We intend to conduct a retrospective cohort study based on the Surveillance, Epidemiology, and End Results (SEER) database to investigate the prognosis of liver transplantation Patients diagnosed with CHC (ICD-O-3:8180/3) and treated with transplantation or hepatectomy were extracted from the SEER database (2000–2018). We utilized Propensity Score Matching to control confounding bias. Kaplan-Meier curve was used for survival analysis, and Cox regression was used to find independent factors associated with prognosis.We identified 123  patients with CHC who were treated between 2004 and 2015. In the entire cohort, survival analysis demonstrated transplantation group was associated with better overall survival and cancer-specific survival . In addition, liver transplantation still conferred better overall and cancer-specific survival than liver resection after Propensity Score Matching . However, this advantage didn't appear in the subgroup, regardless of whether the tumor size was greater than 3 cm or not. . Multivariate analysis validated the finding that liver transplantation was a protective factor for overall survival .Liver transplantation may be an option in individuals with CHC and should be taken into consideration due to its advantages in terms of overall survival and cancer-specific survival. However, a sizable sample is required for future studies to determine which subset of CHC patients may benefit more from liver transplantation. It has Allen et al.  first prDue to the tiny population and changing classification over time, the treatment and prognosis of CHC are still uncertain and controversial. CHC has similar clinical features as HCC, including sex, HBV infection, AFP and PIVKA-Ⅱ, but more frequent lymph node metastasis and vascular invasion compared with HCC . As a revs. LR among CHC patients.So far, the status of liver transplantation in CHC remains much debated . To dete2For this study, data from SEER 18 registries from 2004 to 2015 were gathered . FirstlyThe demographic variables were age, sex, grade of tumor differentiation, race, tumor size, treatment, AJCC TNM stage, regional lymph node status, and extent of regional lymph node surgery. The age of patients was divided into two groups: ≥65 & <65. White, black, and other races were listed as the race. The tumor differentiation was classified into three groups: poor differentiation-undifferentiation (Grade Ⅲ-Ⅳ); well-moderate differentiation (Grade Ⅰ-Ⅱ); and unknown. The extent of regional lymph node surgery refers to the removal, biopsy, aspiration, or absence of these operations. Correspondingly, the regional lymph nodes that underwent the above operations were recorded as “positive” or “negative”, and those that lacked the above procedures were recorded as “not examined”. The American Joint Committee on Cancer (AJCC) stage was categorized as AJCC Ⅰ-Ⅱ and AJCC Ⅲ-Ⅳ according to the 6th cancer staging systems. The treatment code in the SEER database for CHC includes: LR: 20–26, 36–38, 50, 51, 60, LT:61,75.Because all patients’ information in the SEER database has been deidentified and is publicly accessible, the research involving the anonymous data from that database was exempt from medical ethics review and did not require informed consent.The main outcomes were taken to be overall survival (OS) and cancer-specific survival (CSS), which are frequently used to assess prognostic outcomes. The period between the day of diagnosis and any sort of death was defined as OS. The time until death attributed to CHC was referred to as CSS.t-test, and the latter is examined by Χ2 or Fisher exact test. Using the “MatchIt” package, we performed Propensity Score Matching to balance the covariates between the two groups [R software (version 4.2.2) was used for statistical analysis and graph creation in this work. Mean ± SD and Counts are the presentation formats for continuous and categorical variables, respectively. The former is examined by o groups . ConsistThe above steps are implemented through the following two packages, namely “survival” and “survminer” ,20. Vari33.1Forty-nine (39.84 %) of the 123 CHC patients received LT, and seventy-four (60.16 %) received LR in the entire cohort. The patients’ characteristics are shown in 3.23.2.1In the entire cohort, the median OS was 30 and 92 months in the LR and LT groups, respectively, and the median CSS for the LR cohort was 36 months. When the final follow-up occurred, CSS in the LT group was still higher than 50 %. After the median follow-up of 41(0–179) months, the overall patient survival rate for those undergoing LR was 70.3 %, 44.4 %, and 34.5 % at one, three, and five years, respectively and the one-, three-, and five-year CSS was 74.8 %, 50.8 %, and 42.9 %, respectively. For patients undergoing LT, the one-, three-, and five-year OS was 86.2 %, 72.4 %, and 60.3 % respectively; the one-, three-, and five-year CSS was 90 %, 79.0 %, and 69.3 %, respectively. The OS and CSS curves for CHC patients are shown in 3.2.2The median OS was 39 and 114 months in the LR and LT group, respectively, and the median CSS for the LR cohort was 59 months. When the final follow-up occurred, CSS in the LT group was still higher than 50 %. After the median follow-up of 48.5 (0–167) months, overall patient survival for those undergoing LR was 75 %, 52.1 %, and 42.1 % at one, three, and five years, respectively, and the one-, three-, and five-year CSS was 82.7 %, 58.4 %, and 49.8 %, respectively. For patients undergoing LT, the one-, three-, and five-year OS was 90.3 %, 80.6 %, and 63.5 %, respectively; the one-, three-, and five-year CSS was 93.2 %, 83.1 %, and 69.2 % respectively. The OS and CSS curves for CHC patients are shown in 3.3≤3 cm seems to be clinically important, as iCCA patients who meet Milan criteria also appear to have favorable post-LT outcomes [A subset analysis based on tumor size was conducted to further control for disease-related confounding bias between the two groups. Focusing on multiple tumors with a diameter outcomes . Given t3.3.1The tumor burden of 51 patients was no more than 3 cm; 17 of them underwent LR, while 34 of them underwent LT. After the median follow-up of 55(4–167) months, the median OS for LR and LT were 54 and 94 months, respectively, whereas the median CSS for LR and LT was not reached until the end of the follow-up. The one-year, three-year, and five-year OS of liver resection are 82.4 %, 58.0%, and 41.1 %, respectively.  while one-year, three-year, and five-year CSS of liver resection are 85 %, 69 %, and 57.5 %, respectively. . The log-rank test revealed that patients undergoing LT had a similar OS with LR (log-rank p = 0.23), and there was no significant difference between the two groups in terms of CSS (log-rank p = 0.37), as shown in 3.3.2Only 15 CHC patients with tumor burden over 3 cm received transplantation, the other 57 cases all underwent hepatectomy. Since multiple tumors>3 cm exceed Milan criteria, even for individuals with HCC, liver transplantation is not recommended as first-line treatment, and therefore, hepatic resection is preferred in these populations. After the median follow-up of 30.5(0–179) months, the median OS for LR was 27 months, while the median CSS for LR was 30 months. Overall patient survival for those undergoing LR was 66.7 %, 38.2 %, and 33.7 % at one, three, and five years, respectively, and the one-, three-, and five-year CSS was 71.1 %,45.2 %, and 39.9 %, respectively. Due to the small number of cases in the LT group, we didn't report a median survival time. Among patients with tumor size>3 cm, there was no significant discrepancy in long-term survival between hepatic resection and transplant. .3.4vs. LR), tumor size, and AJCC stage (Ⅲ-Ⅳ vs. Ⅰ-Ⅱ) were likely independent factors of OS. Therefore, we selected these variables in the multivariate regression model. Multivariate analysis, which is consistent with survival analysis, revealed that LT confers an advantage for overall patient survival in people with CHC. . In addition, patients with advanced AJCC stage had poorer OS . However, the OS of CHC patients is unaffected by tumor size. .After survival analysis, independent factors affecting the prognosis of CHC patients were further explored. Univariate and multivariate analyses of the impact of patient demographics, tumor features, and clinical parameters on overall patient survival are shown in vs. Ⅰ-Ⅱ) were also positive CSS-related factors , tumor size, and AJCC stage  and some features of advanced tumors like macrovascular invasion, and portal vein tumor thrombus, are unknown. Furthermore, the long-term survival of LT for HCC patients 5In conclusion, liver transplantation confers an advantage in OS and CSS for CHC patients. Liver transplantation may be feasible for patients with CHC and should be taken into consideration. However, a sizable sample is required for future studies to determine which subset of CHC patients may benefit more from liver transplantation.National Key Technologies R&D Program (2018YFC1106800), the 10.13039/501100001809Natural Science Foundation of China , and the 1.3.5 project for disciplines of excellence, 10.13039/501100013365West China Hospital, Sichuan University (ZYJC18008).This work was supported by grants from the Because all patient information in the SEER database has been deidentified and is publicly accessible, the research involving the anonymous data from that database was exempt from medical ethics review and did not require informed consent.www.seer.cancer.gov) SEER*Stat Database: Incidence - SEER Research Data, 18 Registries, Nov 2020 Sub (2000–2018) - Linked To County Attributes - Time Dependent (1990–2018) Income/Rurality, 1969–2019 Counties, National Cancer Institute, DCCPS, Surveillance Research Program, released April 2021, based on the November 2020 submission).The datasets generated during and/or analyzed during the current study are available in the SEER repository. Program (Shizheng Mi: Software, Visualization, Writing – original draft, Writing – review & editing. Ziqi Hou: Formal analysis, Software, Validation. Guoteng Qiu: Formal analysis, Investigation. Zhaoxing Jin: Conceptualization, Software. Jiwei Huang: Conceptualization, Funding acquisition, Methodology.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


A maximum hydrogen yield of 2.34, 2.3, and 2.6 mol H2 mol−1 glucose, respectively, was obtained under the following set of optimal conditions: initial substrate concentration—10,000 mg L−1, initial pH—6.0, and ferrous sulphate concentration—100 mg L−1, respectively. The addition of trace metal to the medium (100 mg L−1 FeSO4·7H2O) enhanced the biohydrogen yield from 2.3 mol H2 mol−1 glucose to 2.6 mol H2 mol−1 glucose. Furthermore, the experimental data was subjected to kinetic analysis and the kinetic constants were estimated with the help of well-known kinetic models available in the literature, namely, Monod model, logistic model and Luedeking-Piret model. The model fitting was found to be in good agreement with the experimental observations, for all the models, with regression coefficient values >0.92.Mangrove sediments host rich assemblages of microorganisms, predominantly mixed bacterial cultures, which can be efficiently used for biohydrogen production through anaerobic dark fermentation. The influence of process parameters such as effect of initial glucose concentration, initial medium pH, and trace metal (Fe Fossil Fuels are the primary energy source for the world's increasing energy consumption. According to a recent survey, total world energy use rises from 524 quadrillion British thermal units (Btu) in 2010 to 630 quadrillion Btu in 2020 and to 820 quadrillion Btu in 2040 [Biohydrogen production through anaerobic fermentation is a sustainable alternate for the energy crisis and green environment –12. FermA kinetic model can adequately describe the relationship among the different state variables and explain the behavior of fermentation quantitatively by providing useful information that can be subsequently used for analysis, design, and operation of any fermentation process –22. The The sediments were collected from the mangrove rhizosphere of Pichavaram, Tamil Nadu, India, at a depth of 100 cm, and later stored in sterile polythene bags. Heat-shock treatment was done on this sediment sample, by constant heating at 110°C for 2 h, in order to stimulate spore germination and eliminate all vegetative cells, particularly methanogens. The coarse particles were removed using a stainless steel mesh, while the finer fractions were stored at 4°C .4Cl—0.5 mg, K2HPO4—0.25 mg, MgCl2·6H2O—0.3 mg, NiSO4—0.016 mg, CoCl2—0.025 mg, ZnCl2—0.0115 mg, CuCl2—0.0105 mg, CaCl2—0.005 mg, and MnCl2—0.015 mg.The nutrient medium (non-sterilized) used in this study had the following chemical composition (per litre): NH2+ concentration, was evaluated by carrying out experiments at different low to high levels of these parameters, and the average values of biohydrogen yield were presented. The pH of the growth medium was adjusted using 1N HCl or 1N NaOH during the start of the experiments. The growth medium was inoculated with 100 g of pretreated sediment under aseptic conditions, and the flasks were incubated at 35°C for fermentation.Batch tests were conducted in duplicate, in 1 L Erlenmeyer flasks (working volume: 0.7 L), fitted air-tightly with rubber septum, and adequately sealed using commercially available fix gels. The effect of process parameters on biohydrogen yield, namely, the influence of initial substrate concentration (glucose), initial pH, and trace metal, Feλmax of 550 nm [The biohydrogen gas was measured using wet gas flow meter . The gas content was analyzed using a gas chromatograph  equipped with a thermal conductivity detector (TCD), and the column was packed with dual packed column. The operating temperatures of the column, detector and injector, were 40°C, 80°C, and 50°C, respectively. Biomass concentration was measured as volatile suspended solid (VSS) and analyzed according to Standard Methods . Glucosef 550 nm . The sluBiohydrogen fermentation reached nearly constant values at the end of 120 h for each batch tests, including their duplicates. Glucose degradation efficiencies, cumulative biohydrogen gas, and hydrogen yields were calculated for each set of experimental condition.−1, the values of cumulative biohydrogen production and glucose degrading efficiencies were 430, 1190, 2600, 2200, and 2099 mL and 75, 83, 90, 80, and 72%, respectively . It was found that, under near neutral pH condition, a significant amount of substrates was consumed by bacterial growth other than hydrogen production which was verified by the higher biomass concentration at higher pH. Thus, it could be stated that the favourable pH for this mixed bacterial culture was 6.0. Similar results of maximum hydrogen production at the pH of 6.0 were reported [The profile of cumulative biohydrogen gas production at various initial medium pH conditions is shown in  enzymes . When thH of 6.0 . Initialreported .2+ concentrations. The values of cumulative biohydrogen production for five different Fe2+ concentrations: 100, 200, 300, 400, and 500 mg L−1 were 3040, 2800, 2610, 2300, and 1180 mL, respectively, and the corresponding substrate degradation efficiencies were 94, 92, 91, 90, and 80%. Hydrogen yields of 2.6, 2.3, 2.1, 1.8, and 0.9 mol H2 mol−1 glucose were obtained for various concentrations of iron as illustrated in −1 of Fe2+ concentration, the biohydrogen production was at its maximum (2.6 mol H2 mol−1 glucose), and it was found to decrease when the Fe2+ concentration was increased , μmax is the maximum specific growth rate (h−1), x is the cell concentration (g L−1), and Ks is the substrate consumption rate constant (g L−1). Equation 1μ=Kμmax and Ks were calculated as 0.166 h−1 and 0.112 g L−1 respectively.x). The Riccatti equation is given by the following equation:β = 1/xmax.The specific growth rate for the logistic curve relates the change of specific growth rate with respect to change in cell concentration , dx/dt is the specific growth rate (h−1), P is the product (biohydrogen production), x is the cell concentration (g L−1), YP/x is the growth associate product yield coefficient, and β is the non-growth associated product yield coefficient.The Luedeking-Piret model shown in  has beenYP/x) was calculated by plotting specific hydrogen production rate versus specific growth rate, and the value was found to be 11.04. From R2 value of 0.999.Scanning electron micrographs showed that the granules had multiple cracks with cavities on the surface . These cFurthermore, considering the practicality of this research work, microbiological analyses are warranted at this stage to characterize the dominant anaerobic consortium responsible for biohydrogen production. In general, kinetic models are applied in order to study and assess the metabolic features of defined cultures. Further studies in this field should be aimed at the following aspects: optimization studies with different innocula, substrates and process parameters, evaluation of the performance, and economics of a continuous biohydrogen production processes (bioreactors).2+ concentration had influence on the biohydrogen yield. Maximum biohydrogen yields were found to be 2.34, 2.3, and 2.5 mol H2 mol−1 glucose at the following conditions: initial substrate concentration—10,000 mg L−1, medium pH—6.0, and Fe2+ concentration—100 mg L−1, respectively. The addition of trace metal to the medium at a concentration of 100 mg L−1 was found to enhance biohydrogen production although higher metal ion concentrations reduced biohydrogen production. The kinetics of batch anaerobic hydrogen production was estimated by fitting the experimental data to the well-known unstructured kinetic models. The Monod model, logistic model, and Luedeking-Piret model were used to describe the kinetics of cell growth rate as a function of substrate, cell concentration, and product formation, respectively, in the hydrogen production process, and the corresponding kinetic constants were estimated. The results showed that high regression co-efficient values (R2) were obtained between the model fitted and the experimental observations for the different models, namely, as 0.976, 0.943, and 0.999, respectively.The results from batch tests showed that initial substrate (glucose) concentration, medium pH, and Fe


Dendrimers and telodendrimer micelles represent two new classes of vehicles for drug delivery that have attracted much attention recently. Their structural characterization at the molecular and submolecular level remains a challenge due to the difficulties in reaching high resolution when imaging small particles in their native media. This investigation offers a new approach towards this challenge, using scanning tunneling microscopy (STM) and atomic force microscopy (AFM). By using new sample preparation protocols, this work demonstrates that (a) intramolecular features such as drug molecules and dendrimer termini can be resolved; and (b) telodendrimer micelles can be immobilized on the surface without compromising structural integrity, and as such, high resolution AFM imaging may be performed to attain 3D information. This high-resolution structural information should enhance our knowledge of the nanocarrier structure and nanocarrier-drug interaction and, therefore, facilitate design and optimization of the efficiency in drug delivery. Amongcarriers –14. Specin alone . in vivo movement. Since polymer molecules are the basic unit within micelles, multifunctionalities may be implemented for individual molecules, and size may be tuned  by varying the conditions of assembly. The amphiphilicity enables the incorporation of hydrophobic drugs such paclitaxel (PTX) enclosed inside the micelles, with a load capacity as high as 7.3 mg/mL [in vitro anticancer activity of PTX loaded PEG5k-CA8 micelles have been performed on human ovarian clear cell carcinoma cells (ES-2) and firefly luciferase-expressing ovarian adenocarcinoma cell lines (SKOV3-luc-D3). PTX-loaded PEG5k-CA8 micelles exhibited equivalent cytotoxic activity in vitro compared with the clinical formulations of PTX, such as Taxol and Abraxane [In vivo antitumor efficacy of PTX loaded PEG5k-CA8 micelles have been tested in nude mice bearing human SKOV3-lue ovarian cancer xenograft, where the results indicated that this vehicle could deliver PTX preferentially to tumor sites via enhanced permeation and retention (EPR) effect, and thus exhibits superior in vivo anticancer effect overall in animal models, compared to Taxol and Abraxane [Complimentary to the “hard” dendrimers discussed above, “soft” nanostructures, such as nanomicelles, made by assembly of biocompatible telodendrimers - (PEG-) block-dendritic oligomers of cholic acid (CA)) in aqueous conditions were also developed recently –21. Thes.3 mg/mL . The in Abraxane . In vivoAbraxane , 17.  To enhance the efficiency of drug delivery, knowledge of the nanocarrier structure and nanocarrier-drug interaction is critical for their design and optimization. In the case of dendrimer systems, the location and the binding of drug molecules to dendrimers are particularly important, as the outcome is directly related to loading capacity and release behavior. While macroscopic information such as solubility –14 and s In the case of telodendrimer micelles, dynamic light scattering (DLS) allows the average diameter and distribution to be determined in the solution phase , 17. Ind5k-CA8 , in aqueous media where micelles form. The results are very encouraging: individual micelles are clearly visualized, from which we can extract the size and geometry of micelles in correlation with the conditions of assembly. The difference between native and drug carrying micelles is clearly visible under AFM, from which the drug carrying capacity can be estimated. In addition, the knowledge of the geometry and size of individual micelles facilitates our understanding of their efficacy and further optimization.In this study, we have tested the feasibility and demonstrated the proof-of-concept of using scanning probe microscopy to image PTX-loaded thiol modified telodendrimer micelles, HS-PEGn-octanethiol  were purchased from Sigma-Aldrich and used as received. 200 proof ethanol (99.99% purity) was purchased from Gold Shield Chemical Co. K2PtCl4  was used as received. Ultrapure water (≥8 MΩ·cm) was obtained using a Millipore Milli-Q filtration system. Ultrapure N2  and H2  were used for drying and flaming, respectively. STM tips were made from W wire . Epoxy glue (Epo-tek 377) was purchased from Epoxy Technology.Paclitaxel was purchased from AK scientific Inc. 4th generation hydroxyl-terminated poly(amidoamine) dendrimers, G4 PAMAM-OH , and 1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-3-indoleacetic acid, commonly known as indomethacin (≥99.0%), were purchased from Sigma-Aldrich and used without further purification. 1-adamantanethiol  and 5k-CA8 was synthesized following the established procedure [5k-CA8 telodendrimer was kept in desiccators before use.BocNH-PEGrocedure . The Boc−7 torr. The evaporation rate was 0.3 nm/sec and the final thickness of Au films was 150 nm. After evaporation, the Au was thermally annealed in situ at 375°C for 30–60 min to increase the size of the Au(111) terraces. After annealing, the Au film was allowed to cool for ≥5 hr. under vacuum. Upon removal, the Au films were stored in a sealed glass container.Au(111) thin films were prepared via thermal evaporation of Au onto freshly cleaved mica (0001) in a high-vacuum evaporator  . The sub2 flame in order to coalesce the gold grains on the mica. Then, the gold thin film was cooled in air to room temperature. A small droplet of epoxy glue was applied to each of the dry glass substrates (coverslips). The coverslips were then placed on the gold substrate with the glue attached side facing down. The glue was then cured at 150°C overnight. After removal from oven, the glass coverslip with gold thin film were peeled off from the mica substrate. The 1-adamantanethiol (AD) SAMs were prepared by immersing the gold films in a 10 mM ethanolic AD solution at room temperature for 24 hours [The gold films described above were used to prepare ultraflat gold films on glass substrates following a method reported previously . Briefly24 hours . The AD 5k-CA8 were dissolved in 3 mL of chloroform in a 10 mL single neck flask to form a homogeneous solution. The solvent was removed by rotavaporation, and the sample was further dried on high vacuum pump for 30 min. Then, 1 mL of phosphate buffered saline (PBS) was added into the flask to disperse the solid film via vortex and further sonication for 30 min to yield a homogenous micelle solution. The particle sizes of the micelles before and after PTX loading were measured with DLS Zetatrac (Microtrac) to be 16 nm and 23 nm, respectively. The drug loading capacity was measured using high-performance liquid chromatography (HPLC) calibrated with PTX solutions in dimethyl sulfoxide (DMSO) with known concentrations.6 mg of PTX and 20 mg of HS-PEG5k-CA8 and PTX-loaded HS-PEG5k-CA8 micelles in SAMs, tapping mode was utilized in aqueous solution. The probe is a MSNL-10 silicon cantilever  with a force constant of k = 0.1 N/m. During AFM tapping, the cantilever was modulated by a driving frequency of 68 kHz and an amplitude range from 0.30 to 0.71 V, with damping from 30 to 70%. When imaging PAMAM dendrimers, a silicon cantilever  was used. The probe has a force constant of k = 1.0 N/m as measured by thermal noise method. During tapping mode imaging, the cantilever was modulated by a driving frequency of 74 kHz and amplitude of 67.0 nm (0.63 V), with the damping set to 85%. For displacing adsorbates such as dendrimers or alkanethiolates, tips were placed in contact with the surface with increasing load beyond threshold [AFM measurements of micelles and dendrimers were performed on a MFP3D AFM . When imaging HS-PEGhreshold , 29. DatThe STM has a walker-type scanner  and was used under ambient conditions for this investigation. The STM tips were cut W wires which were electrochemically etched at 2.0 V in 3.0 M NaOH solutions. A homemade potentiostat monitored the etching process , 37. All5k-CA8 telodendrimer is soluble in water and self-assembles into micelles. PTX is loaded into the micelle via a procedure of solvent evaporation followed by the aqueous dispersion of micelles [ For structural characterization via AFM, micelles must be immobilized on surface supports. Immobilized drug delivery vehicles are the key component in therapies using patches . A potenmicelles . μM. The micelles have noncharged surfaces, the Zeta-potential was measured close to zero [5k-CA8) was used and the exposure time was typically 1 hour. This led to 29.0% surface coverage of PTX loaded micelle on the gold surface. After deposition, the samples were rinsed with Milli Q water and kept in the water solution before AFM measurement. In order to maintain the integrity of micelles on solid surface, gold surfaces were covered by SAMs of AD. The use of AD is based mainly on two considerations: (a) SAMs provide a buffer to dampen collisions and allow full contact between micelles and gold surfaces and (b) AD can be exchanged by alkane thiol functionalities to enable micelles to anchor onto gold surfaces. As illustrated in  to zero . AD SAMs2 AFM topography image of PTX-loaded micelles on ultraflat Au. Each bright protrusion is a single PTX-loaded micelle. The height of a typical PTX-loaded micelle, as shown in cursor 1, is 4.0 nm, measured from the lowest point in the local surroundings to the apex of the micelle. Its lateral boundaries are clearly visible from the AFM phase image shown in Figure  2 AFM topography image of original micelles on ultraflat Au. The measured height of the unloaded micelle is 1.9 nm, as shown in cursor 4. a, b, and h measure 23.7 ± 2.4 nm, 17.2 ± 2.3 nm, and 1.8 ± 0.2 nm, respectively.  Upon immobilization, AFM imaging is carried out in water media. To attain accurate measurements in 3D without significant deformation, tapping mode is utilized, from which height is extracted from topographic images, and lateral boundaries are well defined from phase images. The AFM images in V, of each micelle can be calculated using the simple geometric formula: V = (1/6πh)(3/4ab + h2)  [a = 33.0 nm, b = 28.1 nm, thus V = 1490.1 nm3. From Figures  a = 25.2 nm, b = 17.3 nm, and h = 1.9 nm, which corresponds to V = 328.9 nm3. The average volume of PTX-loaded micelle and unloaded micelle is 1475.8 ± 396.2 nm3 and 295.1 ± 62.6 nm3, respectively. PTX-loaded micelle exhibits a larger volume than unloaded micelle. Our Investigations also reveal that the amounts of PTX uptake affect the volume of micelles.  The volume,  + h2)  . From FiV = 4/3π(D/2)3. Here, V is volume and D is the diameter. The volume of a typical PTX-loaded micelle in Figure  3, the corresponding diameter is 14.2 nm. Among the 49 PTX-loaded micelles measured, the average diameter is 14.2 ± 1.2 nm. The volume of a typical unloaded micelle in Figure  3, the corresponding diameter is 8.6 nm. Among the 50 unloaded micelles measured, the average diameter is 8.2 ± 0.6 nm. The diameter of PTX-loaded and unloaded micelle obtained from the dynamic light scatting (DLS) measurement is 23 ± 8 and 16 ± 4 nm, respectively [ By assuming that the micelle has a spherical shape in water solution, we can estimate the diameter of micelles in solution based on ectively , while ANptx) and telodendrimers (Ntelo) are estimated from two equations below, (a) assuming that the volume of individual components are conserved, based on Connolly solvent-excluded volume [3, and PTX being 0.754 nm3; (b) the mixing follows 7.5: 2.1 = PTX: telodendrimers. Therefore, for a typical PTX loaded micelle in Figure  3 As a bonus, we can estimate the number of PTX molecules based on the volume measurements from AFM. The numbers of PTX 1), Nptx5k-CA8 micelles, AFM images may be attained in their native media. This versatility of imaging in water media at a designed temperature allows direct comparison before and after loading or uptake of drugs. In addition, the accuracy enables quantification, such as the determination of height, lateral dimension, volume, and number of drugs enclosed. Therefore, we encourage researchers to consider the application of AFM in determination of the size and geometry of drug-carrying vehicles in the various synthetic and drug-loading steps.Taken collectively, AFM provides an alternative and more accurate approach to measure the geometry and size of individual drug delivery vehicles. Even for soft systems such as HS-PEGμM aqueous solutions. Second, as illustrated in 2PtCl4 was then added to achieve a molar ratio of 1 : 120, dendrimer: Pt2+. The ratios were guided by the number of tertiary amines (dendrimer branch points) within individual dendrimers, for example; G4 has 62 tertiary amines. Once mixed, the solution was kept at room temperature for 3–5 days, allowing sufficient time for Pt(II)-amine coordination within dendrimers [2+)n-(Indo)m, as represented in Scheme  To visualize intramolecular structure of PAMAM dendrimers using STM, two key steps are involved, surface immobilization and introduction of metal ions to enable the transport of STM current . Detailendrimers . Third, 2 pieces of gold films were H2-flamed [μL drop of G4 PAMAM-OH-(Pt2+)n-(Indo)m dendrimer solution was deposited onto the Au(111) surface and allowed to contact for 1.25 min. After washing with water and ethanol the surface was flooded with a 1.0 mM C8 solution for 2 min. The surface was then washed again with ethanol and dried under N2 before STM and AFM imaging. The formation of C8 SAMs confines dendrimers laterally, thus maintaining the structural integrity, and prevents lateral movement during scanning [ For the surface deposition of dendrimers, as shown in Scheme  2-flamed  and alloscanning . SAMs alhAPP), is a sensitive indicator of surface features, the accuracy is difficult to gauge due to the difficulties in determining the LDOS contribution. Therefore, AFM is frequently utilized for the same sample to determine the height of dendrimers [hAPP and true height with this combined approach [ While STM enables high-resolution imaging and accurate determination of the lateral dimension of individual dendrimers , AFM allndrimers . As illundrimers . Our preapproach , 29.2+)n-(Indo)m dendrimers are taller than the bare G4 dendrimers. Figure  2 STM topograph of G4 PAMAM-OH-(Pt2+)n-(Indo)m dendrimers immobilized on a Au(111) surface. The bright protrusions correspond to individual G4 PAMAM-OH-(Pt2+)n-(Indo)m dendrimer molecules. The STM apparent height, or hAPP, is obtained by measuring the height from the lowest point in the immediate surrounding matrix to the top of the dendrimer. These cursors indicate that dendrimers loaded with indomethacin adopt an elliptical dome shape similar to the base dendrimers reported previously [hAPP in cursor profiles 1 and 2 is 0.43 and 0.48 nm, respectively. In contrast, the hAPP of a typical G4 dendrimer, as shown in Figure  hAPP by 0.08 nm. Among the 102 dendrimers we compared, drug-loaded G4 dendrimers appear 0.09 ± 0.02 nm taller than the dendrimers themselves. The true height is further investigated using AFM as described in the previous section. The typical real height (hREAL) for G4 and indo-G4 complexes are 2.5 ± 0.3 nm and 3.4 ± 0.7 nm, respectively. eviously , 47, 48.V = (1/6πhREAL)(3/4ab + hREAL2), where a and b are the long and short lateral axes, respectively. In a typical case shown in a = 5.6 nm, b = 4.2 nm for the indomethacin-loaded G4 and the height is 3.4 nm, thus V = 52.3 nm3. From Figure  a = 5.7 nm, b = 5.2 nm, and hREAL = 2.2 nm, which corresponds to a V = 31.2 nm3. Among the 102 dendrimers compared, drug-loaded dendrimers are 54% more voluminous than base dendrimers. The average lateral dimensions are a = 6.8 ± 1.2 nm and b = 5.6 ± 0.9 nm for indomethacin-loaded G4 and a = 6.2 ± 0.8 nm and b = 5.1 ± 0.7 nm for unloaded G4. Since the lateral deformation of both loaded and unloaded G4 dendrimers are similar, the height, and thus volume, increases observed with the addition of indomethacin suggest that the addition of indomethacin to the exterior of dendrimers increases the overall structural integrity upon surface immobilization. After measuring the lateral and vertical dimensions, the volume of dendrimers can be accurately determined and compared. Assuming an elliptical cap geometry for all dendrimers, the volume of individual molecules may be calculated using 2+)n-(Indo)m dendrimers (2+)n-(Indo)m dendrimer surface and the inset is a high-resolution image of a single dendrimer in which the intramolecular features are clearly visible. At first glance, these intramolecular features fall into two types of contrast; that is, one appears brighter than the other. Both previous and present studies of G4-dendrimer reveal the apparent height of −OH termini to be below 0.11 nm [2+)n-(Indo)m dendrimers analyzed, indomethacin features have a hAPP range = 0.12–0.25 nm with an average hAPP = 0.16 nm. In contrast, intramolecular feature hAPP measured on indomethacin-loaded and unloaded metal ion-doped G4 PAMAM-OH dendrimers ranged 0.03–0.10 nm, among 40 dendrimers measured previously [2+)n, where intramolecular features, or −OH termini, are clearly visible [2+)n-(Indo)m dendrimers carry 5–7, 2–14, and 2–19 drugs, respectively [ The indomethacin is distinctly recognizable in STM topographs, because they appear taller and usually broader than the −OH termini of dendrimers. ndrimers  appear bndrimers . Figure  0.11 nm , 29. Theeviously  and in t visible . The numThis study demonstrates the significance of using STM and AFM in the fundamental studies of new drug-delivery vehicles, telodendrimer micelles and PAMAM dendrimers. The preliminary results indicate that the exquisitely high-resolution images enable insightful and fundamental information be revealed in the context of molecular level location and load of drug molecules, as well as the stability of drug-carrier complex. The number of drug molecules per carrier can be directly extracted in the case of dendrimers and estimated in the case of telodendrimer micelles. Since those studies are at the individual carrier's level, the results can be directly linked to simulations which shall facilitate the prediction and design of new carriers.


Based on the food Se concentrations and the daily per-capita consumption, the estimated daily Se intake in Shadi was 550 ± 307 µg per capita. Moreover, the Se concentrations in the hairs of local adult residents were 3.13 ± 1.91 mg kg−1 (n = 122) and 2.21 ± 1.14 mg kg−1 (n = 122) for females and males, respectively, suggesting that females might be exposed to higher levels of Se from daily cooking. Although there was no human selenosis occurrence in recent years, the high level of the daily Se intake suggested that the potential risk of selenosis for local residents, especially females, might be a matter of concern.Enshi is a high selenium (Se) region in Hubei, China, where human selenosis was observed between 1958 and 1963. This study investigated the daily dietary Se intake of residents in Shadi, a town located 72 km northeast of Enshi City, to assess the risk of human selenosis in the high Se area. Foods consumed typically by the local residents and their hair samples were analyzed for total Se concentration. Concentrations of Se in different diet categories were as follows: cereals: 0.96 ± 0.90 mg kg Selenium (Se) is an essential trace element for animals and humans . It play−1 in the Keshan disease area [−1 in the selenosis area in Enshi [−1 for both males and females [−1 [et al. [−1 or above, and the symptoms of selenosis occurred at the dietary Se intake of >910 µg day−1. In particular, 550 µg Se day−1 was considered as the high limit of the Se intake safe rate in high Se areas, such as in Enshi [The daily dietary Se intake depends on Se concentrations in foodstuff and the amount of food consumed. Moreover, food Se contents are highly dependent on the soil Se content, as well as the plants’ ability to take up and accumulate Se from the soil . Therefoase area  to 4990 in Enshi . The rec females , while tales [−1 . An earl [et al.  showed tin Enshi . −1, posing a potential chronic selenosis risk for local residents [et al. [−1versus high doses of 200–263 µg Se day−1 for a time period of one to 24 years, demonstrating that there were highly significant correlations between the Se concentration in muscle and that in whole blood (r = 0.90), red blood cells (r = 0.91), blood plasma (r = 0.87), hair (r = 0.89) and toe nails (r = 0.85). Indeed, these biological tissues can be good bio-indicators for assessing the Se status in the human body. Moreover, Gao et al. [There were 477 cases of human selenosis reported between 1923 and 1988 in different villages of Enshi. For example, there were 283 people suffering from hair and nail loss due to unknown etiology-endemic selenosis in 1963. In the Yutangba village, 19 out of 23 people were affected by high levels of Se, and their livestock died from Se poisoning. As a result, the village was evacuated in the late 1960s. Although no human Se toxicity has been reported in recent years, the symptoms of animal Se toxicity have been observed, including hoof and hair loss . Recentlesidents . Seleniuesidents . Behne eIn the present study, to prevent selenosis from happening again in the high Se area, such as Shadi, the commonly consumed foods were collected to determine Se concentrations and to estimate the Se daily intake based on their diet composition, and hair samples were also collected to directly monitor the Se-status in the residents’ bodies. −1 dry weight (DW) and a range of 11.26 to 27.50 mg kg−1 DW [Samples were collected from Shadi, one of the areas with the greatest number of human selenosis cases . Shadi h kg−1 DW . The samn = 66), corn (n = 124), tuber (n = 18), major types of vegetable (n = 69), chicken (n = 12) and egg (n = 12) samples were purchased from local residents of Laoxiongpo, Beifengya and Sangshupo in Shadi. Meanwhile, 122 hair samples from males and 122 hair samples from females were also collected from those local residents. Approximately 1.0 g of hair samples (about 1–3 cm length) was collected using stainless-steel scissors from the nape (or back of the neck) to the scalp  region.Rice  of vegetables were washed in distilled water, air-dried and then ground into fine powder to homogenize. For cereal samples, about 200 g of threshed rice were oven-dried at 55 °C, ground using a stainless steel grinder and then stored at 4 °C till chemical analysis. Chicken tissue samples were rinsed in distilled water, air-dried, blended using a pulp refiner and then stored. Hair samples were washedin acetone and distilled water three times, air-dried, cut into small pieces and stored for acid digestion.The edible parts  was diluted using 5% HCl to 10 mg L−1 Se working standard solution. The 1.0% (w/v) NaBH4 was prepared using 0.2% (w/v) NaOH solution. All used chemicals  were analytical grade or above and manufactured by Sinopharm Chemical Reagent . Ultra pure water from a Millipore Milli-Q system  was used for the preparation of all chemical solutions. A 1000 mg Let al. [3–HClO4  in a 50 mL conical flask at room temperature overnight. The digestion solution was heated on an electrical hot plate at 100 °C for 1 h, 120 °C for 2 h, 180 °C for 1 h and then kept at 210 °C until a white fume formed. The final volume of the digestion solution was approximately 2 mL. After acid digestion, the solution was cooled to room temperature, then 5 mL of concentrated HCl (12 M) were added to reduce Se (VI) to Se (IV) within at least 3 h. The selenite-Se solution was transferred into a 25 mL volumetric flask and made up to 25 mL by Milli-Q water. The Se concentration was determined by Hydride Generation Atomic Fluorescence Spectrometry (HG-AFS) 9230 , using NaBH4 as a reducing agent. Two standard reference materials, shrub leaves (GBW 07603-GSV-2) and hair (GBW 07604-GSV-3), prepared by the National Institute of Minerals of China, were used as the Quality Control/QC samples. The measurement of relative standard deviation (RSD) and the instrument detection limit were 0.76% and 0.08 µg kg−1, respectively.The total Se concentrations in samples were determined according to the method previously reported by Gao et al. . In brieThe data were analyzed by the ANOVA procedure at the 0.05 level of significance. The multiple comparison of means was done by the Scott Knott test.−1 DW and 0.43 ± 0.55 mg kg−1 DW, respectively  Vegetables: ranging from 0.23 ± 1.00 mg kg−1 in carrot to 1.57 ± 1.06 mg kg−1 in kidney bean; (3) Animal proteins: 1.99 ± 1.11 mg kg−1 in chicken and egg. Due to significant variation in soil Se distribution, concentrations of Se in cereals also varied significantly, showing that cereals from Laoxiongpo had the highest levels of Se accumulation. Therefore, compared with other regions of China  for males and 3.13 ± 1.91 mg kg−1 (n = 122) for females. The average Se concentration in female hair was significantly higher than that in male hair (p < 0.05), and the high Se concentrations were frequently observed in adult female hair samples, which might be a result from the fact that women had higher levels of exposure to Se from their daily cooking. Actually, local high Se coals were often used for in-door cooking in the present study area. Earlier studies showed that hair Se content from people with chronic selenosis was 32.2 mg kg−1, but 3.7 mg kg−1 from people without selenosis in the same high Se area [−1, except for some female hair samples. The hair Se concentrations in local residents were 2.21 ± 1.14 mg kg Se area . In this−1 in Beifengya, 3.07 ± 1.52 mg kg−1 in Laoxiongpo and 2.48 ± 2.00 mg kg−1 in Sangshupo (p < 0.05). Meanwhile, there was no significant (ANOVA) correlation between the hair Se and the rice Se . It was possible that the level of Se in hair was dependent on the environmental conditions and the Se intake during a long time period. Although, in the introduction, it was noted that in one study, Se in muscle was correlated even more highly with hair Se than with plasma Se. These findings suggested that Se concentration in hair might not be a good bio-indicator for daily Se intake. Concentrations of Se in hair samples collected from the three villages was 2.40 ± 1.43 mg kgangshupo . Althoug−1 DW [−1 in 1966, three years after the reported selenosis [−1 DW [−1 in corn [−1 during the late 1990s [−1. Similar trends were found in other plant species, such as rice, bean, carrot, garlic, hyacinth bean, Chinese cabbage, pumpkin, eggplant, kidney bean and potato. Such a substantial decrease in Se accumulation in crops in Enshi since 1963 was a result of reducing the utilization of Se-coal stone ash (up to 8390 mg kg−1) [To figure out the change trends, data on Se contents in local corn  from 196−1 DW . The aveelenosis . In the s [−1 DW ,38 and r in corn ,40. Howete 1990s ,35. In tmg kg−1)  on agricmg kg−1) . As showet al. (1996) indicated that the value reduced to 1338 µg in 1985 [In 1966, the daily Se intake in Enshi was 4990 µg with a range of 320 to 6690 µg . Yin et  in 1985 . The pre−1) were significantly higher than that in male hair (2.21 ± 1.14 mg kg−1), suggesting that the high levels of Se exposure for women would be an important matter of concern in future Se risk assessment in Shadi. Although concentrations of Se in foods and Se daily dietary intake have greatly decreased over the past 40 years, the potential threat of selenosis to local residents still exists. More research is needed to further assess the Se-status of local residents, such as Se in plasma.Based on diet composition, the Se daily dietary intake of residents of Shadi was calculated as 550 ± 307 µg per capita, equal to the suggested toxic Se intake dose. Moreover, the Se concentrations in female hair (3.13 ± 1.91 mg kg


P) was investigated using only one descriptor, the semi-empirical electrotopological index (ISET). The predictability of four octanol/water partition coefficient (log P) calculation models was compared using a set of 131 aliphatic organic compounds from five different classes. Log P values were calculated employing atomic-contribution methods, as in the Ghose/Crippen approach and its later refinement, AlogP; using fragmental methods through the ClogP method; and employing an approach considering the whole molecule using topological indices with the MlogP method. The efficiency and the applicability of the ISET in terms of calculating log P were demonstrated through good statistical quality , high internal stability and good predictive ability for an external group of compounds in the same order as the widely used models based on the fragmental method, ClogP, and the atomic contribution method, AlogP, which are among the most used methods of predicting log P.A new possibility for estimating the octanol/water coefficient (log  P) of compounds, which is a measure of hydrophobicity, is widely used in numerous Quantitative Structure-Activity Relationship (QSAR) models for predicting the pharmaceutical properties of molecules | = 0.6319Aμ = 1 + log[1 + (2.6790/0.63191)] = 1.7193ISETO1 = (=O) = AμSETO1 + log AμSETC3 = 1.9507 + log 0.3899 = 1.5416ISETC1 = (−CH3) = SETC1 + log SETC2 = 0.9892 + log 0.9998 = 0.9891ISETC2 = (−CH2−) = SETC2 + log SETC1+ log AμSETC3 = 0.9998 + log 0.9892 + log 0.3899 = 0.5860ISETC3 = (>C<) = AμSETC3 + log SETC2 + log AμSETO1 + log SETC4 = 0.3899 + log 0.9998 + log 1.9507 + log 0.9998 = 0.6799ISETC4 = (−CH2−) = SETC4 + log AμSETC3 + log SETC5 = 0.9998 + log 0.3899 + log 0.8988 = 0.5444ISETC5 = (−CH2−) = SETC5 + log SETC4 + log SETC6 = 0.8988 + log 0.9998 + log 0.9998 = 0.8986ISETC6 = (−CH3) = SETC6 + log SETC5 = 0.9998 + log 0.8988 = 0.9535ISET = 1.5416 + 0.9891 + 0.5860 + 0.6799 + 0.5444 + 0.8986 + 0.9535 = 6.1931P values using ISET are shown in ISET method give good agreement with the experimental partition coefficients. The QSPR models obtained with ISET showed high values for the correlation coefficient (r > 0.99), and the leave-one-out cross-validation demonstrate that the final models are statistically significant and reliable (rcv 2 > 0.98). As can be observed, this model explains more than 99% of the variance in the experimental values for this set of compounds. Among the various classes of compounds the best results obtained with the ISET method are for hydrocarbons  and for alcohols the correlation was greater (s = 0.18). The range of standard deviations obtained verifies the applicability of the present approach to different classes of organic compounds. For alcohols, the earlier approach of Duchowicz et al. [et al. [P for 58 aliphatic alcohols using novel molecular indices based on graph theory, by dividing the molecular structure into substructures obtaining models with good stability and robustness, and values predicted using the multiple linear regression method are close to the experimental values (r = 0.9959 and s = 0.15). The above results show the reliability of the present model calculation based on the semi-empirical calculation of atomic charges and local dipole moments using only one descriptor, ISET.The present results can be compared with those recently published for a new approach based on the Kovats retention indices, which uses multiple linear regressions , where r [et al.  carried P calculation models and ISET for a set of 131 aliphatic organic compounds from five different classes are summarized in r > 0.98 and s < 0.22). The ClogP method offers good predictability (r > 0.99 and s < 0.17), giving larger deviations only in the case of ketones . The MlogP and Ghose/Crippen methods have much larger deviations (r > 0.974 and s < 0.39) in comparison with the other methods.The statistical analysis for the predictive ability of four log P values using ISET and the other four models (and the respective deviations) for an external group of alcohols are shown in P, being unable to distinguish the structural differences between these branched alcohols. The average standard deviation of calculated Log P for the seven alcohols of ISET model is 0.15, whereas for the Ghose/Crippen method it is 0.34. The AlogP method, which is applicable to most neutral organic compounds and selective charged compounds, shows an average standard deviation of 0.26. In contrast, the ClogP method, which uses a large number of parameters and correction factors, results in a standard deviation of 0.17, while for the whole molecule approach the value is 0.24. These results demonstrate that the predictability of the present model for polar aliphatic organic compounds has the same pattern of accuracy as the widely used ClogP model.The experimental and predicted log r2 (q2) alone is insufficient criterion for a QSAR model to be considered highly predictive, and the use of an external set of compounds for the model validation is always necessary. The authors’ state that the correlation coefficient, r, between the predicted and observed activities of compounds from an external test set should be close to 1 [vs. predicted log P values obtaining Y = 1.0273X − 0.1223 with r2 = 0.9858 and Y = 0.9893X (with the intercept set to 0) with r2 = 0.9842. Predicted vs. observed log P values, Y = 0.9596X + 0.1557 with r2 = 0.9858 and Y = 1.008X with r2 = 0.9828 were plotted. The QSPR model has a value of cross-validated (using leave-one-out), rcv 2 = 0.9870 showing that the model has high predictive power.The predictive ability of a QSPR model can be estimated using an external test set of compounds that has not been used for building the model. According to Tropsha and Golbraikh  a high vose to 1 ,41. FollISET in terms of predicting log P using the quantitative structure-activity relationship (QSPR) were demonstrated through the good statistical quality and high internal stability obtained for the studied classes of compounds as well as the good predictive ability for the external group of compounds. The ISET model also has the advantage of simplicity, using only one descriptor, and it has statistical quality of the same order as the widely used models based on the fragmental method, ClogP, and the atomic-contribution method, AlogP. The quality of the results obtained can be considered appropriate for the development of QSPR models for other compounds in the future.The efficiency and the applicability of the descriptor


Salmonella choleraesuis and the Gram (+) organisms Staphylococcus aureus and Bacillus subtilis. The MIC values ranged from 4.96 to 37.32 mg/mL. The Triplaris gardneriana extract presented activity against the three species, with MIC values 18.8, 13.76, and 11.15 mg/mL, respectively. Five extracts presented antioxidant activity, with EC50 values ranging from 69.73 μg/mL (T. gardneriana) to 487.51 μg/mL (Licania rigida). For the anticholinesterase activity, eleven extracts were capable of inhibiting the enzyme activity. From those, T. gardneriana, Parkia platycephala and Connarus detersus presented the best activities, with inhibition values of 76.7, 71.5, and 91.9%, respectively. The extracts that presented antimicrobial activity were tested for hemolytic assay against human A, B, and O blood types and rabbit blood. From those, only the Myracrodruon urundeuva extract presented activity . Infrared spectroscopy of six representative extracts attested the presence of tannins, polyphenols, and flavonoids, which was confirmed by a qualitative phytochemical assay.The antimicrobial, antioxidant, and anticholinesterase activities of ethanolic seed extracts of twenty-one plant species from Brazilian semiarid region were investigated. The extracts were tested for antimicrobial activity against six bacteria strains and three yeasts. Six extracts presented activity against the Gram (−) organism  Plants are considered natural sources of new compounds of medical and biotechnological interest, since they synthesize a large variety of bioactive compounds. An analysis into the sources of new drugs from 1981 to 2007 reveals that almost half of the drugs approved since 1994 were based on natural products. Among these, there are various examples of development of new drugs from plant sources . Well-kn2 in Northeastern Brazil, and out of 1,000 vascular plant species described many are endemic , where As is the absorbance of the solution when the sample has been added at a particular level and ADPPH is the absorbance of the DPPH solution. The percentage of the remaining DPPH was plotted against the sample/standard concentration to obtain the amount of antioxidant necessary to decrease the initial concentration of DPPH by 50% (EC50). Based on the parameter EC50, the result was expressed in terms of μg of seed extract per mL of DPPH in the reaction medium. All determinations were run in triplicate.The capacity to scavenge the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was monitored . The dilμL of 15 mM acetylthiocholine iodide (ATCI) in water, 125 μL of 3 mM DTNB in 50 mM Tris-HCl buffer containing 0.1 M NaCl and 0.02 M MgCl2·6H2O, 50 μL of 50 mM Tris-HCl buffer containing 0.1% BSA, and 25 μL of plant extract sample (5 mg·mL−1 in MeOH diluted ten times with 50 mM Tris-HCl buffer to give a concentration of 0.5 mg·mL−1) were added and the absorbance was measured at 405 nm every 30 s for three times. Then 25 μL of 0.22 U·mL−1 of acetylcholinesterase from electric eel was added and the absorbance was again read every 45 s for eight times. Any increase in absorbance due to the spontaneous hydrolysis of the substrate was corrected by subtracting the rate of the reaction before the addition of the enzyme from the rate of the enzyme reaction. The percentage of inhibition was calculated by comparison of sample rates to a blank.The anticholinesterase activity of the ethanolic extracts was measured as described by , with slμg·mL−1 and reserved. Then, 100 μL of a 1% red blood cells  suspension was added to a new microtube containing 900 μL of each seed extract dilution, which was then taken to incubator at 37°C for 1 h. After that, the tubes were centrifuged at 3,000 ×g for 5 min. The supernatant (200 μL) was placed in a 96-well plate and led to a microplate reader  to measure the absorbance at 540 nm. The cell suspensions of each human blood type or those of rabbits (100 μL) were mixed with distilled water or 0.9% NaCl (900 μL) to obtain the absorbances for 100 and 0% of cell lysis, respectively. The percentage of hemolysis was calculated as follows: % hemolysis = Abstest/Abspc× 100, where Abstest = Abs540 of the 1% cell suspension treated with sample test and Abspc = Abs540 of the 1% cell suspension treated with distilled water. To calculate the relation between percentage of hemolysis and seed extract concentration, the hemolytic activity was expressed as the lowest seed extract concentration (μg·mL−1) capable of causing hemolysis ≥ 20%. All determinations were run in triplicate.The test was performed in 1.5 mL microtubes following the method described by  with somȞmax) were reported in wavenumbers (cm−1). The spectra were used to determine organic groups indicative of the main classes of secondary metabolites.The infrared spectrum was recorded from KBr discs on a FTIR Perkin Elmer model 16 PC spectrophotometer . Absorption maxima , leucoanthocyanidins , flavonoids and xanthones (reaction of magnesium granules with hydrochloric acid), steroids and triterpenes , saponins (foam production after water solubilization and stirring), and alkaloids  . These aE. aerogenes, K. pneumonia, and P. aeruginosa nor against the yeasts C. albicans, C. krusei, and C. tropicalis. MIC values obtained against the Gram (+) organisms S. aureus and B. subtilis, and the Gram (−) organism S. choleraesuis are shown in S. obtusifolia presented the lowest MIC value (4.96 mg·mL−1) against the pathogenic bacteria S. aureus. However, the species T. gardneriana showed the broadest spectrum of activity, being active against B. subtilis, S. choleraesuis, and S. aureus with MIC values of 18.8, 13.76, and 11.15 mg·mL−1, respectively. Although there are a great number of studies reporting antimicrobial activity of plant parts like leaves, roots and stems, antimicrobial screenings using specifically seed extracts are very rare. The reason for that is not easy to explain, since seeds are admittedly a rich source of compounds involved in plant defense [Bixa orellana that has been described to have antimicrobial activity against six strains of bacteria, among these are B. subtilis, S. aureus and P. aeruginosa, and against the yeast C. albicans [Paullinia cupana) seed extracts presented strong antimicrobial activity against a huge number of damaging fungi and bacteria [Syzygium jambolanum seed extracts against the same bacterial strains used in this work , even when compared to the best result observed as described above for S. obtusifolia (MIC 4.96 mg·mL−1). In addition, the S. jambolanum seed extracts were also effective against damaging-fungi strains. The low potency of the ethanolic seed extracts could be due to the chemical composition of the samples, with low concentration of antimicrobial substances. In fact, the results of phytochemical study revealed that all seed extracts with activity against bacteria showed in their composition classes of secondary metabolites widely known as efficient antimicrobial compounds  [The ethanolic seed extracts did not show activity against the Gram (−) organisms  defense . A good albicans . Likewisbacteria . As to tM. urundeuva caused hemolysis in all blood types  employed, being the lowest concentration tested (1.9 μg·mL−1) capable of causing more than 20% hemolysis. These results show that although the antimicrobial extracts are not very potent, they are more specific to bacterial cells, except for M. urundeuva seed extract. It is well reported that some classes of plant secondary metabolites are potent hemolytic compounds [M. urundeuva seed extract in the phytochemical study.The hemolytic activity assay was performed with the seed extracts that were active against the microorganisms used. Among six species tested, only ompounds , such asHymenaea courbaril (EC50 247.95 μg·mL−1), L. rigida (487.51 μg·mL−1), L. tomentosa (216.72 μg·mL−1), and T. gardneriana (69.73 μg·mL−1). The L. tomentosa and T. gardneriana seed extracts were more potent than the positive control vitamin C (EC50 260.27 μg·mL−1) and the second one was just a bit less potent than quercetin (EC50 55.52 μg·mL−1), one of the most potent antioxidant molecules. A previous study [50 value of 111.99 μg·mL−1. This promising result for T. gardneriana could be attributed to components such as tannins, flavones, flavonoids, and especially phenols [The results for antioxidant capacity are also shown in EC50 260. μg·mL−1  phenols , 29. FurT. gardneriana, P. platycephala and C. detersus extracts presented the strongest activity, with inhibition values of, respectively, 76.7, 71.5, and 91.9% at the final concentration of 500 μg/mL. These values are significant when compared to those obtained in previous studies [T. gardneriana showed the presence of chalcones and aurones. Therefore, further studies should be conducted to assess the potential of these extracts and their respective molecules to inhibit AChE enzyme and thus offer a potential to fight neurodegenerative diseases such as Alzheimer's.Concerning the anticholinesterase activity , among t studies , which h studies . Many st studies , but rec studies , flavone studies , and xan studies , as well studies . Thus, tS. obtusifolia, P. platycephala, L. rigida, L. tomentosa, and T. gardneriana) to confirm the results of phytochemical qualitative study. The phytochemical studies with these ethanolic extracts showed the presence of tannins and flavonoids, which were confirmed by infrared spectroscopy  responsible for this activity.This study revealed antibacterial, antioxidant, and anticholinesterase activities in some seed ethanolic extracts of Brazilian semiarid region plants, emphasizing the antioxidant activity presented by


We comp Equally, a perspective can be related to the spatial location of an object; work on frames of reference has focused on people’s sensitivity to whether an object is located above or below, or to the left or the right of someone . Such processing must take into account complex relationships between individuals and objects within their spatial environment. Recently, a number of studies have looked to identify the different processes for visuo-spatial perspective-taking. Difficulty based on angular disparity could be indicative of three different types of rotational strategies. The first is a mental self rotation, which uses an embodied representation of the self that is then rotated to the current bodily position of the target perspective . Such a process uses motor representations to imagine transporting ourselves to another’s position  and thenThe aim of the current study was to test whether embodiment is also used in visual perspective-taking. In the current study, we adapted Kessler and colleagues’  body posParticipants were 40 undergraduate students  from the University catholique de Louvain, Belgium. They all participated in the study in exchange of course credit or a small honorarium of 8 Euros. Participants had an average age of 20.77 years (range 18–25). One participant was not included in the final sample on the basis of performing below chance.Figure 1). The stimuli were created using Blender . The room also contained a single cube, with a numeral written on its top-most face . Within each stimulus, we varied two features orthogonally. Angular disparity between the participant and the avatar was varied through the positioning of the virtual camera in relation to the avatar, creating angles of four different magnitudes: 0°, 60°, 120°, 180°. For angular disparity of both 60° and 120°, separate stimuli were created showing the avatar in clockwise and anticlockwise variants, crucial for evaluating our embodiment hypothesis. We also varied distance, by placing the block at one of two distances from the avatar: “Near” and “Far,” where the Far condition was placed at a distance that was twice as far within the virtual world as the Near condition. In the spatial condition, an equal number of stimuli placed the block/number to the left and to the right of the avatar, at an angle of 45° from the avatar and always in front of him. In the visual condition, stimuli showed the block/number to be directly in front, or directly behind the avatar.In all of the pictures that participants saw, an avatar was placed in the center of a featureless room . Participants were first of all cued with a picture showing a red or blue square with a schematic illustration of a person . The blue picture conveyed the same instruction, but to the right/clockwise. These rotations meant that participants’ own body orientation varied from approximately 60° clockwise to approximately 60° anticlockwise in relation to the screen for every trial. Importantly, though, by keeping their chins on the chin rest, participants’ visual impression did not change (beyond the variations in the stimuli type presented on the screen). Following the rotation cue, participants saw a further screen, asking whether they had made the rotation, this required a mouse click to progress. The experimenter observed a sample of these rotations and saw no cases in which participants made errors in their rotations . Following this stimulus, the standard trial sequence . A fixation cross was followed by a cue . This cue was followed by the picture itself. In response to the picture, participants pressed the left mouse key to indicate that it matched the cue and the right mouse key to indicate that it did not. Participants received feedback during practice, but not during the experiment itself.Participants were randomly divided into two groups, Visual or Spatial. Before the experiment, all participants were given the same basic information, that they would be performing a perspective-taking task. Participants were sat in a rotating chair, with a red rectangle attached to the floor at approximately 60° angle to their right and a blue rectangle at approximately 60° to their left. They placed their chin on a chin-rest (located 50 cm from the screen) on every trial. After further instruction, giving example procedures and the correct answer, all participants completed 16 practice trials without rotation, 8 practice trials with rotation and finally 256 experimental trials divided into four blocks. All trials followed the same basic procedure . An equal number of trials of each of the Angles , of which the angles 60° and 120° were equally often clockwise or anticlockwise rotations. An equal number of stimuli showed each Distance . Finally, an equal number of trials required a left (red) or a right (blue) rotation. Note, that our variable of particular interest- the consistency between avatar and participant rotation- was varied through a combination of Angle and Rotation. Consistent trials occur with a left rotation of the participant and an anticlockwise rotation of the avatar For the Visual condition, trials were again equally divided between Match and Mismatch trials. For Match trials, stimuli were varied exactly as above, save for the fact that the block/number was always directly in front of the avatar. For half of mismatch trials, “number mismatch trials,” the same stimuli were presented, with the block/number directly in front of the avatar, but the preceding cue being a different number to that seen by the avatar in the picture. In the other half, “location mismatch trials,” the cue would have been correct had the avatar been looking at the number, however, it was placed directly behind him. This manipulation meant that participants had to take into account the avatar’s view and could not use the rotation of the number alone as a cue to the correct answer.Figure 3).In summary, the Visual and the Spatial conditions for Match trials  were identical other than features necessary for the specific judgements. Both conditions included a range of angles from 0° to 180°, to confirm that rotation was being used for the task at hand. By manipulating the position of the participant in relation to the screen, and the positioning of the avatar on the screen, we varied consistency between body postures. This was independent of Angle, Distance, Rotation, Task Content , Number, Cue and Direction, so that any influence could only be the result of the congruency between the embodied state of the participant and the avatar , as were incorrect responses.Our first analyses investigated the effects of Angle and Distance on perspective-taking. Particularly important here are the effects of Angle and any interaction between Angle and Content. A linear effect of Angle would be representative of participants using some form of rotation to complete the task. This analysis does not investigate the embodied nature of the process.F = 10.46, p = 0.003, ηp2= 0.216, with shorter avatar-object distances processed more quickly1. There was also a main effect of Angle, F = 37.71, p < 0.001, ηp2 = 0.498, which represented a linear trend. There was also a main effect of content, with Visual judgements being responded to more quickly, F = 12.89, p = 0.001, ηp2 = 0.253.A 4 × 2 × 2 ANOVA with Response Time as a dependent variable, Angle  and Distance  as within subjects factors and content  as a between subjects factor revealed a main effect of distance, F = 7.43, p = 0.004, ηp2 = 0.163, revealed a different relationship with Angle for each Content. For both Visual, F = 41.36, p < 0.001, and Spatial, F = 31.83, p < 0.001, perspective-taking the relationship with Angle fitted a linear trend. We investigated this relationship further, by computing separate t-tests for adjacent angles for each content. For Spatial perspective-taking, the strongest effect was for participants being slower at 120° than 60°, t(19) = 5.67, p < 0.001, with a less strong, but still significant effect of 180° being slower still t(19) = 5.67, p = 0.003. Though responses at 0° were the slowest, these were not significantly slower than at 60°, t(19) = 1.361, p = 0.190. Visual perspective judgements showed a different pattern of performance . Here difference was greatest for judgements at 180° being slower than at 120°, t(19) = 4.75, p < 0.001. There was a trend for an effect of faster judgements at 60° than 0°, t(19) = 1.934, p = 0.068 and no significant effect between 60° and 120°, t(19) = 1.341, p = 0.196, though again the larger angle produced a numerically longer response time. The interaction between Distance and Content, F = 7.00, p = 0.012, ηp2 = 0.156, illustrated that there was an effect of Distance on Visual, F = 20.85, p < 0.001, ηp2 = 0.543, but not Spatial, F = 1.47, p = 0.705, ηp2 = 0.008, judgements.An interaction between Angle and Content, Table 1).Error rates across conditions were generally low and did not contradict the findings from response time  = 25.55, p < 0.001, ηp2 = 0.402, was moderated by an interaction between Angle and Content, F = 10.63, p = 0.002, ηp2 = 0.219. Over this smaller range of angles, the effect was only significant in the Spatial domain, F = 33.07, p < 0.001, ηp2 = 0.635, not the Visual domain, F = 1.69, p = 0.210, ηp2 = 0.081. There was no significant effect of Consistency, F = 1.30, p = 0.261, ηp2 = 0.033, but there was a trend for an interaction between Consistency and Content, F = 3.63, p = 0.064, ηp2 = 0.087. This illustrated a trend for Consistent trials being easier than Inconsistent, but only in the Spatial condition, F = 3.041, p = 0.097, ηp2 = 0.138 .Trials in which angular disparity was either 60° or 120° could be either Consistent or Inconsistent on the basis of whether participants have rotated their body to the left or to the right. Analysing this subset of trials with Consistency as an additional factor can test the role of embodiment. A 2 × 2 × 2 ANOVA was completed with Content as a between subjects factor and Consistency  and Angle  as within subjects factors. A main effect of Angle, In Experiment 1, Visual perspective-taking, but not spatial perspective-taking showed an effect of distance. This was surprising and had not been evidenced in our previous study , in whicper se) and tested only at angles higher than 90°, those which previous studies have found to show clearest embodiment effects  viewpoint rotation process. However, as the trends were non-significant, it also remains possible that our test was insensitive to differing embodied effects  from the University catholique de Louvain, Belgium. They all participated in the study in exchange for a small honorarium of 8 Euros. Participants had an average age of 21.93 years (range 18–26).The design of Experiment 2 was identical to that of Experiment 1, other than the following details. Instead of making responses to a preceding cue, here participants made a forced choice response. For Spatial perspective-taking, this meant pressing the left button on the mouse when the number was located to the left of the avatar and the right when it was to his right , as were trials in which response time was more than 2.5 standard deviations away from the mean .Again, trials in which participants made incorrect responses were excluded  = 19.88, p < 0.001, ηp2 = 0.399, 120° < 150° . This effect was moderated by an interaction with Content, F = 14.20, p = 0.001, ηp2 = 0.321, showing the effect of Angle was significant for Spatial judgements, F = 18.95, p = 0.001, ηp2 = 0.558, but not for Visual judgements, F = 1.11, p = 0.308, ηp2 = 0.069. There was no interaction between Angle and Consistency, or Angle, Consistency and Content, Fs < 1.10, ps > 0.307, ηp2 < 0.035.A 2 × 2 × 2 ANOVA with Content  as a between subjects factor, and Angle  and Consistency  as within subjects factors revealed an effect of Angle, was an effect of Consistency, F = 25.84, p < 0.001, ηp2 = 0.463. This was moderated by a significant interaction, with Content, F = 11.42, p = 0.002, ηp2 = 0.276. Investigating this interaction showed that while the size of this effect was numerically greater in the spatial condition, F = 18.79, p = 0.001, ηp2 = 0.566, it was also significant in the Visual condition, F = 15.31, p = 0.001, ηp2 = 0.505. As in Experiment 1, there was also an effect of Content, F = 9.79, p = 0.004, ηp2 = 0.246, such that Visual perspectives were processed more quickly.Crucially, there Across two experiments, we investigated the degree to which perspective-taking required mental rotation and the degree to which that rotation was embodied. We tested this for two very different kinds of perspective-taking. We found further evidence that an explicitly spatial task recruited mental rotation. When participants judged whether an object was to the left or the right of an avatar it became increasingly more difficult as the angle of the avatar’s body became increasingly more different from the participant’s position. In addition to this, we found evidence that this rotational process was an embodied self rotation, as has previously been shown by and inhibit a salient self-view of a turtle happily upstanding or disarmingly prostrate, we also need to mentally align how we see the world with how it is seen by the person with whom we are interacting. We replicate findings from our previous study  are achieved after judgements of whether or not someone can see something , how we deal with special cases in which other’s perceptual access is compromised and how experts overcome the costly nature of this perspective-taking process.When we interact in complex social environments we undertake complex visuo-spatial reasoning which may or may not involve thinking about the mental states of other people. Taxing judgements of how the world appears to someone else and what things are located to the left or the right of them seem to involve a comparable process of embodied self rotation. We imagine ourselves in the position of a target other. To do this we take as a starting point the current position of our own body as well as the visual input of a scene in front of us. Embodied perspective-taking processes are robust processes effective in generating visual perspectives of anyone whose basic perceptual apparatus is the same as ours and generating spatial perspectives of anyone who shares our basic anatomy. That is not to say that these processes are the same 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.


Candida isolates from patients with oral candidiasis (OC) were compared between studies conducted in 2006–2007 and 2012–2013.The isolation frequency and susceptibility to antifungal agents of oral Candida strain and the susceptibility against antifungal drugs as compared to those evaluated in 2006–2007.A total158 strains was isolated from 112 patients who visited Kagoshima University Hospital for the treatment of OC during the 14-month period from February 2012 and March 2013, and evaluated on the isolation frequency of each Candida isolates were C. albicans and C. glabrata. In addition, the proportion of the latter increased from 12.3% in the 2006–2007 study to 23.4% in the 2012–2013 study, while the proportion of the former decreased from 86.2% to 72.8%, respectively. C. albicans was isolated in almost all patients, while C. glabrata was only isolated concomitantly with C. albicans. Minimal inhibitory concentrations (MICs) were not significantly different between groups with a few exceptions. Candida isolates, of which MICs surpassed break points, apparently increased for miconazole and itraconazole against C. glabrata in the 2012–2013 study, but this was not statistically significant. As a result, more cases of autoimmune disease, a greater number of C. glabrata isolates, and higher resistance to azoles were seen in the 2012–2013 study than in the 2006–2007 study.There was a higher frequency of xerostomia as a chief complaint and of autoimmune disease in the 2012–2013 study than in the 2006–2007 study. More than 95% of C. glabrata infection, a causative fungus of OC, and in C. glabrata resistance to azoles, caution is needed in the selection of antifungal drugs for the treatment of OC.These data indicate that with recent increases in  Candida albicans resistant to fluconazole was reported to be frequently isolated from the oral cavity of acquired immunodeficiency syndrome patients. An epidemiological study was conducted on the susceptibility to antifungal drugs of Candida strains isolated from patients with invasive candidiasis [In the early 1990s, didiasis  and repodidiasis -6. Theredidiasis  and Repodidiasis -10. Howedidiasis .Candida strains is likely to change over time, as shown by the comparisons of previous studies conducted in different time periods. For example, C. glabrata was rarely isolated from AIDS patients with oral candidiasis (OC) or esophageal candidiasis in Sudo’s study published in 1997 [C. glabrata was detected in 12.3% of patients with OC in our study, in which itraconazole (ITCZ) was effective despite the existence of resistant C. glabrata against ITCZ [C. glabrata against ITCZ occurred with high frequencies of 50% and 72.7% in OC and non-OC patients of this study, respectively, [C. glabrata in only 1.0–9.3% of patients with mycosis, mainly deep-seated mycosis [Candida species and their resistant strains varies over time, but no study has reported variations in frequency of OC over time in the same health facility.The isolation frequency of  in 1997 ; in contnst ITCZ . In addictively, , a previ mycosis . Thus, tCandida strains isolated form the oral cavity of OC patients who visited Kagoshima University Hospital in 2012–2013 (14 months) and to assess the isolation frequency of Candida species and their resistant strains in comparison with our previous study conducted in 2006–2007 (24 months) in order investigate the use antifungal drugs in the treatment of OC.The present study was undertaken to evaluate antifungal drugs against This study analyzed 158 strains isolated from 112 patients who visited the Department of Oral Surgery, Kagoshima University Hospital, for the treatment of OC during the 14-month period from February 2012 and March 2013. Strains collected from patients having received antifungal drug therapy within 3 months from collection were excluded from this study.Candida was isolated.An analysis was conducted for age, gender, underlying systemic disease, steroid hormone use, denture use, chief complaints, and oral disease in the 112 patients from whom Candida Culture Medium  and incubated for 48 hours at 36°C.Prior to the diagnosis of OC, the affected area of the oral cavity was wiped with a sterile cotton swab pre-immersed in sterile physiological saline. Fluid sample collected on the swab was smeared onto a CHROMagar Candida species was determined by colorimetric observation of colonies in the CHROMagar Candida culture medium containing synthetic colorimetric substrates which are changed to be chromogenic by enzymes contained in Candida species. Confirmation of the determination of Candida species was then done by PCR based on the method reported from Kanbe et al., briefly fungal cells grown in the CHROMagar Candida culture medium were harvested by centrifugation, washed three times with distilled water, and the DNAs were extracted and purified according to the protocol of a DNA purification kit, Fast DNA Kit . Then, the DNA samples were amplified by PCR and the PCR products were analyzed by agarose gel electrophoresis to determine the kind of Candida species [Positive culture of  species .Fluid collected on the swab from the affected area was transferred onto a glass slide for Gram staining by a routine method. A diagnosis of OC was made when colony formation was noted during incubation and the presence of pseudo mycelium was shown by Gram staining .Candida strains detected by the above-mentioned methods were re-seeded onto Sabouraud agar medium , followed by 48-hour incubation at 36°C. Then the culture was diluted and adjusted to a McFarland factor of 0.5 by measuring the optical density at 530 nm using a spectrophotometer ViSpec II , and applied for the assay to determine the susceptibility. A minimum inhibitory concentration (MIC)of each of AMPH-B, 5-FC, FLCZ, MCZ, ITCZ, and MCFG against these Candida strains were determined with a Yeast-like Fungal Sensitivity Kit ASTY  compatible with the National Committee for Clinical Laboratory Standards (NCCLS) M-27A method [Candida species, were calculated. Resistant strains were determined by the following MIC criteria: AMPH-B, FLCZ, ≥64 μg/mL; 5-FC, ≥32 μg/mL; MCZ, ≥4 μg/mL; MCFG, >2 μg/mL; and ITCZ, ≥1 μg/mL.A method . The kitA method ,16. MIC5All enrolled patients provided written informed consent upon directly providing verbal and written detailed explanation of this investigation. This study was approved by the Human Ethics Committee of the Kagoshima University Medical and Dental Hospital (No.23-18 and 23–27).Candida species (2 × 7), and the incidence of resistant strains between the 2006–2007 study and the 2012–2013 study. The difference in age between the 2 studies was examined by Student’s t-test. P value less than 0.05 was regarded as statistically significant.Fisher’s exact probability test was employed to determine differences in gender (2 × 2), chief complaints (2 × 8), systemic disease , steroid use (2 × 2), presence of dentures (2 × 2), oral candidiasis (2 × 4), composition of isolated As shown in Table Classification of diagnosed OC cases is also shown in Table Candida were isolated in patients in the 2012–2013 study. The most frequently isolated Candida species were Candida albicans , followed by C. glabrata , C. tropicalis , C. parapsilosis , and C. krusei  , while C. glabrata was never found on its own, only concomitantly with C. albicans in 36 patients. More than one kind of Candida species was isolated in 41 patients, with concomitant C. albicans and C. glabrata being the most common combination in approximately 90% of these patients. In contrast, among 65 strains of Candida isolated in patients in the 2006–2007 study, 56 strains of C. albicans (86.2%), 8 strains of C. glabrata (12.3%), and 1 strain of C. tropicalis (1.5%) were identified, and no strains of C. parapsilosis and C. krusei were detected. Thus, in the 2012–2013 study, the number of isolated strains of C. glabrata was almost double that of the 2006–2007 study, and the ratio of C. albicans to all Candida strains was therefore significantly lower in the 2012–2013 study when analyzed on a strain-basis or on a patient-basis. Thus, in both patient-basis and strain-basis analyses, a statistical difference was observed between the 2006–2007 study and the 2012–2013 study (p < 0.01).In total, 158 strains of Candida species other than C. albicans and C. glabrata were isolated in either study, MIC values of antifungal drugs against C. tropicalis, C. parapsilosis, and C. krusei, which were never isolated alone but only in combination with C. albicans and C. glabrata, were not reliable. Therefore, the results of susceptibility testing mainly refer to the MICs of C. albicans and C. glabrata (Table Since few 1 to 3 strains) of the  strains C. albicans in the 2006–2007 study and 1 strain of C. glabrata in the 2012–2013 study demonstrated high MIC of over 16 μg/mL. However, the presence of resistant strains could not be detected in the concentration range used in this analysis. There was no obvious difference in the susceptibility of the 2 main Candida species, C. albicans and C. glabrata, between the 2 studies, with MIC90 values of 0.5 μg/mL and 2.0 μg/mL, respectively, in the 2006–2007 study and of 1.0 μg/mL and 1.0 μg/mL, respectively, in the 2012–2013 study.The MIC was found to be low (≤2 μg/mL) in 63 (96.9%) of the 65 strains in the 2006–2007 study and in 153 (99.4%) of the 154 strains in the 2012–2013 study. Two strains of C. albicans in the 2006–2007 study and 2 strains of C. albicans and 1 strain of C. glabrata in the 2012–2013 study demonstrated high MIC of more than 32 μg/mL. There was no obvious difference in the susceptibility of C. albicans and C. glabrata between studies, with the 2006–2007 study demonstrating MIC90 of 0.5 μg/mL and 16 μg/mL, respectively, and the 2012–2013 study, of 1.0 μg/mL and 8.0 μg/mL, respectively.Two strains of C. albicans, 1 strain was C. glabrata, and 1 strain was C. tropicalis, and in 12 strains (7.6%) in the 2012–2013 study, of which 3 strains were C. albicans and 9 strains were C. glabrata, and which were all found to be FLCZ-resistant strains. A higher proportion of resistant C. glabrata strains were found in the 2012–2013 study  than in the 2006–2007 study . An increase in MIC50 of C. glabrata from 2 μg/mL in 2006–2007 to 16 μg/mL in 2012–2013 was also observed.MIC values were found below 64 μg/mL in 60 (92.3%) of the 65 strains in the 2006–2007 study and in 146 (92.4%) of the 158 strains in the 2012–2013 study. High MIC ≥64 μg/mL was recorded in 5 strains (9.5%) in the 2006–2007 study, of which 3 strains were C. albicans (4.6%) and 2 strains of C. glabrata (25.0%) in 2006–2007, and for 19 strains (12.0%) in 2012–2013, of which 6 strains were C. albicans (5.4%), 11 strains were C. glabrata (30.6%), 1 strain was C. tropicalis (33.3%), and 1 strain was C. krusei (100%). Although MIC90 against C. glabrata in the 2012–2013 study (16 μg/mL) was higher than that in the 2006–2007 study (4.0 μg/mL), no other significant differences were seen between studies. The incidence of resistant C. glabrata was similar between the 2 studies at 25.0% (2/8 strains) and 30.6% (11/36 strains), respectively.The MIC of 60 (92.3%) of the 65 strains in the 2006–2007 study and in 139 (88.0%) of the 158 strains in the 2012–2013 study were below 4 μg/mL. High MIC (≥4 μg/mL) was recorded for 3 strains of C. albicans (10.7%), 4 strains were C. glabrata (50.0%), and 1 strain was C. tropicalis (100%), and in 31 strains (19.6%) in 2012–2013, of which 5 strains were C. albicans (4.5%), 24 strains were C. glabrata (66.7%), and 2 strains were C. tropicalis (66.7%). There was a higher proportion of resistant strains of C. glabrata against ITCZ in the 2012–2013 study  than in the 2006–2007 study , but the difference was not statistically different. The MIC90 against C. glabrata was similar in both studies at >8.0 μg/mL, but that against C. albicans in the 2012–2013 study (0.25 μg/mL) was lower than that in the 2006–2007 study (1.0 μg/mL).The MIC of 54 (83.1%) of the 65 strains in the 2006–2007 study and of 127 (80.4%) of the 158 strains in the 2012–2013 study were less than 1 μg/mL. High MIC (≥1 μg/mL) was recorded in 11 strains (16.9%) in 2006–2007, of which 6 strains were C. albicans and C. glabrata, and 1 strain of C. glabrata in the 2012–2013 study. Although the MIC90 against C. glabrata in 2012–2013 (1.0 μg/mL) was lower than in 2006–2007 (>16 μg/mL), there was no other obvious differences in the susceptibility of C. albicans and C. glabrata between studies.The MIC of 63 (96.9%) of the 65 strains in the 2006–2007 study and of 157 (99.4%) of the 158 strains in the 2012–2013 study were >4 μg/mL. High MIC (≥4 μg/mL) was recorded in only 2 strains (4.6%) in the 2006–2007 study, of which 1 strain each was C. albicans Candida (NCAC) strains have been isolated in increasing numbers in medically compromised patients including those with OC and denture stomatitis. C. albicans remains the most common fungus associated with infection, followed by C. glabrata. In clinically isolated samples from the blood, urine, and tracheal secretion of candidemia patients, the isolation frequency of C. glabrata has been found to be 16–26%, which is the highest among NCAC strains [Candida species was C. albicans (42.1%), followed by C. glabrata (26.7%), and the proportion of candidemia caused by NCAC strains was higher than that caused by C. albicans[C. glabrata, in candidemia has recently been reported, with a higher incidence of C. glabrata found from 2006 to 2010  in Brazil [Candida species may vary by geographic region [Non- strains -20. In t albicans. Such a n Brazil . In addiCandida infections due to C. glabrata[C. glabrata increased from 14.1% in 2006–2007 to 32.1% in 2012–2013 when assessed by the number of patients, indicating a similar tendency observed in Japanese OC patients. The tendency of a shift of NCAC species would also be attributable to frequent and wide use of azole antifungal drugs, such as fluconazole, in the treatment of mycosis [C. glabrata, was reported to increase [C. glabrata became high.However, few reports exist on differences in variation of isolation frequency and its relation to candidemia in oral candidiasis. In contrast, there are several reports on oropharyngeal  glabrata,25. In t mycosis . Moreoveincrease -28. In JC. albicans and C. glabrata as observed in 32.1% of patients in the 2012–2013 study. In fact, cases of mixed infection suggest that C. albicans enhances the invasiveness of C. glabrata, and leads to increased lactate dehydrogenase (LDH) release from a reconstituted human oral epithelium (RHOE) in vitro[By comparing background data of OC patients in the 2012–2013 study and in the 2006–2007 study, the following differences were observed: (1) the frequency of xerostomia as a chief complaint increased from 24.6% to 37.5% of patients, (2) autoimmune disorders as a systemic disease increased from 6.7% to 27.6%, and (3) erythematous OC as a type of OC increased from 19.3% to 46.4%. Differences in the frequency of xerostomia and autoimmune disorders were associated with an increase in patients with Sjögren’s syndrome, a chronic and progressive autoimmune disorder mainly characterized by xerostomia. Differences in erythematous OC may be partly attributed to an increase in simultaneous identification of  in vitro. Further90 values against C. glabrata in both studies. In particular, the MIC90 value of MCZ against C. glabrata increased from 4 μg/mL (2006–2007) to 16 μg/mL (2012–2013). Such high MIC values of azole antifungal drugs, which suggest a decrease in the susceptibility of C. glabrata to these drugs, have been recognized in several studies [C. glabrata isolated from patients with blood stream infections, with a dose-dependent susceptibility rate of 76.5%, while the susceptibility rates of FLCZ were 100% for C. albicans and C. parapsilosis[C. glabrata, resulted from resistance to FLCZ, leading to oropharyngeal Candida infections, which are more severe and more difficult to treat in HIV-infected and cancer patients [C. glabrata was observed against other azoles, such as ITCZ and ketoconazole [C. glabrata increased from 50.0% (4/8) in the 2006–2007 study to 66.7% (24/36) in the 2012–2013 study. In cases of other azole antifungal drugs, FLCZ and MCZ, resistant strains of C. glabrata increased slightly from 12.5% to 25.0% and from 25.0% to 30.6%, respectively. Thus, a tendency for increased isolation frequencies of resistant C. glabrata against azole antifungal drugs, especially ITCZ have been observed. Frequent exposure of antifungal drugs, especially azoles, is known to lead to an increase in resistant Candida strains, which has been reported since fluconazole was clinically available in 1989. After that new azole antifungal drugs, such as itraconazole and voliconazole, have been widely used for deep-seated mycosis. Thus, increases in clinically available antifungal azole drugs and in their clinical usages for the treatment of deep-seated mycosis could lead to an increment of resistant Candida strains. This tendency would be still continuing during the period of 2006–2013.By comparing the susceptibilities of patients in the 2012–2013 study with that of the 2006–2007 study, azole antifungal drugs  demonstrated high MIC studies -19,30. Dapsilosis,20,23. Dconazole . In addiCandida species isolated from OC patients, the ratio of C. glabrata to C. albicans increased in the 2012–2013 study as compared to the 2006–2007 study, accompanying the tendency of increases in tolerant strains of C. glabrata against azole antifungal drugs and in cases of autoimmune disease. The higher isolation frequency of C. glabrata, especially of co-isolation frequency with C. albicans, may be associated with the increase in erythematous OC. Considering the increase in C. glabrata resistant strains against azoles, selection of appropriate antifungal drugs and dosing regimens against resistant C. glabrata, as well as against C. albicans is important.In oral The authors declare that they have no competing interests.KY and MY participated in the design of the study, conducted the study, and conducted the data collection and analyses, and drafted the manuscript. NT, FJ, HD, SR, HT, and SK, participated in the design of the study and carried out the data collection. All authors contributed to the writing of the manuscript and critically reviewed the final version. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6831/14/14/prepub