During a hospital visit at 6 weeks postpartum, a sample of freshly expressed foremilk was collected and information on maternal dietary habits in the postpartum period was obtained through an interviewer-administered food frequency questionnaire. We applied principal component factor analysis to selected nutrients in order to identify maternal dietary patterns, and assessed correlations in human milk macronutrients and fatty acids across levels of dietary patterns. (3) Results: Five dietary patterns were identified, named “Vitamins, minerals and fibre”, “Proteins and fatty acids with legs”, “Fatty acids with fins”, “Fatty acids with leaves”, “Starch and vegetable proteins”. These dietary patterns were correlated with some milk components, namely fatty acids, and in particular ω-3 and its subcomponents. (4) Conclusions: This study showed that overall maternal dietary habits during breastfeeding may influence human milk composition, suggesting the importance of adequate maternal nutrition during lactation not only for the mother herself but also to provide the infant with milk containing adequate amount and quality of nutrients for a balanced nutrition. Human milk provided by healthy and well-nourished mothers is the optimal way of providing nutrients as required by biological processes associated with growth and development . It contThe composition of human milk depends on different environmental and maternal factors ,11,12,13Using data of the Italian MEDIDIET study, we evaluated the association between maternal dietary patterns and human milk macronutrient and fatty acid composition.2), and not on a restriction diet. All mothers signed an informed consent to participate to the study [The MEDIDIET study is an Italian multicentric observational study carried out between 2012 and 2014, including 300 healthy breastfeeding mothers. Study design, inclusion criteria, dietary assessment of breastfeeding mothers, milk collection, analysis of milk samples and description of study characteristics have been provided in detail elsewhere . Brieflyhe study . The EthHuman milk was collected at 6 ± 1 week postpartum. Mothers provided a sample of their milk (30–50 mL) expressed in the morning after breakfast and before lunch. Mothers were instructed to collect their milk at times other than a breastfeeding session, and the times of milk collection as well as of the last breastfeeding were recorded. The freshly expressed foremilk was stirred and divided in sterile 10 mL tubes, overlaid with nitrogen gas to avoid oxidation. Milk samples were then stored at −70 °C.Human milk macronutrient composition  was analysed using mid infrared transmission spectroscopy . The complete profile of FA with chain length between 4 and 24 carbon atoms was analysed as methyl esters by capillary gas chromatography and flame ionization detection (“cGC-FID”) with and without preparative separation of phospholipids by high-performance liquid chromatography and evaporative light-scattering detection (“HPLC-ELSD”) according to the method proposed by Beermann et al. .Maternal dietary intakes from partum to the day of milk collection were obtained by trained interviewers using a validated and reproducible food frequency questionnaire (FFQ) ,28, duriMaternal dietary patterns were identified through an exploratory principal component factor analysis (PCFA) carried out on the intake of 31 selected nutrients as reported in 2). The dominant nutrients were used to label the dietary patterns. Mothers’ factor scores were calculated for each dietary pattern as the sum of individual nutrient intakes weighted for the corresponding factor loadings. Each mother’s factor scores indicate to what extent her diet was adherent to each dietary pattern. To further describe and interpret the identified dietary patterns, the Spearman rank correlation coefficients were calculated between the continuous factor scores derived from PCFA and the weekly intake of selected food groups and added fats.Each nutrient’s factor loading represents how strongly that nutrient is associated with a dietary pattern. We considered as highly associated those nutrients with a factor loading ≥0.63 in absolute value, hereafter named “dominant nutrients”. We set this cut-off because it implies a contribution of the dietary patterns to the original nutrient’s total variance of approximately 40%  using analysis of variance (ANOVA). We also evaluated the relationship between quartiles of the identified dietary patterns and baseline maternal characteristics . All analyses were performed using SAS software, version 9.4 .The energy density ranged from 37.0 to 89.0 kcal/100mL with a mean ± standard deviation (SD) of 57.5 ± 10.5 kcal/100 mL. Regarding macronutrient contents, the lactose content ranged from 6.2 to 7.2 with a mean ± SD of 6.8 ± 0.2 g/100 mL. The corresponding figures were 0.2 (min), 1.4 (max), 0.9 ± 0.2 (mean ± SD) g/100mL for proteins and 0.7 (min), 7.2 (max) and 3.1 ± 1.3 (mean ± SD) g/100 mL for fats. As for the FA contents, the mean ± SD for saturated fatty acids (SFA) was 41.9 ± 4.9% of FA (min = 27.9 and max = 56.1), for monounsaturated fatty acids (MUFA) was 44.1 ± 4.8% of FA (min = 31.1 and max = 60.8) and for polyunsaturated fatty acids (PUFA) was 13.6 ± 2.5% of FA (min = 9.0 and max = 24.3). The mean ± SD contents of ω-6 FA and ω-3 FA were 12.4 ± 2.5 % of FA, and 1.2 ± 0.5 % of FA, respectively.p < 0.0001), and the overall and individual measures of sampling adequacy were satisfactory .Visual inspection showed that the correlation matrix of the 31 maternal nutrient intakes was adequate to carry out the PCFA. The “Proteins and fatty acids with legs” dietary pattern was weakly positively correlated with human milk content of SFA (r = 0.12) and ω-6 (r = 0.12), and with AA/DHA (r = 0.13) and LA/DHA (r = 0.12) ratios, whereas it was negatively correlated with MUFA (r = −0.19).There were positive correlations between “Fatty acids with fins” dietary pattern and ω-3 (r = 0.23), EPA (r = 0.25), DHA (r = 0.25), and DPA (r = 0.19), and ALA (r = 0.13) milk contents. The correlation was negative for AA/DHA ratio (r = −0.27), and LA/DHA ratio (r = −0.27), AA/EPA ratio (r = −0.26), ω-6/ω-3 ratio (r = −0.21), and LA/ALA ratio (r = −0.13).The “Fatty acids with leaves” dietary pattern was positively correlated with MUFA (r = 0.17) and ALA (r = 0.13); it was inversely correlated with SFA (r = −0.20).Lastly, the “Starch and vegetable proteins” pattern seemed to be positively correlated with lactose (r = 0.12).p = 0.0029), EPA (p = 0.0195), DHA (p = 0.0093), and DPA (p = 0.0273), than mothers with a lower adherence. Conversely, the mean ω-6/ω-3 ratio (p = 0.009), AA/EPA ratio (p = 0.0012), AA/DHA ratio (p = 0.0193), and LA/DHA ratio (p = 0.0277) tended to be lower for mothers with a high adherence to this dietary pattern.p = 0.0378). In addition, although there was no correlation for this component, the mean content of AA was significantly higher in mothers with higher adherence to this dietary pattern (p = 0.0473). Although some significant differences emerged for the mean contents of ω-3 (p = 0.0339), ALA (p = 0.0354), and AA/EPA ratio (p = 0.0229) across quartiles of the dietary pattern, the shapes of these relationships were unclear.An increasing trend of the AA/DHA ratio was observed across quartiles of the “Proteins and fatty acids with legs” dietary pattern (p = 0.0038), EPA (p = 0.0004), DHA (p = 0.0013), and DPA (p = 0.0276); conversely, they showed significantly lower ω-6/ω-3 (p = 0.0426), AA/EPA (p = 0.0004), AA/DHA (p = 0.0006), and LA/DHA ratios (p = 0.0012).Mothers in the highest quartile of the “Fatty acids with fins” dietary pattern showed significantly higher contents of ω-3 (p = 0.0322) and ALA (p = 0.0032), and lower mean content of SFA (p = 0.0035) compared to mothers in the lowest quartiles.Mothers in the highest quartiles of the “Fatty acids with leaves” dietary patterns showed significantly higher mean contents of MUFA . Likewise, higher total ω-3, EPA, DHA, and DPA in milk were correlated with higher adherence to the “Fatty acids with fins” pattern. The “Proteins and fatty acids with legs” pattern was inversely correlated with human milk content of MUFA and weakly positively with SFA, ω-6, AA/DHA ratio, and LA/DHA ratio. Conversely, the “Fatty acids with leaves” dietary pattern was inversely related to the SFA content in human milk, and positively related to the MUFA content in milk. Lastly, the “Starch and vegetable proteins” pattern showed only a weak positive correlation with lactose in milk.Macronutrient compounds in milk have been reported to vary within mothers and to depend on lactation period, while they appear stable despite variations in maternal nutrition . ConversTwo previous studies carried out in China have assessed the role of maternal dietary patterns, derived from food groups, on human milk macronutrients ,20 and FIn a seemingly paradoxical way, we found associations between “Vitamins, minerals and fibre” dietary pattern with concentrations of ω-3 in human milk. Accordingly, the pattern is not characterized by fat-rich components, nor by energy-delivering nutrients. This pattern may be characterized by intakes of the maternal ω-3 (ALA) from vegetable sources, and a generally well balanced food intake within a favorable lifestyle . Within PCFA is a well-known technique used to identify a few a posteriori dietary patterns from a larger set of dietary variables, allowing us to consider diet as an overall exposure, taking into account correlations and interactions between different dietary components. This method requires some subjective decisions in different steps of the analysis, including the type  and number of dietary variables to analyse, the number of factors to retain, the choice of applying a rotation method, and the interpretation of the identified dietary patterns . Thus, wThis study has strengths and weaknesses which are inherent to its observational design and general setting. The multicentric approach allowed inclusion of mothers from different Italian regions and living both in inland and coastal areas. Maternal dietary information was self-reported and dependent on participant’s recall. However, it was collected by trained interviewers using a structured FFQ which has been validated and tested for reproducibility in the Italian adult population ,28, and In the present study, we identified five major dietary patterns among Italian breastfeeding mothers and assessed their role on human milk macronutrients and FA profile. We confirmed that some human milk compounds—including FA, and in particular ω-3 and its subcomponents—are influenced by maternal dietary habits during breastfeeding. No association was found between maternal dietary patterns and lactose, protein, fat or energy in human milk. Our results suggest that an adequate maternal nutrition during lactation may be important not only for the mother herself, but also to provide the infant with a milk with adequate amounts and quality of nutrients for an appropriate nutrition.


Danio rerio) is a translational species widely used in behavioral neuroscience, where tricaine methanesulfonate (MS222) is the most acceptable and used drug when conducting routine procedures. Based on this, we investigated the effects of MS222 (100 mg/l) in young adults and aging zebrafish 1, 2, 3, and 7 days after exposure. Animals’ were submitted to the anesthetic procedure until loss of body posture, slowing of opercular movements and lack of response to tail touch with a plastic pipette were achieved, then further left in the drug for 3 min. After that, animals (6 mpf vs. 24 mpf) were transferred to a recovery tank until fully recovered and transferred back to their housing system until further testing in the free movement pattern (FMP) Y-maze, which assesses zebrafish working memory and cognitive lexibility. Young animals had significant impairment in their working memory and cognitive flexibility 1 and 2 days after the exposure to MS222, being fully recovered by day 3 and with no effects 7 days post drug exposure. Increased repetitions were also observed for animals exposed to MS222 which could indicate increased stress-related response in animals up to 2 days after drug exposure. No drug effect was observed in aging animals besides their natural decreased alternations and working memory. Overall, behavioral experiments after routine procedures using MS222 should be performed with caution and need to be delayed, at least 3 days after exposure where working memory, cognitive flexibility, and repetitive behavior are back to normal.Exposure to anesthetic drugs is common in biomedical sciences being part of routine procedures in different translational species, however its impacts on memory and cognition are still debated, having different impacts depending on drug and age. The zebrafish ( Anesthesia is a common procedure used to decrease/avoid pain and distress in both clinics and in a research context  are widely used as a translational model to study memory and cognition . All experiments were carried out following approval from the University of Portsmouth Animal Welfare and Ethical Review Board, and under license from the UK Home Office .Adult zebrafish  were bred in-house and reared in standard laboratory conditions on a re-circulating system . Animals were kept in groups of 10 per 2.8 L (~28.5°C (±1 °C); pH 8.4; 14/10-h light/dark cycle) and fed three times a day with a mixture of live brine shrimp and flake flood. All animals used for this experiment were euthanized using 2-phenoxyethanol from Aqua-Sed —age [6 vs. 24 months post-fertilization (mpf)]; and factor 2 (5 levels)—period of time after exposure to MS222 . Control animals were exposed to home tank water having similar handling as MS222 groups; however, controls were randomly tested during 1, 2, 3, or 7 days after handling. Animals were exposed to MS222 at a concentration of 100 mg/l buffered with sodium bicarbonate  = 0.25, power = 0.99, alpha = 0.05] based on several published articles using zebrafish in the FMP Y-maze task and extensive experience with this protocol in our laboratory  and repetitions (rrrr + llll) were used as proportion of the total number of turns which are highly expressed through 1-h  total percentage use  of each tetragram sequence for the 60 min of exploration, referred to as “global” search strategy; (2) “immediate” search strategy, analyzed search pattern configurations over 10 min time bins throughout the trial, equating to six equal, consecutive time bins. “Global” search strategy was used to measure working memory as repetition of previous turn choices must be remembered for patterns of movement to be repeated over 1 h of exploration, requiring memory of arm entries and order of entry. The second type of strategy was used to assess cognitive flexibility. Animals that are not able to update information gained during exploration of the FMP n = 8–9 per batch/per group, total n = 15–16/per group). The behavioral testing was carried out in a fully randomized order, choosing fish at random from one of five housing tanks for testing. After data collection and screening for extreme outliers , data was analyzed in full. A total of 10 animals out of 170 were excluded due to poor engagement with the task .All behavioral tests were performed between 10:00 and 16:00 h (lights on at 9:00 h) using two independent batches  across a 1-h trial. To analyze the data according to left and right turns in 10-min time bins, raw data was processed using the Zantiks Y-maze Analysis Script created specifically for this purpose . Normality of data and homogeneity of variance were analyzed by Kolmogorov–Smirnov and Bartlett’s tests, respectively. Considering that data were normally distributed, one-way analysis of covariance (ANOVA) with treatment (five levels—control vs. 1 d MS222 vs. 2 d MS222 vs. 3 d MS222 vs. 7 d MS222 for each age group) as the factor, was used to analyze average turns, repetitions, and alternations of each age. Two-way repeated measures ANOVA was used for cognitive flexibility analysis using time (six levels: 6 × 10 min time bins) and treatment (five levels: control vs. 1 d MS222 vs. 2 d MS222 vs. 3 d MS222 vs. 7 d MS222) as factors. For comparison of average turns, alternations, and repetitions between ages two-way ANOVA was performed using treatment (five levels—control vs. 1 d MS222 vs. 2 d MS222 vs. 3 d MS222 vs. 7 d MS222) and age (two levels—6 mpf vs. 24 mpf) as factors. Tukey’s test was used as post hoc analysis, data was represented as mean and error of the mean (SEM), and results were considered significant when p ≤ 0.05.Data from the F = 5.556; p*** = 0.0005), repetitions  = 4.067; p*** = 0.0255). Tukey’s post hoc test revealed that animals had a significant decrease in alternations 1 d (p*** = 0.0066) and 2 d (p*** = 0.0095) after the exposure to MS222 compared to controls but had fully recovered after 3 d (p = 0.3710) and 7 d (p > 0.9999). For repetitions, a significant increase was observed for 1 d (p*** = 0.0117) and 2 d (p*** = 0.0240) after MS222 exposure compared to controls. No post hoc effects were observed for average turns after Tukey’s test analysis  = 11.05; p**** < 0.0001), treatment  = 5.566; p*** = 0.0005) and subject  = 3.028; p*** < 0.0001). No interaction effect  = 0.3700; p = 1.261) was observed for alternations across time. Briefly, both groups control, 3 d and 7 d after MS222 had a peak in their alternations at 40 min compared to their 10 min time bin . When comparing differences between groups, a significant decrease in alternations was specifically observed when comparing 1 d and 2 d vs. controls at 40 min  = 4.067; p*** = 0.0047), time  = 4.067; p*** = 0.0031) and subject effect  = 4.249; p**** < 0.0001) was also observed for repetitions across time with no interaction (time vs. treatment) effect  = 1.122; p = 0.3235). A significant increase in repetitions was observed 1 day after MS222 compared to controls at 10 min (p*** = 0.0481), 20 min (p*** = 0.0350) and 50 min (p*** = 0.0022). Repetitions were also increased for the group 6 mpf + 2 d MS222 at 40 min when compared to controls  = 1.155; p = 0.3381), repetitions  = 1.370; p = 0.4417) and average turns  = 0.8366; p = 0.5065; F = 1.155; p = 0.3381), time  = 1.815; p = 0.1164) or interaction between factors  = 1.013; p = 0.4456) on alternations across time were observed. A significant effect for subject was found  = 2.112; p*** < 0.0001; F = 3.529; p*** = 0.0060) and subject  = 2.779; p*** < 0.0001) with no effect for treatment  = 0.9476; p = 0. 4417) or interaction  = 0.7931; p = 0. 7223) was observed for repetitions across time in animals exposed to MS222 (Aging fish (24 mpf) when treated with MS222 and tested 1 day or 7 days after exposure did not show significant changes for alternations  = 5.654; p*** = 0.0003) and age  = 7.199; p*** = 0.0081) was observed for alternations. No significant effect of treatment per se was observed on the average of alternations  = 2.031; p = 0.0929). For repetitions, no significant effect age  = 4.913e-005; p = 0.9944) was observed. However, a significant treatment  = 2.723; p*** = 0.0317) and interaction between factors effect  = 2.511; p*** = 0.0442) was observed similarly to the findings described in F = 2.110; p = 0.0824), treatment  = 1.670; p = 0.1599) and age  = 1.813; p = 0.1802; Finally, in order to understand the interactions between animals’ age and the exposure to MS222 affecting memory and cognition, two-way ANOVA was performed and a significant effect for interaction  zebrafish had a significant decrease in their working memory, as demonstrated by them showing a lower number of alternations during the 60 min in the FMP Y-maze, together with disrupted patterns of alternations across time, as animals did not show an alternation peak at 40 min compared to control animals. A previous study that focused on the characterization of the FMP Y-maze showed that alternations are the preferred searching strategy used for zebrafish, mice, and humans when navigating the maze  and carp . The neurobiology of repetitive behaviors is directly related to stress and regularly discussed as a coping mechanism to reduce the arousal level of the animal when it is exposed to stressful events  can be found below: The animal study was reviewed and approved by University of Portsmouth Animal Welfare Ethical Review Board.BF and MP designed the experimental procedures and analyzed the data. MP contributed for reagents/analysis tools. BF and NA performed the experiment. BF, NA, and MP contributed for the manuscript writing. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The amounts of idarubicin added, loading medium, loading condition, and drug concentration were investigated as factors influencing drug loading efficiency. The drug loading rate was negatively correlated with the amount drug added and diameter of CalliSpheres® and positively correlated with the drug concentration. Compared to loading in purified water and incubation at room temperature, 5% glucose, heating, and ultrasound could accelerate drug loading. The idarubicin loading efficiency was above 95% after 10 min for all three CalliSpheres® with the optimized method of adding 20 mg of idarubicin at a concentration of 2 mg/mL and incubating at room temperature. The drug leak rate was under 1% within 8 h after mixing with iopamidol. Drug release tests indicated the sustained-release performance of CalliSpheres®, and the time to reach 75% of the release plateau level was 8, 26, and 51 min for 50–150, 100–300, and 300–500 μm CalliSpheres®, respectively. After idarubicin loading, the diameters increased by 12%, 36%, and 38% for 50–150, 100–300, and 300–500 μm CalliSpheres®, respectively, and the surface of CalliSpheres® was observed to become smoother than that before drug loading. All three CalliSpheres® presented satisfactory loading efficiency with the optimized method, as well as proper compatible stability and sustained release performance. Among them, 100–300 μm CalliSpheres® are recommended.This study aimed to investigate the idarubicin loading method, compatible stability with contrast agent, release profiles, and morphological properties of 50–150, 100–300, and 300–500 μm CalliSpheres Transarterial chemoembolization (TACE) is the most commonly used therapy in unresectable hepatocellular carcinoma (HCC) . CompareTM  and HepaSphereTM . However, the detailed IDA loading profiles, such as dosage, concentration, and drug loading time and medium, have not been studied clearly. These features are key issues for clinical application, since unloaded IDA mixed in DEB might cause a rapid release to the systemic circulation [Some previous studies have investigated the ability to load IDA to classic embolic beads , such asculation . In addiculation .®  is a novel DEB, which was approved for clinical use in 2015 in China and obtained FDA 510 (k) certification in 2018 [® have a similar chemical structure and application property to DC Bead, but at a lower price [®, such as doxorubicin [CalliSpheres in 2018 . CalliSper price . Previouorubicin , oxaliplorubicin , irinoteorubicin . However® of different particle size, as well as their compatible stability, release profiles, and morphological changes. The authors aimed to provide solutions for clinical application by this in vitro study.In this present study, detailed IDA loading features and optimized loading methods were investigated for three CalliSpheres®  with sizes of 50–150, 100–300, and 300–500 μm were evaluated. Each package contains 1 g of DEB and 7 of mL normal saline, making up a total volume of about 8 mL. Idarubicin hydrochloride for injection  and iopamidol injection  were kindly provided by Chia Tai Tianqing Pharmaceutical Group Co. Ltd., Nanjing, China. Each IDA package contains hydrochloride powder with 10 mg of active pharmaceutical ingredients (APIs). Idarubicin hydrochloride standard (>99%) was purchased from the National Institutes for Food and Drug Control, China. Methyl alcohol (HPLC grade) was purchased from Sigma-Aldrich, Saint Louis, MO, USA. Phosphate buffered saline  and fetal bovine serum (FBS) were purchased from Gibco, Grand Island, NY, USA. The 0.9% sodium chloride for injection (0.9% NaCl) and the 5% glucose for injection (5% Glu) were purchased from Shijiazhuang No. 4 Pharmaceutical, China. Ultrapure water (H2O) was obtained from a Millipore system . All other chemicals were of analytical grade.CalliSpheres® with different particle sizes of 50–150, 100–300, and 300–500 μm were evaluated in this part. The drug solution was prepared by adding H2O, 0.9% NaCl, or 5% Glu to a package of idarubicin hydrochloride for injection to produce the required concentrations. CalliSpheres® were rinsed by the corresponding medium three times, discarding the liquid supernatant and setting it aside. IDA solution was then added to CalliSpheres®. After gentle agitation, the mixture was incubated at room temperature (37 °C) or in a running ultrasound cleaner . Liquid supernatant was collected at 0, 5, 10, 15, 30, 45, 60, and 90 min, diluted by mobile phase-methyl alcohol and 0.1% phosphoric acid at a ratio of 70:30 (v/v), and tested by an Agilent 1260 high-performance liquid chromatography (HPLC) system , equipped with an Agilent G4212B DAD detector and an Agilent Eclipse Plus C18 column . The mobile phase was methyl alcohol–0.1% phosphoric acid , at a rate of 1.0 mL/min. Other parameters are as follows: column temperature = 25 °C, injection volume = 10 μL, UV wavelength = 254 nm, and running time = 10 min. The efficiency of loading was calculated as follows:The amount IDA added, loading medium, loading condition, and IDA concentration were investigated. CalliSpheres® preparation, while other factors were fixed as follows: loading medium = purified H2O, loading condition = room temperature, and drug concentration = 500 μg/mL. In the screening of loading medium, the experimental groups were set as purified H2O, 0.9% NaCl, and 5% Glu, while the other factors were fixed as follows: amount of IDA to add = 20 mg, loading condition = room temperature, and drug concentration = 500 μg/mL. In the screening of the loading condition, the experimental groups were set as incubation at room temperature (37 °C) and in an ultrasound cleaner, while the other factors were fixed as follows: amount of IDA to add = 20 mg, loading medium = purified H2O, and drug concentration = 500 μg/mL. In the screening of IDA concentration, the experiment groups were set as 0.25, 0.5, 1, 1.5, and 2 mg/mL, while the other factors were fixed as follows: amount of IDA to add = 20 mg, loading medium = purified H2O, and loading condition = room temperature.In the screening of the amount of IDA to add, the experimental groups were set as 5, 10, 15, 20, and 40 mg, for each marketed CalliSpheres®, at an amount of 20 mg for each marketed CalliSpheres® preparation. A 10 mL iopadol injection, equivalent to 3.7 g iopadol, was added, and, after gentle agitation, the mixture was incubated at room temperature. The IDA concentration in the mixed liquid was tested at 0, 10, 20, 30, and 45 min and then at 1, 1.5, 2, 4, and 8 h, by the above-mentioned HPLC method. The IDA leak rate was calculated as follows:IDA was loaded by the optimized loading method to 100–300 and 300–500 μm CalliSpheres®, the drug release rate was tested in PBS and PBS + 10% FBS separately to investigate the influence of FBS. The other parameters were as follows: bath temperature = 37 °C, flow rate = 5.0 mL/min, and medium volume = 200 mL. IDA concentration was evaluated at 10, 20, 30, and 45 min and then at 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 48, and 72 h. Before analysis by HPLC, each solution sample was mixed with methyl alcohol at a volume ratio of 1:1, followed by high-speed centrifugation , for protein precipitation. In addition, free drug of IDA was also evaluated as a control group, in order to correct the drug degradation during the experiment.The IDA release rate was measured by a Pharmacopeia flowthrough apparatus 4 (USP 4) Sotax system . For each size of CalliSpheres® before and after IDA loading were detected by a scanning electron microscope . The sample preparation process was as follows: 50 μL of DEB suspension were first spread on cover slips, dried overnight at room temperature, and metal was sprayed for 2 min by an ion sputtering machine . The SEM parameters were as follows: condition = high vacuum, mode = secondary electrons with E-T detector, and accelerating voltage = 5 kV. Changes in color and size of CalliSpheres® were detected by an inverted fluorescence microscope . Quantitative particle sizes were measured by a laser diffraction particle size analyzer .Morphological changes of CalliSpheresp < 0.05 was considered significant.Data are mainly presented as mean ± standard deviation. Statistical analyses and plotting were performed using SPSS Statistics 23 and GraphPad Prism 8. Comparisons among groups were determined by t-tests or analysis of variance. ®, while the added amount was under 20 mg, whereas the drug-loading speed decreased with an increase in particle sizes. When the amount of IDA added increased to 40 mg, however, only 50–150 μm CalliSpheres® enabled a loading efficiency of 90% at 1 h after the beginning of drug loading. These results imply that the IDA loading ability for CalliSpheres® was lower for larger sizes than for smaller ones.As presented in ® showed poor drug loading efficiency in the objective drug loading amount (20 mg), while the loading efficiency was already over 95% within 15 min in 50–150 and 100–300 μm CalliSpheres®. Thus, the influences of the loading medium, loading condition, and drug concentration were further investigated based on 300–500 μm CalliSpheres®. As shown in According to clinical application, 20 mg of IDA are adequate for a single embolization treatment . However®. An ultimate loading test was also conducted. Upon increasing the IDA amount to 40 mg with all other parameters being the same, the drug loading efficiency exceeded 90% at 30 min .The release profiles of CalliSpheres® increased after being rinsed by H2O and later decreased after loading IDA. From the initial marketed products to the final IDA-loaded DEB, there was a statistically significant diameter increase in all three CalliSpheres®.The results of particle size measurement are shown in ® turned from blue to orange after IDA loading and faded after the drug release. The black circular stripe is the contact surface of CalliSpheres® and glass slide, under the inverted microscope. The surface of 50–150 μm CalliSpheres® was bumpier than the larger ones, which might be ascribed to the preparation technology of CalliSpheres® of different sizes.The morphology before/after drug loading and release under an optical microscope is shown in ® presented small holes before IDA loading, whereas these holes became smooth after drug loading , HepaSphereTM (65% ± 6%), or LifePearlTM (73% ± 3%) [®, 26 min for 100–300 μm CalliSpheres®, and 51 min for 300–500 μm CalliSpheres®. Compared to DC BeadTM with 100–300 μm particle sizes, which takes 24 min to reach 75% of the release plateau level (5 mg IDA loaded) [® is higher. In addition, it was indicated that FBS would accelerate IDA release, probably because some components in FBS weakened the electrostatic interaction.The selection of particle sizes for DEB is another important issue in clinical practice . It is w3% ± 3%) . As for  loaded)  or 37 mi®, there was a variation in the trend of diameters before/after rinsing and IDA loading, which was measured by laser diffraction particle size analyzer, and was consistent with the images produced by an optical microscope. In general, the diameters after IDA loading were larger than those of the original marketed products. Especially for 300–500 μm CalliSpheres®, the mean diameter after IDA loading was over 500 μm. These beads are unable to enter the branch blood vessels supplying a tumor and embolize them, thus 300–500 μm CalliSpheres® are not recommended. IDA has an orange-red color, thus the general conditions of drug loading and release could be observed through the color of CalliSpheres® seen under optical microscope. In SEM images, no drug crystals were observed on the surface of CalliSpheres®, which was different from the cases for some rapid-release drugs such as oxaliplatin [Regarding the physical properties of CalliSpheresliplatin . These r® of three different particle sizes. To better simulate clinical practice, marketed idarubicin hydrochloride for injection was used in this research, as well as APIs. Limited by experiment conditions, however, no other IDA preparations from different manufacturers were evaluated. In addition, although the IDA release rate was tested by the USP 4 method, which is considered to have proper in vivo–in vitro correlation, the results cannot completely reflect the drug eluting process in TACE treatment of HCC.This study optimized the IDA loading method and evaluated the compatible stability, drug release profiles, and physical properties of CalliSpheres®. All three CalliSpheres® of different particle sizes disclosed satisfactory loading efficiency with the optimized method, as well as sustained release performance in vitro. The optimized IDA loading method is of important significance for clinical applications, especially indicating that normal saline might not be an appropriate drug-loading medium. In addition, IDA-loaded CalliSpheres® present great compatible stability with iopamidol within 8 h, meaning that pre-operation drug leaks do not need to be considered in most clinical applications. According to the experiment results and general clinical settings, 100–300 μm CalliSpheres® are recommended.This in vitro study investigated the IDA loading method, release profiles, and morphologic properties of CalliSpheres


Sesbania sesban L. extract, a plant with high anti-inflammatory actions that are beneficial for rheumatoid arthritis treatments.Rheumatoid arthritis, a chronic and progressive inflammation condition in the joints, has significantly reduced the patient quality of life and life expectancy. Crucially, there is no complete therapy for this disease, and the current treatments possess numerous side effects. Thus, novel therapeutic approach is necessary. To that end, this study developed novel silk fibroin in-situ hydrogel containing The hydrogels were manufactured using simple method of spontaneous gelation at different temperature. The gel properties of morphology, gelation time, viscosity, gel strength, stability, drug loading capacity, drug release rate, and in-vitro anti-inflammatory activity were investigated with appropriate methods.Sesbania sesban L. leaf extract with a total phenolic content of 92.8 ± 8.30 mg GAE/g, and sustained the release rate for >20 dạys, followed the Higuchi model. Regarding the in-vitro activities, all formulations were nontoxic to the RAW 264.7 cell line and demonstrated comparable anti-inflammatory activity to the free extract, in terms of the NO reduction levels.The optimal formulation had highly porous structure, with a gelation time of 0.5 h at room temperature and bodily temperature of 37 °C, a viscosity of 2530 ± 50 cP, a gel strength of 1880.14 ± 35.10 g, and a physical stability of >6 months. Moreover, the hydrogel contained the Conclusively, the systems possessed potential properties to be further investigated to become a prospective rheumatoid arthritis treatment. Rheumatoid arthritis (RA), a chronic and progressive inflammation condition in the joints, is an autoimmune disease with complicated pathology , Egyptian riverhemp, Dien dien (in Vietnamese)) has been proposed. SS has been proven, both by ethnopharmacological information and scientific data, to possess several therapeutic activities, especially the anti-inflammatory action that is beneficial for RA treatment , Fetal Bovine Serum (FBS), PenStrep (penicillin + streptomycin), and trypsin + EDTA were bought from Thermo Fisher Scientific, Singapore. MTS -5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay kit was purchased from Promega, USA. Nitric oxide (NO) assay kit was imported from Elabscience, USA. All other chemicals and solvents were of the reagent grades or higher.Approximately 1 kg of each plant part, leaves and flowers, was dried under normal temperature, followed by grinding to 1-mm powder. Then, 1 g of the SS leaves/flowers powder was sonication-assisted extracted with 10 mL of EtOH, at a varied temperature of 30, 50, and 60 °C, for a varied extraction time of 60 and 120 min. All SS extractions (SSE) were freshly prepared prior to utilizations.v/v) and 2 mL of Na2CO3 solution (7.5% w/v). The mixture was then incubated at 37 °C in the dark for 1 h, followed by UV-Vis spectroscopic measured at 765 nm . The total polyphenol concentrations were then calculated based on the standard curve with gallic acid . The total phenolic content of the extracts was calculated following The SSE total phenolic content, at different extraction conditions, was determined utilizing the Folin-Ciocalteu reagent, following the published method with some modifications  Na2CO3, washed with de-ionized water, and air dried. Then, the degummed silk (10 g) was cut into small pieces, dissolved in a hot (85 °C) mixture of CaCl2:H2O:Ca(NO3)2:EtOH (30:45:5:20 w/w/w/w), dialyzed against de-ionized water to remove residual salts, and centrifuged at 10,000 rpm for 30 min. Finally, the fibroin solution was freeze-dried  at 10−4 Torr and −55 °C. The fibroin powder was stored at −20 °C for further experiments.Fibroin was extracted and purified from the raw w/v). Then, the blank (unloaded) fibroin hydrogel was formulated by allowing 10 mL of the fibroin aqueous solution to spontaneously become solid gel at 4 °C, room temperature , or bodily temperature (37 °C). For the SSE loaded fibroin hydrogel, 10 mL of the SSE, which contained 1 g of the total phenolic content of the SS leaves/flowers, was concentrated to 0.1 mL using the solvent evaporation method. Then, this 0.1-mL SSE was added to 10 mL of fibroin aqueous solution, followed by the incubation at 4 °C, RT, or 37 °C. The SSE loaded fibroin hydrogel was formulated gradually.Freeze-dried fibroin powder was dissolved in de-ionized water at various concentrations of 1, 2, and 5%  was determined by the vial inversion method. To this end, 2 mL of the formulations was put in a vial at 4 ± 0.5 °C, 25 ± 0.5 °C (RT), or 37 ± 0.5 °C, the vial was inverted regularly to observe the gel behaviors. The sol- and gel-formation was set as flowing liquid and non-flowing solid, respectively. The sol-gel transition time was determined as the initial time point that the non-flowing solid occurred.The hydrogel viscosity was measured at 25 ± 0.5 °C (RT) by a Brookfield viscometer model DV-E  utilizing a 40-mm parallel plate configuration with a rotation rate of 15 rpm. The samples (1 mL hydrogels) were placed on the plate and subjected to the viscosity test.The hydrogel strength was determined using a texture analyzer . Briefly, 1 mL of the blank/SSE loaded fibroin solutions was subjected to a 5-mL vial and incubated at 25 ± 0.5 °C (RT) to form solid gel. The gel was then compressed by a 50-mm hemispherical probe P/0.5 HS to a fixed depth of 4 mm. The gel strength was calculated and demonstrated by the software included in the machine.Scanning electron microscopy  was used to observe the hydrogel morphology. The gel, after being fixed, was subjected to a hard resin for ultramicrotome cut into ultra-thin 100-nm slices. These slices were mounted on a metal stub using a sticky carbon disk, followed by sputter coating with gold (10-nm thick). Then, the samples were SEM analyzed.The hydrogel physical stability was conducted at both RT and 4 °C. At each time intervals , the gels were re-measured their properties of appearance, viscosity, and gel strength. The hydrogels were considered stable when these characteristics remained similar  to that in the initial time.t (tC) was calculated based on the gallic acid standard curve, and the cumulative drug release percentage was determined followed 0V is the total volume of the release medium (5 mL), 0M is the initial amount of total polyphenol in the hydrogel, iC is the concentrations of released drug at the time point i, and V is the withdrawal sample volume at each time point (0.5 mL).In-vitro cumulative drug release profile of SSE loaded fibroin hydrogel, in terms of total polyphenol, was determined by the shaker method. The hydrogel was designed to be administered directly in the inflamed joint, thus, the drug release study was conducted in a simulated inflammatory joint condition. To this end, 5 mL of SSE loaded fibroin solution  were put in a vial to form hydrogel. Then, 2 mL of the PBS buffer, pH 6.5, was added to the hydrogel. The system was continuously shaken at 50 rpm, at 37 °C. At each specific time point, 0.5 mL of samples was withdrawn and buffer refilled. The amount of the total polyphenol content in the samples was determined using the Folin-Ciocalteu reagent, with similar method described in the section “SS extraction and total phenolic content determination”. The concentration of total polyphenol released (µg/mL) at time R2 value was considered the most fitted model to describe the drug release behaviors.To clarify the drug release mechanism, numerous models including the zero-order, first-order, Higuchi, Hixon-Crowell, and Korsmeyer–Peppas kinetics were tested. The release model possessing the highest 2. The medium was replaced every other day. The confluent cells (70–80%) were washed, trypsinized using trypsin-EDTA 0.25%, and re-seeded into the 96-well plates for cytotoxicity and anti-inflammatory tests.The RAW 264.7 macrophages cell line was cultivated in DMEM supplemented with 10% FBS and 1% PenStrep, at 37 °C and 5% CO3 cells/well. The cells were then incubated for 24 h at 37 °C and 5% CO2, followed by the addition of 20 µL samples at various concentrations of 75, 150, 300, and 500 µg/mL, equivalent to the SSE total phenolic content. The samples included the SSE, the blank fibroin hydrogel, and the SSE loaded fibroin hydrogel. Then, the cells were re-incubated for 24 h at the same condition. Finally, the cells were washed thrice with PBS, and 20 µL of MTS solution was added into each well, followed by incubation for 4 h at 37 °C and 5% CO2. The absorbance was measured using UV-Vis plate reader at 490 nm. Mixture of PBS and MTS without cells served as a blank, and the positive control was the untreated cells (i.e. 100% viability). The percentage of cell viability was calculated based on The cell viability was determined using the MTS assay, with slight modification from a previous study , followed by incubation at 37 °C, 5% CO2 for 24 h. Then, the cells were treated with 100 ng/mL of LPS and the samples at concentrations of 75, 150, 300, and 500 µg/mL, equivalent to the SSE total phenolic content. After another 24-h-incubation, the culture media were collected, centrifuged at 3000 rpm for 10 min, and the supernatants were mixed with the NO assay kit following the manufacturer instruction, followed by UV-Vis spectroscopic measured at 550 nm. The NO concentrations were calculated based on the NO calibration curve. The assay negative and positive controls were the non-LPS-induced RAW 264.7 cells and the LPS-induced RAW 264.7 cells without treatments, respectively.The anti-inflammatory test of the blank fibroin hydrogel, the SSE, and the SSE loaded hydrogel, was conducted using the lipopolysaccharide (LPS) induced RAW 264.7 cells. For this, the cells were seeded in a 96-well plate . One-way ANOVA and Student’s The long-term RA current treatments of NSAID, corticosteroids, and DMARD, cause lots of side effects and inconveniences for patients. Thus, this study developed and characterized the SSE loaded fibroin in-situ hydrogel for the purpose of both finding the novel therapeutic drug/extract for RA, as well as encapsulating it in a biocompatible, biodegradable, and controlled release hydrogel system to enhance the patient compliance.p<.05). Notably, higher temperature of 60 °C and longer extraction time of 120 min did not increase the total phenolic content. Therefore, the best extraction condition was at a temperature of 50 °C and a time of 60 min, which yielded 92.8 ± 8.30 mg GAE/g of the leaf extract. Our result was higher than that of the Indonesian SS leaves, which had a maximum total phenolic content of approximately 51.8 mg GAE/g , first order (R2 = 0.5284), Higuchi (R2 = 0.9973), Hixon-Crowell (R2 = 0.6116), and Korsmeyer–Peppas (R2 = 0.6842), showed that the most fitted one was Higuchi model, with an equation of y = 19.488x − 2.3553. This indicates that the release profile is based on diffusion-controlled mechanism, corresponding to the gradual biodegradability of fibroin in the medium  possessed no potential cytotoxicity to the RAW 264.7 cells, with a cell viability of >80% . This suhydrogel . FurtherSesbania sesban L. extract for rheumatoid arthritis treatment. The optimal hydrogel contained the plant leaf extract with a total phenolic content of 92.8 ± 8.30 mg GAE/g. Physico-chemically, the gel was translucent with a highly porous structure, possessed a gelation time of 0.5 h at bodily temperature of 37 °C, a viscosity of 2530 ± 50 cP, a gel strength of 1880.14 ± 35.10 g, and a physical stability of >6 months. The system could sustain release the extract for >20 dạys, followed the Higuchi model. Furthermore, all formulations were nontoxic to the RAW 264.7 cell line and demonstrated comparable anti-inflammatory activity to the free extract. These data, together with the inherent RA treatment activity of the phenolic compounds, make the product a potential system to be a novel treatment for RA in the future.This study presents the novel silk fibroin in-situ hydrogel containing


T (=ATCC TSD-225T=NCTC 14479T), an isolate from the ruminal content of a dairy cow. Phenotypic and genotypic traits of the isolate were explored. JE7A12T was found to be a strictly anaerobic, catalase-negative, oxidase-negative, coccoid bacterium that grows in chains. The API 50 CH carbon source assay detected fermentation of d-glucose, d-fructose, d-galactose, glycogen and starch. HPLC showed acetate to be the major fermentation product as a result of carbohydrate fermentation. Phylogenetic analysis of JE7A12T based on 16S rRNA nucleotide sequence and amino acid sequences from the whole genome indicated a divergent lineage from the closest neighbours in the genus Ruminococcus. The results of 16S rRNA sequence comparison, whole genome average nucleotide identity (ANI) and DNA G+C content data indicate that JE7A12T represents a novel species which we propose the name Ruminococcus bovis with JE7A12T as the type strain.This study describes JE7A12 Ruminococcus was first described by Sijpesteijn [Ruminococcus flavefaciens as the type strain [Ruminococcus have most frequently been isolated from the rumen and gastrointestinal tract of a wide variety of animals, including humans [Ruminococcus group 1 includes the type strain Ruminococcus flavefaciens, Ruminococcus albus, Ruminococcus bromii and Ruminococcus callidus. Ruminococcus group 2 species have recently undergone taxonomic re-classification with many species being reassigned to different genera [Ruminococcus are the species found in group 1 [The genus pesteijn  with Rue strain . Previoug humans . The gent genera . It is n group 1 .Ruminococci are ubiquitous members of the human gastrointestinal and rumen microbial consortia worldwide where they play a role in the fermentation of cellulose rich feedstuffs and resistant starch [et al. found that species of the genus Ruminococcus were present in all ruminants surveyed and, on average, were found to comprise 3.6 % of the total rumen bacterial community [Ruminococcus is naturally high there is evidence that their functional role is larger than the abundance would suggest. Xia et al. revealed that 70–80 % of the starch degrading bacteria in the barley-fed beef heifers were members of the family Ruminococcaceae [Ruminococcus [Ruminococcus has the potential to elucidate underlying microbial functionality in the rumen and the influence of members of the genus with regards to ruminant feed utilization and nutrition. The following description pertains to the isolation and classification of a novel group 1 amylolytic species, represented by strain JE7A12T, of the genus Ruminococcus.Microbial fermentation plays a prominent role in the utilization of feed by ruminants. In the rumen, bacterial fermentation is known to contribute to the stabilization of ruminal pH, increase volatile fatty acid production, reduce ammonia concentration and improve fibre digestibility . Ruminoct starch . Assessmommunity . While tccaceae . Similarococcus . Thus, cT was recovered from the rumen content of a healthy, Holstein dairy cow obtained from a farm in Tulare, California, USA on a modified chopped meat broth with carbohydrates solid medium (DSMZ Medium 110) at 37 °C in an anaerobic environment . The medium was modified by the removal of fat-free ground meat and casein and the addition of 30.0 g peptone, 15.0 g meat extract, 10.0 g meat peptone, 15.0 g agar and 100 ml clarified rumen fluid [T displayed off-white colonies approximately 0.1–0.3 mm in diameter on supplemented Bacto Tryptic Soy Broth (BD) with 0.4 g l-cysteine hydrochloride, 0.02 g ferric ammonium citrate, 10 µg vitamin K1, 2.0 mg resazurin sodium salt, 10.0 ml vitamin supplement ATCC MDVS (ATCC) and 7.0 g Gelrite (CP Kelco) per litre of medium (TSB+FAC). Gram-staining was performed as described by Beveridge [T is a strictly anaerobic coccoid, commonly found in pairs and chains [T does not require rumen fluid for growth.JE7A12en fluid  per litreveridge . Cell momega X [T was placed in a dendrogram of all type strains of species from the order Clostridiales for which a full length 16S rRNA sequence was available in the RDP database [Ruminiococcus as well as close phylogenetic neighbours , Clostridium leptum (91.2 %) and Caproiciproducens galactitolivorans (89.2 %).The full length 16S rRNA sequence of JE7A12 methods . The fulT and type strains for all current species of the genus Ruminococcus [Clostridium leptum and Caproiciproducens galactitolivorans were included in the ANI analysis due to their close 16S rRNA similarity. Due to bias in ANI algorithms, the ANI of JE7A12T was evaluated utilizing both MUMmer and blast algorithms [blast was to Ruminococcus bromii. However, the two species are genetically distant as their genomes share 72.7 % sequence similarity but at only 20.1 % coverage of the genome  was compared between JE7A12ococcus . Additiogorithms  (Tables gorithms . The bese genome . MUMmer coverage . The onlT were determined using a 3 % (v/v) hydrogen peroxide solution and 1.2 % tetra-methyl-p-phenylenediamine dihydrochloride solution, respectively. Growth temperature ranges were determined in TSB+FAC medium at 25, 30, 37, 40 and 50 °C. Optimal growth was observed at 37 and 40 °C, reduced growth at 30 °C and no growth at 25 and 50°C incubation temperature. No motility was observed. Growth in the presence of salt was studied by supplementing TSB+FAC liquid medium with NaCl (0.5–4.5 % w/v in 0.5 % increments). Hungate tubes were incubated at 37 °C for 72 h and monitored for growth. JE7A12T was capable of growing in salt concentrations of up to 2.5 %. Tolerance to pH (5.0–9.0) was tested on TSB+FAC with pH tested in increments of 0.5 pH units. Hungate tubes were incubated at 37 °C for 72 h and monitored for growth. The optimal pH for growth was pH 7.0–7.5 with reduced growth at pH 6.0–6.5. No growth was observed at pH 8.0–9.0 and pH 5.0–5.5 on TSB+FAC.Catalase and oxidase activities of JE7A12T was qualitatively measured using the API 50CH carbon panel (BioMérieux). JE7A12T cells were grown to late exponential phase and recovered by centrifugation at 3000 g for 10 min. Cells were resuspended and 0.017 % .Carbohydrate fermentation of JE7A12hydrates . JE7A12TT and all current species of the genus Ruminococcus can be found in R. bromii, JE7A12T shows narrow specialization with regards to carbohydrate fermentation, while other members of the genus Ruminococcus generally ferment a wider range of carbohydrates [R. bromii has been reported to ferment most of the same carbon sources as JE7A12T, including galactose, glucose, fructose, maltose, glycogen and starch [R. bromii derived from the bovine rumen are not known to ferment fructose or galactose and are rarely able to ferment glucose. Utilization of these carbon sources have more commonly been observed in human derived R. bromii [T from ruminally derived Ruminococcus bromii.A comparison of carbon source fermentation between JE7A12hydrates . Specifid starch . Despite bromii . Therefo2SO4 at 0.5 ml min−1. Pure standards of acetate, ethanol, glycerol, lactate, butyrate, butanol, propionate, succinate and pyruvate were used for calibration at varying concentrations. JE7A12T produces acetate as a major fermentation product as well as ethanol and glycerol as minor products. No lactate, butyrate, butanol, propionate, succinate or pyruvate is produced. A comparison of metabolite production between JE7A12T and all current species of the genus Ruminococcus can be found in T most closely resembles that of R. bromii and R. gauvreauii which are the only species in the genus that produce acetate, and only acetate, as a major metabolic product. While the other members of the genus produce acetate, they also produce high levels of succinate, lactate and formate.Metabolite production was measured using a Waters Acquity UPLC Q System with RI detector. The column used was a Phenomenex 00 H-0138-K0 Rezex ROA Organic Acid H+ (8 %) operated at 60 °C. The mobile phase was 0.001625 M HRuminococcus bovis (bo'vis. L. gen. n. bovis of the cow)Ruminococcus bovis is an obligately anaerobic, catalase-negative and oxidase-negative bacterium. It is Gram-stain-positive and forms chains of small cocci when cultured in liquid medium. When cultured on TSB+FAC solid medium, it forms small, slightly opaque, off-white, circular colonies with even margins. Fermentation of d-galactose, d-glucose, d-fructose, maltose, glycogen, aesculin/ferric citrate and starch is indicated by API CH50. The major fermentation product is acetate, with ethanol and glycerol as minor products. No lactate, butyrate, butanol, propionate, succinate or pyruvate is produced.T (=ATCC TSD-225T=NCTC 14479T) and was originally isolated from rumen content of a healthy, Holstein cow from Tulare, California, USA. The genomic DNA G+C content of the type strain is 34.6 mol%.The type strain is JE7A12Click here for additional data file.


Objective: This prospective study evaluated the 50% effective dose (ED50) and 95% effective dose (ED95) of nalbuphine combined with propofol during painless gastroscopy.Methods: Seventy-five patients who underwent painless gastroscopy were randomly divided into five groups , with doses of 0, 0.05, 0.1, 0.15, or 0.2 mg/kg nalbuphine in each group. Propofol was given to all groups as the sedative. The bispectral index (BIS) value, propofol dose, examination time, and awakening time were recorded. The number of patients with intolerance indexes  was recorded in each group. The ED50/ED95 of nalbuphine combined with propofol for gastroscopy were calculated.Results: Compared with those of groups N0, N0.05, or N0.1, the propofol dose and awakening time were significantly reduced in group N0.15 or N0.2 (p < 0.05). The successful rate of painless gastroscopy in group N0.15 or N0.2 significantly increased compared to that of group N0 or N0.05 (p < 0.05). When combined with propofol, nalbuphine had an ED50 and ED95 for painless gastroscopy of 0.078 mg/kg  and 0.162 mg/kg , respectively.Conclusion: The ED50/ED95 of nalbuphine combined with propofol are 0.078 and 0.162 mg/kg, respectively, for painless gastroscopy. Nalbuphine at 0.162 mg/kg combined with propofol is effective and safe for painless gastroscopy in adults. Gastros50/ED95 of nalbuphine combined with propofol will help patients have a safe, painless gastroscopy.Nalbuphine is a κ-receptor agonist and partial μ-receptor antagonist. As a κ-receptor agonist, nalbuphine has a unique analgesic effect on visceral pain , which i50/ED95 of nalbuphine combined with propofol during painless gastroscopy, and we determined the feasibility of nalbuphine combined with propofol for painless gastroscopy in adults.In this study, we evaluated the EDThis experiment has been approved by the Medical Ethics Committee of Hainan General Hospital . Informed consent forms were signed by patients.2, and American Society of Anesthesiologists classification I–II. The exclusion criteria included hepatitis and renal failure, habitual sedative or analgesic use, mental illness, and allergy to nalbuphine or propofol.Overall, 75 patients who underwent painless gastroscopy in Hainan General Hospital from April 2020 to July 2020 were selected. The inclusion criteria were as follows: age 18–60 years, body mass index of 18–25 kg/m0), 0.05 mg/kg (group N0.05), 0.1 mg/kg (group N0.1), 0.15 mg/kg (group N0.15), and 0.2 mg/kg (group N0.2). All patients fasted from solids for 8 h and from liquids for 2 h. All patients took the left recumbent position in the operating room, with standard monitoring that included oxygen saturation (SpO2), noninvasive blood pressure, and electrocardiogram. All patients inhaled oxygen using the nasal catheter (3 L/min) for 5 min before anesthesia.The patients were randomly divided into five groups of nalbuphine according to dose, as follows: 0 mg/kg  was injected intravenously at the onset; at 3 min after the onset, propofol  was administrated intravenously at a rate of 50–150 μg/kg/min until the patient lost consciousness, as reflected by a bispectral index (BIS)  value between 50 and 65. Then, the gastroscopy was performed. If the gastroscopy failed , the propofol dose was increased to facilitate completion of the examination. When the heart rate decreased to 50 beats per min, 0.2–0.5 mg atropine was injected. When the mean arterial pressure was less than 60 mmHg, 5–10 mg ephedrine was applied. When SpOObservation indexes included the following: the BIS value at baseline (before the beginning of anesthesia) and at the beginning of gastroscopy, the effect-site concentration of propofol at the beginning of gastroscopy and at the end of gastroscopy, the propofol dose (the amount of propofol), the examination time (the time of gastroscopy), and the awakening time (the time between the end of the gastroscopy and when the patients to awake) were recorded. The number of patients with any coughing, swallowing, retching, or limb movement was recorded in each group. The number of patients who experienced a successful gastroscopy  was recorded in each group. The number of patients with apnea, postoperative pruritus, postoperative nausea/vomiting, or postoperative anxiety/irritability was recorded.50/ED95 of nalbuphine combined with propofol. The patients who required increasing propofol doses were regarded as having a failed gastroscopy.The effect index was the number of patients who experienced a successful gastroscopy, and the index was used to calculate the ED50) and the ED95 as well as the 95% CIs of nalbuphine when combined with propofol were determined by binary regression (probit)  was used for statistical analyses of data. The median effective dose  .0 and group N0.2 were 726.13 ± 123.46 (s) and 245.00 ± 68.74 (s), respectively. A sample size of 15 in each group was required for a beta value of 0.10 and an alpha value of 0.05.The required sample size was calculated using PASS 11.0 . The main indicator considered was the awakening time. The pilot study had 5 cases in each group. The mean ± standard deviation of awakening time in group Np value of <0.05 was considered to be statistically significant.Normally distributed statistics were analyzed as the mean ± standard deviation and by one-way analysis of variance. Categorical variables were presented as proportions (%) and were compared using Fisher’s exact test or the chi-squared test. Trends in the nalbuphine doses were evaluated using the chi-squared test for trend. A 0 and one from group N0.15) because gastric mucosa tissues were taken for observation by pathology and the duration of gastroscopy was more than 10 min. No significant demographic differences were noted among the five groups (p > 0.05). Results are shown in Overall, 77 patients were enrolled in the study, and 75 patients were assessed for eligibility. Two patients were excluded ; groups N0.15 and N0.2 had significantly reduced incidences of coughing, retching, swallowing, and limb movement (p < 0.05). Compared with group N0.05, groups N0.15 and N0.2 had significantly reduced incidences of retching, swallowing, and limb movement (p < 0.05). Results are shown in Compared with group Np > 0.05). Results are shown in There was no significant difference in the incidence of apnea, postoperative pruritus, postoperative nausea/vomiting, or postoperative anxiety/irritability among the five groups ; compared with group N0.05, groups N0.15 and N0.2 had significantly increased incidences of successful gastroscopy (p < 0.01). No significant differences in success were noted among the other groups (p > 0.05). Results are shown in Forty-four patients experienced successful gastroscopies . The incidences of successful gastroscopy in groups N50 of nalbuphine combined with propofol for painless gastroscopy was 0.078 mg/kg , and the ED95 of nalbuphine was 0.162 mg/kg  for the same procedure. Results are shown in The statistics were analyzed by binary regression (probit). The EDp > 0.05). After the nalbuphine dose was increased, the effect-site concentration of propofol, the propofol dose, and the awakening time decreased gradually in groups N0, N0.1, and N0.15 (p < 0.05), whereas no statistically significant differences were noted in these indexes between groups N0.2 and N0.15 (p > 0.05). The examination time for group N0 was significantly longer than that for any other group (p < 0.05), but no significant difference was noted among the other four groups (p > 0.05). All results are shown in No statistically significant differences were observed in the BIS value at baseline or at the beginning of gastroscopy among the five groups . If apnea lasts for more than 30 s, artificial respiration with a hand-controlled balloon was used to maintain blood oxygen saturation greater than 95% until spontaneous breathing was restored. The momentary apnea did not cause adverse effects for the patients. No patients had adverse cardiovascular reactions after receiving the anesthetic drugs; fluctuations of blood pressure and heart rate were within the physiological ranges, and cardiovascular drugs were not used.In this study, six patients had apnea  and 0.162 mg/kg , respectively. Therefore, 0.162 mg/kg of nalbuphine combined with propofol anesthesia is safe and effective for gastroscopy in adults, and it could provide the best conditions for gastroscopy, without any adverse reactions.The probit regression method showed that the ED50/ED95 of nalbuphine combined with propofol for patients with painless gastroscopy are 0.078 and 0.162 mg/kg, respectively. So, nalbuphine at 0.162 mg/kg combined with propofol is an effective and safe way for painless gastroscopy in adults.The EDThis study had some limitations. First, no serum nalbuphine/propofol assays were used. Second, clinical examinations of outpatients were usually incomplete. Last, the hidden diseases and different sensitivities to drugs in individuals might have affected the results.


Data on the long-term effects of neonatal acute kidney injury (AKI) are limited.th percentile) according to gestational age, weight, and postnatal age. Clinical and laboratory features in the neonatal AKI period were recorded for 72 cases; at long-term evaluation (2–12 years), kidney function tests with glomerular filtration rate (eGFR) by the Schwartz formula, microalbuminuria, office and 24-h ambulatory blood pressure monitoring (ABPM), and kidney ultrasonography were performed.We invited 302 children who had neonatal AKI and survived to hospital discharge; out of 95 patients who agreed to participate in the study, 23 cases were excluded due to primary kidney, cardiac, or metabolic diseases. KDIGO definition was used to define AKI. When a newborn had no previous serum creatinine, AKI was defined as serum creatinine above the mean plus two standard deviations (SD) (or above 97.52. Office hypertension (systolic and/or diastolic BP ≥ 95th percentile), microalbuminuria (> 30 mg/g creatinine), and hyperfiltration (> 187 ml/min/1.73 m2) were present in 13.0%, 12.7%, and 9.7% of patients, respectively. ABPM was performed on 27 patients, 18.5% had hypertension, and 40.7% were non-dippers; 48.1% had abnormal findings. Female sex was associated with microalbuminuria; low birth weight  and low gestational age (< 32 weeks) were associated with hypertension by ABPM. Twenty-three patients (33.8%) had at least one sign of microalbuminuria, office hypertension, or hyperfiltration. Among 27 patients who had ABPM, 16 (59.3%) had at least one sign of microalbuminuria, abnormal ABPM (hypertension and/or non-dipping), or hyperfiltration.Forty-two patients (58%) had stage I AKI during the neonatal period. Mean age at long-term evaluation was 6.8 ± 2.9 years (range: 2.3–12.0); mean eGFR was 152.3 ± 26.5 ml/min/1.73 mEven children who experienced stage 1 and 2 neonatal AKI are at risk for subclinical kidney dysfunction. Non-dipping is seen in four out of 10 children. Long-term follow-up of these patients is necessary. The incidence of acute kidney injury (AKI) in newborns is high compared to other age groups of childhood. Its incidence is 20–40% in neonatal intensive care units (NICUs) –3; low bThere is limited knowledge about the long-term effects of neonatal AKI, such as hypertension, decreased kidney function, and tubular malfunction. Kidney dysplasia, obstructive uropathy, cortical necrosis, and cystic kidney diseases are risk factors for progression to CKD in the future . In addiWe aimed to assess the prevalence of long-term (2–12 years) kidney dysfunction , based on clinical indicators including ambulatory blood pressure monitoring (ABPM), and to determine associated factors in a case series of children who had suffered from AKI in the neonatal period .th percentile) according to birth characteristics  and postnatal age [Medical records of patients hospitalized in newborn wards of Hacettepe University Ihsan Dogramaci Children’s Hospital from January 2000 to December 2009 were retrospectively reviewed to identify the patients who suffered from AKI. AKI was defined as an increase in serum creatinine level of 0.3 mg/dl or more within 48 h or 50% or more from the previous lowest value within 7 days, based on the Kidney Disease: Improving Global Outcomes (KDIGO) workgroup’s definition of AKI modified for neonates, as used in previous neonatal studies . We exclatal age –25.Of the patients who had developed neonatal AKI according to the defined criteria, 347 patients survived and were discharged. Of those, 27 patients (7.8%) had died following discharge. Of 302 parents or caregivers who were sent invitation letters, 109 responded by telephone, and 95 agreed to participate in the study. Twenty-three patients with primary kidney, cardiac or metabolic diseases that may affect long-term evaluation were excluded. In total, 72 patients were analyzed, including 46 males (63.9%) and 26 females (36.1%) , AKI features . AKI severity was classified according to KDIGO methods.Patients were asked about their current medical condition, chronic diseases, and medications. Physical examination, weight, height, body mass index (BMI) measurement; laboratory studies including complete blood count, kidney function test, venous blood gas, urinalysis, urinary microalbumin, creatinine, phosphate; kidney ultrasonography (US), kidney Doppler US, auscultatory office blood pressure (BP) measurement, and 24-h ABPM were performed.Estimated glomerular filtration rate (eGFR) was calculated based on the Schwartz formula [Serum creatinine was measured with Jaffe method .  formula .Hyperfiltration: Mean GFR between 2 to 12 years of age is 133 ± 27 ml/min/1.73 m2 [2 (> mean + 2 SD) [/1.73 m2 , 29. GFR + 2 SD) , 27.Microalbuminuria was defined as a urinary microalbumin (mg)/creatinine (g) ratio (ACR) above 30 in a spot urine sample.Tubular phosphorus reabsorption (TPR) was calculated; TmP/GFR was defined as maximum serum phosphorus level adjusted to GFR and measured as TmP/GFR = TPR x serum phosphorus level. The normal range is 4–6 mg/dl.Tubular function disorder was defined as TPR level below 85% and/or venous blood HCO3 level below 20 mmol/L.th percentile was defined as hypertension.Standard procedures were applied for office BP measurement using the auscultatory method; BP was measured three times with an interval of 3 min between measurements and the average of the last two was used. The patient’s BP percentile was determined according to age, gender and height percentile according to 2016 European Society of Hypertension guidelines . The preth percentile by sex and age) were calculated for the 24-h period, daytime (from 08.00 am to 08.00 pm), and nighttime (from midnight to 06.00 am) periods. SD scores (SDS) for SBP, DBP and MAP were determined according to the standard values adjusted by age and gender [th percentile, with normal ABPM profile. Masked hypertension was defined as office BP < 95th percentile and ABPM hypertension [24-h ABPM was performed in patients with height ≥ 120 cm and ≥ 6 years of age; patients with poor cooperation or who were not able to carry the equipment for 24 h were excluded. Patients were not hospitalized during ABPMs and they performed normal daily activities. Spacelabs ABPM devices (Model no: 90207–30) were used. Measurements were performed every 15–20 min during waking hours (day) and every 30 min during sleep periods (night). A valid ABPM profile was defined as follows: 24-h recording with at least 70% of expected measurements, at least 40 to 50 readings for a 24-h period; and at least two valid daytime and one valid nighttime measurement per hour , 30. Thed gender . The prertension .Patients underwent kidney US and kidney Doppler US in the supine position. All evaluations were performed by a single pediatric radiologist (BO). Kidney size, parenchymal thickness, parenchymal echogenicity, and the kidney Doppler indices  were evaluated by US. Kidney vertical lengths were evaluated according to age, sex, weight, and height, and percentiles were determined .2). Three kidney dysfunction sets (KDS) were defined for the evaluation. KDS-1 was defined as the presence of microalbuminuria and/or hypertension by office BP and/or hyperfiltration (eGFR > 187 ml/min/1.73 m2); KDS-2 was defined as the presence of microalbuminuria and/or hypertension with ABPM and/or hyperfiltration. KDS-3 was defined as the presence of microalbuminuria and/or abnormal ABPM (hypertension and/or non-dipping) and/or hyperfiltration. Univariate analysis was used to evaluate the following as independents factors for the presence of proteinuria, hypertension, hyperfiltration, KDS-1, KDS-2, and KDS-3: age, gender, birth weight, gestational age, weight according to gestational age, systemic diseases in the neonatal period, mechanical ventilation, surgical procedure before AKI, age at AKI diagnosis, total fluid intake before AKI, aminoglycoside use, indomethacin use, radiocontrast use, vasopressor drug use, oliguria, AKI stage, and KRT. Since the significance of hyperfiltration for long-term kidney dysfunction is still unclear; we additionally, repeated our analyses excluding hyperfiltration from the composite outcome.Long-term kidney dysfunction was defined as the presence of at least one sign of microalbuminuria (ACR > 30), hypertension (office or ABPM), or hyperfiltration , and the categorical variables were calculated as the number of cases and percentage. The categorical variables were evaluated by Pearson’s chi-square or Fisher’s exact test. Student’s t-test and Mann–Whitney U test were used to determine the significance of the difference between the groups. A p-value of ≤ 0.05 was considered to be statistically significant.Baseline clinical features of the patients are summarized in Table p = 0.247). Clinical and laboratory characteristics of patients at long-term evaluation are summarized in Table 2 (range: 99–229 ml/min/1.73 m2). Mean eGFR levels were 154.0 ± 29.3 ml/min/1.73 m2 in males and 151.2 ± 21.3 ml/min/1.73 m2 in girls (p = 0.785), and they were 161.3 ± 30.8 ml/min/1.73 m2 between the ages of 2 to 6 and 145.9 ± 21.2 ml/min/1.73 m2 between the ages of > 6 to 12 years (p = 0.014).The mean age of the participants at the time of assessment was 6.8 ± 2.9 years (range: 2.3–12.0 years). The mean ages of boys (6.5 ± 2.8 years) and girls (7.4 ± 3.0 years) were comparable (n = 8) or only systolic (n = 1) BP ≥ 95th percentile.Office auscultatory BP measurements were available for 69 patients; BP was not measured in three patients due to poor cooperation. Nine patients (13.0%) had either systolic and diastolic (n = 23), who were not able to stay overnight (n = 17), who gave insufficient cooperation (n = 4), and who did not accept (n = 1).24-h ABPM measurements were available for 27 patients (6.6–12.0 years). ABPM was not performed in patients who were shorter than 120 cm  or elevated BP load  during 24-h or daytime or nighttime. Eleven patients (40.7%) had systolic  or diastolic  non-dipper; only 14 patients (51.9%) had a normal ABPM pattern . When combined with office BP measurement, 19 patients (70.4%) had normal BP, three patients (11.1%) had white coat hypertension, four patients (14.8%) had masked hypertension, and one patient (3.7%) had elevated BP with both office BP and ABPM.Detailed results of the 24-h ABPM are presented in Table nd percentile, IQR 51st percentile for the right kidney; 57th percentile, IQR 48th percentile for left kidney), and the mean parenchymal thickness  had minor abnormal kidney US findings, including unilateral ectopic kidney  and minimal dilatation of the collecting system . Doppler US results were normal in all cases.Kidney and Doppler US were performed in 71 patients. The median kidney longitudinal length  had at least one sign of KDS-1; among 27 patients who had ABPM, 11 (40.7%) and 16 (59.3%) had at least one sign of KDS-2 and KDS-3, respectively , 12.7% (9 of 71), and 9.7% (7 of 72) of patients, respectively. None of the patients had an eGFR < 90 ml/min/1.73 mThe distribution of kidney dysfunction parameters according to AKI stage groups are shown in Fig. p = 0.010). A higher percentage of girls also met KDS-1 criteria compared to boys . The rate of hyperfiltration (GFR > 187 ml/min/1.73 m2) in the large for gestational age (LGA) group was higher with a borderline significance compared to small for gestational age (SGA) and appropriate for gestational age (AGA) . However, analysis using the Bonferroni method indicated that the comparisons between each paired group were not significantly different. ABPM hypertension was more frequent in patients with a birth weight less than 1,500 g , and in patients with a gestational age less than 32 weeks . No significant association was found with other factors.Girls had a higher rate of microalbuminuria than boys , proteinuria (12.7%), and hyperfiltration (9.7%). To our knowledge, this is the first study that analyzed ABPM in a cohort with neonatal AKI, and it was found to be abnormal in approximately 50% of cases.The distribution of gestational age and birth weight were representative. Systemic diseases among the pre-renal causes and risk factors for AKI, such as sepsis, PDA, RDS, and dehydration rates, were found to be high, and these rates were similar to those found in other studies , 34. It n = 20) who weighed < 1,000 g [n = 25) and the non-AKI group (n = 49), the mean GFR was lower in children with very low birth  independent of AKI [Routine serum biochemistry values for kidney function were normal at the re-evaluation period. Abitbol et al. showed high serum creatinine and low GFR values in 9 (45%) patients in a long-term follow-up study of newborns with AKI . There is evidence that hyperfiltration is a marker for kidney dysfunction, and these patients may be considered as stage 1 CKD [2 (> mean + 2SD). In the study performed by Askenazi et al., hyperfiltration (defined as > 150 ml/min/1.73 m2) was present in 31% (9 out of 29 patients) of patients with AKI (age of diagnosis 6.4 ± 5.8 years) after 3–5 years follow-up [Seven patients (9.7%) had hyperfiltration . The prevalence of hypertension in our population was shown to be 6.1% in a population based study covering 3622 children aged 5–18 years (42). One patient had isolated systolic hypertension, and eight patients had both systolic and diastolic hypertension. In other studies, examining the long-term effects of patients with AKI, the frequency of hypertension varied between 3 and 20% %. The pr, 43. Anip = 0.047), and in patients with a gestational age < 32 weeks . As suggested in the Brenner hypothesis, low nephron numbers or nephron deficiency, such as low birth weight, SGA, preterm birth, may increase the risk of hypertension and kidney disease in the long-term into adulthood [p = 0.5); while diastolic (27% vs. 17% p < 0.01) and systolic  BP loads were higher in the very low birth weight group [p < 0.05) [ABPM was performed in 27 patients (37.5%). Five patients (18.5%) had hypertension by ABPM. ABPM hypertension was more frequent in patients with a birth weight < 1,500 g  . We hypoThe non-dipping phenomenon detected by ABPM is known to be associated with metabolic syndrome, diabetes mellitus, obesity, hypertensive end-organ damage, and cardiovascular diseases . In our Although we did not detect any significant anomalies in our study, kidney volume loss was identified as a long-term effect of AKI in the neonatal period , 35, 38.One- to two-thirds of the patients had at least one long-term kidney dysfunction. Notably, the correlation between microalbuminuria, hyperfiltration, and hypertension was not remarkable. Longitudinal studies are needed to understand the evolution of these abnormalities, their interaction, and the significance of the non-dipping pattern.We are aware of the limitations of our study. The cross-sectional design was the first limitation; as a result, we were not able to examine changes over time. Second, eGFR was based on serum creatinine. Third, we were not able to evaluate markers for renal tubular injury other than TPR and metabolic acidosis. Fourth, lack of a non-AKI comparison group is a limitation of the study. Although the follow-up duration in our cohort is comparable to other studies, a longer follow-up could be associated with higher prevalence of the outcomes. However, compared to previous studies, this study was strengthened by a relatively larger patient group, long-term follow , exclusion of diseases that may interfere with long-term outcome, and performance of ABPM.In conclusion, in a cohort of patients mainly with stage 1 and 2 AKI during the neonatal period and excluding those with primary diseases that may affect the kidney outcome, we found an increased rate of office hypertension, glomerular hyperfiltration, and microalbuminuria approximately 7 years after the AKI episode. Moreover, we showed that majority of patients had abnormal ABPM pattern with a very high rate of non-dipping. Long-term and longitudinal follow-up of these patients is necessary to shed light on the significance of these parameters and the value of any intervention.


Sphingobacterium mizutaii LLE5 was isolated from activated sludge. This strain could utilize SMX as its sole carbon source and degrade it efficiently. Under optimal degradation conditions , S. mizutaii LLE5 could degrade 93.87% of 50 mg/L SMX within 7 days. Four intermediate products from the degradation of SMX were identified and a possible degradation pathway based on these findings was proposed. Furthermore, S. mizutaii LLE5 could also degrade other sulfonamides. This study is the first report on (1) degradation of SMX and other sulfonamides by S. mizutaii, (2) optimization of biodegradation conditions via response surface methodology, and (3) identification of sulfanilamide, 4-aminothiophenol, 5-amino-3-methylisoxazole, and aniline as metabolites in the degradation pathway of SMX in a microorganism. This strain might be useful for the bioremediation of SMX-contaminated environment.Sulfamethoxazole (SMX) is the most commonly used antibiotic in worldwide for inhibiting aquatic animal diseases. However, the residues of SMX are difficult to eliminate and may enter the food chain, leading to considerable threats on human health. The bacterial strain  This type of aquaculture leads to the risk of outbreaks of aquatic animal diseases. Sulfonamides are synthetic drugs used as anti-microbial, anti-diabetic, diuretic, anticonvulsant, and herbicidal agents. Among which, sulfamethoxazole (SMX) is one of the most prescribed antibiotics worldwide, can kill aquatic pathogens and can effectively inhibit aquatic animal diseases4. However, SMX cannot be completely utilized and metabolized by aquatic animals because approximately 50% are excreted to the environment without modification. Consequently, SMX has become a widely distributed pollutant in aquatic and domestic waste waters5. Numerous studies have indicated that SMX has adverse ecological effects and may threaten human health after long-term exposure. The World Health Organization has classified SMX as a Category 3 carcinogen in 20176. However, SMX has been proven to be difficult to eliminate by conventional water treatment processes7. The residues of SMX enter the food chain, leading to considerable threats to human health. Therefore, an efficient and reliable degradation method for removing SMX residue from the water environment must be developed8.The amount of aquaculture in China ranks first in the world all year round. The huge output mainly depends on high-density aquaculture9. The physical and chemical methods mainly include anion exchange, nano-filtration, struvite precipitation, and electrodialysis10. However, for technical and economic reasons, physical and chemical techniques may not be feasible11. Microbial biodegradation has advantages in terms of its eco-friendliness, low cost, and scope of implementation and has been proven as an effective method for the remediation of SMX residues in aquaculture waters12. To date, more than 20 strains of SMX-degrading strains have been isolated from different environments, and all of them exhibit SXM-degrading ability under laboratory conditions. Liang et al. isolated Achromobacter sp. JL9, which can use SMX as the sole nitrogen source for growth. The degradation rate of SMX was 90.4%, and the highest reaction rate constant was 0.0384 min−113. Gao et al. found that Phanerochaete chrysosporium has a strong tolerance to sulfamethoxazole in the concentration range of 10–30 mg/L14. At 10 mg/L, the degradation rate reached 53% after 24 h and 74% after 10 days. Jia et al. reported that the sulfate-reducing bacteria (SRB) sludge system shows considerable ability to degrade SMX. When the initial concentration is 25, 50, 100, 150, and 200 mg/ L, the removal rates of SMX by SRB sludge through adsorption and biodegradation are 3.9, 5.6, 13.2, 15.9, and 21.3 mg/L/d15. Other bacteria, including Achromobacter denitrificans PR116, P. chrysosporium17, Pseudomonas stutzeri18, and Acinetobacter sp.19, could serve as resources for the bioremediation of SMX from the polluted environment. Nevertheless, further studies are needed. Previous studies mainly isolated SMX-degrading bacteria through traditional methods, and the degradation efficiency was relatively low. The degradation mechanism of SMX and the metabolic pathway involved are also unclear.SMX removal is mainly by physical, chemical, and microbial methodsThe aims of this study were: (1) to isolate a bacterial strain that can highly degrade SMX by a novel method; (2) to optimize the environmental parameters to improve degradation efficiency; and (3) to deduce the possible degradation pathway of degrading strain and examine the mechanism underlying SMX degradation.Lentimicrobium, Flaviolibacter, Ramlibacter, and Rhodoferax. After enrichment, the bacterial diversity of SMX1 rapidly decreased, 6012 sequences were clustered into only 23 OTUs. The main genera were Pseudomonas, Sphingobacterium, Escherichia-Shigella, Alcaligenes, and Sediminibacter. Pseudomonas became the highest abundance genus in SMX2. After the second passage, with the increase in the SMX concentration, the bacterial diversity of SMX2 decreased continuously, and 6048 sequences were clustered into 20 OTUs. The main genera were Sphingobacterium, Escherichia-Shigella, Alcaligenes, and Sediminibacter, Sphingobacterium replaced Pseudomonas as the genus with the highest abundance, and the proportion of Sphingobacterium reached 85.84%. The result indicates that Sphingobacterium can tolerate higher concentrations of SMX than the other bacteria. As a result, the diversity in the enrichment gradually declined with the increase in SMX concentrations and passage generations. After the third and fourth passages, the 6003 sequences of SMX4 were clustered into 16 OTUs, and the main genera were Sphingobacterium, Alcaligenes, and Sediminibacter. The proportion of Sphingobacterium reached 93.68% and became the dominant genera. This result provides strong evidence that Sphingobacterium contributed to SMX degradation. By contrast, the rapid reduction in the proportion of Pseudomonas and other bacteria indicates their lack of ability to tolerate high SMX concentrations.A total of 5978, 6012, 6048, 6005, and 6003 16S rRNA gene sequences of the distinct V3–V4 regions were obtained from sludge sample (SMXY) and four generations of enrichment cultures (SMX1–4) by high-throughput sequencing, respectively. After statistical analysis and annotation, the results   for the fitted quadratic polynomial model is shown in Table P < 0.05) with R2 = 0.9831 and Adj R2 = 0.9613. The results of regression analysis indicated that A, B, AB, A2 and B2 are significant model terms, whereas the C, AC, BC, C2 are nonsignificant model terms. The same was confirmed from the Pareto chart  and half-life (t/21) were determined by using the first-order kinetic model. Table R2 varied from 0.9572 to 0.9924 indicating that the degradation data reliably fitted with the first-order kinetic model. Degradation rate constants (k) varied from 0.0620 to 0.4247 d−1 that characterized the degradation process of various sulfonamides by strain LLE-5. Theoretical half-life (t1/2) of SMX, sulfaguanidine, sulfamisoxazole, and sulfadimidine was noted as 1.63, 5.22, 6.80, 7.96, and 11.18 days, respectively. These results show that S. mizutaii LLE5 has a broad specificity for the degradation of sulfonamides and has considerable potential for processing sulfonamide pollution in the environment.The degradation characteristics of SMX by S. mizutaii LLE5 in MSM liquid medium were detected by HPLC/MS. According to the chemical structure of SMX and the mass spectrum, four candidate products were identified. These products were sulfanilamide (171 m/z), 4-aminothiophenol (124 m/z), 5-amino-3-methylisoxazole (99 m/z), and aniline (92 m/z). None of these products were detected when the culture medium only contained SMX and without S. mizutaii LLE5, indicating that they are SMX biodegradation metabolites. A possible metabolic pathway for SMX biodegradation by LLE5 was proposed . Their functions will be identified in our future work. According to the degradation products of SMX by S. mizutaii LLE-5, the strain mainly transformed SMX into sulfanilamide and 5-amino-3-methylisoxazole through hydrogenation. Then, sulfanilamide is degraded via desulfurization into aniline and via deamination into 4-aminothiophenol. The results showed that the strain had a clear degradation function of sulfamethoxazole and had a good tolerance. However, SMX could not be completely mineralized in terms of degradation products. This work is the first to report the pathway of SMX degradation by a Sphingobacterium strain and provided a certain reference for the study of microbial degradation of SMX.The existing reports show that most strains of 25. Agar plates were prepared by adding 1.5% (w/v) agar into the liquid media.SMX , HPLC-grade methanol, and acetonitrile were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. All other reagents used in the present study were of analytical grade. Mineral salt medium (MSM) and Luria–Bertani (LB) medium were used as described by Song et al26. The PCR product was sent to Biomark Gene Technology Company for library construction and high-throughput sequencing. The obtained sequence data were analyzed using Arch software (http://www.drive5.com/usearch/). The identity of sequences ≥ 97% was assigned to an operational taxonomic unit (OTU), and each OTU was considered to represent a species. The relative abundance of OTUs in the samples was statistically analyzed using R software (https://www.R-project.org)27.Activated sludge samples were collected from the wastewater treatment pond of a pharmaceutical factory in Liaoyang City, Liaoning Province, China. 10.0 g of sample was suspended in 100 mL of MSM supplemented with 100 mg/L SMX. Then, the sample was incubated on a rotary incubator shaker at 30 °C and 100 r/min in the dark. After 7 days, 10 mL of the sample was transferred into fresh MSM supplemented with 200 mg/L of SMX and incubated for another 7 days. After repeating this process four times, the SMX concentration in the fourth generation increased to 500 mg/L. The activated sludge sample was designated as SMXY, and the four generations of SMX enrichment cultures were designated as SMX1, SMX2, SMX3, and SMX4. The total DNA of SMXY and SMX1-4 were extracted using a FastDNA SPIN kit for soil . After purification, the 16S rRNA distinct regions V3–V4 were amplified with the following primers: 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The polymerase chain reaction (PCR) system was designed as described by Li et alSphingobacterium cells was used for isolation of SMX-degrading bacteria. Then, 100 μL of SMX4 was spread on the MSM solid plate containing 100 mg/L SMX and incubated at 30 °C. The rapidly growing colonies on the plate with different morphologies were selected and restreaked three times to obtain pure cultures. The degradation efficiency of the isolates was determined by a high-performance liquid chromatography (HPLC) system  equipped with an eclipse Plus C18 column. 10 mL of the culture and equal volume of ethyl acetate were added into a 50 mL centrifuge tube and mixed using a vortex mixer for 1 min. Then, the centrifuge tube was placed in a shaker, mixed at 220 r/min for 30 min, and centrifuged at 5000 g for 8 min. The upper phase was filtered with a 0.22 μm membrane for HPLC analysis. The elution comprised a mixture of methanol, formic acid , and distilled water, running at the flow rate of 1.0 mL/min. The injection volume was 10 μL, and the column temperature was 30°C28. The isolate with the highest degradation efficiency was selected for further analysis. Morphology was investigated using a light microscope . The carbohydrate assimilation of isolate was conducted with Biolog GEN3 plates following the analytical methods described by Hobbie et al29. The genomic DNA of the isolate was extracted and was used as a template for 16S rDNA amplification as described by Ruan et al30. The universal primers were 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′ACGGHTACCTTGTTTACGACTT-3′). The purified PCR product was sequenced by Bomad Technology . The obtained sequence was deposited in GenBank. The strain was further identified by performing multiple sequence alignment using Clustal X sofware, and phylogenetic relationships were analyzed via the neighbor-joining (NJ) method with MEGA 6 software31.According to the results of high-throughput sequencing, the fourth-generation enrichment sample SMX4 containing the most S. mizutaii LLE-5 was inoculated into 100 mL of LB medium and incubated at 30 °C on a rotary shaker at 150 rpm. After 12 h, the bacterial cells were harvested by centrifugation at 4000 g for 10 min. The precipitate was washed two times by phosphate buffered saline (PBS) solution and suspended for subsequent studies32.Before the degradation experiment, strain activation was performed. 33: temperature ; initial medium pH ; inoculum amount ; and initial concentration . The sterile distilled water was supplemented every 12 h according to the loss of weight. Selected single environmental factors and their interactions were further optimized via response surface methodology (RSM) analysis based on Box − Behnken design34. The second order polynomial equation is expressed as follows:Yi refers to the predicted response, Xi and Xj are variables, bo is a constant, bi denotes the linear coefficient, bii represents the quadratic coefficient, and bij corresponds to the interaction coefficient.The effect of single environmental factors on the biodegradation efficiency was evaluated by analyzing the following parameters and their rangesS. mizutaii LLE-5 cells were inoculated into 100 mL of MSM supplemented with 50 mg/L of SMX. The residual of SMX and cell numbers of S. mizutaii LLE-5 were detected every day. The control was inoculated with killed cells of S. mizutaii LLE-5. Each test was conducted in triplicate35. The ability of S. mizutaii LLE-5 to degrade other structurally similar sulfonamides, including sulfadiazine, sulfaguanidine, sulfamisoxazole, and sulfadimidine, were evaluated. Analytical methods were the same as above described. The first-order kinetic model .The SMX degradation test was performed under optimal conditions. t1/2) values of different sulfonamides were calculated by Eq. (k is degradation rate constant (d−1).The theoretical half-life . The extraction method was same as described above. The flow rate was 0.2 mL/min, the mobile phase A was 0.1% formic acid (V/V), and B was methanol. The targeted screening gradient elution program was: 0–2 min, 95% A; 2–25 min, 95%–5% A; 25–35 min, 5% A; and 35–40 min, 95% A. The sample injection volume was 20 μL, and the column temperature was 30 °C. The mass spectrometry conditions were: DuoSprayTM ion source, electrospray ionization (ESI), positive ion mode scanning, the ion source temperature was 550 ℃, the spray voltage was 5500 V, and curtain air (CUR) was 35 psi. The accumulation time was 0.25 s, and the collision voltage was (35 ± 15) eV.Intermediates generated during SMX degradation by Supplementary Information.