Fed-batch cultures of recombinant Rchia pastoris were conducted for production of angiostatin. The whole fermentation included a growth phase on glycerol and an expression phase on methanol. When ammonium hydroxide solution was used to adjust pH, the cell growth during the expression phase was inhibited and the highest angiostatin concentration was 9.08 mg/L. Shake-flask cultures were carried out in media containing different quantities of ammonia. The results showed that ammonia had an obvious inhibition effect on the cell growth during the expression phase. Therefore KOH solution was used to adjust pH, and during the expression phase cells were able to grow and the highest angiostatin concentration reached 20 mg/L.

A recombinant strain of Pichia pastoris with a phenotype of Muts was used to produce angiostatin in a 5-L fermentor. The methanol utilization ability of the present strain was weak, which resulted in extremely low growth rate and angiostatin productivity during the expression phase with methanol as the sole carbon source. To enhance the cell density and angiostatin expression level, mixed-carbon-source of glycerol-methanol was used in the expression phase. The methanol concentration was well controlled at 5 g/L by a methanol sensor and control system, and glycerol was continuously fed into the fermentor to achieve a higher cell density. 120 g/L of cells and 39 mg/L of angiostatin were reached at the end of fermentation which lasted 110 h. The mean specific cell growth rate in the expression phase was 0.01 h(-1), and the mean specific angiostatin productivity was 0.006 mg/(g x h). According to the data obtained in several runs of fermentation in which glycerol was fed at different rates, a higher mean specific angiostatin productivity was reached at the mean specific cell growth rate of 0.012 h(-1). To avoid the repression of angiostatin expression caused by residual glycerol and ethanol accumulation due to overfeeding of glycerol, glycerol addition was controlled to produce continuous oscillations in dissolved oxygen, because the change of dissolved oxygen concentration could deliver the information of available carbon source in the fermentation broth. Controlled glycerol feeding also avoided the problem of oxygen limitation brought by high cell density, and thus decreased the cooling requirement of the fermentor. Cell density reached 150 g/L at the end of fermentation, and angiostatin level reached 108 mg/L after an expression period of 96 h when the mean specific growth rate was maintained at 0.012 h(-1) by using the glycerol feeding strategy to result in the oscillations in dissolved oxygen. The mean specific angiostatin productivity was improved to 0.02 mg/(g x h). The apparent cell yield on glycerol and methanol were respectively 0.69 g/g and 0.93 g/g, higher than those in the fermentation without using the feeding strategy with dissolved oxygen as the indicator of metabolism.
Angiostatins ; genetics ; metabolism ; physiology ; Biotechnology ; methods ; Carbon ; metabolism ; Fermentation ; physiology ; Glycerol ; metabolism ; Methanol ; metabolism ; Oxygen ; metabolism ; Pichia ; genetics ; metabolism

Objective To discuss the clinical value of transabdominal sonography after bowl preparation in diagnosis of ileocecal valve syndrome ( IVS) .Methods The ultrasonic features of IVS in 37 cases were summerized and correlated with the follow-up findings after conservative treatment or the pathologic results after operation .Twenty-eight cases were confirmed by follow-up and 9 cases by operative pathology.Results Among the 37 cases of IVS,28 were idiopathic IVS (75.7%,28/37) and 9 were secondary IVS (24.3%, 9/37%).For the secondary cases, the primary diseases included 5 acute appendicitis,2 Meckel diverticulum,1 terminal ileitis and 1 carcinoma of ascending colon .The diagnostic accuracy rate of ultrasound was 89.2%(33/37).Misdiagnosis rate was 10.8%(4/37),including 1 case of idiopathic and 3 cases of secondary IVS .The IVS ultrasonic images coulde be displayed clearly using 7.0-10.0 MHz probes.In fasting examination,three ultrasonic characteristic signss were found in interminal ileum region at the right lower abdomen .And these features were bagel-shaped sign [91.9%(34/37),average size (1.9 ±1.6) cm ×(0.8 ±0.3) cm],short sleevelet-shaped sign [91.9% (34/37,average size (2.1 ± 0.4)cm ×(1.3 ±0.2) cm],and rose-shaped sign [83.8% (31/37),average size (1.4 ±0.2) cm × (1.0 ±0.2) cm].The shapes of some signs were changeable when the probe compressed .In the case of idiopathic IVS ,several pathologic changes could be seen on sonography after intestinal tract filling of oral 20%mannitol,including slight thickened mucosa and submucosa of erminalileum ,enlarged ieocecal valve and the crocodile-mouth sign.Conclusions Transabdominal ultrasonic examination with high frequency probe after bowl preparation plays an important role in diagnosis of IVS .The method is simple and accurate and should be recommended and applied clinically .

Objective To explore the incidence and risk factors of perioperative ischemic stroke in non-cardiac and non-neurosurgical surgeries and its correlation with preoperative risk assessment of cerebrovascular events,so as to guide perioperative risk management.Methods A retrospective study was conducted on 40 patients aged≥18 years who underwent non-cardiac and non-neurosurgical surgeries and experienced perioperative ischemic stroke in the First Affiliated Hospital of Henan University of Science and Technology from January 2015 to January 2022,forming the stroke group.A control group of 160 patients without perioperative ischemic stroke was selected in a 1:4 case-control ratio,matched for gender,age,date of operation,and the surgeon.Clinical data and preoperative risk assessment of cerebrovascular events(including the single or combined application of head CT/MRI,transcranial Doppler ultrasound,carotid ultrasound,and neurological consultation)of the two groups of patients were collected and statistically analyzed.Multiple logistic regression analysis was used to identify risk factors associated with perioperative ischemic stroke.Results The incidence of perioperative ischemic stroke was 0.042%.Multiple logistic analysis results showed that hypertension(OR=7.858,95%CI 2.175-28.388,P=0.002),hyperlipidemia(OR=4.457,95%CI 1.320-15.049,P=0.016),renal insufficiency(OR=8.277,95%CI 1.480-46.282,P=0.016),and intraoperative hypotension(OR=3.862,95%CI 1.211-12.317,P=0.022)were independent risk factors for perioperative ischemic stroke in non-cardiac and non-neurological surgeries;preoperative cerebrovascular risk assessment(OR=0.130,95%CI 0.031-0.542,P=0.005)was a protective factor against it.Conclusions The incidence of perioperative ischemic stroke in non-cardiac and non-neurosurgical surgery is low but has a poor prognosis.Hypertension,hyperlipidemia,renal insufficiency,and postoperative hypotension are risk factors for perioperative ischemic stroke,while preoperative cerebrovascular event risk assessment is beneficial to reducing its incidence.

The aim of this study was to investigate the effects of polarization program on the ability of macrophages to regulate iron metabolism. M1 and M2 macrophages were propagated in vitro from porcine alveolar macrophages 3D4/2 and polarized by cytokines. The 3D4/2 macrophages were treated with 20 ng/mL interferon gamma (IFN-γ) and 10 ng/mL interleukin-4 (IL-4) combined with 10 ng/mL macrophage colony-stimulating factor (M-CSF) to induce polarization to M1 and M2, respectively. After incubation for 24 h, the expression levels of inflammatory factors and iron-metabolism genes were determined using real-time qPCR, Western bot and immunofluorescence. The M1/M2 macrophages culture media supernatant was collected and used to treat porcine intestinal epithelial cells IPEC-J2. The proliferation ability of IPEC-J2 was detected using CCK-8 assay kit. Following exogenous addition of ammonium ferric citrate (FAC) to M1/M2 macrophages, the phagocytic function of macrophages was detected using fluorescein isothiocyanate-dextran (FITC-dextran) and flow cytometry. The results showed that, compared with control, M1 macrophages had higher mRNA levels of iron storage proteins (ferritin heavy and light polypeptide, i.e. FtH and FtL), hepcidin and lipocalin-2, as well as iron content. Moreover, iron enhanced the ability of M1 macrophages to phagocytize FITC-dextran. There was no significant change in these mRNA expression levels in M2 macrophages, but the mRNA expression levels of ferroportin and transferrin receptor were up-regulated. In addition, the conditioned media supernatant from M2 macrophages promoted cell proliferation of IPEC-J2. These findings indicate that M1 macrophages tend to lock iron in the cell and reduce extracellular iron content, thereby inhibiting the proliferation of extracellular bacteria. While M2 macrophages tend to excrete iron, which contributes to the proliferation of surrounding cells and thus promotes tissue repair.
Animals ; Cytokines ; Ferritins ; Iron/metabolism* ; Macrophages/metabolism* ; Macrophages, Alveolar/metabolism* ; Swine

BACKGROUNDThe domestic prevalence of chronic hepatitis B (CHB) in China is 7.18% in 2006, imposing great societal healthcare burdens. Nucleot(s)ide analogues (NUCs) anti-hepatitis B virus (HBV) therapies are widely applied despite the relatively low rate of seroconversion and high risk of drug-resistant mutation. More effective treatments for CHB deserve further explorations. Combined therapy of NUCs plus Chinese herbal medicine (CHM) is widely accepted in China, which is recognized as a prospective alternative approach. The study was primarily designed to confirm the hypothesis that Tiaogan-Yipi Granule (, TGYP) or Tiaogan-Jianpi-Jiedu Granule (, TGJPJD) plus entecavir tablet (ETV) was superior over ETV monotherapy in enhancing HBeAg loss rate.

METHODSThe study was a nationwide, large-scale, multi-center, double-blind, randomized, placebo-controlled trial with a designed duration of 108 weeks. A total of 16 hospitals and 596 eligible Chinese HBeAg positive CHB patients were enrolled from November 2012 to September 2013 and randomly allocated into 2 groups in 1:1 ratio via central randomization system: experimental group (EG) and control group (CG). Subjects in EG received CM formulae (TGYP or TGJPJD, 50 g per dose, twice daily) plus ETV tablet (or ETV placebo) 0.5 mg per day in the first 24 weeks (stage 1), and CHM granule plus ETV tablet (0.5 mg per day) from week 25 to 108 (stage 2). Subjects in CG received CHM Granule placebo plus ETV tablet (0.5 mg per day) for 108 weeks throughout the trial. The assessments of primary outcomes (HBV serum markers and HBV-DNA) were conducted by a third-party College of American Pathologists (CAP) qualified laboratory. Adverse effects were observed in the hospitals of recruitment.

DISCUSSIONThe study was designed to compare the curative effect of CM plus ETV and ETV monotherapy in respect of HBeAg loss, which is recognized by the European Association for the Study of the Liver as "a valuable endpoint". We believe this trial could provide a reliable status for patients' "journey" towards durable responses after treatment discontinuation. The trial was registered before recruitment on Chinese Clinical trial registry (No. ChiCTR-TRC-12002784, Version 1.0, 2015/12/23).