HBV DNA Integration into Telomerase or MLL4 Genes and TERT Promoter Point Mutation as Three Independent Signatures in Subgrouping HBV-Related HCC with Distinct Features.

Introduction: A set of genetic mutations to classify hepatocellular carcinoma (HCC) useful to clinical studies is an unmet need. Hepatitis B virus-related HCC (HBV-HCC) harbors a unique genetic mutation, namely, the HBV integration, among other somatic endogenous gene mutations. We explored a combination of HBV DNA integrations and common somatic mutations to classify HBV-HCC by using a capture-sequencing platform. Methods: A total of 153 HBV-HCCs after surgical resection were subjected to capture sequencing to identify HBV integrations and three common somatic mutations in genomes. Three mutually exclusive mutations, HBV DNA integration into the TERT promoter, HBV DNA integration into MLL4, or TERT promoter point mutation, were identified in HBV-HCC. Results: They were used to classify HBV-HCCs into four groups: G1 with HBV-TERT integration (25.5%); G2 with HBV-MLL4 integration (10.5%); G3 with TERT promoter mutation (30.1%); and G4 without these three mutations (34.0%). Clinically, G3 has the highest male-to-female ratio, cirrhosis rate, and associated with higher early recurrence and mortality after resection, but G4 has the best outcome. Transcriptomic analysis revealed a grouping different from the published ones and G2 with an active immune profile related to immune checkpoint inhibitor response. Analysis of integrated HBV DNA provided clues for HBV genotype and variants in carcinogenesis of different HCC subgroup. This new classification was also validated in another independent cohort. Conclusion: A simple and robust genetic classification was developed to aid in understanding HBV-HCC and in harmonizing clinical studies.

Hepatitis B Virus DNA Integration Drives Carcinogenesis and Provides a New Biomarker for HBV-related HCC.

Hepatitis B virus (HBV) DNA integration is an incidental event in the virus replication cycle and occurs in less than 1% of infected hepatocytes during viral infection. However, HBV DNA is present in the genome of approximately 90% of HBV-related HCCs and is the most common somatic mutation. Whole genome sequencing of liver tissues from chronic hepatitis B patients showed integration occurring at random positions in human chromosomes; however, in the genomes of HBV-related HCC patients, there are integration hotspots. Both the enrichment of the HBV-integration proportion in HCC and the emergence of integration hotspots suggested a strong positive selection of HBV-integrated hepatocytes to progress to HCC. The activation of HBV integration hotspot genes, such as telomerase (TERT) or histone methyltransferase (MLL4/KMT2B), resembles insertional mutagenesis by oncogenic animal retroviruses. These candidate oncogenic genes might shed new light on HBV-related HCC biology and become targets for new cancer therapies. Finally, the HBV integrations in individual HCC contain unique sequences at the junctions, such as virus-host chimera DNA (vh-DNA) presumably being a signature molecule for individual HCC. HBV integration may thus provide a new cell-free tumor DNA biomarker to monitor residual HCC after curative therapies or to track the development of de novo HCC.

Cell-Free Virus-Host Chimera DNA From Hepatitis B Virus Integration Sites as a Circulating Biomarker of Hepatocellular Cancer.

BACKGROUND AND AIMS: Early recurrence of hepatocellular carcinoma (HCC) after surgical resection compromises patient survival. Timely detection of HCC recurrence and its clonality is required to implement salvage therapies appropriately. This study examined the feasibility of virus-host chimera DNA (vh-DNA), generated from junctions of hepatitis B virus (HBV) integration in the HCC chromosome, as a circulating biomarker for this clinical setting. APPROACH AND RESULTS: HBV integration in 50 patients with HBV-related HCC was determined by the Hybridization capture-based next-generation sequencing (NGS) platform. For individual HCC, the vh-DNA was quantified by specific droplet digital PCR (ddPCR) assay in plasma samples collected before and 2 months after surgery. HBV integrations were identified in 44 out of 50 patients with HBV-related HCC. Tumor-specific ddPCR was developed to measure the corresponding vh-DNA copy number in baseline plasma from each patient immediately before surgery. vh-DNA was detected in 43 patients (97.7%), and the levels correlated with the tumor sizes (detection limit at 1.5 cm). Among the plasma collected at 2 months after surgery, 10 cases (23.3%) still contained the same signature vh-DNA detected at baseline, indicating the presence of residual tumor cells. Nine of them (90%) experienced HCC recurrence within 1 year, supporting vh-DNA as an independent risk factor in predicting early recurrence. Analysis of circulating vh-DNA at recurrence further helped identify the clonal origin. A total of 81.8% of recurrences came from original HCC clones sharing the same plasma vh-DNA, whereas 18.2% were from de novo HCC. CONCLUSIONS: vh-DNA was shown to be a circulating biomarker for detecting the tumor load in majority of patients with HBV-related HCC and aided in monitoring residual tumor and recurrence clonality after tumor resection.

The complete mitochondrial genome of Meretrix lusoria (Bivalvia: Veneroida: Veneridae) from Kumamoto, Japan.

We sequenced and assembled the complete mitochondrial genome sequence of the Meretrix lusoria, from Kumamoto, Japan. The length of mitogenome is 20,180 bp, including 13 protein-coding genes, two ribosomal RNA genes, and 22 transfer RNA genes. The nucleotide composition of the mitogenome was 25.73% for A, 42.41% for T, 9.35% for C, and 22.49% for G. The AT and GC skewness of mitogenome sequence are -0.245 and 0.412, showing the T-skew and G-skew. The reconstructed phylogenetic relationships of 25 Bivalvia species based on 12 protein-coding genes were highly supported and the clade of all Meretrix clams included had a support value of 99%. Our results shall provide a better understanding in the evolutionary histories of the Veneroida and relative species.

Neutrophil Extracellular Traps Drive Mitochondrial Homeostasis in Tumors to Augment Growth.

Neutrophil infiltration and neutrophil extracellular traps (NET) in solid cancers are associated with poorer prognosis, but the mechanisms are incompletely understood. We hypothesized that NETs enhance mitochondrial function in tumor cells, providing extra energy for accelerated growth. Metastatic colorectal cancer tissue showed increased intratumoral NETs and supranormal preoperative serum MPO-DNA, a NET marker. Higher MPO-DNA correlated with shorter survival. In mice, subcutaneous tumor implants and hepatic metastases grew slowly in PAD4-KO mice, genetically incapable of NETosis. In parallel experiments, human cancer cell lines grew slower in nu/nu mice treated with DNAse, which disassembles NETs. PAD4-KO tumors manifested decreased proliferation, increased apoptosis, and increased evidence of oxidative stress. PAD4-KO tumors had decreased mitochondrial density, mitochondrial DNA, a lesser degree of ATP production, along with significantly decreased mitochondrial biogenesis proteins PGC1α, TFAM, and NRF-1. In vitro, cancer cells treated with NETs upregulated mitochondrial biogenesis-associated genes, increased mitochondrial density, increased ATP production, enhanced the percentage of cancer cells with reduced mitochondrial membrane potential, and increased the oxygen consumption rate. Furthermore, NETs increased cancer cells' expression of fission and fusion-associated proteins, DRP-1 and MFN-2, and mitophagy-linked proteins, PINK1 and Parkin. All of which were decreased in PAD4-KO tumors. Mechanistically, neutrophil elastase released from NETs activated TLR4 on cancer cells, leading to PGC1α upregulation, increased mitochondrial biogenesis, and accelerated growth. Taken together, NETs can directly alter the metabolic programming of cancer cells to increase tumor growth. NETs represent a promising therapeutic target to halt cancer progression. SIGNIFICANCE: Neutrophils through the release of NETs facilitate the growth of stressed cancer cells by altering their bioenergetics, the inhibition of which induces cell death.

The complete mitochondrial genome of Montipora aequituberculata (Scleractinia, Acroporidae).

The complete mitogenome of the hexacorallia, Montipora aequituberculata has been amplified and sequenced. The mitogenome consists of 17,886 bp, with 13 protein-coding genes, 2 ribosomal RNA genes, 2 transfer RNA genes and a control region. It has been observed that ND5 gene is split into two parts by a large fragment of genes, which commonly presented in scleractinian coral. The overall base composition of the H-strand was A, 24.91%; G, 24.1%; C, 14.2%; and T, 36.8%, with a slight AT bias of 61.7%. The control region was 627 bp in length and located between 12S rRNA and COIII gene. Based on the neighbour-joining (NJ) tree, M. aequituberculata was grouped with M. cactus, Anacropora matthai and Acropora tenuis, and formed a clade of Acroporidae. In conclusion, the complete mitogenome of M. aequituberculata data may provide more informative for phylogenetic approach for corals phylogeny.

Complete mitochondrial genome of the palemargin grouper Epinephelus bontoides (Pisces: Perciformes).

The complete mitogenome of the palemargin grouper, Epinephelus bontoides, was presented in this study. This mitochondrial genome consists of 16 903 bp, follow the typical gene arrangement with 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a non-coding control region (CR). The overall base composition was A, 28.7%; G, 16.1%; C, 28.0%; and T, 27.2%. The control region was 1200 bp in length, which located between tRNAPro and tRNAPhe, rich in A + T (69.2%) content. Based on the Neighbor-Joining (NJ) tree, E. bontoides was grouped with E. trimaculatus, E. quoyanus, E. areolatus, and E. bleekeri, and then combined with E. merra formed a clade. This complete mitogenome of E. bontoides can provide essential phylogenetic information of Epinephelus.

Alteration of colonic epithelial cell differentiation in mice deficient for glucosaminyl N-deacetylase/N-sulfotransferase 4.

Glucosaminyl N-deacetylase/N-sulfotransferases (NDSTs) are the first enzymes that mediate the initiation of heparan sulfate sulfation. We previously identified NDST4 as a putative tumor suppressor in human colorectal cancer. In the study, we generated an Ndst4 knockout (Ndst4-/-) mouse strain and explored its phenotypic characteristics, particularly in the development of colonic epithelial homeostasis. The Ndst4-deficient mice were viable and fertile, and their life spans were similar to those of wild-type littermates. No gross behavioral or morphological differences were observed between the Ndst4-/- and wild-type mice, and no significant changes were determined in the hematological or serum biochemical parameters of the Ndst4-/- mice. Ndst4 RNA transcripts were expressed in the brain, lung, gastrointestinal tract, pancreas, and ovary. However, Ndst4-null mice exhibited no gross or histological abnormalities in the studied organs, except for the colon. Although no alterations were observed in the crypt length or number of proliferating cells, the Ndst4-/- mice exhibited an increased number of goblet cells and a decreased number of colonocytes in the proximal colon compared with the wild-type mice. Moreover, Ndst4 deficiency increased the basal level of apoptosis in the colonic epithelium. Taken together, we established, for the first time, an Ndst4-/- mouse strain and revealed the involvement of Ndst4 in the development and homeostasis of colonic epithelium. Accordingly, NDST4 in human colon might direct the biosynthesis of specific heparan sulfate proteoglycans that are essential for the maintenance of colonic epithelial homeostasis. Thus, the loss of its function may result in the tumorigenesis and progression of colorectal cancer.

DNA barcoding reveals that the common cupped oyster in Taiwan is the Portuguese oyster Crassostrea angulata (Ostreoida; Ostreidae), not C. gigas.

The Pacific cupped oyster, Crassostrea gigas, is one of the major aquacultural shellfish species that has been introduced to Europe and America from its native source in the West Pacific. In Taiwan, the cultivated cupped oysters along the west coast have been identified as C. gigas for over centuries; however, several molecular phylogenetic studies have cast doubt upon the existence of this species in Taiwan and adjacent waters. Indeed, our analyses of mitochondrial cytochrome oxidase I (COI) sequences from 313 Crassostrea collected from 12 locations along Taiwanese and southern Chinese coastlines confirm that all samples were the Portuguese oyster, C. angulata, rather than C. gigas. Multiple lines of evidence, including haplotypic and nucleotide diversity of the COI gene, demographic history, and population genetics, suggest that Taiwanese C. angulata is unique, probably experienced a sudden population expansion after the Last Glacial Maxima around 20,000 years ago, and has a significantly limited genetic connectivity across the Taiwan Strait. Our study applies an extended sampling and DNA barcoding to confirm the absence of C. gigas in natural and cultivated populations in Taiwan and southern China, where we only found C. angulata. We highlight the importance of conserving the gene pool of the C. angulata population in Taiwan, particularly considering the current threats by large-scale environmental disturbances such as marine pollution, habitat destruction, and climate change.

Mitochondrial DNA sequence of Conus textile (Neogastropoda: Conidae).

The cone snail Conus textile belongs to the family Conidae. It is a kind of molluscivorous species. The complete mitochondrial DNA sequence was constructed by next-generation sequencing in this study. The mitogenome of C. textile is 15,765 bp in length, including 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes and 1 control region. The base composition was 27.3% A, 37.9% T, 15.7% C and 19.1% G. The phylogenetic tree of C. textile with the other 6 Conus species and 15 Neogastropoda sea snails was built. It provides fundamental data for further research of phylogeny and biogeography with this genus.

The complete mitochondrial genome of Conus capitaneus (Neogastropoda: Conidae).

The complete mitochondrial genome sequence of cone snail Conus capitaneus, a kind of worm-hunting sea snails, was performed by next-generation sequencing. The mitogenome is 15,829 bp in length, including 13 protein-coding genes, 22 tRNA genes, 2 ribosomal RNA genes (12S and 16S rRNA) and 1 control region. It has an overall base composition of A (25.6%), T (36.6%), C (16.3%) and G (21.5%). It shows 79.8% identity with C. tribblei, which also belongs to worm-hunting sea snail. The phylogenetic analysis was conducted with 21 closely related species to assess their phylogenetic relationship. The complete mitogenome of the C. capitaneus provides important DNA molecular data for further phylogeography.

The complete mitochondrial genome of Conus striatus (Neogastropoda: Conidae).

Conus striatus is a kind of piscivorous cone snail. We have sequenced it by next generation sequencing method. We used de novo assembly and reference mapping methods to assemble mitogenome. The mitochondrial genome is 15,738 bp, containing 13 protein coding genes, 22 transfer RNAs and 2 ribosomal RNAs genes. The overall base composition of C. striatus is 25.9% for A, 16.3% for C, 20.8% for G and 38.6% for T. The phylogenetic analysis was conducted with 18 related species and confirmed the classification status. The complete mitogenome of the C. striatus provides an essential and important DNA molecular data for further phylogeography and evolutionary analysis for cone snail phylogeny.

Complete mitochondrial genome of Junceella fragilis (Gorgonacea, Ellisellidae).

In this article, the complete mitogenome of the Octocorallia, zooxanthellate, Junceella fragilis has been amplified and sequenced. This mitochondrial genome consists of 18,724 bp, with 14 protein-coding genes, 2 ribosomal RNA genes, 1 transfer RNA genes, no intron was observed. It has been observed that a mitochondrial mismatch repair (mtMutS) gene was present in all octocorals. The overall base composition of the heavy strand was A, 29.1%; G, 20.4%; C, 33.0%; and T, 17.5%, with a slight AT bias of 62.1%. The complete mitogenomic data may provide more informative for phylogenetic approach for soft corals phylogeny especially for octocorallian species.

Complete mitochondrial genome of the sixblotch hind Cephalopholis sexmaculata (Pisces: Perciformes).

The complete mitogenome of the sixblotch hind, Cephalopholis sexmaculata was presented in this study. This mitochondrial genome consists of 16,589 bp, with 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and a noncoding control region (CR), and its gene arrangement is identical to most vertebrates. The overall base composition of the heavy strand is A, 29.35%; G, 16.08%; C, 28.56%; and T, 26.01%. The COI gene started with GTG codon and the ATP6 gene started with CTG codon. The complete mitogenomic data may provide informative for further phylogenetic approach of species of Cephalopholis and related genera belong to the Epinephelidae groupers.

The complete mitochondrial genome of Conus tulipa (Neogastropoda: Conidae).

The complete mitogenome sequence of the cone snail Conus tulipa (Linnaeus, 1758) has been sequenced by next-generation sequencing method. The assembled mitogenome is 16,599 bp in length, including 13 protein-coding genes, 22 transfer RNA genes and 2 ribosomal RNA genes. The overall base composition of C. tulipa is 28.7% A, 15.2% C, 18.4% G and 37.7% T. It shows 81.1% identity to the cone snail C. consors, 78.5% to C. borgesi and 77.5% to C. textile. Using the 13 protein-coding genes and 2 ribosomal RNA genes of C. tulipa in this study, together with 18 other closely species, we constructed the species phylogenetic tree to verify the accuracy and utility of new determined mitogenome sequence. The complete mitogenome of the C. tulipa provides an essential and important DNA molecular data for further phylogeography and evolutionary analysis for cone snail phylogeny.

DNA Hypermethylation of SHISA3 in Colorectal Cancer: An Independent Predictor of Poor Prognosis.

BACKGROUND: Shisa3 is a novel tumor suppressor identified in lung cancer. However, its antitumor activity in other human cancers and the mechanism of gene inactivation remain unknown. METHODS: SHISA3 expression was measured by reverse transcription-PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR). DNA methylation was determined by bisulfite sequencing and pyrosequencing. RESULTS: Down-regulation of SHISA3 expression was observed in all of 11 colorectal cancer (CRC) cell lines and was further confirmed in 34 (65.4 %) of 52 colorectal carcinomas by RT-qPCR. Four of six CRC cell lines could restore SHISA3 expression after treatment with 5-aza-2'-deoxycytidine. Tumor-specific methylation of five CpG sites in the first intron of SHISA3 was identified by bisulfite sequencing, and their methylation levels were quantified in 127 pairs of primary CRC tissues by bisulfite pyrosequencing. The methylation levels of SHISA3 in tumors were noticeably higher than that in their matched normal mucosae. In addition, SHISA3 hypermethylation was significantly associated with an increased risk of disease recurrence in patients with stage II and III disease (P = 0.007) and was an independent predictor of poor overall survival [hazard ratio (HR) 2.9, 95 % confidence interval (CI) 1.5-5.8; P = 0.002] and disease-free survival (HR 4.0, 95 % CI 1.6-10.2; P = 0.003) of CRC patients. CONCLUSIONS: SHISA3 gene is epigenetically inactivated in a substantial fraction of CRC, and its hypermethylation is of prognostic significance in predicting clinical outcome. The quantitative bisulfite pyrosequencing assay established could be a cost-effective tool for providing a potential biomarker of adverse prognosis in CRC.

Complete mitochondrial genome of the amphidromous, red-tailed goby Sicyopterus lagocephalus (Pallas) (Teleostei, Gobiidae).

In this article, the complete mitogenome of the amphidromous, red-tailed goby, Sicyopterus lagocephalus has been amplified and sequenced by long polymerase chain reaction. This mitochondrial genome consists of 16,500 bp, with 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a non-coding control region (CR), and its gene arrangement is identical to those of most vertebrates. The CR (841 bp) is located between tRNA(Pro) and tRNA(Phe). The overall base composition of the heavy strand is A, 28.9%; G, 16.4%; C, 28.3%; and T, 26.4%, with a slight AT bias of 55.3%. The complete mitogenomic data may provide more informative for phylogenetic approach for gobioid phylogeny especially for Sicydiine gobies.

Unusual distribution of floating seaweeds in the East China Sea in the early spring of 2012.

Floating seaweeds play important ecological roles in offshore waters. Recently, large amounts of rafting seaweed have been observed in the East China Sea. In early spring, juveniles of commercially important fish such as yellowtail accompany these seaweed rafts. Because the spatial distributions of seaweed rafts in the spring are poorly understood, research cruises were undertaken to investigate them in 2010, 2011, and 2012. Floating seaweed samples collected from the East China Sea during the three surveys contained only Sargassum horneri. In 2010 and 2011, seaweed rafts were distributed only in the continental shelf and the Kuroshio Front because they had become trapped in the convergence zone of the Kuroshio Front. However, in 2012, seaweed was also distributed in the Kuroshio Current and its outer waters, and massive strandings of seaweed rafts were observed on the northern coast of Taiwan and on Tarama Island in the Ryukyu Archipelago. Environmental data (wind, currents, and sea surface height) were compared among the surveys of 2010, 2011, and 2012. Two factors are speculated to have caused the unusual distribution in 2012. First, a continuous strong north wind produced an Ekman drift current that transported seaweed southwestward to the continental shelf and eventually stranded seaweed rafts on the coast of Taiwan. Second, an anticyclonic eddy covering northeast Taiwan and the Kuroshio Current west of Taiwan generated a geostrophic current that crossed the Kuroshio Current and transported the rafts to the Kuroshio Current and its outer waters. Such unusual seaweed distributions may influence the distribution of fauna accompanying the rafts.

Protocadherin 10 suppresses tumorigenesis and metastasis in colorectal cancer and its genetic loss predicts adverse prognosis.

Protocadherin 10 (PCDH10), a novel tumor suppressor gene in human cancers, is located in a common deleted region at chromosome 4q28 in colorectal cancer (CRC). This study aimed to ascertain the genetic loss of PCDH10 and its clinical relevance in CRC and to explore the tumor suppressor function of PCDH10. The genetic deletion of PCDH10 was determined in 171 pairs of primary tumors and corresponding normal mucosae by loss of heterozygosity study. In total, 53 carcinomas were positive for allelic loss of PCDH10. The genetic aberration was significantly associated with tumor progression and distant metastasis (p = 0.021 and p = 0.018, respectively) and was an independent predictor of poor survival for CRC patients (p = 0.005). Expression of PCDH10 gene was silenced or markedly down-regulated in all of 12 CRC cell lines tested and in 41 of 53 colorectal carcinomas compared with their matched normal mucosae. Ectopic expression of PCDH10 suppressed cancer cell proliferation, anchorage-independent growth, migration and invasion in vitro. Subcutaneous injection of PCDH10-expressing CRC cells into SCID mice revealed the reduction of tumor growth compared with that observed in mock-inoculated mice. Furthermore, through intrasplenic implantation, the re-expression of PCDH10 in silenced cells restrained liver metastasis and improved survival in SCID mice. In conclusion, PCDH10 is a pivotal tumor suppressor in CRC, and the loss of its function promotes not only tumor progression but also liver metastasis. In addition, the genetic deletion of PCDH10 represents an adverse prognostic marker for the survival of patients with CRC.