Optimization of the fermentation condition for human apolipoproteinA-I expression in recombinant Escherichia coli was investigated. The recombinant plasmid pBV220-ApoA-I was transformed respectively into different E.coli hosts such as JM109, BL21(DE3),DH5?, BMH7118,and TG1. The best host E.coli was DH5? in which the recombinant ApoA-I expression percentage was 21.2% corresponding to that in BL21(DE3) in flask shaker cultivation,while the ApoA-I expressed percentage in E.coli TG1 was 11%.Fed-batch cultivation was performed in FMG-5L fermentor,the optimum fermentation cultivation conditions were as following :optimum pH value was 7.0 in growth phase and 7.4 in the expression phase. The initial glucose concentration in batch phase was 3 g?L -1.The optimum C/N ratio was 2∶1.The recombinant ApoA-I reached about 40% of the total protein, and concentration of ApoA-I was 2.86 g?L -1.

The temperature effect on the recombinant protein production formation was investigated in present study. The culture temperature of growth phase is 30℃, and the culture temperature of induction phase was arranged according to three modes. Hign cell-density and high expression culture of E.coli to product recombinant human apolipoprotein A-I Milano by two temperature-shifted induction . Two temperature-shifted induction was carried out high density and high expression recombinant human ApoA-1 Milano. The recombinant protein ApoA-I Milano reached 4.8 g?L -1 with the final cell density of OD 600 150. And the two temperature-shifted induction avoided the acetic acid successfully to the influence of the high density and high expression. Two temperature-shifted induction was viable in high density culture and high expression of heterogenous protein in recombination E.coli.The sduty provides a basic work for production of recombinant ApoA-I Milano in scale.

OBJECTIVETo study the short- and long-term results for acute deep vein thrombosis (DVT) of the lower extremity treated by surgical thrombectomy and interventional thrombectomy.

METHODSOne hundred and thirty-six acute DVT cases treated by surgical thrombectomy or interventional thrombectomy from March 2000 to August 2008 were reviewed. There were 80 patients treated by surgical thrombectomy. Among them, 30 cases were male, 50 cases were female, aged from 26 to 81 years with a mean of (58 ± 14) years. The other 56 cases were treated by interventional thrombectomy. Among them, 25 cases were male, 31 cases were female, aged from 22 to 92 years with a mean of (57 ± 17) years. All the 136 patients received district anticoagulation with heparin and thrombolysis with urokinase after operation.

RESULTSAfter operation, the circumference difference between bilateral thigh in intervention group were less than that in surgical group [(0.8 ± 1.3) cm vs. (1.5 ± 1.7) cm, P = 0.002]. The circumference difference of bilateral calf had no significant difference [(0.7 ± 1.1) cm vs. (1.0 ± 1.1) cm, P = 0.152]. The average hospital stay in intervention group was shorter than that in surgical group [(7 ± 4) d vs. (15 ± 7) d, P = 0.000]. The morbidity of complications in intervention group was less than that in surgical group (8.9% vs. 32.5%, P = 0.000). One hundred and eight patients were followed up, who was 79.4% of the total patients, and were followed up for an average of (46 ± 29) months. The circumference difference of thigh and calf, the symptom grade, the pigmentation, varicose veins, intermittent claudication and ulceration between the two groups had no significant difference (P > 0.05).

CONCLUSIONCompared with surgical group, intervention group has a better short-term effect, shorter hospital stays, less complications and similar long-term result.

Acute Disease ; Adult ; Aged ; Aged, 80 and over ; Female ; Follow-Up Studies ; Humans ; Lower Extremity ; blood supply ; Male ; Middle Aged ; Retrospective Studies ; Thrombectomy ; methods ; Venous Thrombosis ; surgery ; therapy ; Young Adult

AIMTo study the effects of hydrocortisone sodium succinate on sodium current in human atrial myocytes and in guinea pig ventricular myocytes.

METHODSSingle cardiac myocytes were isolated by enzyme. The effects of hydrocortisone sodium succinate on sodium current (INa) were assessed by applying whole-cell patch clamp techniques.

RESULTSHydrocortisone sodium succinate (1, 3, 10 micromol x L(-1)) was shown to inhibit INa of both human atrial myocytes and guinea pig ventricular myocytes in concentration dependent manner and the IC50 were 6.97 and 8.74 micromol x L(-1), respectively. The inhibition effects acted quickly (1-3 min) and the maximal activating voltage of INa was not changed in both human and guinea pig cardiac myocytes.

CONCLUSIONHydrocortisone sodium succinate can exhibit inhibitory effects on INa in both human and guinea pig cardiac myocytes, and its inhibitory effects act rapidly, which are not consistent with genomic effects, so there may be nongenomic effects.

Adolescent ; Adult ; Animals ; Cell Separation ; Child ; Child, Preschool ; Guinea Pigs ; Heart Atria ; pathology ; Heart Defects, Congenital ; pathology ; Heart Ventricles ; cytology ; Humans ; Hydrocortisone ; analogs & derivatives ; pharmacology ; Myocytes, Cardiac ; drug effects ; physiology ; Patch-Clamp Techniques ; Sodium Channels ; drug effects

Apolipoprotein AI (apo AI), the major protein component of human high-density lipoprotein (HDL), is a single-chain polypeptide of 243 amino acids. Several epidemiological studies have shown that the plasma concentrations of HDL has the role of reverse cholesterol transport (RCT) and inversely correlated with the incidence of coronary artery disease. Because apo AI lacks post-translational modifications, it is convenient to express human apo AI in Escherichia coli expression system. However, there is a poor stability of the mRNA and the apo AI protein in E. coli, it is difficult to express mature apo AI in recombinant bacteria, moreover, even as a fusion protein, apo AI is still sensitive to degradation and can not be cleaved efficiently from the fusion tags. In contrast, proapolipoprotein AI (proapo AI, having an additional polypeptide containing the amino acids Arg-His-Phe-Trp-Gln-Gln at the amino-teminal of the mature protein) proved stable and undegraded in Escherichia coli, and therefore, in this research, an expression system of E. coli including a plasmid of P(R)P(L) tandem promoter was adapted to produce proapo AI. Furthermore, site-directed mutagenesis of the proapo AI cDNA was performed to generate a Clu8Asp mutation in the amino-terminal sequence of proapo AI which created an acid labile Asp-Pro peptide bond between amino acid 8 and 9, and permitted specific chemical cleavage to remove pro-peptide. After inducing with a shift of temperature, yields of recombinant proapo AI achieved about 40% of total cell protein and the recombinant proapo AI expressed proved as a form of inclusion body in cells, so protein need to renature. First of all, the protein was dissolved in buffer with denaturant, and renaturation was carried out on a hydrophobic interaction column (Phenyl Sepharose), ion-exchange chromatography and gel-filtration chromatography were then used to further purify the protein. The purified recombinant apo AI was detected by a set of tests including Western-blotting, Circular dichroism spectra and lipid-binding test, the results shown that recombinant apo AI has similar structural and lipid-binding properties identical to those of native plasma apo AI, which facilitates further research and application.
Apolipoprotein A-I ; biosynthesis ; genetics ; Chromatography, Ion Exchange ; methods ; Escherichia coli ; genetics ; metabolism ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Protein Precursors ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

Objective To evaluate the effects of antiretroviral therapy(ART)for the prevention of mother-to-child transmission(PMTCT)of acquired immune deficiency syndrome(AIDS)on the growth and development of 18-month-old children born by human immunodeficiency virus(HIV)-positive pregnant women in Lingshan County,Guangxi Zhuang Autonomous Region,and provide scientific evidence for improving the ART medication plan for PMTCT.Methods Lingshan County,ranking the first in the HIV-epidemic counties of Guangxi,was selected as the research site.According to the design of retrospective case-control study,we assigned all the subjects into the case group and the control group:(1)The case group included the HIV-positive pregnant women who had received ART for PMTCT and their HIV-negative infants in Lingshan County from 2010 to 2017.The historical cards and PMTCT data of them were collected from the national PMTCT database.(2)The control group included the healthy pregnant women and their healthy babies born in the Lingshan Maternity and Infant Hospital in 2017,and the children's growth and development data were collected.The stunted growth in children was defined as at least one of the three main indicators of body height,body weight,and head circumference below the normal range.Results The number of HIV-positive mothers and their infants in the case group was 391 and 368,respectively,and 87.21%(341/391)and 95.38%(351/368)of mothers and infants respectively received ART medication.The HIV positive rate,mortality rate,and mother-to-child transmission rate of 18-month-old children were 1.36%(5/368),4.35%(16/368),and 2.01%(5/249),respectively.The incidence of stunted growth of 18-month-old children in the case group and the control group was 42.12%(155/368)and 23.06%(101/438),respectively,with significant difference(
Case-Control Studies ; China/epidemiology* ; Female ; Growth and Development ; HIV ; HIV Infections/prevention & control* ; Humans ; Infant ; Infectious Disease Transmission, Vertical/prevention & control* ; Mothers ; Pregnancy ; Pregnancy Complications, Infectious/drug therapy* ; Retrospective Studies

OBJECTIVE: To evaluate the effectiveness and safety of Rotarex catheter system in treating femoropopliteal artery stenosis accompanied with thrombosis. METHODS: From Jun. 2017 to Dec. 2019, the clinical data of 32 femoropopliteal artery stenosis accompanied with thrombosis cases treated with Rotarex catheter system were retrospectively analyzed. There were 23 males and 9 females aged from 50 to 89 years and the mean age was (70.7±10.3) years. Six cases had acute course of disease (≤2 weeks), 17 cases had subacute course of disease (>2 weeks, ≤3 months), and 9 cases had chronic course of disease (>3 months). Mean lesion length was (23.4±13.7) cm, mean occlusion length was (19.9±13.3) cm, and in-stent occlusion 7 cases. The superficial femoral artery (SFA) was involved in 13 cases, the popliteal artery (PA) was involved in 8 cases, and both SFA and PA were involved in the other 11 cases. All the cases were treated with Rotarex catheter system. When necessary, suction with large lumen catheter was enabled. Residual stenosis was treated with percutaneous transluminal angioplasty (PTA). Drug-coated balloon (DCB) was only used in patients with financial status, and stent was used only when it was necessary. Heparin was used for 24 h after procedures, and after that, antiplatelet agents were used. Doppler ultrasonography was taken during the followed-up. RESULTS: Technical success was 100%, and mean procedure time was (107.4±21.5) min. 8F (1F≈0.33 mm) and 6F Rotarex catheter were used in 27 and 5 cases respectively. In 27 cases, forward flow was obtained immediately after debulking with Rotarex catheter, and in the other 5 cases, suction with large lumen catheters were used. PTA was used in all 32 cases. DCB were used in 8 cases, of which 4 were used in in-stent stenosis. Twelve cases were implanted stents. There were no perioperative deaths. The only one procedure related complication was distal embolism. We took out the thrombus with guiding catheter. In all cases, mean hospital stay were (4.6±1.5) d. The ankle brachial index increased from 0.32±0.15 to 0.86±0.10 after treatment (t=-16.847, P < 0.001). The Rutherford stages decreased significantly (Z=-4.518, P < 0.001). All the patients were followed up for 6.0-36.0 months, and the median time was 16.0 months. 2 cases stopped antiplatelet agents, which resulted in acute thrombosis. Another percutaneous mechanical thrombectomy and PTA were taken in one of them. Two cases died of cardiovascular disease during the follow-up, and no amputation was observed. Target lesion restenosis occurred in 7 cases during the follow-up, and target lesion revascularization (TLR) was taken in two of them. CONCLUSION In treating femoropopliteal artery stenosis accompanied with thrombosis, Rotarex catheter can remove thrombus effectively, and that can expose underlying lesions and reduce stent use and complications rates. It is a safe and effective method.
Male ; Female ; Humans ; Middle Aged ; Aged ; Aged, 80 and over ; Femoral Artery/surgery* ; Retrospective Studies ; Constriction, Pathologic ; Platelet Aggregation Inhibitors ; Treatment Outcome ; Thrombosis ; Catheters

OBJECTIVE: To evaluate the role of Rotarex mechanical thrombectomy system in treating instent restenosis of peripheral artery disease (PAD). METHODS: The clinical data of 7 in-stent restenosis (ISR) cases of lower extremity PAD from June 2017 to Dec 2018 were retrospectively analyzed. There were 5 males and 2 females and the mean age was (70.0±7.6) years from 59.0 to 76.0 years. All the cases were treated by Rotarex mechanical thrombectomy system. In the 7 cases, time interval from the previous stent implantation to ischemia recurrence was 1.0 to 72.0 months, and the median time was 6.0 months. The period from ischemia recurrence to endovascular therapy was 3 days to 2 years, and the median time was 62 days. Rotarex mechanical debulking catheter and percutaneous transluminal angioplasty (PTA) were used in all the cases, and the stent was used only when it was necessary. Anticoagulation was used for 24 hours after procedures and then antiplatelet agents were used as usual. Doppler ultrasonography was taken during the followed-up. RESULTS: All the 7 cases were successful in technology, 3 of which were implanted with new stents for the fracture of the old ones. while for the other four cases, no new stent was implanted. The ankle-brachial index (ABI) increased from 0.31±0.08 to 0.86±0.08 after treatment (t=-12.84, P < 0.001). Thrombectomy was applied urgently in one case because of acute thrombosis in the stent, and the result was good. There was no other complications in hospital. All the patients were followed up for 5.0-22.0 months, and the median time was 14.0 months. No death and amputation occurred during the follow-up. One patient stopped antiplatelet agents because of gastrointestinal bleeding, which resulted in acute thrombosis. in-stent restenosis reappeared in 3 cases. CONCLUSION Debulking using Rotarex catheter is safe and effective in treating in-stent restenosis of PAD, especially in reducing stents implantation, but is not good at dealing with old thrombus and proliferating intima, and can do nothing about fractured stents and hyperplasia of intima, so it needs to be combined with stents and drug coated balloons.
Aged ; Arteriosclerosis Obliterans/surgery* ; Coronary Restenosis ; Female ; Femoral Artery ; Humans ; Lower Extremity ; Male ; Middle Aged ; Recurrence ; Retrospective Studies ; Stents ; Thrombectomy ; Treatment Outcome

OBJECTIVE: To evaluate the effectiveness and safety of Rotarex mechanical thrombectomy system in treating acute lower limb ischemia. METHODS: From December 2017 to December 2019, the clinical data of 23 acute lower limb ischemia cases treated with Rotarex mechanical thrombectomy system were retrospectively analyzed. There were 14 males and 9 females from 53- to 84-year-old patients and the mean age was (69.1±9.1) years. Duration of symptoms was 6 hours to 14 days (median time 7 days). In the study, 8 acute thromboembolism cases and 15 acute thrombosis cases were included (In which, there was one thromboangiitis obliterans case and two in-stent restenosis cases). In 5 cases, the lesions were located above the groin; in 16 cases, the lesions were located below the groin, and in the other 2 cases, the lesions were located both above and below the groin. All the cases were treated with Rotarex mechanical thrombectomy system. When residual stenosis was greater than 50%, percutaneous transluminal angioplasty (PTA) was used, and stent was used only when it was necessary. Heparin was used 24 h after the procedure, and after that, antiplatelet agents were used in acute thrombosis cases, and oral anti-coagulants were used in acute thromboembolism cases. Doppler ultrasonography was taken during the follow-up. RESULTS: In all the 23 cases, there were 22 successful cases and 1 unsuccessful case, the mean procedure time was (68.2±15.6) min. Percutaneous transluminal angioplasty was used in 18 cases, 7 of which were implanted stents (3 stents were implanted in iliac artery and 4 in superficial femoral artery). There were 3 procedure related complications. The first one was arterial wall injury which resulted in contrast medium extravazation, and in this case, we solved it with prolonged balloon inflation. The second one was distal embolism. We took out the thrombus with guiding catheter. The last one was acute occlusion in a stent, and thrombectomy was applied urgently, and the result was good. Mean hospital stay were (3.6±1.7) days. The ankle brachial index (ABI) increased from 0.25±0.10 to 0.85±0.16 after treatment (t=12.901, P < 0.001). All the patients were followed up for 4.0-28.0 months, and the median time was 12.0 months. One patient stopped antiplatelet agents, which resulted in acute thrombosis 2 months later. Another percutaneous mechanical thrombectomy and PTA were taken. In the failed case, the patient suffered amputation above the knee 3 months later and in another case, the patient died of heart failure 8 months after the procedure. Two target lesion restenosis occurred during the follow-up. Because the patients' symptom was not sever, no procedure was taken. CONCLUSION Percutaneous mechanical thrombectomy using Rotarex catheter is safe and effective in treating acute lower limb ischemia. For one side, it can restore blood flow to the affected limbs quickly, and for the other, it has the characteristics of minimally invasive and good repeatability. So it should be considered that this me-thod can be widely used for acute lower limb ischemia.
Aged ; Aged, 80 and over ; Humans ; Ischemia ; Middle Aged ; Retrospective Studies ; Thrombectomy

Background:The Food and Drug Administration recently announced that the use of morcellation may cause fibroids or pelvic dissemination and metastasis of uterine sarcoma;therefore,the use of morcellation is limited in the USA.A large sample study is necessary to assess the proportion of uterine malignant tumors found in patients with laparoscopic myomectomy.Methods:A national multicenter study was performed in China.From 2002 to 2014,33,723 cases were retrospectively selected.We calculated the prevalence and recorded the clinical characteristics of the patients with malignancy after morcellation application.A total of 62 cases were finally pathologically confirmed as malignant postoperatively.Additionally,the medical records of the 62 patients were analyzed in details.Results:The proportion of postoperative malignancy after morcellation application was 0.18％ (62/33,723) for patients who underwent laparoscopic myomectomy.Nearly 62.9％ (39/62) of patients had demonstrated blood flow signals in the uterine fibroids before surgery.And,23 (37.1％) patients showed rapid growth at the final preoperative ultrasound.With respect to the pathological types,38 (61.3％) patients had detectable endometrial stromal sarcoma,13 (21.0％) had detectable uterine leiomyosarcoma,only 3 (3.2％) had detectable carcinosarcoma,and 5 (8.1％) patients with leiomyoma had an undetermined malignant potential.Conclusions:The proportion of malignancy is low after using morcellation in patients who undergo laparoscopic myomectomy.Patients with fast-growing uterine fibroids and abnormal ultrasonic tumor blood flow should be considered for malignant potential,and morcellation should be avoided.