Identification and Validation of Human Papillomavirus Encoded microRNAs

Peer reviewe

Genomic convergence between Akkermansia muciniphila in different mammalian hosts

Background Akkermansia muciniphila is a member of the human gut microbiota where it resides in the mucus layer and uses mucin as the sole carbon, nitrogen and energy source. A. muciniphila is the only representative of the Verrucomicrobia phylum in the human gut. However, A. muciniphila 16S rRNA gene sequences have also been found in the intestines of many vertebrates. Results We detected A. muciniphila-like bacteria in the intestines of animals belonging to 15 out of 16 mammalian orders. In addition, other species belonging to the Verrucomicrobia phylum were detected in fecal samples. We isolated 10 new A. muciniphila strains from the feces of chimpanzee, siamang, mouse, pig, reindeer, horse and elephant. The physiology and genome of these strains were highly similar in comparison to the type strain A. muciniphila Muc(T). Overall, the genomes of the new strains showed high average nucleotide identity (93.9 to 99.7%). In these genomes, we detected considerable conservation of at least 75 of the 78 mucin degradation genes that were previously detected in the genome of the type strain Muc(T). Conclusions The low genomic divergence observed in the new strains may indicate that A. muciniphila favors mucosal colonization independent of the differences in hosts. In addition, the conserved mucus degradation capability points towards a similar beneficial role of the new strains in regulating host metabolic health.Peer reviewe

Genomic convergence between Akkermansia muciniphila in different mammalian hosts

Abstract Background Akkermansia muciniphila is a member of the human gut microbiota where it resides in the mucus layer and uses mucin as the sole carbon, nitrogen and energy source. A. muciniphila is the only representative of the Verrucomicrobia phylum in the human gut. However, A. muciniphila 16S rRNA gene sequences have also been found in the intestines of many vertebrates. Results We detected A. muciniphila-like bacteria in the intestines of animals belonging to 15 out of 16 mammalian orders. In addition, other species belonging to the Verrucomicrobia phylum were detected in fecal samples. We isolated 10 new A. muciniphila strains from the feces of chimpanzee, siamang, mouse, pig, reindeer, horse and elephant. The physiology and genome of these strains were highly similar in comparison to the type strain A. muciniphila MucT. Overall, the genomes of the new strains showed high average nucleotide identity (93.9 to 99.7%). In these genomes, we detected considerable conservation of at least 75 of the 78 mucin degradation genes that were previously detected in the genome of the type strain MucT. Conclusions The low genomic divergence observed in the new strains may indicate that A. muciniphila favors mucosal colonization independent of the differences in hosts. In addition, the conserved mucus degradation capability points towards a similar beneficial role of the new strains in regulating host metabolic health

<i>In situ</i> hybridization for HPV16-miR-H1-1 and HPV16-miR-H2-1.

<p>(A) Hematoxylin-eoxin (HE) staining, immunohistochemical staining for p16, and <i>in situ</i> (italics) hybridization for scramble (negative control), U6 (positive control), human miR-205 (positive control for cervical tissue), HPV16-miR-H1-1 (16-miR-H1-1) and HPV16-miR-H2-1 (16-miR-H2-1). Shown are two cervical intraepithelial neoplasia grade 1 (CIN 1) samples (samples 10 and 28), one squamous cell carcinoma (SCC) (sample 102), as well as normal squamous epithelium (SE) (sample 104) and normal columnar epithelium (CE) (sample 104). Arrows point to positive signals. (B) Areas of selected picture fields shown in higher magnification to depict localization and positive hybridization signal. Same numbering is used as in (A).</p

Locations and putative target sites of HPV 16 encoded microRNAs.

<p>Locations of HPV16-miR-H1-1 and HPV16-miR-H2-1 in the viral genome are shown as black bars and the predicted secondary structures are given next to the bars. For each miRNA, the seed sequences and predicted target sequences in the HPV genome are shown.</p

Predicted viral miRNA candidate.

<p>Each row presents one candidate miRNA with name, reference genome, pre-miRNA location in the genome, total read counts of pre-miRNA, viral gene annotation in corresponding region, strand information and mature miRNA sequence. Some miRNAs were shown in more than one isolate/subtype papillomavirus genomes. ^§ Prediction results from second round.</p

Summary of miRNA <i>in situ</i> hybridization (ISH), DNA PCR and p16 staining results.

<p>Expression of HPV16-miR-H1-1 was shown in all disease tissues. Expression of HPV16-miR-H2-1 was shown in only one carcinoma sample 102. CIN1-3, cervical intraepithelial neoplasia 1-3; SCC, squamous cell carcinoma; AIS, adenocarcinoma <i>in situ</i>; AC, adenocarcinoma; SE, squamous epithelium; CE, columnar epithelium. NA, not analyzed.</p

Mapped results of the twelve small RNA sequencing libraries.

<p>For each library, total reads from SOLiD small RNA sequencing and reads mapped to the papillomavirus reference genome are presented. Names of cell lines or histology of tissue samples are given in library description. Percentage of reads mapped to human miRNAs and to the human genome, respectively, are given. CIN, cervical intraepithelial neoplasia.</p

Summary of TaqMan miRNA qPCR, DNA PCR and p16 staining results, as well HPV detection and/or genotyping results by LDR, Luminex and Hybrid Capture 2.

<p>Obtained positive results in TaqMan qPCR out of performed runs are given. HPV6-miR-H1 did not give any positive results. Positive signals were obtained for HPV16-miR-H1, HPV16-miR-H2, HPV38-miR-H1 and HPV68-miR-H1 from cell lines and patient samples. Positive control U6 gives positive result in every reaction. The presence of pre-miRNA coding regions for HPV16-miR-H1 and HPV16-miR-H2 was confirmed by DNA PCR in all cell lines and many tissue samples. Results of p16 tissue staining, as well as results of HPV genotyping by LDR or Luminex, and high risk HPV detection by Hybrid Capture 2 are given. NA, not analyzed. ND, not done (inadequate sample). CIN1-3, cervical intraepithelial neoplasia 1–3; Cond pl, condyloma planum; SCC, squamous cell carcinoma; AIS, adenocarcinoma <i>in situ</i>; AC, adenocarcinoma; SE, squamous epithelium; CE, columnar epithelium; LDR, ligase detection reaction based HPV genotyping assay; Luminex, Luminex based HPV genotyping assay; HC2, Hybrid Capture 2 HPV detection assay; *, numbers are HPV types; NA, not analyzed; ND, no data (not enough material for testing).</p