Plasma cell free DNA methylation markers for hepatocellular carcinoma surveillance in patients with cirrhosis: a case control study.

BACKGROUND: Hepatocellular carcinoma (HCC) is the leading cause of death in patients with cirrhosis, primarily due to failed early detection. HCC screening is recommended among individuals with cirrhosis using biannual abdominal ultrasound, for earlier tumor detection, administration of curative treatment, and improved survival. Surveillance by imaging with or without biomarkers such as alpha-fetoprotein (AFP) remains suboptimal for early stage HCC detection. Here we report on the development and assessment of methylation biomarkers from liquid biopsies for HCC surveillance in cirrhotic patients. METHODS: DNA methylation markers including the HCCBloodTest (Epigenomics AG) and a DNA-methylation panel established by next generation sequencing (NGS) were assessed using a training/testing design. The NGS panel algorithm was established in a training study (41 HCC patients; 46 cirrhotic non-HCC controls). For testing, plasma samples were obtained from cirrhotic patients (Child class A or B) with (60) or without (103) early stage HCC (BCLC stage 0, A, B). The assays were then tested using blinded sample sets and analyzed by preset algorithms. RESULTS: The HCCBloodTest and the NGS panel exhibited 76.7% and 57% sensitivities at 64.1% and 97% specificity, respectively. In a post-hoc analysis, a combination of the NGS panel with AFP (20 ng/mL) achieved 68% sensitivity at 97% specificity (AUC = 0.9). CONCLUSIONS: Methylation biomarkers in cell free plasma DNA provide a new alternative for HCC surveillance. Multiomic panels comprising DNA methylation markers with other biological markers, such as AFP, provide an option to further increase the overall clinical performance of surveillance via minimally invasive blood samples. TRIAL REGISTRATION: Test set study-ClinicalTrials.gov (NCT03804593) January 11, 2019, retrospectively registered.

Transcriptome analysis of enriched Golovinomyces orontii haustoria by deep 454 pyrosequencing.

Powdery mildews are phytopathogenic ascomycetes that have an obligate biotrophic lifestyle and establish intimate relationships with their plant hosts. A crucial aspect of this plant-fungus interaction is the formation of specialized fungal infection structures termed haustoria. Although located within the cell boundaries of plant epidermal cells, haustoria remain separated from the plant cytoplasm by a host plasma membrane derivative, the extrahaustorial membrane. Haustoria are thought to represent pivotal sites of nutrient uptake and effector protein delivery. We enriched haustorial complexes from Arabidopsis thaliana plants infected with the powdery mildew fungus Golovinomyces orontii and performed in-depth transcriptome analysis by 454-based pyrosequencing of haustorial cDNAs. We assembled 7077 expressed sequence tag (EST) contigs with greater than 5-fold average coverage and analyzed these with regard to the respective predicted protein functions. We found that transcripts coding for gene products with roles in protein turnover, detoxification of reactive oxygen species and fungal pathogenesis are abundant in the haustorial EST contigs, while surprisingly transcripts encoding presumptive nutrient transporters were not highly represented in the haustorial cDNA library. A substantial proportion (∼38%) of transcripts coding for predicted secreted proteins comprises effector candidates. Our data provide valuable insights into the transcriptome of the key infection structure of a model obligate biotrophic phytopathogen.

A small, variable, and irregular killer cell Ig-like receptor locus accompanies the absence of MHC-C and MHC-G in gibbons.

The killer cell Ig-like receptors (KIRs) of NK cells recognize MHC class I ligands and function in placental reproduction and immune defense against pathogens. During the evolution of monkeys, great apes, and humans, an ancestral KIR3DL gene expanded to become a diverse and rapidly evolving gene family of four KIR lineages. Characterizing the KIR locus are three framework regions, defining two intervals of variable gene content. By analysis of four KIR haplotypes from two species of gibbon, we find that the smaller apes do not conform to these rules. Although diverse and irregular in structure, the gibbon haplotypes are unusually small, containing only two to five functional genes. Comparison with the predicted ancestral hominoid KIR haplotype indicates that modern gibbon KIR haplotypes were formed by a series of deletion events, which created new hybrid genes as well as eliminating ancestral genes. Of the three framework regions, only KIR3DL3 (lineage V), defining the 5' end of the KIR locus, is present and intact on all gibbon KIR haplotypes. KIR2DL4 (lineage I) defining the central framework region has been a major target for elimination or inactivation, correlating with the absence of its putative ligand, MHC-G, in gibbons. Similarly, the MHC-C-driven expansion of lineage III KIR genes in great apes has not occurred in gibbons because they lack MHC-C. Our results indicate that the selective forces shaping the size and organization of the gibbon KIR locus differed from those acting upon the KIR of other hominoid species.

A novel system of polymorphic and diverse NK cell receptors in primates.

There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in "higher" primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire.

Deep sequencing of naupliar-, cyprid- and adult-specific normalised Expressed Sequence Tag (EST) libraries of the acorn barnacle Balanus amphitrite.

In order to improve the genetic characterisation of the barnacle Balanus amphitrite, normalised EST libraries for the developmental stages, viz. nauplius (a mix of instars I and II), cyprid and adult, were generated. The libraries were sequenced independently using 454 technologies and 575,666 reads were generated. For adults, 4843 unique isotigs were estimated and 6754 and 7506 in the cyprid and naupliar stage, respectively. It was found that some of the previously proposed cyprid-specific bcs genes were also expressed during the naupliar and adult stage. Furthermore, as lectins have been hypothesised to influence settlement cue recognition in barnacles, the database was searched for lectin-like isotigs. Two proteins, uniquely expressed in either the cyprid or the adult stage, matched a mannose receptor, and their nucleotide sequences were 33% and 31% identical to a lectin (BRA-3) isolated from Megabalanus rosa. Further characterisation of these genes may suggest their involvement in settlement.

Genome comparison of the epiphytic bacteria Erwinia billingiae and E. tasmaniensis with the pear pathogen E. pyrifoliae.

BACKGROUND: The genus Erwinia includes plant-associated pathogenic and non-pathogenic Enterobacteria. Important pathogens such as Erwinia amylovora, the causative agent of fire blight and E. pyrifoliae causing bacterial shoot blight of pear in Asia belong to this genus. The species E. tasmaniensis and E. billingiae are epiphytic bacteria and may represent antagonists for biocontrol of fire blight. The presence of genes that are putatively involved in virulence in E. amylovora and E. pyrifoliae is of special interest for these species in consequence. RESULTS: Here we provide the complete genome sequences of the pathogenic E. pyrifoliae strain Ep1/96 with a size of 4.1 Mb and of the non-pathogenic species E. billingiae strain Eb661 with a size of 5.4 Mb, de novo determined by conventional Sanger sequencing and next generation sequencing techniques. Genome comparison reveals large inversions resulting from homologous recombination events. Furthermore, comparison of deduced proteins highlights a relation of E. billingiae strain Eb661 to E. tasmaniensis strain Et1/99 and a distance to E. pyrifoliae for the overall gene content as well as for the presence of encoded proteins representing virulence factors for the pathogenic species. Pathogenicity of E. pyrifoliae is supposed to have evolved by accumulation of potential virulence factors. E. pyrifoliae carries factors for type III secretion and cell invasion. Other genes described as virulence factors for E. amylovora are involved in the production of exopolysaccharides, the utilization of plant metabolites such as sorbitol and sucrose. Some virulence-associated genes of the pathogenic species are present in E. tasmaniensis but mostly absent in E. billingiae. CONCLUSION: The data of the genome analyses correspond to the pathogenic lifestyle of E. pyrifoliae and underlines the epiphytic localization of E. tasmaniensis and E. billingiae as a saprophyte.

European sea bass genome and its variation provide insights into adaptation to euryhalinity and speciation.

The European sea bass (Dicentrarchus labrax) is a temperate-zone euryhaline teleost of prime importance for aquaculture and fisheries. This species is subdivided into two naturally hybridizing lineages, one inhabiting the north-eastern Atlantic Ocean and the other the Mediterranean and Black seas. Here we provide a high-quality chromosome-scale assembly of its genome that shows a high degree of synteny with the more highly derived teleosts. We find expansions of gene families specifically associated with ion and water regulation, highlighting adaptation to variation in salinity. We further generate a genome-wide variation map through RAD-sequencing of Atlantic and Mediterranean populations. We show that variation in local recombination rates strongly influences the genomic landscape of diversity within and differentiation between lineages. Comparing predictions of alternative demographic models to the joint allele-frequency spectrum indicates that genomic islands of differentiation between sea bass lineages were generated by varying rates of introgression across the genome following a period of geographical isolation.

Onset of immune senescence defined by unbiased pyrosequencing of human immunoglobulin mRNA repertoires.

The immune system protects us from foreign substances or pathogens by generating specific antibodies. The variety of immunoglobulin (Ig) paratopes for antigen recognition is a result of the V(D)J rearrangement mechanism, while a fast and efficient immune response is mediated by specific immunoglobulin isotypes obtained through class switch recombination (CSR). To get a better understanding on how antibody-based immune protection works and how it changes with age, the interdependency between these two parameters need to be addressed. Here, we have performed an in depth analysis of antibody repertoires of 14 healthy donors representing different gender and age groups. For this task, we developed a unique pyrosequencing approach, which is able to monitor the expression levels of all immunoglobulin V(D)J recombinations of all isotypes including subtypes in an unbiased and quantitative manner. Our results show that donors have individual immunoglobulin repertoires and cannot be clustered according to V(D)J recombination patterns, neither by age nor gender. However, after incorporating isotype-specific analysis and considering CSR information into hierarchical clustering the situation changes. For the first time the donors cluster according to age and separate into young adults and elderly donors (>50). As a direct consequence, this clustering defines the onset of immune senescence at the age of fifty and beyond. The observed age-dependent reduction of CSR ability proposes a feasible explanation why reduced efficacy of vaccination is seen in the elderly and implies that novel vaccine strategies for the elderly should include the "Golden Agers".

Analysis of single nucleotide polymorphisms in three chromosomes of European sea bass Dicentrarchus labrax.

Single nucleotide polymorphisms (SNPs) are believed to contain relevant information and have been therefore extensively used as genetic markers in population and conservation genetics, and molecular ecology studies. This study reports on the identification of potential SNPs in a diploid European sea bass Dicentrarchus labrax genome by using reference sequences from three assembled chromosomes and mapping all WGS datasets onto them (3× Sanger, 3× 454 and 20× SOLEXA). A total of 20,779 SNPs were identified over the 1469 gene loci and intergenic space analysed. Within chromosomes the occurrence of SNPs was the lowest in exons and higher in introns and intergenic regions, which may be explained by the fact, that coding regions are under strong selective pressure to maintain their biological function. The ratio of nonsynonymous to synonymous mutations was smaller than one for all the chromosomes, suggesting that most of deleterious nonsynonymous mutations were eliminated by negative selection. SNPs were not uniformly distributed over the chromosomes. Two chromosomes exhibited large regions with extremely low SNP density, which might represent homozygous regions in the diploid genome. The results of this study show how SNP detection can take profit from sequencing a single diploid individual, but also uncover the limits of such an approach. SNPs that have been identified will support marker development for genetic linkage mapping, population genetics and aquaculture related questions in general.

Analyses of the vrl gene cluster in Desulfococcus multivorans: homologous to the virulence-associated locus of the ovine footrot pathogen Dichelobacter nodosus strain A198.

Major parts of the virulence-associated vrl locus known from the gammaproteobacterium Dichelobacter nodosus, the causative agent of ovine footrot, were analyzed in the genome of the sulfate-reducing deltaproteobacterium Desulfococcus multivorans. In the genome of D. multivorans 13 of the 19 vrl genes described for D. nodosus are present and highly conserved with respect to gene sequence and order. The vrl locus and its flanking regions suggest a bacteriophage-mediated transfer into the genome of D. multivorans. Comparative analysis of the deduced Vrl proteins reveals a wide distribution of parts of the virulence-associated vrl locus in distantly related bacteria. Horizontal transfer is suggested as driving mechanism for the circulation of the vrl genes in bacteria. Except for the vrlBMN genes D. multivorans and Desulfovibrio desulfuricans G20 together contain all vrl genes displaying a high degree of similarity. For D. multivorans it could be shown that guanine plus cytosine (GC) content, GC skew, di-, tri- or tetranucleotide distribution did not differ between the vrl locus and its flanking sequences. This could be a hint that the vrl locus originated from a related organism or at least a genome with similar characteristics. The conspicuous high degree of conservation of the analyzed vrl genes may result from a recent transfer event or reflect a function of the vrl genes, which is still unknown and not necessarily disease associated. The latter is supported by the evidence for expression of the vrl genes in D. multivorans, which has not been described as pathogen or to be associated to any disease pattern before.

Whole genome analysis of the marine Bacteroidetes'Gramella forsetii' reveals adaptations to degradation of polymeric organic matter.

Members of the Bacteroidetes, formerly known as the Cytophaga-Flavobacteria-Bacteroides (CFB) phylum, are among the major taxa of marine heterotrophic bacterioplankton frequently found on macroscopic organic matter particles (marine snow). In addition, they have been shown to also represent a significant part of free-living microbial assemblages in nutrient-rich microenvironments. Their abundance and distribution pattern in combination with enzymatic activity studies has led to the notion that organisms of this group are specialists for degradation of high molecular weight compounds in both the dissolved and particulate fraction of the marine organic matter pool, implying a major role of Bacteroidetes in the marine carbon cycle. Despite their ecological importance, comprehensive molecular data on organisms of this group have been scarce so far. Here we report on the first whole genome analysis of a marine Bacteroidetes representative, 'Gramella forsetii' KT0803. Functional analysis of the predicted proteome disclosed several traits which in joint consideration suggest a clear adaptation of this marine Bacteroidetes representative to the degradation of high molecular weight organic matter, such as a substantial suite of genes encoding hydrolytic enzymes, a predicted preference for polymeric carbon sources and a distinct capability for surface adhesion.