ABSTRACT
Objective@#To explore the influence of different inner dressings in negative-pressure wound therapy (NPWT) on escharectomy wound of full-thickness burn rabbits.@*Methods@#Eighteen Japanese white rabbits were inflicted with full-thickness burn on unilateral back. They were divided into polymer dressing group (PD), biological dressing group (BD), and silver biological dressing group (SBD), according to the random number table, with 6 rabbits in each group. On 3 days post burn, the wounds were performed with escharectomy, and then wounds of rabbits in group PD were covered with polyurethane foam. Wounds of rabbits in group BD were covered with porcine acellular dermal matrix (ADM) and wounds of rabbits in group SBD were covered with silver porcine ADM. Then continuous NPWT was performed on rabbits of the three groups for 7 days. Immediately after surgery and on post surgery day (PSD) 7, general observation of wound was conducted and tissue around the wound was harvested for determination of dry to wet weight ratio. The content of bacteria was counted and the content of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6 in wound was determined by enzyme-linked immunosorbent assay. Fibroblasts in wound were counted after Masson staining and number of microvessels was counted after CD31 antibody immunohistochemical staining. Data were processed with analysis of variance for repeated measurement, LSD-t test, paired samples t test, and Bonferroni correction.@*Results@#(1) Immediately after surgery, there was no granulation tissue in basal wound of rabbits in the three groups, with rich blood supply and obvious edema. On PSD 7, much granulation tissue was found in basal wound of rabbits in the three groups, with no or mild edema and no obvious redness and swelling in wound edge. (2) There were no significant differences in dry to wet weight ratios of tissue around the wound among and within the three groups immediately after surgery and on PSD 7 (with F values respectively 0.70 and 0.09, t values from 0.17 to 0.52, P values above 0.05). (3) Immediately after surgery, the content of bacteria in wounds of rabbits in groups PD, BD, and SBD was respectively (603.0±146.0) ×104, (573.0±63.0) ×104, and (590.0±100.0)×104 colony-forming unit (CFU)/g, with no significant difference among them (F=0.13, P>0.05). On PSD 7, the content of bacteria in wounds of rabbits in groups PD, BD, and SBD were respectively (5.4±0.8) ×104, (4.6±0.9) ×104, and (3.5±0.9)×104 CFU/g. Among them, the content of bacteria in wounds of rabbits in group SBD was lower than that in groups PD and BD, respectively (with t values respectively 3.78 and 2.29, P<0.05 or P<0.01). The content of bacteria in wounds of rabbits in the three groups on PSD 7 was decreased compared with that immediately after surgery (with t values from 10.05 to 21.81, P values below 0.01). (4) There was no significant difference in content of TNF-α, IL-1β, and IL-6 in wounds of rabbits in the three groups immediately after surgery and on PSD 7 (with F values from 0.10 to 1.89, P values above 0.05). The content of TNF-α in wounds of rabbits in the three groups on PSD 7 was significantly higher than that immediately after surgery (with t values from 2.93 to 5.01, P<0.05 or P<0.01). (5) There was no significant difference in amount of fibroblasts in wounds of rabbits in the three groups immediately after surgery and on PSD 7 (with F values respectively 0.01 and 0.81, P values above 0.05). The amount of fibroblasts in wounds of rabbits in the three groups on PSD 7 was larger than that immediately after surgery (with t values from 4.78 to 11.58, P values below 0.01). (6) There was no significant difference in number of microvessels in wounds of rabbits in the three groups immediately after surgery and on PSD 7 (with F values respectively 2.42 and 2.49, P values above 0.05). The number of microvessels in wounds of rabbits in the three groups on PSD 7 was larger than that immediately after surgery (with t values from 7.17 to 11.14, P values below 0.01).@*Conclusions@#SBD is better at inhibiting the growth of bacteria. PD, BD, and SBD have almost the same effects on reducing tissue edema and inflammatory reaction, and on promoting the accumulation of collagen fibers and tissue vascularization.
ABSTRACT
We have developed a rapid and high throughput lipase-ANS (8-Anilino-l-naphthalenesulfonic acid) assay to evaluate the thermo-stability of lipases based on the ANS fluorescence signal's increasing and shifting when this small fluorescence probes binds to lipase. The testing lipase samples were incubated at a temperature range of 25 degrees C to 65 degrees C for 30 min before mixed with ANS solution (0.20 mg/mL lipase and 0.05 mmol/L ANS in the buffer of 20 mmol/L Tris-HCl, 100 mmol/L NaCl, pH 7.2) in a cuvette or microplate. Fluorescence signals of the samples were measured at EX 378 nm, EM 465 nm with a fluorescence photometer or a plate reader, and Tm was calculated with the software of GraphPad Prism5.0. The Tm values of several mutants of Penicillium expansum lipase (PEL) were measured with this ANS assay and conventional method simultaneously and the results show that Tm values are comparative and consistent between these methods, suggesting that the lipase-ANS assay is a reliable, rapid and high throughput method for lipase thermo-stability measurement.
Subject(s)
Anilino Naphthalenesulfonates , Chemistry , Enzyme Stability , High-Throughput Screening Assays , Methods , Hot Temperature , Lipase , Metabolism , Spectrometry, FluorescenceABSTRACT
One of the important strategies for improving recombinant protein expression in Pichia pastoris is optimizing control during the high density fermentation process. Some fermentation control has been used and got obviously higher expression level:optimization of basal salt medium composition; using two stage pH and two stage temperature control during cell growth and induction stage; appropriately increasing dissolved oxygen; selecting optimal cell density prior to induction (CWPTI) and specific growth rate prior to induction (?PTI),using limit amount of initial glycerolin the basic media and feeding it during the fermentation with exponential fed-batch Model,selecting appropriate methanol feeding strategy by methanol-limited feeding batch (MLFB),oxygen-limited feeding batch (OLFB),methanol-unlimited feeding batch (MNLFB) or temperature-limited feeding batch (TLFB). Some research suggested that the expression level of the recombinant protein increase 9 times after optimizing the fermentation control.