Lysostaphin (Lysn) is an antibacterial metalloendopeptidase that cleaves the pentaglycin bridges in the cell wall of Staphylococci. Although many studies have demonstrated its high activity in vitro, the medical application of Lysn has been hampered by its short half-life in vivo. In order to enhance its stability in vivo without significantly suppressing the enzymatic activity, we designed and tested eight single cysteine substitutions in Lysn for covalent attachment of polyethylene glycol chains (PEGylation). The purified mutants, fully reduced by Dithiothreitol (DTT), were treated with mPEG-MAL(20 kDa). The PEG modification efficiency was above 70% as determined by reverse-phase high-pressure liquid chromatography (HPLC) analysis. The PEG-Lysn proteins were further purified by cation exchange chromatography (MacroCap SP), reaching at least 95% purity. The activities of the PEG-Lysn proteins were determined by the turbidity and minimum inhibitory concentration (MIC) assays. We found that the PEGylated V240C and T244C mutants retained about 50% of the original antibacterial activity of Lysn. Overall, this study will help develop highly stable and active PEG-Lysn to treat systemic S. aureus infections.
Amino Acid Substitution ; Lysostaphin ; chemistry ; Polyethylene Glycols ; chemistry ; Protein Engineering ; Recombinant Proteins ; chemistry ; Staphylococcus aureus

The development and progression of atherosclerosis and thrombosis are closely related to changes of hemodynamics parameters. Ultrasonic pulse wave Doppler technique is normally used for noninvasively blood flow imaging. However, this technique only provides one-dimensional velocity and depends on the angle between the ultrasound beam and the local velocity vector. In this study, ultrasonic particle image velocimetry method was used to assess whole field hemodynamic changes in normal blood vessels. By using the polynomial fitting method, we investigated the velocity gradient and assessed the shear in different blood flow velocity of 10 healthy rats. It was found that using four polynomial fitting could result in optimal measurement results. The results obtained by ultrasonic particle image velocimetry accorded with the results obtained using Doppler technique. The statistical average of cyclical vessel wall shear stress was positively related to the locational mean velocity. It is proven that ultrasonic particle image velocimetry method could be used to assess directly the real-time whole field hemodynamic changes in blood vessels and was non-invasively, and should be a good prosperous technique for monitoring complex blood flow in stenotic ar- teries.
Algorithms ; Animals ; Arteries ; diagnostic imaging ; pathology ; Blood Flow Velocity ; Echocardiography, Doppler ; Hemodynamics ; Models, Cardiovascular ; Rats ; Rheology ; Stress, Mechanical ; Ultrasonics

Objective To explore the characteristics of gait of old hemiplegic patients. Methods 22 old hemiplegic patients were investigated with Motion Analysis. Results The bilateral support phase increased, especially for the affected lateral. The swing phase decreased, as well as the frequency, step length, stride length and walking speed. Their heads flexed forward, but trunks remained upright. The motion of range of shoulder and elbow decreased in the affected lateral (P<0.05), and the angle of flexion and adduction in the affected shoulder and flexion in elbow increases (P<0.05). The motion of range of the affected hip, knee and ankle decreased (P<0.05). Conclusion The walking ability impaired in the old hemiplegic patients, with upper limbs spasticity, vision compensation, etc.

Objective To compare the efficacy and safety of two kinds of homemade sirolimus-eluting stents (Firebird and Excel) for treatment of acute ST segment elevation myocardial infarction (STEMI) in patients who underwent percutaneous coronary intervention (PCI). Methods The 249 consecutive patients with STEMI who underwent PCI were randomly divided into two groups: Excel group (n=136) and Firebird group (n=113). They were followed up for 6-24 months, and coronary angiography was reviewed average 12 months later. The primary endpoints were major adverse cardiac events, including death, reinfarction and target vessel revascularization. The second endpoints included late luminal loss and restenosis 12 months after treatment. Results There were no significant differences in baseline data, coronary arterial lesion before operation, and immediateness condition after PCI between the two groups (all P＞0.05). Within follow-up, there were 2 (1.47%) death cases and 1 (0.88%) death case, 1 (0.74%) and 1 (0.88%) nonfatal myocardial infarction case, 2 (1.47%) and 2 (1.77%) target vessel revascularization cases in the two groups respectively (all P＞0.05). There were no significant differences in late luminal loss of in-stent and in-segment, the rates of in-stent restenosis, in-segment restenosis and stent thrombosis, the in-stent minimal lumen diameter and in-segment minimal lumen diameter between the two groups (all P＞0.05). Conclusions The two kinds of homemade sirolimus-eluting stents may have similar efficacy and safety in patients with STEMI treated with primary PCI.

Objective To determine whether field triage would reduce median contact-to-device ( C2D ) time in patients with ST-segment elevation acute myocardial infarction ( STEMI ) . Methods Consecutive patients with STEMI underwent primary percutaneous coronary intervention( PCI) from March 2010 to February 2014 in Shanghai Pudong Gongli Hospital were analyzed. Patients were divided into two groups. A total of 121 patients were admitted by field triage and 101 patients by non-field triage. The primary study point was C2D time and the study points secondary included ( door-to-balloor, D2B) time, peak Troponin I ( TnI) levels, hospital mortality and 30 days follow-up mortality. Results Baseline and procedural characteristics between the two groups were comparable. Comparing to non-field triage group, the C2D time was reduced [(92. 0 ± 56. 0)min vs. (131. 0 ± 61. 0)min,P﹤0. 01]. The D2B time was lower in the field triage group vs. the non-field triage group [(55. 0 ±26. 0)min vs. (96. 0 ±31. 0)min,P﹤0. 01]. The percentage of patients with C2D time less than 90 minutes increased significantly from 85. 1% to 98. 3%( P﹤0. 01 ) in the field triage group. Peak TnI level was significantly reduced in the field triage group [(23. 5 ±22. 0) μg/L vs. (43. 5 ± 39. 0) μg/L,P﹤0. 01]. In-hospital mortality and 30 days follow-up mortality did not significantly differ between the 2 groups (3. 3% and 3. 0%, P=0. 885;3. 3% and 5. 0%, P=0. 544, respectively). Conclusions In STEMI patients, field triage was associated with significantly reduced C2D and D2B times.

Objective To investigate the effect of arsenic trioxide on expression VEGF of lymphoma cells. Methods The VEGF mRNA expression was analysed by by Real-time PCR, and VEGF protein expression in Raji and Jurkat lymphoma cell lines by ELISA. Results ATO can inhibit lymphoma cells by inducing apoptosis. ATO induced lymphoma cell apoptosis was due to time.With the period of ATO effecting on cells goes, the expression of VEGF mRNA and the protein were down-regulated significantly (after 24, 48, 72 h). There were, the VEGF mRNA △△Ct data of treated with ATO, at 12 h, for Raji: 0.75±0.15, 72 h, Jurkat: 1.67±0.13. After 72 h, Raji: 8.95±0.38; Jurkat: 9.09±0.16 (f =3.54, P <0.01; t =3.65, P <0.01). And about the VEGF protein, at 12 h, Raji: 198.38±4.37; Jurkat: 563.11±3.81. After 72 h, Raji: 23.55±2.06; Jurkat: 57.11 ±3.88 (t =2.48, P <0.05; t =2.59, P <0.05). Conclusion ATO can inhibit the proliferation of lymphoma cells by down-regulating the expression of VEGF mRNA and its protein.

Objective To investigate the effects of basal and early phase insulin secretion on plasma glucose level in type 2 diabetes. Methods Plasma glucose and true insulin levels were measured at 0, 30, 60, 120 min during standard meal test in 81 patients with type 2 diabetes. Insulin sensitivity index (ISI) and insulin secretion index (?I 30 /?G 30 ) were calculated for evaluating the insulin sensitivity. Contributions of basal and early insulin secretion to plasma glucose level were evaluated by multivariate regression analysis with SAS software. Results ISI and ?I 30 /?G 30 showed nearly equal effects on plasma glucose levels by multivariate regression analysis. Among insulin levels of different time points during standard meal test, basal and postprandial 60 min insulin levels played important roles in changes of plasma glucose levels. The effect of fasting insulin on the area under plasma glucose curve was stronger than that of ?I 30 /?G 30 . Conclusion Both basal and early insulin secretions greatly contribute to glycemic control.

Objective To explore the 3D gait analysis of stroke hemiplegic patients. Methods 28 stroke hemiplegic patients were examined with the 3D gait analysis system before and after 6-week rehabilitation. Results After rehabilitation, support phase time of the affected lower limb increased and swing phase time decreased (P<0.05). Step length, stride length and walking speed increased (P<0.05). There was significantly difference in range of motion (ROM) of hip deduction/abduction, flexion/extension, and knee flexion/extension, and ankle internal/external rotation, varus/eversion, dorsiflexion/plantar flexion before and after rehabilitation (P<0.05). Conclusion 3D gait analysis system can evaluate the patient's gait objectively and quantitatively, and provide the basis for rehabilitation assessment.

Objective To investigate the phenotypic and genetic characteristics of the lysostaphin‐resistant Staphylococcus aureus variants induced by recombinant lysostaphin in vitro .Methods Three clinical isolates of S . aureus ,including two resistant to methicillin (MRSA ) and one susceptible to methicillin (MSSA ) were induced by treatment with sub‐MIC of recombinant lysostaphin via one‐step selection in vitro .Susceptibility of the variants to antibiotics were determined and compared with their parental strains .The full length of femABX genes was amplified by polymerase chain reaction and sequenced to identify the potential mutation sites in these genes .The growth‐curve in liquid medium and virulence in a mouse systemic infection model of both parental and variant strains were observed . Results The frequency of lysostaphin resistance in S . aureus was between 10-4 to 10-8 following induction by lysostaphin . Resistance to lysostaphin was associated with a significant decrease in growth rate in vitro and virulence in vivo ,as well as increased susceptibility toβ‐lactams evidenced by the M IC of β‐lactams against the variants as low as 1/4 000 to 1/2 of the M IC against their parental strains . Sequencing of f emA BX genes showed mutation in femA gene in both variants ,which resulted in a premature termination codon .Conclusions Resistance of S . aureus to lysostaphin may develop following induction by recombinant lysostaphin in vitro . The lysostaphin‐resistant S . aureus variants are characteristic of lower growth rate , decreased virulence ,and higher susceptibility to β‐lactams .

OBJECTIVETo evaluate efficacy of continuous blood purification (CBP) in childhood severe sepsis through the analysis of organ function, inflammatory mediators and prognosis.

METHODForty-seven children with severe sepsis aged 29 days -16 years who were treated in PICU of Shanghai and Zhejiang five hospitals during October 1, 2011 and September 30, 2012 were enrolled; 30 cases treated with CBP were recorded as logged group , 17 cases without CBP as unlogged group. Changes in the cardiovascular, respiratory function, renal function, inflammatory markers, PRISM score III, PCIS and survival were observed and compared between the two groups at baseline (d0), first days (d1), second days (d2), third days (d3), fifth days (d5).

RESULT(1) Cardiovascular function: In d3 and d5, heart rate (HR) and mean arterial pressure (MAP) were improved as compared to unlogged group (121, 119 vs. 138, 137; 71, 80 mmHg vs. 63, 62 mmHg, P < 0.05), with no statistical significance in arterial blood lactate concentration. (2) Oxygenation index (PaO₂/FiO₂) and arterial oxygen saturation (SaO₂) increased as compared to unlogged group, but did not reach statistical significance. (3) Blood urea nitrogen (BUN) and creatinine (Cr) were improved as compared with unlogged group from d1 (P < 0.05). (4) Inflammatory mediators did not show significant differences. (5) Twenty-eight days survival rate: logged group was 70.0%, unlogged group was 52.9%, but the difference was not statistically significant (P = 0.242).

CONCLUSIONCBP can improve circulatory function, oxygenation, and renal function in children with severe sepsis. No evidence was found that CBP could decrease the level of inflammatory mediators, improve critical score and 28 days survival rate.

Adolescent ; Blood Pressure ; Blood Urea Nitrogen ; Child ; Child, Preschool ; Creatinine ; blood ; Female ; Heart Rate ; Hemofiltration ; methods ; Humans ; Infant ; Infant, Newborn ; Intensive Care Units, Pediatric ; Male ; Oxygen ; blood ; Oxygen Consumption ; Prognosis ; Prospective Studies ; Sepsis ; physiopathology ; therapy ; Survival Rate ; Treatment Outcome