[Sequencing and Proteomic Analysis of Exosomes from Apheresis Platelets in Different Storage Periods].

OBJECTIVE: To investigate the changes of gene sequencing and proteomics of apheresis platelet (AP) exosomes in different storage periods and predict the function of AP exosomes in different storage periods. METHODS: Platelets at different storage periods of 0 day (D0), 3 day (D3) and 5 day (D5) were collected, exosomes were extracted with Gradient centrifugation; gene sequencing and proteomic analysis were used to analyze the exosomes, and biological functions of platelet exosomes were analyzed and predicted by bioinformatics. Liquid mass spectrometry (LMS) was used to detect the changes and function prediction of exosomes proteins. The small RNA sequencing library was prepared, and the constructed library was sequenced and bioinformatics technology was used for data analysis. RESULTS: AP exosome iTRAQ protein analysis showed that AP exosomes stored in D3 with 55 up-regulated proteins and 94 down-regulated proteins (P<0.05, FC<0.83 or FC>1.2), while AP exosomes stored in D5 with 292 up-regulated proteins and 53 down-regulated proteins (P<0.05, FC<0.83 or FC>1.2) as compared with D0. KEGG pathway analysis showed that the proteins were mainly involved in transport and metabolism, immune system, cancer, membrane transport and other processes. There were statistically significant differences between AP exosome miRNAs in different storage days (P<0.01). The number of miRNA up-regulated and down-regulated was 374 and 255 as compared with the number of platelet exosomes miRNA stored in D3 and D0, while that was 297 and 242 in D5 and D0, and 252 and 327 in D5 and D3, respectively. The target genes of differential platelet exosome miRNAs were analyzed by GO enrichment. Target genes of differential miRNA were mainly involved in membrane composition, mainly played molecular functions binding to proteins, and participated in biological processes of transcriptional regulation. CONCLUSION: The exosome differential proteins and miRNAs in D5 are significantly different from those in the D0 of APs, and they are involved in various biological processes.

Changes in Complement Levels and Activity of Red Blood Cells, Fresh Frozen Plasma, and Platelet Concentrates During Storage.

Complement cascade plays an important role in the field of transfusion medicine. The study aimed to detect the complement levels of different blood components and different blood types to explore the risk of transfusion of stored blood. The samples including red blood cells (n = 110), fresh frozen plasma (n = 120), and platelet concentrates (n = 104) from healthy blood donors in our center were collected. Complement components (C3, C4, C3b, C3d, and CH50) were assayed to evaluate the activation of complement. The complement levels of various blood components at different storage times were observed. The differences in complement levels of four blood types in various blood components were compared. The complement levels of red blood cells in storage were low, with no significant changes (P > 0.05). C3b and C3d levels in platelets began to significantly increase after storage for 3 days (P < 0.05). The fresh frozen plasma during storage had higher complement levels, and the concentrations of C3 and C4 decreased and C3b and C3d increased at month 4 (P < 0.05). The differences in complement levels of four blood types in various blood components did not significantly change (P > 0.05), but the C3b and C3d levels of AB fresh frozen plasma remained stable during storage, which different from other blood types. The transfusion of red blood cells was relatively safe in terms of complement activation. The activation of complement proteins occurred during the storage of platelet and plasma, except group AB plasma.

G-CSF inhibits LFA-1-mediated CD4+ T cell functions by inhibiting Lck and ZAP-70.

In this study, we showed that G-CSF mobilization increased the frequency of T cells, specifically CD3+CD4+ T cells. G-CSF mobilization decreased the secretion of inflammatory cytokines of CD4+ T cells through the LFA-1/ICAM-1 signaling pathway, whereas it did not alter the TH1/TH2 ratio. We found that G-CSF mobilization inhibited LFA-1-mediated CD4+ T cell polarization and motility. In vitro, G-CSF stimulation also attenuated the polarization and adhesiveness of CD4+ T cells through the LFA-1/ICAM-1 interaction. Further investigation revealed that G-CSF mobilization suppressed LFA-1 signaling by down-regulating Lck and ZAP-70 expression in CD4+ T cells, similar results was also confirmed by in-vitro studies. These findings suggested that G-CSF directly suppressed LFA-1-mediated CD4+ T cell functions through the down-regulation of Lck and ZAP-70. The immunosuppressive effect of G-CSF mobilization deepened our understanding about peripheral blood hematopoietic stem cell transplantation. LFA-1/ICMA-1 pathway may become a potential target for graft-versus-host disease prophylaxis.

Influenza virus vaccine expressing fusion and attachment protein epitopes of respiratory syncytial virus induces protective antibodies in BALB/c mice.

Respiratory syncytial virus (RSV) is an important viral pathogen that causes life-threatening respiratory infections in both infants and the elderly; no vaccines are at present available. In this report, we examined the use of influenza virus as a vehicle for production of an experimental RSV vaccine. We used reverse genetics to generate a recombinant influenza A virus with epitopes from the RSV fusion (F) and attachment (G) proteins (rFlu/RSV/F+G) in the influenza virus nonstructural (NS1) protein gene. Expression of RSV F+G epitope proteins was confirmed by Western blotting, and no changes in viral morphology were evident following examination by electron microscopy. BALB/c mice immunized intranasally with rFlu/RSV/F+G showed viral-specific antibody responses against both influenza and RSV. Total IgG, IgG1, IgG2a and IgA were measured in mice immunized with rFlu/RSV/F+G, revealing robust cellular and mucosal immune responses. Furthermore, we found that rFlu/RSV/F+G conferred protection against subsequent influenza and RSV challenges, showing significant decreases in viral replication and obvious attenuation of histopathological changes associated with viral infections. These findings suggest that rFlu/RSV/F+G is a promising vaccine candidate, which should be further assessed using cotton rat and primate models.

A novel biodegradable nicotinic acid/calcium phosphate composite coating on Mg-3Zn alloy.

A novel biodegradable composite coating is prepared to reduce the biodegradation rate of Mg-3Zn alloy. The Mg-3Zn substrate is first immersed into 0.02 mol L(-1) nicotinic acid (NA) solution, named as vitamin B3, to obtain a pretreatment film, and then the electrodeposition of calcium phosphate coating with ultrasonic agitation is carried out on the NA pretreatment film to obtain a NA/calcium phosphate composite coating. Surface morphology is observed by scanning electron microscopy (SEM). Chemical composition is determined by X-ray diffraction (XRD) and EDX. Protection property of the coatings is evaluated by electrochemical tests. The biodegradable behavior is investigated by immersion tests. The results indicate that a thin but compact bottom layer can be obtained by NA pretreatment. The electrodeposition calcium phosphate coating consists of many flake particles and ultrasonic agitation can greatly improve the compactness of the coating. The composite coating is biodegradable and can reduce the biodegradation rate of Mg alloys in stimulated body fluid (SBF) for twenty times. The biodegradation process of the composite coating can be attributed to the gradual dissolution of the flake particles into chippings.

[Research of aeration with bio-film technology to treat urban landscape water].

Research of the aeration with bio-film technology was carried out to treat scenic water of a sanatorium in Beijing. The aim of the research was improving the water habitat by increasing the transparency and reducing the concentration of N and P. The equipments were set in a 5,000 m2 water area, which combined the plug flow jet aerator with the elastic biological filler. The research indicated that the transparency increased from 25 cm to 120 cm by the technology. The removal efficiencies of NH4(+)-N, NO3(-)-N and TP were 86.6% , 90% and 73.3%, but there was only 22.4% for TN. The concentration of DO increased from 4.3 mg/L to 7 mg/L. In a word, the aeration with bio-film technology was an effective measure to improve the water habitat by increasing the transparency.

[Pilot-scale comparison research of different constructed wetland types to treat eutrophic lake water].

Comparison research of different constructed wetland types to treat lake Wulihu water was carried out. Under the condition of the loading rates 0.8 m3/(m2 x d), the removal efficiencies of the vertical flow wetland (VFW), subsurface flow wetland (SFW) and free surface wetland(FSW) had the following results: To ammonia nitrogen (NH4(+)-N) the average removal rates were 33.2%, 27.4% and 14.1%, respectively; To total nitrogen (TN) the average removal rates were 52.3% , 50.1% and 19.2%, respectively; To total phosphorus (TP) the average removal rates were 58.8%, 57.9% and 26.3%, respectively; To permanganate index the average removal rates were 37.2%, 38.3% and 14.8%, respectively; To chlorophyll a (Chl-a) the average removal rates were 86.9%, 96.1% and 55.3%, respectively. Obviously, VFW and SFW are more effective than FSW at treating eutrophicated water such as Lake Wulihu which with characters of low organically pollution and with high nitrogen and phosphorus pollution, and the VFW is the most effective on the removal of NH4(+)-N, TN and TP. SFW is the most effective on the removal of permanganate index and Chl-a. The effluent stability of VFW is better than SFW, and the SFW is better than FSW.