Effect of hemX gene deletion on heme synthesis in Bacillus amyloliquefaciens / 生物工程学报

Heme, which exists widely in living organisms, is a porphyrin compound with a variety of physiological functions. Bacillus amyloliquefaciens is an important industrial strain with the characteristics of easy cultivation and strong ability for expression and secretion of proteins. In order to screen the optimal starting strain for heme synthesis, the laboratory preserved strains were screened with and without addition of 5-aminolevulinic acid (ALA). There was no significant difference in the heme production of strains BA, BAΔ6 and BAΔ6ΔsigF. However, upon addition of ALA, the heme titer and specific heme production of strain BAΔ6ΔsigF were the highest, reaching 200.77 μmol/L and 615.70 μmol/(L·g DCW), respectively. Subsequently, the hemX gene (encoding the cytochrome assembly protein HemX) of strain BAΔ6ΔsigF was knocked out to explore its role in heme synthesis. It was found that the fermentation broth of the knockout strain turned red, while the growth was not significantly affected. The highest ALA concentration in flask fermentation reached 82.13 mg/L at 12 h, which was slightly higher than that of the control 75.11 mg/L. When ALA was not added, the heme titer and specific heme production were 1.99 times and 1.45 times that of the control, respectively. After adding ALA, the heme titer and specific heme production were 2.08 times and 1.72 times higher than that of the control, respectively. Real-time quantitative fluorescent PCR showed that the expressions of hemA, hemL, hemB, hemC, hemD, and hemQ genes at transcription level were up-regulated. We demonstrated that deletion of hemX gene can improve the production of heme, which may facilitate future development of heme-producing strain.

Retrospective cohort study on subtotal colonic bypass plus colostomy with antiperistaltic cecoproctostomy in the treatment of senile slow transit constipation / 中华胃肠外科杂志

Objective@#To investigate the clinical efficacy of laparoscopic subtotal colonic bypass plus colostomy with antiperistaltic cecoproctostomy (SCBCAC) in the treatment of senile slow transit constipation.@*Methods@#A retrospective cohort study was performed. Clinical data of 30 colonic slow transit constipation patients aged ≥70 years old undergoing laparoscopic SCBCAC from July 2012 to October 2016 (bypass plus colostomy group), and 28 patients undergoing laparoscopic subtotal colonic bypass with antiperistaltic cecoproctostomy (SCBAC) from February 2009 to June 2012 (bypass group) at our institute were collected. Efficacy was compared between the two procedures. Inclusion criteria: (1) meeting the Rome III diagnosis criteria for constipation; (2) confirmed diagnosis of slow transit constipation; (3) age ≥ 70 years old; (4) receiving non-surgical treatment for more than 5 years, and Wexner constipation score > 15; (5) follow-up for more than 2 years. Those with psychiatric symptoms or previous psychiatric history, obvious signs of outlet obstructive constipation, organic diseases of the colon and life-threatening cardiovascular diseases or cancer were excluded. In the bypass plus colostomy group, laparoscopy was performed via five trocars. The ileocecal junction and the ascending colon were mobilized and the ileocecal junction was pulled down to the pelvic inlet. The ascending colon was transected and the appendix was excised. The lateral peritoneum of the sigmoid colon and the rectal mesentery were dissected and the upper rectum was transected. The avil of a circular stapler was placed in the bottom of the cecum. The shaft of the stapler was placed in the rectum via the anal canal to complete end-to-side anastomosis (end rectum to lateral cecum). The end of the rectal-sigmoid colon was used for colostomy via an extraperitoneal approach to complete the operation. The following efficacy indexes were collected before surgery and 3, 6, 12, and 24 months after surgery: the number of daily bowel movements, the Wexner incontinence scale (WIS, 0-20, the lower the better), the Wexner constipation scale (WCS, 0-30, the lower the better), the gastrointestinal quality of life index (GIQLI, 0-144, the higher score, the better), abdominal pain intensity indicated by the numerical rating scale (NRS, 0-10, the lower score, the better), and the abdominal bloating score (ABS, 0-4, the lower score, the better). The complications defined as Clavien-Dindo class II or above were observed and recorded.@*Results@#No significant differences in preoperative WCS, WIS, GIQLI, NRS, and ABS were observed between bypass plus colostomy group and bypass group (all P>0.05). All the patients successfully underwent laparoscopic surgery and no patient in either group experienced postoperative fecal incontinence. WCS and GIQLI were significantly improved (all P<0.001) at 3, 6, 12, and 24 months after surgery in both groups. At 12 months after surgery, the number of bowel movements was significantly less in bypass plus colostomy group than that in bypass group [(2.4±0.7) times vs. (3.4±1.2) times, t=4.048, P<0.001]. At 3, 6, 12 and 24 months after surgery, the improvement of GIQLI in bypass plus colostomy group was significantly better than that in bypass group (all P<0.001). At 24 months after surgery, GIQLI in bypass plus colostomy group and bypass group was 122.3±5.3 and 92.8±16.6, respectively, with a significant difference (t=9.276, P<0.001). At 12 and 24 months after surgery, NRS in bypass plus colostomy group was significantly better than that in bypass group (both P<0.001). At 24 months after surgery, NRS in bypass plus colostomy group was 0.9±0.7, while that in bypass group was 3.7±2.7. There was a significant difference between two groups (t=5.585, P<0.001). At 6, 12 and 24 months after surgery, the improvement of ABS in bypass plus colostomy group was also significantly better than that in bypass group. At 24 months after surgery, ABS in bypass plus colostomy group was 0.6±0.6, while that in bypass group was 2.5±1.0, with a significant difference between two groups (t=8.797, P<0.001). At 1 year after surgery, barium enema examination was performed in all the patients of both groups. The barium emptying time was (21.2±3.8) hours and (95.8±86.2) hours in bypass plus colostomy group and bypass group respectively. The former group was significantly better than the latter group (t=4.740, P<0.001).@*Conclusions@#Laparoscopic SCBCAC is an effective and safe procedure for the treatment of senile slow transit constipation and can significantly improve prognosis. Its clinical efficacy is better than laparoscopic SCBAC.