Red-based recombineering has been widely used in Escherichia coli genome modification through electroporating PCR fragments into electrocompetent cells to replace target sequences. Some mutations in the PCR fragments may be brought into the homologous regions near the target. To solve this problem in markeless gene deletion we developed a novel method characterized with two-step recombination and a donor plasmid. First, generated by PCR a linear DNA cassette which comprises a I-Sec I site-containing marker gene and homologous arms was electroporated into cells for marker-substitution deletion of the target sequence. Second, after a donor plasmid carrying the I-Sec I site-containing fusion homologous arm was chemically transformed into the marker-containing cells, the fusion arms and the marker was simultaneously cleaved by I-Sec I endonuclease and the marker-free deletion was stimulated by double-strand break-mediated intermolecular recombination. Eleven nonessential regions in E. coli DH1 genome were sequentially deleted by our method, resulting in a 10.59% reduced genome size. These precise deletions were also verified by PCR sequencing and genome resequencing. Though no change in the growth rate on the minimal medium, we found the genome-reduced strains have some alteration in the acid resistance and for the synthesis of lycopene.
Chromosomes, Bacterial ; genetics ; DNA ; Endonucleases ; metabolism ; Escherichia coli ; genetics ; Genetic Engineering ; methods ; Recombination, Genetic ; Sequence Deletion

[Objectice] To study the relationship between the livestock trade and schistosomiasis transmission and to provide an evidence for making a strategy of schistosomiasis control in mountainous areas. [Methods] A retrospective survey and analysis was conducted to investigate the prevalence of schistosomiasis in both humans and livestock (cattle, horses, mules, donkeys and pigs), and the number and migration of livestock in Weishan County, Yunnan Province in 1980～ 1991. [Results] A positive correlation was found between the infection rate of residents and the numbers of livestock migration (R=0. 9151, P＜0.01). During 1980 to 1991 the infection rate was increased gradually along with the development of livestock husbandry, especially, from the economic reforms since 1984. In 1984 there was positive correlation in the infection rate both human and livestock (R=0. 8458, P＜0.05). The results show that the infection rates of livestock on sale including cattle, horses, mules, donkeys and pigs are 9.54%, 29.39%, 16.38%, 14.47%, 25.73% and 11.11%, respectively. [Conclusion] The infection rate of human and livestock arises by parallel. The high frequency of livestock trade resulted in serious spreading of the infection source of schistosomiasis. The migration of the infected livestock might be an important factor in transmitting schistosomiasis.

Objective To retrospectively analyze management of chronic lateral ankle instability and evaluate its clinical results. Methods There were 74 patients including 43 males and 31 females at age range of 15-63 years (mean 39 years). All patients had at least half year of disease history, more than twice repeated strains and over six weeks of conservative management. Of all, 41 patients were trea-ted with surgical operations including modified Brostrom in 12 patients, Myerson in eight and Chrisman-Snook in 21. The aasoeiated pathological problems should be treated at the same time, ie, tenedesis for the peroneal tendon subluxations in six patients, Achilles tendon lengthening in nine, lateral shift of cal-caneal osteotomies in eight and clearance and holes-making for the osteochondral lesions in 13. Results Of all, functional instability was alleviated in 21 patients through conservative treatment before surgery. Of 53 patients with either functional or mechanical instability, 10 patients showed symptomatic relief, two refused surgical operation and the remained 41 patients were treated surgically. Thirty-nine patients trea-ted surgically and 28 treated conservatively were followed up for 6-91 months. The Roos Functional Out-come Score was used for validating the results. The average value of the ankle functional outcome was 86.24 and 97.34 respectively following operation and conservative management. Conclusions The full examination is needed to remove any possible pathological factors for the patients with chronic lateral ankle instability. The surgical operation can obtain satisfactory functional recovery of the ankle if the conserva-tive management is failed.

with PsA in Chinese Han population from Shandong Province.

Objective To explore the CT and MR features of malignant peripheral nerve sheath tumor (MPNST) of adults.Methods The CT and MRI findings of 20 patients of MPNST confirmed by pathology were analyzed retrospectively.Results Among 20 cases,the lesions were located in the lower extremities (n=4),shoulder and spine (n=4),abdomen (n=3),chest wall (n=2),mediastinum (n=2),breast (n=1),prostate (n=1),scrotum (n=1),scalp (n=1),left maxillary sinus and orbital bottom (n=1).Nineteen cases appeared as solid masses and 1 case appeared as diffuse lesion.The maximum cross section of the lesions were about 1.41 cm× 1.42 cm-17.10 cm× 18.08 cm.Sixteen cases were ill defined margin and 4 cases were well-defined margin.CT scan showed 14 cases were lower density or isodensity and with patchy inhomogeneous density.Multiple calcification were found in 2 cases and osteolytic destruction were found in 7 cases.Enhanced CT of 10 cases showed solid component and gradually delayed enhancement,while cystic lesion and necrosis were not enhancement.The tortuous arteries were displayed in 7 cases.Seven cases were performed MRI and the lesions appeared as hypo-intensity on T1WI and hypo-intensity on T2WI with obviously high intensity of cystic component.Enhanced MRI of 3 cases showed significantly heterogeneous enhancement,cyst and necrosis had no enhancement.MR dynamic enhancement of 1 case showed time-signal intensity curve was platform type.DWI showed high signal.Conclusion MPNST has certain CT and MR characteristics which are helpful to improve the diagnostic accuracy.

Objective To evaluate the prevalence of extraosseous findings on integrated CT images of routine SPECT/CT bone imaging and its clinical incremental values. Methods A total of 843 patients (470 males, 373 females, age range: 26-92 years) who underwent SPECT/CT bone imaging during May 2013 to December 2015 were enrolled in this retrospective study. A modified C-RADS was used to classify the extraosseous findings to E1, E2, E3 and E4. χ2 test was used for data analysis. Results The CT images in 78.6%(663/843) of patients were normal or with no additional clinical significance (E1 and E2), and those in 21.4%(180/843) of patients might need further assessment (E3 and E4). The rate of E4 extraosseous findings in patients with malignancy was higher than that in patients without malignancy: 9.5%(59/622) vs 5.0%(11/221); χ2=4.352, P<0.05. There was no significant difference of the rate between genders: 8.5%(40/470) in males vs 8.0%(30/373) in females; χ2=0.510, P>0.05. With age increasing, the prevalence of E4 finding increased and the rate was the highest in the patients over 80 years old (125%, 16/128). Seventy patients had E4 findings and chest masses and nodules were the most common, followed by the abdominal or pelvis lymph node enlargements. Conclusions Potentially important extraosseous findings are common on SPECT/CT. Systematic reviewing CT images and communicating the important unexpected findings to clinical physicians could enhance its clinical incremental values.

Objective To investigate the killer cell immunoglobulin-like receptor (KIR) genes, KIR2DS4 and its variant KIR1D for an association with syphilis in the comparison between syphilis patients and unrelated healthy subjects. Methods One hundred and ninety syphilis patients and 192 unrelated healthy subjects were performed to determine the KIR genotypes by PCR-SSP method. The gene frequencies of KIR2DS4 and KIR1D were analyzed for an association with syphilis in the patients and healthy people who belonged to KIR gene haplotype A. Results Of 192 healthy individuals, 187 were identified with a KIR2DS4 gene. And 91 individuals were classified as homozygous haplotype A with the percent of 48.7% (91/187) in 187 KIR2DS4 positive individuals. Of 190 syphilis patients, 181 were identified with a KIR2DS4 gene. And 89 individuals were classified as homozygous haplotype A with the percent of 49.2% (89/181) in 181 KIR2DS4 positive individuals. The frequency of KIR1D/KIR1D in syphilis patients classified as haplotype A was 16.9%, and was significantly higher than that in the control group (6.6%, P=0.032). However, there was no significant difference for the frequencies of KIR2DS4/KIR2DS4 and KIR2DS4/KIR1D between the two groups (P>0.05). Conclusion KIR1D/KIR1D might be associated with syphilis in the comparison between syphilis patients and unrelated healthy controls who were classified as homozygous haplotype A.

To investigate the efficacy and safety of minimal y invasive endovascular techniques treatment of hydronephrosis in pregnancy complicated by infection.
65 cases of pregnancy hydronephrosis coinfected patients with minimal y invasive endovascular treatment. Patients were fol owed up for 3-12 months, compared with 48 patients with the same period of conservative treatment .
Ureteral perforation and other surgery-related complications occurred in 65 patients in the observation group has not occurred. The patient's duration of hospitalization、the recurrence rate of postoperative infection, antibiotic duration of treatment, fever days were significantly lower than the control group(P<0.05).
Compared with conservative treatment, minimal y invasive endovascular technical treatment of gestational hydronephrosis co-infection with faster recovery, shorter hospital stay, can significantly reduce the number of days of fever patients and reduce the number of days of antibiotic use, can be used as conventional means of treatment of gestational hydronephrosis infection combined conventional.

Objective To investigate the changes and the clinical significance of serum concentration of soluble Fas (sFas) and Fas ligand (FasL) in the elderly. Methods Fifty elderly subjects and forty-seven adults were recruited. Serum concentration of sFas and EasL were detected by enzyme-linked immunosorbent assay (ELISA) . Psychosocial stress and coping styles were also evaluated. Results The sFas level of the elderly was significantly higher than that in control group,whereas the EasL level was decreased with ageing (P<0.01). A positive correlation between sFas level and psychosocial stress, and a negative correlation between sFas level and positive coping were also found (all P<0. 05). Conclusions Age-related changes occur in serum sEas and EasL levels.The interaction of sFas/FasL system with psychosocial stress and coping styles seems to play important roles in immunosenescence. Our results also suggest that during aging a subtle balance between sFas and FasL could exist.

OBJECTIVE:To investigate the effects of low molecular weight heparin calcium and rivaroxaban combined with atorvastatin on related indexes in patients with acute pulmonary embolism(APE). METHODS:The data of 72 APE patients were analyzed retrospectively. According to treatment plan,the patients were divided into group A(21 cases),group B(26 cases)and group C(25 cases). Group A was treated with intramuscular injection of Low molecular heparin calcium injection immediately after admission;2 days later,they were given Warfarin sodium tablets;7 days later,Low molecular heparin calcium injection was stopped while warfarin was still administrated,lasting for 3-6 months. Group B was given Low molecular heparin injection(same usage and dose as group A);2 days later,they were additionally treated with Rivaroxaban tablets;7 days later,Low molecular heparin calcium injection was stopped while rivaroxaban was still administrated,lasting for 3-6 months. Based on the treatment in group B,group C was treated with Atorvastatin calcium tablets 20 mg orally,once a day in the evening,lasting for 3-6 months. The time of dyspnea,chest pain and cyanosis disappearance were observed in 3 groups as well as the levels of HR,pa(O2),pa(CO2), CRP,D-dimer,TNF-α,IL-1,IL-6,ET-1 and NO before and after treatment. The occurrence of clinical outcome events and ADR were also observed. RESULTS:The time of dyspnea,chest pain and cyanosis disappearance in group A were longer than group B, and the group B was longer than the group A,with statistical significance(P<0.05). Before treatment,there was no statistical sig-nificance in the levels of HR,pa(O2),pa(CO2),CRP,IL-1,IL-6,TNF-α,D-D,ET-1 and NO among 3 groups(P>0.05). After treatment ,HR of 3 groups were all lower than before ,and they showed group Agroup B>group C,with statistical signif-icance(P<0.05). After treatment,CRP,IL-1,IL-6,TNF-α and D-D levels in 3 groups were significantly lower than before,and they showed group C>group A and group B,with statistical significance (P<0.05);but there was no statistical significance be-tween group A and B(P>0.05). ET-1 levels in 3 groups were significantly lower than before,and the levels showed group group group B>group A,with statistical significance (P<0.05). The incidence of clinical outcome events in group A was significantly higher than group C, the incidence of ADR in group B was significantly lower than that group A,with statistical significance(P<0.05). CONCLUSIONS:Low molecular weight heparin calcium and rivaroxaban combined with atorvastatin can significantly im-prove clinical symptoms and blood gas indexes,relieve vascular endothelial damage,reduce the levels of inflammatory cytokines and improve the prognosis of patients with APE,without increasing the incidence of ADR.