Extraction of exosome by gel electrophoresis microfluidic chip and determination of miRNA-21 in exosome of human plasma / 生物工程学报

We developed a high-efficiency microfluidic chip for extracting exosomes from human plasma. We collected peripheral blood from normal human, designed and fabricated a microfluidic chip based on nanoporous membrane and agarose gel electrophoresis to isolate exosomes. The extracted exosomes were characterized by transmission electron microscopy, nanosight and Western blotting, the morphology, concentration and particle size of exosomes were identified and analyzed. Meanwhile, we used ultracentrifugation and microfluidic chip to isolate exosomes separately. The particle size and concentration of the exosomes extracted by two methods were compared and analyzed, and their respective extraction efficiency was discussed. Finally, the expression level of miRNA-21 in exosomes was analyzed by RT-PCR. The microfluidic chip isolated (in 1 hour) high-purity exosomes with size ranging from 30-200 nm directly from human plasma, allowing downstream exosomal miRNA analysis. By comparing with ultracentrifugation, the isolation yield of microfluidic chip was 3.80 times higher than ultracentrifugation when the volume of plasma sample less than 100 μL. The optimized parameters for exosome isolation by gel electrophoresis microfluidic chip were: voltage: 100 V; concentration of agarose gel: 1.0%; flow rate of injection pump: 0.1 mL/h. The gel electrophoresis microfluidic chips could rapidly and efficiently isolate the exosomes, showing great potential in the research of exosomes and cancer biomarkers.

Preliminary results of SARS-CoV-2 detection in sewerage system in Niterói municipality, Rio de Janeiro, Brazil

This study presents preliminary results from a sewage-based surveillance to monitor the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the municipality of Niterói, State of Rio de Janeiro, Brazil. By using ultracentrifugation method associated to quantitative reverse transcription polymerase chain reaction (RT-qPCR) we detected SARS-CoV-2 in 41.6% (5/12) of raw sewage samples obtained from sewage treatment plants and sewers network in the city. This pioneer study carried out in Brazil aims to subsidise information for health surveillance concerning the viral circulation in different areas of the city and, revealed the insertion and importance of environmental virology in health public policies.

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.

Peripheral blood exosomes from patients with multiple myeloma mediate bortezomib resistance in cultured multiple myeloma cells / 南方医科大学学报

OBJECTIVE@#To investigate the role of exosome in mediating bortezomib (Btz) resistance in multiple myeloma cells and explore the underlying mechanisms.@*METHODS@#Peripheral blood samples were collected from 15 patients with multiple myeloma with Btz tolerance, and serum exosomes were isolated by ultracentrifugation and identified with electron microscopy, NTA and Western blotting. cultured multiple myeloma cells were treated with gradient concentrations of Btz to determine the optimal drug concentration for subsequent experiment. The cells were pretreated with different concentrations of exosomes, and their sensitivity to BTZ was assessed using MTS assay. We searched the exosome database Exocarta and used STRING to generate the network map and the protein interaction graph.@*RESULTS@#The diameters of the vesicles isolated from the peripheral blood of the patients were mostly below 200 nm with a mean particle size of 153 nm and a mode of 140.1 nm. The results of Western blotting showed that the isolated exosomes expressed the marker proteins CD63, Tsg101 and Alix. In cultured multiple myeloma cells, pretreatment with exosomes resulted in a decreased sensitivity of the cells to bortezomib, and longer treatment durations and higher exosome concentrations consistently enhanced the resistance of the cells to the same Btz concentration. Analysis of the Exocarta database identified human serum exosomal proteins ABCB1, ABCB4, PDCD6IP, and EGFR, among which EGFR served as a network node.@*CONCLUSIONS@#Exosome within a specific concentration range may serve as a signal carrier to mediate the resistance of multiple myeloma cells to Btz. EGFR likely plays a key role to promote exosome-mediated Btz resistance in myeloma cells.

Metodología para la obtención y evaluación de un extracto proteínico de una cepa autóctona de Helicobacter pylori / Methodology to obtain and evaluate a protein extract of an autochthonous Helicobacter pylori strain

Helicobacter pylori es una bacteria gram negativa que posee numerosos antígenos que juegan un importante papel en la patogénesis de las enfermedades gastroduodenales. Debido a la necesidad de métodos de diagnóstico estandarizados con antígenos locales u autóctonos nos propusimos el diseño de una estrategia para la obtención de extractos de antígenos con reactividad frente a sueros de pacientes infectados por H. pylori. Dos cepas de H. pylori, una autóctona (IPK196A) y una de referencia ATCC 43504, se cultivaron en un medio líquido modificado. Se sometieron a los protocolos de ruptura por ultrasonido aplicándose tres variantes de precipitación y al fraccionamiento celular mediante ultracentrifugación diferencial. Los extractos proteínicos se visualizaron mediante electroforesis en gel de poliacrilamida y se transfirieron para la detección de antígenos inmunorreactivos a sueros de pacientes con infección por H. pylori e individuos sanos. La variante de ultrasonido y precipitación con Coomasie fue la más efectiva para concentrar las muestras. El método de ultracentrifugación mejoró la resolución de las proteínas reactivas y permitió separarlas según su localización subcelular. El sistema de transferencia húmedo fue ideal para la inmunodetección de los antígenos obtenidos por ultrasonido mientras que el sistema semiseco permitió detectar las proteínas de membrana obtenidas por ultracentrifugación diferencial. La introducción de una metodología en el laboratorio para la obtención y evaluación de extractos proteínicos antigénicos a partir de cepas autóctonas de H. pylori, constituye la antesala para el diseño de futuros diagnosticadores y candidatos vacunales(AU)

Helicobacter pylori is a gram-negative spiral-shaped bacterium, which has many antigens that play an important role in the pathogenesis of gastroduodenal diseases. Due to the lack of standardized methods from native or autochthonous antigens, we proposed in this study, the design of a strategy for extracting and obtaining immunoreactive antigens against H. pylori infected-patient sera. Two H. pylori strains, one autochthonous (IPK196A) and one reference ATCC 43504, were cultured in a modified liquid medium. Both strains were subjected to the ultrasound rupture protocols applying three precipitation variants and cell fractionation by differential ultracentrifugation. Protein extracts were visualized by polyacrylamide gel electrophoresis and transferred for the detection of immunoreactive antigens to sera from patients with H. pylori infection and healthy individuals. The precipitation with Coomasie was the most effective variant. The ultracentrifugation extraction method optimized the resolution of the proteins, which could be separated according to their subcellular location. The wet transfer system was ideal for the immunodetection of the antigens obtained by ultrasound, while the semi-dry system allowed detecting the membrane proteins by differential ultracentrifugation. The introduction of a methodology in the laboratory for obtaining and evaluating antigenic antibodies from autochthonous strains of H. pylori, is the prelude to the design for future diagnostics and vaccine candidates(AU)

Removing Lipemia in Serum/Plasma Samples: A Multicenter Study

BACKGROUND: Lipemia, a significant source of analytical errors in clinical laboratory settings, should be removed prior to measuring biochemical parameters. We investigated whether lipemia in serum/plasma samples can be removed using a method that is easier and more practicable than ultracentrifugation, the current reference method. METHODS: Seven hospital laboratories in Spain participated in this study. We first compared the effectiveness of ultracentrifugation (108,200×g) and high-speed centrifugation (10,000×g for 15 minutes) in removing lipemia. Second, we compared high-speed centrifugation with two liquid-liquid extraction methods—LipoClear (StatSpin, Norwood, USA), and 1,1,2-trichlorotrifluoroethane (Merck, Darmstadt, Germany). We assessed 14 biochemical parameters: serum/plasma concentrations of sodium ion, potassium ion, chloride ion, glucose, total protein, albumin, creatinine, urea, alkaline phosphatase, gamma-glutamyl transferase, alanine aminotransferase, aspartate-aminotransferase, calcium, and bilirubin. We analyzed whether the differences between lipemia removal methods exceeded the limit for clinically significant interference (LCSI). RESULTS: When ultracentrifugation and high-speed centrifugation were compared, no parameter had a difference that exceeded the LCSI. When high-speed centrifugation was compared with the two liquid-liquid extraction methods, we found differences exceeding the LCSI in protein, calcium, and aspartate aminotransferase in the comparison with 1,1,2-trichlorotrifluoroethane, and in protein, albumin, and calcium in the comparison with LipoClear. Differences in other parameters did not exceed the LCSI. CONCLUSIONS: High-speed centrifugation (10,000×g for 15 minutes) can be used instead of ultracentrifugation to remove lipemia in serum/plasma samples. LipoClear and 1,1,2-trichlorotrifluoroethane are unsuitable as they interfere with the measurement of certain parameters.

Vesículas extracelulares derivadas de macrófagos alteram o potencial de invasão, proliferação e migração de linhagens celulares do carcinoma de células escamosas de língua oral / Macrophage-derived extracellular vesicles alter the potential for invasion, proliferation, and migration of oral tongue squamous cell carcinoma cell lines

O câncer é composto pelas células malignas em proliferação associadas às diferentes células circunjacentes, formando o microambiente tumoral (TME), onde há uma constante troca de informações. Uma das formas de comunicação entre os diferentes tipos celulares do TME se dá por meio da liberação de vesículas extracelulares (EVs), um campo de estudo ainda pouco explorado. O presente estudo se propôs a avaliar os efeitos das EVs liberadas por macrófagos do TME, células altamente plásticas em seu fenótipo (M1 ­ perfil antitumoral; M2 ­ perfil pró-tumoral), em diferentes linhagens do carcinoma de células escamosas de língua oral (CCELO) no tocante à capacidade invasiva, proliferativa e migratória. Foi observado que as amostras de EVs extraídas dos macrófagos eram relativamente puras em EVs, porém subtipo inespecíficas. No ensaio de invasão em miomas, quando colocadas as células inflamatórias em cocultura com as células HSC-3, as células M1 inibiram a invasão e M2 aumentaram a capacidade invasiva das células malignas. Por outro lado, o tratamento com M1 EVs aumentou a capacidade invasiva das células HSC-3 e o tratamento com EVs de M2 inibiu a invasão dessas células, sendo observado um perfil semelhante nas células SCC-25 e SAS quando submetidas aos mesmos tratamentos. Na análise do marcador Ki-67 nos miomas, tanto as células HSC-3 quanto SCC-25 e SAS apresentaram o mesmo padrão de proliferação independentemente do tratamento utilizado, quando comparados com os respectivos controles negativos. Quando analisada a proliferação das células malignas no IncuCyte®, tratadas com EVs dos diferentes tipos de macrófagos em diferentes concentrações, foi identificado um aumento na capacidade proliferativa de células HSC-3 e SAS tratadas com M1 EVs em um padrão dose dependente. Um aumento da capacidade proliferativa seguindo um padrão dose dependente também ocorreu quando as células SAS foram tratadas com M2 EVs. Nos demais ensaios de proliferação no IncuCyte® também foram identificados efeitos na capacidade proliferativa, no entanto um padrão dose dependente não foi observado. No ensaio de migração no IncuCyte®, foram identificadas diferenças significativas na capacidade migratória de células SCC-25 e SAS tratadas com diferentes tipos de EVs nas diferentes concentrações, quando comparadas ao controle negativo. Os achados deste estudo sugerem que as EVs derivadas de macrófagos são fatores importantes na tumorigênese do CCELO, bem como abre discussões sobre os diferentes efeitos das células inflamatórias no TME a depender do tipo de comunicação celular executada (AU).

Cancer is an entity composed of proliferating malignant cells associated with the different types surrounding cells, forming the tumor microenvironment (TME), where there is a constant exchange of information. One of the ways of communicating between different types of TME cells is through the release of extracellular vesicles (EVs), a field of study that remains poorly understood. The aim of the present study was to evaluate the effects of EVs released from TME macrophages, which are cells highly plastic in their phenotype (M1 showing an anti-tumor profile and M2 exhibiting a pro-tumor profile) in different cell lines of tongue squamous cells carcinoma (TSCC) regarding to invasive, proliferative and migratory capacity. It was observed that EVs samples obtained from macrophages were relatively pure in EVs, although they were non-specific subtypes. In the myoma invasion assay, it was observed that when inflammatory cells were co-cultured with HSC-3 cells, M1 cells inhibited invasion and M2 increased the invasive ability of the malignant cells. On the other hand, treatment with M1 EVs increased the invasive capacity of HSC-3 cells, and treatment with M2 EVs inhibited the invasion of these malignant cells, and a similar profile was observed in SCC-25 and SAS cells when they were submitted to the same treatments. In the analysis of the Ki-67 marker in myomas, HSC-3, SCC-25 and SAS cells showed the same proliferation pattern regardless the type of the treatment used when compared to the respective negative controls. When it was analyzed the proliferation of malignant cells in IncuCyte® treated with EVs derived from different types of macrophages at different concentrations, an increase in the proliferative ability of HSC-3 and SAS cells treated with M1 EVs was observed in a dosedependent pattern. An increase in proliferative ability in dose-dependent profile was also observed when SAS cells were treated with M2 EVs. In the other proliferation assays performed in IncuCyte®, effects on proliferative capacity were also highlighted, however a dose-dependent pattern was not observed. In the IncuCyte® migration assay, significant differences were observed in the migration capacity of SCC-25 and SAS cells treated with different types of EVs at different concentrations when compared to the negative control. The findings of this study suggest that macrophages-derived EVs are pivotal factors in TSCC tumorigenesis, as well as permits discussions on the different effects of inflammatory cells on TME depending on the type of cell communication performed (AU).

Preparation and immunogenicity of influenza virus-like particles using nitrocellulose membrane filtration

PURPOSE: Nitrocellulose membrane–based filtration system (NCFS) is widely used for protein concentration. In this study, we applied NCFS for production of virus-like particle (VLP) as a vaccine candidate and evaluated yield property and immunogenicity. MATERIALS AND METHODS: Influenza VLPs were generated by baculovirus-insect cell protein expression system. NCFS and sucrose gradient ultracentrifugation were used for purification of VLP. Immunogenicity of VLP was evaluated by animal experiment. RESULTS: Influenza VLPs expressing hemagglutinin (HA) and neuraminidase proteins derived from highly pathogenic influenza virus (H5N8) were effectively produced and purified by NCFS. HA activity of VLP which correlated with antigenicity was well conserved during multiple purification steps. This NCFS based purified VLPs induced influenza virus–specific antibody responses. CONCLUSION: Our results indicate that the influenza VLP vaccine could be prepared by NCFS without loss of immunogenicity and elicit antigen-specific immune responses.

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.

Necessidades e preocupações dos pais em diferentes etapas do ciclo vital / Parents' needs and concerns at different stages of the life cycle / Necesidades y preocupaciones de los padres en diferentes etapas del ciclo vital

Objetivo: identificar as necessidades e as preocupações prioritárias, manifestadas pelos pais no desempenho do seu papel, em três etapas do ciclo vital: adolescência, idade produtiva e idade madura. Metodologia: estudo exploratório com abordagem qualitativa, desenvolvido com quatorze pais residentes em um município no extremo sul do Brasil. Os dados foram coletados entre maio e agosto de 2011, por meio de entrevista em profundidade. Através da técnica da análise textual discursiva e da matriz construída com base na teoria bioecológica de Bronfenbrenner, foram construídas três categorias: Necessidades/preocupações do pai, geradas em sua relação com o mundo do trabalho; Necessidades/preocupações que emergem da relação de cuidado com os filhos e Preocupações dos pais com relação ao futuro dos filhos. Conclusão: identificou-se que a preocupação com o futuro dos filhos foi apontada por pais de todas as faixas-etárias investigadas. .

Objective: this study aimed to identify priority needs and concerns expressed by fathers in the performance of their role in three stages of the life cycle: adolescence, productive age, and mature age. Methodology: this is an exploratory study with a qualitative approach, conducted with fourteen fathers residing in a municipality in the extreme south of Brazil. The data were collected between May and August 2011 by means of the in-depth interview. Through the technique of written discourse analysis and the array built upon Bronfenbrenner's bioecological theory, we obtained three categories: fathers' needs/concerns, generated in their relationship with the world of work; needs/concerns that emerged from the relationship of care with the children; and fathers' concerns about the future of the children. Conclusions: we identified that the concern with the future of the children was pointed out by fathers of all age groups investigated. .

Objetivo: identificar las necesidades y preocupaciones prioritarias, manifestadas por los padres en el desempeño de su función, en tres etapas del ciclo de vida: adolescencia, edad productiva y edad madura. Metodología: estudio exploratorio con abordaje cualitativo, desarrollado con catorce padres residentes en un municipio en el extremo sur de Brasil. Los datos fueran colectados entre mayo y agosto de 2011, a través de entrevistas en profundidad. A través de la técnica de análisis textual y discursiva e de la matriz construida basada en la teoria bioecologica de Bronfenbrenner, fueran construidas tres categorías: Necesidades/ preocupaciones de lo padre, generado en suya relación con el mundo de lo trabajo; Necesidades/preocupaciones que emergen de la relación de cuidado con hijos e preocupaciones de los padres con lo futuro de los hijos. Conclusión: Se identifico que la preocupación con el futuro de los hijos fue apuntado por los padres de todas las edades averiguadas. .

Efeito da temperatura das amostras de leite na concentração de cálcio solúvel e de beta-caseína: interferência no teste de estabilidade frente ao etanol / Effect of milk sample temperature on the concentration of soluble calcium and beta-casein: interference with the ethanol stability test

O objetivo deste trabalho foi avaliar a variação do perfil proteico e do cálcio solúvel na coagulação do leite pelo etanol nas temperaturas de 4ºC, 10ºC, 15ºC e 20ºC. Amostras de leite de 61 animais foram avaliadas quanto à estabilidade ao etanol nas concentrações de 66 a 92 por cento (v/v) nas temperaturas de 4ºC, 10ºC, 15ºC e 20ºC. Três amostras, após 24 horas de armazenamento a 4ºC, foram ultracentrifugadas em quadruplicata (40.000 x g) a 4ºC e a 20ºC, respectivamente, por 60 minutos. Em seguida, o sobrenadante foi retirado e submetido à análise do cálcio solúvel pela técnica via úmida (digestão nitroperclórica) e leitura em espectrofotômetro de absorção atômica. O perfil proteico foi analisado pela técnica de eletroforese capilar empregando kit específico para determinação proteica. Os resultados mostraram uma correlação positiva entre o aumento da temperatura das amostras e a estabilidade do leite frente às diferentes concentrações de etanol. A porcentagem de cálcio solúvel no sobrenadante após ultracentrifugação foi maior nas amostras tratadas a 4ºC (P<0,05). As amostras ultracentrifugadas na temperatura de 4ºC apresentaram quantidades superiores de β-caseína no sobrenadante em comparação com as amostras tratadas a 20ºC. O abaixamento da temperatura favoreceu a migração da β-caseína e do cálcio coloidal para a fase solúvel do leite, o que possivelmente favoreceu o aumento da instabilidade das amostras no teste do etanol. Os resultados sugerem que a temperatura ideal para a realização de teste de estabilidade do leite frente ao etanol deveria ser de 21ºC...

The aim of this study was to evaluate the variation in protein profile and soluble calcium in milk coagulation by ethanol at 4ºC, 10ºC, 15ºC and 20ºC. Milk samples from 61 dairy cows were evaluated for stability of ethanol concentrations from 66 to 92 percent (v/v) at temperatures of 4°C, 10°C, 15°C and 20°C. Three samples were ultracentrifuged (40,000 x g) after 24 hours of storage at 4°C and 20°C, respectively, for 60 minutes. Their supernatants were removed and subjected to analyses of soluble calcium through nitro-perchloric digestion and atomic absorption spectrophotometry. The protein profiles were determined by capillary electrophoresis using a specific kit for protein determination. The results showed a positive correlation between the increase in temperature of the samples and the stability of milk against various concentrations of ethanol. The percentage of soluble calcium in the supernatant after centrifugation was higher in samples treated at 4°C (P<0.05). The samples ultracentrifuged at 4°C showed higher amounts of β-casein in the supernatant compared with samples stored at 20°C. The lowering of the temperature favored the migration of β-casein and colloidal calcium to the soluble phase of milk, which may also have favored the instability of milk in the ethanol test. According to the results, the milk sample temperature for the ethanol stability test should be 21ºC...

Mechanism of the Rpn13-induced activation of Uch37

Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other's ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37Δ(Hb,Hc,KEKE), a truncation removal of the C-terminal extension region (residues 256-329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270-407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.

Immune modulatory function of abundant immune-related microRNAs in microvesicles from bovine colostrum

Colostrum provides essential nutrients and immunologically active factors that are beneficial to newborns. Our previous work demonstrated that milk contains large amounts of miRNA that is largely stored in milk-derived microvesicles (MVs). In the present study, we found that the MVs from colostrum contain significantly higher levels of several immune-related miRNAs. We hypothesized that the colostrum MVs may transfer the immune-related miRNAs into cells, which contribute to its immune modulatory feature. We isolated colostrum MVs by ultracentrifugation and demonstrated several immune modulation features associated with miRNAs. We also provide evidence that the physical structure of milk-derived MVs is essential for transfer miRNAs and following immune modulation effect. Moreover, we found that colostrum powder-derived MVs also contains higher levels of immune-related miRNAs that display similar immune modulation effects. Taken together, these results show that MV-containing immunerelated miRNAs may be a novel mechanism by which colostrum modulates body immune response.

Effect of Omega-3 Fatty Acids on Low Density Lipoprotein Subfraction, Adiponectin and Apolipoprotein B in Type 2 Diabetic Patients

BACKGROUND: Omega-3 fatty acids derived from fish oil have been reported to exert a beneficial effect on reducing cardiovascular disease. Reports about their mechanism have generated several interesting findings, including a change in small dense low density lipoprotein (sdLDL) cholesterol proportion, adiponectin, and apolipoprotein B (apoB), in addition to changes in the lipid profile. The principal objective of our study was to evaluate the effects of omega-3 fatty acids on plasma sdLDL, adiponectin, apoB100, and B48 in type 2 diabetic patients with hypertriglyceridemia. METHODS: We randomized 28 type 2 diabetic patients in a placebo-controlled, double-blind trial to receive either omega-3 fatty acids or placebo, both administered at a dose of 4 g daily for 12 weeks. LDL subfractions prior to and after treatment were separated via low-speed ultracentrifugation and analyzed via immunoelectrophoresis. Adiponectin, apoB100, and B48 levels were measured using an ELISA kit. RESULTS: sdLDL proportions were reduced in the omega-3 fatty acids group by 11% after 12 weeks of treatment (n = 17, P = 0.001), and were reduced by 4% in the control group (n = 11, P = 0.096). The patients receiving the omega-3 fatty acids evidenced a significant reduction in the levels of triglyceride (P = 0.001), apoB100, and B48 after 12 weeks (P = 0.038 and P = 0.009, respectively) relative to the baseline. Omega-3 fatty acids supplementation increased fasting blood glucose (P = 0.011), but the levels of HbA1c in each group did not change to a statistically significance degree. The adiponectin value was not reduced in the omega-3 fatty acids group (P = 0.133); by way of contrast, the placebo group evidenced a significant reduction in adiponectin value after 12 weeks (P = 0.002). CONCLUSION: Omega-3 fatty acid treatment proved effective in the reduction of atherogenic sdLDL and apoB in type 2 diabetic patients (Clinical trials reg. no. NCT 00758927, clinicaltrials.gov).

Study on best preparation procedure of Lumbricus for anticoagulated blood region in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>Applying for the activity of enzyme in vitro,the research optimized the best preparation procedure for the anticoagulated blood region from Lumbricus.</p><p><b>METHOD</b>All through our experiment, the content of protein and theactivity of enzyme were examined. The extraction process, the refining technology, concentration processes of Lumbricus were optimized with single factor checking and orthogonal design method.</p><p><b>RESULT</b>At 37 degrees C, the coarse powder of Lumbricus soaking with 15 fold of 0.9% sodium chloride and ultrasonic extracting 40 minites for three times was the best ultrasonic extraction. Utrafiltration membrane with molecular weights of 30 x 10(3) for refining and 10 x 10(3) for concentrating were selected.</p><p><b>CONCLUSION</b>Ultrasonic extraction and membrane separation technology, to well improve the effect of purification for the anticoagulant site of Lumbricus, is conducive to further study.</p>

Separación de poblaciones de glóbulos rojos senescentes por gradientes preformados de Percoll / Separation of populations of senescent red blood cells by Percoll performed gradients

Los mecanismos que determinan la senescencia de los eritrocitos han sido extensamente estudiados, sin embargo, no se han logrado conclusiones definitivas debido a la ausencia de una técnica que permita el aislamiento de grupos etáreos bien definidos. Los métodos más comúnmente empleados se basan en el aumento de densidad de los eritrocitos durante el envejecimiento. En este trabajo desarrollamos una técnica para la separación de glóbulos rojos de distintas edades empleando gradientes preformados de Percoll, un polímero sintético con propiedades fisicoquímicas adecuadas para trabajar con células vivas. En las suspensiones eritrocitarias obtenidas se realizaron determinaciones hematológicas, actividades de enzimas antioxidantes y el ensayo de eritrofagocitosis. Los valores de los parámetros hematológicos evaluados fueron significativamente mayores en las suspensiones de glóbulos rojos jóvenes. Las actividades enzimáticas mostraron una disminución de la capacidad antioxidante en las poblaciones de eritrocitos senescentes. Este proceso favorecería la interacción de los hematíes envejecidos con las células fagocíticas, demostrada median­te el ensayo de eritrofagocitosis. Los resultados obtenidos indican que el método de gradientes de Percoll permite una adecuada separación de las suspensiones eritrocitarias de distintas edades, con una eficiencia comparable a la observada en la técnica de centrifugación diferencial considerada de referencia.

The mechanisms that determine the senescence of the erythrocytes have been extensively studied; however. definitive conclusions have not been achieved mainly because of the lack of a technique that allows the isolation of well-defined etarian groups. The methods most commonly used for separating erythrocytes from different ages are based on the increase in density that these cells present during their aging. In the present work we have developed a technique for obtaining red blood cells from different ages using Percoll preformed gradients, a synthetic polymer with adequate physic-chemic properties to work with lives cells. In the erythrocytes suspensions we have made hematological determinations. activities of antioxidants enzymes and the essay of erythrophagocytosis. The values of the hematological parameters were significantly higher in the suspensions of young red blood cells. In the measurements of the enzymatic activity we observed a decrease of the antioxidant capacity in the populations of senescent erythrocytes. This process would promote the interaction between the old erythrocytes and the phagocyte cells, demonstrated by the erythrophagocytosis essay. The results obtained indicate that the method Percoll density gradients allows an appropriate separation of the erythrocytes suspensions of different ages with a comparable efficiency to that observed in the technique differential centrifugation, considered as reference.

Isolation and characterization of photosystem II from the filamentous sporophyte of Porphyra yezoensis.

Thylakoid membranes were isolated and purified from diploid filamentous sporophytes of Porphyra yezoensis Ueda using sucrose density gradient ultracentrifugation (SDGUC). After thylakoid membranes were solubilized with SDS, the phtosystem II (PSII) particles with high 2, 6-dichloroindophenol (DCIP) photoreduction activity were isolated by SDGUC. The absorption and fluorescence spectra, DCIP photoreduction activity and oxygen evolution activity of the thylakoid membranes and PSII particles were determined. The polypeptide composition of purified PSII particles was distinguished by SDS-PAGE. Results showed that PSII particles of sporophytes differed from the gametophytes in spectral properties and polypeptide composition. Apart from 55 kDa D1-D2 heterodimer, CP47, CP43, 33 kDa protein, D1, D2, cyt b559 and 12 kDa protein were identified from PSII particles from sporophytes; a new 102 kDa protein was also detected. However, cyt c-550, 20 kDa, 14 kDa and 16 kDa proteins found in PSII particles from gametophytes were not detected in the sporophytes.

[ relationship of the first/third trimester hematocrit level with the birth weight and preeclampsia]

Cells and the volume of blood separated by a centrifuge. Evaluation of hematocrit is one of the routine tests during pregnancy. Researches have shown that there is a significant relationship between both high and low levels of hematocrit and adverse outcomes of pregnancy. This study aimed at examining the relationship between first and third trimester hematocrit, and birth weight and preeclampsia. Hematocrit is defined as the relationship between the volume of pregnant women who referred to health centers in Esfahan during research period. Data were collected by questionnaire through interview with subjects and medical records. Data were analyzed with T-test and one-way ANOVA. A descriptive and analytic research was performed on 322 and preeclampsia was found [P < 0.001 and P < 0.04, respectively], but, there was not a statistically significant association between first and third trimester hematocrit and birth weight [P > 0.05]. A statistically significant relationship between first and third trimester hematocrit preeclampsia, so, further study is suggested. It seems that there is a U-shaped relationship between hematocrit level and preeclampsia, so, further study is suggested

Glicación in vitro de lipoproteínas de baja densidad y aterogenicidad en cultivos celulares / In vitro glycation of low-density lipoproteins and atherogenicity in cell culture

La hiperglucemia sostenida incrementa la glicación de proteínas. En particular, las modificaciones en las lipoproteínas de baja densidad (LDL) aumentan su potencial aterogénico. En este trabajo se comparan las modificaciones producidas por la glicación in vitro de LDL aisladas por dos métodos: precipitación selectiva (PS) y ultracentrifugación (UC). Para ello, se determinó el incremento de fructosamina, el consumo de los grupos e-amino de lisina, guanidinio de arginina y la disminución de residuos de triptofano. Para todos los analitos, los resultados cinéticos indicaron diferencias significativas con relación al basal (p<0,05), coincidentes para ambos métodos en el tiempo de aparición y en el porcentaje de variación. La aterogenicidad de las LDL glicadas separadas por PS fue estudiada en cultivos de macrófagos RAW 264.7 evaluando la formación de células espumosas y cuantificando la incorporación de LDL por tinción de los depósitos lipídicos con Oil Red. Los resultados indican que la captación de LDL modificadas aumentó con el tiempo de incubación, siendo mayor la aterogenicidad de las LDL glicadas respecto de las nativas (p<0,001, 1 h a 37 °C). El procedimiento de PS seleccionado, accesible al laboratorio bioquímico clínico, permite evaluar las modificaciones por glicación que sufren las LDL en pacientes diabéticos.

Long-term hyperglycemia increases protein glycation. In particular, modifications in the low-density lipoproteins (LDL) increase their atherogenic potential. In this study, the modifications caused by in vitro glycation of LDL separated by two methods: selective precipitation (SP) and ultracentrifugation (UC) were compared. Increase fructosamine level, decrease of e-amino group of lysine, guanidinio of arginine and triptophan fluorescence were determined. Results showed significant differences vs. basal (p<0.05) for all the tested parameters, with coincidence for the two separation methods both in time and grade of modifications. The atherogenicity of glycated LDL separated by SP was studied in macrophages RAW 264.7 in culture, through the formation of foam cells and the quantification of the dye taken up by the cellular storage lipids. Results show that the uptake of modified LDL by macrophages increased with the time of incubation, being the atherogenicity of glycated LDL greater than native LDL (p<0.001, 1 h at 37 °C). The selected SP procedure, within the facilities of routine biochemical laboratory, enables the evaluation of the modifications caused by glycation in the LDL of diabetic patients.