Metabolism of minor isoforms of prion proteins Cytosolic prion protein and transmembrane prion protein

Transmissible spongiform encephalopathy or prion disease is triggered by the conversion from cellular prion protein to pathogenic prion protein. Growing evidence has concentrated on prion protein confi...     

Genome-wide high resolution parental-specific DNA and histone methylation maps uncover patterns of imprinting regulation in maize

Genetic imprinting is a specific epigenetic phenomenon in which a subset of genes is expressed depending on their parent-of-origin. Two types of chromatin modifications, DNA methylation and histone modification, are generally believed to be involved in the regulation of imprinting. However, the genome-wide correlation between allele-specific chromatin modifications and imprinted gene expression in maize remains elusive. Here we report genome-wide high resolution allele-specific maps of DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3) in maize endosperm. For DNA methylation, thousands of parent-of-origin dependent differentially methylated regions (pDMRs) were identified. All pDMRs were uniformly paternally hypermethylated and maternally hypomethylated. We also identified 1131 allele-specific H3K27me3 peaks that are preferentially present in the maternal alleles. Maternally expressed imprinted genes (MEGs) and paternally expressed imprinted genes (PEGs) had different patterns of allele-specific DNA methylation and H3K27me3. Allele-specific expression of MEGs was not directly related to allele-specific H3K27me3, and only a subset of MEGs was associated with maternal-specific DNA demethylation, which was primarily located in the upstream and 5′ portion of gene body regions. In contrast, allele-specific expression of a majority of PEGs was related to maternal-specific H3K27me3, with a subgroup of PEGs also associated with maternal-specific DNA demethylation. Both pDMRs and maternal H3K27me3 peaks associated with PEGs are enriched in gene body regions. Our results indicate highly complex patterns of regulation on genetic imprinting in maize endosperm.Genetic imprinting is an epigenetic phenomenon, where genes are expressed in a parent-of-origin dependent manner in many plant species and mammals. Although first discovered in plants (Kermicle and Alleman 1990), research on genetic imprinting is much more advanced in mammals in terms of the number of imprinted genes identified and the understanding of their regulatory mechanisms (Koerner and Barlow 2010; Barlow 2011; Bartolomei and Ferguson-Smith 2011). Only a small number of imprinted genes were observed in plants for a long time since the phenomenon is highly specific to the triploid endosperm (Raissig et al. 2011). However, recent studies have indicated that genetic imprinting in plants is much more prevalent than previously thought, with hundreds of genes shown to be imprinted in several plant species (Gehring et al. 2011; Hsieh et al. 2011; Luo et al. 2011; Waters et al. 2011; Zhang et al. 2011). In contrast to mammals, where the regulation of genetic imprinting has been extensively studied (Koerner and Barlow 2010; Barlow 2011; Abramowitz and Bartolomei 2012), the understanding of regulation of parental imprinting in plants is highly limited.DNA methylation is one of the primary modifications reported to be associated with genetic imprinting. In Arabidopsis, DNA methylation around several maternally expressed imprinted protein-coding genes (MEG) including FWA, FIS2, and MPC was shown to be important for their maternally preferred expression, as all these genes exhibited biallelic expression in endosperm fertilized with met1 pollen (Kinoshita et al. 2004; Jullien et al. 2006; Tiwari et al. 2008). Two studies using RNA-seq in Arabidopsis also showed that a number of MEGs exhibited biallelic expression in paternal met1 endosperm, and the maternal alleles of dozens of paternally expressed imprinted genes (PEGs) were reactivated in maternal dme endosperm (Hsieh et al. 2011; Wolff et al. 2011). In maize, five confirmed endosperm MEGs (Fie1, Fie2, Mez1, Meg1, and Mee1) contain differentially methylated regions (DMRs) (Gutierrez-Marcos et al. 2004; Gutierrez-Marcos et al. 2006; Haun et al. 2007), and activation of the Fie1 maternal allele in the endosperm requires DNA demethylation of the maternal allele (Hermon et al. 2007).Another modification associated with genetic imprinting involves histone methylation. Polycomb repressive complex 2 (PRC2) is known to mediate the trimethylation of histone H3 lysine 27 (H3K27me3) (Schuettengruber and Cavalli 2009). Results on PHE1, the only well-studied PEG in plants, indicated that silencing of its maternal allele depends on a functional PRC2 complex in addition to DNA demethylation (Kohler et al. 2003, 2005; Makarevich et al. 2008). Recently, a number of MEGs and PEGs were shown to be biallelically expressed in maternal fie or fis2 endosperm (Hsieh et al. 2011; Wolff et al. 2011).Although the studies above suggest that DNA methylation and the PRC2 complex could be responsible for monoallelic expression of imprinted genes, there is not yet any general rule for the function of DNA and histone methylation on the regulation of genetic imprinting in plants. A high resolution genome-wide map of allele-specific DNA methylation and allele-specific histone modification will be crucial to gain better understanding of the regulation of genetic imprinting. Recently, several genome-wide studies have provided evidence that allele-specific patterns of DNA methylation or parent-of-origin dependent differentially methylated regions (pDMRs) are associated with some imprinted genes in mice and plants (Zhang et al. 2011; Ibarra et al. 2012; Xie et al. 2012; Rodrigues et al. 2013). Several studies in mammals also suggest a mutually exclusive relationship between allele-specific DNA methylation and histone modification or among different histone modifications (Xin et al. 2001; Fournier et al. 2002; Carr et al. 2007; Lindroth et al. 2008; Singh et al. 2010; Hon et al. 2012).Here we report a genome-wide analysis of allele-specific DMRs, H3K27me3, and imprinted gene expression in maize endosperm. Thousands of pDMRs and allele-specific H3K27me3 peaks were identified. Correlation of pDMRs, allele-specific H3K27me3 profile, and the expression of imprinted genes showed that MEGs and PEGs have different patterns of DNA methylation and H3K27me3. This study reveals complex patterns of genetic imprinting regulation in maize endosperm.     

Immunogenicity and therapeutic effects of recombinant Ag85AB fusion protein vaccines in mice infected with Mycobacterium tuberculosis

The immune function of tuberculosis (TB) patients is disordered. By using immune regulators to assist chemotherapy for TB the curative effect might be improved. In this study, a vaccine containing Mycobacterium tuberculosis (M. tuberculosis) recombinant Ag85AB fusion protein (rAg85AB) was constructed and evaluated. The mice were immunized intramuscularly three times at two-week intervals with Ag85AB fusion protein combined with Corynebacterium parvum adjuvant (rAg85AB+CP). In comparison to control mice that received either CP alone or saline, the mice that received rAg85AB+CP had significantly higher number of T cells secreting IFN-γ and higher levels of specific antibodies of IgG, IgG1 and IgG2a isotypes in sera. The specific antibodies also had higher ratios of IgG2a to IgG1, indicating a predominant Th1 immune response. To test for immunotherapy of TB, M. tuberculosis infected mice were given three intramuscular doses of 20 μg, 40 μg or 60 μg of rAg85AB in rAg85AB+CP, or phosphate-buffered saline (PBS), or CP or Mycobacterium phlei (M. Phlei) F.U.36. Compared with the PBS group, 20 µg, 40 µg and 60 µg rAg85AB+CP and M. phlei F.U.36 groups reduced the pulmonary bacterial loads by 0.13, 0.15, 0.42 and 0.40 log₁₀, and the liver bacterial loads by 0.64, 0.64, 0.53 and 0.61 log₁₀, respectively. Pathological changes of lungs were less, and the lesions were limited to a certain extent in 40 µg and 60 µg rAg85AB+CP and M. phlei F.U.36 groups. These results showed that rAg85AB+CP had immunotherapeutic effect on TB, significantly increasing the cellular immune response, and inhibiting the growth of M. tuberculosis.     

Dynamics of HCV genotype 4 resistance-associated variants during virologic escape with pIFN/RBV + daclatasvir: A case study using ultra deep pyrosequencing

BackgroundDaclatasvir (DCV) is an approved NS5A inhibitor with potent anti-HCV activity and broad genotype coverage. DCV resistance-associated variants (RAVs) have been described for patients infected with genotype (GT) 1, but increased GT4 prevalence in European countries as a result of immigration has boosted interest in this genotype.ObjectivesEstablishment of NS5A variability in treatment-naive patients with HCV genotype 4 infection and a case study of the dynamics of resistance-associated variants in a virologic failure receiving pIFN/RBV + DCV, as assessed by ultra-deep sequencing.Study designFive treatment-naïve GT4 patients (GT4a [n = 1], GT4d [n = 3], GT4o [n = 1]) were evaluated for inclusion in the COMMAND-4 study and treatment with pIFN/RBV ± DCV.ResultsPatient (Pt) 1 received pIFN/RBV; Pts2–4 received pIFN/RBV + DCV; Pt5 was a screening failure. Pt1 relapsed; Pt2 experienced breakthrough at Wk4; Pts3 and 4 achieved a sustained virologic response. No substitutions associated with DCV-resistance were detected at baseline. In terms of viremic time points for Pts1 and 2, the extent of NS5A diversity pre-treatment was not significantly related to viral load (r = −0568; p = 0.035). In Pt2, multiple substitutions associated with DCV-resistance were observed after breakthrough at NS5A amino acid positions 28, 31 and 93. These substitutions were frequently observed on the same haplotype (L28S + M31I = 55.52, 82.50, and 99.36% at Wk4, 8 and 9; L28S + M31I + Y93H = 11.77, 5.01 and <0.6% at Wk4, 8 and 9).ConclusionsThis is the first report to describe DCV-resistance in patients infected with GT4d, supporting a possible role for a recently described RAV (L28S), and presenting the dynamics of HCV quasispecies during therapy failure, with indications of changes of diversity and association of mutations.     

Recovery of duck hepatitis A virus 3 from a stable full-length infectious cDNA clone

Recently, duck hepatitis A virus 3 (DHAV-3) with genetically distinct characteristics from DHAV-1 and DHAV-2 was recognized in South Korea and China. In this short communication, we successfully constructed a stable full-length infectious cDNA clone derived from DHAV-3 by solving instability of cloned full-length cDNA in Escherichia coli (E. coli). The cDNA fragments amplified from the genome of DHAV-3 were assembled and inserted into a low-copy-number plasmid. Finally, a full-length cDNA clone containing an engineered SacII site that served as a genetic marker was obtained. The cDNA clone showed stable by serial passages in E. coli when propagated at 25°C under low level of antibiotic selection. BHK-21 cells were transfected with transcribed RNA from the full-length cDNA clone; infectious viral particles were rescued, showing its fatality to 10-day-old duck embryos. The results indicated that the constructed full-length cDNA clone of DHAV-3 is infectious. By various virological assays, our results indicated that the rescued virus exhibited similar biological properties with the parental virus. Animal experiments revealed that the rescued virus retained the high pathogenicity to 1-day-old ducklings and could induce a fatal hepatitis indistinguishable from its parental virus. Our present studies provide a useful tool for future research on genomic functions and molecular pathogenesis of DHAV-3.     

A novel strain of <Emphasis Type="Italic">Beet western yellows virus</Emphasis> infecting sugar beet with two distinct genotypes differing in the 5′-terminal half of genome

The complete genomic sequences of two distinct Beet western yellows virus (BWYV) genotypes infecting sugar beet in Beijing, named as BWYV-BJ^A and BWYV-BJ^B (GenBank accession number HM804471, HM804472, respectively), were determined by RT-PCR sub-cloning approach. BWYV-BJ^A and BWYV-BJ^B were 5674 and 5626nt in length, respectively. BWYV-BJ^B was 48nt shorter than BWYV-BJ^A in the regions 1589–1615 and 1629–1649nt. Sequence alignment analysis showed that the full length of BWYV-BJ^A and BWYV-BJ^B shared 93% nucleotide sequence identity, with relatively high variability within ORFs 0, 1, 2 (at the nucleotide level was 86.3–88.8%) and high conservation within ORFs 3, 4, 5 (at the nucleotide level was 99.3–99.5%). The complete nucleotide sequences of BWYV-BJ^A and BWYV-BJ^B were most related to BWYV-US (80.6 and 79.0%, respectively). ORFs 1, 2 of BWYV-BJ^A and BWYV-BJ^B shared the highest homology with BWYV-US (nucleotide identity 91.2–93.3, 86.7–89.5%, respectively) and their ORFs 3, 4 were more closely related to BWYV-IM. However, their ORF5 were more closely related to that of Cucurbit aphid-borne yellows virus China strain (CABYV-CHN), with 68.1 and 68.5% nucleotide identity, respectively. Based on the sequence and phylogenetic analysis, we proposed that BWYV-BJ was at least a novel strain of BWYV, and BWYV-BJ^A, BWYV-BJ^B were two distinct genotypes of BWYV-BJ. In addition, phylogenetic analysis and recombination analysis suggested that BWYV-BJ^A and BWYV-BJ^B might be recombinant viruses.     

Polymorphism of prion protein gene in sheep of Inner Mongolian,China

Susceptibility to natural scrapie in sheep is associated with polymorphisms at codons 136, 154 and 171 of the prion protein (PrP) gene. To assess the risk of scrapie in sheep raised in China, DNA from 30 sheep of two breeds was isolated, amplified and sequenced for the PrP gene. The ovine PrP gene was found to be highly homogenous. The genotype associated with high susceptibility to scrapie (VRQ) was absent, whereas that associated with the resistance (ARR) was present in 6.7% of sheep examined. ARK was also rare (6.7%). ARQ that is associated with an intermediate susceptibility was the genotype observed in the most of sheep examined (86.6%). These data suggest that Chinese sheep of Mongolian sheep breed are susceptible to scrapie.     

Lectins but not antifungal proteins exhibit anti-nematode activity

A variety of lectins and antifungal proteins were tested for toxicity against the plant parasitic nematodes Ditylenchus dipsaci and Heterodera glycines. It was found that lectins from the mushrooms Xylaria hypoxylon, Agrocybe cylindracea and Tricholoma mongolicum (TML-1) were the most potent against D. dipsaci, with EC₅₀ being 4.7, 9, and 20 mg/ml, respectively. Lectins from Pseudostellaria heterophylla, samta tomato, and the mushrooms T. mongolicum (TML-2), Ganoderma lucidum, and Boletus edulis, and antifungal proteins from Ginkgo biloba toward D. dipsaci and pumpkin Cucurbita moschata had much lower anti-nematode potencies and could be considered as inactive for practical purposes. All lectins except that from P. heterophylle were potent against H. glycines.     

Distribution and molecular diversity of three cucurbit-infecting poleroviruses in China

Cucurbit aphid-borne yellows virus (CABYV) and Melon aphid-borne yellows virus (MABYV) have been found to be associated with cucurbit yellowing disease in China. Our report identifies for the first time a third distinct polerovirus, tentatively named Suakwa aphid-borne yellows virus (SABYV), infecting Suakwa vegetable sponge. To better understand the distribution and molecular diversity of these three poleroviruses infecting cucurbits, a total of 214 cucurbitaceous crop samples were collected from 25 provinces in China, and were investigated by RT-PCR and sequencing. Of these, 108 samples tested positive for CABYV, while 40 samples from five provinces were positive for MABYV, and SABYV was detected in only 4 samples which were collected in the southern part of China. Forty-one PCR-amplified fragments containing a portion of the RdRp gene, intergenic NCR and CP gene were cloned and sequenced. Sequence comparisons showed that CABYV isolates shared 78.0–79.2% nucleotide sequence identity with MABYV isolates, and 69.7–70.8% with SABYV. Sequence identity between MABYV and SABYV was 73.3–76.5%. In contrast, the nucleotide identities within each species were 93.2–98.7% (CABYV), 98.1–99.9% (MABYV), and 96.1–98.6% (SABYV). Phylogenetic analyses revealed that the polerovirus isolates fit into three distinct groups, corresponding to the three species. The CABYV group could be further divided into two subgroups: the Asia subgroup and the Mediterranean subgroup, based on CP gene and partial RdRp gene sequences. Recombination analysis suggested that MABYV may be a recombinant virus.     

Molecular characterization of the genome of duck enteritis virus

The genomic sequence of a strain of duck enteritis virus (DEV) was determined and analyzed in this study. The size of its genome is 158,091 bp in length and the genome is predicted to encode 78 putative proteins and resembles the members of the Alphaherpesvirinae in genomic organization and gene composition. The genome of the virus is composed of a unique long (UL) region, a unique short (US) region, a unique short internal repeat (IRS) region and a unique short terminal repeat (TRS) region. Its genomic arrangement pattern (UL-IRS-US-TRS) corresponds to D-type herpesvirus and is consistent with the members of Varicellovirus and Iltovirus genera. Sequence analysis reveals that the genome of the virus contains 67 genes having homologs in most members of the Alphaherpesvirinae. Out of these genes, one gene has a homolog in cercopithecine herpesvirus 8 which is a virus of Betaherpesvirinae, and 5 genes have homologs in avian herpesviruses. Furthermore, the genome possesses three unique genes without homologs in any other herpesviruses. Like most members of the Alphaherpesvirinae, the genes in the UL region of its genome are well conserved, whereas the gene arrangement of IRS-US is similar to that of Marek's disease virus and equine herpesviruses 1. Therefore, our data based on the genomic analysis suggest that DEV represents an osculant taxonomic entity within the Alphaherpesvirinae.