ObjectiveTo prepare a recombinant PETase with a dual-catalytic-triad and to evaluate its efficiency in the biodegradation of polyethylene terephthalate （PET）. MethodsBased on the crystal structure of wild-type PETase， point mutations （T88H/L117D） were introduced via site-directed mutagenesis. The recombinant protein was prepared using prokaryotic expression and chromatography purification techniques. The enzymatic hydrolysis of the mutant PETase was assessed by relatively quantifying the products mono （2-hydroxyethyl） terephthalate （MHET） and terephthalic acid （TPA）. ResultsBoth wild-type and mutant PETases accumulated as inclusion bodies， accounting for approximately 20% of the total bacterial protein. After solubilization in urea， the proteins were eluted at 300 mmol/L imidazole during affinity chromatography purification， with concentrations of 1.824 and 1.833 mg/mL and purities of 83.11% and 84.32%， respectively. Subsequent anion-exchange chromatography yielded highly pure enzymes in the 200 mmol/L NaCl fraction： 2.776 mg/mL （96.86% purity） for the wild type and 1.967 mg/mL （95.13% purity） for the mutant. Following refolding， the final concentrations were 0.484 mg/mL for the wild type and 0.991 mg/mL for the mutant. Hydrolysis assays revealed that the mutant released MHET and TPA at （237.67±17.00）% and （197.33±12.01）% of the wild-type levels， respectively. ConclusionThe T88H/L117D dual-catalytic-triad PETase is successfully prepared and it significantly enhanced PET-degrading activity， thus， it′s a promising biocatalyst for PET bioremediation.

Objective:To design and develop a cone-beam CT imaging system for small animals with continuously adjustable spatial resolution.Methods:The imaging system used an X-ray source with a focal spot size of 30 μm and a flat panel detector with a pixel size of 100 μm. On this premise, a " stepping-focusing-rotating" image acquisition mode was proposed, in which the " focusing" and " stepping" systems were sequentially embedded in the " rotating" system. In this acquisition mode, the X-ray source and flat panel detector were relatively stationary to form the " focusing" system. When the " stepping" system accurately transported the object to the scanning position, the " focusing" system could achieve adjustable spatial resolution by making linear motion around the object to be scanned according to different experimental requirements. Finally the " rotating" system achieve high-quality imaging.Results:The variable spatial resolution of small animal CBCT ranges from 35.7 μm to 71.4 μm, and the FOV ranges from 39.6 mm to 108.0 mm. The conversion time for the limit spatial resolution is 19.125 s, which allowed accurate 3D reconstruction of normal mice at different resolutions with high reproducibility.Conclusions:A cone-beam CT suitable for small animals has been developed, whose spatial resolution and FOV can be adjusted arbitrarily within a certain range, which can meet the different imaging requirements in rodent experiments.

Objective:To design and develop a cone-beam CT imaging system for small animals with continuously adjustable spatial resolution.Methods:The imaging system used an X-ray source with a focal spot size of 30 μm and a flat panel detector with a pixel size of 100 μm. On this premise, a " stepping-focusing-rotating" image acquisition mode was proposed, in which the " focusing" and " stepping" systems were sequentially embedded in the " rotating" system. In this acquisition mode, the X-ray source and flat panel detector were relatively stationary to form the " focusing" system. When the " stepping" system accurately transported the object to the scanning position, the " focusing" system could achieve adjustable spatial resolution by making linear motion around the object to be scanned according to different experimental requirements. Finally the " rotating" system achieve high-quality imaging.Results:The variable spatial resolution of small animal CBCT ranges from 35.7 μm to 71.4 μm, and the FOV ranges from 39.6 mm to 108.0 mm. The conversion time for the limit spatial resolution is 19.125 s, which allowed accurate 3D reconstruction of normal mice at different resolutions with high reproducibility.Conclusions:A cone-beam CT suitable for small animals has been developed, whose spatial resolution and FOV can be adjusted arbitrarily within a certain range, which can meet the different imaging requirements in rodent experiments.

Objective: To investigate the effects of sodium butyrate on intestinal barrier of the severe scald mice and the related mechanism. Methods: Eighteen C57BL/6 female mice, aged eight to twelve weeks, were divided into sham scald group, pure scald group, and scald+ sodium butyrate group according to random number table, with 6 mice in each group. Back of each mouse in pure scald group and scald+ sodium butyrate group were immersed into 90 ℃ water for 9 s, causing full-thickness scald of 30% total body surface area, while back of each mouse in sham scald group were immersed into 37 ℃ water for 9 s, causing sham injury. All of the mice in 3 groups were intraperitoneally injected with 1 mL sterile lactated Ringer′s solution immediately after injury. Besides, mice in scald+ sodium butyrate group were intraperitoneally injected with 300 mg/kg sodium butyrate at 30 min before injury and immediately after injury, while mice in sham scald group and pure scald group were intraperitoneally injected with the same volume of sterile phosphate buffer solution. At post injury hour (PIH) 24, portal vein of mice in 3 groups was harvested, intestinal permeability was measured by fluorescin isothiocyanate-dextran fluorescence probe tracing method, then lileal tissue of mice in 3 groups was harvested, protein expressions of zonula occludens l (ZO-1), occludin, claudin-1, claudin-2, nucleotide-binding oligomerization domain-containing protein-like receptor family pyrin domain containing 3 (NLRP3), interleukin-1β (IL-1β), and IL-18 were detected by Western blotting, and distribution of ZO-1 in intestinal mucosa was observed by indirect immunofluorescence. Data were processed with one-way analysis of variance, least-significant difference test, and Bonferroni correction. Results: (1) At PIH 24, the intestinal permeability of mice in sham scald group, pure scald group, and scald+ sodium butyrate group was 0.88±0.19, 2.62±0.48, 1.23±0.16, respectively. Compared with that in sham scald group, the intestinal permeability of mice in pure scald group was significantly elevated (P<0.01), while the intestinal permeability of mice in scald+ sodium butyrate group showed no obvious change (P>0.05). Compared with that in pure scald group, the intestinal permeability of mice in scald+ sodium butyrate group was significantly decreased (P<0.01). (2) At PIH 24, compared with those in sham scald group, the protein expressions of ZO-1, occludin, and claudin-1 of mice in pure scald group and scald+ sodium butyrate group were significantly decreased (P<0.05), while the protein expression of claudin-2 was significantly increased (P<0.05). At PIH 24, compared with those of pure scald group, the protein expressions of ZO-1 and occludin of mice in scald+ sodium butyrate group were significantly elevated (P<0.05), while the protein expression of claudin-2 was significantly decreased (P<0.05), the protein expression of claudin-1 showed no significant difference (P>0.05). (3) At PIH 24, compared with those in sham scald group, the protein expressions of NLRP3, IL-1β, and IL-18 of mice in pure scald group and scald+ sodium butyrate group were significantly increased (P<0.05). Compared with those of pure scald group, the protein expressions of NLRP3, IL-1β, and IL-18 of mice in scald+ sodium butyrate group were significantly decreased (P<0.05). (4) At PIH 24, ZO-1 in intestinal mucosa of mice in sham scald group was distributed smoothly, continuously and homogeneously along the membrane. ZO-1 in intestinal mucosa of mice in pure scald group was distributed unsmoothly with breaks. The distribution of ZO-1 in intestinal mucosa of mice in scald+ sodium butyrate group was ameliorated compared with that in pure scald group. Conclusions Sodium butyrate can inhibit the activation of NLRP3 inflammasome and decrease the production of IL-1β and IL-18 in intestinal mucosa of severe scald mice, which protects the intestinal barrier function by alleviating the alteration of tight junction protein expression and localization.

Objective: To investigate the significance of intestinal fatty acid binding protein (IFABP) in the evaluation of intestinal barrier dysfunction of mice at the early stage of severe burn injury. Methods: Thirty-six 8-week-old C57BL/6 male mice were collected and divided into normal control group (n=6) and scald group (n=30) according to random number table. Back of each mouse in scald group was placed into hot water of 90 ℃ for 10 s, causing full-thickness scald (hereinafter refer to as burn) of 30% total body surface area, while mice in normal control group were not inflicted with burns. Six mice in normal control group were taken, and 6 mice in scald group at 1, 2, 6, 12, and 24 h post injury were taken respectively. The portal vein blood of each mouse was extracted and the plasma was separated to measure intestinal permeability with fluorescin isothiocyanate-dextran fluorescence probe tracing method and plasma IFABP content by enzyme-linked immunosorbent assay. The distal ileum tissue of mice in normal control group and scald group at each time point post injury was collected to observe the morphology of the intestinal mucosa tissue by hematoxylin-eosin staining. Data were processed with one-way analysis of variance and Student-Newman-Keuls test, and pearson correlation test was used to analyze the correlation between intestinal permeability and plasma IFABP content of burned mice. Results: (1) At 1, 2, 6, 12, and 24 h post injury, the intestinal permeability of mice in scald group was 2.7±0.8, 5.4±2.5, 7.3±4.2, 12.4±6.1, 1.4±0.7, respectively, obviously higher than 1.0±0.4 of normal control group (P<0.05 or P<0.01). The intestinal permeability of mice in scald group showed an increasing trend post injury, reaching the peak at 12 h post injury, and rapidly falling back at 24 h post injury. (2) At 1, 2, 6, 12, and 24 h post injury, the plasma IFABP content of mice in scald group was (64±11), (59±12), (76±18), (111±22), and (66±10) ng/mL, obviously higher than (35±8) ng/mL in normal control group (P<0.05 or P<0.01). The plasma IFABP content of mice in scald group showed an increasing trend post injury, reaching the peak at 12 h post injury, and rapidly decreasing at 24 h post injury. (3) Uniform thickness of mucosa, intact epithelia, regularly arranged villi, and no inflammatory cell infiltration were observed in ileum of mice in normal control group. In ileum of mice in scald group, shortened villi of mucosa with different degrees, edema of lamina propria, and infiltration of neutrophils were observed at 1 and 2 h post injury; obviously damaged and partially exfoliated ileal mucosa, disorderly arranged and broken villi, degenerated and necrotic epithelial cells, dilated central lacteal, and infiltration of lymphocytes and neutrophils were observed at 6 and 12 h post injury; the damage of ileal mucosa was alleviated, and basically intact epithelia, dilated central lacteal, and infiltration of inflammatory cells were observed at 24 h post injury. (4) There was a significantly positive correlation between the intestinal permeability and the plasma IFABP content of burned mice (r=0.841, P<0.05). Conclusions The plasma IFABP can be used as a good biological indicator for the evaluation of intestinal barrier dysfunction of mice at the early stage of severe burn injury.

Objective: To investigate the effects of short chain fatty acid (SCFA) on barrier disruption of human intestinal epithelial cell induced by endotoxin/lipopolysaccharide (LPS) and the related mechanism. Methods: The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer-cells. Cells were divided into control group, LPS group, and SCFA+ LPS group according to the random number table. Cells in control group were only routinely cultured with DMEM medium. Cells in LPS group were cultured with DMEM medium and LPS with final mass concentration of 10 μg/mL. Cells in SCFA+ LPS group were cultured with DMEM medium, LPS and SCFA (consisting of 0.5 mmol/L acetate, 0.01 mmol/L propionate, and 0.01 mmol/L butyrate) with final mass concentration of 10 μg/mL. At post culture hour (PCH) 0, 1, 2, 6, 12, and 24, transepithelial electrical resistance (TER) of cells was determined with an ohmmeter, with sample number of 72. Another portion of cells were divided and treated as above, and then Western blotting was employed to detect the protein expressions of zonula occludens 1 (ZO-1), occludin, and claudin-1 at PCH 24, with sample number of 6. Another portion of cells were divided and treated as above and then immunofluorescence was used to observe cellular morphology and distribution of ZO-1. Data were processed with analysis of variance of factorial design, one-way analysis of variance, least-significant difference test, and Bonferroni correction. Results: (1) Compared with that in control group, TER of cells in LPS group was significantly reduced from PCH 1 to 24 (P<0.01), while TER of cells in SCFA+ LPS group showed no obvious change (P>0.05). TER of cells in SCFA+ LPS group was significantly higher than that in LPS group from PCH 1 to 24 (P<0.01). (2) Compared with the protein expressions of ZO-1, occludin, and claudin-1 of cells in control group (1.25±0.10, 1.17±0.04, and 1.24±0.20), those of cells in LPS group (0.74±0.23, 0.76±0.11, and 0.77±0.11) at PCH 24 were significantly decreased (P<0.05), while those of cells in SCFA+ LPS group (1.23±0.46, 1.05±0.09, and 1.01±0.13) showed no significant differences (P>0.05). Protein expressions of occludin and claudin-1 of cells in SCFA+ LPS group were significantly higher than those in LPS group at PCH 24 (P<0.05). Protein expression of ZO-1 of cells in SCFA+ LPS group was higher than that in LPS group at PCH 24 with no significant difference (P>0.05). (3) At PCH 24, cells in control group were compact in arrangement with pebble-like appearance, and ZO-1 was distributed smoothly and continuously along the cell membrane. In LPS group, cells were sparse in arrangement with change in appearance, and ZO-1 was distributed uncontinuously along the cell membrane with curls and breaks. In SCFA+ LPS group, the appearance of cells and distribution of ZO-1 were remarkably ameliorated compared with those in LPS group. Conclusions SCFA can alleviate the barrier disruption of human intestinal epithelial cell induced by LPS through interdicting the abnormal distribution of ZO-1 and decrease of TER and tight junction proteins′ expressions.

BACKGROUND:Zinc-modified calcium silicate (CaSiO3) bioceramics coating on the titanium surface prepared in preliminary experiments has good chemical stability and antibacterial property. OBJECTIVE:To observe the effects of zinc-modified CaSiO3 bioceramics coating on osteointegration. METHODS:MC3T3-E1 cels were respectively cultured on the titanium with zinc-modified CaSiO3 bioceramics coating (experiment group), titanium with CaSiO3 bioceramics coating (control group) and pure titanium (blank control group). Then, cel adhesion, proliferation, calcification rate and the expression of type I colagen and osteocalcin were detected. The implant materials mentioned above were respectively inserted into the femurs of New Zealand white rabbits, and after 1.5 months, the osteoproliferation and osteointegration between the implants and the host were tested. RESULTS AND CONCLUSION:In vitro experiment: The number of adhesive cels at 12 hours after co-culture was significantly increased in the experimental group compared with the control group and blank control group (P < 0.05). At 14 days after co-culture, cel proliferation ability and ability of calcium nodule formation in the experiment group were significantly better than those in the other groups (P < 0.05). At 21 days after co-culture, there was no significant difference in the expression of type I colagen, but the expression of osteocalcin in the experiment group was higher than that in the control group and blank control group (P < 0.05).In vivo experiment: In the experiment group, a large amount of bone substances were detected, the coating materials directly contacted with the bone interface, new bone tissues and little fibrous tissues were observed at the interface. In contrast, there was a small amount of bone hyperplasia in the control group and almost no bone hyperplase in the blank control group. Moreover, a small part of the implant directly contacted with the bone interface and the most part was separated from bone trabeculae by fibrous tissues. These findings indicate that zinc-modified CaSiO3 bioceramics coating can enhance the ability of osteointegration between titanium implants and the host.

A novel method for quantification of astragaloside IV in Radix Astragli was developed by using online SPE liquid chromatography coupled with corona charged aerosol detection( CAD) . The sample solution
was loaded into Acclaim Polar AdvantageⅡC18(50 mm×4. 6 mm, 3 μm) which was selected as online SPE column. Then the cleaning process was done by using the Right one of dual gradient pumps with Methanol-water as mobile phase. Acclaim C18(150 mm×4. 6 mm, 5 μm) was selected as analytical column with acetonitrile-water as mobile phase at a flow rate of 1. 0 mL/min. The eluate containing the target from SPE column was transferred into the analytical column by heart-cutting mode. The temperature of nebulizer of corona CAD was set at 30 ℃ and the nitrogen pressure was 241. 3 kPa. The baseline separation of astragaloside IV from matrix components has been achieved. There was a good linear correlation in the range of 4 . 0-80 mg/L for astragaloside IV and the correlation coefficient was 0 . 9998 . The standard addition average recovery of astragaloside IV in Radix Astragli was 97 . 6%. It has been validated that astragaloside IV in Radix Astragli and its preparations can be quantified rapidly and accurately with this method.

Objective To summarize the clinical experience of one stage hybrid operation for aortic arch replacement and explore the indication. Methods From July,2009 to March,2010, 22 consecutive patients received one stage hybrid operation in our hybrid suite for aortic dissection or aortic aneurysm involving aortic arch. Two operative methods are used. (1)Bypass from ascending aorta to brachiocephalic arteries using midstemotomy and normothermia with antegrade aortic arch endovascular stented graft implantation. (2) Ascending aorta replacement and/or aortic valve replacement and/or coronary artery bypass grafting using midstemotomy and cardiopulmonary bypass with antegrade aortic arch endovascular stented graft implantation. Results All patients were technically successful. Angiography during the operation showed 100% patency of all the bypass grafts and no obvious translocation or endoleak of the stents. One patient in the first group died on sixth day after operation due to distal dissection rupture. There was one case of mediastinal lymph effusion in the second group and one case of death due to renal failure and respiratory failure 12 days after operation in the second group. The ICU stay and hospital stay were obviously shorter in hybrid open chest group than that in traditional open chest operation group(P ＜0.05). The blood product consumption and expenditure were also obviously less in hybrid open chest group than that in traditional open chest operation group (P ＜0.05). All the patients were followed up with a mean period of (14.45 ±2.33) months (range: 12 -20 months). All other patients were recovered with normal social life. CT showed neither endoleak nor translocation of the stented grafts. Faulse lumen closure rate at stented-graft segment is 100%. There was no obvious change of distal part of the dissection three months after operation except some thrombosis formation in some of the false lumen. Conclusion One stage hybrid operation for aortic arch replacement is safe and effective in shortening the duration of the operation and reducing the surgical trauma and risk of interval between procedures, shortening the hospital stay and reducing the blood product consumption compared with conventional operation with satisfactory early results. The midterm and long term results are still needed to be followed up.