AIM: To establish a new sensitive microplate-based method to determine alpha-glucosidase inhibiting activity and provide a reliable high-throughput way for screening alpha-glucosidase inhibitors in vitro.METHODS: The fitting combination of enzyme and substrate in a certain reaction was tested.Acarbose,the most popular alpha-glucosidase inhibitor in clinical use was used to validate the established method.Calibration curve,wavelength fidelity and kinetic analysis,together with the effect of altered incubation time,temperature and pH were then studied.RESULTS:The details of assay procedure and evaluation of factors affecting the measurement are described.As little as 160 μl assay system was performed in a 96-well plate.The optimal action was finally achieved by incubated at 37 ℃,pH 7.0 for 15 min and measured at 400 nm.Results from the validation exercises by Acarbose strongly demonstrated the accuracy and reliability of the proposed approach.CONCLUSION: This method reported in the current paper makes it possible to rapidly examine large numbers of samples for the presence of alpha-glucosidase inhibitors in very small sample volumes.Such action may help to pace the development of potential oral medications from natural products protecting patients against postprandial hyperglycemic toxicity and therefore treating diabetes mellitus and the related complications.

Objective To investigate effect of continuous veno-venous hemofiltration on treatment of children with acute re-spiratory distress syndrome .Methods A total of 84 cases of children with acute respiratory distress syndrome were divided into control group (n= 42) and observation group (n = 42) .Control group was given conventional therapy including etiological treat-ment ,mechanical ventilation ,fluid management and anti-inflammatory treatment .Besides conventional therapy similar to control group ,observation group was administrated continuous veno-venous hemofiltration .Gender ,age ,admission critical illness score (PICS) ,clinical manifestations ,blood gas indexes (PaO2 ,SaO2 ) and oxygenation index (OI = PaO2 /FiO2 ) before and after treat-ment ,duration of mechanical ventilation ,pediatric intensive care unit (ICU) hospitalization days and mortality in above two groups were analyzed retrospectively .Results There were not significant difference in gender ,age and admission critical illness score (PICS) of two groups(P> 0 .05) .PaO2 ,SaO2 and OI of observation group were higher than that of control group in the third and fifth day after treatment (P < 0 .05) .The average ICU hospitalization day of observation group was shorter than that of control group (P< 0 .05) .Mortality of observation group was lowered compared with control group (P< 0 .05) .Conclusion Veno-venous hemofiltration could effectively reduce duration of mechanical ventilation and mortality in treatment of children with acute respirato -ry distress syndrome ,which is worth clinical popularization .

Objective To study the local irritation of repeated intrathecal injection of Ziconotide Acetate,and to provide reference for irritancy evaluation ofintrathecal injection.Methods Sixteen New Zealand rabbits were assigned into two groupsat random:Control group and Ziconotide Acetate group,eight animals each group.Totally 50 μL saline or Ziconotide Acetate (100 μg/mL) were administrated by repeated lumbar intrathecal injection once daily for 7 d.Animal behavior was observed every day,and four animals in each group were sacrificed 2 d later after the last injection,the lumbar spinal cord was removed for histopathological examination and irritancy evaluation.The remaining animals were sacrificed for initancy evaluation 14 d later after the last injection.Results Only one animal died after anesthesia on day three in saline group,while no obvious adverse reactions were observed in other rabbits during the entire study,and no intrathecal irritant reactions of histopathological examination were found in both groups.The reversible minor mechanical damage was observed at the injection point,2 d after the last administration.Conclusion For 7 d repeated lumbar intrathecal injection in rabbits,no intrathecal irritant reactions observed in Ziconotide group,and the New Zealand rabbit could be used as a local irritation evaluation model.

Objective To evaluate the feasibility of using pure laparoscopic radical nephrectomy and thrombectomy to treat renal tumor with renal vein and vena caval thrombus. Methods Two ca-ses o{ right renal tumor with renal vein and vena caval thrombus were reported. Contrast-enchanced CT showed renal tumor extended into renal vein and vena cava in 1 case, and filling defect was found in right renal vein and extended to vena cava in the other. Both patients received pure laparoscopic ra-dical nephrectomy and thrombectomy through retroperitoneal approach. Four trocars were placed du-ring the operation, and the renal artery was dissected before the vena cava was mobilized circumferen-tially above and below the renal vein, a faparoscopic vessel blockage clamp was used to partly occlude the vena cava containing the thrombus. The vena cava was repaired after the intact tumor thrombus was extracted. Results The tumor thrombus extended 0.3 cm and 1.0 cm above the renal vein, re-spectively. Both patients were discharged 5 d after operation. Pathological examinations showed that tumors were epithelioid renal angiomyolipoma and grade Ⅰ-Ⅱ clear cell carcinoma separately. Both patients were free of local recurrence and metastasis 5 months after operation. Conclusion Pure la- paroscopic radical nephrectomy and thrombectomy for renal tumor with vena caval and renal vein thrombus is feasible in carefully selected patients.

Objective:To express fusion protein of mouse thymic stromal lymphopoietin (TSLP) and HIV-1 gp120BAL V1/V2 subdomain in 293F cell.Methods:Full length of the V1V2 sequence from BAL isolate was fused with the C-terminus of mouse thymic stromal lymphopoietin (TSLP) and sub-cloned into pCEP-Pu vector to construct the eukaryotic expression plasmid-pCTV1V2BAL.The recombinant plasmid was confirmed by enzyme digestion and sequencing , then transfected into 293 F cells using PEI as a transfection reagent .The fusion protein was purified by metal chelate affinity chromatography and characterized by SDS -PAGE and Western blot . The epitopes of V1/V2 in fusion protein were identified by ELISA .Results:The SDS-PAGE and Western blot results showed that there were highly heterogeneous glycoprotein bands at the site between 35 kD and 55 kD, which reacted with anti-mTSLP rabbit polyclonal antibody and anti-His tag mouse monoclonal antibody .The ELISA analysis showed that antibodies to V 1/V2BAL existed in the sera of HIV-1 positive patients.Conclusion:The mTSLP-V1/V2 fusion protein was successfully expressed in 293F cells, which may be useful for HIV-1 vaccine research .

OBJECTIVE:To establish the quality standard of Smilacina japonica. METHODS: S. japonica was identified qualitatively in respects of original plant morphology,character,microscopic identification,TLC,etc. The moisture,ash and extract of medicinal materials were determined. The content of(25S)-17α-hydroxy-5α-spirostane-9-ene-3β-O-β-D-glucopyranoyl-(1→2)-[ β-D-xylanosyl(1→3)]-β-D-glucopyranoyl(1→4)-β-D-galactopyranoside(SJ-13) was determined by HPLC. The determination was performed on Waters SunFire C18column with mobile phase consisted of acetonitrile-water(35∶65,V/V)at the flow rate of 1.0 mL/min. The detection wavelength was set at 203 nm and the column temperature was 20 ℃. The sample size was 10 μ L. RESULTS:The original plant was perennial herbal,with height of 30-60 cm. The surface of the medicinal material was brown to brownish brown,with wrinkle and 1 row cells in epidermis. The powder of medicinal material was grayish yellow and contained large amount of acicular crystal. TLC spots were clear and well-separated. The content of moisture was 5.26%-8.88%;the content of total ash was 4.60%-28.86%;the acid-insoluble ash was 1.56%-23.39%,water extract was 23.84%-51.26% and alcohol extract was 22.65%-57.36%. The linear range of SJ-13 were 8.92-31.22 μg(r＝0.999 9). RSDs of precision,stability and reproducibility tests were all lower than 1%. The average recoveries were 97.0%-99.2%(RSD＝0.8%,n＝6). RSD of durability test was lower than 1%. The content of SJ-13 was 4.40-29.80 mg/g in 10 batches of medicinal samples. CONCLUSIONS:The content of water, total ash,acid insoluble components should not exceed 11%,35%,28%;the content of water extract,alcohol extract and SJ-13 should not be less than 19%,18% and 4.40 mg/g,respectively. Established standard can be used for the quality control of S. japonica.

Objective:To investigate the circadian rhythm of blood pressure and morning blood pressure surge (MBPS) in children with neurally mediated syncope (NMS).Methods:From July 2018 to June 2019, 135 cases [aged 3-16 years old (10.12±2.53) years old, with 74 males and 61 females] with unexplained syncope, presyncope, and symptoms such as headache, dizziness, chest pain, and chest tightness were collected in the Second Xiangya Hospital, Central South University for the first time.The 24 hour ambulatory blood pressure monitoring (24 h ABPM) was completed on the same day of the head-up tilt test (HUTT). Patients were divided into HUTT negative and positive groups, and dippers and non-dippers groups. MBPS (sleep-trough surge) was calculated and compared respectively.Results:(1) There were 51 patients in the HUTT positive group, including 27 patients with vasovagal syncope, 23 patients with postural orthostatic tachycardia syndrome, and 1 patient with orthostatic hypotension. In HUTT positive group, there were 22 cases (43.14%) of dippers and 29 cases of non-dippers.There were 84 patients in the HUTT negative group, there were 32 cases (38.10%) of dippers and 52 cases of non-dippers. There were no statistical significances in the dipper proportion between HUTT positive and negative group ( χ2=1.305, P>0.05). (2) Sleep-trough systolic blood pressure (SBP) surge was 1-45 mmHg [(15.97±8.03) mmHg](1 mmHg=0.133 kPa), and sleep-trough diastolic blood pressure (DBP) surge was -6-43 mmHg[(14.05±7.97) mmHg]. There were no statistical significances in sleep-trough surge between the HUTT negative and positive group (all P>0.05). (3) The age in the dipper group was higher than that in the non-dipper group [(10.72±2.20) years old vs. (9.72±2.66) years old, t=2.288, P<0.05]. The daytime average SBP [(110.20±8.33) mmHg vs.(105.54±7.51) mmHg, t=3.381, P<0.01], and morning peak SBP [(109.99±10.19) mmHg vs.(106.63±8.71) mmHg, t=2.045, P<0.05] of the dipper group were higher than those of the non-dipper group.The nighttime average SBP[(95.41±7.50) mmHg vs.(98.59±6.88) mmHg, t=2.540, P<0.01], nighttime average DBP[(48.61±4.52) mmHg vs.(52.28±4.65) mmHg, t=4.547, P<0.01], nocturnal minimum SBP[(89.62±8.18) mmHg vs.(93.60±7.38) mmHg, t=2.940, P<0.01], and nocturnal minimum DBP[(44.99±5.32) mmHg vs.(49.01±5.54) mmHg, t=4.205, P<0.01] of the dipper group were lower than that of the non-dipper group.Nocturnal SBP reduction rate [(13.42±2.68)% vs.(6.48±2.49)%, t=15.384, P<0.01], nocturnal DBP reduction rate[(19.98±4.92)% vs.(12.46±5.05)%, t=8.561, P<0.01], sleep-trough SBP surge[(20.37±8.30) mmHg vs.(13.03±6.36) mmHg, t=5.800, P<0.01], and sleep-trough DBP surge[(16.91±8.06) mmHg vs.(12.13±7.36) mmHg, t=3.554, P<0.01] of the dipper group were higher than those of the non-dipper group. Conclusions:Nocturnal blood pressure reduction and sleep-trough surge decreased in NMS children, and there is a circadian rhythm disorder of blood pressure.

Background: Classical swine fever virus (CSFV), the causative agent of classical swine fever (CFS), is a highly contagious disease that poses a serious threat to Chinese pig populations. Objectives: Many provinces of China, such as Shandong, Henan, Hebei, Heilongjiang, and Liaoning provinces, have reported epidemics of CSFV, while the references to the epidemic of CSFV in Yunnan province are rare. This study examined the epidemic characteristics of the CSFV in Yunnan province. Methods: In this study, 326 tissue samples were collected from different regions in Yunnan province from 2015 to 2021. A reverse transcription-polymerase chain reaction (RT-PCR), sequences analysis, and phylogenetic analysis were performed for the pathogenic detection and analysis of these 326 clinical specimens. Results: Approximately 3.37% (11/326) of specimens tested positive for the CSFV by RTPCR, which is lower than that of other regions of China. Sequence analysis of the partial E2 sequences of eleven CSFV strains showed that they shared 89.0–100.0% nucleotide (nt) and 95.0–100.0% amino acid (aa) homology, respectively. Phylogenetic analysis showed that these novel isolates belonged to the subgenotypes 2.1c and 2.1d, with subgenotype 2.1c being predominant. Conclusions The CSFV was sporadic in China’s Yunnan province from 2015 to 2021. Both 2.1c and 2.1d subgenotypes were found in this region, but 2.1c was dominant.

Objective:To provide a new morning check method for the output dose stability of the multileaf collimator (MLC) of the CyberKnife M6 (CK-M6) system.Methods:The CT images of a verification phantom with a size of 20 cm × 20 cm × 10 cm were transmitted into the Precision Treatment Plan ning System (ver. 1.1.1.1). The high-precision alignment between the accelerator output front and the fixed position of the phantom surface was achieved using the fiducial tracking method. A 10 cm × 10 cm radiation field was formed by the MLC and a DailyCheck plan with an output of 200 MU was designed. The repeatability, sensitivity, and accuracy of the DailyCheck plan were measured, and the CK-M6 system was continuously tested for one month using the artificial fixed method and the DailyCheck plan designed in this study. Results:The average and the standard deviation of 10 repeated measurements by the DailyCheck plan were 492.28 pC and 0.09, respectively, indicating good stability. There was a linear correlation between the measured values and the output dose, with a correlation coefficient of R2 > 0.999. Moreover, there was a position deviation of 2 mm between the phantom and the accelerator output front, and the result ant effect on the measured values was equivalent to a dose deviation caused by an output of 1.24 MU. The result from the continuous measurement of both the artificial fixed method and the DailyCheck plan fell within permissible limits, showing high consistency. Conclusions:The DailyCheck plan established through the fiducial tracking of a verification phantom can achieve the convenient, quick, and accurate daily detection of the output dose stability of the MLC of CK-M6. Therefore, this method can be widely applied in the clinical quality control of the CK-M6 system.

Objective To identify the influencing factors of operation time of hand-assisted laparoscopic living donor nephrectomy, and to analyze the relationship between influencing factors and the severity of postoperative complications. Methods Clinical data of 91 donors who underwent hand-assisted laparoscopic nephrectomy were retrospectively analyzed. The correlation between preoperative baseline data of donors and operation time was analyzed. The relationship between operation time and postoperative complications was assessed and the threshold of operation time was determined. Results Multiple donor renal arteries, thick perirenal and posterior renal fat, metabolic syndrome, high Mayo adhesive probability (MAP) score and Clavien-Dindo score prolonged the operation time. By analyzing the receiver operating characteristic (ROC) curve, we found that when the operation time was ≥138 min, the incidence of postoperative complications of donors was significantly increased (P<0.05). Conclusions For donors with multiple renal arteries, thick perirenal and posterior renal fat, metabolic syndrome and high MAP score and Clavien-Dindo score, experienced surgeons should be selected to make adequate preoperative preparation and pay close attention after surgery, so as to timely detect postoperative complications and reduce the severity of complications, enhance clinical prognosis of the donors.