Mangifera indica L. and root of Carica papaya L. Toxicological assessment of the preparation was carried out in this study.The herbal preparation DAS-77, used for the treatment of various ailments in Nigeria, contains the milled bark of p.o. up to 20 g/kg in divided doses and i.p. at 250–3000 mg/kg. Mortality within 24 h was recorded. In the chronic toxicity study, rats were treated p.o. for 90 days at doses of 80, 400  and 2000 mg/kg. By 90 days, animals were sacrificed and blood samples collected for hematological and biochemical analysis. Organs were harvested for weight determination, antioxidants and histopathological assessments.In the acute toxicity study, DAS-77 was administered to mice p.o. up to 20 g/kg in divided doses but the i.p. LD50 was 1122.0 mg/kg. At TD, DAS-77 produced significant (p < 0.05) reductions in body weight, food intake and K+, and increases in ovary weight, neutrophils and HDL, which were reversible. Histopathological presentations were generally normal. Effects at the other doses were comparable to those at TD except for reversible increases in antioxidants in the liver, kidney and testes, and sperm abnormality, and reductions in liver enzymes, sperm motility and count.DAS-77 did not produce any lethality administered in vivo antioxidant activity. However, possibly reversible side-effects include electrolyte imbalance and sterility in males.Findings in this study revealed that DAS-77 is relatively safe with the potential for enhancing  Herbal medicines as preparations derived from naturally occurring plants with medicinal or preventive properties are a major component in all indigenous peoples’ traditional medicine, including Ayurvedic, homeopathic, naturopathic, traditional oriental, Native American  and Afripersonal communication by the President of WASP, Prof. Helen Kwanashie).Considering the challenges confronting the appropriate delivery of official health care to millions of people in remote and rural communities which serve as abode for over 70% of the population, including socio-economic demands for adequate pharmaceutical supplies, prevalent transportation difficulties, shortage of needed expertise for rational use of drugs, shortage and cost of orthodox products , the mosMangifera indica Linn., Anacardiaceae) and the dried root of pawpaw . It is used to treat diverse ailments in Nigeria including ulcers, diarrhea, sexually transmitted infections, dysmenorrhea, cholera, stomach disorders, dysentery and hemorrhoids. Due to multiplicity of usage and tendency for prolonged use, the herbal preparation was investigated in this study for possible chronic toxicity effects using a 90 day administration schedule.DAS-77 is a herbal preparation that contains the milled dried callous bark of mango (Mangifera indica) and the dried root extract of pawpaw (Carica papaya) (1:1). The product is a light brown, coarse powder with a pungent smell, and solution pH 8.5. DAS-77 was constituted in distilled water before administration to experimental animals.The herbal preparation DAS-77 is a product of Doynik Ventures, Ijoko-Lemode, via Sango Ota, Ogun State, Nigeria. It contains the milled young callous bark of mango  and had free access to standard rodent pellet diet  and water Research .et al. [Preliminary phytochemical screening  of DAS-77 was done according to the methods of Harborne , Trease et al. .p.o.) at a dose of 20 g/kg in divided doses. Six other groups of mice (n = 5) separately received distilled water 10 ml/kg and DAS-77 at doses of 250, 500, 1000, 2000, and 3000 mg/kg intraperitoneally (i.p.). Mice were generally observed for toxic symptoms and behavioural changes  for 2 h post-administration. The median lethal dose (LD50) was estimated by the log dose-probit analysis method [Mice were generally fasted for 12 h prior to the commencement of this test. A group of animals received DAS-77 orally  and DAS-77 at doses of 80, 400, and 2000 mg/kg  [in vivo antioxidants and malondialdehyde (MDA). The remnants of the organs were preserved in 10% formol-saline for histopathological assessment.Rats were weighed weekly and observed for behavioral changes, feeding and drinking habits, and general morphological changes. At the end of the 90 day treatment period, 10 rats from each group were anaesthetized by ne (w/v) . Blood sne (w/v) . After sMortality in each treatment group was recorded during the course of the 90 day administration of DAS-77 and reversibility period of 30 days in which animals were not treated with the herbal preparation. At the end of the reversibility period, rats were sacrificed and assays conducted in the main study were done.Blood samples were analyzed using established procedures and automated hematology analyzer. Parameters evaluated include packed cell volume (PCV), red blood cell (RBC) count, hemoglobin (Hb), platelet count, total and differential white blood cell (WBC) count, mean cell hemoglobin concentration (MCHC), mean red cell volume (MCV), and mean cell hemoglobin (MCH).Serum samples were analyzed for creatinine, urea, albumin, total protein, cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), triglycerides (TG), glucose, uric acid, alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), total bilirubin, and direct bilirubin using Roche and Cobas commercial kits and Roche/Hitachi 904 automated analyzer. Serum electrolytes concentration was determined by established methods; sodium and potassium concentration by flame photometry, chloride and bicarbonate concentration by titrimetric method, and calcium concentration by cresol phthalein complexone method -24.Sperm analysis to assess seminal fluid for motility, count and morphology was carried out according to the methods of Cheesbrough  and OgliSemen (10–15 μl) was placed on a slide in a way that the spermatozoa were evenly distributed and covered with a glass. After appropriate focus, several fields of the specimen were assessed for motility using the 40 × objective of the microscope. The number of motile cells was noted out of a total of 100 spermatozoa.Using sodium bicarbonate-formalin diluting fluid, a 1 in 20 dilution of semen was carried out with thorough mixing. An improved Neubauer ruled chamber was filled with well-mixed diluted semen using a Pasteur pipette. The number of spermatozoa in an area of 2 sq mm was counted using the 10 × objective of the microscope after 3–5 min. Estimation of the number of spermatozoa in 1 ml of fluid was done by multiplication of the number counted by 100 000.A thin smear of the liquefied well-mixed semen, made on a slide, was fixed with 95% v/v ethanol while still wet for 5–10 min. This was allowed to air-dry after which it was washed with sodium bicarbonate-formalin solution. This was to remove any present mucus. Rinsing with water was carried out many times. Subsequently, the smear was covered with dilute carbon fuchsin (1 in 20). This was allowed to stain for 3 min and the stain was washed off with water. Dilute Loeffler’s methylene blue (1 in 20) was used to cover the smear for 2 min to achieve counterstaining. This was washed off with water, drained and allowed to dry. Normal and abnormal spermatozoa were examined using the 40 × objective of the microscope. Estimation of percentage normal and abnormal morphology was done from the counting of hundred spermatozoa.Reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and MDA were determined according to established methods -29. The The various tissues obtained from experimental animals fixed in 10% formol-saline were dehydrated in graded alcohol, embedded in paraffin, and cut into 4–5 μm thick sections. Hematoxylin-eosin was used to stain the sections for photomicroscopic assessment using a Model N-400ME photomicroscope  ,32. Slidp < 0.05.Results are expressed as mean ± SEM. Data analysis was carried out using One-way ANOVA followed by Dunnet’s and Bonferroni posttests using GraphPad Prism 5 . Significance was considered at values of Preliminary phytochemical screening of DAS-77 showed the presence of tannins (3.26%), saponins (2.32%), phenols (1.31%), flavonoids (0.54%) and alkaloids (0.04%). Percentages represent the crude yield of the phytoconstituents.p.o. up to 20 g/kg in divided doses. Administered i.p., mortality was 0% at the lowest dose of 250 mg/kg and 100% at the highest dose of 3000 mg/kg, with the LD50 graphically estimated to be 1122.0 mg/kg. Writhing, grooming, increased locomotor activity, and convulsion were the behavioral manifestations observed with the i.p. route.No mortality and visible signs of toxicity were observed upon administration of DAS-77 p < 0.01, 0.001) in change in body weight on days 63 and 70. The same trend of effect was obtained with DAS-77 at doses of 400 mg/kg (p < 0.05) and 2000 mg/kg (p < 0.001) but on days 42 and 90, and 84 and 90 respectively  in change in body weight on days 84 and 90. At the dose of 400 mg/kg, the herbal remedy elicited significant reduction  on days 28 and 56. DAS-77 at the dose of 2000 mg/kg did not produce any significant effect on change in body weight all through the treatment period  on average daily food intake at the dose of 2000 mg/kg only. At this dose, there were reductions in average daily food intake on days 14, 21, 42 and 84 compared to control  in average daily water intake on day 21 compared to control. The herbal remedy at doses of 80 and 2000 mg/kg did not elicit any significant effect all through the treatment period  in weight change (3.85 ± 1.61 g) compared to control (10.46 ± 0.97 g) while effects were not significant at 80 and 2000 mg/kg. The herbal preparation also elicited a significant reduction  in food intake at doses of 400 and 2000 mg/kg  relative to control (289.80 ± 4.46 g). DAS-77 did not produce any significant effect on water intake  sperm motility  and sperm count , and increased sperm abnormality . These effects were reversed  in weight (2.41 ± 0.10 g vs. 2.84 ± 0.05 g for control) at the dose of 2000 mg/kg and the ovaries in which there were significant increases (p < 0.01) in weight (0.14 ± 0.02 g and 0.14 ± 0.01 g respectively vs. 0.08 ± 0.00 g for control) at doses of 400 and 2000 mg/kg (Table p < 0.05) was observed in the reversibility study (0.88 ± 0.03 g vs. 0.73 ± 0.06 g for control).DAS-77 did not produce any significant effect on the weight of vital organs except in the case of the liver in which there was a significant reduction (kg Table . The effp < 0.05) in the group treated with the herbal preparation at the dose of 2000 mg/kg (5.20 ± 0.58 × 10³/μl) when compared with the group treated with distilled water (9.40 ± 0.98 × 10³/μl)  in the proportion of neutrophils in the groups treated with DAS-77 at doses of 80 mg/kg (46.10 ± 2.16%), 400 mg/kg (56.30 ± 1.54%) and 2000 mg/kg (60.90 ± 1.38%) compared to control (39.80 ± 1.95%). The effects of DAS-77 on WBC count and proportion of neutrophils were reversed after 30 days of cessation of administration  in the concentration of albumin in the group treated with the herbal preparation at the dose of 80 mg/kg (37.30 ± 0.78 g/L) when compared with the group treated with distilled water (33.20 ± 1.03 g/L) (Table p < 0.05) in the concentration of HDL in the groups treated with DAS-77 at doses of 80 mg/kg (1.15 ± 0.05 mmol/L) and 400 mg/kg (1.11 ± 0.10 mmol/L) when compared with the group treated with distilled water (0.87 ± 0.05 mmol/L). The concentrations of AST and ALT were significantly reduced (p < 0.001) in the groups treated with the formulation at the dose of 2000 mg/kg (74.50 ± 8.61 u/L and 18.00 ± 2.12 u/L respectively) when compared with the group treated with distilled water (143.70 ± 11.34 u/L and 36.20 ± 4.56 u/L respectively). The effects on albumin, HDL, AST, and ALT were reversed after 30 days of cessation of administration of DAS-77  in the concentration of K+ in the groups treated with the formulation at the doses of 80 mg/kg (5.98 ± 0.25 mmol/L), 400 mg/kg (5.04 ± 0.10 mmol/L), and 2000 mg/kg (5.19 ± 0.14 mmol/L) when compared with the group treated with distilled water (7.52 ± 0.47 mmol/L) (Table p < 0.05) in the concentration of Ca2+ in the group treated with DAS-77 at the dose of 2000 mg/kg (1.25 ± 0.04 mg/dL) when compared with the distilled water treated group (1.09 ± 0.05 mg/dL). These effects were reversed after 30 days of cessation of administration of DAS-77  in the level of catalase in the groups treated with the formulation at doses of 80 mg/kg (58.21 ± 13.80 u/mg) and 2000 mg/kg (44.41 ± 4.75 u/mg) compared with the distilled water treated group (17.66 ± 5.22 u/mg)  in the level of MDA in the groups treated with DAS-77 at doses of 80 mg/kg (0.11 ± 0.03 nm/mg), 400 mg/kg (0.07 ± 0.02 nm/mg), and 2000 mg/kg (0.13 ± 0.03 nm/mg) compared with the group treated with distilled water (0.27 ± 0.08 nm/mg). The effects on catalase and MDA in the kidney were reversed upon discontinuation of treatment for 30 days  in the levels of GSH (3.69 ± 0.55 vs. 1.45 ± 0.22 nm/mg in control), SOD (5.82 ± 0.72 vs. 3.24 ± 0.33 u/mg in control), catalase (26.87 ± 3.33 vs. 14.94 ± 1.54 u/mg in control), and peroxidase (1.83 ± 0.23 vs. 0.10 ± 0.10 u/mg in control).There were significant differences in the level of antioxidant enzymes in the liver across the treatment groups after a 30 day cessation of therapy Table . At the There was no significant difference in the levels of the various antioxidant enzymes and MDA in the brain in the reversibility study Table .p < 0.01) in the levels of GSH (3.58 ± 0.75 nm/mg), SOD (5.75 ± 0.82 u/mg), catalase (26.55 ± 3.79 u/mg), and peroxidase (1.80 ± 0.26 u/mg) compared with the distilled water treated groups   Table .A summary of the histopathological presentations in the various organs is shown in Table There were no adverse histopathological presentations observed in the distilled water and DAS-77 80 mg/kg) treatment groups. The liver appeared normal with preserved hepatic architecture, hepatocytes arranged as radial plates, and having eosinophilic cytoplasm and central nuclei. No cytoplasmic inclusions were seen and no portal inflammation Figures  and 6. C0 mg/kg tThere were no adverse histopathological presentations observed in all the treatment groups. Normocellular glomerular tufts were displayed on a background containing tubules. No necrosis was observed.Generally, there were no adverse histopathological presentations observed in the treatment groups but cerebral oedema was observed in the 400 mg/kg group Figures  and 12. The lungs were normal in all the treatment groups. The alveolar air spaces were surrounded by interstitium containing few blood vessels and inflammatory cells.The heart was normal in all the treatment groups. The cardiac myocytes were arranged in interlacing and parallel array. Their nuclei were spindle shaped and elongated.Presentations in the distilled water group were normal. The spleen was congested in all the DAS-77 treatment groups with the background splenic stroma showing engorgement of sinusoids by red blood cells Figures  and 16.The pancreas was normal in all the treatment groups. Closely packed acini, separated by delicate fibrocollagenous stroma that transmits blood vessels were observed.Normal histopathological presentations were observed in all the treatment groups. Histologic section shows seminiferous tubules lined by germ cells in various stages of development (the spermatogenic series), and containing luminal spermatozoa. Leydig cells were seen in the interstitium.Normal histopathological presentations were observed in all the treatment groups. Histologic section of ovary shows follicles at various stages of development as well as corpora lutea, displayed on a fibrocellular stroma.Mortality recorded in the distilled water and DAS-77  treatment groups within 30, 60 and 90 days, and the reversibility study period were 0/20, 1/20, 1/20 and 0/20 (10%); 0/20, 1/20, 0/20 and 0/20 (5%); 1/20, 1/20, 0/20 and 0/20 (10%); and 1/20, 2/20, 0/20 and 1/20 (20%), respectively.Worldwide, various medicinal plants and botanical drugs have been widely adapted as primary therapeutic agents or supplements for treating various human ailments . Based o50 when DAS-77 was administered intraperitoneally was estimated to be 1122.0 mg/kg. The behavioral changes observed during acute exposures through the intraperitoneal route were writhing, grooming, increased locomotor activity and convulsion.DAS-77 did not induce lethality in mice when administered orally up to 20 g/kg in divided doses. Hence, the herbal preparation can be said to be safe when administered orally according to the assertion of Clarke and Clarke  that a sConsidering the therapeutic dose of DAS-77 (400 mg/kg), the hematological analysis showed an increase in neutrophils which possibly suggest a boost in defense, mainly acute response to infections or antigens. This is based on the fact that neutrophils are essential first line defense of the body against infections or introduction of antigens and neutropenia (decrease in neutrophils) makes an individual highly susceptible to infections . Other hIn respect of the subtherapeutic dose (80 mg/kg), DAS-77 had no significant effect on organ weights compared to control. There were no significant changes in food and water intake, and weight of animals. Hematological findings showed increase in the percentage of neutrophils which possibly suggests a boost in the immune response . There wDAS-77 at the supratherapeutic dose (2000 mg/kg) also produced an increase in the size of the ovaries. This may be due to preparation for ovulation in view of the fact that histopathological examinations showed normal presentation without any inflammation. There was also a decrease in food intake which possibly suggests anorexia. This may be an indication of impact on the CNS at this dose. There was a decrease in the WBC count which could be that the formulation suppresses immunity at this dose. Differential WBC analysis revealed an increase in the percentage of neutrophils possibly suggesting a boost in the acute immune response of the body . There wHistopathological presentations in this study give credence to the results obtained in the assessment of hematological and biochemical parameters with the 90 day administration of DAS-77. These findings may suggest that the mortality recorded in the course of this study may be administration related.Findings in the acute toxicity test suggest that DAS-77 is practically non-toxic when administered orally. The results obtained in respect of the chronic toxicity study suggest that DAS-77 is relatively safe when administered orally for an extended period at subtherapeutic and therapeutic doses. The phytomedicine at these doses showed potential for boosting components of the immune system and protecting the kidneys, liver and cardiovascular system. These findings provide a justification for specifically designed studies to further investigate these possible beneficial activities. However, DAS-77 at these doses showed a tendency to reversibly cause electrolyte imbalance. Observations in this study also revealed that the supratherapeutic dose of DAS-77 is endowed with the potential beneficial effects observed with the lower doses but with the risk of reversible electrolyte imbalance also and additionally, reversible sterility in males.50: Median lethal dose; EDTA: Ethylenediamine-tetra Acetate; MDA: Malondialdehyde; PCV: Packed Cell Volume; RBC: Red Blood Cell; Hb: Hemoglobin; WBC: White Blood Cell; MCHC: Mean Cell Hemoglobin Concentration; MCV: Mean Red Cell Volume; MCH: Mean Cell Hemoglobin; HDL: High Density Lipoprotein; LDL: Low Density Lipoprotein; TG: Triglycerides; ALP: Alkaline Phosphatase; AST: Aspartate Transaminase; ALT: Alanine Transaminase; GSH: Reduced Glutathione; SOD: Superoxide Dismutase; CAT: Catalase; GPx: Glutathione Peroxidase.LDThe authors declare that there are no conflicts of interest in respect of this study.AJA and OA designed the study and assisted SOA in its conduct, CCA was responsible for the histopathological assessment, SOA and AJA drafted the manuscript, and OOA coordinated the study and revised the manuscript. All authors reviewed and interpreted the data, read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/12/79/prepub


This study utilized HAP and HAP treated with 10%, 20%, 30%, 40%, 50%, or 60% phosphoric acid solution for 10 minutes at 25°C, followed by rinsing 3 times with ultrapure water. The 30% phosphoric acid etching process that provided a Ca/P ratio of 1.50, without destruction of the grain boundary of HAP, was selected as a surface-modification procedure. Additionally, HAP treated by the 30% phosphoric acid etching process was stored under dry conditions at 25°C for 12 hours, and the Ca/P ratio approximated to 1.00 accidentally. The initial adhesion, proliferation, and differentiation  activity and relative mRNA level for ALP) of MC3T3-E1 cells on the modified surfaces were significantly promoted (P < 0.05 and 0.01). These findings show that the 30% phosphoric acid etching process for the nanostructured HAP surface can alter the Ca/P ratio effectively and may accelerate the initial adhesion, proliferation, and differentiation of MC3T3-E1 cells.The purpose of this study was to establish an acid-etching procedure for altering the Ca/P ratio of the nanostructured surface of hydroxyapatite (HAP) by using surface chemical and morphological analyses  and to evaluate the  In vivo bioresorbability of BCP can be controlled by phase composition because the reactivity of BCP increases as the β-TCP/HAP ratio increases [ Hydroxyapatite [Ca10PO6(OH)2] (ncreases . Recentlβ-TCP (Ca/P ratio = 1.50)   solution for 10 minutes at 25°C, followed by rinsing 3 times with ultrapure water (MilliQ water: >18 MΩcm) .HAP plates   were used in this study. HAP plates were treated with 10%, 20%, 30%, 40%, 50%, or 60% phosphoric acid [Hin vacuo (≤10−7 Pa) with Al-Kα monochromatic X-rays at a source power of 150 W. The AXIS-HS was equipped with an electron flood gun for charge compensation. Wide- and narrow-scan spectra were acquired at pass energies of 80 and 40 eV, respectively. Peak positions were calibrated by referencing a value of 284.6 eV for the peaks corresponding to C–C and C–H in the C 1s spectrum. After smoothing the narrow scans, a straight line  was applied in the quantification. The relative sensitivity factors used to calculate the atomic ratios from the peak area ratios were 0.278 for C 1s, 0.780 for O 1s, 1.833 for Ca 2p, and 0.486 for P 2p. Reproducibility was guaranteed by obtaining 10 measurements per experimental sample. The data were analyzed using one-way ANOVA and Tukey's test for multiple comparisons (P < 0.05). HAP, HAP—10% PA, HAP—20% PA, HAP—30% PA, HAP—40% PA, HAP—50% PA, and HAP—60% PA plates were mounted individually onto stubs with insulating tape. The surfaces of the plates were chemically analyzed using an X-ray photoelectron spectroscopy (XPS) instrument . The measurements were performed α radiation at 40 kV and 50 mA between 10 and 90 (2θ) degrees at a step size of 0.04 degrees and a scan rate of 2 degrees/minute. The results were compared to the ICDD standard for HAP (no. 01-072-1243). The thin films of the HAP and HAP—30% PA plates were analyzed using an XRD instrument . Samples were scanned with Cu-KA HAP—30% PA plate that was stored under dry conditions at 25°C for 12 hours (HAP—30% PA—12 h) was added as a new sample. The surface characteristics of HAP, HAP—30% PA, and HAP—30% PA—12 h were evaluated by the following methods.P < 0.05).Samples were analyzed using an XPS instrument at incidence angles of 90, 75, 60, 45, 30, and 15 degrees. The measurements were obtained under the above-mentioned conditions. The data were analyzed using one-way ANOVA and Tukey's test for multiple comparisons (μm) was defined as the average value of 5 areas (100 μm × 100 μm) with 9 measuring points. The wettability of sample surfaces was measured using a contact angle meter  with the sessile drop method and 3.6 μL ultrapure water; Young's equation was used for data analysis. The data were analyzed using one-way ANOVA and Tukey's test for multiple comparisons (P < 0.05).An SEM  was used to observe the surface topography of samples. The surface roughness (Ra) of samples was determined using a confocal laser scanning microscope . The Ra value ) were cultured in Dulbecco's Modified Eagle Medium (D-MEM)  containing 10% fetal bovine serum  and 1% penicillin-streptomycin  at 37°C in a humidified atmosphere of 5% CO4) were seeded onto HAP, HAP—30% PA, and HAP—30% PA—12 h plates sterilized by ultraviolet (UV) irradiation for 4 hours. The cells were incubated at 37°C and 5% CO2. Initial cell adhesion was evaluated at 0.5 and 24 hours and cell proliferation at 1, 4, and 7 days. Cells were seeded on tissue culture plates and incubated as controls over each incubation period.MC3T3-E1 cells  of a 96-well microplate. The absorbance was read at 450 nm by using a microplate reader . The data were analyzed using two-way ANOVA and Tukey's test for multiple comparisons (P < 0.05).Initial cell adhesion and cell proliferation were analyzed by a Cell Counting Kit-8 assay , a colorimetric assay that uses a tetrazolium salt, WST-8, to determine viable cell number. After each incubation period, the growth medium in each well was removed by aspiration and replaced with a mixture of WST-8 and D-MEM in a 1 : 10 ratio. After 2 hours of incubation at 37°C and 5% CO4) were seeded onto HAP, HAP—30% PA, and HAP—30% PA—12 h plates as described previously. Alkaline phosphatase (ALP) activity was evaluated at 7, 14, and 21 days after obtaining a confluent cell monolayer. Real-time polymerase chain reaction (PCR) analysis of ALP was carried out at 14, 21, and 28 days after obtaining a confluent cell monolayer. MC3T3-E1 cells , which is optimized to detect placental ALP activity and uses p-nitrophenyl phosphate (pNPP) as a colorimetric phosphatase substrate. After each incubation period, the growth medium in each well was removed by aspiration, and each well was rinsed twice with 1 mL of lysis buffer. A mixture (1 mL) of 10 mL lysis buffer and 20 μL Triton X-100 was added to each well, the cells were scraped and suspended, and 1,000 μL of the cell suspension was transferred to a new well and incubated at 37°C and 5% CO2 for 10 minutes. An aliquot of the cell suspension (300 μL) was then mixed with 300 μL of the pNPP reaction mixture in a separate well. After 60 minutes of incubation, 300 μL of stop solution was added to the cell suspension, followed by shaking for 1 minute. A 150 μL aliquot of the mixture was dispensed to each well (n = 5) in a 96-well microplate. The absorbance was read at 405 nm by using the microplate reader.ALP activity was determined by the SensoLyte μL by using a NanoDrop 1000 system . First-strand cDNA was synthesized from total RNA (10 ng/μL) by using a ReverTra Ace qPCR RT Kit . For each gene, a cycle curve experiment was performed, and the optimal number of PCR cycles was selected according to the results. Real-time PCR analysis was performed for ALP and GAPDH . The sequences of the primers used in this analysis were as follows: forward, 5′-AGGGTGGACTACCTCTTAGGTCT-3′ and reverse, 5′-TGGTCAATCCTGCCTCCTTCC-3′ for ALP amplification; and forward, 5′-GCCAGCCTCGTCCCGTAG-3′ and reverse, 5′-CAAATGGCAGCCCTGGTGAC-3′ for GAPDH amplification. The primers were purchased from Sigma. The data were analyzed using two-way ANOVA and Tukey's test for multiple comparisons (P < 0.05). Total RNA was extracted using an RNeasy Mini Kit . The total RNA concentration was adjusted to 10 ng/P > 0.05). The XPS-determined Ca/P ratio (1.65 ± 0.02) of HAP matched the theoretical value (1.67). The Ca/P ratios for HAP—10% PA, HAP—20% PA, HAP—30% PA, HAP—40% PA, HAP—50% PA, and HAP—60% PA were significantly smaller than that for HAP (P < 0.01). The Ca/P ratio (1.50 ± 0.03) of HAP—30% PA was the smallest and matched the theoretical value of TCP (1.50), although its value was not significantly different from that of HAP—20% PA (P > 0.05). The XPS-determined binding energies (eV) of Ca 2p, P 2p, and Δ; atomic concentrations (at.%); and Ca/P ratios for HAP, HAP—10% PA, HAP—20% PA, HAP—30% PA, HAP—40% PA, HAP—50% PA, and HAP—60% PA are shown in  The XRD spectra of HAP and HAP—30% PA thin films, referenced to the ICDD standard for HAP (no. 01-072-1243), are shown in P < 0.05), and there was no significant difference between the binding energies of HAP and HAP—30% PA (P > 0.05). As for the Ca/P ratios, there were significant differences among samples (P < 0.01), and the values of HAP, HAP—30% PA, and HAP—30% PA—12 h were 1.65 ± 0.04, 1.45 ± 0.01, and 1.09 ± 0.02, respectively. In each sample, changes in the binding energies and the Ca/P ratios were not recognized by the different incidence angles of the X-ray.The XPS-determined binding energies (eV) of Ca 2p, P 2p, and Δ and the Ca/P ratios for HAP, HAP—30% PA, and HAP—30% PA—12 h at each incidence angle of the X-ray  are shown in SEM images for HAP, HAP—30% PA, and HAP—30% PA—12 h are shown in μm) was significantly larger than the Ra values of HAP and HAP—30% PA . The contact angle of HAP—30% PA—12 h (13.67 ± 3.54 degrees) was significantly smaller than the contact angles of HAP and HAP—30% PA . The surface roughness and wettability for HAP, HAP—30% PA, and HAP—30% PA—12 h are shown in P < 0.05). At 24 hours, the number of adherent cells for HAP—30% PA—12 h was significantly higher than that for HAP (P < 0.01) and HAP—30% PA (P < 0.05).The initial cell adhesion of MC3T3-E1 cells to HAP, HAP—30% PA, and HAP—30% PA—12 h at 0.5 and 24 hours is shown in P < 0.01); the difference between the growth on HAP—30% PA and HAP—30% PA—12 h was not statistically significant (P > 0.05).The proliferation of MC3T3-E1 cells on HAP, HAP—30% PA, and HAP—30% PA—12 h at 1, 4, and 7 days is shown in P < 0.01). The ALP activity at 14 days on HAP—30% PA—12 h was higher than that on HAP—30% PA, but the ALP activity at 21 days was lower than that on HAP—30% PA. Although the ALP activities on HAP and HAP—30% PA gradually increased from 7 to 21 days, the ALP activity on HAP—30% PA—12 h reached its peak at 14 days. The ALP activity of MC3T3-E1 cells on HAP, HAP—30% PA, and HAP—30% PA—12 h at 7, 14, and 21 days after obtaining a confluent cell monolayer is shown in P > 0.05).  Relative mRNA levels for ALP in MC3T3-E1 cells on HAP, HAP—30% PA, and HAP—30% PA—12 h at 14, 21, and 28 days after obtaining a confluent cell monolayer are shown in P > 0.05). Since the 30% phosphoric acid etching process did not break the grain boundary of HAP and effectively brought the Ca/P ratio to 1.50, this process was selected as a surface-modification procedure. Modification to the HAP surface by using etching with 10%, 20%, 30%, 40%, 50%, and 60% phosphoric acid altered the Ca/P ratio from 1.50 to 1.58. The Ca/P ratio of HAP—30% PA was the smallest and matched the theoretical value of TCP (1.50). However, there were no significant differences among the binding energies of Δ for all samples  for HAP and HAP—30% PA was assumed to be the same. This finding does not illustrate that the changed Ca/P ratio leads to alteration of the chemical composition of the surface. Thus, the nanostructured surface of HAP—30% PA has not yet been confirmed as being modified to TCP, although the Ca/P ratio was 1.50.P < 0.05). The Ca/P ratio of HAP—30% PA—12 h (1.09) was significantly smaller than that of HAP—30% PA (1.50) (P < 0.01). The SEM results showed a greater degree of scabrousness at the surface of HAP—30% PA—12 h than HAP—30% PA. The Ca/P ratio of HAP—30% PA—12 h was approximated to the Ca/P ratio of DCPD (1.00), but this finding was obtained accidentally. The XPS depth profiling analyses for HAP, HAP—30% PA, and HAP—30% PA—12 h were performed to evaluate the chemical compositions of their nanostructured surfaces. The binding energy of Δ of HAP—30% PA—12 h was significantly smaller than that of HAP and HAP—30% PA ( The reasons for reduction of the Ca/P ratio of the modified surface were (i) Ca defect and (ii) P abundance. P abundance could not be considered because the surface treated with 30% phosphoric acid solution was rinsed with ultrapure water, and the expansion of the break of the grain boundary of HAP was not observed in the SEM image of HAP—30% PA—12 h. The 30% phosphoric acid etching process promotes decalcification of the surface, resulting in the reduction of the Ca/P ratio by Ca defects. As for the reduction of the Ca/P ratio of HAP—30% PA—12 h to 1.00, it is believed that the Ca in the outer layer of the nanostructured surface is supplied to the deep layer for 12 hours to equilibrate the amorphous calcium phosphate. Reduction in the Ca/P ratio leads to high solubility of the surface. Additionally, surface modification induces the rougher and wetter surface of HAP. Generally, a contact angle of more than 65 degrees indicates a hydrophobic surface, and a contact angle of less than 65 degrees indicates a hydrophilic surface. Since the contact angle of HAP (102.10 degrees) is greater than 65 degrees, the hydrophobic surface becomes more intense with an increase in surface roughness. However, in this study, the contact angle grew smaller with an increase in surface roughness, resulting in a super hydrophilic surface. These findings demonstrate that the surface might have been modified both morphometrically and chemically.in vitro responses of osteoblast-like cells to HAP—30% PA and HAP—30% PA—12 h would be better than those to HAP since the high biodegradation rate of TCP should improve early cell attachment [P < 0.01 for both). MC3T3-E1 cells reached a proliferation peak around day 7 on all substrates, and the cell differentiation process is reflected by ALP activity [P < 0.01) and, in particular, the ALP activity on HAP—30% PA—12 h reached its peak (at 14 days) faster than that on HAP and HAP—30% PA. Moreover, at 14 days, the mRNA expression on HAP—30% PA—12 h and HAP—30% PA was estimated in sequence, but there was no expression on HAP. Intracellular signal transduction during the initial adhesion stage might play a crucial role in cell differentiation and mineralization on calcium phosphate-related biomaterials [We expected that the tachment , 15. Duractivity , 17. The peak at  days fasaterials .in vivo and involve bone resorption and calcium release.Furthermore, we will investigate the response of osteoclasts to HAP—30% PA and HAP—30% PA—12 h because osteoclasts regulating bone metabolism are coupled with osteoblasts in a direct or indirect manner in vitro response of MC3T3-E1 osteoblast-like cells to the modified surfaces. The 30% phosphoric acid etching process for obtaining a Ca/P ratio of 1.50, without destruction of the grain boundary of HAP, was selected as a surface-modification procedure. Additionally, HAP was treated with 30% phosphoric acid solution and stored under dry conditions at 25°C for 12 hours, and the Ca/P ratio approximated to 1.00 accidentally. The acid-etching procedure for HAP significantly promoted the initial adhesion, proliferation, and differentiation of MC3T3-E1 cells. In future studies, the Ca/P ratio of HAP scaffolds used clinically will be modified, and the usefulness of the scaffolds will be evaluated by in vivo studies. In this study, we established the acid-etching procedure to alter the Ca/P ratio of the nanostructured surface of HAP by surface chemical and morphological analyses and evaluated the


To evaluate the prevalence and associated factors of steep cornea/keratoconus in the adult Chinese population.The population-based Beijing Eye Study 2011 included 3468 individuals with a mean age of 64.6±9.8 years (range: 50–93 years). A detailed ophthalmic examination was performed including optical low-coherence reflectometry. Steep cornea/keratoconus were defined as an anterior corneal refractive power exceeding 48 diopters.P<0.001), smaller interpupillary distance (P = 0.038), lower best corrected visual acuity (P = 0.021), higher cylindrical refractive error (P<0.001) and more myopic refractive error (P<0.001). It was not significantly associated with gender, body height, psychic depression, cognitive function, blood concentrations of glucose, lipids, creatinine and C-reactive protein, blood pressure and quality of life score, nor with intraocular pressure, dry eye feeling, and lens thickness.Mean refractive power of the cornea was 43.16±1.45 diopters  and 43.98±1.52 diopters . A steep cornea/keratoconus defined as corneal refractive power of ≥48 diopters and ≥49 diopters was detected in 27 subjects  and 6 (0.2± 0.1%) subjects, respectively. Presence of steep cornea/keratoconus was associated with shorter axial length (A steep cornea/keratoconus defined as corneal refractive power of 48+ diopters has a prevalence of 0.9±0.2% among Chinese aged 50 years and above. Its prevalence was significantly associated with the ocular parameters of shorter axial length, smaller interpupillary distance, higher cylindrical and myopic refractive error and lower best corrected visual acuity, however, with none of the systemic parameters tested. Keratoconus is a conical deformation of the anterior corneal surface leading to refractive myopia, irregular astigmatism and eventually to a marked reduction in visual acuity The Medical Ethics Committee of the Beijing Tongren Hospital approved the study protocol and all participants gave informed written consent, according to the Declaration of Helsinki.The Beijing Eye Study 2011 is a population-based cross-sectional study in Northern China All examinations were carried out in the communities, either in schoolhouses or in community houses. All study participants underwent an interview with standardized questions on their family status, level of education, income, quality of life, psychic depression, physical activity, known major systemic diseases such as arterial hypertension and diabetes mellitus, and quality of vision. Cognitive function was assessed using the MMSE  scale Using the biometric measurements of the anterior corneal curvature, the corneal refractive power was calculated assuming a refractive index of the cornea of 1.3315 as proposed by Olsen P-values were 2-sided and were considered statistically significant when the values were less than 0.05.Statistical analysis was performed using a commercially available statistical software package . In a first step, we examined the mean values (presented as mean±standard deviation) of the systemic and ocular parameters in the steep cornea/keratoconus group versus the non-keratoconus group. The Gaussian distribution of the parameters was tested using the Kolmogorov-Smirnov test. We calculated the significance of the differences between both groups using the student-t-test for non-paired samples for the parametric parameters, and the Mann-Whitney-U test for non-paired samples for the non-parametric parameters. In a second step, we performed a binary logistic regression analysis with the presence of steep cornea/keratoconus as dependent parameter and those parameters as independent parameters which were significantly associated with steep cornea/keratoconus in univariate analysis. 95% Confidence intervals (CI) and odds ratios (OR) were presented. All P<0.001) older did not vary significantly in gender (P = 0.994).Out of the 3468 participants, keratometric measurements were available for 3166 (91.3%) subjects (1792 (56.6%) women). The mean age was 64.2±9.8 years , the mean refractive error  was −0.19±2.13 diopters . The group of subjects without keratometric measurements as compared with the group of subjects with keratometric measurements was significantly  and in the steepest meridian 7.55±0.26 mm . Correspondingly, the mean refractive power of the cornea in the flattest meridian was 43.16±1.45 diopters  and it was in the steepest meridian 43.98±1.52 diopters . A steep cornea/keratoconus  was detected in 27 subjects. The prevalence rate was 0.9±0.2% . A steep cornea/keratoconus with a corneal refractive power of ≥49 diopters was detected in 6 subjects resulting in a prevalence rate of 0.2±0.1% . Two subjects showed a corneal refractive power of ≥50 diopters .P = 0.32 for flattest meridian; P = 0.09 for steepest meridian).In the whole study population, 2 eyes had undergone perforating keratoplasty for corneal ulceration and trauma. Corneal refractive power in these 2 eyes ranged between 43.9 and 44.5 diopters. Cataract surgery had been performed in 162 subjects of the study population. Their mean corneal refractive power was 43.04±1.52 diopters  in the flattest meridian and 44.20±1.69 diopters  in the flattest meridian. In two of the eyes after cataract surgery, the corneal refractive power exceeded 48 diopters. The eyes after cataract surgery and the phakic eyes did not differ significantly in corneal refractive power (P = 0.006), shorter body height (P<0.001), lower level of education (P<0.001) and lower income (P<0.001), rural versus urban region of habitation (P = 0.008), lower cognitive score (P = 0.029), higher psychic depression score (P = 0.012), shorter axial length (P<0.001), smaller pupil diameter (P = 0.031), shorter interpupillary distance (P<0.001), smaller central corneal thickness (P = 0.033), larger cylindrical refractive error (P = 0.014), and lower best corrected visual acuity (P = 0.004) (P = 0.99), body mass index (P = 0.33), systolic (P = 0.23) or diastolic (P = 0.46) blood pressure, quality of life index ( P = 0.33), blood concentrations of glucose (P = 0.87), cholesterol (P = 0.84), creatinine (P = 0.51) and C-reactive protein (P = 0.92), smoking (P = 0.11) or alcohol consumption (P = 0.39), dry eye feeling (P = 0.15), intraocular pressure readings (P = 0.37), lens thickness (P = 0.12) and optic disc size (P = 0.39)  , 2. The  = 0.39) ,2.P = 0.006), but not with age (P = 0.79). In a second step of the analysis with the additional parameters of body height and weight, only shorter body height (P = 0.001) was significantly associated with steep cornea/keratoconus, while age (P = 0.46), gender (P = 0.65) and body weight (P = 0.73) were not significantly associated. If body mass index was added, the result was similar. In a next step, the parameters of age, gender, body weight, or body mass index were dropped and the level of education was added. It revealed significant associations between the presence of steep cornea/keratoconus and lower body height (P = 0.001) and lower level of education (P = 0.04). If the region of habitation was added, prevalence of steep cornea/keratoconus was associated only with lower body height (P<0.001) and rural region (P = 0.04), while level of education was no longer significantly (P = 0.38) associated. In the next step of the statistical analysis, the parameter of package years of smoking was added. It showed that the presence of steep cornea/keratoconus was only associated with lower body height (P = 0.001) and rural region of habitation (P = 0.004), while package years of smoking (P = 0.152) was not significantly correlated with steep cornea/keratoconus. In a similar manner, the depression score (P = 0.10) and the cognitive score (P = 0.69) were not significantly associated with the presence of steep cornea/keratoconus. In a last step of the multivariate analysis, those ocular parameters were added which were significantly different between the steep cornea/keratoconus group and the non-keratoconus group in the univariate analysis. It revealed that after adjustment for body height and region of habitation, presence of steep cornea/keratoconus was significantly associated shorter axial length (P<0.001), smaller interpupillary distance (P = 0.038), lower best corrected visual acuity (P = 0.021), higher cylindrical refractive error (P<0.001) and more myopic refractive error (P<0.001) (P = 0.49) and area of habitation (P = 0.59) were no longer significantly associated with the prevalence of steep cornea/keratoconus.The multivariate binary logistic regression analysis was carried out in several steps. A first step included the presence of steep cornea/keratoconus as dependent parameter and age and gender as independent parameters. It confirmed that the presence of steep cornea/keratoconus was significantly associated with female gender (P<0.001) . In thatIf steep cornea/keratoconus was defined as a corneal refractive power of 49+ diopters or of 50+ diopters, the study groups were statistically too small for a meaningful multivariate analysis.If the corneal refractive power was calculated assuming a refractive index of the cornea of 1.3375 instead of 1.3315, the mean refractive power of the cornea in the flattest meridian was 43.9±1.5 diopters  and it was in the steepest meridian 44.8±1.5 diopters . Using the corneal refractive index of 1.3375, a steep cornea/keratoconus  was detected in 71 subjects , a steep cornea/keratoconus as corneal refractive power of ≥49 diopters in 23 subjects , and a steep cornea/keratoconus as corneal refractive power of ≥50 diopters in 5 subjects .Steep cornea/keratoconus defined as corneal refractive power of 48+ diopters had a prevalence of 0.9±0.2% among Chinese aged 50+ years and living in Greater Beijing. In multivariate analysis, the presence of steep cornea/keratoconus was significantly associated with shorter axial length, smaller interpupillary distance, higher cylindrical and myopic refractive error and lower best corrected visual acuity. It was not significantly associated with any systemic parameter tested, including age, gender, body height and body mass index, consumption of alcohol and smoking, psychic depression and cognitive function, level of education and self-reported income, quality of life, blood pressure and blood concentrations of lipids, creatinine, glucose and C-reactive protein. A steep cornea/keratoconus defined as corneal refractive power of >49 diopters was detected in 6 (0.2%) eyes, and a steep cornea/keratoconus defined as corneal refractive power of >50 diopters was detected in 2 (0.06%) eyes.Since the present study is the first population-based investigation on the prevalence of steep cornea/keratoconus in a population outside of rural central India, the results of our study cannot directly be compared with findings of other studies. Most investigations on the frequency of keratoconus examined the relative frequency of keratoconus as reason for penetrating keratoplasty or assessed the prevalence of the diagnosis of keratoconus in the Medicare population of the U.S.A 2) and the Beijing Eye Study with a markedly more urban population with an average body mass index of 25.6±3.8 kg/m2, one may infer that the prevalence of steep cornea/keratoconus may not show marked inter-ethnic differences.In the Central India Eye and Medical Study CIEMS, the prevalence of steep cornea/keratoconus defined as a corneal refractive power of ≥48 diopters  was 2.3±0.2%  P = 0.004), and in which the presence of a steep cornea/keratoconus was not significantly associated with alcohol consumption (P = 0.99) or smoking (P = 0.08) nor with questions relating to the psychiatric status. In contrast to the present study, steep cornea/keratoconus in the CIEMS was additionally associated with lower body height (P<0.001) and lower level of education (P = 0.03). In previous studies, associations between keratoconus and Down syndrome, Leber’s congenital amaurosis, and mitral valve prolapse were reported In the present study, presence of steep cornea/keratoconus was significantly associated with shorter axial length, smaller interpupillary distance, higher cylindrical and myopic refractive error and lower best corrected visual acuity. Similar results were obtained in the CIEMS, in which the prevalence of steep cornea/keratoconus was significantly  associated with higher myopic refractive error . Again, it is in agreement with the CIEMS, however not in agreement with a recent hospital-based study by Spoerl and colleagues, in which a significant correlation was reported between non-smokers and keratoconus (P<0.001) In the present study, the prevalence of steep cornea/keratoconus was not related with smoking  and in false negative results (eyes with mild keratoconus). Third, in a similar manner, another limitation is the definition of steep cornea/keratoconus as a corneal refractive power of 48+ diopters. One has, therefore, to keep in mind that the reported prevalence figures and the associations refer to this definition.In conclusion, in adult Chinese aged 50+ years and living in Greater Beijing, a steep cornea/keratoconus defined as corneal refractive power of 48+ diopters had a prevalence of 0.9±0.2%. Prevalence of steep cornea/keratoconus was significantly associated with the ocular parameters of shorter axial length, smaller interpupillary distance, higher cylindrical and myopic refractive error and lower best corrected visual acuity, however, with none of the systemic parameters tested.


Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detected at 50°C/pH 4. rEG A retained 100% of activity when incubated at 45 and 55°C for 72 h. Purified rEG A kinetic parameters towards CMC were determined as Km = 27.5 ± 4.33 mg/mL, Vmax = 1.185 ± 0.11 mmol/min, and 55.8 IU /mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass conversion into products of commercial importance, such as second-generation fuel ethanol. One of the major challenges of modern society is to promote economic growth in a sustainable model. Global demands of energy consumption stimulate the research on alternative fuels, aiming the reduction of the dependence on nonrenewable energy sources. For some decades now, Brazil and the USA have successfully produced bioethanol from sugarcane and corn, respectively. Nonetheless, plant biomass generated by extensive cultures, and which is not totally converted into useful by-products such as fertilizers and animal feed, tends to accumulate and cause environmental problems. Numerous efforts have been made in order to develop biotechnological routes to produce the so-called second-generation bioethanol from agriculture residues such as corn stover, rice straw, sorghum bagasse, corncobs, wheat bran, wheat straw, and sugarcane bagasse. The limiting step of this process is the availability of low-cost efficient enzymes to convert lignocellulose into fermentable glucose units.β-1,4 bonds, to oligosaccharides and glucose. Based on model organisms from the genera Trichoderma and Phanerochaete, fungi cellulolytic enzymes acting in synergism have been classified as (1) endoglucanases or endo-β-1,4-glucanases (EC 3.2.1.4), responsible for the random attack of internal glycosidic bonds of the cellulose amorphous region, generating oligosaccharides of various sizes and new chain ends for the action of a second class of enzymes, (2) cellobiohydrolases (EC 3.2.1.91), which processively degrade the reducing and nonreducing ends of amorphous or crystalline cellulose regions, releasing glucose or cellobiose, and (3) β-glucosidases, which hydrolyze cellobiose and other small oligosaccharides into glucose. More recently, swollenins, which are proteins homologous to plant expansins, were reported to cooperate in cellulose hydrolysis by fungi such as T. reesei, Trichoderma pseudokoningii, Trichoderma asperellum, and Aspergillus fumigatus.Filamentous fungi can produce and secrete enzymes which efficiently degrade cellulose, a linear polymer of glucopyranose units connected by Aspergillus have been described as efficient cellulases producers. Aspergillus nidulans produces three endoglucanases, four cellobiohydrolases and one β-glucosidase. Lockington et al. }  was used. Bacteria were grown at 37°C, in LB medium  supplemented with the appropriate antibiotics, when necessary.For cloning experiments and plasmid manipulations, A. nidulans total RNA was extracted using the Trizol reagent (Invitrogen). Synthesis and amplification of the eglA cDNA were performed by RT-PCR using the specific primers EGA-SnaBI (5′-TACGTAGCTTTCACATGGTTTGG-3′) and EGA-AvrII (5′-CCTAGGTTATTGACTTCCCACG-3′), whose design was based on the eglA gene sequence described by Chikamatsu et al. , in 1-L flasks, incubated at 30°C at 250 rpm until the culture reached an OD600 value of 1.0. Cells were then harvested and washed two times with distilled water, resuspended in 100 mL of BMMY medium in 1-L flasks and incubated under the same conditions for additional 48 h, with the addition of methanol to a final concentration of 0.5% (v/v) at every 24 h in order to maintain the induction condition. Finally, cells were centrifuged at 12,000 g/4°C for 15 min, the supernatant was collected, and 0.2 g/L sodium azide was added.An isolated colony of the 2, at 10°C. The MW50 eluted fraction was concentrated on a MW10 ultrafiltration membrane with the molecular weight cut-off of 10,000 Da  and then submitted to gel filtration chromatography in a Sephadex G50 column (60.0 × 2.7 cm) equilibrated with 0.5 M sodium phosphate buffer pH 7.0, 25 mM NaCl, at a 20 mL/h flux, at 28°C. The eluted fractions were tested for CMCase activity and protein concentration (A280 nm). Fractions presenting CMCase activity were then pooled, dialyzed , and applied onto an ionic exchange column (Q-Sepharose 15.0 × 2.5 cm) previously equilibrated with 0.5 M sodium phosphate buffer pH 7.0, at a 20 mL/h flux, at 28°C. The eluted fractions displaying CMCase activity were pooled and employed for the recombinant enzyme biochemical characterization.The rEG A purification procedure was based on two systems of ultrafiltration membranes followed by a two-step chromatographic protocol. Initially, the supernatant was applied into an ultrafiltration system employing a membrane with molecular weight cut-off of 50,000 Da , under pressure of 2.5 kgf/cmThe SDS-PAGE protocol was performed according to Sambrook and Russel  employinThe CMCase activity, employing CMC as substrate, was determined by the method described by Mandels et al.  and modie ), using glucose as standard. One unit of enzyme activity was established as the amount of enzyme that released 1 μmol of reducing sugar per minute per mL, expressed as IU/mL.The amount (mg/mL) of reducing sugars produced in each reaction was determined by the DNS method  measuredEnzyme activity was evaluated at temperatures ranging from 30 to 80°C. Optimal pH was established with the following buffers: 50 mM sodium acetate (pH 4.0–pH 6.5), 50 mM sodium phosphate (pH 6.0–pH 7.0), and 50 mM Tris-Cl (pH 6.5–pH 8.0). The determined optima temperature and pH were employed in the subsequent experiments.The evaluation of the rEG A thermostability was performed by enzyme preincubation at 45°C, 55°C, 70°C, and 80°C for 3, 12, 24, 48, and 72 h.2 concentration which causes 50% inhibition of the rEG A CMCase activity) of the following reagents: AlCl3, CaCl2, ZnSO4, NaCl, CoCl2, CuCl2, KCl, FeCl3, EDTA, SDS, beta-mercaptoethanol, and 1,4-dithio-DL-threitol (DTT).The effect of metal ions and other chemicals on the endoglucanase activity was assayed by the addition to the reaction system of 18 mM (the HgClβ-D-glucoside (pNPG) as substrates. The final concentration of reducing sugars was determined as described previously. In order to evaluate the rEG A activity towards the substrate 4-methylumbelliferyl-β-D-cellobioside (MUC), a qualitative analysis was performed employing UV light to detect the fluorescent digestion product. rEG A substrate specificity was performed using CMC, filter paper , xylan, microcrystalline cellulose , and p-nitrophenyl-In all experiments, the values for CMCase activity represent the averages of experimental triplicate.Km and Vmax), CMC (concentration ranging from 0 to 35 mg/mL) was employed as substrate in a reaction mixture containing 775 μg purified protein in 50 mM sodium acetate pH 4.0 at 50°C for 30 min. The obtained data were analyzed using the program EnzFitter Windows .To determine the rEG A Michaelis-Menten kinetic parameters (P. pastoris CE (1.25 U/mL FPAse activity) at the proportions of 25, 50, 75, and 100% in the final volume of 6 mL completed with distilled water. Sodium azide (0.2 g/L) was added to avoid contamination by microorganisms. Reaction mixtures were incubated for 24, 48, 72, and 96 h at 50°C/150 rpm. Aliquots of 0.5 mL were periodically collected.rEG A capacity to hydrolyze lignocellulosic substrates derived from agriculture was assayed in 50 mL flasks containing 2/3 of substrate solution  and 1/3 of A. nidulans grown with 10 g/L SCB as the sole carbon source produced a 1.2 kb cDNA fragment, compatible to the size predicted from the splicing of the four putative introns [P. pastoris pPIC9 expression vector under control of the inducible promoter AOX1.The RT-PCR assay using total RNA from  introns . This cDP. pastoris recombinant clone was selected for the next experiments. One clone harboring the empty pPIC9 vector was used as negative control.Based on the highest CMCase, a P. pastoris clones were grown under induction conditions, and culture supernatants were evaluated for enzyme activity during a 120 h period. The P. pastoris clone containing the eglA/pPIC9 construct presented the highest CMCase activity from 24 h of growth; the same activity was maintained throughout the cultivation period. No enzyme activity was detected for the negative control.P. pastoris strain was grown upon induction for 48 h, and the supernatant was applied into a ultrafiltration system employing a membrane with a molecular weight cut-off of 50,000 Da  followed by a cut-off membrane of 10,000 Da . The obtained sample was named CONCMW10 and was subsequently purified by a two-step separation protocol. After passage through a gel filtration column, an isolated peak of CMCase activity, distinct from the one presenting the highest protein concentration, was obtained (data not shown). Samples corresponding to this activity peak were pooled and submitted to ionic exchange chromatography, which resulted in a sharp peak (data not shown) corresponding to a single protein band of 35 kDa, coincident with the CMC degradation spot in the activity gel , xylan from oat spelts (Sigma), p-nitrophenyl-beta-D-glucopyranoside , and 4-methyl-beta-umbelliferyl D-cellobioside  were employed for the rEG A substrate specificity assay . Filter A. nidulans purified recombinant endoglucanase Km and Vmax values. With aid of the EnzFitter program, rEG A Km and Vmax values were determined as 27.5 ± 4.33 mg/mL and 1.185 ± 0.11 mmol/min, respectively.Increasing CMC concentrations were employed for the determination of the P. pastoris recombinant strain CE was assayed for the capacity of hydrolyzing the natural substrates banana stem, ball-milled steam-exploded sugarcane bagasse, soybean residues, and corn stover. Aliquots from the reaction mixtures were collected after 24, 48, 72, and 96 h of incubation, and released total reducing sugars (TRSs) were quantified. The 72 h incubation period was identified as the most efficient for the release of TRS when the rEG A CE was added at the proportion of 100%  presenting a CMCase specific activity more than seven times higher than the concentrated one (CONCMW50). The same effect was not observed for the second ultrafiltration probably because the enzyme passed through the membrane pore. The ability of CMCase to penetrate an ultrafiltration membrane may be due to its compact structure and/or nonuniformity of membrane pore size.The chromatographic step consisted in gel filtration which separated two distinct enzymatic peaks (data not shown). In the fractions present in the first peak, specific activity value increased almost 45 times . The oveP. pastoris [rEG A optimal temperature range 40–60°C,  indicatepastoris –18, and Aspergilli display higher activity in the acidic pH range [The optimum pH 4.0) we described for rEG A does not match the value (pH 6.5) observed for the partially purified native EG A [.0 we despH range , 20. AccpH range  and DashpH range , acidophβ-mercaptoethanol and DTT provoked 80 and 32% increase in enzyme activity, respectively  is higher than the value observed for another fungal endoglucanase expressed in P. pastoris for biotechnological purposes, the Trametes versicolor recombinant enzyme (35–40 IU/mg) [The specific activity we have determined for 0 IU/mg) . When co0 IU/mg) . These dP. pastoris CE was assayed for the degradation of natural lignocellulosic biomass: banana stem, steam-exploded sugar cane bagasse, soybean residue, and corn stover. The degradation of residues by holocellulases is an efficient and inexpensive process to obtain products with high added value, such as the ones derived from pulp and paper industry, second-generation biofuels, composting, food and feed, among others [The recombinant g others . Howeverg others , 35. rEGg others , the effrEG A activity towards different agriculture residues may also be related to the lignin content of these substrates cell wall. According to Howard et al. , lignin In addition, degradation efficiency by rEG A enzyme could be optimized by different pretreatment schemes or/and the association of other enzymes, such as cellobiohydrolases and xylanases, in the context of enzyme cocktails.A. nidulans endoglucanase A had been partially characterized in a previous study [Although native us study , most of


Trans fatty acids (TFA) lower HDL and increase triglyceride concentrations while polyunsaturated fatty acids (PUFA) lower triglycerides and may decrease HDL concentrations. The effect of the interaction between trans fat and PUFA on lipids is uncertain.Men and women (n = 1032) in the Genetics of Lipid-Lowering Drugs and Diet Network (GOLDN) study were included. Fatty acids in erythrocyte membranes were measured with gas chromatography while data on potential confounders were obtained from questionnaires. To test the interaction between total erythrocyte PUFA (ePUFA) and TFA (eTFA) on lipid concentrations we distributed eTFA into tertiles and dichotomized ePUFA at the median concentration.st, 2nd and 3rd tertiles of eTFA, multivariate-adjusted means±s.e.m for HDL were 46.2±1.1, 46.3±1.1 and 45.5±1.0 mg/dL among those with low ePUFA, respectively, while they were 50.0±1.1, 46.9±1.1 and 44.7±1.1 mg/dL among those with high ePUFA, respectively (P for interaction = 0.01). For the 1st, 2nd and 3rd tertiles of eTFA, multivariate-adjusted means±s.e.m for triglycerides were 178.6±11.3, 144.7±10.9 and 140.8±10.6, respectively, among those with low ePUFA, while they were 133.8±11.3, 145.7±10.9 and 149.3±11.5, respectively, among those with high ePUFA (P for interaction = 0.005). Results for VLDL were similar to those for triglycerides. No significant interactions were observed for LDL or total cholesterol.For the 1The relation between trans fat and HDL, VLDL and triglycerides may depend on PUFA. The benefit of avoiding trans fat may be greater among individuals with higher PUFA intake. Supplementation with PUFA among individuals with relatively high trans fat intake may have limited benefits on lipid profiles. Dietary fat has varying effects on plasma lipoproteins In contrast trans fatty acids, e.g., elaidic acid, up-regulate cholesterol ester transfer protein with concomitant increase in very low-density lipoprotein (VLDL) cholesterol, increase inflammation and down-regulate LPL activity, even in presence of linoleic acid The metabolic effects of individual dietary fats may also be modified by the overall fatty acid composition of the diet. Indeed fatty acid patterns have been associated with dyslipidemia and the metabolic syndrome in cross-sectional and prospective studies This study was approved by the institutional review boards at the University of Alabama at Birmingham, Tufts University, University of Minnesota and University of Utah. All patients gave written informed consent.The participants in this study were 1328 white men and women in the Genetics of Lipid Lowering Drugs and Diet Network (GOLDN) family study that enrolled patients from two genetically homogeneous centers in Minneapolis, MN and Salt Lake City, Utah Habitual dietary intake was assessed with a validated National Cancer Institute diet history questionnaire (DHQ) All samples were centrifuged within 20 minutes of collection at 2000×g for 15 min at 4°C and stored frozen at −70°C. For each analyte, specimens from each participant were assayed in the same batch to eliminate inter-assay imprecision.Biochemical analyses were performed as previously described Fatty acids in the erythrocyte membranes were extracted with a mixture of chloroform:methanol  collected in heptane and injected onto a capillary Varian CP7420 100 m column using a Hewlett Packard 5890 gas chromatograph equipped with a HP6890A autosampler. The initial temperature of 190°C was increased to 240°C over 50 minutes to separate fatty acids from 12∶0 through 24∶1n9 SAS Software version 9.2  was used for statistical analyses. From the 1328 men and women screened for the GOLDN study, we excluded all subjects who did not meet our inclusion criteria P≤0.05. The means, standard errors and P-values reported for multivariate analyses are from models that included an interaction term for total eTFA and ePUFA.Lipid profiles, namely HDL, LDL, VLDL, total cholesterol or triglycerides were the outcome variables in mixed multivariate models that simultaneously included all four fat subtypes in erythrocyte membranes, covariates and pedigree as a random effect. The covariates included in the final models were age, sex (men vs. women), BMI, study site, smoking , alcohol intake status (current drinker vs. non-current drinker) and physical activity (as quartiles). Income and education did not change the models appreciably and were excluded from the final analyses. The means and standard errors reported are adjusted for age, sex, BMI, study site, smoking, alcohol intake status, physical activity and pedigree. The differences in the distribution of potential confounders across fatty acid categories were considered significant at P<0.05) younger, had lower waist circumference, blood pressure and fasting glucose when compared to those in the lowest tertile. In the group with higher PUFA concentrations, individuals in the top tertile of trans fat were significantly (P<0.05) younger, had lower BMI, waist circumference, were less likely to be alcohol drinkers and had lower fasting glucose compared to those in the lowest tertile. In both the lower and higher groups of ePUFA concentrations, erythrocyte saturated fat concentrations decreased with increase in trans fat intake.st, 2nd and 3rd tertile of total trans fat, respectively. Thus, individuals in the top tertile had 84% higher trans fat on average compared to those in the lowest tertile. The amount of energy from trans fat estimated from a food frequency questionnaire was also higher in top tertile compared to the lowest tertile of erythrocyte trans fat in both the low and high erythrocyte membrane PUFA groups  erythrocyte membrane trans fat content as a percentage of total erythrocyte membrane fat was 1.21±0.19, 1.64±0.12 and 2.23±0.37 for the 1A groups . As expeA groups .P = 0.01) but no association was observed for LDL (P = 0.46), triglycerides (P = 0.79) or VLDL (P = 0.51). In the same model, ePUFA showed a significant inverse association with triglycerides (P = 0.01) and VLDL (P = 0.004) but no association was observed between PUFA and LDL (P = 0.91) or HDL (P = 0.15).In fully adjusted models that included main effects and an interaction term for eTFA and ePUFA, erythrocyte total trans fat concentrations showed a significant inverse association with HDL  (panel A).P for interaction = 0.99) between eTFA, ePUFA and LDL cholesterol concentrations ( panel B).There was no significant interaction (( panels C and D). For the 1st, 2nd and 3rd tertiles of eTFA, multivariate-adjusted means ± s.e.m for triglycerides were 178.6±11.3, 144.7±10.9 and 140.8±10.6, respectively, among those with low ePUFA, while they were 133.8±11.3, 145.7±10.9 and 149.3±11.5, respectively, among those with high ePUFA concentrations, (P for interaction = 0.005) ( panel C). As for triglycerides, we observed a significant inverse association (P = 0.004) between ePUFA and VLDL, no main effect for eTFA (P = 0.51) and a significant interaction between eTFA and ePUFA (P = 0.01) ( panel D). A non-significant inverse association between eTFA and VLDL was observed among individuals in the low ePUFA group i.e., multivariate-adjusted VLDL means ± s.e.m of 138.7±9.9, 112.5±9.6 and 110.9±9.3 for the low, moderate and high eTFA concentrations, respectively. A non-significant positive association between eTFA and VLDL was observed among individuals in the high ePUFA group i.e., 100.3±9.9, 111.4±9.6 and 115.0±10.1, for the low, moderate and high eTFA concentrations, respectively. No significant eTFA or ePUFA main effects or interactions were observed for total cholesterol concentrations  and use of objectively measured independent (erythrocyte fatty acids) and dependent variables (lipids). Use of erythrocyte membrane fatty acids measured by gas chromatography greatly enhances the quality of trans fat and PUFA assessments. Furthermore, our analyses adjusted for various potential confounders including age, sex, study site, body mass index, physical activity, alcohol intake, smoking, erythrocyte monounsaturated fat, erythrocyte saturated fat and pedigree as a random effect.When the erythrocyte PUFA concentration is above the median in our study population, we see the expected associations The exact reasons for these observations are not clear but could be related to trans fat inhibition of LPL activity and up-regulation of CETP activity Our study has a number of limitations. It was a cross-sectional design thus we are unable to determine whether the observed associations may have been affected by reverse causality since individuals with dyslipidemia may have changed their diet. Secondly, we did not measure LPL or CETP activities so as to better understand the underlying mechanisms. Nonetheless, these findings are interesting in that they show for the first time that the effect of trans fat or PUFA vary depending on relative concentrations of other fatty acids. Our study is unique in that lipids were measured three weeks after patients suspended use of their lipid-lowering drugs. Thus our findings are not confounded by lipid-lowering drugs.These findings will need to be replicated in prospective studies with lipids and/or cardiovascular events as end-points. Other human or animal studies are needed to elucidate on the mechanism underlying the observed interaction between trans fat, PUFA and lipids .The association between trans fat and lipids  may vary depending on PUFA. The benefit of avoiding trans fat may be greater among individuals with higher PUFA intake. Supplementation with PUFA among individuals with relatively high trans fat intake may have limited benefits on lipid profiles.Table S1Interaction between erythrocyte trans fat and polyunsaturated fat on HDL, LDL, total cholesterol and triglyceride concentrations.(DOC)Click here for additional data file.


Actinoplanes. The addition of C7N-aminocyclitols for enhanced production of acarbose and concurrently reduced formation of impurity C by cultivation of A. utahensis ZJB-08196 in 500-mL shake flasks was investigated, and validamine was found to be the most effective strategy. Under the optimal conditions of validamine addition, acarbose titer was increased from 3560 ± 128 mg/L to 4950 ± 156 mg/L, and impurity C concentration was concurrently decreased from 289 ± 24 mg/L to 107 ± 29 mg/L in batch fermentation after 168 h of cultivation. A further fed-batch experiment coupled with the addition of validamine (20 mg/L) in the fermentation medium prior to inoculation was designed to enhance the production of acarbose. When twice feedings of a mixture of 6 g/L glucose, 14 g/L maltose, and 9 g/L soybean flour were performed at 72 h and 96 h, acarbose titer reached 6606 ± 103 mg/L and impurity C concentration was only 212 ± 12 mg/L at 168 h of cultivation. Acarbose titer and proportion of acarbose/impurity C increased by 85.6% and 152.9% when compared with control experiments. This work demonstrates for the first time that validamine addition is a simple and effective strategy for increasing acarbose production and reducing impurity C formation.Commercial production of acarbose is exclusively via done microbial fermentation with strains from the genera of  It is one of the latest successful products of bacterial secondary metabolism to be introduced into the pharmaceutical world market [α-glycosidases [The d market –5. The sosidases –8.Actinoplanes. Many experiments including producer strain mutagenesis and screening, media formula, fermentation conditions, and acarbose isolation and purification have been conducted in order to increase acarbose yield because of its high commercial value [Actinoplanes sp. A56 and developed an optimized industrial fermentation processes for acarbose production, as a result about 5000 mg/L of acarbose was obtained. Our group has extensively studied the production of acarbose [A. utahensis ZJB-08196 was isolated by mutagenesis and screening method [A. utahensis ZJB-08196 at elevated osmolality via intermittently feeding of necessary components regarding acarbose formation afforded a peak acarbose titer of 4878 mg/L [S-adenosylmethionine (100 μM) after 12 h inoculation in fed-batch fermentation, the maximal titer of acarbose was obtained at 6113 mg/L [So far, commercial production of acarbose is exclusively via microbial fermentation with strains from the genera of al value , 9–11. Hal value , 13 repoacarbose –18. A hig method . Fed-bat878 mg/L . By exogActinoplanes sp. commonly produces an extensive series of acarviosyl containing acarbose-like components, including impurity A-H [α-1,4-D-glycosidic linkage of the maltose moiety in acarbose is replaced with a 1,1-linkage in impurity C  according to the method as described previously [Stenotrophomonas maltophilia CCTCC M 204024 [Acarbose standard was purchased from Sigma. Maltose, glucose, sucrose, L-Tyr, monosodium glutamate, glycerol, Keviously . ValienaM 204024 –26. The A. utahensis ZJB-08196, which was screened by our lab and previously deposited at the China Center for Type Culture Collection  as CCTCC M 209022, was used for acarbose production [2HPO4·3H2O 1.0, KCl 0.5, MgSO4·7H2O 0.5, FeSO4 0.1, and agar 20.0, and the initial pH value was adjusted to 7.0. For inoculum, a colony of about 1 × 1 cm2 size from a freshly prepared agar plate was inoculated into a 500-mL shake flask containing 100 mL of seed medium and cultivated at 28°C and 200 rpm for 72 h. The seed medium consisted of (g/L) corn starch 15, soybean flour 40, glycerol 20, CaCO3 2.0, and K2HPO4 0.5, with pH unadjusted. Batch and fed-batch fermentations were carried out by inoculating 10% (v/v) of the seed culture into 500-mL shake flasks containing 50 mL of fermentation medium at 28°C and 200 rpm. The basal medium for acarbose fermentation consisted of (per L) maltose syrup  270 mL, glucose 40.0 g, soybean flour 17.0 g, sodium glutamate 5.0 g, CaCO3 2.5 g, CaCl2 2.5 g, FeCl3 0.5 g, K2HPO4 1.0 g, and glycerol 5.0 g, and the initial pH value was adjusted to 7.0.The mutant strain oduction . This st7N-aminocyclitols  were added into the fermentation medium, respectively, to investigate the effect of different C7N-aminocyclitols addition on acarbose fermentation. The adding concentration and adding time of validamine on acarbose production were optimized. On the basis of above experiments, an optimal feeding strategy, coupled with exogenous validamine addition (20 mg/L), was performed for maximum acarbose production.Sterile-filtered aqueous solutions of CSamples (5 mL) were centrifuged at 9,000 ×g for 15 min. The cell precipitates were dried at 80°C to a constant weight for determining dry cell weight (DCW) as the biomass. Acarbose titer and impurity C concentration in the supernatant were analyzed by using a Shimadzu CT0-10ASVP HPLC system according to the method as described previously . Maltoset-test. The difference between contrasting treatments was considered significant when P is <0.05 in a two-tail analysis. Each run of fermentation experiments was duplicated three times to ensure reliability and accuracy. Data are the average of three independent sample measurements. The error bars indicate the standard deviation (SD) from the mean of triplicates. Data were analyzed with Student's A. utahensis ZJB-08196 were firstly investigated. Cultivations were performed in 500-mL shake flasks containing 50 mL of fermentation medium at 28°C and 200 rpm, and lasted for 216 h. The results were shown in A. utahensis ZJB-08196, different from Actinoplanes sp. CKD485-16 [A. utahensis ZJB-08196 converts the α-1,4-glycosidic bond to α-1,1 of the reducing end glucose moiety of acarbose. At the end of cultivation (216 h), the impurity C concentration reached approximately 450 ± 51 mg/L. According to the fermentation profiles, when the acarbose reached its highest titer (at 168 h), the concentration of impurity C was 289 ± 24 mg/L. It can be calculated that the proportion of acarbose/impurity C was only 12.32. As a byproduct, impurity C seems to be unavoidable. However, the higher concentration of impurity C produced in the fermentation added much pressure to downstream processing due to impurity C structural resemblance to acarbose. The US Food and Drug Administration and other regulatory agencies set strict limits on the content of impurity C (<1.5 percent). To satisfy the mandatory requirements, the purification methods utilize complex process in which a step using a combination of the cation exchanger and the anion exchanger is repeatedly performed and a chromatographic step using the cation exchanger is further conducted [The time courses for acarbose accumulation, substrate consumption, cell growth, and impurity C formation in batch culture of KD485-16 , 20. Aftonducted . The hig7N-aminocyclitols exhibited physiological roles in almost all aspects of microorganisms due to their glycosidase and glucosyltransferase inhibitory activity [7N-aminocyclitols on acarbose fermentation was investigated. Validamycin A, validamycin B, validamycin D, validamycin E, valienamine, and validamine were added to the fermentation medium of A. utahensis ZJB-08196 at a final concentration of 80 mg/L prior to inoculation, respectively. The fermentations were carried out for 168 h in 500-mL shake flasks with 50 mL of fermentation medium at 28°C. The results were shown in A. utahensis ZJB-08196. In the following experiments, the addition conditions were investigated about the positive effect of validamine on acarbose fermentation.The Cactivity , 21. Theactivity . Our groactivity –26. In tEffect of different concentrations of validamine added in the fermentation medium on the formation of acarbose and impurity C was evaluated. Fermentations were carried out at 28°C and 200 rpm for 168 h with the exogenous addition of validamine in fermentation medium in the range from 0 to 160 mg/L prior to inoculation. The results were shown in A. utahensis ZJB-08196.Validamine was added at 0 h, 24 h, 48 h, 72 h, 96 h, and 120 h at final concentrations of 20 mg/L and 40 mg/L to study the effect of its adding time on acarbose fermentation. The results were shown in A. utahensis ZJB-08196 in fermentation medium with the addition of validamine at 20 mg/L at the beginning of fermentation were shown in A. utahensis ZJB-08196 had a broad osmolality optimum between 309 mOsm/kg and 719 mOsm/kg [Actinoplanes sp. SE50/110 [Actinoplanes sp. CKD485-16 [A. utahensis ZJB-08196 may have no effect on the acarbose production and impurity C formation. Time courses of acarbose formation, maltose and glucose consumption, cell growth, and impurity C accumulation during batch cultivation of  mOsm/kg , which iA. utahensis ZJB-08196 accelerated the accumulation of acarbose greatly. A maximal acarbose titer of 6606 ± 103 mg/L was obtained at 168 h, which is increased by more than 33.4% compared to the batch culture. Impurity C began to accumulate after 72 h and reached 212 ± 12 mg/L at 168 h of cultivation.Fed-batch culture is a common strategy in fermentation process to achieve good production . Effect A. utahensis ZJB-08196 fermentation. On one hand, the addition of validamine in the fermentation broth promoted the consumption rates of glucose and maltose. The specific consumption rates of glucose and maltose increased by 4.4% and 15.1% in batch fermentation compared with the control experiments in the absence of validamine. In fed-batch fermentation, the specific consumption rates of glucose and maltose increased by 47.8% and 71.5%. On the other hand, the addition of validamine in the fermentation increased the maximum acarbose titer, acabose productivity, and proportion of acarbose/impurity C greatly. Compared with control experiments, the maximum acarbose titer, acarbose productivity, and proportion of acarbose/impurity C increased by 39.0%, 39.0%, and 275.5% in batch fermentations, respectively. As for fed-batch fermentations, the maximum acarbose titer, acarbose productivity, and proportion of acarbose/impurity C increased by 85.6%, 85.6%, and 152.9%, respectively. The comparison of the kinetic parameters of acarbose fermentation with different methods is shown in A. utahensis ZJB-08196 may be explained from two respects. On one hand, validamine is a glucosyltransferase inhibitor, which can inhibit the α,α-1,4-linkage in the maltose moiety of acarbose to convert to an α,α-1,1-linkage in the trehalose moiety of impurity C, thus reducing the formation of impurity C. On the other hand, validamine is involved with biosynthetic pathway of the acarbose as an activator. In order to discover the real mechanism of the effects of validamine on acarbose production and impurity C formation by A. utahensis ZJB-08196, we are working on the analysis of the transcription level of structural genes and global regulatory genes of acarbose and impurity C with the addition of validamine in fermentation medium by the cultivation of A. utahensis ZJB-08196.The regulative mechanism about validamine on acarbose production and impurity C formation by μg/mL broth, and its cost is very low. This shows that validamine can be obtained with cheap price. In addition, the amount of validamine added to the fermentation medium is very low (20 mg/L). Therefore, the method of using validamine to enhance the production of acarbose and concurrently reduce the formation of impurity C is potential and hopeful in industrial application.The prevalence of diabetes, especially type II diabetes mellitus, is markedly increasing throughout the world . Facing Actinoplanes sp. in a fermentation process. A higher acarbose titer and proportion of acarbose/impurity C in fermentation broth would facilitate the downstream process. The current study reveals for the first time that the addition of validamine is useful for the enhanced production of acarbose and concurrently reduced formation of impurity C by the cultivation of A. utahensis ZJB-08196, which would bring more convenience to product separation. Remarkable improvements on acarbose titer, acarbose productivity, and proportion of acarbose/impurity C were successfully achieved when 20 mg/L of validamine was added in the fermentation medium prior to inoculation both in batch and fed-batch cultures. The acarbose titer and acarbose productivity obtained in fed-batch culture in this work are the highest that have been reported for all the submerged fermentations of A. utahensis [Acarbose, one of the most popular pharmaceuticals in the therapy of diabetes type II, is primarily manufactured using tahensis . This si


Data from 16 patients who had complete measurements during both treatment phases were analysed. The change in IGF1 concentrations during CIPII treatment was 10.4 μg/l  and during s.c. insulin treatment was −2.2 μg/l . When taking the effect of treatment order into account, the estimated change in IGF1 concentrations was found to be 12.6 μg/l  with CIPII treatment compared with that with s.c. insulin treatment. IGFBP1 concentrations decreased to −100.7 μg/l  with CIPII treatment. During CIPII treatment, parts of the GH–IGF1 axis changed compared with that observed during s.c. insulin treatment. This supports the hypothesis that the i.p. route of insulin administration is of importance in the IGF1 system.In type 1 diabetes mellitus (T1DM), low concentrations of IGF1 and high concentrations of IGF-binding protein 1 (IGFBP1) have been reported. It has been suggested that these abnormalities in the GH–IGF1 axis are due to low insulin concentrations in the portal vein. We hypothesized that the i.p. route of insulin administration increases IGF1 concentrations when compared with the s.c. route of insulin administration. IGF1 and IGFBP1 concentrations in samples derived from an open-label, randomized cross-over trial comparing the effects of s.c. and i.p. insulin delivery on glycaemia were determined. T1DM patients were randomized to receive either 6 months of continuous i.p. insulin infusion (CIPII) through an implantable pump  followed by 6 months of s.c. insulin infusion or  Insulin and insulin-like growth factor 1 (IGF1) are structurally and functionally closely related peptides. IGF1, mainly synthesized in the liver after stimulation of the GH receptor, plays a central role in cell metabolism and growth regulation Through an up-regulation of hepatic GH receptor expression, insulin increases the hepatic sensitivity of GH stimulation and subsequently increases IGF1 production Supplementary Figures 1, 2, and 3, see section on With continuous i.p. insulin infusion (CIPII), insulin is infused directly into the i.p. space and is almost entirely absorbed in the portal system, resulting in higher portal insulin concentrations, higher hepatic uptake and lower peripheral plasma insulin concentrations compared with those observed upon s.c. insulin administration The full study design has been published previously Insulin  was administered with an implantable pump . The CIPII pump was implanted under general anaesthesia at the start of the CIPII phase in all the subjects. For subjects who received s.c. insulin during the second treatment phase, the CIPII pump was filled with an inert fluid at the end of the first treatment phase. S.c. insulin was delivered with either MDIs or CSII, according to what was used before the study.Patients treated with MDIs continued to follow their own insulin regimen, i.e. rapid-acting insulin analogues before meals and a daily dose of long-acting insulin. Between both treatment phases of 6 months, a cross-over phase of 4 weeks was instituted to minimize the carry-over effects of CIPII treatment. During the cross-over phase, insulin was administered subcutaneously.If the subject was using more than 40 IU of s.c. insulin per day before starting the CIPII phase of the study, his or her starting dose was set at 90% of the prior s.c. dose. Subjects using <40 IU of s.c. insulin received a starting dose of 80% of the prior s.c. dose. Initially, the dose was equally divided between a basal rate (50%) and a bolus before meals. During all study visits, the seven-point glucose readings were used to adjust the dose regimen if necessary to achieve preprandial glucose levels between 4.0 and 7.0 mmol/l and postprandial levels between 4.0 and 9.0 mmol/l. The patients were instructed not to start a specific diet or weight reduction programme during the trial.Measurements were carried out at baseline, the end of the qualification phase, and at the start, at the halfway point, and at the end of both treatment phases. HbA1c concentrations were measured using a Primus Ultra2 using HPLC . IGF1 and IGFBP1 concentrations, reported as μg/l, were measured in 1.5 cc serum samples collected at random and non-fasting at the start and end of each treatment phase and stored at −80 °C until analysis in 2011, carried out at the Department of Clinical and Experimental Medicine of the Linköping University, Linköping, Sweden. Total IGF1 concentration was measured using a one-step ELISA after acid–ethanol extraction from its binding protein using a commercial kit (Human IGFI Quantikine ELISA Kit R&D Systems) post hoc analysis was the difference in IGF1 concentrations between the two treatment phases. Secondary outcomes were changes in IGFBP1 concentrations during both treatment phases, changes in IGF1 and IGFBP1 concentrations in patients with and without detectable C-peptide and correlations of changes in HbA1c concentrations, total insulin dose, C-peptide concentrations with IGF1 and IGFBP1 concentrations.The primary outcome of this Q–Q plots. In addition, Q–Q plots were used to determine whether the tested variable had a normal distribution. Correlations were investigated using the Pearson product–moment correlation coefficient or, when appropriate, non-parametric Spearman's ρ. Comparisons between outcomes during both treatment modalities were made using t-test for paired comparisons for IGF1 and Wilcoxon match-pair signed-rank tests for IGFBP1. Patients with and without detectable C-peptide were compared with unpaired t-test. IGFBP1 concentrations had a skewed distribution (right tail), and they are presented as median and interquartile range (IQR). Differences in IGFBP1 concentrations were normally distributed. Besides the linear mixed models, all the analyses were carried out using SPSS version 18.0, Inc. A (two-sided) P value of <0.05 was considered to be statistically significant.To calculate the mean difference with a 95% CI, the Hills–Armitage approach was used, which accounts for any period effect. Linear mixed models  were used to test differences, taking treatment order into account. The assumption of normal distribution of the residuals was examined using The study was carried out in accordance with the Declaration of Helsinki. Informed consent was obtained from all the patients for the initial study. The protocol was approved by the Medical Ethics Committee of the Isala Clinics in Zwolle. Additional informed consent was obtained for the present study.s.d.) concentrations at the start of the s.c. and i.p. insulin treatment phases were respectively 83.7±31.9 and 76.3±24.5 μg/l.The study sample consisted of 16 patients, six males and ten females, with a median (IQR) age of 42.4 (30.4–49.4) years and diabetes for a duration of 21.7 (10.4–30.5) years. Three patients used MDIs and 13 CSII before the study, the qualification phase and the s.c. treatment phase. Mean IGF1 (±P=0.33) and IGFBP1 (P=0.83) concentrations between both treatment phases.Results obtained for the IGF1 and IGFBP1 measurements during the different treatment modalities are summarized in P=0.06) and during the s.c. treatment phase was −2.2 μg/l . When taking the effect of treatment order into account, the estimated difference in concentrations between the i.p. treatment phase and the s.c. treatment phase was found to be 12.6 μg/l .The estimated mean change in IGF1 concentrations during the CIPII phase was 10.4 μg/l , but not during the s.c. treatment phase, 9.4 μg/l . The estimated difference between both phases was −110.4 μg/l .IGFBP1 concentrations decreased significantly during the i.p. treatment phase, −100.7 μg/l . HbA1c concentrations improved to −10.0 mmol/mol  with CIPII treatment than with s.c. insulin treatment. During the i.p. treatment phase, changes in HbA1c concentrations correlated with changes in IGF1 concentrations , but not with those in IGFBP1 concentrations .HbA1c concentrations decreased with CIPII treatment from 68±16.5 to 60±6.6 mmol/mol after 3 months and remained stable at 6 months (61±9.9 mmol/mol). During the s.c. treatment phase, there was no change in glycaemic control. No significant carry-over effects were observed between both treatment phases (P=0.71) compared with s.c. insulin treatment. Spearman's correlation coefficient indicated a non-significant association between the mean difference in insulin dose and IGF1 concentrations during the i.p. treatment phase . Changes in IGFBP1 concentrations did not correlate with changes in total insulin dose  during the i.p. treatment phase. Changes in IGF1 and IGFBP1 concentrations during the CIPII phase did not exhibit any significant correlation .Mean daily insulin dose decreased with i.p. treatment with −2.0 IU/day  and detectable  C-peptide: 12.6±22.2 vs 3.7±22.1 ng/ml (P=0.45). IGFBP1 concentrations were −49.5  and −57.7  μg/l respectively. The association between the concentrations of C-peptide and the change in IGF1 concentrations during the i.p. or s.c. treatment phase was also not significant: r=−0.02 (P=0.94) and r=−0.16 (P=0.56).There was no significant difference in the change in IGF1 concentrations during the i.p. treatment phase between patients with undetectable  and/or the duration of the present study. In the study carried out by Hanaire-Broutin, IGF1 concentrations tended to increase even after 6 months. In severely uncontrolled diabetes, IGF1 concentrations are low et al. As there is  no insulin production in patients with T1DM, it has been hypothesized that low insulin concentrations in the portal vein cause decreased IGF1 concentrations/bioactivity through both GH receptor- and IGFBP1-mediated mechanisms P<0.001). Therefore, we hypothesize that the high IGFBP1 concentrations in these five individuals represent an acute effect in the start-up phase of i.p. insulin. It has been reported that insulin withdrawal for 8 h in patients with type 1 diabetes treated with CSII increased IGFBP1 concentrations sixfold, and it is conceivable that the high IGFBP1 values could be due to a lag in insulin delivery post hoc analysis of patients with IGFBP1 concentrations <150 μg/l still indicated that the change in IGFBP1 concentrations during the i.p. treatment phase remained significant  and, as a right skew could influence the estimated difference between the treatment modalities, that the estimated difference between the treatment groups still remained at −49.9 μg/l . When paired comparisons of IGFBP1 concentrations were made during treatment at 3 and 6 months, IGFBP1 concentrations were found to be lower with CIPII treatment than with CSII treatment. The lowering of IGFBP1 concentrations suggests an increase in free IGF1 concentrations, i.e. IGF1 bioactivity by CIPII treatment At the start of the CIPII treatment phase, several patients had very high IGFBP1 values. Due to these outliers, the IGFBP1 concentrations at the start of the i.p. treatment phase were high. Of interest, all five patients with IGFBP1 concentrations >150 μg/l (range: 181.2–330.0 μg/l) were in the ‘i.p. first’ cross-over group. It was remarkable that additional analysis indicated a significantly longer median duration between pump implantation and IGFBP1 measurement for these five patients compared with the other patients (0.5 vs 0.0 years; For the interpretation of the results of this study, it must be acknowledged that the original study was powered to detect differences in hypoglycaemic events between i.p. and s.c. insulin and not in IGF1 or IGFBP1 concentrations. In contrast to the studies carried out by Shishko and Hanaire-Broutin Although the clinical significance of low IGF1 concentrations in patients with T1DM remains unclear at present, CIPII could have an additional benefit on top of glycaemic control by altering the dysregulated GH–IGF system by increasing portal insulin concentrations. This is a hypothesis worth testing in future research.http://dx.doi.org/10.1530/EC-13-0089.This is linked to the online version of the paper at P v D was responsible for study design and drafting and reviewing the manuscript and carried out the statistical analyses; S L was responsible for study design, inclusion, and drafting and reviewing the manuscript; N K and H B were responsible for study design and reviewed the manuscript, K G carried out the statistical analyses and reviewed the manuscript; and H A was responsible for drafting and reviewing the manuscript.