Extraction, identification and relative quantification of outer membrane vesicles of Klebsiella pneumoniae / 中华检验医学杂志

Objective:Based on the modified ultracentrifugation method, the outer membrane vesicles (OMV) secreted by Klebsiella pneumoniae were rapidly separated, identified and quantified. Methods:Standard strains of classic Klebsiella Pneumoniae (cKP) purchased from the Clinical Laboratory Center of the National Health Commission, and hypervirulent Klebsiella pneumoniae (hvKP) which was donated by Taiwan University were cultured in M9 basal media for 9 hours, and the OMV were extracted by modified ultracentrifugation. The shape and size of OMV were identified by transmission electron microscopy (TEM), relative quantification by Stewart phospholipids analysis method. Two groups were compared using independent samples t test. Results:It was observed under the TEM that most of the OMV secreted by cKP and hvKP showed spherical vesicle structure and a small part were irregular. The diameter of OMV ranged from 20 to 250 nm, multiple vesicles could be seen in clusters. Relative quantification found that the number of OMV secreted by hvKP were more than cKP ( P<0.05). Conclusions:This study successfully achieved the extraction, identification and quantification of OMV from Klebsiella pneumoniae through the modified ultracentrifugation method, which provided a foundation for further study about the function and mechanism of OMV, and also provided new ideas for the treatment of bacteria. Based on the ultracentrifugation method, the OMV secreted by Klebsiella pneumoniae were rapidly separated and extracted, then identified and quantified.

Capillary ultracentrifugation technology in accurate identification of Rh phenotype / 中国输血杂志

【Objective】 To explore the application of capillary ultracentrifugation technology in accurate identification of Rh phenotype and clinical blood transfusion. 【Methods】 132 samples, presenting mixed field agglutination during Rh phenotyping in our laboratory from May 2019 to February 2020, were separated using capillary ultracentrifugation technology, and the proximal and distal red blood cells were taken for Rh phenotype test, and then blood donors with the same Rh phenotype as the proximal cells were selected for cross matching. 【Results】 132 samples were subjected to capillary ultracentrifugation, and new red blood cells were successfully isolated from the proximal side in 128 (96.97%)cases, and the Rh phenotype was accurately identified, i.e. CcDEe in 47 cases (36.72%), CcDee in 12(9.38%). ), ccDEEin 11 (8.59%), CCDee in 52(40.63%), ccDEe in 5 (3.91%), and ccDee in 1(0.78%). 4 cases of mixed field reaction remained after centrifugation, and all of them had a history of blood transfusion within 2 days. For the 128 patients whose Rh phenotype was accurately identified, blood donors with the same type of Rh phenotype were selected, and 4 patients whose Rh phenotype could not be determined, donors with CCDee phenotype were selected based on the frequency of phenotype distribution and the principle of reducing antigen exposure. The cross-matched blood of all patients were consistent. 【Conclusion】 Capillary ultracentrifugation technology can effectively separate the new red blood cells, improve the accurate identification of Rh phenotype, and provide safety guarantee for clinical targeted blood transfusion.

Mechanism of Astragali Radix Vesicle-like Nanoparticles for Reducing Blood Glucose in db/db Diabetic Mice by Regulating Gut Microbiota / 中国实验方剂学杂志

Objective:To study the extraction method and characteristics of vesicle-like nanoparticles （VLNs） in Astragali Radix decoction， and to explore the mechanism of the VLNs in reducing blood glucose by regulating the gut microbiota of db/db diabetic mice. Method:Ultracentrifugation and size exclusion chromatography were used to enrich VLNs from Astragali Radix decoction， and the morphology， particle size and concentration of the VLNs were analyzed by transmission electron microscope and nanoparticle tracking analyzer. The db/db diabetic mice were randomly divided into model group， Astragali Radix VLNs high-， medium- and low-dose （21.1， 10.6， 5.3 g·kg^-1） groups and metformin group （0.25 g·kg^-1） according to their blood glucose levels. There were 7 mice in each group， and another 7 C57BL/6 mice were set as the normal group. The mice were given intragastrically for 3 weeks （once a day）， and the changes of fasting blood glucose were observed every week. Hematoxylin-eosin （HE） staining was used to observe the pathological morphology of liver and pancreas of diabetic mice. The feces of mice were collected for 16S rRNA diversity detection of intestinal microbes. Result:The size of the nanoparticles obtained by the two methods was about 200 nm. Astragali Radix VLNs extracted by ultracentrifugation had a typical saucer-like shape with the concentration of 3.0×10¹¹ particles·mL^-1. The morphology of Astragali Radix VLNs obtained by size exclusion chromatography was relatively poor with the concentration of 2.2×10¹¹ particles·mL^-1. After 3 weeks of administration， compared with the model group， Astragali Radix VLNs high-， medium- and low-dose groups could significantly reduce the fasting blood glucose of diabetic mice （<italic>P</italic><0.05， <italic>P</italic><0.01）. The VLNs could improve the gut microbiota dysbiosis， significantly decrease the ratio of Firmicutes/Bacteroidota， and increase the relative abundance of beneficial bacteria and reduce the relative abundance of harmful bacteria. Conclusion:Astragali Radix VLNs may reduce the blood glucose of db/db diabetic mice by adjusting the ratio of Firmicutes/Bacteroidota in the intestinal flora.

Isolation of Small Extracellular Vesicles From Human Serum Using a Combination of Ultracentrifugation With Polymer-Based Precipitation

Methods for reproducibly isolating and enriching small extracellular vesicles (EVs) from blood are essential for clinical utilization of small EVs in cancer patients. We combined ultracentrifugation (UC) with polymer-based precipitation (ExoQuick [EQ] or Total Exosome Isolation [TEI] kit) to isolate small EVs (diameter, 30–150 nm) from the serum of breast cancer patients. We compared the performance of four cycles of UC (UC4x) with that of two cycles of UC followed by enrichment using the EQ (UC2x→EQ) or TEI (UC2x→TEI) kits. The mean concentration of small EVs isolated from 1 mL of serum using UC2x→EQ (139.0±29.1 µg) and UC2x→TEI (140.4±5.0 µg) did not differ from that obtained using UC4x (141.8±26.9 µg). The mean number of EV particles obtained using UC4x was 29.2±9.9×109 per mL of serum, whereas UC2x→EQ and UC2x→TEI yielded higher numbers of EVs (50.7±17.0×10⁹ and 59.3±20.6×10⁹, respectively). Concentrations of EV microRNAs, including miR-21 and miR-155, did not differ between the three methods. In conclusion, performing UC prior to the use of polymer-based precipitation kits could be feasible for isolating small EVs from human serum in large sample-based translational researches.

Ultracentrifugation, ExoPerfectTM-MU and PEG6000 for the extraction and identification of seminal plasma exosomes: A comparative study / 医学研究生学报

Objective　No studies have been reported on the comparison of ultracentrifugation, ExoPerfectTM-MU and PEG6000 in extracting seminal plasma exosomes. This article aimed to compare the three methods for the extraction and identification of seminal plasma exosomes. Methods　Semen samples were obtained from 30 healthy donors and randomly divided into three portions, followed by extraction of exosomes from the seminal plasma by ultracentrifugation, ExoPerfectTM-MU, and 8%PEG6000, respectively. The size of the extracted exosomes was measured with the nanoparticle tracking analyzer (NTA), their morphology observed under the transmission electron microscope (TEM), and their protein biomarkers detected by Western blot.　Results　Significantly higher expressions of CD63 and TSG101 were found in the exosomes extracted by ultracentrifugation than in those extracted by ExoPerfectTM-MU and 8%PEG6000 (P0.05). Compared with the 8%PEG6000 group, the ultracentrifugation and ExoPerfectTM-MU groups showed significantly higher concentrations ([11.90±1.78] vs [21.20±0.98] and [19.74±1.45]×108/mL, P<0.01) and numbers of seminal plasma exosomes under TEM (4.7±1.7 vs 7.0±1.6 and 6.0±1.6, P< 0.01).　Conclusion　Ultracentrifugation, ExoPerfectTM-MU and 8%PEG6000 are all capable of successful extraction and identification of seminal plasma exosomes, but the former two yield more exosomes, the latter one gives a higher purity, and ExoPerfectTM-MU is simple and convenient in operation.

Determination of serum high- and low-density lipoprotein cholesterol by ultracentrifugation-high performance liquid chromatography--improvement and verification of β-quantification method / 中华检验医学杂志

Objective To propose and validate a reduced volume β-quantification method to measure serum high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C). Methods The reduced volume β-quantification method involved separation of LDL and HDL by ultracentrifugation and preparation of HDL by chemical precipitation. The sampling and reconstitution of the bottom fractions were performed gravimetrically and sample volume was thus decreased from 5 to 0.8 ml. High performance liquid chromatography was used to determine the cholesterol concentration of bottom fractions and HDL-C in the supernatant. Serum levels of LDL-C depended on a calculation of bottom fractions cholesterol minus HDL-C. Results The total CVs for HDL-C and LDL-C were 0.65％ -1.75％ and 0.63％ -1.11％. The results of the developed method were consistent with the current reference method and well within the allowable bias for Cholesterol Reference Method Laboratory Network surveys. Conclusion A new method for the measurement of HDL-C and LDL-C has been established. This method requires a small amount of serum and is easy to operate, exhibiting a desirable precision and accuracy. It is reliable and can be used as a candidate reference method for HDL-C and LDL-C.

Current status of the reference system for measurement of serum high- and low-density lipoprotein cholesterol / 中华检验医学杂志

Decreased serum high-density lipoprotein cholesterol (HDL-C) and increased low-density lipoprotein cholesterol (HDL-C) concentrations are important risk factors for cardiovascular diseases. Achieving accurate and comparable HDL-C and LDL-C results is the primary requirement for the prevention and treatment of cardiovascular diseases, in which reference system including reference method, reference materials and standardization programs are needed. The current reference method for HDL-C and LDL-C measurement is the β-quantification method developed by the United States Center for Disease Control and Prevention (CDC), a multi-step procedure involving ultracentrifugation, chemical precipitation, and cholesterol analysis by CDC reference method. This method uses a large amount of serum and is technically demanding which limited its application in standardization. The reduced volume β-quantification method uses a small volume of serum sample, is simple and high through put and is more applicable. However, both methods are sensitive to serum matrix. Commutability of reference materials should be evaluated when they are used to calibrate routine methods. Fresh (non-frozen) serum samples should be used for a split sample comparison between reference and direct methods. In addition, development of reference measurement procedure based on ultracentrifugation and preparation of commutable reference materials are the important work in lipoprotein standardization.

Comparison of different separation protocols for exosomes derived from urine of patients with aortic dissection / 第二军医大学学报

Objective To explore a suitable separation protocol for exosomes derived from urine of patients with aortic dissection. Methods Urine samples were collected from the patients with aortic dissection. The exosomes were extracted and purified by ultracentrifugation, dialysis-ultrafiltration and precipitation kit methods. The biological morphology of exosomes was observed under transmission electron microscope. The particle size, distribution and concentration of the exosomes were detected by nanoparticle tracking analysis. Results The exosomes extracted with ultracentrifugation method had half cup-like or concave-hemispherical morphology; the particle size was large and well-distributed; the range of the particle size was wide with a mean value of (236.4±46.5) nm; and the concentration was low, being (2.82±0.21)×1012 per 1 mL urine. The exosomes extracted by dialysis-ultrafiltration method had normal morphology and large quantity; the particle size was small and concentrated; the range of the particle size was narrow with a mean value of (128.7±6.3) nm; and the concentration was high, being (2.16±0.15)×1014 per 1 mL urine. There were no exosomes in the extractive by precipitation kit method under transmission electron microscope. Conclusion Dialysis-ultrafiltration method is a suitable method for extracting exosomes from urine of the aortic dissection patients and can yield a high concentration, while it is not suitable when exosomes of very high purity are required.

Comparision of serum exosomes and exosomal RNA isolation methods / 中华检验医学杂志

Objective The effect of serum exosome isolation method on obtained exosomal RNA is not yet clear .The aim of this study was to provide evidences to selected exosome isolation methods .Methods This was a method comparison study to assess three methods .Vein blood samples were collected from 3 healthy donors from Nanfang hospital , 4 ℃ 12 h was taken to make blood natural coagulated and the supernatant was taken as the serum . Exosomes extracted by Ultracentrifugation , ExoQuick and Total Exosome Isolation Reagent ( TEI ) kits from serum were assessed by transmission electron microscopy , nanoparticle tracking analysis and protein analysis to identify morphology , protein expression , concentration and size distribution of particles .ExoRNA extracted by Trizol-LS, SeraMir, Total Exosome RNA Isolation ( TER) and exoRNeasy were assessed by Bioanalyzer 2100 and qPCR to ascertain RNA distribution and miRNA expression.Results For exosomes isolation , two commercial kits ( ExoQuick and TEI ) showed higher exosomes recovery and protein concentration than ultracentrifugation , but exosome isolated by ultracentrifugation expressed abundant protein makers mostly .For exo-RNA isolation, the distribution of RNA and expression of miRNAinfluenced by three methods , but there was no effect on the relative expression trend of miRNA.ExoRNeasy resulted in the highest yield and most narrow size distribution pattern of small RNA with higher level of miRNA expression .Results of TEI with TER kits showed no obvious bands of small RNA and moderate miRNA expression among methods .Ultra with Trizol-LS or ExoQuick with SeraMir showed low concentration measurable bands around 100 nt, with changeable miRNA profiling irregularly . Conclusions Extraction of exosomes using traditional ultracentrifugation method is applicable for proteomics research work .Extraction using ExoQuick and TEI kits is suitable for preparing exosomes from scarce samples such as serum.

Trypanosoma evansi is alike to Trypanosoma brucei brucei in the subcellular localisation of glycolytic enzymes

Trypanosoma evansi, which causes surra, is descended from Trypanosoma brucei brucei, which causes nagana. Although both parasites are presumed to be metabolically similar, insufficient knowledge of T. evansi precludes a full comparison. Herein, we provide the first report on the subcellular localisation of the glycolytic enzymes in T. evansi, which is a alike to that of the bloodstream form (BSF) of T. b. brucei: (i) fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hexokinase, phosphofructokinase, glucose-6-phosphate isomerase, phosphoglycerate kinase, triosephosphate isomerase (glycolytic enzymes) and glycerol-3-phosphate dehydrogenase (a glycolysis-auxiliary enzyme) in glycosomes, (ii) enolase, phosphoglycerate mutase, pyruvate kinase (glycolytic enzymes) and a GAPDH isoenzyme in the cytosol, (iii) malate dehydrogenase in cytosol and (iv) glucose-6-phosphate dehydrogenase in both glycosomes and the cytosol. Specific enzymatic activities also suggest that T. evansi is alike to the BSF of T. b. brucei in glycolytic flux, which is much faster than the pentose phosphate pathway flux, and in the involvement of cytosolic GAPDH in the NAD+/NADH balance. These similarities were expected based on the close phylogenetic relationship of both parasites.

Purification of coxsackievirus A16 viral particles and preparation and identification of neutralizing monoclonal antibody against coxsackievirus A16 / 国际检验医学杂志

Objective To establish the rapid purification of Coxsackievirus A16 using ultracentrifugation .And To prepare and i‐dentify the neutralizing monoclonal antibody against CA16 .Methods The CA16 culture supernatant was harvested and then con‐centrated by 100K capsule .The concentration of CA16 was purified by cesium chloride ultracentrifugation .Purification of CA16 were identified by transmission electron microscopy .BALB/c mice were immunized with inactivated CA16 .Spleen cells were harves‐ted and fused with SP2/0 myeloma cells ,hybridoma cell strain secreting mAb against CA16 were objected to screening .Character‐ization of the prepared mAb were analyzed by ELISA and microneutralization assay .Results The purified CA16 method of cesium chloride gradient ultracentrifugation was established ,TEM analysis was showed that CA16 particles have icosahedral structure ,the diameters of the viral particles were approximately 20-30 nm .Two hybridoma cell strains secreting mAb against CA16 were ob‐tained ,the subtypes of two mAbs were IgG2a ,the binding titers of Anti/CA16/5 and Anti/CA16/10 were 103 and 104 respectively . Neutralizing titer of the two mAbs were 1∶256 and 1∶1 024 respectively .Conclusion Establishment method of cesium chloride gradient ultracentrifugation was performed to purify CA16 ,the two mAbs with neutralizing ability to against CA16 may become ap‐plication of treatment and vaccine .

Properties of Lipid Rafts from Cerebral Cortex Based on HPLC MS/MS Proteomics / 天津医药

Objective To verify the validity of the sucrose density gradient ultracentrifugation method for lipid rafts from cerebral cortex. Methods Extract lipid rafts from cerebral cortex in mouse were extracted by the sucrose density gradi-ent ultracentrifugation method. The properties of lipid rafts were detected by Western blotting method, double enzyme and light scattering methods. HPLC MS/MS proteomics and bioinformatics were used to locate proteins of lipid rafts in cells. Re-sults Lipid rafts from cerebral cortex were provided with the model properties of lipid rafts such as high light scattering and cholesterol and high expression of Flotillin-1. HPLC MS/MS proteomics identified total 647 proteins. Most of these pro-teins were from plasma membrane, endoplasmic reticulum, cytoskeleton and cytosol, however, there were 21% proteins among total 647 proteins were from nucleus, mitochondria and ribosomes. Conclusion The sucrose density gradient ultra-centrifugation method is a effective method to extract lipid rafts from cerebral cortex, however, the properties of mixture should be considered.

The comparison among extraction methods of exosomes from T cells of rats / 重庆医学

Objective The purification methods of the exosomes derived form T cells were established in order to get high quan-tity exosomes .Methods Exosomes from T cells culture supernatants were purified by ExoQuick Precipitation ,ultrafiltration and sucrose gradient centrifugation ,differential ultracentrifugation ,and confirmed via using transmission electron microscopy .The pro-tein expression of the exosomes were analyzed by SDS-PAGE electrophoresis .Western blotting was used to test the expression of IL-2 .Results The protein concentration of the exosomes purified through ExoQuick Precipitation ,ultrafiltration and sucrose gradi-ent centrifugation were higher than through differential ultracentrifugation (P<0 .05) .SDS-PAGE displayed the difference among the exosome purified by three methods .Three kinds of exosomes all expressed IL-2 .Conclusion ExoQuick Precipitation ,ultrafiltra-tion and sucrose gradient centrifugation technique can obtain high purity and complete exosome sample .

The Influence of Food Ingestion and Sample Storage on Direct LDL-Cholesterol Measurement by Immunoseparation Method / 대한임상병리학회지

BACKGROUND: Elevated level of low density lipoprotein-cholesterol (LDL-C) is one of the major risk factors for the development of coronary heart disease. Direct LDL-C determination method by immunoseparation (DLDL-C) recently developed is claimed not to be influenced by food ingestion. We re-evaluated the effects of diet and storage conditions for this method. METHODS: Samples were collected from thirty-two medical college students before and after meal to study the effects of diet on this method. We compared the difference of LDL-C of filtered samples between refrigerated and frozen state. We also compared direct and indirect calculated measurements of LDL-C with ultracentrifugal beta-quantification (BQLDL-C) method. RESULTS: Morning 2-hour-postprandial specimen can be acceptable with no minimal significant bias, but afternoon 2-hour or 4-hour-postprandial specimen cannot be recommended due to significant negative bias (8.6-9.6%). Storage of filtered samples showed no significant difference between frozen and refrigerated state. Calculated LDL-C when triglyceride level is more than 400 mg/dL was not reliable due to large proportional and constant bias. In contrast, DLDL-C showed good accuracy comparing with BQLDL-C (y=0.909x+3.3, r=0.869, n=9, x=BQLDL-C, y=DLDL-C). CONCLUSION: In conclusion, morning two-hour postprandial specimens can be acceptable for DLDL-C, but afternoon postprandial specimens may not be recommended due to significant negative bias. DLDL-C seems to be reliable and useful especially for hypertriglyceridemic patients or follow-up cases of hypercholesterolemia with normal triglyceride or HDL-C levels.