Production of bioethanol using starch as raw material has become a very prominent technology. However, phytate in the raw material not only decreases ethanol production efficiency, but also increases phosphorus discharge. In this study, to decrease phytate content in an ethanol fermentationprocess, Saccharomyces cerevisiae was engineered forheterologous expression of phytase on the cell surface. The phy gene encoding phytase gene was fused with the C-terminal-half region of α-agglutinin and then inserted downstream of the secretion signal gene, to produce a yeast surface-display expression vector pMGK-AG-phy, which was then transformed into S. cerevisiae. The recombinant yeast strain, PHY, successfully displayed phytase on the surface of cells producing 6.4 U/g wet cells and its properties were further characterized. The growthrate and ethanol production of the PHY strain were faster than the parent S. cerevisiae strain in the fermentation medium by simultaneous saccharification and fermentation. Moreover, the phytate concentration decreased by 91% in dry vinasse compared to the control. In summary, we constructed recombinant S. cerevisiae strain displaying phytase on the cell surface, which could effectively reduce the content of phytate, improve the utilization value of vinasse and reduce the discharge of phosphorus. The strain reported here represents a useful novel engineering platform for developing an environment-friendly system for bioethanol production from a corn substrate.
6-Phytase ; metabolism ; Biofuels ; Ethanol ; chemistry ; Fermentation ; Industrial Microbiology ; Saccharomyces cerevisiae ; metabolism ; Starch ; chemistry ; Zea mays ; chemistry

BACKGROUND: Based on previous technique prepared for encapsulating living cells with alginate-polysine- alginate (APA) microcapsules, it has been confirmed that microencapsulated chromaffin cells have good analgesic effects. The immunoisolated effects of such microcapsule materials need to be evaluated. OBJECTIVE: This study aimed to investigate the immunological rejections of APA microencapsulated chromaffin cells transplanted into rat anterior chamber of eyes and tendon of feet, and to evaluate the immunoisolated effect of microencapsulation.DESIGN: A randomized controlled animal experiment. SETTING: Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: Forty-eight female SD rats, with the age of 3 months, were provided by the Laboratory Animal Center, Tongji Medical College, Huazhong University of Science and Technology. The protocol was carried out in accordance with ethical guidelines for the use and care of animals. Alginate and polylysine used in the experiment were the products of Sigma Company, USA. Microcapsule generator was gifted by Germany. METHODS: This study was performed at the Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology from September 2002 to September 2003. Suprarenal medulla was taken from 6 healthy adult cadavers of brain death. After isolated, digested and cultured, suprarenal medulla was prepared into chromaffin cell suspension. Written informed consents were obtained from the family members of donors, and the protocol was given approval by the Ethics Committee of the hospital. Empty microcapsules and microencapsulated cells were prepared by APA. The 48 rats were randomly divided into the human chromaffin cell (HCC) group, the empty microcapsule group and the microencapsulated HCC (ME-HCC) group. In each group, there were two transplanted regions of anterior chamber of eyes and tendon of feet, with 8 rats used for each region. Each rat in the HCC group was perfused 2×1010 L-1 cell suspension into the anterior chamber of eyes and tendon of feet. Those in the empty microcapsule group and the ME-HCC group were perfused 100 empty capsules and ME-HCCs (100 microcapsules, 400-500 HCCs per microcapsule) into the same regions, respectively. MAIN OUTCOME MEASURES: On day 7 after transplantation, serum interleukin (IL)-2 level was determined by ELISA. Serum IgG and IgM levels were determined with a laser turbidimeter. On day 28 after transplantation, rat right eyeball and left feet were harvested, routinely sliced and stained by haematoxylin-eosin (HE). Histo-morphological structure was observed under a 40×light microscope. RESULTS: Forty-eight rats were included in the final analysis. Serum IL-2, IgG and IgM levels were significantly lower in the empty microcapsule group and ME-HCC group than in the HCC group (t=8.544-21.64, P < 0.01). A lot of lymphocyte and neutrophile infiltration could be found in the anterior chamber of eyes and tendon of feet of rats in the HCC group, but a little seen in that of the empty microcapsule group and ME-HCC group. CONCLUSION: APA microencapsulation has an effective immunoisolated effect on immunological rejection due to its good biocompatibility and mechanical stability.

Objective To study the efficacy and safety of Solifenacin on female overactive bladder (OAB) symptoms secondary to uncomplicated lower urinary tract infection. Methods Seventy-three adult female patients who had clinically diagnosed as OAB symptoms secondary to uncomplicated lower urinary tract infection were randomly divided into treatment group (41 cases) and control group (32 cases). Treatment group received Solifenacin , 5 mg orally qd , as well as OAB behavioral therapy , but control group was given only OAB behavioral therapy. The overactive bladder syndrome score (OABSS) was evaluated before and after antibiotic treatment in all the patients , and the treatment or observation time lasted four weeks after the antibiotic treatment were deactivated. Then OABSS scoring and the cure rate between two groups were compared. Results OABSS score of treatment group decreased significantly after receiving Solifenacin treatment one week , and the cure rate reached 56.10%, and two weeks later, the cure rate reached 92.68%. OABSS score of control group had no significant change one week after treatment and the cure rate was only 6.25%, but at the fourth week OABSS had decreased significantly and the cure rate reached 37.50%, which was still significantly lower than that of treatment group. There were no drug adverse events during treatment in both groups. Conclusions There is obvious clinical effect of solifenacin on female OAB symptoms secondary to uncomplicated lower urinary tract infection, which is safe and could significantly shorten the course of treatment.

HSF1 is the major heat shock transcription factor that binds heat shock element (HSE) in the promoter of heat shock proteins (HSPs) and controls rapid HSP induction in cells subjected to various stresses such as elevated temperature, chemicals, or exposure to toxins. Although at least four members of the vertebrate HSF have been cloned, details of their individual physiological roles remain relatively obscure. To clarify the exact in vivo functions of HSF1 and assess whether HSF1 exhibits redundant or unique roles, we have created homozygous Hsf1-/- mice using standard gene targeting techniques and isolated Hsf1-/- embryonic fibroblasts. Here we demonstrate that heat shock response (HSR) was not attainable in Hsf1-/- embryonic fibroblasts, and this response was required for thermotolerance and protection against heat-induced apoptosis, and that homozygous Hsf1-/- mice, which survived to adulthood according to genetic background, exhibited multiple phenotypes including: (1) placental defects that reduced embryonic viability after late midgestation (day 13.5); (2) growth retardation; (3) female infertility caused by preimplantation lethality, and (4) increased mortality (+/+ vs -/-, P＜0.05) and exaggerated production of proinflammatory cytokine, TNF α (+/-　vs -/-, P＜0.05) after endotoxin challenge. Interestingly, although Hsf1-/- mice exhibited placental defects and embryonic death, basal HSP expression is not appreciably altered during embryonic development by the HSF1 null mutation, suggesting this factor might be involved in regulating some non-HSP genes or signaling pathways which may be important for development. Taken together, our results established direct causal effects for the HSF1 transactivator in regulating diverse physiological and pathophysiological conditions such as developnent, growth, reproduction, apoptosis and sepsis. The present work also provided a useful mammalian model for further investigating the implications of Hsf1 and its target genes (HSPs and other possible non-HSP genes) in various physiological and pathophysiological processes.

Objective:To investigate the role and mechanism of high mobility group box 1(HMGB1) in the injury of Caco-2 intestinal epithelial barrier induced by lipopolysaccharide (LPS).Methods:The Caco-2 cellular monolayer barrier was established with Transwell chamber. After the Caco-2 monolayer model was established, the transepithelial electrical resistance (TEER) values were measured. When the TEER value reached 500 Ω·cm 2, the cells were divided into 3 groups: control group, LPS treatment group, and LPS+ ethyl pyruvate (EP) treatment group. The concentration of LPS and EP were 100 μg/mL, 50 μg/mL, separately. Then TEER values were measured at 12, 24, 48 and 72 h, and FITC-dextran permeability was detected at 24 h. The cells were seeded on 6-well plates. After cell density reached 80%, treatments were given as the above. The real-time polymerase chain reaction (RT-PCR) and Western blot were used to measure the changes in the protein and mRNA expressions of Occludin, HMGB1, and nuclear factor-κB (NF-κB). Results:Compared with the control group, the TEER values (Ω·cm 2) reduced at 12, 24, 48 and 72 h in the LPS treatment group [(514.22±12.59) vs (304.96±9.69), (521.65±13.35) vs (276.21±7.82), (523.99±8.18) vs (206.64±15.85), (491.21±6.72) vs (156.33±10.83), all P<0.05]. The FITC-dextran permeability increased significantly at 24 h [(2.58±0.07) vs (1.04±0.06), P<0.05]. The expression levels of Occludin protein and mRNA were decreased (all P<0.05), while the expression levels of HMGB1 and NF-κB protein and mRNA were significantly increased (all P<0.05). Compared with the LPS treatment group, the TEER values (Ω·cm 2) increased significantly at 12, 24, 48 and 72 h in the EP treatment group [(519.00±5.66) vs (304.96±9.69), (504.69±8.57) vs (276.21±7.82), (453.65±10.74) vs (206.64±15.85), (385.28±7.57) vs (156.33±10.83), all P<0.05]. The FITC-dextran permeability decreased at 24 h [(1.23±0.11) vs (2.58±0.07), P<0.05]. The expression level of Occludin protein and mRNA were increased ( P<0.05), while the expression levels of HMGB1 and NF-κB protein and mRNA were significantly decreased (all P<0.05). Conclusions:LPS can injure intestinal barrier directly in vitro and reduces the expression of tight junction proteins between cells. The mechanism may be related to the increased expression of HMGB1 and NF-κB protein.

AIM: To investigate the effect of nucleolin on diabetic cardiomyopathy in mice.METHODS: A type II diabetic cardiomyopathy mouse model was prepared using a cardiac-specific nucleolin-overexpressing transgenic mice.The mice were divided into wild-type mouse control group, nucleolin transgenic mouse control group, wild-type mouse diabetes group and nucleolin transgenic mouse diabetes group.Wheat germ agglutinin (WGA) fluorescent dye, Masson staining and PowerLab system detection were used to further clarify the role of nucleolin on cardiac hypertrophy, fibrosis and cardiac function in type II diabetic cardiomyopathy mice.RESULTS: Compared with wild-type mouse control group, no significant increase in blood glucose level was found, while genetical myocardial cell hypertrophy was significantly attenuated in nucleolin transgenic mouse diabetes group.The collagen fibers were also significantly reduced, and hemodynamic indexes ± dp/dtmax, left ventricular end-diastolic pressure, left ventricular systolic pressure and heart rate were also improved.The above differences were statistically significant.CONCLUSION: Nucleolin may reduce the occurrence of myocardial hypertrophy and fibrosis, thus improving the cardiac function of diabetic cardiomyopathy mice.

Objective To determine if polydatin inhibited oxidative stress and inflammatory response in rats with sepsis-induced acute kidney injury (AKI).Methods Seventy-two rats (weighing 180-220 g) were randomly divided into the following groups: sham group, cecal ligation and puncture (CLP) CLP+normal saline group (group CN), group CLP+vehicle (group CV), and group CLP+polydatin (group CD) (n=18 each).Rats in groups CN, CV and CD underwent CLP to mimic sepsis-induced AKI.In sham group, the cecum was not ligated or punched, and the remaining procedures were the same as in group CLP.Normal saline, vehicle, and 30 mg/kg polydatin were administered at 6, 12, and 18 hours after CLP via the tail vein.At 24 hour post CLP, two clinically used markers of AKI, blood urea nitrogen (BUN), and creatinine (Cr) were tested, pathological changes of kidney tissue was observed under light microscopy in each group.Renal tubular damage assessment was carried out.Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) content of renal tissue, serum cytokines such as TNF-α, IL-1β and IL-6, were also measured in each group at 24 hours after CLP.Results Compared with sham group, multiple indexes such as BUN, Cr, tubular injury scores, MDA content of renal tissue, and serum cytokines incluing TNF-α, IL-1β and IL-6 increased significantly (P<0.01), while SOD and GSH levels of renal tissue significantly decreased in groups CN and CV (P<0.01).Compared with groups CN and CV, the indicators such as BUN, Cr, tubular injury scores, MDA content of renal tissue, and serum cytokines such as IL-1β and IL-6 significantly decreased (P<0.05);while SOD and GSH levels of renal tissue significantly increased (P<0.05).Conclusion Sepsis caused by sepsis cecal ligation and puncture can cause acute kidney injury.Polydatin could alleviate kidney damage by attenuating systemic inflammatory response and inhibiting oxidative stress of renal tissue.

Objective To explore appropriate organization and financing models of urban employees supplementary medical insurance under the conditions of the market economy. Methods Employee surveys on willingness to participate in, willingness to pay for, and actual enrolment in supplementary medical insurance were conducted in Shanghai, Wenzhou and Chengdu and nationally different operational models were compared so as to analyze prospectively coverage and operational models of feasible supplementary medical insurance options. Results Characteristics of the employees needs for various supplementary medical insurance options were garnered and appropriate coverage and operational models were put forward. Conclusion It is suggested that the role of the government in the development of supplementary medical insurance be clearly defined, the development of supplementary medical insurance be promoted through policy support and guidance, and the supervision of supplementary medical insurance be strengthened.

Objective To investigate the risk factors and countermeasure of residual stones after single‐channel percutaneous nephrolithotomy for higher stone‐free rate and better operation result .Methods All patients who underwent single‐channel percu‐taneous nephrolithotomy in our hospital from June 2011 to December 2013 were retrospected and the cause of residual stones were analyzed .Results There were 42 patients who had residual stones after operation among total 262 patients undergone single‐chan‐nel PCNL .21 patients had residual stones because the stones they burdened were too complex .7 patients were concerned with com‐plications such as intraoperative hemorrhage .The stone fragments scattered into the calices in 7 patients with overlarge stone during fragmentation .The other causes concerned with stone residue included anatomic structural abnormalities of the kidneys(3 patients) , operation itself inherent limitations(3 patients) ,insufficient practice and experience in operation(1 patients) .Conclusion The main causes concerned with residual stones of single‐channel PCNL are complexity of urinary calculi ,bleeding ,scattering of stone frag‐ments and anatomic structural abnormalities of the kidney .

Objective To observe the effects of nAChR antagonistα-conotoxin Eb1.6 on ther-mal pain threshold and spinal IL-1βexpression levels and astrocytes activation in rats using L5 spinal nerve transaction (SNT)model.Methods Fifty male Sprague-Dawley rats were randomly assigned into 5 groups with each group 10 rats:sham group,different doses of α-CTX Eb1.6 (0.1 5,1.5 and 1 5 nmol/kg)groups and the saline group after SNT.Saline solution or different doses of Eb1.6 were intraperitoneally injected seven days after the surgery when the model was stable and the treatment continued for seven days.Measured the TWLs of all groups of the rats 1,2,4,7,12 hours after the in-jection on 7 d and 13 d.The rats were sacrificed and L5 spinal cord tissues were collected immediately after the behavioral tests on 13 d.The expression of GFAP and IL-1βwere assessed by Western blot assay and enzyme linked immunosorbent assay (ELISA)separately.Results Groups E1,E2,E3 and C had shorter TWL before the injection on 7 d and 13 d than group N(P <0.05).The TWLs of the rats in groups E1,E2 and E3 of 1 h,2 h and 4 h after the injection on 7 d were significantly higher than that before the injection(P <0.05)with 2 h after the injection showed the most obvious change.The TWL of 1 h,2 h,4 h and 7 h after the injection of the rats in group E1,E2 and E3 and those of 12 h after the injection of the rats in group E2 and E3 on 13 d were significantly higher than that before the injection(P <0.05 )and also higher than TWL of the respective time points on 7 d(P < 0.05 ),also with 2 h after the injection showed the most obvious change.The TWLs of 2 h after the injection a-mong group E1,E2 and E3 showed significant differences both on 7 d and 13 d (P <0.05).Rats spi-nal IL-1βand GFAP expression levels of group E1,E2,E3 and C were significantly higher than those of group N(P <0.05).Rats spinal IL-1β and GFAP expression levels of groups E1,E2,E3 signifi-cantly decreased compared with group C(P <0.05).There were significant differences among the spi-nal IL-1βand GFAP expression levels of group E1,E2 and E3(P <0.05).Conclusion Eb1.6 dose-de-pendently reduced the thermal hyperalgesia induced by L5 spinal nerve transection.Repeated treat-ment of Eb1.6 could produce better analgesic effect,which might be partly attribute to the inhibition of spinal IL-βlevels and astrocytes activation.