OsJMJ718, a histone demethylase gene, positively regulates seed germination in rice.

Seed vigor has major impact on the rate and uniformity of seedling growth, crop yield, and quality. However, the epigenetic regulatory mechanism of crop seed vigor remains unclear. In this study, a (jumonji C) JmjC gene of the histone lysine demethylase OsJMJ718 was cloned in rice, and its roles in seed germination and its epigenetic regulation mechanism were investigated. OsJMJ718 was located in the nucleus and was engaged in H3K9 methylation. Histochemical GUS staining analysis revealed OsJMJ718 was highly expressed in seed embryos. Abiotic stress strongly induced the OsJMJ718 transcriptional accumulation level. Germination percentage and seedling vigor index of OsJMJ718 knockout lines (OsJMJ718-CR) were lower than those of the wild type (WT). Chromatin immunoprecipitation followed by sequencing (ChIP-seq) of seeds imbibed for 24 h showed an increase in H3K9me3 deposition of thousands of genes in OsJMJ718-CR. ChIP-seq results and transcriptome analysis showed that differentially expressed genes were enriched in ABA and ethylene signal transduction pathways. The content of ABA in OsJMJ718-CR was higher than that in WT seeds. OsJMJ718 overexpression enhanced sensitivity to ABA during germination and early seedling growth. In the seed imbibition stage, ABA and ethylene content diminished and augmented, separately, suggesting that OsJMJ718 may adjust rice seed germination through the ABA and ethylene signal transduction pathways. This study displayed the important function of OsJMJ718 in adjusting rice seed germination and vigor, which will provide an essential reference for practical issues, such as improving rice vigor and promoting direct rice sowing production.

Relationship between Protein, MicroRNA Expression in Extracellular Vesicles and Rice Seed Vigor.

Plant extracellular vesicles are non-self-replicating particles released by living plant cells and delimited by a lipid bilayer. They contain a large amount of lipids, RNA, and proteins. Seed vigor plays an important role in agricultural production and preservation of germplasm resources. Extracellular vesicles with cross-species communication with bioactive molecules can resist pathogens, exhibit anti-aging properties, and perform other functions; however, its potential influence on seed vigor has not been reported. In this study, rice seeds with different germination percentages were used to extract extracellular vesicles, endogenous proteins, and RNA. Protein qualitative identification and miRNA differential analysis were performed to analyze the regulatory mechanism of extracellular vesicles on seed vigor. Results: The profiles of four miRNA families were found to be significantly different: osa-miR164, osa-miR168, osa-miR166, and osa-miR159. Protein correlation analysis predicted that extracellular vesicles might mediate the synthesis of the seed cell wall; glyoxic acid cycle and tricarboxylic acid cycle; non-specific lipid transfer; mitochondrial quality control; and other biological processes to regulate rice seed viability. In addition, cupin protein, phospholipase D, aldehyde dehydrogenase, seven heat shock proteins (especially BiP1 and BiP2), protein disulfide isomerase-like (PDI), thioredoxin, calnexin and calreticulin, glutathione transferase, and other proteins found in extracellular vesicles were closely related to seed vigor. This provides a novel direction for the study of the regulation mechanism of seed vigor.

Pan-cancer analysis and experimental validation of DTL as a potential diagnosis, prognosis and immunotherapy biomarker.

BACKGROUND: DTL has been found to be related with multiple cancers. However, comprehensive analyses, which identify the prediction value of DTL in diagnosis, prognosis, immune infiltration and treatment, have rarely been reported so far. METHODS: Combined with the data online databases, the gene expression, gene mutation, function enrichment and the correlations with the immunity status and clinical indexes of DTL were analyzed. Expression of DTL and the degree of immune cell infiltration were examined by immunofluorescence (IF) and immunohistochemistry (IHC) and analyzed by statistical analysis. Furthermore, the influences of DTL on the cell cycle, cell proliferation and apoptosis were detected by live cell imaging, IF and flow cytometric (FC) analysis. Genomic stability assays were conducted by chromosome slide preparation. RESULTS: DTL was widely expressed in various cells and tissues, while it was overexpressed in tumor tissues except acute myeloid leukemia (LAML). Pan-cancer bioinformatics analysis showed that the expression of DTL was correlated with the prognosis, immunotherapy, and clinical indexes in various cancers. In addition, gene set enrichment analysis (GSEA) uncovered that DTL was enriched in oocyte meiosis, pyrimidine metabolism, the cell cycle, the G2M checkpoint, mTORC1 signaling and E2F targets. Furthermore, the overexpression of DTL, and its association with immune cell infiltration and clinical indexes in liver hepatocellular carcinoma (LIHC), bladder urothelial carcinoma (BLCA) and stomach adenocarcinoma (STAD) were verified in our study. It was also verified that overexpression of DTL could regulate the cell cycle, promote cell proliferation and cause genomic instability in cultured cells, which may be the reason why DTL plays a role in the occurrence, progression and treatment of cancer. CONCLUSIONS: Collectively, this study suggested that DTL is of clinical value in the diagnosis, prognosis and treatment of various cancers, and may be a potential biomarker in certain cancers.

Improvement of irradiation-induced fibroblast damage by α2-macroglobulin through alleviating mitochondrial dysfunction.

CONTEXT: α2-Macroglobulin (α2-M) is believed to be a potential anti-irradiation agent, but related mechanisms remains unclear. OBJECTIVE: We investigated the irradiation protective effect of α2-M. MATERIALS AND METHODS: A total of 10 Gy dose of irradiation was used to damage human skin fibroblasts. The influence of α2-M (100 µg/mL) on the proliferation, migration, invasion and apoptosis of fibroblasts was observed using Cell Counting Kit-8 (CCK8), wound healing, transwell, and flow cytometry. Malondialdehyde, superoxide dismutase and catalase was measured using related ELISA kits. The levels of mitochondrial membrane potential and calcium were detected using flow cytometry. The expression of transient receptor potential melastatin 2 (TRPM2) was investigated through western blotting and immunofluorescence staining. RESULTS: High purity of α2-M was isolated from Cohn fraction IV. α2-M significantly increased cell proliferation, migration, invasion, but suppressed cell apoptosis after irradiation. The promotion of cell proliferation, migration and invasion by α2-M exceeded over 50% compared group irradiation. The increased cell ratio in the S phase and decreased cell ratio in the G2 phase induced by irradiation were remarkably reversed by α2-M. α2-M markedly suppressed the increased oxidative stress level caused by irradiation. The mitochondrial damage induced by irradiation was improved by α2-M through inhibiting mitochondrial membrane potential loss, calcium and TRPM2 expression. DISCUSSION AND CONCLUSIONS: α2-M significantly promoted the decreased fibroblast viability and improved the mitochondria dysfunction caused by irradiation. α2-M might present anti-radiation effect through alleviating mitochondrial dysfunction caused by irradiation. This study could provide a novel understanding about the improvement of α2-M on irradiation-induced injury.

Identification of human parvovirus 4 genotypes 1 and 2 in Chinese source plasma pools.

Human parvovirus B19 (B19V) and human parvovirus 4 (PARV4) are known to infect humans and transmit through contaminated blood and blood products. Globally, three genotypes of B19V, as well as PARV4, have been identified, respectively. The existence of different B19V genotypes in Chinese plasma donors has been investigated, however, the data regarding PARV4 were not available. The main objective of this study is to identify the genotypes of PARV4 circulating in Chinese plasma donors. By using a duplex quantitative polymerase chain reaction assay adapted for all genotypes of B19V and PARV4, 78 source plasma pools for fractionation were screened and quantified. Results showed that positive rates of B19V and PARV4 DNA in plasma pool samples were 25.64% and 14.10%, respectively. PARV4 sequences in two positive samples were next genotyped, and these two sequences belonged to PARV4 genotypes 1 and 2, respectively. In conclusion, the data present demonstrate the existence of PARV4 genotypes 1 and 2 in Chinese plasma donors for the first time and also show the relatively lower prevalence and level of PARV4 DNA in Chinese plasma donors in comparison with that of B19V DNA.

LcMYB4, an unknown function transcription factor gene from sheepgrass, as a positive regulator of chilling and freezing tolerance in transgenic Arabidopsis.

BACKGROUND: Sheepgrass (Leymus chinensis (Trin.) Tzvel) is a perennial forage grass that can survive extreme freezing winters (- 47.5 °C) in China. In this study, we isolated an unknown function MYB transcription factor gene, LcMYB4, from sheepgrass. However, the function of LcMYB4 and its homologous genes has not been studied in other plants. RESULTS: The expression of the LcMYB4 gene was upregulated in response to cold induction, and the LcMYB4 fusion protein was localized in the nucleus, with transcriptional activation activity. Biological function analysis showed that compared with WT plants, LcMYB4-overexpressing Arabidopsis presented significantly increased chilling and freezing tolerance as evidenced by increased germination rate, survival rate, and seed setting rate under conditions of low temperature stress. Furthermore, LcMYB4-overexpressing plants showed increased soluble sugar content, leaf chlorophyll content and superoxide dismutase activity but decreased malondialdehyde (MDA) under chilling stress. Moreover, the expression of the CBF1, KIN1, KIN2 and RCI2A genes were significantly upregulated in transgenic plants with chilling treatment. These results suggest that LcMYB4 overexpression increased the soluble sugar content and cold-inducible gene expression and alleviated oxidative damage and membrane damage, resulting in enhanced cold resistance in transgenic plants. Interestingly, our results showed that the LcMYB4 protein interacts with fructose-1,6-bisphosphate aldolase protein1 (LcFBA1) and that the expression of the LcFBA1 gene was also upregulated during cold induction in sheepgrass, similar to LcMYB4. CONCLUSION: Our findings suggest that LcMYB4 encodes MYB transcription factor that plays a positive regulatory role in cold stress.

A MYB-related transcription factor from sheepgrass, LcMYB2, promotes seed germination and root growth under drought stress.

BACKGROUND: Drought is one of the most serious factors limiting plant growth and production. Sheepgrass can adapt well to various adverse conditions, including drought. However, during germination, sheepgrass young seedlings are sensitive to these adverse conditions. Therefore, the adaptability of seedlings is very important for plant survival, especially in plants that inhabit grasslands or the construction of artificial grassland. RESULTS: In this study, we found a sheepgrass MYB-related transcription factor, LcMYB2 that is up-regulated by drought stress and returns to a basal level after rewatering. The expression of LcMYB2 was mainly induced by osmotic stress and was localized to the nucleus. Furthermore, we demonstrate that LcMYB2 promoted seed germination and root growth under drought and ABA treatments. Additionally, we confirmed that LcMYB2 can regulate LcDREB2 expression in sheepgrass by binding to its promoter, and it activates the expression of the osmotic stress marker genes AtDREB2A, AtLEA14 and AtP5CS1 by directly binding to their promoters in transgenic Arabidopsis. CONCLUSIONS: Based on these results, we propose that LcMYB2 improves plant drought stress tolerance by increasing the accumulation of osmoprotectants and promoting root growth. Therefore, LcMYB2 plays pivotal roles in plant responses to drought stress and is an important candidate for genetic manipulation to create drought-resistant crops, especially during seed germination.

bHLH92 from sheepgrass acts as a negative regulator of anthocyanin/proanthocyandin accumulation and influences seed dormancy.

LcbHLH92, a pleiotropic gene from sheepgrass, negatively regulates anthocyanins/proanthocyandins and reduces seed dormancy in transgenic Arabidopsis.

LcFIN2, a novel chloroplast protein gene from sheepgrass, enhances tolerance to low temperature in Arabidopsis and rice.

Adverse environmental stresses affect plant growth and crop yields. Sheepgrass (Leymus chinensis (Trin.) Tzvel), an important forage grass that is widely distributed in the east of Eurasia steppe, has high tolerance to extreme low temperature. Many genes that respond to cold stress were identified in sheepgrass by RNA-sequencing, but more detailed studies are needed to dissect the function of those genes. Here, we found that LcFIN2, a sheepgrass freezing-induced protein 2, encoded a chloroplast-targeted protein. Expression of LcFIN2 was upregulated by freezing, chilling, NaCl and abscisic acid (ABA) treatments. Overexpression of LcFIN2 enhanced the survival rate of transgenic Arabidopsis after freezing stress. Importantly, heterologous expression of LcFIN2 in rice exhibited not only higher survival rate but also accumulated various soluble substances and reduced membrane damage in rice under chilling stress. Furthermore, the chlorophyll content, the quantum photochemistry efficiency of photosystem II (ΦPSII), the non-photochemical quenching (NPQ), the net photosynthesis rate (Pn) and the expression of some chloroplast ribosomal-related and photosynthesis-related genes were higher in the transgenic rice under chilling stress. These findings suggested that the LcFIN2 gene could potentially be used to improve low-temperature tolerance in crops.

Transcriptomic Analysis Reveals a Comprehensive Calcium- and Phytohormone-Dominated Signaling Response in Leymus chinensis Self-Incompatibility.

Sheepgrass (Leymus chinensis (Trin.) Tzvel.) is an economically and ecologically important forage in the grass family. Self-incompatibility (SI) limits its seed production due to the low seed-setting rate after self-pollination. However, investigations into the molecular mechanisms of sheepgrass SI are lacking. Therefore, microscopic observation of pollen germination and pollen tube growth, as well as transcriptomic analyses of pistils after self- and cross-pollination, were performed. The results indicated that pollen tube growth was rapidly inhibited from 10 to 30 min after self-pollination and subsequently stopped but preceded normally after cross-pollination. Time course comparative transcriptomics revealed different transcriptome dynamics between self- and cross-pollination. A pool of SI-related signaling genes and pathways was generated, including genes related to calcium (Ca2+) signaling, protein phosphorylation, plant hormone, reactive oxygen species (ROS), nitric oxide (NO), cytoskeleton, and programmed cell death (PCD). A putative SI response molecular model in sheepgrass was presented. The model shows that SI may trigger a comprehensive calcium- and phytohormone-dominated signaling cascade and activate PCD, which may explain the rapid inhibition of self-pollen tube growth as observed by cytological analyses. These results provided new insight into the molecular mechanisms of sheepgrass (grass family) SI.

MADS-box family genes in sheepgrass and their involvement in abiotic stress responses.

BACKGROUND: MADS-box genes are categorized into A, B, C, D and E classes and are involved in floral organ identity and flowering. Sheepgrass (Leymus chinensis (Trin.) Tzvel) is an important perennial forage grass and adapts well to many adverse environments. However, there are few studies on the molecular mechanisms of flower development in sheepgrass, especially studies on MADS-domain proteins. RESULTS: In this study, we cloned 11 MADS-box genes from sheepgrass (Leymus chinensis (Trin.) Tzvel), and phylogenetic analysis of the 11 genes with their homologs revealed that they are divided into nine subclades. Tissue-specific expression profile analysis showed that most of these MADS-box genes were highly expressed in floral organs. LcMADS1 and LcMADS3 showed higher expression in the stamen than in the other tissues, and LcMADS7 showed high expression in the stamen, glume, lemma and palea, while expression of LcMADS2, LcMADS9 and LcMADS11 was higher in vegetative organs than floral organs. Furthermore, yeast two-hybrid analyses showed that LcMADS2 interacted with LcMADS7 and LcMADS9. LcMADS3 interacted with LcMADS4, LcMADS7 and LcMADS10, while LcMADS1 could interact with only LcMADS7. Interestingly, the expression of LcMADS1 and LcMADS2 were significantly induced by cold, and LcMADS9 was significantly up-regulated by NaCl. CONCLUSION: Hence, we proposed that LcMADS1, LcMADS2, LcMADS3, LcMADS7 and LcMADS9 play a pivotal role in sheepgrass sexual reproduction and may be involved in abiotic stress responses, and our findings provide useful information for further exploration of the functions of this gene family in rice, wheat and other graminaceous cereals.

Analysis of Protein Expression in Human Cells Cocultured with Porcine Peripheral Blood Mononuclear Cells.

OBJECTIVE: Porcine endogenous retroviruses (PERV) involved in pig to human xenotransplantation have raised great concerns because of their ubiquitous nature in pigs and their ability of infecting human cells in vitro. Although no significant cytopathic effect attributed to PERV was evident on PERV-infected human embryonic kidney 293 (HEK293) cells, we did proteomic analysis to investigate the differences of protein profile in order to further characterize the effect of PERV infection. METHODS: HEK293 cells were cocultured with porcine peripheral blood mononuclear cells (PBMCs). Protein profiles of PERV-infected and -noninfected HEK293 cells were analyzed by two-dimensional gel electrophoresis (2-DE). Protein spots with at least 1.5-fold alteration were identified by high-definition mass spectrometry (HDMS) analysis. Then real-time RT-PCR and Western blotting were performed to validate the proteomic results. RESULTS: Differential analysis of PERV-infected and -noninfected HEK293 cells by 2-DE revealed ten differentially regulated proteins. The proteins identified by HDMS were involved in various cellular pathways including signal transduction, cell apoptosis, and protein synthesis. CONCLUSION: The results of this study revealed differentially expressed proteins in HEK293 cells cocultured with porcine PBMCs and implied that these changes were probably induced by PERV infection. These results provide clues and potential links to understanding the molecular effect of the infection by human-tropic PERV.

Large-scale purification of high purity α1-antitrypsin from Cohn Fraction IV with virus inactivation by solvent/detergent and dry-heat treatment.

α1-Antitrypsin (AAT) is widely used to treat patients with congenital AAT deficiency. Cohn Fraction IV (Cohn F IV) is normally discarded during the manufacturing process of albumin but contains approximately 33% of plasma AAT. We established a new process for large-scale purification of AAT from it. liquid chromatography-electrospray ionization-tandem mass spectrometry and high-performance liquid chromatography were applied for qualitative identification and composition analysis, respectively. Stabilizers were optimized for AAT activity protection during lyophilization and dry-heat. Virus inactivation by dry-heat and solvent/detergent (S/D) was validated on a range of viruses. AAT with purity of 95.54%, specific activity of 3,938.5 IU/mg, and yield of 26.79%, was achieved. More than 95% activity was reserved after S/D. More than 96% activity was obtained after lyophilization or dry-heat. After S/D, pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) were inactivated below detectable level within 1 H. Virus titer reductions of more than 5.50 log10 and 5.38 log10 were achieved for PRV and VSV, respectively. Porcine parvovirus and encephalomyocarditis virus were inactivated by 3.17 log10 and 5.88 log10 reduction after dry-heat. The advantages of this process, including suitability for large-scale production, high purity, better utilization of human plasma, viral safety, commercial and inexpensive chromatography medium, may facilitate its further application.

Simultaneous detection and differentiation of human parvovirus B19 and human parvovirus 4 by an internally controlled multiplex quantitative real-time PCR.

Human parvovirus B19 (B19V) and human parvovirus 4 (PARV4) are two parvoviruses known to infect humans and transmit through blood and plasma derived medicinal products (PDMPs). Inactivation of the two parvoviruses has proven to be difficult and nucleic acid testing (NAT) would be an efficient means to exclude viruses. In this study, an internally controlled multiplex quantitative real-time PCR (qPCR) assay for B19V and PARV4 simultaneous detection and quantification was established and evaluated. The optimized multiplex qPCR assay allowed for simultaneous detection of all of the genotypes (1-3) of B19V and PARV4, with equal limit of quantification (LOQ) of 5 copies/µL, rather than other blood-borne viruses. It had a wide dynamic range of reliable amplification linearity of at least 8 orders of magnitude. Low standard deviations (SD) of quantification cycle (Cq) values and low coefficients of variation (CV) of copy numbers for both B19V and PARV4 suggested a high level of repeatability and reproducibility for the multiplex qPCR assay. This multiplex qPCR assay can be served as a readily applicable approach to screen plasma units intended for further manufacturing into PDMPs to reduce the risk of parvoviruses infection by such products and may also be useful for the detection of B19V/PARV4 co-infection or co-existence.

Inactivation of viruses by pasteurization at 60 °C for 10 h with and without 40% glucose as stabilizer during a new manufacturing process of α2-Macroglobulin from Cohn Fraction IV.

Pasteurization is regularly used to inactivate viruses for the safety of plasma derivatives. Influence of pasteurization at 60 °C for 10 h on α2-Macroglobulin activity and virus inactivation were studied. With 40% sugar as stabilizers more than 70% α2-Macroglobulin activity was reserved after pasteurization compared with 20% in control. Glucose presented a better activity protection effect than sucrose and maltose. By pasteurization without stabilizer the virus titers of pseudorabies virus, Sindbis virus, porcine parvovirus and encephalomyocarditis virus were reduced more than 5.88 log10, 7.50 log10, 4.88 log10, and 5.63 log10 respectively within 2 h. By pasteurization with 40% glucose vesicular stomatitis virus was inactivated more than 5.88 log10 within 1 h. Only 2.71 log10 reduction was achieved for encephalomyocarditis virus after 10 h. 40% glucose protected α2-M activity and viruses simultaneously from pasteurization. Other viral inactivation methods need to be incorporated to ensure viral safety of this manufacturing process of α2-Macroglobulin.

Overexpression of a novel cold-responsive transcript factor LcFIN1 from sheepgrass enhances tolerance to low temperature stress in transgenic plants.

As a perennial forage crop broadly distributed in eastern Eurasia, sheepgrass (Leymus chinensis (Trin.) Tzvel) is highly tolerant to low-temperature stress. Previous report indicates that sheepgrass is able to endure as low as -47.5 °C,allowing it to survive through the cold winter season. However, due to the lack of sufficient studies, the underlying mechanism towards the extraordinary low-temperature tolerance is unclear. Although the transcription profiling has provided insight into the transcriptome response to cold stress, more detailed studies are required to dissect the molecular mechanism regarding the excellent abiotic stress tolerance. In this work, we report a novel transcript factor LcFIN1 (L. chinensis freezing-induced 1) from sheepgrass. LcFIN1 showed no homology with other known genes and was rapidly and highly induced by cold stress, suggesting that LcFIN1 participates in the early response to cold stress. Consistently, ectopic expression of LcFIN1 significantly increased cold stress tolerance in the transgenic plants, as indicated by the higher survival rate, fresh weight and other stress-related indexes after a freezing treatment. Transcriptome analysis showed that numerous stress-related genes were differentially expressed in LcFIN1-overexpressing plants, suggesting that LcFIN1 may enhance plant abiotic stress tolerance by transcriptional regulation. Electrophoretic mobility shift assays and CHIP-qPCR showed that LcCBF1 can bind to the CRT/DRE cis-element located in the promoter region of LcFIN1, suggesting that LcFIN1 is directly regulated by LcCBF1. Taken together, our results suggest that LcFIN1 positively regulates plant adaptation response to cold stress and is a promising candidate gene to improve crop cold tolerance.

Inactivation of viruses during a new manufacturing process of α2-macroglobulin from Cohn Fraction IV by dry-heat treatment.

BACKGROUND: α2-Macroglobulin (α2-M) has a curative effect on radiation injury. Virus transmission through plasma derivatives is still not risk-free. Effect of dry heat on α2-M activity and virus inactivation by dry heat in a new manufacturing process of α2-M were studied. STUDY DESIGN AND METHODS: Effects of 100°C for 30 minutes, 80°C for 72 hours, and lyophilization on α2-M activity were detected, and stabilizing agents were optimized. Effect of a treatment at 100°C for 30 minutes has been tested on a range of viruses and characteristics change of α2-M was investigated. RESULTS: More than 90 and 80% α2-M activity recovery were reserved after treatment at 100°C for 30 minutes and 80°C for 72 hours, respectively. A concentration of 0.05 mol/L histidine presented a better protecting effect for α-M activity. No substantial changes were observed in the characteristics of α2-M compared with the untreated. By lyophilization and dry-heat treatment at 100°C for 30 minutes, murine encephalomyocarditis virus and pseudorabies virus (PRV) were inactivated below detectable level within 5 minutes (virus titers reduction ≥ 5.75 log) and 30 minutes (virus titers reduction ≥ 6.00 log), respectively. Bovine viral diarrhea virus and porcine parvovirus were inactivated by 4.29 and 2.46 log reduction, respectively. CONCLUSION: Treatment at 100°C for 30 minutes could improve the virus safety of α2-M with a slight activity loss.

Existence of various human parvovirus B19 genotypes in Chinese plasma pools: genotype 1, genotype 3, putative intergenotypic recombinant variants and new genotypes.

BACKGROUND: Human parvovirus B19 (B19V) is a frequent contaminant of blood and plasma-derived medicinal products. Three distinct genotypes of B19V have been identified. The distribution of the three B19V genotypes has been investigated in various regions or countries. However, in China, data on the existence of different B19V genotypes are limited. METHODS: One hundred and eighteen B19V-DNA positive source plasma pool samples collected from three Chinese blood products manufacturers were analyzed. The subgenomic NS1/VP1u region junction of B19V was amplified by nested PCR. These amplified products were then cloned and subsequently sequenced. For genotyping, their phylogenetic inferences were constructed based on the NS1/VP1-unique region. Then putative recombination events were analyzed and identified. RESULTS: Phylogenetic analysis of 118 B19V sequences attributed 61.86 % to genotype 1a, 10.17 % to genotype 1b, and 17.80 % to genotype 3b. All the genotype 3b sequences obtained in this study grouped as a specific, closely related cluster with B19V strain D91.1. Four 1a/3b recombinants and 5 new atypical B19V variants with no recombination events were identified. CONCLUSIONS: There were at least 3 subtypes (1a, 1b and 3b) of B19V circulating in China. Furthermore, putative B19V 1a/3b recombinants and unclassified strains were identified as well. Such recombinant and unclassified strains may contribute to the genetic diversity of B19V and consequently complicate the B19V infection diagnosis and NAT screening. Further studies will be required to elucidate the biological significance of the recombinant and unclassified strains.

Prevalence of human parvovirus B19 in Chinese plasma pools for manufacturing plasma derivatives.

BACKGROUND: Human parvovirus B19 (B19V) is a frequent contaminant of blood and plasma-derived medicinal products. To ensure the quality and safety of plasma-derived products, European regulations, Plasma Protein Therapeutics Association (PPTA) standard and FDA guidelines require testing of manufacturing plasma for parvovirus B19 DNA to limit the load of this virus. In China, however, there have been no related documentation and technical guiding principles for monitoring B19V, moreover, an adequate level of information on the prevalence of B19V in Chinese plasma donations is not available. FINDINGS: By using an in-house quantitative polymerase chain reaction (qPCR) assay adapted for all three genotypes of B19V, 235 source plasma pools from three regional different Chinese manufacturers of blood products were screened and quantified. Results showed that 71.91 % (169/235) of plasma pools were contaminated by B19V, with the concentrations of 5.18 × 10(2)-1.05 × 10(9) IU/mL. Approximately 31.95 % of the DNA-positive plasma pools were only moderately contaminated (<10(4) IU/mL), while 68.05 % contained >10(4) IU/mL. CONCLUSIONS: The high level of B19V in plasma pools could present a great risk in plasma derivatives. Therefore, the implementation of B19V NAT (Nucleic Acid Testing) assays capable of detecting all B19V genotypes and discard donations with high titer B19V DNA for Chinese blood products manufacturers seems to be necessary.

A Simple Method to Extract Whole Apolipoproteins for the Preparation of Discoidal Recombined High Density Lipoproteins as Bionic Nanocarriers for Drug Delivery.

PURPOSE: To develop a simple method to extract the whole apolipoproteins (apo) including apoA-I in native high density lipoproteins (HDLs) and prepare discoidal Tanshinone IIA-loaded reconstituted HDL (TA-rHDLs) as a dual functional drug delivery system with plaque-site target and therapeutic promises in atherosclerotic lesions. METHODS: A method based on isoelectric precipitation coupled with organic solvent precipitation was developed to isolate the whole apolipoproteins (apos). TA-rHDLs were prepared by incubating the resultant apos with liposomes and the incubation conditions were optimized using fluorescence quenching experiment. TA-rHDLs were characterized in terms of size, zeta potential, morphology, interaction between lipid and apos, safety, and bionic function. RESULTS: The extraction results showed that the yield of the HDL apos was 82.4%, with 59% being apoA-I type, similar ratio of apoA-I in the native apos. TA-rHDL prepared were disc-like with an average diameter of 157.6 ± 4.8 nm, zeta potential of -20.90 ± 0.15 mV, and entrapment efficiency of (90.13 ± 1.4) %. The interaction between the lipids and apos was electrostatic and hydrophobic force and was associated with amino acid sequence. Haemolysis and cytotoxicity assays showed good biocompatibility of TA-rHDL. Sterol efflux assay from macrophages mediated by TA-rHDLs and structure remodeling behavior from discs to spheres proved that TA-rHDL could resemble the biological activity of native nascent HDL irrespective of the size. CONCLUSIONS: The simple approach to isolate apos may provide a convenient and economical resource to support the development of rHDL as a potential targeting nanocarrier for lipophilic cardiovascular drugs.