them was very low in all investigated tissues. In addition, the expression of the PtPHT genes was verified by quantitative real-time PCR in four tissues of P. simonii. Transcripts of 7 PtPHT genes were detected in all four tested tissues of P. simonii. Most PtPHT genes were expressed in the roots of P. simonii at high levels. Further, PtPHT1.2 and PtPHO9 expression was increased under drought conditions, irrespective of the phosphate levels. In particular, PtPHT1.2 expression was significantly induced by approximately 90-fold. However, the transcriptional changes of some PtPHT genes under drought stress were highly dependent on the phosphate levels. These results will aid in elucidation of the functions of PtPHT in the growth, development, and stress response of the poplar plant.Inorganic phosphate is one of key macronutrients essential for plant growth. The acquisition and distribution of phosphate are mediated by phosphate transporters functioning in various physiological and biochemical processes. In the present study, we comprehensively evaluated the phosphate transporter (PHT) gene family in the latest release of the  Phosphorus is a major macronutrient required for plant growth and development. It constitutes up to 0.2% of the dry weight of plant cells and consequently is required in significant quantities ; in addition, we analyzed the phylogeny, gene structures, genomic locations, and cis elements of these genes. We also investigated the expression patterns of all of the PHT genes in various tissues, as well as their expression profiles at different Pi levels  in P. simonii. Moreover, we examined the transcriptional profiles of PHT genes in P. simonii roots under drought conditions. The results of this study may provide a basic foundation for future functional genomic studies of the PHT family in poplar.Poplar is a model woody tree species used in functional genomic studies due to its small genome, easy transformation, fast growth, and vegetative propagation  were rooted in pots (10 L) filled with fine sand. The plants were cultivated in a glasshouse  and irrigated with 50 ml nutrient solution  containing Pi at one of three different levels  separately every other day. Each Pi treatment was applied to 12 plants for a period of 8 weeks. In addition, a phosphate-free solution was prepared by replacing KH2PO4 with K2SO4. Six plants for each Pi treatment were subjected to drought stress for 2 weeks before harvesting, and the other six plants for each treatment were not subjected to drought stress and served as controls. After the treatments, whole plantlets were harvested and separated into roots, stems, new leaves, and old leaves. Then, samples from the same tissues were pooled for each treatment, frozen in liquid nitrogen for further analysis.The cuttings of Arabidopsis and Oryza sativa were used as query sequence with E-value cutoff set as 1e−10 to perform a local BLASTP against P. trichocarpa JGI gene catalog , respectively. The choice of candidate phosphate transporters was based on the E-value, the sequence homology value (>60%) and the value of score (>450). All corresponding DNA and protein sequences of poplar phosphate transporters were obtained from Phytozome 11.0  and also verified with the Pfam database (http://pfam.xfam.org/). Various splicing variants of one gene and incomplete genes with short length were discarded. In addition, redundant sequences were removed, and the remaining sequences were used in further analyses.To identify phosphate transporters in poplar, the reported protein sequences from both http://web.expasy.org/protparam/). Further, TMpred (http://www.ch.embnet.org/software/TMPRED_form.html) was used to predict the transmembrane domains (TMDs) of each PHT protein. In addition, the subcellular location of each PHT was predicted using WoLF PSORT (http://www.genscript.com/wolf-psort.html). Multiple amino acid alignments of the PHTs were generated using ClustalW .For each putative protein, the grand average of hydropathicity (GRAVY) was calculated using ProtParam  program . The cis-regulatory elements were scanned in PLACE  of the PtPHT genes were calculated using the 2−ΔΔCT method  according to the manufacturer's instructions. The first-strand cDNA was synthesized using a PrimeScript RT Reagent Kit  following the removal of trace genomic DNA using DNase I . The specific primers Table  for quanPtPHT genes, including 12 PHO, 14 PtPHT1, 2 PtPHT2, 6 PtPHT3, and 8 PtPHT4 genes from the genome of P. trichocarpa according to the full AtPHT family in Arabidopsisf  and a TATA-box-like element (TATABOX2) was detected in 22 and 34PtPHT genes, respectively; A PHR1 element (P1SB) was observed in 20 PtPHT genes; and a helix–loop–helix element, (CATATGGMSAUR) was present in 11 PtPHT genes. Furthermore, 17 PtPHT genes had typical cis elements that were present especially in the respective genes, and the total number of these cis elements was 32.Some Pi-response and other stress-related PtPHT genes in 12 various tissues of Populus tremula using a public database, PopGenIE (http://popgenie.org/)  Figure . These tPtPHT genes in the roots, stems, young leaves, and mature leaves of P. simonii. Transcripts of 35 PtPHT genes were detected in the examined tissues   were detected in all four tested tissues  with improved annotation, and they were classified into five clusters: PHT1, PHT2, PHT3, PHT4, and PHO. In a subsequent study, Loth-Pereda et al. (Populus genome (v1), and we expanded upon their findings with the identification of 14 PHT1 family members in the latest version of Populus genome (v3). Notably, the PHT1 family is larger in poplar (14 genes) than in Arabidopsis (9 genes), barley (8 genes), rice (13 genes), and tomato (8 genes)  were detected in poplar. These two genes are located on different chromosomes (Chr8 and Chr10), and the lengths of their encoded proteins greatly differ which is constituent with the previous report that AtPHT2.1 cDNA encodes a 61-kD protein that is structurally similar but different from the PHT1 family proteins in Arabidopsis .The presence of a large number of genes within a family reflects the successive expansion and rearrangement of the genome by the extensive duplication and diversification that frequently occur over the course of evolution  is also similar to that of the ABRE element (ABREOSRAB21), which is present in 6 of the 42 PtPHT promoters, proteins in the bZIP class of transcription factors might bind to them  to potentially regulate the phosphate starvation response in Arabidopsis  were not expressed in any tissues examined. PtPHT2.1 was highly expressed in the roots, cambium-phloem, mature petiole, and dormant buds, while PtPHT2.2 was mainly expressed in the roots, cambium-phloem, and mature leaves. Six homologous PtPHT3 genes exhibited substantial differences in their expression in the various tissues examined. Three of them  were highly expressed in the roots of P. tremula, while the others  were expressed at very low levels. Although most PHT4 family members, excluding PtPHT4.3 and PtPHT4.6, were highly expressed in the roots and leaves, PtPHT4.1 was also highly expressed in the cambium-phloem, dormant buds, and dormant flowers. Further, PtPHO5 and PtPHO6 were highly expressed in nine tissues examined, while PtPHO7 and PtPHO8 were exclusively highly expressed in only a few of the tissues. Expression of the PtPHT genes varied among the different tissues examined, suggesting that these genes may have different functions in Pi uptake, transport, and storage. These results suggest that the PHT genes may have different functions since they expressed in a wide range of tissues, which may be partly crucial for the growth of perennial species.Homologous PtPHT genes in different tissues might be related to their subcellular locations  and HvPHT1;6 (385 μM) in barley (Rae et al., PHT genes in poplar require further study, including assessment of the heterologous transformation of Xenopus oocytes and gene over-expression studies using plant cell cultures.The expression of PtPHT genes, such as PtPHT1.2 and PtPHO9, were up-regulated under drought conditions, irrespective of the Pi level, suggesting that their expression was not affected by the Pi level under these conditions. However, the changes in expression of the other PtPHT genes, including PtPHT3.3b, PtPHT4.3, and PtPHT4.5a, under drought stress were largely dependent on the phosphate level (Figure PtPHT genes induced by a low level of Pi may contribute to drought tolerance of poplar plants. These results suggest that under drought conditions, Pi uptake in plants likely changes in association with alterations in PHT gene expression. This altered uptake in the presence of different levels of phosphate might be related to the drought resistance of plants.The expression levels of some l Figure . EspeciaCZ and ZZ conceived and designed the experiments. SM and CZ performed the experiments. CZ, ML, and SM analyzed the data. CZ, ML, and ZZ wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Panax ginseng and a patented drug Liuwei Dihuang Wan (LDW) for evaluating our database. By comparison of different databases, as well as checking against literature, we have demonstrated the completeness, effectiveness, and accuracy of our database.With the advancement of systems biology research, we have already seen great progress in pharmacology studies, especially in network pharmacology. Network pharmacology has been proven to be effective for establishing the “compounds-proteins/genes-diseases” network, and revealing the regulation principles of small molecules in a high-throughput manner, thus would be very effective for the analysis of drug combinations, especially for TCM preparations. In this work, we have proposed the TCM-Mesh system, which records TCM-related information collected from various resources and could serve for network pharmacology analysis for TCM preparations in a high-throughput manner (http://mesh.tcm.microbioinformatics.org/). Currently, the database contains 6,235 herbs, 383,840 compounds, 14,298 genes, 6,204 diseases, 144,723 gene-disease associations, 3,440,231 pairs of gene interactions, 163,221 side effect records and 71 toxic records, and web-based software construct a network between herbs and treated diseases, which will help to understand the underlying mechanisms for TCM preparations at molecular levels. We have used 1,293 FDA-approved drugs, as well as compounds from an herbal material  And particular attention should be focused on the materials and the progress of preparation to ensure its efficacy and safety.Traditional Chinese Medicine (TCM) has been widely used in clinics in China for at least 3,000 years. Through continuous development and innovation, ancient physicians select the essence and discard the dross of TCM, the TCM theory forms a unique academic system finally. The traditional Chinese system of medicine is a complex system, which not only contains medicine plants but also includes animal and mineral materials. With their respective dosages by the guidance of Chinese medicine theory and the rule of “King, Vassal, Assistant and Delivery servant”4. For example, Ganoderma lucidum, called ‘Ling-Zhi’ in China, has been used as a health-preserving and therapeutic agent. Pharmacological studies have shown that G. lucidum possesses medicinal properties including anti-cancer, anti-diabetic, anti-hepatotoxic and immunomodulatory8. The polysaccharides of G. lucidum, as the major bioactive ingredients, are responsible for the therapeutic uses. Additionally, Panax ginseng is a traditional herbal medicine that has been used therapeutically for more than 2,000 years. As the most valuable of all medicinal plants, especially in China, Korea, and Japan, ginseng exert a wide range of medicinal efficacy. Currently, it has been used to treat various diseases such as cardiovascular diseases10, Alzheimer disease11, sleep disorder and diabetes13. Ginseng contains a considerable amount of different compounds, of which the ginsenoside plays a major role14. In addition to single traditional herbs, Liuwei Dihuang Wan (LDW), a representative famous Chinese medicine preparation, is consist of six herbs16: Rehmannia glutinosa libosch., Cornus officinalis Sieb., Dioscorea oppositifolia L., Paoenia ostii, Alisma orientale Juz and Poria cocos (Schw.) Wolf, with a dose proportion of 8:4:4:3:3:318. So far, LDW has remained one of the most popular TCM preparations prescribed for primary or adjuvant treatment of many kinds of diseases, such as diabetes, hypertension, diabetic retinopathy and nephropathy24.Nowadays, many traditional Chinese herbs are famous for their therapeutic effect and low toxicity around the world and have increasingly arisen people’s attention3. As analytical theory and technology advanced rapidly, the TCM studies must keep up the pace of modern science to remain powerful. Since the effects on human cells and genes by the chemical ingredients from TCM remain unclearly, deep mining of related information is necessary. There is no doubt that the investigation of the molecular basis of herbal formulae will increase the acceptance of TCM worldwide25.The usage for TCM preparations has been reasonable and extensive in recent years, but many problems remain to be resolved, such as quality control and therapeutic targets evaluation. Unlike modern drugs discovered by targeting a specific protein, understandings of the molecular basis of TCM are still limited, it is a serious challenge for the modernization of TCM28. The network pharmacology approach has been abstracted as the interpretation of the “compound-proteins/genes-diseases” pathways (chains). And it is capable of describing complex interactions among biological systems of the human body, drugs, and diseases from a network perspective. For instance, if a compound and an approved drug target the same protein, the compound may be the most promising candidate for the discovery of the new drug, and if a compound and a disease share the same protein, the TCM that contains the compound as main constituent may have the same effect on this disease. Since the holistic philosophy of TCM shares the key ideas with network pharmacology, a new interdisciplinary termed TCM network pharmacology has been proposed30, which provides a new research paradigm for translating TCM from an experience-based medicine to an evidence-based medicine system.With the development of the big data research in biomedicine, network pharmacology has emerged as a systematic approach for drug-target analysis, and it has gradually pushed the paradigm shift for drug discovery: the shift from “one target, one drug” mode to “network target, multiple component therapeutics” modeFor the treatment of disease, TCM functions as a whole. That is, each herbal formula has its corresponding syndromes. While in the modern science, the synergy between diverse compounds of a medicine is the basis of its function. Network pharmacology studies can help people understand the role of a herbal formula in molecular level. Moreover, by analyzing the “drug-target-disease” interaction network in medication process, the researchers can reveal the effects of the herbal formula systematically. In summary, the TCM network pharmacology proposed has not only become an appropriate entry point for modern TCM research but also provided a strong theoretical support for the discovery of new indications of TCM.31, TCM@taiwan32, ChEMBL33 and TCMID34 are mainly for compounds of TCMs; STITCH35 is for compound-protein interactions; STRING36 is for protein-protein interactions; OMIM37 and GAD38 are for protein-disease associations. However, network pharmacology analysis is limited by several aspects of currently available databases. For example, database such as TCMID is not complete enough, whose compounds are much less than STITCH. In addition, a large amount of redundancies from databases including OMIM and STITCH have to be trimmed to improve the efficiency of the database. Finally and most importantly, the independent database can’t fully reflect the medicinal compounds’ effects on human. Therefore, more comprehensive and optimized database and analytical system for TCM are needed, which will help us better understand the therapeutic mechanism of the TCM preparation. And for the comprehensive understanding of the effects of TCMs, toxicity39 and side effects41 databases are crucial for the safety assessment: For many herbs, it’s their toxicity that is used to treat diseases; for other herbs, although they are non-toxicity or low toxicity, overdosing will lead to serious side reaction. For example, it was reported that 121 people died in the past 60 years due to the overdose of Ephedra42. Therefore, it’s of great importance for deciphering the toxicity mechanism of TCMs.Currently, various databases have been used for TCM analysis: Super Natural IIIn this work, we have proposed the TCM-Mesh platform, which was designed as an integration of database and a data-mining system for network pharmacology analysis of TCM preparations. The TCM-Mesh system has realized the network pharmacology analysis in a comprehensive, one-stop manner. Thus it could well serve for the network pharmacology analysis, especially for TCM preparations. TCM-Mesh recorded about > 6,000 herbs, >380,000 compounds, >14,000 genes, >6,000 diseases, >140,000 gene-disease associations, >160,000 side effect records and >70 compound toxic records. Through the online interface offered by the TCM-Mesh web portal, users could make an inquiry that facilitated the information mining of TCMs from multiple perspectives, as well as downloading those information for further analysis locally.44, and proteins/genes with combined score higher than 700 were regarded as compound’s predicted functional partners. Notice that TCM-Mesh was mainly developed for finding the “compounds-proteins/genes-diseases” pathways, the herb-related compounds without Can SMILE string and CID number were removed from our database, and as a result, the number of herbs was reduced inevitably.Through data integrating and filtering, TCM-Mesh now contained up to 6,235 herbs, 383,840 compounds, 4,518,065 compound-gene interactions, 14,298 genes, 3,440,231 gene interactions, 6,204 diseases, 144,723 gene-disease associations, 163,221 side effect records and 71 toxicity records. The combined score (0–999), obtained from the STITCH, represented the strength of the links between the compounds and the different proteins. The computing method was recorded in the literatureWe compared TCM-Mesh with TCMID and STITCH databases. Now TCMID records about 46,929 prescriptions, 8,203 herbs, 43,413 compounds and 3,854 diseases according to the home page of TCMID. We have found that the data size in TCM-Mesh were much more than in the TCMID . For 5-fluorouracil, it is effective on colorectal cancer46 and breast cancer47. And its related genes were ABCC5, CASP3, CASP8, CYP2C9, DHFR, DPYD, TP53, TYMP, TYMS and UPP1 respectively according to the TCM-Mesh, all genes were related to colorectal cancer except UPP1. Hydrocortisone was used to treat various kinds of inflammatory disease such as arthritis49. Its related genes were CRH, CYP19A1, CYP3A4, HSD11B1, HSD11B2, HSD3B2, NR3C1, POMC, SERPINA6 and SHBG, of which CRH, CYP19A1 and SERPINA6 were related to arthritis, as well as HSD11B1 and POMC were related to inflammation. Regorafenib was reported to cure gastrointestinal stromal tumors51, its related genes were BRAF, CSF1R, FLT1, KDR, KIT, PDGFRA, RAF1 and RET respectively. According to the TCM-Mesh, more than half of them were related to colorectal cancer and gastrointestinal stromal tumors. Capecitabine was used to cure colorectal cancer and breast cancer52, and its related genes were CDA, CES1, CYP2C9, CYR61, DPYD, ERBB3, HIF1A, HRAS TYMP and TYMS respectively, seven of which were related to colorectal cancer and breast cancer. Sulfasalazine was used for the treatment of inflammatory bowel disease53, whose related genes were ABCG2, ALOX5, CRP, IL1B, MPO, NAT2, NFKBIA, PTGS2, SLC46A1 and SLC7A11 respectively. And six of them were related to the indication of Sulfasalazine. Prednisone was a synthetic corticosteroid drug that was used to treat certain inflammatory disease , as well as some autoimmune diseases and several types of cancers (at higher doses)55. Its related genes were ABCB1, ALB, CRP, CYP3A4, HLA-A, HLA-C, HSD11B1, INS, NR3C1 and SERPINA6 respectively. And out of these genes, NR3C1 was related to atopic asthma, CRP was associated with inflammation, ALB was a C-reactive protein which was regarded as a marker of inflammation, HLA-A and HLA-C were related to autoimmune disease, as well as CYP3A4 was related to prostate cancer. Oxaliplatin, whose ten related genes were BIRC5, CASP3, CCND1, ERCC1, GSTP1, MTOR, PARP1, SRC, TYMP and VEGFA respectively, was used for curing colon cancer52. And seven of these genes were related to the indication.We used the drug name to search with the “compounds-proteins/genes-diseases” network been retrieved from the TCM-Mesh. For 5-fluorouracil and Hydrocortisone, they were not available in our database, as we used their respective synonyms: 5-FU and Cortisol for the alternatives. For each drug, we retained the 10 genes with the highest combined score for simplicity. And we aimed to figure out whether the diseases extracted from our database matched with the indication of the drug, and furthermore, which gene was the candidate target for the drug. The detailed information of candidate genes of drugs was shown in Table As shown in Fig. 56. Firstly, 1,293 compounds were used to search in the TCM-Mesh with 1,108 compounds and 103,428 related proteins been retrieved from the database. Secondly, as too many results were listed when we set combined score to 700, we set combined score to 800 for simplicity. We used proteins for searching with 16,061 approved genes  been collected. For each of drugs, we have chosen 10 genes with the highest score. By counting manually, we found that for these 1,015 FDA approved small molecular drugs, 63.1% (640) of whose highest scoring gene was associated with the indications of the drug , and 75.9%, 80.6%, 81.6% of whose top3, top5, top10 highest scoring genes were related to the indications of the drug respectively  were searched in the TCM-Mesh and no candidate target were retrieved might be due to two reasons: Firstly, we have found that some drugs were not used for treatment of diseases, but used as auxiliary agents such as anesthetic and contrast medium, thus these kinds of drugs could not be searched in the TCM-Mesh, which was focus on the diseases; moreover, for each compound we chose 10 genes for simplicity, therefore, possible targets might be filtered.We have compared TCM-Mesh database with several public available databases, and we have found that our database obtained the same searching results with STITCH while our database was much conciser. Furthermore, our database was relatively more complete compared to TCMID. As a database mainly focusing on TCM ingredient analysis, TCM-Mesh’s comparable results with those based on the general database such as STITCH has shown its superiority for TCM ingredient interpretations.Based on the TCM-Mesh system, we have tested and interpreted the functions of a famous traditional herb ginseng and a widely used TCM preparation: Liuwei Dihuang Wan, from the network perspective, and we have found the candidate pathway for bioactive components of TCMs to exert the respective pharmacological effect.The TCM-Mesh system has its web portal for searching of TCM preparations, small molecules, as well as genes and diseases. Results have shown that comprehensive information on herbs, compounds, genes, diseases, as well as relationship between them, would be provided. And the downloadable result report for TCM preparation and drugs were also offered for the users.79. Only by integration of gut microbiota, could we obtain a full picture of the TCM effectiveness map of TCM preparations.TCM quality control, as well as better understanding of the regulation of TCM preparations on diseases, would be essential for the application of TCMs. Firstly, we used the combined score in the current version of TCM-Mesh for simplicity, which was still primitive and did not reflect the fidelity of the “compound-proteins/genes-diseases” pathways, and refined scoring function will be added in the future. Secondly, with the advancement of weighted network analysis method, we believe that deeper mining of key chains in “small molecule-proteins/genes-diseases” network would be critical, which will be integrated into our next version. Thirdly, a holistic view about network pharmacology should be established as soon as possible: gut microbial communities act both as targets of drugs, as well as drugs themselves that could affect human as the hostTCM-Mesh was composed of four data fields Table , namely 56. Then we compared the indications with the disease from the network. If one of the diseases matched with recorded indications, we might find a candidate pathway for the drug to exert its effect.For a small molecular drug, we could set a threshold of combined score with the “compound-proteins/genes-disease” network obtained from our database. If there were more than 10 related genes in our result, we only retained the ten genes with the highest combined score for simplicity. The information of the drug and its corresponding indications were collected from the Drugbank32 and TCMID34.Traditional Chinese plant consisted of a great number of different small molecules. The chemical constituents of traditional Chinese medicine were obtained from the TCM Database@TaiwanAfter obtaining all the processed compound files, we used the Can SMILE string or name of small molecules to search with the related genes and corresponding potential related-disease retrieved from our database. Unlike the small molecular drug, we took all the traditional Chinese medicine related compounds as a whole. It relied on different compounds’ synergistic effects to exert its effect. Different compounds may act on the same pathway.Traditional Chinese medicine preparation consisted of several different traditional Chinese medicine. The different traditional Chinese medicine related molecule data were also obtained from the TCM Database@Taiwan and TCMID. The detailed data analysis process was the same as that for traditional Chinese herb.The information of herb-ingredients such as herb and its related compounds with CID number were collected from TCMID. The information of compounds and their targets, diseases and their related proteins were obtained from STITCH and OMIM respectively. As the target ID used by STITCH and OMIM were different from each other, the data from these resources were inconsistent. To overcome the barriers, the information of proteins aliases and approved gene symbol were retrieved from OMIM to unite the data. The protein interactions were obtained from STRING. The toxic and side-effect records of compounds were derived from TOXNET and SIDER. And the gene-disease associations were collected from GAD for facilitating the analysis. The FDA-approved compounds used in our study were retrieved from DrugBank, while the chemical constituents of Ginseng were collected from the TCM Database@Taiwan and chemical constituents of LDW were collected from TCM Database@Taiwan and TCMID.80 to convert both mol2 and SMILE formats into CAN formats. To further unify the format, the symbols represented configuration and chirality like “/”, “\”, “@”, “” and “H” were removed from the CAN format in both the database and the herb related compounds. With the unified format obtained, the Can SMILE string of compounds could be used to search with hits from the TCM-Mesh.The compounds of ginseng and LDW that we downloaded from TCM Database@Taiwan were mol2 formats while the formats we used in STITCH were SMILES formats. To improve compatibility of data, we converted all of them into a standard generalized format: Canonical SMILE format (Can). We used Open BabelWe integrated data from various data sources Fig. , to makeSecondly, target ID was different in various databases as we mentioned above. To unite our data, we used the protein aliases from OMIM to establish relation between the proteins and the genes, thus for most of proteins with the corresponding genes of proteins been retrieved from our database. The original table named protein aliases and approved gene were merged into one new table called “protein-gene links”, which contained 14,298 genes. And protein interactions were also converted to the genes interactions. Thus the amount of data was reduced from 4,274,001 entries to 3,440,231 entries as many proteins was searched with no results return.Finally, the GAD contained 167,130 entries, as many diseases were redundant in the database, we processed our data manually and removed the duplicate records, as a result the amount of data were reduced to 144,723 entries. Similarly, the disease entries of OMIM were reduced from 7,086 to 6,204.The TCM-Mesh database was built by MySQL. The main goal of our database was to build the connection between the herb and diseases. And the various data fields in our database were connected with their intrinsic relations Fig. .Firstly, we used the information of herb supplied by TCMID to connect herbs and their respective ingredients. We used the name and the CID numbers of compounds to search with the toxicity and the side effect of the compounds, as well as the related proteins of the compounds been retrieved from the TCM-Mesh. Secondly, we used the proteins to search and corresponding gene, gene interactions were retrieved. Finally, we used the genes to search in the database and obtained the candidate related-disease and the gene-disease associations. We have used combined score between compound and protein as a threshold to filter the results.81, an open source platform for complex network analysis and visualization, which would help to display the network in a more interactive manner. The compound in the network was shown as yellow square, the gene in the network was shown as blue circle while the disease was shown as the red triangle. And the association between genes was indicated by a solid line with 50% transparency while the links between compounds, genes and diseases were indicated by a gray solid line. The “yellow square-blue circle-red triangle” chain was the potential pathway for the compound to act on the disease by targeting the gene.The “compound-gene-disease” network was visualized by using Cytoscapehttp://mesh.tcm.microbioinformatics.org/). Currently, TCM-Mesh web portal has several major functions: (1) User can obtain the “herb-compounds-genes-diseases” network from the TCM-Mesh by inputting the English/pin yin name of herb. (2) Similarly, user can obtain the “compound-genes-diseases” network by inputting the name/CAN string of compound. (3) User can obtain gene related herbs, gene related compounds and gene related diseases by inputting the gene. (4) User can obtain disease related gene and disease-related researches by inputting the disease. (5) Searching suggestion is available when inputting the English/pin yin name of the herb. (6) In addition to the search function, browse, as well as download functions were made available for user. We have used “ren shen”(ginseng) as an example for testing our web service, the results webpages were shown in Fig. The web portal of TCM-Mesh was also established (


Firmicutes (44.62%), Bacteroidetes (38.49%), and Proteobacteria (4.11%) were the three most abundant phyla present in the GIT of the sheep. The present study also revealed the core genera of Prevotella, Bacteroides, Ruminococcus, Oscillospira, Treponema, and Desulfovibrio in the GIT. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States indicated that the metabolic pathway related to carbohydrate metabolism was the richest in the sheep GIT. In addition, a series of metabolic pathways related to plant secondary metabolism was most abundant in the stomach and large intestine than in the small intestine. Overall, the present study provides insight into the microbial community composition in GIT of the Chinese Mongolian sheep which is highly diverse and needs to be studied further to exploit the complex interactions with the host.Chinese Mongolian sheep are an important ruminant raised for wool and meat production. However, little is known about the microbiota of the gastrointestinal tract (GIT) of Chinese Mongolian sheep. To increase our understanding of the microbial community composition in the GIT of Chinese Mongolian sheep, microbiota of five sheep is investigate for the first time using the Illumina MiSeq platform. High microbial diversity was obtained from the GIT, and the microbiota exhibited a higher biodiversity in the stomach and large intestine than in the small intestine. The online version of this article (doi:10.1186/s13568-017-0378-1) contains supplementary material, which is available to authorized users. Microbiota of mammalian gastrointestinal tract (GIT) is a complex ecosystem constitute of diverse bacterial populations . These animals were reared and maintained in Gansu, China, in accordance with the standard livestock management practices. A diet of corn silage was provided in accordance with the agricultural industry standard of the People’s Republic of China (NYT816-2004). Animals were butchered in accordance with the approved by the Sichuan Agricultural University Committee on Ethics in the Care and Use of Laboratory Animals (Permit No. DKY-S20123517). Fresh samples (20 g) were collected from different segments of the GIT, namely, they were the stomach , small intestine , and large intestine . Finally, 50 samples were placed in sterile centrifuge tubes and immediately frozen in liquid nitrogen containers. The samples were then stored at −80 °C until further analysis. All samples were analyzed within a month.Bacteria DNA was extracted from GIT samples (100 mg each) in accordance with the instructions of the EZNA Stool DNA Kit (Omega Bio-tek). Sterile zirconia beads were used to increase the extraction yield and the quality of the bacteria DNA . The bacterial sequence reads were assembled using mothur v 1.32 . Gene prediction was performed using PICRUSt 1.0.0 and Greengenes database v13.5 . Meanwhile, the beta diversity showed a degree of diversity discrepancy in all samples.Using the Illumian MiSeq platform of 16S rRNA gene amplicons, a total of 557,657 sequences with a median length of 252 base pairs (bp) (V4~533–786 bp) assigned to 16,252 OTUs were obtained from all samples , 19 classes , 20 orders , 38 families , 40 genera , and 18 species .According to the SILVA taxonomic database, all sequences of the samples were classified from phylum to species by using the QIIME program. In this study, the classifications of relative abundance of bacterial OTUs from phylum, class, order, family, genus, and species were shown with heatmaps  from Bacteroidetes, two genera (Ruminococcus and Oscillospira) from Firmicutes, one genus (Treponema) from Spirochaetes, and one genus (Desulfovibrio) from Proteobacteria. We found four unique bacterial genera  between the stomach and small intestine. In addition, two unique bacterial genera  were observed between the small intestine and large intestine. However, we did not find unique bacterial genera between the stomach and large intestine.We detected unique and shared bacterial genera along the GIT from sheep at the genus level by using our sequencing data. The genera with an average abundance >0.1% were analyzed using Venn diagrams Fig. a. SurpriOscillospira was observed only in the rumen, reticulum, and abomasum. Succinivbrio was observed only in the rumen and abomasum, and Fibrobacter was discovered only in the reticulum and abomasum. In the small intestine . Among these 24 KEGG pathways, eight  primarily related to carbohydrate metabolism and bacterial flagellar assembly were more abundant in the stomach and small intestine than in the large intestine; three  primarily related to the metabolism of cofactors and vitamins were significantly abundant only in the stomach; and two  were significantly abundant only in the small intestine. Careful analysis of each segment showed that 23 KEGG pathways were significantly more abundant in the rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, colon, and rectum . Three KEGG pathways  were significantly abundant only in the reticulum. However, five KEGG pathways  were significantly abundant only in the jejunum.Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) was used to predict the functional composition of the gut microbiota genomes in Chinese Mongolian sheep. The functional profiles are shown in Fig. This study aimed to provide new insights into the diverse symbiotic bacterial communities along the GIT of Chinese Mongolian sheep. The gut microbiota co-developed with the host from birth is involved in the regulation of mammal’s immune function, digestion, physiology, and disease treatment  of relative abundance of bacterial OTUs from phylum, class, order, family, genus, and species, bacteria from 2.80% phyla, 2.90% class, 2.90% order, 19.92% family, 6.88% genera, and 63.42% species were unknown. Although we obtained a huge number of microbiota members (>93.12%) along the GIT of sheep on the level of genus, a large number of microbiota members (>63.42%) cannot be classified or remain unknown on the level of species. Recently, a metagenomic data analysis of the human gut has shown extensive strain-level variation across species, and differences in gene copy number affect specific adaptive functions (Greenblum et al. In summary, we successfully for the first time characterized the bacterial taxa and metabolic pathways in all intestinal segments of Chinese Mongolian sheep by using Illumina MiSeq. Nevertheless, the obtained functional profiles are merely a prediction; detailed analyses are still needed to elucidate this aspect. Further studies are warranted to determine the contributions of the bacterial taxa and metabolic pathways to the health, development, and physiology of Chinese Mongolian sheep.


Escherichia and Gardnerella were not found in the diabetes plus hyperlipidemia cohort but they were found in all of the other cohorts. Cetobacterium was only present in the diabetes plus hypertension cohort. The most abundant bacteria in the diabetes only and diabetes plus hyperlipidemia cohorts was Lactobacillus, while Prevotella was the most abundant bacteria in the diabetes plus hypertension and diabetes plus hypertension and hyperlipidemia cohorts. Moreover, the relative abundance of Lactobacillus was significantly lower in the diabetes plus hypertension cohort than in the diabetes only and diabetes plus hyperlipidemia cohorts. Several bacteria were correlated with the participants' fasting blood glucose, blood pressure, and blood lipids. In conclusion, hypertension and/or hyperlipidemia and other patient factors can affect the composition of the urinary microbiota in those with diabetes. The insights from this study could be used to develop microbiota-based treatment for comorbid conditions, including urinary tract infections, in those with diabetes.Evidence shows urine specimens from different women have different populations of bacteria. The co-occurrence of hypertension and hyperlipidemia in those with diabetes may alter the composition of urine and the microenviroment of the bladder in which bacteria live. The aim of this study was to characterize the urinary microbiota in women with type 2 diabetes mellitus only and those with diabetes plus hypertension and/or hyperlipidemia, and to explore whether the composition of the urinary microbiota is affected by fasting blood glucose, blood pressure, and blood lipids. We enrolled 28 individuals with diabetes only, 24 with diabetes plus hypertension, 7 with diabetes plus hyperlipidemia, and 11 with diabetes plus both hypertension and hyperlipidemia. Modified midstream urine collection technique was designed to obtain urine specimens. Bacterial genomic DNA was isolated using magnetic beads and the urinary microbiota was analyzed using the Illumina MiSeq Sequencing System based on the V3-V4 hypervariable regions of the 16S rRNA gene. Among the four cohorts, the diabetes plus hypertension cohort had the highest relative abundance of Proteobacteria. In contrast, the diabetes plus hyperlipidemia cohort had the lowest relative abundance of Proteobacteria. In addition,  The number of people around the world with diabetes has risen from 108 million in 1980 to 422 million in 2014 , blood pressure (BP), blood lipids (BL), asymptomatic bacteriuria (according to urine cultures) and nitrite-positive samples (which are indicative of urinary tract infections).Seventy individuals with T2DM were divided into four cohorts: 28 were allocated to the T2DM only (DM) cohort, 24 to the T2DM plus hypertension (DM+HT) cohort, 7 to the T2DM plus hyperlipidemia (DM+HLP) cohort, and 11 to the T2DM plus both hypertension and hyperlipidemia (DM+HT+HLP) cohort. The participants had been previously diagnosed (with T2DM only or with T2DM plus hypertension and/or hyperlipidemia) by clinicians in local provincial or municipal hospitals in China. They were enrolled at the Endocrinology Department of the First Affiliated Hospital, School of Medicine, Zhejiang University from June 2015 to January 2016. Written informed consent was obtained from the participants prior to enrollment, and approval for the study was obtained from the Ethics Committee of the First Affiliated Hospital (reference number: 295). The following exclusion criteria were applied: a urinary tract infection (UTI) in the previous month, antibiotic use in the previous 3 months, inability to complete the questionnaire, menstruation, urinary incontinence, known anatomical abnormalities of the urinary tract , and use of a urinary catheter. Each participant's most recent FBG, BP, and BL values were obtained from their medical records. Water intake was assessed using the Chinese Food Frequency Questionnaire  collection technique that involved disinfection and a four-tube collection method. The MMSU collection technique involved the following steps: (a) A polyvinylpyrrolidone-iodine antiseptic  was poured onto sterile cotton balls, which were then placed into a 40 mL sterile sputum cup. (b) Four 50-mL sterile centrifuge tubes  were opened and the lids were placed upwards so that the participant would not touch the interior or top edge of either the tubes or the lids. (c) The participant pulled her underwear down to her knees and squatted on a squat toilet, with legs spread apart. (d) The participant disinfected the thumb, middle finger, and index finger of each hand twice with the polyvinylpyrrolidone-iodine antiseptic. (e) The participant used her dominant hand to pick up one of the cotton balls covered with antiseptic, cleaned the far labial fold, starting from above the urinary meatus down toward the rectum. The cotton ball was then discarded without crossing the sterile field that had previously been disinfected. The near labial fold was then disinfected by cleaning down the center of the urinary meatus. The labia were held apart to prevent the labia minora from falling back over the urinary meatus. (f) After disinfection, the participant urinated into tube 1 until half the tube was filled. Then, without stopping the flow of urine, the participant urinated into tubes 2, 3, and 4 in that order. The redundant urine was voided to the toilet. All the tubes were filled up to the halfway mark except for tube 2, which was filled up to the labeled line (representing 40 mL of urine). After training, we asked the participant demonstrating the MMSU technique to assess their competency to perform the procedure. The collection procedure was supervised by a senior nurse.5 CFU/mL of the same bacterial strain in two consecutive MMSU specimens , NaCl, 0.5 M EDTA (pH 7.97), and SDS. The tube was kept at −80°C until DNA extraction. The method of magnetic beads isolation of genomic DNA from bacteria was based on the manufacturer's protocol with minor modifications ; its integrity and size were checked by 1.0% agarose gel electrophoresis containing 0.5 mg/mL ethidium bromide. The DNA complex was placed at −20°C until PCR amplification. We failed to extract sufficient DNA for sequencing from the sample of one participant, so this participant was replaced by another who had similar demographic characteristics.Forty mL urine was aspirated from tubes 2 and 3, separated into three sections and injected into three 15 mL sterile centrifuge tubes. Each of them was pelleted by centrifugation at 4,000 × The 16S rRNA gene V3-V4 regions were PCR-amplified from microbial genome DNA using primers  . For continuous variables, Independent Table We obtained 5,175,509 raw sequences from the 70 samples, with a median read length of 2 × 300 and 443 base pairs. After filtering and removing the chimeric sequences, we obtained 3,981,519 reads for further analysis, which accounted for 76.93% of the valid reads, with a mean of 56,878.84 reads  per sample. The mean read length was 438 bp (ranging from 423 to 436). The value of Good's coverage estimator was 98%.As shown in Table The numbers of bacterial phyla detected in the DM, DM+HT, DM+HLP, and DM+HT+HLP cohorts were 40, 37, 30, and 26, respectively. The predominant bacterial phyla in the DM cohort were Proteobacteria (58.27%), Firmicutes (20.83%), Bacteroidetes (10.14%), Actinobacteria (7.52%), and Fusobacteria (1.06%). The DM+HT cohort was dominated by Proteobacteria (60.18%), Firmicutes (20.60%), Bacteroidetes (11.20%), Actinobacteria (5.36%), and Synergistetes (0.54%). The DM+HLP cohort was dominated by Proteobacteria (50.26%), Firmicutes (24.43%), Bacteroidetes (13.36%), Actinobacteria (9.21%), and Acidobacteria (1.18%). The DM+HT+HLP cohort was dominated by Proteobacteria (37.29%), Firmicutes (22.78%), Bacteroidetes (22.47%), Actinobacteria (15.33%), and Fusobacteria (0.82%).The numbers of bacterial genera in the DM, DM+HT, DM+HLP, and DM+HT+HLP cohorts were 320, 303, 236, and 225, respectively. The genera detected in each of the cohorts are shown in Table Lactobacillus, Prevotella, and Acinetobacter . In addition, as shown in Figure Oscillospira was capable of being used as a diagnostic factor for distinguishing those in the DM+HLP cohort from those in the DM cohort.The levels of t Figure . Comparet Figure . In addit Figure . The levt Figure . The relTo better understand the relationships between urinary microbiota and individual characteristics such as FBG, BP, and BL, correlation analyses were carried out. The bacteria that were significantly correlated with the three factors are listed in Tables Lactobacillus iners, Acinetobacter rhizosphaerae, and Acinetobacter schindleri than those in the DM cohort . In contrast, they also had a higher relative abundance of Kocuria palustris compared to those in the DM cohort . Interestingly, Eggerthella lenta, which was not detected in the DM cohort, was found in the DM+HT cohort. In addition, Deinococcus aquatilis, which was detected in the DM cohort, was absent in the DM+HT cohort. Additionally, A. rhizosphaerae was significantly lower in the DM+HT+HLP cohort compared to in the DM cohort . A. rhizosphaerae was also significantly lower in the DM+HT+HLP cohort compared to in the DM+HLP cohort .At the species level, those in the DM+HT cohort had significantly lower relative abundances of r < 0.7, p < 0.05; Tables To explore the association between nutrients intake and the relative abundance of bacterial genus in urinary microbiota and between nutrients intake and medicine intake, we compared the difference of nutrients intake among DM, DM+HT, DM+HLP, and DM+HT+HLP group, and there were difference in energy, protein, fat, carbohydrate, vitamin B1, vitamin B2, vitamin C, vitamin E, potassium, sodium, magnesium, selenium, copper, manganese, monounsaturated fatty acid, polyunsaturated fatty acid and aspirin intake among groups. We have found that some nutrients intake were weak or mild positively or negatively associated with urinary microbiota  Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria. This is somewhat similar to the findings of several previous studies on urinary microbiota profiles, as in these previous studies, one of the most abundant bacteria was Proteobacteria, which were generally ranked as the second or third most abundant bacteria, though they sometimes ranked lower  that help to combat the inflammatory responses caused by diabetes, hypertension, and hyperlipidemia in order to maintain healthy urinary conditions.Several bacteria were not present in the DM+HLP cohort, but were detected in the three other cohorts such as Lactobacillus, which is similar to the findings of previous studies  had a lower relative abundance of Allobaculum than controls  has a large impact on the reproduction of the microflora. Therefore, it is important to further investigate the mechanisms behind the association of urinary microbiota with hypertension, hyperlipidemia, and urinary tract infections, as this may be useful for developing microbiota-based treatment to control hypertension, hyperlipidemia, and bladder infections in those with diabetes.This is the first study to investigate the association between urinary microbiota profiles and comorbid hypertension and hyperlipidemia in those with T2DM. It demonstrated that the co-occurrence of hypertension and hyperlipidemia in those with T2DM affects the relative abundance of various bacteria in urine. UTIs may be affected by the relative abundances of Proteobacteria, LL, WW, YX, and FL conceived and designed the study. ZL generated the sequencing data. FL, LZ, and PJ collected the samples. QY and FL conducted urine cultures and the urinalysis. FL, BW, and LZ extracted the bacterial DNA. FL, ZL, and YX analyzed the data, carried out the computational analysis, interpreted the data, and drafted the manuscript.This study was funded by the Opening Foundation of China's State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (grant number: 2015KF05).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purpose. The aim of this scanning electron microscope (SEM) study was to investigate the interface between the bone and a novel nanostructured calcium-incorporated dental implant surface in humans. Methods. A dental implant  with a nanostructured calcium-incorporated surface , which had been placed a month earlier in a fully healed site of the posterior maxilla (#14) of a 48-year-old female patient, and which had been subjected to immediate functional loading, was removed after a traumatic injury. Despite the violent trauma that caused mobilization of the fixture, its surface appeared to be covered by a firmly attached, intact tissue; therefore, it was subjected to SEM examination. The implant surface of an unused nanostructured calcium-incorporated implant was also observed under SEM, as control. Results. The surface of the unused implant showed a highly-structured texture, carved by irregular, multi-scale hollows reminiscent of a fractal structure. It appeared perfectly clean and devoid of any contamination. The human specimen showed trabecular bone firmly anchored to the implant surface, bridging the screw threads and filling the spaces among them. Conclusions. Within the limits of this human histological report, the sample analyzed showed that the nanostructured calcium-incorporated surface was covered by new bone, one month after placement in the posterior maxilla, under an immediate functional loading protocol. For over thirty years, dental implants have been an effective solution for the restoration of the masticatory function, in the case of partially and completely edentulous patients ,2,3,4. TThe original protocol for the placement of dental implants was based on a submerged and undisturbed healing before functionalization, i.e., connection of the prosthetic abutment and placement of the provisional or definitive restoration ,6. This However, in recent years, patients have become more demanding and require early or immediate prosthetic restoration of the implants to avoid annoying periods with provisional removable prostheses ,7,8. ConSuch variations of the classical surgical and prosthetic protocol “ad modum Branemark” ,6 undoubTo meet the needs of patients and clinicians, a whole range of new micro-rough implant surfaces, produced by various subtractive ,15 or adHistological and histomorphometrical studies in humans have shown that micro-rough surfaces can accelerate bone healing by stimulating the deposition of new bone on the fixture and thereby increasing the percentage of bone-to-implant contact, when compared to the conventional machined surfaces ,16,19,20More recently, to further enhance and speed up bone healing mechanisms, dental implant companies have introduced new surfaces with a controlled nanophotography  ,22,23,24Among these surfaces are those coated with nanoparticles and calcium ions ,25,26. TAmong the nanostructured calcium-incorporated surfaces are those obtained by discrete calcium-phosphate deposition (DCD) ,28, thos® surface  is a nanostructured surface enriched with calcium ions through a hydrothermal method [The Xpeedl method ,32,33. HHowever, there is not yet a literature study on the scanning electron microscope (SEM) evaluation of the interface between the human bone and this new nanostructured calcium-incorporated surface. Hence, the aim of this study is to investigate the interface between the bone and this new nanostructured calcium-incorporated surface in humans, through scanning electron microscopy.®, Megagen Implant Co., Gyeongbuk, South Korea) has a novel calcium-incorporated surface (Xpeed®). This nanostructured, calcium-incorporated surface is obtained as previously described [2. With this procedure, a nanolayer of Ca2+ ions is incorporated onto the RBM surface, giving a CaTiO3 nanostructure. Previous studies [The implant evaluated in the present study  had been placed in the posterior maxilla to replace a first premolar (#14) of a female patient who was 48 years old. The patient was a smoker (10 cigarettes/day) but with good oral hygiene. The implant was inserted with a conventional surgical protocol; that is, in a fully healed ridge . However, to meet the patient’s specific requests, the implant had been loaded immediately after placement with a single, temporary acrylic resin crown. The patient gave her informed consent for inclusion in the present study, which was conducted in accordance with the Declaration of Helsinki for medical research involving human subjects of 1975 (revision of 2008). The study protocol was approved by the Ethics Committee of Insubria University .® screw was simply mounted on appropriate stubs with conductive glue without further treatments and observed as above, as a control.After one month of functional loading, unfortunately, the patient was the victim of an accidental trauma. Following a road accident, she fell from the motorcycle and hit her face against the ground violently. The trauma caused fractures and mobilization of some natural teeth (#13 and #15), but it also affected the implant-supported restoration (#14): the fixture lost its stability and had therefore to be removed. Despite the violent trauma that caused the mobilization of the implant, immediately after removal, the surface of the fixture appeared to be covered by tissue. This tissue was firmly attached to the surface and appeared intact. Therefore, the fixture and the surrounding tissue were subjected to SEM examination. All procedures carried out in the present study were in full compliance with the principles described in the Declaration of Helsinki for medical research involving human subjects of 1975 (revision of 2008). The implant was fixed overnight in Karnowski, dehydrated with graded ethanol and hexamethyldisilazane, mounted on suitable stubs with conductive glue and gold-coated with an Emitech K550 sputter-coater . The specimen was then observed with a FEI XL-30 FEG high resolution SEM  operated at an acceleration voltage of 7 kV using secondary electron imaging (SE). Pictures were directly obtained in digital format as 1424 × 968, 8 bpp TIFF grayscale files. Subsequently the same specimen was embedded in glycolmethacrylate , sectioned along its longitudinal axis with a high precision diamond-coated disk and polished flat. The specimen was then mounted as above, carbon coated with an Emitech K250 flash evaporator  and observed with the same FEG SEM operated at an acceleration voltage of 15 kV using backscattered electron (BSE) imaging. An unused AnyRidgeThe surface of a new, unused screw showed a highly-structured texture, with nested nooks and cavities of a variety of sizes and orientations. Although not really fractal, the surface showed a sort of self-similarity with extends down to the grain structure of the metal A,B. The The lateral surface of the implant , as wellBone is a natural nanostructured material , made of organic compounds (mainly collagen) reinforced with inorganic compounds (hydroxyapatite). It is this natural nanostructure that nanotechnology aims to emulate for dental applications ,22,34,35® [Among the nanostructured, calcium-incorporated surfaces, there are surfaces enriched with calcium ions by a hydrothermal method, like Xpeed® ,32,33.®) can have high survival and success rates, at least in the short term [Although several clinical studies have shown that dental implants with a nanostructured surface enriched with calcium ions through the hydrothermal method  ,41,42; sThe surface enriched with calcium ions through the hydrothermal method has been studied in more detail, and recently two human histological and histomorphometrical studies have evaluated the early bone response to nanostructured, calcium-incorporated implants ,33.®) with that determined by the insertion of implants with a machined surface [test) and the other a machined surface (control) [test and control implants were compared [control group), the histomorphometrical evaluation revealed a mean BIC% (±SD) and BD% (±SD) of 21.2% (±4.9) and 29.8% (±7.8), respectively [test group), the histomorphometrical analysis revealed a mean BIC% (±SD) and BD% (±SD) of 39.7% (±8.7) and 34.6% (±7.2), respectively [p < 0.001) [In a randomized controlled histological/histomorphometrical study in the human posterior maxilla, the authors compared the early (eight weeks) bone response following the insertion of implants with a nanostructured calcium-incorporated surface (Xpeed surface . In thiscontrol) . All impcompared . In the ectively . In the ectively . A stati< 0.001) . Therefo< 0.001) .test) and one a cylindrical implant with a self-tapping thread design and a sandblasted surface (control) [test and control implants were compared [test implants, a mean BIC% and BD% of 35.9 (±9.1) and 31.8 (±7.5) were reported, whereas with control implants, a mean BIC% and BD% of 29.9 (±7.6) and 32.5 (±3.9) were seen [p = 0.16) was found in the BIC% between test and control implants [p = 0.9) [In another human histological and histomorphometrical study , the autcontrol) . All impcontrol) . Once agcompared . With teere seen . Althougimplants . Finallyp = 0.9) . The autp = 0.9) . Our present SEM report seems to confirm the evidence emerging from these studies. In fact, one month after the placement and immediate loading in the posterior maxilla, the surface of the nanostructured calcium-incorporated fixture was covered by newly formed bone tissue, capable of filling the spaces between the threads and even creating “bridges” between them. The osseointegration process was relatively mature even after a short period (one month), with the different phases of bone maturation in evidence. It is important to emphasize that this result was achieved in the posterior maxilla one month after implant placement, under the immediate functional loading of a standard-sized fixture. The posterior maxilla is generally characterized by a rather poor bone quality compared to other anatomic sites, and is therefore one of the most dangerous sites in the positioning of dental implants ,12,13,18Our present study has limitations. First, it is only a case report, and logically it would be advisable to evaluate a larger number of samples to draw more specific conclusions about the effectiveness of this nanostructured calcium-incorporated surface in accelerating and stimulating the deposition of new bone in the early healing phases. Moreover, the fact that the implant was removed after a trauma might have somehow affected our SEM assessment, and it would have been preferable to intentionally remove the fixture (and not as a result of a motorcycle accident), to better evaluate an untouched bone-implant interface. For all these reasons, further SEM studies on a larger sample of patients and with a different design  are needed to confirm the positive outcomes emerging from our present report.