Expression of HPV16 E5 Produces Enlarged Nuclei and Polyploidy through Endoreplication

Anogenital cancers and head and neck cancers are causally-associated with infection by high-risk human papillomavirus (HPV). The mechanism by which high-risk HPVs contribute to oncogenesis is poorly understood. HPV16 encodes three genes (HPV16 E5, E6, and E7) that can transform cells when expressed independently. HPV16 E6 and E7 have well-described roles causing genomic instability and unregulated cell cycle progression. The role of HPV16 E5 in cell transformation remains to be elucidated. Expression of HPV16 E5 results in enlarged, polyploid nuclei that are dependent on the level and duration of HPV16 E5 expression. Live-cell imaging data indicate these changes do not arise from cell-cell fusion or failed cytokinesis. The increase in nuclear size is a continual process that requires DNA synthesis. We conclude HPV16 E5 produces polyploid cells by endoreplication. These findings provide insight into how HPV16 E5 can contribute to cell transformation

The First Illumina-Based De Novo Transcriptome Sequencing and Analysis of Safflower Flowers

BACKGROUND: The safflower, Carthamus tinctorius L., is a worldwide oil crop, and its flowers, which have a high flavonoid content, are an important medicinal resource against cardiovascular disease in traditional medicine. Because the safflower has a large and complex genome, the development of its genomic resources has been delayed. Second-generation Illumina sequencing is now an efficient route for generating an enormous volume of sequences that can represent a large number of genes and their expression levels. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the genes and pathways that might control flavonoids and other secondary metabolites in the safflower, we used Illumina sequencing to perform a de novo assembly of the safflower tubular flower tissue transcriptome. We obtained a total of 4.69 Gb in clean nucleotides comprising 52,119,104 clean sequencing reads, 195,320 contigs, and 120,778 unigenes. Based on similarity searches with known proteins, we annotated 70,342 of the unigenes (about 58% of the identified unigenes) with cut-off E-values of 10(-5). In total, 21,943 of the safflower unigenes were found to have COG classifications, and BLAST2GO assigned 26,332 of the unigenes to 1,754 GO term annotations. In addition, we assigned 30,203 of the unigenes to 121 KEGG pathways. When we focused on genes identified as contributing to flavonoid biosynthesis and the biosynthesis of unsaturated fatty acids, which are important pathways that control flower and seed quality, respectively, we found that these genes were fairly well conserved in the safflower genome compared to those of other plants. CONCLUSIONS/SIGNIFICANCE: Our study provides abundant genomic data for Carthamus tinctorius L. and offers comprehensive sequence resources for studying the safflower. We believe that these transcriptome datasets will serve as an important public information platform to accelerate studies of the safflower genome, and may help us define the mechanisms of flower tissue-specific and secondary metabolism in this non-model plant

Safflower flower phenotypes and schematics of the transcriptome sequencing analysis.

<p>(A) Phenotypes of the red/orange (left) and white (mutant; right) flowers of <i>Carthamus tinctorius</i> L. used in this study. (B) Overall workflow of the experiment. (C) Overall workflow of the data assembly. (D) Overall workflow of the bioinformatic analysis.</p

Summary of the sequence assembly after Illumina sequencing.

<p>Summary of the sequence assembly after Illumina sequencing.</p

qRT-PCR analysis of 12 flavonoid biosynthetic pathway-related candidate unigenes in white and red flowers.

<p>W2, flowering day 2 of the white flower; W4, flowering day 4 of the white flower; R2, flowering day 2 of the red flower; R4, flowering day 4 of the red flower. The numbers after the gene name means the serial number of unigene. The gene sequences used for qRT-PCR analysis are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038653#pone.0038653.s005" target="_blank">File S5</a>.</p

Overview of the safflower flower tissue transcriptome assembly.

<p>(A) The size distribution of the contigs obtained from our <i>de novo</i> assembly of high-quality clean reads. (B) The size distribution of the unigenes produced from further assembly of contigs (i.e., contig joining, gap filling, and scaffold clustering). (C) The size distribution of the CDS produced by searching unigene sequences against various protein databases (Nr, SwissProt, KEGG and COG, in order) using BLASTX (E-value <10⁻⁵). (D) The size distribution of the proteins predicted from the CDS sequences. (E) And (F) Size distributions of the ESTs and proteins obtained from the ESTScan results. For unigene CDS that had no hits in the databases (Nr, SwissProt, KEGG and COG), the BLAST results were subjected to ESTScans and then translated into peptide sequences.</p

Predicted flavonoid biosynthetic pathway-related genes and products in the safflower flower.

<p>The pathways leading to the biosynthesis of different flavonoids. Abbreviations: CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone3-hydroxylase; F3′H, flavonoid 3′-hydroxylase;F3′5′H, flavonoid 3′,5′-hydroxylase; FLS, flavonol synthase; DFR, dihydroflavonol 4-reductase; LAR, leucoanthocyanidin reductase; 3GT, flavonol 3-O-glucosyltransferase; and 3GRT, flavonol-3-O-glucoside L-rhamnosyltransferase. The dotted arrow indicates that the involved enzymes are not yet clear.</p

Phylogenetic analysis and virulence characteristics of methicillin-resistant Staphylococcus aureus ST764-SCCmec II: an emerging hypervirulent clone ST764-t1084 in China

ABSTRACTPrevious studies have shown that the increased prevalent ST764 clone in China, Japan, and other Asian areas. However, the knowledge of the genetic features and virulence characteristics of methicillin-resistant Staphylococcus aureus (MRSA) ST764 in China is still limited. In this study, we identified 52 ST764-SCCmec type II isolates collected from five cities in China between 2014 and 2021. Whole genome sequencing showed that the most common staphylococcal protein A (spa) types of ST764 in China were t002 (55.78%) and t1084 (40.38%). Virulence assays showed that ST764-t1084 isolates had high haemolytic activity and α-toxin levels. Of the critical regulatory factors affecting α-toxin production, only the SaeRS was highly expressed in ST764-t1084 isolates. Mouse abscess model indicated that the virulence of ST764-t1084 isolates was comparable to that of S. aureus USA300-LAC famous for its hypervirulence. Interestingly, ST764-t002 isolates exhibited stronger biofilm formation and cell adhesion capacities than ST764-t1084 isolates. This seems to explain why ST764-t002 subclone has become more prevalent in China in recent years. Phylogenetic analysis suggested that all ST764 isolates from China in Clade III were closely related to KUN1163 (an isolate from Japan). Notably, genomic analysis revealed that the 52 ST764 isolates did not carry arginine catabolic mobile element (ACME), which differed from ST764 isolates in Japan. Additionally, most ST764 isolates (69.23%) harboured an obvious deletion of approximately 5 kb in the SCCmec II cassette region compared to KUN1163. Our findings shed light on the potential global transmission and genotypic as well as phenotypic characteristics of ST764 lineage